More resistant tendons obtained from the association of Heteropterys aphrodisiaca and endurance training

Juliana C Monteiro
Marcos Lm Gomes
Tatiana C Tomiosso
Wilson R Nakagaki
Mariana M Sbervelheri
Danilo L Ferrucci
Edson R Pimentel
Heidi Dolder
More resistant tendons obtained from the association of Heteropterys aphrodisiaca and endurance training
R E S E A R C H A R T I C L E Open Access
Background: Popular Brazilian medicine uses Heteropterys aphrodisiaca infusion as a tonic or stimulant, for the treatment of nervous debility and breakdown and for muscle and bone weakness. This study investigated the effects of Heteropterys aphrodisiaca infusion on the tendon properties and extracellular matrix of rats under endurance training. Methods: Wistar rats were grouped as follows: CS-control sedentary, HS-H. aphrodisiaca sedentary, CT-control trained, HT-H. aphrodisiaca trained. The training protocol consisted in running on a motorized treadmill, five times a week, with weekly increase in treadmill speed and duration. Control groups received water while the HS and HT groups received H. aphrodisiaca infusion, daily, by gavage for the 8 weeks of training. Achilles tendons were frozen for biochemical and biomechanical analysis or preserved in Karnovsky's fixative, then processed for histomorphological analysis with light microscopy. Results: Biomechanical analysis showed significant increase in maximum load, maximum stress, modulus of elasticity and stiffness of the HT animals' tendons. The metalloproteinase-2 activity was reduced in the HT group. The compression region of HT animals' tendons had a stronger and more intense metachromasy, which suggests an increase in glycosaminoglycan concentration in this region of the tendon. The most intense birefringence was observed in both compression and tension regions of HT animals' tendons, which may indicate a higher organizational level of collagen bundles. The hydroxyproline content increased in the HT group. Conclusions: The association of endurance training with H. aphrodisiaca resulted in more organized collagen bundles and more resistant tendons to support higher loads from intense muscle contraction. Despite the clear anabolic effects of Heteropterys aphrodisiaca and the endurance exercise association, no side effects were observed, such as those found for synthetic anabolic androgenic steroids.

Background

Heteropterys aphrodisiaca O. Mach. (Malpighiaceae), also known as "nó-de-cachorro", "nó-de-porco" and "cordão-de-São-Francisco", was described by Hoehne in 1920 as a plant with stimulative and aphrodisiac properties. Also, Brazilian traditional medicine uses H. aphrodisiaca root infusion as a tonic or stimulating treatment and for nervous debility, nervous breakdown and for muscle and bone weakness. This plant is found mainly in the "Cerrado" regions (a savanna-like biome) of Mato Grosso and Goiás States (Brazil) [1,2].

Previous studies with H. aphrodisiaca suggested that the root extract could increase corporal and testicular weight, as well as Leydig cell volume within the rat testis [3,4]. Leydig cells produce testosterone, thus regulating muscle protein metabolism, erythropoiesis, plasma lipids, bone metabolism and cognitive functions [5]. Since testosterone regulates growth, structure and functions of accessory sex organs [6] and the treatment with H. aphrodisiaca infusion showed no alterations of these organs' weights, Chieregato [3] suggested this hormone resulted in weight gain since it acts on the skeletal muscle mass.

Higher doses of anabolic androgenic steroids (AAS), when combined with a training program, have been claimed to increase muscle strength and mass [7]. However, some reports have mentioned that tendons do not follow the same rate of protein gain as achieved by the muscle bulk, and therefore high intensity and frequency of training might lead to tendon ruptures [8,9]. Some studies using animals suggested that steroids modify the collagen crimp pattern and tendon biomechanical properties [10]. Steroid use may cause collagen dysplasia in the flexor digitorum tendon of mice [11], increasing tendon stiffness and diminishing both elongation and energy absorption [12,13]. Thus, such tendons are more likely to fail during strenuous activities. In addition, AAS treatment can impair tendon tissue remodeling by down regulating matrix metalloproteinase (MMP) activity in animals undergoing physical exercise, hence increasing the potential for tendon injury [14]. In human beings, morphological changes at the muscle-tendon junction, as well as tendon ruptures were found when anabolic steroids were associated with exercise [15]. High intensity loads during exercise seem to play a role in the process that allows the deleterious effects of steroids to be manifested. In the above studies, the groups that had been treated with both steroids and exercise suffered the worst injuries.

The response of tendons to exercise and some treatments may be analyzed regarding structural, chemical and mechanical aspects. However, most studies have been limited to analysis of only one or two of these aspects [16]. The present study aggregates biochemical, structural and biomechanical data in order to precisely investigate the effects of H. aphrodisiaca infusion on the tendon properties under long term endurance training. This study is part of a comprehensive study dealing with the effects of H. aphrodisiaca administration and intensive endurance exercise on some biological systems.

Methods

Animals

Adult Wistar rats, 90 days old, were obtained from the Center for Biological Investigation -CEMIB (State University of Campinas, Campinas, SP, Brazil). The rats were housed, three per cage, under standard conditions with 12 h L:12 h D cycle. Animals were provided with commercial rat feed and water ad libitum. The Institutional Committee for Ethics in Animal Care and Use of this University approved the experimental protocol (process n°1233-1).

Medicinal Plant

H. aphrodisiaca roots were collected in February 2007, in Mato Grosso State, Brazil. The species was identified by comparison with the voucher herbarium specimen of the plant at the Herbarium of the Mato Grosso Federal University, Brazil (number 23928). The roots were dried at room temperature, crushed and powdered using a grinding mill. The infusion was routinely prepared by pouring 100 mL of boiling water over 25 g of powdered roots, which was allowed to steep for 4 hours, then filtered by using filter paper. The yield was an infusion of 68.66 mg of dry extract (6.866% w/v) and a yield of 6.832% (w/w) in terms of initial crude dry weight of plant material. The infusion was prepared every four days and stored in the refrigerator. The doses of H. aphrodisiaca were selected according to previous studies [4].

Study groups and experimental protocol

Forty-eight male rats were divided into four groups (n = 12/group): two sedentary (CS and HS) and two submitted to involuntary running on a motorized treadmill (CT and HT). All groups received either 0.5 mL of distilled water (CS and CT, control groups) or H. aphrodisiaca infusion (HS and HT). Either water or infusion (104 mg/day) was administered by gavage during the 8 weeks of both training and sedentary periods. Trained rats (group CT and HT) were allowed to adapt to treadmill running for a 3 week period, prior to the beginning of the experimental protocol, which consisted of low to moderate level exercise carried out daily for 5 days a week (Table 1). After adaptation, trained rats were subjected to 8 weeks of intensive aerobic exercise training (treadmill running), also on a weekly cycle of 5 consecutive exercising days followed by a two day rest, as shown in Table 1, adapted from [17][18][19][20]. This program is a form of endurance training and does not compare with power training [18]. Forty-eight hours after the last training, the rats were anesthetized with xylazine chloride (Anasedan, Vetbrands, São Paulo, Brazil) and ketamine chloride (Cetamin, Syntec, Cotia, Brazil) (5 and 80 mg/Kg body weight, respectively). The right and left tendons were excised and frozen for biochemical, Since proteoglycans have many closely placed negative radicals, sections with high concentrations of proteoglycans, stained with toluidine blue, determine closely arranged and interacting molecules of this stain, replacing the blue with a violet coloring.

Extraction procedures

The tendons (n = 4/group) were treated with 25 volumes of 4 M guanidine chloride (GuHCl), containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 20 mM ethylenediamine tetraacetic acid (EDTA) in 50 mM sodium acetate buffer, pH 5.8 (Heinegård and Sommarin, 1987) at 4°C for 24 h. The mixture was then centrifuged (20,000 × g, 4°C, 30 min), the supernatant precipitated in acetate-ethanol and used in the biochemical analyses.

Electrophoresis (SDS-PAGE)

SDS-PAGE was performed according to Zingales [21], using gradient gels (4-16%). The tendon extracts (50 μL) from all experimental groups were precipitated with 100 μL of 50 mM acetate buffer, pH 7.4, and 9 volumes of absolute ethanol, for 24 hr at 4°C. These samples were analyzed by SDS-PAGE (50 μL). Gels were stained with Coomassie Brilliant Blue R-250. The relative molecular masses were estimated by comparisons with protein standard molecular mass markers.

Quantitative analysis

The amount of proteins in the extract of GuHCl was measured by the Bradford method [22], using bovine serum albumin (BSA) (1 mg/mL) as a standard. The readings were performed in a micro-plate reader at 595 nm. Sulfated glycosaminoglycans (GAG) of GuHCl extracts were quantified by the dimethylmethylene blue method (DMMB) [23] using chondroitin sulphate (1 mg/mL) as a standard, and readings were performed in a micro-plate reader at 526 nm. To quantify hydroxyproline, tendon fragments (n = 4) were dehydrated in acetone for 48 h and, subsequently, for another 24 hours in a mixture of chloroform and ethanol, at a ratio of 2:1. The tendon fragments were then hydrolyzed in 6 N HCl (10 mg of tissue/mL), for 18 h at 120°C, and the hydrolysate was neutralized with 6N NaOH. Samples were treated with chloramine T solution and perchloric acid/aldehyde, as described by Stegemann and Stalder [24]. After incubation for 15 min at 60°C, the material was cooled and absorbance was measured at 550nm in a spectrophotometer, Ultrospec 2100 (Pro Amersham Biosciences, England). The amount of hydroxyproline in the sample was calculated by comparison with a standard curve of hydroxyproline, and expressed as mg/g of wet tissue.

Gelatin zymography

Tendon fragments (n = 4/group) were incubated in 0.3 mL of extraction buffer (10 mM cacodylic acid, pH 5.0, 0.15 M NaCl, 1 μM ZnCl 2 , 20 mM CaCl 2 , 1.5 mM NaN 3 , 0.01% Triton X-100 [v/v]), at 4°C for 24 hours. After this period, the solution was submitted to centrifugation for 10 minutes (20,000 × g at 4°C). Samples were dried and resuspended in the same extraction buffer (pH 5.0). Zymography assays were performed on 10% polyacrylamide electrophoresis gels containing 0.1% gelatin, using 8 μg and 50 μg of protein per sample to indentify MMP-2 and MMP-9, respectively. After electrophoresis, the gels were washed with 2.5% Triton X-100 at room temperature and incubated overnight in a solution of 50 mM Tris-HCl, pH 8.0, 5 mM CaCl 2 and 0.02% NaN 3 at 37°C for 20 hours. Finally, the gels were stained with Coomassie Brilliant Blue. The protein bands corresponding to gelatinolytic activity were observed after washing the gels with a solution containing 30% methanol and 10% acetic acid. The gel was evaluated by band densitometry using the Scion Image program. Each sample was analyzed individually and the experiments were repeated four times, with three gels used for MMP activity quantification while the fourth was incubated in EDTA (20 mM) to confirm the presence of MMPs. The total MMP-2 activity corresponds to the sum of each isoform's activity.

Biomechanical test

After euthanasia, the right paw with the gastrocnemius and soleus muscles and Achilles tendon was excised. Five tendons of each group were used for the mechanical test. They were kept in physiological solution until tested, to prevent drying of the tissues. For the test, the specimens were clamped in a mechanical support with the myotendinous junction and the phalanges at opposite extremities, as employed by Nakagaki and colleagues [25]. The clamp-to-clamp distance was maintained at 7 mm for all tests. Each tendon was submitted to a pre-conditioning test, with 10 cycles of loading-unloading from 0 to 0.5 mm, at a speed of 20 mm/ min [26] and then submitted to the uniaxial tensile test. During the test, the tendon was subjected to a gradual increase in load at a constant displacement rate of 20 mm/min using a load cell of 1 kN, until the tendon broke [26]. The data were digitized, displayed and stored in a computer. These data were used to calculate the structural properties (maximum load and displacement at maximum load) and the material properties (maximum stress, strain at maximum stress and modulus of elasticity or Young's modulus) of the tendons in each group [27]. The maximum stress was obtained from the relation between load (N) and cross-section area (CSA) (mm 2 ) and expressed in MPa (megapascal). The CSA was determined according to Goodship and Birch [28]. Each tendon had its shape cast using an alginate dental impression paste (Avagel) manufactured by Dentsply. The CSA mould obtained was cut transversally and photographed. These images were then analyzed with the software Image Pro-Plus. The strain was calculated using the formula ε = ΔL/L 0 , where ΔL = L-L 0 (L = final length and L 0 = initial length). Strain was calculated based on clamp-to-clamp displacement. The elastic modulus of each group was measured in the interval corresponding to the most linear region of the stress-strain curve for each sample. The extrinsic stiffness was calculated as the slope of the linear region of the load-displacement curve (in N/mm). The experiments used a machine specifically designed for testing the mechanical properties of materials (MTS, model TETSTAR II) designed by the Laboratory of Mechanical Properties, School of Mechanical Engineering, UNICAMP.

Statistical analysis

Statistica software (v 8.0) (Tulsa, OK, USA) was used for the statistical analysis. All data were presented as mean ± standard deviations (S.D.), and a value of p < 0.05 was considered significant. The statistical comparison among the control and treated groups was determined using one-way ANOVA followed by the post hoc test of Tukey. In addition, two-way ANOVA was used, when appropriate, to determine how H. aphrodisiaca treatment and/or exercise training affected the results, and whether there was interaction between these two conditions.

Results

Biochemical assay

Analysis in SDS-PAGE of the Guanidinium chloride extract showed the presence of faint bands of collagen and noncollagenous proteins (NCP) in extracts of animals of the CT group, compared with other groups. This result showed that exercise by itself markedly reduced the presence of collagen and NCP. However H. aphrodisiaca treatment in sedentary rats maintained the same bands as displayed in CS, and also a few additional ones. Furthermore, exercise associated with H. aphrodisiaca (HT group) accentuated the appearance of NCP (Figure 1). The NCP levels, measured by protein analysis with the Bradford method, were significantly decreased (p < 0.05) in animals of the control trained group (CT) when compared to other groups (Figure 2a), confirming the SDS-PAGE analysis. Two-way ANOVA showed interaction between training and the treatment with plant infusion for tendon protein content (p = 0.0008). The sulfated glycosaminoglycan levels were statistically similar among all groups (Figure 2b).

Hydroxyproline is an indicator of collagen concentration in tissues. Its content was the highest in HT group (Figure 2c). Two-way ANOVA showed that there was interaction between endurance training and treatment with H. aphrodisiaca for hydroxyproline levels in the tendon (p = 0.005).

Zymography analysis

The zymography (Figure 3a and 3b) data demonstrated a tendency towards reduction of the activity of MMP-2 isoforms for the CT and HT groups (Figure 3c), but the difference between them was not statistically significant. The total MMP-2 activity was much reduced in the HT group (Figure 3e), when compared with other groups. MMP-9 activity was not statistically different for any of the experimental groups (Figure 3d).

Morphology

In order to detect proteoglycans within the compression region of the tendon, sections of different experimental groups were stained with Toluidine Blue (Figure 4). Metachromasy was observed in the territorial matrix of the compression region of all groups as a violet coloring, which indicates higher concentrations of negative charges. The trained tendons (Figure 4c and 4d) had a larger and more intensely stained area compared to sedentary rats (CS and HS) (Figure 4a and 4b). This staining pattern suggests an increase in the proteoglycan concentration in this region.

The analysis of trained tendons with polarized light microscopy showed intense birefringence in both compression (Figure 5e and 5g) and tension regions ( Figure  5f and 5h), indicating highly organized collagen bundles.  However, the HT group showed the most intense birefringence in both compression and tension regions of the tendon (Figure 5g and 5h). Both sedentary groups (Figure 5a, 5b, 5c and 5d) had the same morphological pattern and their collagen bundles were not as well aligned as observed in the trained ones.

Biomechanical Parameters

The cross-sectional area increased 27% in HT tendons when compared with CS tendons (means: 3174.00 mm 2 ±425.50 and 2493.2 mm 2 ±373.14, respectively). The other groups did not show alterations in the tendon cross-sectional area. Biomechanical analysis

Discussion

The current study shows the biochemical, biomechanical and morphological alterations in Achilles tendon of rats concurrently submitted to endurance training and H. aphrodisiaca treatment.  (g, h). The stronger birefringence is due to the increased aggregation and longitudinal organization of the collagen bundles. Scale bar = 30 μm.

The implications of endurance training [29][30][31][32] and AAS treatment [10,13,14] on tendon properties have been studied by several investigators. The use of AAS has resulted in a stiffer tendon that failed when submitted to less elongation than was achieved by the control Wistar rats. Exercise did not significantly alter tendon elongation in athlete animals. However, the combination of exercise and steroids significantly increased stiffness and decreased elongation, as well as the energy that the tendon could absorb at tendon failure [13]. In all cases the maximum load that the tendon could withstand did not seem to be affected. However, the use of steroids in the presence of exercise increased the cross-sectional area and reduced the flexibility of the tendon [13]. Wood et al. [10] in similar study, reported alterations in collagen content and the toe-limit strain in rats that had been treated with anabolic steroids and exercise.

In the present study, the maximum stress, modulus of elasticity and stiffness were higher in tendons of trained and treated rats, which also exhibited higher hydroxyproline content and increased cross-sectional area. These data showed that HT animals have more resistant tendons, differing from other studies in which the combination of AAS and exercise did not improve the tendon's biomechanical properties [10,13]. Moreover, the interaction between strenuous exercise and H. aphrodisiaca promoted significant increase in the material properties (maximum stress and modulus of elasticity) and of collagen content, resulting in stronger tendons able to support intense muscular contraction. Tendons may show a faster response to the number of loading cycles, rather than to the magnitude of the load [16]. Simonsen et al. [33] found that a strength-training regimen (high force with few loading cycles) did not stimulate increase in strength of the Achilles tendon of rats; however, lowforce endurance training (e.g. swimming) resulted in stronger tendons. They suggested that the tendons may respond better to the number of muscle contractions that occur during training rather than the absolute tension exerted by the muscle. In this case, the increased tendon mechanical resistance observed during endurance training and H. aphrodisiaca treatment may represent a mechanism to prevent tendon damage due to mechanical fatigue. This biomechanical behavior could be due to the increase of the collagen content, to the fiber orientation and to the interaction between collagen and ground substance [34]. There is a relationship between mechanical properties and collagen content [16], since more resistant tissues have either more collagen per area or collagen fibers with larger diameters [34].

The biomechanical results corroborate the results obtained by polarized microscopy. The organizational aspect of the fibers is better understood when slides are analyzed under polarized microscopy, due to the birefringence properties of collagen bundles. This observation is important because it shows micro-morphological details hidden within these bundles. In the present study, this technique revealed high birefringent brightness due to the condensation and highly tidy fiber array in the trained group. Besides, in the trained group that also received the plant infusion (HT) the results were even more prominent, showing brighter collagen fibers, possibly indicating highly compacted bundles. The lower birefringence found in sedentary animals reflects less organized collagen bundles in these groups. Some observations in the compression region showed that, in HT animals, there was an increase in the round cell population (stereological data not shown), as well as in the metachromasy intensity, which indicates a greater proteoglycan accumulation due to the increased compressive forces during endurance exercise. However, this result was not confirmed by the GAG dosage, which used the whole tendon. It is important to say that microscopical analysis of sections stained with toluidine blue specifically evaluate a region of the tendon where there is greater accumulation of proteoglycans due to the presence of localized compressive forces, as has been observed in tendons of rats [35] and pigs [36]. Nevertheless, proteoglycans, mainly the low weight ones, are distributed all over the tendon, associated to the collagen fibers [37], probably regulating collagen fibrillogenesis [38].

The degradation of collagen, as well as of a great number of other extracellular matrix compounds, is initiated by metalloproteinases (MMPs). An increase in net MMP activity is likely to indicate matrix degradation and accelerated remodeling [39,40]. Increased MMPs activity was verified in human tendons after acute running exercise [39] and in exercised rats (jumping in water) [14]. In the present study, zymography showed that MMP-2 activity in CT animals was similar in the sedentary groups, differing from other studies in which exercise increased the MMPs levels in tendons [14,39]. It is noteworthy that the MMP activity in the above studies was analyzed after only 3 days and 6 weeks of training, respectively.

In the present study, the animals were killed after 11 weeks of training, which means that the MMP activity was evaluated after a long training period. The biomechanical and morphological data demonstrated that the tendons of treated and trained animals had undergone adaptation to the increased demand. Thus, we suggest that there was a period of increased MMP activity to permit tissue remodeling, followed by a period of reduced activity when the tendons were already adapted to the load required. In this case, the mechanical load did not represent a stimulus for the synthesis of pro-MMPs and their eventual activation. Also, the MMP-9 activity did not alter in the exercised animals, which confirms the tendons' adaptation, since high activity of this protein is associated to the presence of immune cells during the initial inflammatory process [41], which occurs in response to tissue damage caused by exercise. In human tendons, protein synthesis and degradation were chronically elevated 4 weeks after the beginning of the training period, whereas protein synthesis remained high throughout a 12-week training cycle, while the degradation was slowly reduced. This suggests that there was probably an early period in the exercise program when collagen turnover in tendons was increased in order to restructure and readapt the tendon to the increased loading pattern [21].

Marqueti et al. [14] showed that the MMPs activity strongly decreased in AAS-treated animals. The inhibition could be due to a decrease in MMP synthesis or inhibition of activation of latent pro-MMPs. Also, that exercise by itself was not enough to compensate the inhibition of MMP activity induced by AAS treatment. In the present investigation the plant infusion without exercise did not inhibit MMP activity. The data obtained suggests that the infusion associated to exercise could have increased the MMPs' activity in the initial training period (1-3 weeks) when the highest mechanical loading occurred, considering that in the HT group the MMP-2 activity was reduced compared to all other groups, and that in this group the tendons were more resistant, according to the biomechanical results, in relation to the CT group. Therefore, we suggest that remodeling was more efficient in the HT group. However, further research is necessary to evaluate the effect of the plant infusion on MMP activity in exercised animals.

The present study faced some limitations regarding to the applied methodology, such as the lack of specific phytochemical data considering the active compounds present in H. aphrodisiaca infusion. Infusion preparation was based on previous studies that have been performed by our group using the same plant species [3,4,42], attempting to mimic the infusion traditionally used by folk medicine. Results of administering H. aphrodisiaca without exercise are not significantly different from the control, which would suggest a mandatory exercise program for efficacy of the plant extract altering rat tendons.

Common side effects due to long term endurance exercise together with synthetic AAS intake have been described as: collagen dysplasia, greater stiffness, reduction of strain, impaired tissue remodeling and others, none of which were noticed in this study after the training protocol and plant infusion administration. Scarce but important data lead us believe that there are no deleterious side effects related to H. aphrodisiaca intake, which was proved by evaluating blood biochemical parameters as well as kidney and liver morphology after long term administration of the plant infusion (data not published). Based on the promising data presented so far, future experiments crossing the variables of infusion dosages and time could be performed in order to potentiate H. aphrodisiaca effects along with endurance exercise. Additionally, phytochemical studies using the same infusion concentration are being carried out to reveal which active components could be related to the anabolic effects shown in this study.

Figure 1
1SDS-PAGE of Guanidinium chloride extract of the Achilles tendon. Analysis in SDS-PAGE showed faint bands of collagen and other proteins in control trained animals (CT). Note that H. aphrodisiaca treatment (HS) maintained the same bands displayed in control sedentary animals (CS) and also exhibited other NCP bands. In treated and exercised animals (HT) the appearance of NCP was accentuated. LMW-Low molecular weight standard.

Figure 2
2Quantifications of noncollagenous protein (NCP), sulfated glycosaminoglycans and hydroxyproline in the Achilles tendon. The NCP levels (a) were significantly decreased with exercise by itself, whereas H. aphrodisiaca treatment did not alter protein levels. The sulfated glycosaminoglycan levels (b) were statistically similar in the four groups. Observe the remarkable effect of training plus H. aphrodisiaca treatment on the hydroxyproline content (c). CS-control sedentary; HS-H. aphrodisiaca sedentary; CT-control trained; HT-H. aphrodisiaca trained. The columns are the mean ± SD. * Differences were significant for p < 0.05 (ANOVA) compared with other groups, by Tukey test.

Figure 3
3Zymography of Achilles tendon extracts. Each lane represents a sample of one animal. a, b-Gelatin zymography gel used to quantify activities of MMP-2 and MMP-9, respectively; c, d and e-Densitometry analysis of MMP-2, MMP-9 and total MMP-2, respectively. CScontrol sedentary; HS-H. aphrodisiaca sedentary; CT-control trained; HT-H. aphrodisiaca trained. In the HT group the MMP-2 activity was reduced when compared with CS. MMPs activity was similar in both HS and CS groups, indicating the H. aphrodisiaca treatment did not inhibit MMP activity. MMP-9 latent (92 kDa), MMP-9 active (82 kDa), MMP-2 latent (72 kDa), MMP-2 intermediate (68 kDa), MMP-2 active (62 kDa). The columns are the mean ± SD. * Differences were significant for p < 0.05 (ANOVA) compared with CS group, by the Tukey test.

Figure 4
4Longitudinal sections of tendons stained with Toluidine Blue. a-control sedentary; b-H. aphrodisiaca sedentary; c-control trained; d-H. aphrodisiaca trained. Observe intense metachromasy in trained groups (c, d). The treatment with H. aphrodisiaca accentuated metachromasy in sedentary (b) as well as trained (d) rats, compared with their respective controls. Scale bar = 25 μm.

showed significant increase in maximum load (Figure 6a), stiffness (Figure 6c), maximum stress (Figure 6d) and modulus of elasticity (Figure 6f) of the tendons of animals trained and treated with H. aphrodisiaca (HT). Two-way ANOVA analysis showed that there was interaction between training and treatment with plant infusion only for the maximum stress (p = 0.006). The displacement (Figure 6b) and strain (Figure 6e) were similar in all groups, with no significant difference among them.

Figure 5
5Polarized light microscopy of Toluidine Blue-stained sections. a, b-correspond to control sedentary; c, d-H. aphrodisiaca sedentary; e, f-control trained; and g, h-H. aphrodisiaca trained. The left column corresponds to the compression region and right column corresponds to the tension region. Observe a higher organization of the collagen bundles in tendons of trained (e-h) versus sedentary (a-d) animals. However, the most intense birefringence was found in tendons of trained rats and also those treated with the plant infusion

Figure 6
6Biomechanical properties of Achilles tendons from control and H. aphrodisiaca sedentary and trained rats. The maximum load (a), stiffness (c), maximum stress (d) and modulus of elasticity (f), but not displacement (b) and strain (e), were higher in H. aphrodisiaca trained rats. CS-control sedentary; HS-H. aphrodisiaca sedentary; CT-control trained; HT-H. aphrodisiaca trained. The columns are the mean ± SD. * Differences were significant for p < 0.05 (ANOVA) compared with other groups, by the Tukey test. # Differences were significant for p < 0.05 (ANOVA) compared with CS and HS, by the Tukey test.

Table 1
1Exercise protocol in treadmill runningEvent
Week
Velocity (m/min)
Duration (min)

Treadmill
1
10.68
5
adaptation
2
12.42
7,5

3
14.16
10

1
14.16
20

Training
2
19.62
30

3
19.62
40

4-8
22.92
45

Monteiro et al. BMC Complementary and Alternative Medicine 2011, 11:51 http://www.biomedcentral.com/1472-6882/11/51
Monteiro et al. BMC Complementary and Alternative Medicine 2011, 11:51 http://www.biomedcentral.com/1472-6882/11/51 Page 7 of 11
Monteiro et al. BMC Complementary and Alternative Medicine 2011, 11:51 http://www.biomedcentral.com/1472-6882/11/51 Page 8 of 11
Monteiro et al. BMC Complementary and Alternative Medicine 2011, 11:51 http://www.biomedcentral.com/1472-6882/11/51 Page 9 of 11
AcknowledgementsThis work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Grant numbers: 06/06132-2 and 08/05610-3) and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-PROEX). The authors thank Dr. Gerson E. R. Campos for providing the treadmill to train the animals, Andrea Aro for helping in the discussion and Rodrigo Leite for English reviewing.ConclusionsAccording to the results found in the present study, it can be concluded that endurance training associated with H. aphrodisiaca infusion increases the material properties of tendons. Rather, the association resulted in more resistant tendons, due to the increase in collagen molecules and the corresponding organizational increase. Despite the clearly anabolic effects of H. aphrodisiaca and associated endurance training, no side effects were observed, such as those found after synthetic AAS use. Therefore, H. aphrodisiaca associated with endurance training contributed to more efficient remodeling of the extracellular matrix, resulting in more resistant tendons to support high loads from intense muscle contraction. These findings suggest that H. aphrodisiaca infusion is a potential aid to optimize tendon remodeling in athletes, where the disparity of the faster physiological muscle adjustment in relation to the tendon often leads to lesions, because the tendons may not resist the increased tension produced by the stronger muscles.  Authors' contributions JCM participated in all aspects of the work. The co-authors participated in the discussion of the results and revision of the manuscript, as well as in the following contributions: MLMG and MMS participated in the training program and the processing of the material; TCT participated in the morphological and biochemical analyses; WRN participated in the biomechanical analysis; DLF participated in the zymographical analysis; ERP and HD participated in supervision and as sponsors. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
Pio Corrêa M: Dicionário de Plantas Úteis do Brasil e das Exóticas Cultivadas. Rio de JaneiroPio Corrêa M: Dicionário de Plantas Úteis do Brasil e das Exóticas Cultivadas. Rio de Janeiro. 1984.

Plantas do Pantanal. Empresa Brasileira de Pesquisa agropecuária do Pantanal, Corumbà. A Pott, V J Pott, Pott A, Pott VJ: Plantas do Pantanal. Empresa Brasileira de Pesquisa agropecuária do Pantanal, Corumbà. 1994.

Efeito do tratamento crônico com extratos de Heteropterys aphrodisiaca O.Mach e Anemopaegma arvense (Vell.) Stellf no testículo de ratos Wistar adultos. Viçosa: UFV. L C Chieregato, Chieregato LC: Efeito do tratamento crônico com extratos de Heteropterys aphrodisiaca O.Mach e Anemopaegma arvense (Vell.) Stellf no testículo de ratos Wistar adultos. Viçosa: UFV; 2005.

Heteropterys aphrodisiaca infusion reduces the collateral effects of cyclosporine A on the testis. J C Monteiro, F S Predes, S L Matta, H Dolder, Anat Rec. 2917Monteiro JC, Predes FS, Matta SL, Dolder H: Heteropterys aphrodisiaca infusion reduces the collateral effects of cyclosporine A on the testis. Anat Rec (Hoboken) 2008, 291(7):809-817.

Anabolic steroid abuse: physiological and anaesthetic considerations. P C Kam, M Yarrow, Anaesthesia. 607Kam PC, Yarrow M: Anabolic steroid abuse: physiological and anaesthetic considerations. Anaesthesia 2005, 60(7):685-692.

D M Creasy, Pathogenesis of male reproductive toxicity. Toxicologic pathology. 29Creasy DM: Pathogenesis of male reproductive toxicity. Toxicologic pathology 2001, 29(1):64-76.

Abuse of anabolic androgenic steroids and related substances in sport and exercise. Current opinion in pharmacology. M S Bahrke, C E Yesalis, 4Bahrke MS, Yesalis CE: Abuse of anabolic androgenic steroids and related substances in sport and exercise. Current opinion in pharmacology 2004, 4(6):614-620.

Triceps rupture. A case report and literature review. The American journal of sports medicine. B R BachJr, R F Warren, T L Wickiewicz, 15Bach BR Jr, Warren RF, Wickiewicz TL: Triceps rupture. A case report and literature review. The American journal of sports medicine 1987, 15(3):285-289.

Asynchronous bilateral Achilles tendon ruptures and androstenediol use. The American journal of sports medicine. V Battista, J Combs, W J Warme, 31Battista V, Combs J, Warme WJ: Asynchronous bilateral Achilles tendon ruptures and androstenediol use. The American journal of sports medicine 2003, 31(6):1007-1009.

The effect of exercise and anabolic steroids on the mechanical properties and crimp morphology of the rat tendon. T O Wood, P H Cooke, A E Goodship, American Orthopaedic Society for Sports Medicine. 153Wood TO, Cooke PH, Goodship AE: The effect of exercise and anabolic steroids on the mechanical properties and crimp morphology of the rat tendon. American Orthopaedic Society for Sports Medicine; 1988:16:153.

Tendon injuries induced by exercise and anabolic steroids in experimental mice. H Michna, International orthopaedics. 112Michna H: Tendon injuries induced by exercise and anabolic steroids in experimental mice. International orthopaedics 1987, 11(2):157-162.

The effects of anabolic steroids on rat tendon. An ultrastructural, biomechanical, and biochemical analysis. The American journal of sports medicine. P D Inhofe, W A Grana, D Egle, K W Min, J Tomasek, 23Inhofe PD, Grana WA, Egle D, Min KW, Tomasek J: The effects of anabolic steroids on rat tendon. An ultrastructural, biomechanical, and biochemical analysis. The American journal of sports medicine 1995, 23(2):227-232.

The effect of anabolic steroids on the biomechanical and histological properties of rat tendon. J W Miles, W A Grana, D Egle, K W Min, J Chitwood, The Journal of bone and joint surgery. 19923Miles JW, Grana WA, Egle D, Min KW, Chitwood J: The effect of anabolic steroids on the biomechanical and histological properties of rat tendon. The Journal of bone and joint surgery 1992, 74(3):411-422.

Androgenic-anabolic steroids associated with mechanical loading inhibit matrix metallopeptidase activity and affect the remodeling of the Achilles tendon in rats. The American journal of sports medicine. R C Marqueti, N A Parizotto, R S Chriguer, S E Perez, H S Selistre-De-Araujo, 34Marqueti RC, Parizotto NA, Chriguer RS, Perez SE, Selistre-de-Araujo HS: Androgenic-anabolic steroids associated with mechanical loading inhibit matrix metallopeptidase activity and affect the remodeling of the Achilles tendon in rats. The American journal of sports medicine 2006, 34(8):1274-1280.

Pectoralis maior tendon rupture and anabolic steroids in anamnesis-a case review. J Marz, P Novotny, Rozhl Chir. 877Marz J, Novotny P: Pectoralis maior tendon rupture and anabolic steroids in anamnesis-a case review. Rozhl Chir 2008, 87(7):380-383.

Effects of exercise on the biomechanical, biochemical and structural properties of tendons. Comparative biochemistry and physiology. C I Buchanan, R L Marsh, 133Buchanan CI, Marsh RL: Effects of exercise on the biomechanical, biochemical and structural properties of tendons. Comparative biochemistry and physiology 2002, 133(4):1101-1107.

Short-term exercise improves myocardial tolerance to in vivo ischemia-reperfusion in the rat. H A Demirel, S K Powers, M A Zergeroglu, R A Shanely, K Hamilton, J Coombes, H Naito, J Appl Physiol. 915Demirel HA, Powers SK, Zergeroglu MA, Shanely RA, Hamilton K, Coombes J, Naito H: Short-term exercise improves myocardial tolerance to in vivo ischemia-reperfusion in the rat. J Appl Physiol 2001, 91(5):2205-2212.

Adverse effect of the anabolic-androgenic steroid mesterolone on cardiac remodelling and lipoprotein profile is attenuated by aerobic exercise training. K Fontana, H C Oliveira, M B Leonardo, C A Mandarim-De-Lacerda, M A Da Cruz-Hofling, International journal of experimental pathology. 895Fontana K, Oliveira HC, Leonardo MB, Mandarim-de-Lacerda CA, da Cruz- Hofling MA: Adverse effect of the anabolic-androgenic steroid mesterolone on cardiac remodelling and lipoprotein profile is attenuated by aerobic exercise training. International journal of experimental pathology 2008, 89(5):358-366.

Treadmill running produces both positive and negative physiological adaptations in Sprague-Dawley rats. A Moraska, T Deak, R L Spencer, D Roth, M Fleshner, Am Physiological Soc. 1321Moraska A, Deak T, Spencer RL, Roth D, Fleshner M: Treadmill running produces both positive and negative physiological adaptations in Sprague-Dawley rats. Am Physiological Soc; 2000:279:1321.

HSP72 as a complementary protection against oxidative stress induced by exercise in the soleus muscle of rats. M B Smolka, C C Zoppi, A A Alves, L R Silveira, S Marangoni, L Pereira-Da-Silva, J C Novello, D V Macedo, Am Physiological Soc. 1539Smolka MB, Zoppi CC, Alves AA, Silveira LR, Marangoni S, Pereira-Da-Silva L, Novello JC, Macedo DV: HSP72 as a complementary protection against oxidative stress induced by exercise in the soleus muscle of rats. Am Physiological Soc; 2000:279:R1539.

Analysis of protein by sodium dodecyl sulphateprolyacrylamide gel electrophoresis. B Zingales, J Biol Chem. 44Zingales B: Analysis of protein by sodium dodecyl sulphate- prolyacrylamide gel electrophoresis. J Biol Chem 1984, 44:4406-4412.

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. M M Bradford, Analytical biochemistry. 72Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry 1976, 72:248-254.

Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. R W Farndale, D J Buttle, A J Barrett, Elsevier883Farndale RW, Buttle DJ, Barrett AJ: Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Elsevier; 1986:883:173-177.

Determination of hydroxyproline. H Stegemann, K Stalder, Clinica chimica acta; international journal of clinical chemistry. 182Stegemann H, Stalder K: Determination of hydroxyproline. Clinica chimica acta; international journal of clinical chemistry 1967, 18(2):267-273.

Biomechanical and biochemical properties of chicken calcaneal tendon under effect of age and nonforced active exercise. Connective tissue research. W R Nakagaki, A Biancalana, G P Benevides, L Gomes, 48Nakagaki WR, Biancalana A, Benevides GP, Gomes L: Biomechanical and biochemical properties of chicken calcaneal tendon under effect of age and nonforced active exercise. Connective tissue research 2007, 48(5):219-228.

The effects of stress enhancement on the extracellular matrix and fibroblasts in the patellar tendon. H Tohyama, K Yasuda, Journal of biomechanics. 335Tohyama H, Yasuda K: The effects of stress enhancement on the extracellular matrix and fibroblasts in the patellar tendon. Journal of biomechanics 2000, 33(5):559-565.

Meniscofemoral ligaments-structural and material properties. C M Gupte, A Smith, N Jamieson, A M Bull, R D Thomas, A A Amis, Journal of biomechanics. 3512Gupte CM, Smith A, Jamieson N, Bull AM, Thomas RD, Amis AA: Meniscofemoral ligaments-structural and material properties. Journal of biomechanics 2002, 35(12):1623-1629.

Cross sectional area measurement of tendon and ligament in vitro: a simple, rapid, non-destructive technique. A E Goodship, H L Birch, Journal of biomechanics. 383Goodship AE, Birch HL: Cross sectional area measurement of tendon and ligament in vitro: a simple, rapid, non-destructive technique. Journal of biomechanics 2005, 38(3):605-608.

The effect of overuse activity on Achilles tendon in an animal model: a biomechanical study. T F Huang, S M Perry, L J Soslowsky, Annals of biomedical engineering. 323Huang TF, Perry SM, Soslowsky LJ: The effect of overuse activity on Achilles tendon in an animal model: a biomechanical study. Annals of biomedical engineering 2004, 32(3):336-341.

Tensile strength properties of Achilles tendon systems in trained and untrained rabbits. A Viidik, Acta orthopaedica Scandinavica. 402Viidik A: Tensile strength properties of Achilles tendon systems in trained and untrained rabbits. Acta orthopaedica Scandinavica 1969, 40(2):261-272.

Anisotropic and biomechanical properties of tendons modified by exercise and denervation: aggregation and macromolecular order in collagen bundles. R Vilarta, C Vidal Bde, Matrix. 91Vilarta R, Vidal Bde C: Anisotropic and biomechanical properties of tendons modified by exercise and denervation: aggregation and macromolecular order in collagen bundles. Matrix (Stuttgart, Germany) 1989, 9(1):55-61.

The biomechanical and biochemical properties of swine tendons-long term effects of exercise on the digital extensors. S L Woo, M A Ritter, D Amiel, T M Sanders, M A Gomez, S C Kuei, S R Garfin, W H Akeson, Connective tissue research. 7Woo SL, Ritter MA, Amiel D, Sanders TM, Gomez MA, Kuei SC, Garfin SR, Akeson WH: The biomechanical and biochemical properties of swine tendons-long term effects of exercise on the digital extensors. Connective tissue research 1980, 7(3):177-183.

E B Simonsen, H Klitgaard, F Bojsen-Moller, The influence of strength training, swim training and ageing on the Achilles tendon and m. soleus of the rat. 13Simonsen EB, Klitgaard H, Bojsen-Moller F: The influence of strength training, swim training and ageing on the Achilles tendon and m. soleus of the rat. Journal of sports sciences 1995, 13(4):291-295.

Biomechanics of knee ligaments. The American journal of sports medicine. S L Woo, R E Debski, J D Withrow, M A Janaushek, 27Woo SL, Debski RE, Withrow JD, Janaushek MA: Biomechanics of knee ligaments. The American journal of sports medicine 1999, 27(4):533-543.

D Z Covizi, S L Felisbino, L Gomes, E R Pimentel, H F Carvalho, Regional adaptations in three rat tendons. 33Covizi DZ, Felisbino SL, Gomes L, Pimentel ER, Carvalho HF: Regional adaptations in three rat tendons. Tissue & cell 2001, 33(5):483-490.

Optical anisotropy of a pig tendon under compression. V Feitosa, B C Vidal, E R Pimentel, Journal of anatomy. Pt 1Feitosa V, Vidal BC, Pimentel ER: Optical anisotropy of a pig tendon under compression. Journal of anatomy 2002, 200(Pt 1):105-111.

Tendon response to tensile stress: an ultrastructural investigation of collagen: proteoglycan interactions in stressed tendon. A M Cribb, J E Scott, Journal of anatomy. 187Pt 2Cribb AM, Scott JE: Tendon response to tensile stress: an ultrastructural investigation of collagen: proteoglycan interactions in stressed tendon. Journal of anatomy 1995, 187(Pt 2):423-428.

Proteins in the tensile region of adult bovine deep flexor tendon. K G Vogel, A B Meyers, Clinical orthopaedics and related research. 367SupplVogel KG, Meyers AB: Proteins in the tensile region of adult bovine deep flexor tendon. Clinical orthopaedics and related research 1999, , 367 Suppl: S344-355.

Physical exercise can influence local levels of matrix metalloproteinases and their inhibitors in tendon-related connective tissue. Soa Koskinen, K M Heinemeier, J L Olesen, H Langberg, M Kjaer, Am Physiological Soc. 861Koskinen SOA, Heinemeier KM, Olesen JL, Langberg H, Kjaer M: Physical exercise can influence local levels of matrix metalloproteinases and their inhibitors in tendon-related connective tissue. Am Physiological Soc; 2004:96:861.

Gene expression and matrix turnover in overused and damaged tendons. G P Riley, 15Scandinavian journal of medicine & science in sportsRiley GP: Gene expression and matrix turnover in overused and damaged tendons. Scandinavian journal of medicine & science in sports 2005, 15(4):241-251.

Matrix metalloproteinases in inflammatory pathologies of the horse. A L Clutterbuck, P Harris, D Allaway, A Mobasheri, Vet J. 1831Clutterbuck AL, Harris P, Allaway D, Mobasheri A: Matrix metalloproteinases in inflammatory pathologies of the horse. Vet J 2008, 183(1):27-38.

Efeitos da infusão de nó-de-cachorro (Hetereopterys aphrodisiaca, O. Mach.) sobre a morfologia e estrutura testicular de ratos Wistar adultos, submetidos a treinamento físico. Mlm Gomes, Campinas: UNICAMP. Gomes MLM: Efeitos da infusão de nó-de-cachorro (Hetereopterys aphrodisiaca, O. Mach.) sobre a morfologia e estrutura testicular de ratos Wistar adultos, submetidos a treinamento físico. Campinas: UNICAMP; 2011.


Non-Invasive Optical Biosensor for Probing Cell Signaling
2007

Ye Fang email@example.com.
Biochemical Technologies, Science and Technology Division
Corning Incorporated
Sullivan Park14831CorningNYUSA

Non-Invasive Optical Biosensor for Probing Cell Signaling

Sensors
72007Received: 24 September 2007 / Accepted: 15 October 2007 /sensors ReviewOptical biosensorresonant waveguide grating biosensorcell signalingG protein-coupled receptordynamic mass redistribution
Cell signaling mediated through a cellular target is encoded by spatial and temporal dynamics of downstream signaling networks. The coupling of temporal dynamics with spatial gradients of signaling activities guides cellular responses upon stimulation. Monitoring the integration of cell signaling in real time, if realized, would provide a new dimension for understanding cell biology and physiology. Optical biosensors including resonant waveguide grating (RWG) biosensor manifest a physiologically relevant and integrated cellular response related to dynamic redistribution of cellular matters, thus providing a non-invasive means for cell signaling study. This paper reviews recent progresses in biosensor instrumentation, and theoretical considerations and potential applications of optical biosensors for whole cell sensing.

Introduction

Optical biosensor is a well-established analytical tool for biomolecular interaction analysis [1,2]. The biosensor typically consists of a biological component, a detector element, and a transducer associated with both components. The biological component is generally a protein target (i.e., bioreceptors) immobilized on the surface of the transducer. Coupled with microfluidics which introduces an analyte as well as separates unbound and bound molecules, the biosensor becomes a powerful biophysical tool for quantitatively characterizing how biomolecular complexes form and dissociate apart over time. However, the ability of optical biosensors to examine living cells was largely unrealized until recent years [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. Advancements in instrumentation, particularly large scale screening platforms [9,14], enable the discovery of specific types of cellular target-dependent optical signals in cells [9,15], thus opening a new possibility for whole cell sensing, in particular, cell signaling analysis [18,19,20]. This article reviews theoretical, instrumental and experimental progresses of optical biosensor for probing cell signaling.

Optical Biosensors

The past decade or so has seen an exponential increase in the number and range of available biosensors based on different optical phenomena. Figure 1 shows operating principles of three types of commercially available optical biosensors: surface plasmon resonance (SPR), and resonant waveguide grating (RWG), and interferometric biosensors.

SPR relies on a prism to direct a wedge of polarized light, covering a range of incident angles, into a planar glass substrate having a gold thin film to excite surface plasmons (Figure 1a). The SPR is a charge-density oscillation or wave that may exist at the interface between two media with dielectric constants of opposite signs (i.e., the gold and a dielectric). The resonant angle at which a minimal in intensity of reflected light occurs is a function of the local refractive index at or near the gold surface [1,2].

RWG biosensor utilizes the resonant coupling of light into a waveguide by means of a diffraction grating. A polarized light, covering a range of incident wavelengths, is used to illuminate the waveguide; light at specific wavelengths is coupled into and propagate along the waveguide ( Figure  1b). The resonant wavelength at which a maximum incoupling efficiency is achieved is a function of the local refractive index at or near the sensor surface [9,21]. An RWG biosensor typically consists of a thin film (~75-100nm) of Nb 2 O 5 with a high refractive index (~2.36) on a glass substrate with an index of ~1.50 [13].

Interferometry biosensor uses a spectrometer to capture interference patterns in the reflected light from the biosensor interface ( Figure 1c). As exampled in this single optical fiber-based interferometric biosensor, a broadband light resource through an optical fiber is directly used to illuminate at the solution-surface interface. The two types of reflected light waves originated from the two reflecting surfaces, the interface with the optical layer and the surface of bio-receptors, can propagate within the fiber and interact each other, leading to an interference pattern. This interference is captured by a spectrometer as a pattern of intensity variation by wavelength with a characteristic profile of peaks and troughs. When there is binding of biological molecules to the biosensor surface, the thickness of bioreceptor layer increases, which in turn leads to a change in the interference pattern at the spectrometer [20].

Theories for Living Cell Sensing with Optical Biosensor

Common to all optical biosensors is that they measure changes in local refractive index at or very near the sensor surface. Almost all optical biosensors are applicable for cell sensing in principle -they can employ an evanescent wave to characterize ligand-induced changes in cells. The evanescent-wave is an electromagnetic field, created by the total internal reflection of light at a solution-surface interface, which typically extends a short distance (~hundreds of nanometers) into the solution with a characteristic depth, termed as penetration depth or sensing volume. This section specifically discusses theoretical considerations for whole cell sensing using RWG biosensor.

Ramsden et al. described a theory to study cell adhesion and spreading on the surface of a RWG biosensor [3]. This theory assumes a 3-layer waveguide system [21], wherein the cells undergo shape changes with well-defined geometrical parameters and steps upon adhesion and spreading on the biosensor surface. Such model allows both the number of cells at the surface and a parameter characterizing their shape to be accurately determined.

Recently, we had developed theoretical and mathematical models that describe the parameters and nature of optical signals measured in living cells in response to stimulation with ligands [13]. These models, based on a 3-layer waveguide system in combination with known cellular biophysics, provide a link from ligand-induced optical signals to several cellular processes mediated through a receptor.

To probe cell signaling, cells are brought to in close contact with the sensor surface, mainly through cell culture. Given that the biosensor measures an averaged response of cells located at the area illuminated by the incident light, a cell layer of highly confluency is used in order to achieve optimal assay results. Because of the large dimension of cells compared to the short penetration depth (~100-200nm) of a biosensor, the sensor configuration is considered as a non-conventional three-layer system: a substrate, a waveguide film with a grating structure, and a cell layer ( Figure 2). Thus, a ligand-induced change in effective refractive index (i.e., the detected signal) is, to first order, directly proportional to the change in refractive index of the bottom portion of cell layer [13]:
c n C S N Δ = Δ ) ((1)
where S(C) is the sensitivity to the cell layer, and Δn c the ligand-induced change in local refractive index of the cell layer sensed by the biosensor. Because the refractive index of a given volume within a cell is largely determined by the concentrations of bio-molecules such as proteins [22], Δn c can be assumed to be directly proportional to ligand-induced change in local concentrations of cellular targets or molecular assemblies within the sensing volume. Considering the exponentially decaying nature of the evanescent wave extending away from the sensor surface, the ligand-induced optical signal is governed by [13]:
Figure 2.
A three-layer waveguide system for detecting ligand-induced dynamic mass redistribution signals in living cells using RWG biosensor. Cells are anchored to the sensor surface through interaction with the extracellular matrix (ECM).

Broadband light Reflected light

Waveguide glass cell ECM ∑ ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ − Δ = Δ Δ − Δ − + i Z z Z z i C i C i e e C d C S N 1 ) ( α (2)
where ΔZ c is the penetration depth into the cell layer, α the specific refraction increment (about 0.18/mL/g for proteins) [23,24], z i the distance where the mass redistribution occurs, and d an imaginary thickness of a slice within the cell layer. Here the cell layer is divided into an equal-spaced slice in the vertical direction. Eq.2 indicates that the ligand-induced optical signal is a sum of mass redistribution occurring at distinct distances away from the sensor surface, each with an unequal contribution to the overall response. Furthermore, the detected signal, in terms of wavelength or angular shifts, is primarily sensitive to mass redistribution occurring perpendicular to the sensor surface. Because of its dynamic nature, it is also referred to as dynamic mass redistribution (DMR) signal. (a) Angular interrogation system. A laser light is split into an array of 7x7 light beams, such that each illuminates a biosensor located within one well of 96well SBS standard microplate. The reflected lights are received with a CCD camera as an image of 7x7 resonant bands. (b) Wavelength interrogation system. A linear array of 16 illumination/detection heads is used to scan a 384well microplate, one column at a time. In each bundled head, there is an optical fiber to send a laser light to illuminate a biosensor located in each well, and a second optical fiber to receive the reflected light.

High Throughput Optical Biosensor Systems

Although there is great difference in operating principle, throughput, sample delivery process and application, most commercially available label free optical systems are configured for bio-molecular interaction analysis at the laboratory scale -the measurements of a small set of samples at one time [20]. For example, Biacore SPR-based systems, the most popular systems, have relative low to medium throughput because limited channels are available for sample analysis, and measurements have to be done in series. However, the applications of optical biosensors in a high throughput screening setting, particularly using living cells, are limited. To address these unmet needs, we have been developing label free optical systems tailoring towards high throughput applications. We are primarily interested in optical systems that are suited for assays using the Society for Biomolecular Sciences (SBS; http://www.sbsonline.org) standard microtiter plates, such as 96well and 384well microplates. The RWG biosensor is chosen, particularly because this type of biosensor with appropriate designs allows lights at nominally normal incident angle to illuminate the waveguide thin film. This is important for illuminating large numbers of biosensors simultaneously. Figure 3 illustrates two types of optical systems developed at our company. In the angular interrogation system (Figure 3a), the total internal reflection is observed at a critical angle of incidence at the interface of the waveguide film and the cell layer. This system consists of a launch system to generate an array of light beams such that each illuminates a RWG biosensor with a dimension of ~200 μm x 3000 μm, and a CCD camera-based receive system to record changes in resonant angle of the reflected light [9]. The array of light beams is obtained using a beam splitter and diffractive optical lenses. This system allows an array of 7x7 biosensor wells in a 96well microplate to be simultaneously sampled at every 3 seconds. This system does not have any moving parts, except of a liquid handling system for compound addition.

In the wavelength interrogation system (Figure 3b), the total internal reflection is achieved at a resonant wavelength of a broadband light source illuminated at the waveguide. This system, commercialized in 2006, consists of a detector with a temperature-controlled environment, an external liquid handling system, and 384well standard SBS microtiter plates [15]. The detector system is centred on integrated fiber optics to measure any wavelength shift of the reflected lights. A broadband light source, generated through a fiber optic and a collimating lens, is used to illuminate a small region of the biosensor through the bottom of a microplate. A detection fiber for recording the reflected light is bundled with the illumination fiber. A series of illumination/detection heads are arranged in a linear fashion, so that reflection spectra are collected from all 16 wells within the same column of a 384well microplate simultaneously. The whole plate is scanned so that each sensor is addressed multiple times, and each column is addressed in sequence. The temperature-controlling unit is built in to minimize any temperature fluctuation.

Probing Cell Signaling with Optical Biosensor

Cells rely on multiple cellular pathways or machineries to process, encode and integrate the information received. Unlike the affinity analysis with optical biosensors that specifically measures the binding of analytes to a protein target, living cells are much more complex and dynamic. This section discusses the cellular mechanisms that provide the basis for cell signaling studies with optical biosensors, as well as the applications of optical biosensor in cell signaling.

Cell-surface interactions

To study cell signaling, cells are brought to contact with the surface of a biosensor, which can be achieved through cell culture. These cultured cells are attached onto the biosensor surface through three types of contacts: focal contacts, close contacts and extracellular matrix contacts, each with its own characteristic separation distance from the surface [25,26]. As a result, the basal cell membranes are generally distant away from the surface by ~10-100nm. Since the penetration depths or sensing volumes of most optical biosensors are in the range of 100-200nm, these biosensors are able to sense the bottom portion of cells.

Cells, in many cases, exhibit surface-dependent adhesion and proliferation. In order to achieve robust cell assays, the biosensor surface may require a coating to enhance cell adhesion and proliferation. On the other hand, the surface properties could have direct impact on cell biologysurface-bound ligands may influence the response of cells [27], while the mechanical compliance of a substrate material, which dictates how it will deform under forces applied by the cell, is influential [28]. Together with the culture conditions (time, serum concentration, confluency, etc.), cellular status obtained may be distinct from one surface to another, and from one condition to another. Thus, special attentions to control cellular status are necessitated for developing biosensor-based cell assays [9, 10, 13].

Cells under un-stimulated conditions

Cells are dynamic objects with relatively large dimensions -typically tens of microns. Even without stimulation, cells constantly undergo micromotion -a dynamic movement and remodeling of cellular structure, as observed in tissue culture by time lapse microscopy at the sub-cellular resolution, as well as by bio-impedance measurements at the nanometer level [29].

Under un-stimulated conditions the cells generally give rise to an almost net-zero DMR response, as examined with RWG biosensor (Figure 4) [9,10,16]. This is partly because of the low spatial resolution of optical biosensors, as determined by the large size of the laser spot and the long propagation length of the coupled light. The size of the laser spot determines the size of the area studied -usually only one analysis point can be tracked at a time. Thus, the biosensor typically measures an averaged response of a large population of cells located at the light incident area. Although cells undergo micromotion at the single cell level, the large populations of cells examined give rise to a net-zero DMR response in average. Furthermore, it is known that intracellular macromolecules are highly organized [11,30] and spatially restricted to appropriate sites in mammalian cells [31,32]. The localization of proteins is tightly controlled in order for cells to regulate the specificity and efficiency of proteins interacting with their proper partners, to spatially separate protein activation and deactivation mechanisms, and thus to determine specific cell functions and responses. Thus, under un-stimulated conditions, the local mass density of cells within the sensing volume can reach an equilibrium state, thus leading to a net-zero optical response. It is worthy noting that the cells examined often have been cultured under conventional culture condition for a period of time such that most of the cells have just completed a single cycle of division.

Cellular responses upon stimulation

Living cells have exquisite abilities to sense and respond to exogenous signals. Cell signaling was originally thought to function via linear routes where an environmental cue would trigger a linear chain of reactions resulting in a single well-defined response. However, amassing evidences show that cellular responses to external stimuli are much more complicated. It has become apparent that the information the cells received is processed and encoded into complex temporal and spatial patterns of phosphorylation and topological relocation of signaling proteins [32,33]. The spatial and temporal targeting of proteins to appropriate sites is crucial to regulate the specificity and efficiency of proteinprotein interactions, thus dictating the timing and intensity of cell signaling and responses. Pivotal cellular decisions, such as cytoskeletal reorganization, cell cycle checkpoints and apoptosis, depend on the precise temporal control and relative spatial distribution of activated signal-transducers [34][35][36]. Thus, cell signaling mediated through a cellular target such as G protein-coupled receptor (GPCR) typically proceeds in an orderly and regulated manner, and consists of a series of spatial and temporal events, many of which lead to changes in local mass density or redistribution in local cellular matters of cells [18]. These changes or redistribution when occurring within the sensing volume can be followed directly in real time using optical biosensors [13]. GPCRs represent the largest family of cell membrane receptors [37], and the largest of druggable targets in human genome [38]. Their primary function is to transduce external signals into an array of cellular responses, through both G protein-dependent and independent pathways. Using panels of agonists targeting many GPCRs, three classes of DMR signals in native human epidermoid carcinoma A431 cell were identified [15] (Figure 4). Since each is correlated with the activation of a class of GPCRs depending on G protein with which the receptor is coupled, the DMR signals obtained are named G q -, G s -and G i -DMR signals, respectively. These DMR signals exhibit distinct kinetic and dynamic characteristics, reflecting the unique signaling integration mediated through different classes of GPCRs. Interestingly, the G q -type DMR signal appears to be rapid, whereas the G s -type DMR signal is comparatively slow.

Characteristics of endogenous GPCR DMR signals

To date, we have found that both the G q and G s -DMR signals are quite universal across multiple cell lines tested, while the G i -DMR signals exhibit strong dependence on the cellular context as well as the receptors. However, for the same class of DMR signals mediated through the activation of different receptors in the same cell line or the activation of the same receptor in different cell lines, there are often noticeable differences in fine features such as the amplitudes and kinetics, reflecting the important roles of cellular context in GPCR signaling as well as the uniqueness in signaling of each receptor. The amplitudes of DMR signals for several endogenous G q -coupled receptors in A431 cells were found to be correlated well with their corresponding Ca 2+ mobilization signals, suggesting that a ligand-induced DMR signal is a function of the expression level of its corresponding receptor, as well as the coupling efficiency of the receptor with its G proteins [15,16].

Cellular mechanisms of GPCR ligand-induced DMR signals

Historically, optical biosensors have found little use in whole cell sensing. The primary reason is that any optical responses of cells measured with a label free biosensor are often considered as nonspecific. In our view, understanding the nature of ligand-induced optical signals is crucial for cell sensing. In the recent years, we have adopted several approaches including chemical biology, cell biology and biophysics approaches to study the cellular mechanisms accounted for ligand-induced DMR signals.

Recent chemical biology and orthogonal fluorescence imaging studies suggest that the G q -DMR signal is downstream of Ca 2+ mobilization [16], whereas the G s -DMR signal is downstream of cAMP accumulation [17; and unpublished data]. Following classical receptor biology, a numerical analysis suggests that protein translocation and receptor internalization are two primary resources for the G q -DMR signal [13]. Central to G q -coupled receptor signaling is the rapid and dynamic translocation of its signaling components, including protein kinase C, GPCR kinases, β-arrestin, phosphatidylinositol phosphate-binding proteins, and diacylglycerol-binding proteins, to name but a few [39].

On the other hand, for the G s -DMR signal, the increase in cell adhesion is the major contributor to the P-DMR event, as shown in the epinephrine-induced responses in A431 cells [17; unpublished data]. Epinephrine is a natural agonist for β 2 adrenergic receptors (β 2 ARs) endogenously expressed in A431. Here the P-DMR event refers to the DMR phase with an increased signal, while the N-DMR refers to the phase with a decreased signal. The occurrence of the initial N-DMR event reflects the fact that the majority of downstream signaling components involved in the β 2 AR signaling complexes, with the exception (thus far) of A-kinase anchoring proteins and β-arrestins, are already compartmentalized at or near the cell membrane [40][41][42]. Thus, for G s -coupled receptor the recruitment of intracellular targets to the activated receptors is much less pronounced than G q -coupled receptor, and is overwhelmed by other cellular events leading to an initial decrease in local mass density in cells.

Applications of optical biosensors in cell signaling

Optical biosensors can follow receptor signaling in real time, and offer a kinetic optical signal in a ligand-dependent manner. A ligand-induced DMR signal is an integrated and quantifiable response of living cells, containing contributions of many downstream signaling events. Chemical biology studies, based on the modulation profiles of various modulators on a ligand-induced DMR signal, suggest that a DMR signal can be treated as a unique readout for systems cell biology of receptor signaling [18]. Such approach has been applied to map the signaling pathways and network interaction of EGF receptor (EGFR) [9,13], as well as to study the cellular functions of cholesterol in quiescent A431 cells [12]. Results showed that the EGF-induced DMR signal is saturable to EGF concentration, and sensitive to the activity of EGFR tyrosine kinase, MEK, and dynamin [9]. These optical biosensor cell assays link the EGF-induced DMR signal of quiescent A431 cells mainly to the Ras/mitogen-activated protein kinase pathway, which primarily proceeds through MEK and leads to cell detachment [43]. The bradykinin-induced DMR responses of cells pre-treated with the buffer only (HBSS) were in comparison with those pretreated with 500nM GF109203x (a protein kinase C inhibitor) (a), or 1 μM KT5720 (a protein kinase B inhibitor) (b). The bradykinin concentration was 16nM, and the cells were starved with 0.1% fetal bovine serum medium for 18hrs before assays. The solid arrows indicate the time when the agonist solution is introduced. Data was reproduced with permission from ref. 10.

GPCR signaling is sophisticated -a receptor may couple simultaneously to more than one G protein subtype, and may interact with other signaling molecules and scaffolding proteins such as arrestins [36]. The DMR signal imitates the complexity of GPCR signaling, as evidenced by bradykinin B 2 receptor signaling in A431. Bradykinin, a vasoactive nonapeptide, is implicated in the regulation of many physiological and pathophysiological responses, including vascular permeability, inflammation, pain and neurotransmitter release [44]. Endogenous bradykinin B 2 receptor in A431 cells is mitogenic or anti-mitogenic, dependent on the cellular states as well as the doses of bradykinin [10,45]. In proliferating states, low doses (<100nM) of bradykinin trigger G s -mediated signaling, while high doses (>100nM) of bradykinin favors G q -signaling. On the other hand, bradykinin between 0.5nM and about 100nM mediated both G s and G q -pathways in quiescent A431 cells obtained with 0.1% fetal bovine serum for about 20hours [10]. As summarized in Figure 5, chemical biology study showed that in those quiescent cells, blockage of protein kinase C activity only partially attenuates the initial P-DMR, but potentiates the retarded event, as indicated by the broken arrows in Figure 5. Conversely, blockage of protein kinase A activity increases the initial P-DMR event, but suppresses the retarded event.

These results suggest that the activation of B 2 receptors leads to both G q and G s signaling, and both pathways negatively cross-regulated each other [10,45]. DMR signal is a novel physiological response of living cells, and can be used to study ligand pharmacology. When a ligand is specific to a receptor expressed in a cell system, the ligand-induced DMR signal is also receptor-specific, dose-dependent and saturable [9,10,15,16]. Furthermore, the DMR signal exhibits expected desensitization patterns [16]. Desensitization and re-sensitization is common to almost all GPCRs [46]. Figure 6 compares the desensitization patterns of A431 cells in response to repeated stimulation with distinct combinations of two agonists obtained using the biosensor with those measured with Ca 2+ mobilization assays. Results showed that both technologies lead to almost identical desensitization patterns. Furthermore, the DMR signal also maintains the fidelity of GPCR ligands, similar to those obtained using conventional technologies. The biosensor can distinguish full agonists, partial agonists, inverse agonists, antagonists, and allosteric modulators [10, 16, 17; unpublished data]. Figure 6. GPCR agonist-induced DMR signals follow classical desensitization patterns, similar to those measured with Ca 2+ mobilization assays. A431 cells responded to repeated stimulation with distinct combinations of two agonists: bradykinin and trypsin, for endogenous B 2 receptor and protease activated receptor subtype 2 (PAR 2 ), respectively. Data was reproduced with permission from ref. 16

Conclusion

The ability of examining living cells is vital to understand the biological functions of cellular targets, and to the success of drug discovery and development. Although more complex and less specific than biomolecular interaction analysis, cell-based assays show superior ability to facilitate the measurements of mode of action, pathway activation, toxicity, and phenotypic responses of cells induced by exogenous stimuli. Together with the commercial availability of high throughput instruments, the newly discovered capability for studying cell signaling will accelerate wide acceptance of optical biosensors in both fundamental researches and drug discovery processes, beyond biomolecular interaction analysis.

Figure 1 .
1Principles of three types of optical biosensors for biomolecular interaction analysis. (a) SPR. The receptor (Y) is covalently coupled to the derivatized surface of a thin layer of gold, which is deposited onto a glass substrate. The light incident on the gold layer is directed by a prism. The reflectance of the gold surface is a function of incident angle. The resonance angle shifts when target molecules ( ) in solution bind to the immobilized receptors. (b) RWG biosensor. The coupling of incident light into the waveguide is achieved by the diffraction grating. The intensity of reflected light is a function of the resonant wavelength. The binding of target molecules in a sample to the immobilized receptors results in a shift in the resonant wavelength. (c) Interferometric biosensor. A broadband light resource is directly incident at the solution-surface interface. Two types of reflected light waves originated from the two reflecting surfaces, the interface with the optical layer and the surface of bio-receptors, interact each other, leading to interference patterns. The binding of biomolecules to the bio-receptors alters the inference pattern.

Figure 3 .
3Two detection schemes of RWG biosensors for high throughput applications.

Figure 4 .
4GPCR signaling and three major classes of DMR signals in living cells using RWG biosensor. (a) G q -signaling and its DMR signal (protease activated receptor subtype 1 in Chinese hamster ovary cells, 10 unit/ml thrombin). (b) G s -signaling and its DMR signal (β 2 -adrenergic receptors in A431 cells, 2nM epinephrine). (c) G i -signaling and its DMR signal (lysophophatidic acid (LPA) receptor subtype 1 in A431, 100nM LPA). The broken arrows indicate the time when an agonist solution is introduced. Data was generated using the angular interrogation system. 1 unit equals to 100 pm shift in resonant wavelength. Figure was reproduced with permission from Ref.15.

Figure 5 .
5Chemical intervention reveals dual signaling of bradykinin B 2 receptor in A431 cells.

.trypsin
bradykinin

bradykinin
trypsin

25%
2min

trypsin
trypsin

1unit

30min

trypsin

bradykinin

braykinin trypsin

trypsin trypsin

Ca 2+ mobilization
RWG biosensor

© 2007 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.

Analysis of molecular recognition using optical biosensors. R J Leatherbarrow, P R Edwards, Curr. Opin. Chem. Biol. 3Leatherbarrow, R.J.; Edwards, P.R. Analysis of molecular recognition using optical biosensors. Curr. Opin. Chem. Biol. 1999, 3, 544-547.

Optical biosensors in drug discovery. M A Cooper, Nature Rev. Drug Discov. 1Cooper, M.A. Optical biosensors in drug discovery. Nature Rev. Drug Discov. 2002, 1, 515-528.

Kinetics of adhesion and spreading of animan cells. J J Ramsden, S.-Y Li, J E Prenosil, E Heinzle, Biotechnol. Bioeng. 43Ramsden, J.J.; Li, S.-Y.; Prenosil, J.E.; Heinzle, E. Kinetics of adhesion and spreading of animan cells. Biotechnol. Bioeng. 1994, 43, 939-945.

Imaging of cell/substrate contacts of living cells with surface plasmon resonance microscopy. K.-F Giebel, C Bechinger, S Herminghaus, M Riedel, P Leiderer, U Weiland, M Bastmeyer, Biophys. J. 76Giebel, K.-F.; Bechinger, C.; Herminghaus, S.; Riedel, M.; Leiderer, P.; Weiland, U.; Bastmeyer, M. Imaging of cell/substrate contacts of living cells with surface plasmon resonance microscopy. Biophys. J. 1999, 76, 509-516.

Monitoring of living cell attachment and spreading using reverse symmetry waveguide sensing. R Horath, H C Pedersen, N Skivesen, D Selmeczi, N B Larsen, Appl. Phys. Lett. 71101Horath, R.; Pedersen, H.C.; Skivesen, N.; Selmeczi, D.; Larsen, N.B. Monitoring of living cell attachment and spreading using reverse symmetry waveguide sensing. Appl. Phys. Lett. 2005, 86, e071101.

Development and application of surface plasmon resonance-based biosensors for the detection of cell-ligand interactions. J G Quinn, S O'neill, A Doyle, C Mcatamney, D Diamond, B D Maccraith, R Kennedy, Anal. Biochem. 281Quinn, J.G.; O'Neill, S.; Doyle, A.; McAtamney, C.; Diamond, D.; MacCraith, B.D.; O'Kennedy, R. Development and application of surface plasmon resonance-based biosensors for the detection of cell-ligand interactions. Anal. Biochem. 2000, 281, 135-143.

Realtime analysis of ligand-induced cell surface and intracellular reactions of living mast cells using a surface plasmon resonance-based biosensor. M Hide, T Tsutsui, H Sato, T Nishimura, K Morimoto, S Yamamoto, K Yoshizato, Anal. Biochem. 302Hide, M.; Tsutsui, T.; Sato, H.; Nishimura, T.; Morimoto, K.; Yamamoto, S.; Yoshizato, K. Real- time analysis of ligand-induced cell surface and intracellular reactions of living mast cells using a surface plasmon resonance-based biosensor. Anal. Biochem. 2002, 302, 28-37.

Feasibility study of an online toxicological sensor based on the optical waveguide technique. J Voros, R Graf, G L Kenausis, A Bruinink, J Mayer, M Textor, E Wintermantel, N D Spencer, Biosensors & Bioelectronics. 15Voros, J.; Graf, R.; Kenausis, G.L.; Bruinink, A.; Mayer, J.; Textor, M.; Wintermantel, E.; Spencer, N.D. Feasibility study of an online toxicological sensor based on the optical waveguide technique. Biosensors & Bioelectronics 2000, 15, 423-429.

Characteristics of dynamic mass redistribution of EGF receptor signaling in living cells measured with label free optical biosensors. Y Fang, A M Ferrie, N H Fontaine, P K Yuen, Anal. Chem. 77Fang, Y.; Ferrie, A.M.; Fontaine, N.H.; Yuen, P.K. Characteristics of dynamic mass redistribution of EGF receptor signaling in living cells measured with label free optical biosensors. Anal. Chem. 2005, 77, 5720-5725.

Optical biosensor provides insights for bradykinin B 2 receptor signaling in A431 cells. Y Fang, G Li, J Peng, FEBS Lett. 579Fang, Y.; Li, G.; Peng, J. Optical biosensor provides insights for bradykinin B 2 receptor signaling in A431 cells. FEBS Lett. 2005, 579, 6365-6374.

Probing cytoskeleton modulation by optical biosensors. Y Fang, A M Ferrie, G Li, FEBS Lett. 579Fang, Y.; Ferrie, A.M.; Li, G. Probing cytoskeleton modulation by optical biosensors. FEBS Lett. 2005, 579, 4175-4180.

Cellular functions of cholesterol probed with optical biosensors. Y Fang, A M Ferrie, G Li, N H Fontaine, Biochim. Biophys. Acta. 1763Fang, Y.; Ferrie, A.M.; Li, G.; Fontaine, N.H. Cellular functions of cholesterol probed with optical biosensors". Biochim. Biophys. Acta 2006, 1763, 254-261.

Resonant waveguide grating biosensor for living cell sensing. Y Fang, A M Ferrie, N H Fontaine, J Mauro, J Balakrishnan, Biophys. J. 91Fang, Y.; Ferrie, A.M.; Fontaine, N.H.; Mauro, J.; Balakrishnan, J. Resonant waveguide grating biosensor for living cell sensing. Biophys. J. 2006, 91, 1925-1940.

Label-free profiling of endogenous G protein-coupled receptors using a cell-based high throughput screening technology. G Li, A M Ferrie, Y Fang, J. Assoc. Lab. Automat. 11Li, G.; Ferrie, A.M.; Fang, Y. Label-free profiling of endogenous G protein-coupled receptors using a cell-based high throughput screening technology. J. Assoc. Lab. Automat. 2006, 11, 181- 187.

Non-invasive optical biosensor for assaying endogenous G proteincoupled receptors in adherent cells. Y Fang, G Li, A M Ferrie, J. Pharmacol. Toxicol. Methods. 55Fang, Y.; Li, G.; Ferrie, A.M. Non-invasive optical biosensor for assaying endogenous G protein- coupled receptors in adherent cells. J. Pharmacol. Toxicol. Methods 2007, 55, 314-322.

Optical biosensor differentiates signaling of endogenous PAR 1 and PAR 2 in A431 cells. Y Fang, A M Ferrie, BMC Cell Biol. 812Fang, Y..; Ferrie, A.M. Optical biosensor differentiates signaling of endogenous PAR 1 and PAR 2 in A431 cells. BMC Cell Biol. 2007, 8, 24:1-24:12.

Systems biology and pharmacology of β 2 adrenergic receptors in A431. Y Fang, A M Ferrie, J Lahiri, Trends in Signal Transduction Research. Meyers, J.N., Ed; New YorkNova Science Publishers IncFang, Y.; Ferrie, A.M.; Lahiri, J. Systems biology and pharmacology of β 2 adrenergic receptors in A431. In Trends in Signal Transduction Research, Meyers, J.N., Ed.; Nova Science Publishers Inc.: New York, 2007; pp. 145-171.

Label-free cell-based assays with optical biosensors in drug discovery. Assays and Drug Development Technologies. Y Fang, 4Fang, Y. Label-free cell-based assays with optical biosensors in drug discovery. Assays and Drug Development Technologies 2006, 4, 583-595.

Optical biosensors: where next and how soon. M A Cooper, Drug Discov. Today. 11Cooper, M.A. Optical biosensors: where next and how soon. Drug Discov. Today 2006, 11, 1061- 1067.

Higher-throughput, label-free, real-time molecular interaction analysis. R L Rich, D G Myszka, Anal. Biochem. 361Rich, R.L.; Myszka, D.G. Higher-throughput, label-free, real-time molecular interaction analysis. Anal. Biochem. 2007, 361, 1-6.

Sensitivity of grating couplers as integrated-optical chemical sensors. K Tiefenthaler, W Lukosz, J. Opt. Soc. Am. B. 6Tiefenthaler, K.; Lukosz, W. Sensitivity of grating couplers as integrated-optical chemical sensors. J. Opt. Soc. Am. B 1989, 6, 209-220.

Refractometry of living cells. Part I. basic principles. Quart. R Barer, S Joseph, J. Microsc. Science. 95Barer, R.; Joseph, S. Refractometry of living cells. Part I. basic principles. Quart. J. Microsc. Science 1954, 95, 399-423.

Ellipsometry as a tool to study the adsorption behavior of synthetic and biopolymers at the air-water interface. J A De Feijter, J Benjamins, F A Veer, Biopolymers. 17De Feijter, J.A.; Benjamins, J.; Veer, F.A. Ellipsometry as a tool to study the adsorption behavior of synthetic and biopolymers at the air-water interface. Biopolymers 1978, 17, 1759-1772.

Interferometric sensor for detection of surface-bound bioreactions. A Brandenburg, R Krauter, C Kunzel, M Stefan, H Schulte, Appl. Optics. 39Brandenburg, A.; Krauter, R.; Kunzel, C.; Stefan, M.; Schulte, H. Interferometric sensor for detection of surface-bound bioreactions. Appl. Optics 2000, 39, 6396-6405.

Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. K Burridge, K Fath, T Kelly, G Nuckolls, C Turner, Annu. Rev. Cell Biol. 4Burridge, K.; Fath, K.; Kelly, T.; Nuckolls, G.; Turner, C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu. Rev. Cell Biol. 1988, 4, 487-525.

Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts. W T Chen, S J Singer, J. Cell Boil. 95Chen, W.T.; Singer, S.J. Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts. J. Cell Boil. 1982, 95, 205-222.

Tissue cells feel and respond to the stiffness of their substrate. D E Discher, P Janmey, Y Wang, Science. 310Discher, D.E.; Janmey, P.; Wang, Y. Tissue cells feel and respond to the stiffness of their substrate. Science 2005, 310, 1139-1143.

Rewiring cell adhesion. M Kato, M Mrksich, J. Am. Chem. Soc. 126Kato, M.; Mrksich, M. Rewiring cell adhesion. J. Am. Chem. Soc. 2006, 126, 6504-6505.

Micromotion of mammalian cells measured electrically. I Giaever, C R Keese, Proc. Natl. Acad. Sci. Natl. Acad. SciUSA88Giaever, I.; Keese, C.R. Micromotion of mammalian cells measured electrically. Proc. Natl. Acad. Sci. USA 1991, 88, 7896-7900.

Organization of mammalian cytoplasm. A Hudder, L Nathanson, M P Deutscher, Mol. Cell Biol. 23Hudder, A.; Nathanson, L.; Deutscher, M.P. Organization of mammalian cytoplasm. Mol. Cell Biol. 2003, 23, 9318-9326.

Location, location, location: membrane targeting directed by PX domains. T K Sato, M Overduin, S D Emr, Science. 294Sato, T.K.; Overduin, M.; Emr, S.D. Location, location, location: membrane targeting directed by PX domains. Science 2001, 294, 1881-1885.

Four-dimensional organization of protein kinase signaling cascades: the roles of diffusion, endocytosis and molecular motors. B N Kholodenko, J. Exper. Biol. 206Kholodenko, B.N. Four-dimensional organization of protein kinase signaling cascades: the roles of diffusion, endocytosis and molecular motors. J. Exper. Biol. 2003, 206, 2073-2082.

Physicochemical modelling of cell signalling pathways. B B Aldridge, J M Burke, D A Lauffenburger, P K Sorger, Nat. Cell Biol. 8Aldridge, B.B.; Burke, J.M.; Lauffenburger, D.A.; Sorger, P.K. Physicochemical modelling of cell signalling pathways. Nat. Cell Biol. 2006, 8, 1195-1203.

Trafficking of the ErbB receptors and its influence on signaling. H S Wiley, Exper. Cell Res. 284Wiley, H.S. Trafficking of the ErbB receptors and its influence on signaling. Exper. Cell Res. 2003, 284, 78-88.

Trafficking of signaling modules by kinesin motors. B J Schnapp, J. Cell Sci. 116Schnapp, B.J. Trafficking of signaling modules by kinesin motors. J. Cell Sci. 2003, 116, 2125- 2135.

β-arrestins: traffic cops of cell signaling. R J Lefkowitz, E J Whalen, Curr. Opinion Cell Biol. 16Lefkowitz, R.J.; Whalen, E.J. β-arrestins: traffic cops of cell signaling. Curr. Opinion Cell Biol. 2004, 16, 162-168.

The repertoire of G-protein-coupled receptors in fully sequenced genomes. R Fredriksson, H B Schioth, Mol. Pharmacol. 67Fredriksson, R.; Schioth, H.B. The repertoire of G-protein-coupled receptors in fully sequenced genomes. Mol. Pharmacol. 2005, 67, 1414 -1425.

G protein-coupled receptor microarrays for drug discovery. Y Fang, J Lahiri, L Picard, Drug Discov. Today. 8Fang, Y.; Lahiri, J.; Picard, L. G protein-coupled receptor microarrays for drug discovery. Drug Discov. Today 2003, 8, 755-761.

Translocation of diacylglycerol kinase θ from cytosol to plasma membrane in response to activation of G protein-coupled receptors and protein kinase C. J Van Baal, J De Widt, N Divecha, W J Van Blitterswijk, J. Biol. Chem. 280van Baal, J.; de Widt, J.; Divecha, N.; van Blitterswijk, W.J. Translocation of diacylglycerol kinase θ from cytosol to plasma membrane in response to activation of G protein-coupled receptors and protein kinase C. J. Biol. Chem. 2005, 280, 9870-9878.

Structure, localization, and regulation of cGMPinhibited phosphodiesterase (PDE3). E Degerman, P Belfrage, V C Manganiello, J. Biol. Chem. 272Degerman, E.; Belfrage, P.; Manganiello, V.C. Structure, localization, and regulation of cGMP- inhibited phosphodiesterase (PDE3). J. Biol. Chem. 1997, 272, 6823-6826.

The scaffold protein gravin (cAMP-dependent protein kinase-anchoring protein 250) binds the β 2 -adrenergic receptor via the receptor cytoplasmic Arg-329 to Leu-413 domain and provides a mobile scaffold during desensitization. G Fan, E Shumay, H Wang, C C Malbon, J. Biol. Chem. 276Fan, G.; Shumay, E.; Wang, H.; Malbon, C.C. The scaffold protein gravin (cAMP-dependent protein kinase-anchoring protein 250) binds the β 2 -adrenergic receptor via the receptor cytoplasmic Arg-329 to Leu-413 domain and provides a mobile scaffold during desensitization. J. Biol. Chem. 2001, 276, 24005-24014.

Gravin-mediated formation of signaling complexes in β 2 -adrenergic receptor desensitization and resensitization. F Lin, H.-Y Wang, C C Malbon, J. Biol. Chem. 275Lin, F.; Wang, H.-Y.; Malbon, C.C. Gravin-mediated formation of signaling complexes in β 2 - adrenergic receptor desensitization and resensitization. J. Biol. Chem. 2000, 275, 19025-19034.

Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase. Z Lu, G Jiang, P Blume-Jensen, T Hunter, Mol. Cell Biol. 21Lu, Z.; Jiang, G.; Blume-Jensen, P.; Hunter, T. Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase. Mol. Cell Biol. 2001, 21, 4016-4031.

Bradykinin receptor ligands: therapeutic perspectives. F Marceau, D Regoli, Nat. Rev. Drug Discov. 3Marceau, F.; Regoli, D. Bradykinin receptor ligands: therapeutic perspectives. Nat. Rev. Drug Discov. 2004, 3, 845-852.

Dual bradykinin B 2 receptor signalling in A431 human epidermoid carcinoma cells: activation of protein kinase C Is counteracted by a G s -mediated stimulation of the cyclic AMP pathway. C Liebmann, A Graness, B Ludwig, A Adomeit, A Boehmer, F.-D Boehmer, B Nürnberg, R Wetzker, Biochem. J. 313Liebmann, C.; Graness, A.; Ludwig, B.; Adomeit, A.; Boehmer, A.; Boehmer, F.-D.; Nürnberg, B.; Wetzker, R. Dual bradykinin B 2 receptor signalling in A431 human epidermoid carcinoma cells: activation of protein kinase C Is counteracted by a G s -mediated stimulation of the cyclic AMP pathway. Biochem. J. 1996, 313, 109-118.

Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. S S G Ferguson, Pharmacol. Rev. 53Ferguson, S.S.G. Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol. Rev. 2001, 53, 1-24.