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In rat, chorda tympani nerve taste responses to Na+ salts increase between roughly 10 and 45 days of age to reach stable, mature magnitudes. Previous evidence from in vitro preparations and from taste nerve responses using Na+ channel blockers suggests that the physiological basis for this developmental increase in gustatory Na+ sensitivity is the progressive addition of functional, Na+ transduction elements (i.e., amiloride-sensitive Na+ channels) to the apical membranes of fungiform papilla taste receptor cells. To avoid potential confounding effects of pharmacological interventions and to permit quantification of aggregate Na+ channel behavior using a kinetic model, we obtained chorda tympani nerve responses to NaCl and sodium gluconate (NaGlu) during receptive field voltage clamp in rats aged from 12–14 to 60 days and older (60+ days). Significant, age-dependent increases in chorda tympani responses to these stimuli occurred as expected. Importantly, apical Na+ channel density, estimated from an apical Na+ channel kinetic model, increased monotonically with age. The maximum rate of Na+response increase occurred between postnatal days 12–14 and 29–31. In addition, estimated Na+ channel affinity increased between 12–14 and 19–23 days of age, i.e., on a time course distinct from that of the maximum rate of Na+response increase. Finally, estimates of the fraction of clamp voltage dropped across taste receptor apical membranes decreased between 19–23 and 29–31 days of age for NaCl but remained stable for NaGlu. The stimulus dependence of this change is consistent with a developmental increase in taste bud tight junctional Cl− ion permeability that lags behind the developmental increase in apical Na+ channel density. A significant, indirect anion influence on apical Na+ channel properties was present at all ages tested. This influence was evident in the higher apparent apical Na+ channel affinities obtained for NaCl relative to NaGlu. This stimulus-dependent modulation of apical Na+ channel apparent affinity relies on differences in the transepithelial potentials between NaCl and NaGlu. These originate from differences in paracellular anion permeability but act also on the driving force for Na+ through apical Na+channels. Detection of such an influence on taste depends fundamentally on the preservation of taste bud polarity and on a direct measure of sensory function, such as the response of primary afferents.
Numerous behavioral, neurophysiological, and electrophysiological studies indicate that the amiloride-sensitive Na+ channel is a transduction pathway for taste system Na+ stimuli in numerous mammals (Brand et al. 1985; DeSimone et al. 1981; Gilbertson et al. 1993; Heck et al. 1984; Hill et al. 1990; Schiffman et al. 1983; reviewed in Stewart et al. 1997a) including some primates (Hellekant et al. 1997a,b). In mammals where this Na+ sensing system is functional, passive movement of stimulus Na+ ions across the taste cell apical membrane causes membrane depolarization and, consequently, release of neurotransmitter onto primary taste afferents (reviewed in Roper 1992). Notably in several species, including rat and hamster, neural responses to NaCl are not completely suppressed by amiloride (Brand et al. 1985;DeSimone and Ferrell 1985; Heck et al. 1984; Hettinger and Frank 1990). The unsuppressed part of the neural response to NaCl has been termed the amiloride-insensitive component (Formaker and Hill 1988;Ye et al. 1993a).
The amiloride-insensitive component of the neural taste response to NaCl is highly dependent on the stimulus anion (Formaker and Hill 1988) and is thought to result from activation of transduction sites below taste cell tight junctions (Ye et al. 1991, 1993a). Specifically, when Na+salts with small, highly permeant anions, such as Cl−, are applied to the lingual surface, electroneutral diffusion of Na+ and the stimulus anion through taste cell tight junctions is facilitated. In contrast, when Na+ salts with larger, tight junction-impermeant anions are applied to the tongue, electroneutral diffusion of Na+ and the stimulus anion is severely limited. Consequently, neural responses to Na+ salts with large, organic anions, such as acetate or gluconate, are transduced entirely by the apical pathway and completely inhibited by amiloride (Formaker and Hill 1988; Ye et al. 1993a). Therefore the composite neural response to NaCl comprises distinct apical, voltage-sensitive and basolateral, voltage-insensitive components.
Interestingly, significant developmental alterations in gustatory Na+ responses have been documented in several mammalian species (Ferrell et al. 1981; Hill 1988; Hill and Almli 1980; Mistretta and Bradley 1983; Ninomiya et al. 1991). In rats, impressive age-dependent increases in the magnitude of whole chorda tympani nerve responses to Na+ (and Li+) stimuli occur (Ferrell et al. 1981; Hill and Almli 1980). Specifically, when expressed relative to an NH4Cl reference response, NaCl and LiCl response magnitudes increased progressively between ∼10 and 45 days postnatal. By ∼45 days of age, NaCl and LiCl response magnitudes reached stable, adult levels. Analyses of single chorda tympani fiber responses during development have established clearly that the developmental increase in rat whole chorda tympani sensitivity to NaCl and LiCl is due specifically to increases in single chorda tympani fiber responses to these salts (Hill et al. 1982). In contrast, responses to acids, ammonium chloride and other monochloride salts are robust in early postnatal rats and remain so during development. The fact that Na+exhibits a unique developmental time course among taste stimuli suggests that underlying physiological changes originate at the level of the receptor cells responsible for transduction of the Na+ taste signal. More recent work has examined the potential mechanisms that underlie developmental changes in neural sensitivity to Na+ stimuli.
Changes in amiloride-sensitive Na+ channel function have been proposed to account for the developmental increase in gustatory system Na+ sensitivity. For example,Hill and Bour (1985) showed that the sensitivity of whole chorda tympani NaCl responses to the suppressive effects of amiloride increased in parallel with developmental increases in sensitivity to NaCl and LiCl. In particular, chorda tympani responses to NaCl and to LiCl in 12- to 13-day-old rats were unaffected by 100 μM amiloride. However, in rats aged 29–31 and 90–110 days, chorda tympani responses were potently suppressed by amiloride. Furthermore the degree of response inhibition caused by amiloride was proportional to overall taste system sensitivity to these stimuli at these age points. An implication of these results is that the apical Na+ transduction pathway develops postnatally, while the basolateral Na+ transduction pathway appears to be in place and functional around the time of birth. These findings were later replicated by Sollars and Bernstein (1994). Hill and Bour (1985) concluded that the concomitant, progressive increases in gustatory sensitivity to NaCl (and LiCl) and to amiloride were due to an orderly increase in the functional expression of amiloride-sensitive Na+channels in taste cell apical membranes. With this notion in mind,Stewart et al. (1995) hypothesized that the developmental increase in gustatory Na+sensitivity was due to a progressive addition of newly synthesized, functional amiloride-sensitive Na+ channels. They tested this hypothesis by correlating the presence of Na+ channel-like antigen in fungiform papilla taste buds with the developmental increase in gustatory Na+ and amiloride sensitivity.
Surprisingly, although the rat chorda tympani does not exhibit significant amiloride sensitivity before 7–10 days postnatal, antigenic determinants of amiloride-sensitive Na+channel are observed in taste cells of fungiform papilla taste buds as early as the day after birth (Stewart et al. 1995). However, limited microscopic resolution in that study precluded definitive localization of Na+-channel-like immunoreactivity to the apical membrane. Likewise, Kossel and co-workers (1997) used whole cell recordings to identify amiloride-blockable currents in isolated, single taste cells of fungiform papilla taste buds from rats as young as 2 days postnatal. They determined that the percentage of neonatal rat taste cells that exhibit amiloride-blockable currents is stable throughout development; the normalized density of amiloride-sensitive channels in taste cells is stable throughout development; and the amiloride-inhibition constant of taste cell Na+ channels is stable throughout development. The authors suggest that redistribution of fully functional channels from the basolateral to apical domain and/or maturation (i.e., progressive opening) of taste pores could account for the developmental increase in neural Na+ and amiloride sensitivity in rats. On the other hand, progressive pore opening would be expected to influence the apparent development of neural responses to all stimuli and not only those to Na+ salts. Nonetheless these electrophysiological and histochemical observations suggest that the Na+ transduction apparatus is functionally mature well before 7–10 days of age and therefore diverge considerably from earlier neurophysiological data.
In an attempt to clarify the nature of this apparent discrepancy, we have used in the present study in vivo lingual receptive field voltage clamping with simultaneous whole chorda tympani recordings to resolve age-related changes in taste cell apical Na+channel function. This approach offers several advantages over the use of either isolated, in vitro preparations or conventional neurophysiological recordings. First, the topological arrangement of taste buds and, therefore segregation of apical and basolateral transduction pathways, remains intact. Second, aggregate apical membrane Na+ channel properties can be studied quantitatively without the use of amiloride. This is especially important because it has been suggested that restricted access of amiloride to taste cell apical membranes caused by unopened taste pores may contribute to the apparent lack of amiloride sensitivity in young rats (Kossel et al. 1997). Finally, the combination of epithelial voltage clamp with neural recording provides a measure of how taste cells "report" exclusively taste-related membrane events. We hypothesized that the developmental increase in gustatory Na+ sensitivity in rats is due to the progressive addition of functional amiloride-sensitive Na+channels to taste cell apical membranes. The results we obtained are consistent with such an age-related increase in the apparent number of functional, amiloride-sensitive Na+ channels in taste cell apical membranes. In addition, the results provide entirely new evidence that the apparent affinity of the aggregate apical Na+ transduction pathway also increases during postnatal development. Finally, our results indicate that these age-related changes in apical Na+ channel function are temporally distinct. Therefore the onset of mature gustatory Na+ sensing in rat appears to rely on developmental sequelae that are more complicated than a simple, monotonic increase in the number of functional Na+ transduction sites. Portions of these findings have been presented previously in preliminary form (Hendricks et al. 1998; Stewart et al. 1997b).
Sprague-Dawley rats at 12–14 days of age, 19–23 days of age, 29–31 days of age, or 60 days of age and older (hereafter termed 60+ day old) were used in these studies. Rats were bred and raised in our colony with the exception of several rats in the 60+ day old group, which were obtained directly from the supplier. These adult animals, and all breeding stock, were obtained from Harlan Sprague-Dawley. Rats were provided standard laboratory chow (Purina) and water ad libitum and maintained on a 12 h:12 h light:dark photoperiod (lights on at 0700). Breeding harems (3 females and 1 male) were established, and the male remained in the cage for 14 days. After 14 days, the male was removed, and the females were segregated into individual maternity cages. Maternity cages were checked for births each day at 0800 and 1800 h. The day of birth (postnatal day 0) was recorded as the date when litters were first observed. If necessary, on the day after birth, litters were culled to 10 pups (5 female, 5 male, when possible); litters with fewer than 8 pups were not used for these studies. Generally, a single pup from each litter was sampled for each age range noted in the preceding text. However, each age range includes two data sets obtained from litter mates. Remaining offspring were weaned to the preceding diet at 21 days of age and then transferred to sex-segregated cages no later than 45 days of age.
Chorda tympani recording
Rats at the ages noted in the preceding text were deeply anesthetized by intraperitoneal injection with pentobarbital (50–65 mg/kg). Supplemental injections (20–30 mg/kg) were given as needed to maintain surgical anesthesia. Body temperature was maintained with a circulating water heating pad (K-MOD 100, Allegiance Healthcare, McGaw Park, IL). Bilateral hypoglossectomy to prevent tongue movement was performed after blunt dissection of surrounding tissue, and the trachea was cannulated to facilitate free breathing. Following placement of the animal into a nontraumatic head holder, the left or right chorda tympani nerve was exposed via an epimandibular approach. The nerve was freed of surrounding connective tissue, cut near its entrance into the tympani bulla, desheathed, and placed onto a 28-gauge platinum wire electrode. A reference electrode was placed in nearby tissue. The wound was filled with a mixture of petroleum jelly and mineral oil to prevent drying of the nerve. Neural activity was fed to a fixed gain isolation amplifier (BioAmp 100, Axon Instruments, Foster City, CA) and then to a Grass P511 preamplifier (AstroMed, West Warwick, RI). The amplified signal was monitored with an oscilloscope and audio loudspeaker. Amplified neural activity was RMS-rectified and integrated with a time constant of 1–2 s. The integrated output was recorded on one channel of a Linseis L6514 4-channel rectilinear chart recorder (Linseis, Princeton Junction, NJ).
Lingual receptive field voltage clamp
Simultaneous lingual epithelial voltage clamping and chorda tympani neural recordings have been described in detail (Stewart et al. 1996; Ye et al. 1993a). Briefly, a portion of the anterior dorsal lingual epithelium was enclosed within a vacuum-applied cast acrylic stimulation chamber. For rats aged 29–31 and 60+ days of age, the chamber enclosed a 28-mm2 patch of dorsal tongue, while the chamber used for 12–14 and 19–23 day old rats enclosed a 12.5-mm2 patch. The outer vacuum groove in the chamber creates a mechanically stable and electrically tight seal between the chamber and lingual epithelium. Stimulus and rinse solutions (see following text) were injected (4 ml; 1 ml/s) into the chamber via tubing fitted to a dedicated port (dead space: ∼0.5 ml). The chamber was fitted with separate Ag-AgCl electrodes for measurement of current and potential, while reference electrodes were placed noninvasively on the ventral lingual epithelium. The current-passing electrode in the chamber served as virtual ground, ensuring that only current passing through the stimulated patch was collected. Command currents and potentials were delivered by a voltage-current clamp amplifier (VCC600; Physiologic Instruments, San Diego, CA). Transepithelial potential and current responses were recorded on two channels of a Linseis L6514 4-channel rectilinear chart recorder.
Stimulus solutions and stimulus application
All solutions used were made from reagent grade chemicals (Sigma, St. Louis, MO; or Fisher, Pittsburgh, PA) dissolved in glass-distilled H2O. The rinse solution contained 15 mM KHCO3 (pH 8.7). A Na+-depleted Krebs-Henseleit solution (DKH) that contained (in mM) 6 KCl, 2 CaCl2, 1.2 MgSO4, 1.3 NaH2PO4, 25 NaHCO3, and 5.6 glucose (pH 7.5) was applied after each stimulus series to help maintain a stable transepithelial potential. Transepithelial potential in the presence of 150 mM NaCl rarely fluctuated more than ±5 mV during the course of an experiment. In addition, those preparations that exhibited absolute transepithelial potentials of greater than ±20 mV in the presence of 150 mM NaCl were excluded from analysis. Stimuli were concentration series (50, 100, 200, and 500 mM) of NaCl and Na+ gluconate (NaGlu). Sodium gluconate was selected as a stimulus in addition to NaCl primarily because the lower shunt permeability of gluconate relative to chloride significantly reduces the voltage-independent (i.e., basolateral) Na+ response component (Ye et al. 1991, 1993a). Therefore the chorda tympani response to NaGlu should derive primarily from voltage-dependent, apical transduction of Na+.
Stimuli were applied under zero-current clamp (equivalent to open circuit conditions), and steady-state potentials were recorded from the front panel display of the voltage-current clamp amplifier. Next, chorda tympani responses to the stimuli were obtained under voltage clamp at +60 and −60 mV relative to the zero-current clamp potential recorded for each stimulus. In this way, neural responses to concentration series were obtained under three membrane potential conditions: effective open circuit, and +60 and −60 mV voltage clamp. Each stimulus series was bracketed by the application of a 500 mM NH4Cl reference stimulus under zero-current clamp, and intervening data were retained for analysis only when the magnitude of the NH4Cl reference responses before and after the stimulus series varied by <10%. Steady-state response magnitudes, measured as the height of each integrated chorda tympani response 30 s after application of the stimulus, were expressed relative to the mean NH4Cl response magnitude bracketing a stimulus series. Ammonium chloride was selected as a reference stimulus for several reasons. First, expression of integrated neural data relative to NH4Cl provides a valid and reliable measure for comparisons of multiunit taste responses among subjects and between treatment groups (Beidler 1953). Importantly, because the responsiveness of the rat taste system to NH4Cl does not change during postnatal development (Hill et al. 1982), it also provides an appropriate, unchanging reference response for comparisons of Na+ responses among age groups.
The protocol for stimulation of the lingual epithelium was generally as follows. The reference NH4Cl stimulus was applied and allowed to remain in the chamber for 40–50 s. The solution was then rinsed from the chamber by repeated application of the KHCO3 rinse solution for ≥60 s. Then, the stimuli of interest (NaCl or NaGlu) were applied as a concentration series with each concentration allowed to remain in the chamber for 40–50 s before being rinsed from the chamber repeatedly with KHCO3, as noted in the preceding text. About 60 s after the final stimulus in a concentration series was rinsed from the chamber, the reference NH4Cl solution was reapplied then rinsed. At this point, DKH was injected into the chamber, allowed to remain on the tongue for 60 s, and then rinsed from the chamber with repeated applications of the KHCO3 rinse solution. Last, another reference stimulus was applied and rinsed, and the next concentration series was begun.
Statistical analyses
Data are expressed as means ± SE unless otherwise noted. Age-dependent differences in zero-current clamp chorda tympani response magnitudes at each stimulus concentration were determined using ANOVA with Student-Newman-Keuls (SNK) post hoc comparisons where appropriate.
Development of chorda tympani responses to NaCl
Relative to the NH4Cl reference response, integrated chorda tympani responses to NaCl and to NaGlu under zero-current clamp increased in a progressive manner between 12–14 days of age and adulthood. Table 1provides summary NaCl and NaGlu response magnitude data. Because we were primarily interested in determining whether the present results were consistent with previously published data regarding Na+ response development, we examined age-related differences in response magnitude only under the zero-current clamp condition, which is equivalent to conventional chorda tympani recording. Significant age-related differences in NaCl response magnitude under zero-current clamp were detected for NaCl concentrations 50 mM (ANOVA, P < 0.05) (Table 1). Student-Newman-Keuls posthoc comparisons indicated a significant, progressive, age-dependent increase in mean response magnitudes to 100, 200, and 500 mM NaCl ≤29–31 days of age (SNK, P < 0.05) (Table 1). At 29–31 days of age, mean chorda tympani response magnitudes to these stimuli did not differ significantly from 60+-day-old rat response magnitudes.
Results obtained for NaGlu were in general very similar to those obtained for NaCl. However, significant, age-related differences in zero-current clamp response magnitudes to NaGlu were detected only at 200 and 500 mM (ANOVA, P < 0.05). Post hoc comparisons revealed a significant age-related difference in response magnitudes to 200 mM NaGlu between 12–14 and 60+ day old rats (SNK,P < 0.05), while significant age-related differences in chorda tympani response magnitudes to 500 mM NaGlu were detected among all groups, except 29- to 31- and 60+-day-old rats. The results for zero-current clamp responses to both Na+salts are consistent with previously published data regarding the development of chorda tympani NaCl responses in rats (Ferrell et al. 1981; Hill and Almli 1980).
In conjunction with the developmental increase in Na+ response magnitude, the sensitivity of chorda tympani NaCl responses to applied lingual epithelial voltage perturbations increased with age. For example, Fig.1 depicts integrated chorda tympani responses to 100 mM NaCl under zero-current clamp and +60 and −60 mV voltage clamp obtained from a 12- to 14- and a 29- to 31-day-old rat. It is clear that the effect of voltage clamp on responses to this NaCl concentration is considerably greater in the 29- to 31-day-old rat. In general, and consistent with previously published results (Ye et al. 1993a,b), chorda tympani responses to NaCl at all concentrations were elevated under submucosal negative voltage clamp, which increases the electrochemical driving force for Na+ into the taste receptor cell, and suppressed under submucosal positive voltage clamp, which decreases the driving force for Na+ into the taste receptor cell. However, at 250 and 500 mM NaCl, the influence of voltage clamp appeared blunted in 29- to 31- and 60+-day-old rats (Fig.2). Chorda tympani responses to all concentrations of NaGlu obtained under voltage clamp also exhibited marked sensitivity to applied voltage perturbations, and the magnitude of this sensitivity appeared to increase with age. In contrast to the NaCl case, the effect of voltage clamp on NaGlu chorda tympani response magnitudes remained robust at higher concentrations and at all ages studied (Fig. 2). The effects of lingual epithelial voltage clamp on chorda tympani response-concentration functions for NaCl and NaGlu are shown in Figs. 3 and4, respectively.
Figures 3 and 4 clearly show that Na+ salt chorda tympani responses are subject to modulation by imposed transepithelial voltage perturbations even at the earliest age-point studied (12–14 days of age). By considering ion channel flow kinetics (Mintz et al. 1986; Ye et al. 1993a) and by assuming that depolarizing Na+ ion influx into taste cells is the rate-limiting event in the evoked neural response to Na+, we obtain the apical channel model equation, which is a modified form of the Beidler taste equation (Beidler 1954) Equation 1where, K m is the concentration of the NaCl that yields a half-maximal chorda tympani response, andCT max is the maximum value ofR, which is the neural response obtained at a given Na+ electrochemical concentration,c e. It is in this last term that the major difference between the apical channel model (Eq. 1 ) and the Beidler taste equation is found. Although the apical channel model equation closely resembles the Beidler taste equation, it takes into account the assumption that depolarizing Na+flux through apical channels into taste receptor cells, which will depend on electrical potential as well as ion concentration, is the rate-limiting step in the generation of the neural taste response to Na+ stimuli. That is, Na+flux through apical channels depends on electrochemical concentration, which is written as Equation 2where, c is the stimulus Na+concentration and φ is FΔV/RT, where ΔV is the applied clamp voltage (J · C− 1), at 35°C (i.e., 308°K), the approximate tongue surface temperature. In this instance,F = 9.65 × 104 C · mol− 1 andR = 8.314 J · mol− 1 · K− 1. Finally, δ is the fraction of φ that is "sensed" by Na+ at taste cell apical membranes (Palmer 1984; Ye et al. 1993a). For each age group, the chorda tympani NaCl and NaGlu response data were fit with this equation.
Figures 3 and 4 depict for each age group the best-fit parameters obtained with the apical channel model for NaCl and NaGlu response data, respectively, as well as plots of the theoretical functions that follow from it. As expected for both stimuli, the channel model equation predicted an age-related, monotonic increase inCT max, which is highly consistent with the postulated developmental increase in apical Na+ channel number. Surprisingly, predicted values of K m decreased in an age-related manner. This unexpected finding is also entirely consistent with the well-reported age-related increase in rat gustatory Na+ sensitivity. An interesting feature of these changes is that the leftward shift inK m along the concentration axis (i.e., an increase in channel affinity for Na+) occurs between12–14 and 19–23 days of age and is superimposed on a rapid increase in CT max that occurs primarily between 12–14 and 29–31 days of age. Finally, and much to our surprise, a shift in the predicted value of δ occurred between 19–23 and 29–31 days of age when NaCl was the stimulus. In contrast, when NaGlu was the stimulus, this parameter appeared to remain stable across the developmental period studied. Using the parameters obtained in the preceding text, the chorda tympani response data shown in Figs.3 and 4, were replotted as a function of NaCl electrochemical concentration, c e. That is, the NaCl and NaGlu response data were replotted after transforming the independent variables of clamp voltage and stimulus concentration into a single variable, c e (Eq.2 ).
Figures 5 and6 depict the resulting response-electrochemical concentration functions for NaCl and NaGlu, respectively. It can be seen clearly that across developmental age the three response-concentration functions (which correspond to the 3 clamp voltage conditions) shown in each panel of Figs. 3 and 4 collapse onto a single curve for each age group. This transformation of the data reveals that even at the earliest age points studied the actual stimulus intensity for Na+ isc e, i.e., the driving force for the ion through a passive channel. Moreover, for both NaCl and NaGlu, the collapsed response-concentration functions for all four age groups manifest the age-dependent increase inCT max and the age-dependent decrease in K m, which is evidenced by the increasing initial slope of the theoretical functions. Although the channel model accounts for most of the variance in the data (minimumR 2 ≥ 0.89), a notably larger deviation is observed for the 60+-day olds.
Such a deviation could arise from a secondary voltage-independent transduction process and could lead to errors in estimatingCT max. In an attempt to eliminate this as a source of error, the data were replotted as the voltage sensitivity index (VSI, Eq. 3 ). Because the VSI is defined as a difference in chorda tympani responses at two voltages, contributions from voltage-independent processes cancel out. The remaining, voltage-sensitive part of the chorda tympani response to NaCl (or NaGlu) should then arise exclusively from transduction by amiloride-sensitive Na+ channels (Ye et al. 1993a). Consequently, the VSI can be used to provide a more accurate estimate of the relative densities of apical Na+ channels among experimental groups (Ye et al. 1993b). In the present case the VSI is Equation 3where R(c, −60) is the mean chorda tympani response magnitude of a stimulus at concentration,c, and a clamp voltage of −60 mV, andR(c, +60) is the mean chorda tympani response magnitude of a stimulus at concentration, c, and a clamp voltage of +60 mV. The theoretical VSIs for NaCl and NaGlu responses were determined by substitution of Eq. 1 intoEq. 3 with the values of K m and δ obtained above. An "apical R max" (renamed from CT max to emphasize that it is the estimated maximum response component that arises exclusively from voltage-sensitive transduction processes) was obtained for each age group. The resulting theoretical curves for NaCl and NaGlu are shown in Fig. 7, A and B,respectively, while predicted apicalR max values are provided in Table2. For NaCl, when apicalR max values for each age group are expressed as a ratio of the apicalR max obtained for 60+-day-old rats, it can be inferred that fungiform papilla taste cell apical membranes in 12- to 14-day-old rats have ∼25% the mature density of functional amiloride-sensitive Na+ channels (0.42/1.68), those of 19- to 23-day-old rats have ∼47% the mature density of functional channels (0.79/1.68), while those of 29- to 31-day-old rats have ∼84% the mature density of apical Na+channels (1.41/1.68). Apical R maxestimates obtained for NaGlu are in excellent agreement with those obtained for NaCl (Table 2).
In the present study, simultaneous in vivo lingual epithelial voltage clamping and chorda tympani recording were used to examine the increase in taste system Na+ sensitivity that occurs during postnatal development in rat. The primary finding from these experiments is that the age-related increase in the chorda tympani Na+ response results from the development of a transduction mechanism that is driven by stimulus electrochemical concentration. This observation implicates a set of apical membrane ion channels as the stimulus transducer and, furthermore, indicates that the postnatal increase in chorda tympani Na+responses is due primarily to an increase in transducer (i.e., ion channel) relative density. These results confirm previous reports, which, based on amiloride pharmacology, concluded that a progressive increase in the number of functional amiloride-sensitive Na+ channels was probably the underlying mechanism for the developmental increase in taste system Na+ sensitivity in rat (Hill and Bour 1985; Sollars and Bernstein 1994). Our current results also extend those findings by revealing that in addition to the age-dependent increase in the relative density of taste receptor cell functional Na+ channels, the apparent Na+ binding affinity of the amiloride-sensitive Na+ channel (corresponding to a decrease inK m) increases as a function of postnatal age. Together, these two age-related changes in the intrinsic functional properties of the Na+ sensing apparatus contribute to the postnatal maturation of anterior tongue taste receptor Na+ sensitivity in rat. An especially interesting feature of these age-dependent changes in Na+ channel properties is that they are temporally distinct.
Results from our kinetic analyses of chorda tympani responses to NaCl and NaGlu during the postnatal period indicated differences in the timing of changes in apparent density and affinity of apical, amiloride-sensitive Na+ channels. Specifically, the age-related decrease in K m values for both Na+ salts was observed to occur between 12–14 and 19–23 days of age. EstimatedK m for NaCl decreased by ∼169 mM over this period and K m for NaGlu by ∼120 mM. Thereafter, K m values for both salts remained stable into adulthood (Fig.8 A). In contrast, for both Na+ salts, CT maxvalues increased rapidly between 12–14 and 29–31 days of age, at which point the rate of growth inCT max slowed considerably as mature, unchanging CT max values were approached. The age-related trends in these parameters are considered in greater detail in the following sections.
Development of chorda tympani responses to NaCl and NaGlu: parallel trends
As described in results, the quantitative trends in chorda tympani response development are in general similar for these two Na+ salts, and this similarity was fully expected at the outset of these studies. However, inspection of the data presented in results clearly indicates that there are important quantitative differences in the parameters that describe the development of the Na+ sensing apparatus with respect to these two salts. Consideration of these differences provides important insight regarding the development not only of apical, amiloride-sensitive Na+ channel function but also the development of taste bud polar epithelial topology.
By describing our data with a channel model equation, we were able to follow, operationally, the development of the apical, voltage-dependent Na+ sensing apparatus by obtaining changing values in the system parameters CT max,K m, and δ. The predictive utility of this model requires that changes in Na+ response magnitude be the consequence of changes in apical (i.e., amiloride-sensitive) Na+ channel function. For NaGlu, across all age points examined, it is clear that CT response magnitude is a single-valued function of the electrochemical Na+ concentration (e.g., Fig. 6). Moreover, values of R max (the estimate of relative channel density eliminating possible voltage-independent processes) for NaGlu predicted from consideration of the VSI revealed that the relative density of apical channels increases rapidly from ∼25% of the mature density at 12–14 days of age to ∼84% the mature density at 29–31 days of age. Because of the accelerating increase observed between these age points, we attempted to describe the age-dependent trend in whole systemCT max values (predicted from Eq.1 ) using a bounded-growth model Equation 4where CT max is the maximum chorda tympani response value predicted by the channel model equation andt is the mean age in days for each age group studied, whileb is the time in days required for the initial response magnitude at t = 0 days to increase by a factor ofe, and t h is the time at which the response magnitude has reached half the mature, unchanging, steady-state response magnitude, a. Values ofCT max obtained for NaGlu were fit with this model, and Fig. 8 A shows the data plotted with the resulting predicted line. The best fit of the model to the data was obtained when a = 1.37, b = 7.4 days, and t h = 20.6 days; the model accounts for nearly all of the variance in the data (R 2 = 0.99). The predicted value for maximum system response to NaGlu is exactly equal to theCT max value obtained from the channel model equation for 60+-day-old rats, which is consistent with the operational assumption that 60 postnatal days is sufficient time to achieve asymptotic values (t = ∞). The time to half-maximal system response, t h, corresponds closely to the time of weaning. When predicted values ofCT max derived from this model are extrapolated to zero days of age (i.e., t = 0), a value of 0.08 is obtained, which suggests that, at birth, fungiform papilla taste receptor cell apical membranes contain a relative Na+ channel density that is ∼6% that of mature rats (0.08/1.37). By ∼30 days of age, the developmental increase inCT max has slowed considerably, and thereafter gradually approaches the unchanging steady-state maximum (i.e., mature adult) value. This model is fully consistent with the hypothesis that the postnatal increase in taste system Na+ sensitivity is due to an increase in the number of functional, apical amiloride-sensitive Na+ channels. It furthermore suggests that activation of inactive channels is accelerated by the presence of some channels already activated. Not surprisingly, whenCT max data for NaCl were fit with the bounded-growth model, a similarly accurate description of the developmental trend in gustatory Na+ sensitivity was obtained.
Figure 8 A also shows the theoretical bounded growth function for the development of NaCl responseCT max. The best fit of the model to the data were obtained when a = 1.54, b= 7.7 days, and t h = 19.0 days (R 2 = 0.98). This predicted maximum system NaCl response is nearly identical to the value ofCT max for 60+-day-old rats obtained from the channel model equation. Moreover, the values of band t h are comparable to those obtained for NaGlu, and extrapolation ofCT max values to zero days of age, yields a relative channel density of ∼8% the adult density. This zero-day value is of particular interest with respect to the findings of Ye et al. (1993b), who, using the apical channel model, determined that fungiform taste receptors of adult rats raised under developmental dietary Na+ restriction express a relative density of apical Na+ channels that is th the density in the taste receptor cells of control animals reared on a Na+ replete diet. However, the effect of developmental Na+restriction appears to be unique to channel density, as no diet-related difference in apparent K m was reported by Ye et al. (1993b). Those results, in conjunction with the current findings, underscore the notion that channel density and affinity are subject to independent regulation, and, further, raise the intriguing possibility that developmental Na+restriction causes truncated development of the gustatory Na+ sensing apparatus. That is, with respect to Na+ sensing, fungiform taste receptor cells in developmentally Na+-restricted rats attain a state of maturity functionally equivalent to that of a newborn. It should be emphasized again, however, that these data relate only to the apical Na+ sensing machinery.
With respect to the entire Na+ sensing apparatus (i.e., apical and basolateral pathways), an important distinction between the bounded growth functions for NaGlu and NaCl relates to the parallel nature of CT max values across development. That is, system CT maxvalues predicted by the channel model equation and by the bounded growth model, as well as the apicalR max values predicted by the VSI function, are larger for NaCl than for NaGlu at all age points. This reliable difference in the magnitudes of NaCl and NaGlu responses was expected and corresponds to the presence of the amiloride-insensitive component of the chorda tympani response to NaCl. It is interesting to note that despite the presence of this amiloride-insensitive response component in NaCl responses from the earliest age points studied, the fundamental hypothesis that the postnatal increase in gustatory Na+ sensitivity is due to an orderly increase in the density of functional, apical (and, therefore voltage-sensitive) Na+ channels is nonetheless validated by the apical channel model. In addition to its contribution to stimulus-dependent differences in measures of maximum response magnitude, the anion-dependent properties of the paracellular shunt in the intact taste bud contribute to differences between NaCl and NaGlu with respect to other Na+ channel kinetic parameters.
In addition to parallel, age-dependent trends in chorda tympaniCT max for NaCl and NaGlu, parallel, age-related changes in the apparent affinity (K m) of Na+ for the apical Na+ channel were seen to occur (Fig.8 B). Specifically, K m for both salts decreased from relatively high values at 12–14 days of age to lower values at 19–23 days of age, and then remained stable across succeeding age points. The similar trends inK m for both salts clearly suggest that the affinity of the apical channel for Na+ ions increases developmentally and, thus, contributes to the overall increase in gustatory Na+ sensitivity that is seen during the postnatal period. We speculate that the change in channel K m is most probably due to developmental alteration in intrinsic channel properties. It is possible, however, that changes in the "affinity" of processes intermediate to Na+ binding/influx and the generation of the neural response (e.g., taste cell neurotransmitter release) are responsible for the apparent shift in channel affinity. Consistent with such a view, Hill et al. (1982) observed a developmental decrease in the latency of chemically-stimulated neural responses in single chorda tympani fibers. On the other hand,Hill and colleagues (1982) found no difference in single chorda tympani fiber response latency in rats aged 10–14 versus those aged 24–35 days of age. In the present study, the apparent shift inK m occurs between 12–14 and 19–23 days of age, long before Hill et al.'s latency shift, which occurred between 24–35 days of age and adulthood. Regardless of the source of this apparent shift, the large differences in predictedK m values observed for the two salts raise an important question: if K mdescribes the affinity of Na+ for a Na+ specific channel, why then are the predicted values of K m for the two salts so divergent? That is, why, in the presence of NaCl, is the apparent affinity of Na+ for Na+channels so much greater than when NaGlu is the stimulus?
From the present experiments, and from previous work (Stewart et al. 1996), it is clear that the lingual transepithelial potential (TEP) that attends application of NaGlu is considerably more submucosa positive compared with the potential that attends application of an equimolar concentration of NaCl. This stimulus-dependent difference in TEP results from the lower shunt permeability of the gluconate anion relative to Cl−. Given the polarized epithelial topology of the taste bud, it is predicted that the lower TEP evoked by NaCl results in a greater depolarization of the basolateral taste receptor cell membrane and in a relatively larger hyperpolarization across the apical taste receptor cell membrane compared with NaGlu (cf. Lindemann 1996; Ye et al. 1994). Importantly, this hyperpolarization at the apical membrane is "seen" by Na+ ions as an increased intracellular electronegative potential and thus a relative increase in the driving force for Na+ ions into the taste cell across the apical amiloride-sensitive conductance. In contrast, when gluconate is the stimulus anion, the more submucosa positive potential creates a relative depolarization of the apical membrane and thus a relative decrease in driving force for Na+ ions into the taste cell. In our model system, the net effect of these stimulus- and, hence, TEP-dependent differences in the driving force for apical Na+flux appears to be manifested as stimulus-dependent shifts inK m. That is, the aggregateK values predicted by our model depend on anion- and, hence, stimulus-related differences in TEP.
The idea that stimulus-dependent TEP can potently influence apparentK m values for the intact system has been demonstrated previously (Stewart et al. 1996). Such stimulus-related modulation of the channel native Michaelis constant, here called K, is the unique consequence of applying the apical channel model to the intact taste system and illustrates how the polar topology of the taste bud imposes unavoidable constraints on the function of membrane effectors, such as ion channels. Moreover stimulus-dependent modulation of an apparent intrinsic channel property such as K emphasizes the importance of studying function under conditions that maintain the essential polarity of the biological system. Given these observations and the likelihood that Na+ salts modulate their own apparent Na+ channel affinity, it is perhaps most appropriate to assign individual Na+ salt stimuli specific K values (e.g.,K NaCl andK NaGlu) when such parameters are determined in the intact system. For the present study, it is critical to bear in mind that the consistent, stimulus-dependent difference between K NaCl andK NaGlu is superimposed on a large developmental change in the Na+ channel "native" K. In addition to this clear and important influence of the stimulus anion on apparent systemK m, the stimulus anion also contributes to differences between NaCl and NaGlu with respect to other system properties.
As noted in results, age-dependent changes in the fraction of the clamp voltage, δ, that is sensed by Na+ions as they traverse the apical Na+ channels were observed. However, in contrast to parallel trends in other system parameters, age-dependent changes in δ were observed for NaCl only. Specifically, and unexpectedly, estimated δ for NaCl declined from comparable values at 12–14 and 19–23 days of age (i.e., 0.44 and 0.45, respectively) to considerably lower values at 29–30 and 60+ days of age (i.e., 0.25 and 0.22, respectively). By comparison, estimated δ values for NaGlu were largely stable throughout the time period studied. For all age groups, δ values for NaCl are smaller than are those for NaGlu. The consistent difference in δ for the two stimuli is a consequence of the higher shunt permeability of Cl− relative to that of gluconate. Moreover, the decline in δ for NaCl in the two oldest age groups implies that taste bud shunt properties change considerably as a function of postnatal age. Specifically, the decrease in δ for NaCl between 19–23 and 29–31 days of age suggests that tight junctional permeability to mobile anions, such as Cl−, actuallyincreases with advancing age. That is, taste bud tight junctions do not become tighter with advancing postnatal age, they become leakier. This decrease in tight junction resistance appears to be unique to taste buds, as measurements of relative transepithelial resistance and conductance did not vary as a function of postnatal age (data not shown). The dissociation between changing δ for NaCl and stable δ for NaGlu is not inconsistent and probably reflects the fact that even a considerable increase in the shunt permeability for smaller ions has no evident impact on permeability to large ions with low tight junctional mobility (e.g., gluconate). This remarkable developmental change in the topological properties of the taste bud has direct functional correlates in the present findings.
The age-related change in δ for NaCl may explain in large part the failure of the theoretical VSI function to fit accurately the data for NaCl responses in 29- to 31- and 60+-day-old rats. While there was excellent agreement between the observed and predicted VSIs for NaCl for 12- to 14- and 19- to 23-day-old rats, by 29–31 days of age and continuing into adulthood, the predicted VSI underestimated the observed VSI at concentrations of NaCl below 200 mM and overestimated the observed VSI at the highest NaCl concentration (500 mM) (Fig.7 A). The degradation in the predictive accuracy of the model probably derives from the loss of voltage sensitivity in chorda tympani responses to higher concentrations of NaCl in 29- to 31- and 60+-day-old rats. A striking example of this can be seen in Fig. 2. Not surprisingly, the timing of the loss of chorda tympani response voltage sensitivity at higher NaCl concentration coincides with the timing of the decrement in δ observed for NaCl.
The critical parameter in the VSI of the chorda tympani response to NaCl is δ. That is, as δ approaches unity, the observed effect of voltage perturbations on chorda tympani responses becomes greater. In contrast to the case of NaGlu, where δ is stable and relatively large, δ appears to decline as a function of increasing NaCl concentration, especially in the older age groups. It is possible that in the presence of high NaCl concentrations, the paracellular diffusion potential of the stimulus ions is increased, and, under voltage clamp conditions, this increase in paracellular electroneutral diffusion potential for Na+ and Cl−provides a "sink" for current injected to maintain command voltage. Essentially, the paracellular shunt becomes a lower resistance pathway for ion movement (i.e., current) than is the apical, amiloride-sensitive Na+ channel, and so a relatively larger fraction of the clamp voltage is dropped across the paracellular shunt as NaCl concentration increases. The consequences of this age-dependent increase in the paracellular shunt permeability (observed as a decline in δ) are two. One probable consequence, which is observed, is that dissipation of clamp voltage across the paracellular shunt diminishes the measurable voltage sensitivity of chorda tympani response to NaCl at high concentrations but only in older rats that have developed the lower paracellular shunt resistance. Another likely consequence is that as the rat matures, the relatively increased electroneutral diffusion of Na+ and the stimulus co-anion through the tight junctions increases the magnitude of the voltage-independent and amiloride-insensitive response component, particularly at high stimulus concentrations. Such a change in shunt properties could also contribute to Na+response development, although available evidence suggests that the magnitude of the amiloride-insensitive component of chorda tympani NaCl responses is stable during development in rats (Hill and Bour 1985; Sollars and Bernstein 1994). This important developmental change in taste bud topological properties, which exerts influences on several fundamental parameters that describe system function, could only be appreciated through examination of the intact, functioning taste system.
It is important to note that the apical channel model assumes that δ is a constant. However, the present data clearly indicate that the value of δ necessarily depends on the potential for paracellular electroneutral diffusion of Na+ and Cl−, which is determined jointly by NaCl concentration and the resistance of the shunt pathway (i.e., tight junctions) (Ye et al. 1993a). It should also be emphasized here that for Na+ salt stimuli that contain large, shunt-impermeant anions, such as gluconate, modulation of δ would not be observed. That is, modulation of δ is essentially anion dependent.
Potential mechanisms for developmental increases in apical Na+ channel number and affinity
Our current findings indicate that temporally distinct, age-dependent changes in the density of apical, amiloride-sensitive Na+ channels and in the apparent affinity of Na+ for those channels together provide the basis for postnatal development of mature gustatory Na+sensitivity of the rat chorda tympani nerve. Our results confirm previous hypotheses (e.g., Hill and Bour 1985;Sollars and Bernstein 1994) regarding the postnatal increase in chorda tympani responses to Na+. The cellular basis for the increase in the density of functional channels remains to be elucidated, but several mechanisms could be involved. For example, a simple increase per taste bud in the number of taste cells that possess a fixed number of apical Na+channels could contribute to an apparent increase in the number of apical amiloride-sensitive Na+ channels. There is, in fact, ample evidence that fungiform papilla taste bud size increases postnatally (Krimm and Hill 1998) and, furthermore, that this size increase may be due proliferation of taste cells as opposed to hypertrophy of individual cells (Kossel et al. 1997). On the other hand, recent developmental studies of taste cell proliferation revealed lower rates of cell addition to taste buds in rats aged zero and 29–31 days compared with those aged 60+ days (Hendricks and Hill 2000). Another obvious mechanism for an increase in the density of apical Na+ channels would be a progressive increase in the de novo synthesis and insertion into the apical membrane of functional Na+ channels. However, previous immunohistochemical results provided qualitative evidence that the fungiform papilla taste cells of 1-day-old rats contained significant amounts of apical Na+ channel-like immunoreactive material (Stewart et al. 1995). That result raises the possibility that from early in development, rat fungiform papilla taste receptor cells synthesize amiloride-sensitive Na+channels, and these extant channels gradually become functional during the postnatal period. For example, it is possible that channels are synthesized and immediately inserted into taste cell apical membranes but that they remain inactive. Subsequent, developmentally regulated activation processes (e.g., covalent modification) (cf. Garty and Palmer 1997; Shimkets et al. 1998) might then act to increase the relative density of functional channels. Alternatively, it is possible that a redistribution of Na+ channels between basolateral and apical membrane compartments occurs during the early postnatal period. This potential mechanism was favored by Kossel and colleagues (1997) and by Stewart et al. (1995), who suggested that such membrane reallocation might be promoted by maturation of, and subsequent association of apical membrane channels with, taste cell cytoskeletal elements. Interestingly, cytoskeletal elements interact with and modulate the function of apical Na+ channels (Ismailov et al. 1997; Smith and Benos 1996; Smith et al. 1991). This intriguing possibility has so far evaded elucidation in the taste system, but recent advances in the molecular biology of apical Na+ channels in other tissues should facilitate its evaluation (Benos et al. 1997;Garty and Palmer 1997).
Strides in the understanding of the apical Na+channel may also provide insights into the mechanism for the considerable increase in the affinity of Na+ for the apical channel that was observed to occur between 12–14 and 19–23 days of age. In the past several years, molecular biological techniques have allowed the cloning of distinct genes that encode the primary amino acid sequences for three homologous subunit proteins that in complementation comprise an amiloride-sensitive, Na+-selective conductance with biophysical properties similar to those of purified, native amiloride-sensitive Na+ channels characterized from several source tissues (Benos et al. 1997; Canessa et al. 1994; Garty and Palmer 1997). Specifically termed the epithelial Na+ channel, or ENaC, the heterotrimeric channel consists of alpha, beta, and gamma subunit proteins, all of which have been detected immunohistochemically in rat fungiform papilla taste cells (Kretz et al. 1999;Lin et al. 1999). When expressed alone, the alpha subunit comprises an amiloride-sensitive conductance, characterized by low maximum Na+ current and low amiloride binding affinity. However, co-expression of all three subunits results in a highly Na+-selective, amiloride-blockable conductance with large maximum Na+ current. It is notable that subunit complementation patterns can significantly influence, for example, the amiloride binding affinity and ion selectivity of the ENaC, in addition to the maximum Na+ current that can be sustained by the channel complex (Benos et al. 1997). Clearly, an age-related alteration in subunit complementation pattern could contribute to the developmental increase in gustatory Na+sensitivity. One striking, though fully speculative, possibility with respect to the current results is that the age-related change in apparent channel affinity is the result of "immature" subunit complementation patterns. However, Kossel et al. (1997)present evidence that the inhibition constant of amiloride for blockade of whole cell inward currents in neonatal taste cells is comparable to that obtained for adult taste cells. Given other results that suggest alterations in subunit complementation can strongly influence ENaC amiloride-binding affinity (Benos et al. 1997), this result tends to argue against an altered subunit complementation pattern in young rat taste receptor cells. On the other hand, it is possible that in taste receptor cells apical Na+channel affinities for Na+ ions and for amiloride are subject to different and distinct regulatory effectors (cf.Garty and Palmer 1997). Clearly, an important area for future investigation includes resolving, in the intact, developing taste system, whether the interaction of amiloride with the apical Na+ channel changes significantly as a function of postnatal age. Results from such experiments, in combination with more molecular studies that directly assess taste receptor cell ENaC subunit expression and complementation patterns during postnatal development, should provide important evidence regarding the cellular mechanisms that underlie the developmental increase in apical Na+ channel affinity.
This work was supported by National Institute on Deafness and Other Communication Disorders Grants DC-00122 and DC-02422 (to J. A. DeSimone), DC-00407 and DC-02406 (to D. L. Hill), and DC-03499 (to R. E. Stewart).
Address for reprint requests: R. E. Stewart, Dept. of Psychology and Neuroscience Program, Washington and Lee University, Lexington, VA 24450 (E-mail: rstewart{at}wlu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2000 The American Physiological Society
As shown in the results, the chorda tympani response to Na+ is a saturating function of the Na+ electrochemical concentration (cf. Eq.1 ), i.e., the driving force for the flux of Na+ through a conductive pathway. This suggests that the form of Eq. 1 is the result of a kinetic process involving the influx into taste receptor cells of Na+ ions across apical membrane ion channels. This can be demonstrated using the Na+ channel model proposed by Mintz et al. (1986) in their computational simulation of sodium transport across tight epithelia. It is assumed that the apical channels have pores connecting the mucosal solution phase with the cell interior and that each pore contains a single Na+ ion binding site. In addition, each Na+ ion entering the channel from either side must overcome a potential energy barrier to reach the binding site. While the binding site may be located at any point within the pore, we assume here that it is located at the pore exit into the cell interior. In this way, a Na+ ion entering the channel from the stimulating solution will encounter the entire electrical potential gradient across the apical membrane. The binding association reaction between a Na+ ion originating from the mucosal side (m) and the pore site has rate constantf m; the similar association reaction involving a Na+ ion originating from the cellular side (c) of the channel has rate constantf c. The binding dissociation reaction releasing Na+ into the mucosal solution has rate constant k m and that releasing Na+ into the cellular medium has rate constant k c. Because Na+ ions entering channels from the mucosal side encounter the additional potential energy of the apical membrane potential, f m andk m can be written respectively as:f mo exp[−θ/2] andk mo exp[θ/2], where θ is the dimensionless apical membrane potential relative to the mucosal solution, and f mo andk mo are rate constants exclusive of electrical potential effects. The values of the pairsf mo andk mo andf c andk c may differ in magnitude depending on the size of the energy barriers on each side of the pore region, but the ratios,k mo/f moandk c/f care equal, defining the thermodynamic dissociation constant,K e, between Na+ions and their channel binding sites.
Let N be the total number of channels per unit area of apical membrane, p o, the fraction of pore binding sites that are unbound, andp 1, the fraction of sites with a bound Na+ ion. Conservation of total binding sites requires: p o + p 1 = 1. In the steady state, the net Na+ flux, J Na is Equation A1 Equation EA1 in conjunction with the total binding site conservation relation noted above permits determination ofp o andp 1 as functions of the rate constants, the Na+ concentration in the mucosal solution,c m, the Na+concentration in the cell, c c, and the apical membrane potential. Accordingly we obtain the following equation for J Na Equation A2where k = k c/k mo. Because the cell electrical potential is electronegative with respect to the mucosal solution and the cell Na+ concentration is kept at a low value by the Na-K pump,c c exp[θ/2] can be neglected compared with c m exp[−θ/2]. We assume that the rate constant for dissociation of Na+ into the cell is ≥10 times greater than the rate constant for dissociation into the mucosal solution, i.e.,k ≥10. K e can be expected to be of the same order as c c (i.e., from 10 to 30 mM), so K e exp[θ/2] can also be expected to be small compared withk(c c + K e).The sodium flux can be, therefore approximated without loss of accuracy by Equation A3Under voltage-clamp conditions, equation A3 becomes Equation A4If the CT response is assumed to be proportional toJ Na, then comparing Eq. EA4 with Eq. 1 in results, we can conclude thatCT max is proportional to the density of channels, N, and the rate constant for dissociation of Na+ from the channel to the cell interior,k c. In terms of this model, we also note that K m in Eq. 1 is Equation A5This suggests the likely source of the anion dependence that we observed in K m , i.e.,the reason why K NaCl is much smaller than K NaGlu. We note that k, c c, and K eshould not change between NaCl and NaGlu. However, the potential across the apical membrane will be affected by the anion present because gluconate has a much lower paracellular shunt permeability than chloride. NaGlu, with its greater transepithelial potential relative to NaCl, should therefore also have a significantly more positive apical membrane potential because changes in the transepithelial potential will produce changes in potential across both the apical and basolateral taste receptor cell membranes. Substituting NaGlu for NaCl therefore effectively reduces the Na+ influx accounting for the poorer CT stimulation at a given Na+ concentration and, likewise, the apparently higher K m. If we take theK m values for NaGlu and NaCl at 60+ days (373.6 and 180.1 mM, respectively), Eq. EA5 suggests that the apical membrane potential for NaGlu is ∼38 mV more positive than for NaCl (2(26) ln [373.6/180.1]). Reasonable values of parameters that would approximately account for theseK m values are: k = 25,K e = 10 mM, andc c = 10 mM for both salts, and θ ≈ −2, (−52 mV) for NaCl, and −0.6 (−15.6 mV) for NaGlu. These give 183.9 mM for NaCl and 370.4 mM for NaGlu and an apical potential difference between the salts of 36.4 mV. Thus although the Na taste transducer is a Na+ channel, the anion present in the Na stimulus strongly modulates the density of depolarizing Na+ current into the receptor cells.


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Claire Martin & Richard Rodney Bennett: When Lights Are Low
Although Martin usually performs with a full group (including a rhythm section), this album is prepared for a cabaret-type performance, which indeed occurred in September, 2005 in Greenwich Village. In a rare New York appearance, the singer was accompanied by Bennett, who also provided all of the arrangements.
Claire Martin is clearly a musical offspring of the 1950s cool vocal style of jazz. Her recordings indicate less of an aggressive style and more the influence of my own two personal benchmarks of the era, Chris Connor and June Christy. The pieces selected for this album are indeed cabaret-oriented and consist of tunes that are not readily identifiable from previous recordings. Listeners who've got to know what they're listening to may be disappointed, but not with the delivery and execution. Those folks would be best off with vocal duet numbers (eg. "When Lights Are Low," "The Very Thought Of You," "We'll Be Together Again") for more familiarity.
On his vocals, Richard Rodney Bennett visits the Nat King Cole Trio songbook ("I Was A Little Lonely," I Keep Going Back to Joe's") and others, while Martin is content to dazzle with songs like "My Mood is You," "What I Was Warned About," "World Weary" and a super version of "Any Place I Hang My Hat(s) Is Home."
Welcome back!
Track Listing: My One And Only; I Was A Little Too Lonely; My Mood Is You; World Weary; When Lights Are Low; Fools Fall In Love; I Got A Right to Sing The Blues; Baby Plays Around; The Very Thought of You; What I Was Warned About; Baby;Don't You Quit Me Now; No Love No Nothing; Not Exactly Paris; Any Place I Hang My Hat Is Home; I Keep Going Back to Joe's; We'll Be Together Again.
Personnel: Claire Martin: vocals; Richard Rodney Bennett: piano, vocals.
About Claire Martin
CD/LP/Track Review
- Time And Place by Dan Bilawsky
- A Modern Art by Bruce Lindsay
CD/LP/Track Review
- When Lights Are Low by Michael P. Gladstone
- Every Now and Then: The Very Best of Claire Martin by Dave Nathan
- Perfect Alibi by Craig Jolley
- Take My Heart by Ian Nicolson
Claire Martin Events
The 606 Club's 40th Anniversary: Claire Martin Feat. Gareth Williams (8:00pm) :: Hamish Stuart :: Samara Feat. Imaani
606 Club
London, UK
Ian And Claire's Christmas Cracker
Turner Sims
Southampton, UK

subset: commoncrawl


text:
Well I tried to install my new LI last night but I couldnt get it installed in the location I wanted (and Cliff said would be the best) without it looking completely stupid. It got really late too so I just figured Id try it another day. I have the CPU and Escort speaker installed and ready to go.
Here is a pic that I sent to Cliff and then he sent back to me. He said the red marks would be the first choice then the green.
Problem is for one the grills outer "ring" is about two inches wide (its an E&G grill). So I have to cut hole in it to slide the brackets in. then theres all kinds of other crap behind it like rings that the grill clips go into, a bar with brackets and bolts that hold the grill flush.
I cut the holes for the first bracket, and got the head in. But when I put the bumper cover back on to make sure I could align and level it, I realized that the front of my car comes to a point at the front which means that one end of the sensor has to stick out WAY too much, almost four inches.
I will have someone cut holes in my bumper cover before I have some bracket (which I would have to make one) sticking out 4 inches.
So I was thinking that maybe I could get away with mounting them in the green location. What do you guys think? I could even mount them with the diodes facing inwards right below the headlight in the top of the turn signal hole. It would leave a bigger spot in the middle but does that matter as much since my car is black?
I am also looking for a LIDAR gun. Are LTI marksman or Laser Atlanta guns easy or hard to jam? I was thinking that If I did go with the green locations I could test them with one of those guns.
Thanks guys


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Mother sues Lauderhill over son's fatal shooting by policeSun Sentinel
Mar. 24, 2013
FACT CHECK: Hillary Said 90% of Clinton Foundation Donations go to Charity. Actual Number? 5.7%
Jorge Ramos: "The Future of This Country Will be Composed Solely by Minorities," It's "Beautiful"
John Podesta Wasn't 'Hacked,' He Fell For An Email Phishing Scam
Illegal Immigrant Child Molester Flees Guatemala for U.S., Goes on Molestation Spree
'The System IS Rigged!' Pat Buchanan Goes Off On CNN Propagandist!
Nadege Lanoue still does not know who shot and killed her 21-year-old son two years ago.
Lauderhill police do, but they're not saying, because it was one or more of the department's own officers.
Read More


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Seagate NAS HDD (ST4000VN000) 4000GB HDD Review
PCMark 8 - Hard Disk Tests
Version and / or Patch Used: 1.0.0
Developer Homepage: http://www.futuremark.com
Product Homepage: http://www.futuremark.com/benchmarks/pcmark8
PCMark 8 is the latest version in our popular series of PC benchmarking tools. Improving on previous releases, PCMark 8 includes battery life measurement tools and new tests using popular applications from Adobe and Microsoft. Whether you are looking for long battery life, or maximum power, PCMark 8 helps you find the devices that offer the perfect combination of efficiency and performance for your needs.
Launching our PCMark 8 testing we have the NAS HDD coming in with a total score of 2486, just behind the Desktop counterpart. Even with the single platter design of our WD Red 1TB, it just doesn't compete with the NAS HDD here.
In WoW the NAS HDD was capable of 114.1 seconds, very close to the Desktop HDD and a full 57 seconds quicker than the WD Red 1TB.
Battlefield 3 has the NAS HDD pulling ahead of the Desktop HDD .15, coming in at 305 seconds.
Utilizing Photoshop under light use, the NAS HDD comes in at 195.7 seconds.
A heavy use scenario brings all our drives closer together. In this test we found the NAS HDD slightly quicker than the Desktop HDD, with the Barracuda with its two platter design and 7200RPM spindle speed way out in front.
The Excel application trace puts the NAS HDD in front of the Desktop HDD .15 by a very thin margin.
The PowerPoint trace put the NAS HDD at 22.3 seconds.
The Word trace puts the Barracuda .14 at the top of the charts, followed very closely by the NAS HDD at 43.7 seconds.
PRICING: You can find products similar to this one for sale below.
United States: Find other tech and computer products like this over at Amazon's website.
United Kingdom: Find other tech and computer products like this over at Amazon UK's website.
Canada: Find other tech and computer products like this over at Amazon Canada's website.
- Page 1 [Introduction and Specifications, Pricing and Availability]
- Page 2 [Seagate NAS HDD (ST4000VN000) 4000GB HDD]
- Page 3 [Benchmarks - Test System Setup and ATTO Baseline Performance]
- Page 4 [Benchmarks - Sequential Performance]
- Page 5 [Benchmarks - AIDA64 Random Access Time]
- Page 6 [Benchmarks - Anvil Storage Utilities]
- Page 7 [Benchmarks - PCMark 8 Hard Disk Tests]
- Page 8 [Benchmarks - BootRacer and Resume from S4 Hibernation]
- Page 9 [Final Thoughts]
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Home Capital Group Inc (TSE:HCG) – Research analysts at Cormark dropped their Q3 2016 earnings estimates for shares of Home Capital Group in a research report issued on Thursday. Cormark analyst J. Fenwick now anticipates that the firm will earn $1.02 per share for the quarter, down from their previous estimate of $1.04. Cormark has a "Buy" rating on the stock. Cormark also issued estimates for Home Capital Group's Q4 2016 earnings at $1.04 EPS, Q1 2017 earnings at $1.01 EPS, Q2 2017 earnings at $1.04 EPS, Q3 2017 earnings at $1.05 EPS, Q4 2017 earnings at $1.06 EPS and FY2017 earnings at $4.16 EPS.
Other research analysts also recently issued reports about the company. Royal Bank Of Canada reduced their target price on Home Capital Group from C$35.00 to C$33.00 in a research note on Thursday, July 28th. National Bank Financial dropped their price target on Home Capital Group from C$35.00 to C$31.00 in a research report on Sunday, July 31st. Scotiabank reiterated a "sector perform" rating and set a C$40.00 price target on shares of Home Capital Group in a research report on Thursday, July 28th. TD Securities dropped their price target on Home Capital Group from C$45.00 to C$40.00 and set a "buy" rating for the company in a research report on Thursday, July 28th. Finally, RBC Capital Markets dropped their price target on Home Capital Group from C$35.00 to C$33.00 in a research report on Thursday, July 28th. Four investment analysts have rated the stock with a hold rating and two have assigned a buy rating to the company's stock. The stock presently has a consensus rating of "Hold" and an average price target of C$34.14.
Shares of Home Capital Group (TSE:HCG) traded down 0.77% during mid-day trading on Friday, reaching $24.55. The company had a trading volume of 157,523 shares. The company has a market capitalization of $1.61 billion and a PE ratio of 6.22. The stock's 50-day moving average price is $27.68 and its 200 day moving average price is $31.53. Home Capital Group has a 1-year low of $22.87 and a 1-year high of $39.84.
In other Home Capital Group news, Director John M. Marsh sold 36,000 shares of the company's stock in a transaction on Thursday, September 8th. The stock was sold at an average price of C$29.50, for a total value of C$1,062,000.00.
Home Capital Group Company Profile
Home Capital Group Inc is a Canada-based holding company, which operates primarily through its subsidiary, Home Trust Company (Home Trust). The Company, through its subsidiary, offers residential and non-residential mortgage lending, securitization of insured residential first mortgage products, consumer lending and credit card products.
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Around February-March in two years' time, New Zealand will host the 2015 ICC Cricket World Cup.
Nelson hopes to have a slice of the action, with its splendid oval at Saxton Field meeting many of the criteria.
Three months later, another international sporting event, the Fifa under-20 Football World Cup, will kick off - provisionally on June 19. There is also a prospect of Nelson being among a handful of host cities.
The million-dollar questions include, what is in it for this region, what will it cost, and will taxpayers wear another significant spendup of this nature?
The Nelson City Council has already committed to spending up to $500,000 in making a bid for a share of the Fifa tournament. This includes staff time.
However, councillors were told this week that this falls well short of New Zealand Football expectations of what it will take to get the nod as a host city. A winning bid is more likely to require a commitment of up to twice that amount.
Even then, NZ Football believes too much of the $500,000 set aside is soaked up in staff time. It would rather see the money allocated to actual tournament costs, such as temporary seating. That is an area in which the Tasman District Council contributed last year for the rugby cup, and surely it would play its part again in the event of a successful bid.
Councillors discussed pulling the plug now and finding some other use for what's left of the $500,000 or keeping the option alive in the meantime, even if the chances of a successful bid look slim.
They voted eight to three to keep their bid on the table. Though this decision will not thrill those in the no-frills, essential-spending-only camp, it was the right one for now, until more information is available.
Presumably, all that will be lost between now and a firm decision either way is a little more on staff time as they continue to seek more detailed information from NZ Football. Arguably, they could spend that time doing something more productive, but their wages will be being paid regardless.
To pull out now would represent a total loss of whatever has been spent to date. More information is needed before the council makes a final decision: Whether to go for broke and seek more funding via the annual plan process, or kick the idea into touch and cut their losses.
It does no harm to keep the ball on the field of play until February, when tournament organising officials are scheduled to visit the city. That is the time to seek specific advice on what is needed, draw up detailed costings and make a decision.
Yes, last year's Rugby World Cup participation gave the city a welcome boost during a sustained flat patch, even if visitor spending might have been patchier than some businesses hoped for and the full expected benefits of any international exposure have yet to be realised.
However, an under-20 football tournament is in a different league, and we need to know more about costs and benefits. Councillors have kept the hosting idea as a goal in the meantime - but they must be ready to withdraw quickly if it looks like turning into an own-goal.
- The Nelson Mail
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Makos team selected for NPC final
Tasman Makos fans show their support ahead of Mitre 10 Cup final


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Royal Mirage Fes Hotel enjoys a prime location within the heart of Fès with easy access to the city's business districts and many of the city's museum and cultural attractions.
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277 reviews
Palais Shéhérazade is located in the heart of Fez's Medina. The property is a XIXth century palace and a former Vizir residence. It is accessible by car.
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In the heart of Fes medina (historic quarter), this Riad is a a 25 minute drive from Fes International Airport.
"Location of the hotel is right in the old medina."
868 reviews
In the heart of Fes, 5 minutes from the train station and 25 minutes from the airport, the 5-star hotel Ramada Fes is in a luxurious and elegant setting.
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133 reviews
Perched on the hills of Fez, Les Mérinides hotel invites you to explore the historic wonders of a city full of heritage. It features a terrace with swimming pool and rooms with free Wi-Fi.
"A luxury hotel with great views over Fes. Restaur…ing. All staff very efficient, but also friendly"
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186 reviews
Situated in the heart of the mysterious and rich city of Fes, the Sofia Hotel welcomes you in the bigger medina of the world near the international airport of Fes-Saïss.
"good service, location, breakfast and close to the gare of fes!"
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167 reviews
The ten-floor Hotel Wassim is located in the heart of the new town of Fez and is close to Fez Saiss Airport and the Royal Golf. The 104 rooms are comfortable and well equipped.
"Spacious room, comfortable bed, clean, good location and nice stuff."
10 reviews
Located near the Batha Museum and Bab Boujloud, this Riad Hotel features a rooftop terrace with a view of the city of Fez.
47 reviews
The Riad Salam Fes has spacious rooms with air-conditioning, satellite TV and free Wi-Fi. Each room has a balcony and guests can enjoy the hammam and relaxation area.
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57 reviews
Palais Amani is located within the walls of Fez's ancient medina (historic quarter), just a few minutes' walk from the Golden Triangle. It features free Wi-Fi internet access and has a hammam.
"It is wonderful and amazing, I have garden and the…staff are so friendly. The breakfast was amazing."
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Silver/Deming Drill, 1 5/16, HSS, 118 Deg
CHICAGO-LATROBE |Zoro #: G1856136|Mfr #: 52484
Zoro #: G1856136Mfr. Part #: 52484
Zoro Customers Also Bought
Technical Specifications
|Zoro #: G1856136 Mfr #: 52484
|Drill Bit Material: High Speed Steel
|Drill Bit Dimension Type: Fractional Inch
|Overall Length: 6"
|Point Type: Split Point
|Shank Type: Round with Flats
|Item: Reduced Shank Drill Bit
|Drill Bit Finish: Black Oxide
|Application: For Alloy And Carbon Steel, Cast Iron And Wood
|List Number: 190F
|Grade: General Purpose
|Flute Length: 3-1/8"
|Shank Dia.: 1/2"
|Drill Bit Size: 1-5/16"
|Country of Origin (subject to change): China
Product Description
Reduced Shank Drill Bit, Fractional Inch, Drill Bit Size 1-5/16 In., Decimal Equivalent 1.3125, Drill Bit Material High Speed Steel, Drill Bit Finish Black Oxide, Shank Type Round with Flats, Grade General Purpose, Overall Length 6 In., Shank Dia. 1/2 In., Flute Length 3-1/8 In., Point Type Split Point, List Number 190F, For Alloy And Carbon Steel, Cast Iron And Wood


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All hotels in Omaha, Nebraska, United States of America
1 room, 2 adults
10308 Sapp Brothers Dr, Omaha, NE, 68138, United States of America, 800-491-6126
Home-Towne Lodge Omaha, Omaha
Lowest price$57
available on 11/01/16
Price Guarantee
No-frills motel in Chalco
- Free WiFi and free parking
Main amenities
- 96 guestrooms
- Daily housekeeping
- Self-serve laundry
For families
- Microwave
- Refrigerator
- Daily housekeeping
- Laundry facilities
- DVD player
- Cable TV service
What's around
- In Chalco
- Chalco Hills Recreation Area (2.6 mi / 4.1 km)
- Tiburon Golf Course (3 mi / 4.8 km)
- Werner Park (3.8 mi / 6.1 km)
- Defy Gravity Indoor Trampoline Park (4.9 mi / 7.9 km)
- SkateDaze (5.1 mi / 8.2 km)
See all 19 Hotels.com reviews
Value for money
Best value for money
Quite clean
Room needs to be redecorated
More comfort would be nice
TripAdvisor Traveller Rating: 3.5
From 32 reviews
More great choices
Home-Towne Lodge Omaha, Omaha
Hotel Info: 866-539-8117from $57
- Standard Room, 2 Queen Beds, Non Smoking
- Standard Room, 2 Queen Beds, Non Smoking
- Standard Room, 1 King Bed
- Double Room, 2 Full Beds, Non Smoking
- Double Room, 2 Double Beds, Non Smoking
- Standard Room, 1 King Bed
- Standard Room, 1 King Bed, Non Smoking
- Standard Room, 2 Queen Beds
- Double Room, 2 Double Beds
- Double Room, 2 Full Beds
- Standard Room, 2 Queen Beds
- Standard Room, 1 King Bed, Non Smoking
At a glance
Key facts
Hotel size
- This hotel has 96 rooms
- Check-in time starts at 2 PM
- Check-out time is noon
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- Pets allowed
- Free WiFi in public areas
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- Free self parking
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Nearby Two Star Omaha hotels
In the hotel
- Laundry facilities
In the room
Be entertained
- Pay movies
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I needed this mug to arrive before the 2 July for a birthday gift so contacted the seller. They were most helpful and arranged for this to happen and the package arrived on the 2 July, in plenty of time. Brilliant!!!
reviewed by Mrs H
10th July 2016
Thank you to the seller it's quality
reviewed by Ruzbi Nayel
7th June 2016
Swift response and quick delivery:another satisfied customer.
reviewed by RCV 75f MCMURRAY
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Service was so efficient I shall not hesitate to use them again.
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Very well packed. Arrived earlier than quoted. Excellent service" Lin M, 26 July 201
reviewed by Lin M
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Technical Deep Dive: SAP HANA Cloud Platform for SAP Cloud for Customer
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With the help of our special guest host Danny Trejo, Matt and Kerry recreat...
Any evil dead fans out there? This week Odin shows you how to make the famo...
The new season of The Walking Dead is just around the corner, and after tha...
Coming Out
Coming out of your paycheck!
Are You Coming
Woman stepping out of the closet
Here are friends that help this guy come out the closet. He's too buys play...
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These two never saw it coming.
Get out of the way dolphin! America coming through!
Might want to set up your watching spot a little further away next time.
Winter clothes are packed away and now the ladies are out to play.
And the last thing they heard was the click click of of chomping teeth.
You should see what the anchor is coming out of.
He Had It Coming
The weird thing is they were coming out.
The Harry and Malfo connection
Edited wikipedia information


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Ms Thomson, Lanarkshire
I could not have asked for a better marketing approach. I put my confidence in you and you delivered.
It is important to note that this mortgage calculator is for guidance purposes only. Mortgage lenders have their own affordability criteria therefore it is important you speak to one of our Mortgage Advisers for further information and a more accurate indication of what products may be available to you and what your monthly mortgage payments are likely to be.
This is a lovely flat. Set in a quiet corner with a private front garden yet close to amenities on Queen Margaret Drive including Tesco and an appealing parade of local shops. The flat itself has been well maintained and is presented in good order with an atmosphere which will appeal to many.
The accommodation comprises security entry to common close and access to the flat. There is a welcoming hallway, spacious lounge with bay window with well fitted kitchen within the double recess. There is a double bedroom to the front and a second bedroom to the rear. There is also a well fitted bathroom with white suite. The flat also has double glazing and gas central heating.
The property enjoys a convenient location with a host of Westend amenities available on Queen Margaret Drive, Great Western Road and Byres Road including retail shopping, bars, bistros and restaurants. There is excellent underground rail, bus services and road access to the nearby networks. EER D
Contact branch for relevant Energy Performance Certificate


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Home » Fiction & Literature » Aubrey De Vere » Legends of the Saxon Saints
Legends of the Saxon Saints
Many years ago a friend remarked to me on the strangeness of the circumstance that the greatest event in the history of a nation, its conversion to Christianity, largely as it is often recorded in national legends, has never been selected as a theme for poetry. That event may indeed not supply the materials necessary for an Epic or a Drama, yet it can hardly fail to abound in details significant and pathetic, which especially invite poetic illustration. With the primary interest of that great crisis, many others, philosophical, social, and political, generally connect themselves. Antecedent to a nation's conversion, the events of centuries have commonly either conduced to it, or thrown obstacles in its way; while the history as well as the character of that nation in the subsequent ages is certain to have been in a principal measure modified by that event. Looking back consequently on that period in which the moral influences of ages, early and late, are imaged, a people recognises its own features as in a mirror, but sees them such as they were when their expression was still undetermined; and it may well be struck by the resemblance at once to what now exists, and also by the dissimilitude. Many countries have unhappily lost almost all authentic records connected with their conversion. Such would have been the fate of England also, had it not been for a single book, 'Bede's Ecclesiastical History.' In the following poems I have endeavoured to walk in the footsteps of that great master. Their scope will best be indicated by some remarks upon the character of that wonderful age which he records.


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Sporting Clube humble Churchill BrothersTeam Herald email@example.com DULER: Sporting Clube de Goa demolished Churchill Bros SC 4 -0 in their first match of the Goa Professional league, at Duler Stadium, Mapusa, on Tuesday. When the two teams met a week ago in the Police Cup, ... Goa Herald, 2 months ago
Sporting begin with big win over Churchill - Navhind Times, 2 months ago
Buzz in the air on eve of kickoff
Panaji: There is growing uncertainty over Goa's participation in the I-League but before there is more clarity on India's premier football competition, there is a sudden buzz in Goan football ahead of the Goa Pro League. Much of that excitement ...Times of India, 2 months ago
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