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Mice with smooth muscle (SM)-specific knockout of Na+/Ca2+ exchanger type-1 (NCX1SM−/−) and the NCX inhibitor, SEA0400, were used to study the physiological role of NCX1 in mouse mesenteric arteries. NCX1 protein expression was greatly reduced in arteries from NCX1SM−/− mice generated with Cre recombinase. Mean blood pressure (BP) was 6–10 mmHg lower in NCX1SM−/− mice than in wild-type (WT) controls. Vasoconstriction was studied in isolated, pressurized mesenteric small arteries from WT and NCX1SM−/− mice and in heterozygotes with a global null mutation (NCX1Fx/−). Reduced NCX1 activity was manifested by a marked attenuation of responses to low extracellular Na+ concentration, nanomolar ouabain, and SEA0400. Myogenic tone (MT, 70 mmHg) was reduced by ∼15% in NCX1SM−/− arteries and, to a similar extent, by SEA0400 in WT arteries. MT was normal in arteries from NCX1Fx/− mice, which had normal BP. Vasoconstrictions to phenylephrine and elevated extracellular K+ concentration were significantly reduced in NCX1SM−/− arteries. Because a high extracellular K+ concentration-induced vasoconstriction involves the activation of L-type voltage-gated Ca2+ channels (LVGCs), we measured LVGC-mediated currents and Ca2+ sparklets in isolated mesenteric artery myocytes. Both the currents and the sparklets were significantly reduced in NCX1SM−/− (vs. WT or NCX1Fx/−) myocytes, but the voltage-dependent inactivation of LVGCs was not augmented. An acute application of SEA0400 in WT myocytes had no effect on LVGC current. The LVGC agonist, Bay K 8644, eliminated the differences in LVGC currents and Ca2+ sparklets between NCX1SM−/− and control myocytes, suggesting that LVGC expression was normal in NCX1SM−/− myocytes. Bay K 8644 did not, however, eliminate the difference in myogenic constriction between WT and NCX1SM−/− arteries. We conclude that, under physiological conditions, NCX1-mediated Ca2+ entry contributes significantly to the maintenance of MT. In NCX1SM−/− mouse artery myocytes, the reduced Ca2+ entry via NCX1 may lower cytosolic Ca2+ concentration and thereby reduce MT and BP. The reduced LVGC activity may be the consequence of a low cytosolic Ca2+ concentration.
- mesenteric arteries
- myogenic tone
- patch clamp
- calcium sparklets
- Bay K 8644
the sodium/calcium exchanger (NCX), which can mediate both Ca2+ exit and Ca2+ entry in cells (5), has been postulated to play a central role in arterial smooth muscle (SM) cell (ASMC) Ca2+ handling and in the maintenance of arterial tone and blood pressure (BP) (4). The block of NCX by the specific inhibitor, SEA0400 (25), in small arteries preconstricted with a low-dose ouabain lowers the cytosolic Ca2+ concentration ([Ca2+]Cyt) and induces vasodilation (21, 48). Moreover, the SM-specific overexpression of NCX elevates BP and induces salt-dependent hypertension, and SEA0400 lowers BP in several salt-dependent animal models (21). Indeed, NCX type-1 (NCX1) is upregulated in mesenteric arteries from rats with ouabain-induced hypertension (33) and pulmonary arteries from humans with primary pulmonary hypertension (49). Whether NCX is involved in maintaining normal BP, however, has not been resolved. Here we employ a SM-specific knockout approach to examine the role of NCX in arterial contractility and the maintenance of vascular tone and BP.
Three isoforms of NCX have been identified: NCX1 is abundant in the heart and is ubiquitously expressed, whereas NCX2 and NCX3 are both restricted to brain and skeletal muscle (34). NCX1 is the only isoform present in ASMCs, where two splice variants, NCX1.3 and NCX1.7, are expressed (27, 35). In cardiac myocytes, NCX1 makes a major contribution to Ca2+ extrusion during diastole (3, 11). Nevertheless, cardiac-specific NCX1 knockout mice thrive: the loss of NCX1 is compensated by a reduced L-type voltage-gated Ca2+ channel (LVGC) current and a shortened action potential because of accelerated Ca2+-dependent LVGC inactivation and augmented transient outward K+ current (18, 31, 32). Importantly, cardiac NCX1-mediated Ca2+ flux must be inward during depolarization and systole (5). The situation is complicated, however, because exchanger-mediated fluxes depend on the relative activation of the exchanger, governed by cytosolic Na+ and Ca2+ (26), as well as on the net driving force, governed by the membrane potential (Vm) and the Na+ and Ca2+ electrochemical gradients (5).
ASMC NCX1, too, can play an important role in Ca2+ extrusion when [Ca2+]Cyt is suddenly elevated (23, 40, 41). Nevertheless, under near steady-state conditions, when ASMC activation and the Vm are both relatively constant, NCX1 should tend to operate close to its reversal potential (ENa/Ca). This is determined by the Na+ and Ca2+ electrochemical potentials, ENa and ECa, respectively, and the 3 Na+:1 Ca2+-coupling ratio: i.e., ENa/Ca = 3ENa − 2ECa (5). The driving force on Ca2+ (Vm − ENa/Ca) is governed by changes in Vm and cytosolic Na+ and Ca2+. The exchanger can move (net) Ca+ either outward when (Vm − ENa/Ca) is negative or inward when (Vm − ENa/Ca) is positive.
The inhibitory effects of SEA0400 on [Ca2+]Cyt and myogenic tone (MT) suggested that NCX1 mediates (net) Ca2+ entry in ASMCs within pressurized, tonically constricted small arteries (21, 36, 48). The present report shows that MT is also reduced, as is BP, in SM-specific knockout of NCX1 (NCX1SM−/−) mice. This is consistent with the view (21) that NCX1 normally mediates Ca2+ entry in tonically constricted small arteries. Reduced ASMC NCX1 expression is, however, as in heart (32), accompanied by a functional reduction in Ca2+ entry via LVGCs. The latter is normally a major contributor to MT (8, 20, 47). But, in contrast to cardiac muscle where elevated [Ca2+]Cyt in cardiac muscle-specific NCX1 knockout mice accelerates LVGC inactivation, the reduced MT and low BP suggest that [Ca2+]Cyt may be low in NCX1SM−/− mice. This could explain the reduced protein kinase C-dependent phosphorylation of LVGCs and, thus, reduced LVGC activation in ASMCs (37).
Experimental Animals
Ethical approval.
All mouse protocols were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.
Generation of NCXSM−/− mice.
NCXSM−/− mice were generated by crossing homozygous NCX1 LoxP [floxed (Fx)] mice (NCX1Fx/Fx) (18) with hemizygous SM-specific Cre recombinase mice (CreSM+) (46). These genetically altered mice are on a C57BL/6J background [denoted as wild-type (WT)] and are congenic with their littermates. The NCX1 exon 11 in NCX1Fx/Fx mice is flanked by two LoxP sites (Fig. 1A) (18). CreSM+ mice were generated by inserting the Cre gene, under the control of the SM myosin heavy chain (MHC) promoter, randomly into the mouse genome. This construct also contained an internal ribosomal entry site and enhanced green fluorescent protein (eGFP) downstream from the Cre recombinase (Fig. 1A) to enable simultaneous transcription, but independent translation, of these genes. When the SM-MHC promoter is activated in SM cells, both Cre and eGFP are expressed; the GFP can be visualized by its fluorescence. In these myocytes, Cre excises the genomic exon 11 DNA between the LoxP sites, leaving one intact LoxP site and the gene for a truncated, nonfunctional NCX1.
When Cre is expressed in a tissue-specific manner in mice that are homozygous for a floxed gene, both copies of the gene should, theoretically, be excised from the genome only in the target cells to generate a tissue-specific knockout mouse. Although the SM-MHC promoter restricts the adult expression to SM (46), it is also active transiently during male gametogenesis (9, 10, 16), resulting in the recombination of NCX1 exon 11 in meiotic cells. Thus crosses of female NCX1Fx/Fx mice with male NCX1Fx/+-CreSM+ mice (i.e., floxed heterozygotes with a SM-specific Cre gene) produced either NCX1Fx/− (-Cre−) or NCX1Fx/−-CreSM+ (=NCX1SM−/−) mice. The latter contain one globally deleted allele and one SM-specific-deleted allele; i.e., they were SM-specific NCX1 knockout mice. Controls for NCX1SM−/− mice were NCX1Fx/− littermates as well as C57BL/6J WT mice. Genomic DNA, obtained from tail biopsies, was used for genotyping by PCR. Primers for the floxed exon 11 were designed to distinguish the WT, floxed, and deleted alleles (Fig. 1B).
BP Measurements
Telemetric BP measurement.
WT, NCX1Fx/−, and NCX1SM−/− mice (∼16 wk old) were anesthetized with isoflurane supplemented with 100% O2. The right common carotid artery was exposed and ligated via an anterior neck midline incision. Telemetric BP sensors (DSI TA11PA-C10, Data Science International, Minneapolis, MN) were used. The catheter of the BP sensor was inserted into a small hole proximal to the ligature, the tip was passed to the origin of the carotid at the aortic arch, and the catheter was fixed in place with a suture and the hole sealed with adhesive (Vetbond, 3M, St. Paul, MN). The body of the sensor was passed through a subcutaneous tunnel to a subcutaneous pocket in the abdominal wall. Following 7–10 days of recovery from surgery, 24-h BPs were recorded with DSI receivers and software.
Intrafemoral BP measurement.
WT, NCX1Fx/−, and NCX1SM−/− mice (∼18 wk old) were anesthetized with isoflurane supplemented with 100% O2; the core temperature was maintained at 37.5–38°C. The right femoral artery was surgically isolated and cannulated with a 1.4-Fr Mikro-tip pressure catheter (Millar Instruments, Houston, TX). BP was measured under 1.5% isoflurane anesthesia (22); data were calculated off-line (BioPac System, Santa Barbara, CA). After the experiment, the animal was deeply anesthetized with isoflurane and euthanized by cervical dislocation.
The data collection for both BP methods was performed double blinded: the individual measuring the BP did not know the genotype. Animal code numbers were matched with genotype and BP after the data on all the mice had been collected.
Arterial Diameter and [Ca2+]Cyt and GFP Measurements
Arterial diameter, myogenic reactivity and tone, and evoked vasoconstriction.
WT, NCX1Fx/−, and NCX1SM−/− mice (12–16 wk of age) were euthanized with a CO2 overdose followed by cervical dislocation. Small arteries from the superior mesenteric artery arcade were isolated and cannulated at both ends. Arterial diameter, myogenic reactivity, MT, and evoked vasoconstriction were measured in the isolated, pressurized arteries as described (48). MT at 70 mmHg pressure was expressed as a percentage of the passive external diameter (PD ≈ 135 μm at 70 mmHg), measured in Ca2+-free physiological salt solution (Ca2+-free PSS) at the end of each experiment. Myogenic reactivity is shown as the steady-state arterial diameter in PSS, as a function of intralumenal pressure, compared with the PD (in Ca2+-free PSS) at the same pressure.
[Ca2+]Cyt and GFP fluorescence measurements.
For simultaneous [Ca2+]Cyt and diameter measurements, pressurized arteries from WT mice were loaded with fluo-4. Details of the dye-loading procedure and imaging methods are published (48). Similar methods were used for GFP fluorescence measurements in NCX1SM−/− arteries. Briefly, the pressurized arteries were imaged with a Nipkow-Yokogawa spinning disc confocal imaging system (CSU-10; Solamere Technology, Salt Lake City, UT) mounted on a Nikon Eclipse TE2000-U inverted microscope (×60; numerical aperture, 1.2; water immersion objective). Fluorescence was excited at 488 nm (argon ion laser). Optical sections of the arterial wall (106 μm × 70 μm, and containing 20–35 cells) were imaged with a Stanford XR-Mega 10 camera (Stanford Photonics, Palo Alto, CA) at 1–30 frames/s using a video-capture board (PCI 1407, National Instruments, Austin, TX).
Immunoblot Analysis
Aorta, main mesenteric artery, urinary bladder, heart, and brain were dissected, minced, and homogenized in homogenization medium, and the membrane fractions were prepared to assay NCX protein expression. Tissue extracts were analyzed (48) with specific monoclonal NCX1 antibody (R3F1, from K. D. Philipson, University of California, Los Angeles, CA). Band intensities were quantified with ID image analysis software (Eastman Kodak, Rochester, NY) (17) and normalized with β-actin or glyceraldehyde-3-phosphate dehydrogenase to control for protein loading.
Arterial Myocyte Isolation
Male WT, NCX1Fx/−, and NCX1SM−/− mice (12–16 wk of age) were euthanized with CO2 followed by cervical dislocation. The main mesenteric artery was removed, cleaned, and used for myocyte isolation. The artery was digested for 35 min at 37°C in low-Ca2+ PSS containing (in mg/ml) 1.5 collagenase type XI, 0.16 elastase type IV, and 1 bovine serum albumin, and individual myocytes were dissociated by trituration (2, 47). Only cells with elongated morphology (i.e., relaxed cells) were studied.
Patch-Clamp Recording
Whole cell, patch-clamp configuration was applied to record membrane currents using an Axopatch 200 patch-clamp amplifier (Axon Instruments, Union City, CA), as described (47). Fire-polished micropipettes (1–3 MΩ resistance) were manufactured from borosilicate capillary tubing (Garner Glass, Claremont, CA) using a micropipette puller (model P-97, Sutter Instruments, Novato, CA). An Ag-AgCl reference electrode was connected to the bath using an agar salt bridge containing 1 M KCl. Before the pipette approached the cell, the chamber was perfused with Ca2+-free PSS containing 1.4 mM extracellular Mg2+ concentration to prevent cell contraction caused by ATP diffusing from the pipette. After seal formation, the cells were superfused with normal PSS. The holding potential was −70 mV in all experiments. The leak and capacity transients were subtracted using a P/4 protocol. The series resistance was compensated to give the fastest possible capacity transient without producing oscillations. The membrane currents were recorded with 12-bit analog-to-digital converters (Digidata 1322A, Axon Instruments). Data were sampled at 500 kHz (unless otherwise stated), filtered at 5 kHz with a 902LPF low-pass Bessel filter (Frequency Devices, Haverhill, MA), and stored for subsequent analysis. All records were obtained at room temperature (25–26°C).
Macroscopic currents were recorded using 15-ms voltage clamp steps to a range of potentials from −50 to +90 mV. Preliminary experiments showed that LVGC currents carried by Ca2+ were small and difficult to measure accurately; therefore, we used Ba2+ as the current carrier. To construct current-voltage curves, the current during the pulse (IBa) is plotted as a function of pulse voltage. Because the amplitude of the tail current following the pulse (−Itail) is a measure of the conductance activated during the pulse, conductance-voltage (G–V) curves are obtained by plotting −Itail as a function of pulse voltage.
Recording of Ca2+ Sparklets
Detailed methods for recording Ca2+ sparklets are published (28, 29). Briefly, Ca2+ sparklets were recorded using a total internal reflection fluorescence microscope (Olympus, South Windsor, CT). Cells were loaded with the Ca2+ indicator Fluo-5F. Images were acquired at 30–90 Hz. Background-subtracted fluorescence signals were converted to concentration units using the Fmax equation (28, 47). Ca2+ sparklets were detected and defined for analysis using an automated algorithm written in Interactive Data Language software (IDL, Research Systems, Boulder, CO). Ca2+ sparklets had an amplitude equal to or larger than the mean basal [Ca2+]Cyt plus three times its standard deviation. For a [Ca2+]Cyt elevation to be considered a sparklet, a grid of 3 × 3 contiguous pixels had to have a [Ca2+]Cyt value at or above the amplitude threshold.
Reagents and Solutions
The artery dissection solution contained (in mM) 145 NaCl, 4.7 KCl, 1.2 MgSO4·7H2O, 2 MOPS, 0.02 EDTA, 1.2 NaH2PO4, 2 CaCl2·2H2O, 5 glucose, and 2.0 pyruvate and 1% albumin (pH 7.4 at 5°C). The PSS perfusion solution contained (in mM) 112 NaCl, 25.7 NaHCO3, 4.9 KCl, 2.5 CaCl2, 1.2 MgSO4·7H2O, 1.2 KH2PO4, 11.5 glucose, and 10 HEPES (adjusted pH to 7.3–7.4 with NaOH). High (10–75 mM) extracellular K+ concentration ([K+]o) solution was made by replacing NaCl with an equimolar KCl of normal PSS. Ca2+-free solution was made by omitting Ca2+ and adding 0.5 mM EGTA. Low (25.7 mM) extracellular Na+ concentration ([Na+]o) solution was made by replacing 112 mM NaCl with equimolar LiCl; 25.7 mM NaHCO3 was retained to minimize the perturbation of the acid-base balance. Solutions were gassed with 5% O2-5% CO2-90% N2 (48).
The tissue homogenization medium contained (in mM) 140 NaCl, 10 NaH2PO4, 2 EDTA, and 10 NaN3 and 2.5× concentrated Roche (Hoffmann-La Roche, Nutley, NJ) complete protease inhibitor cocktail.
To isolate Ba2+ currents (IBa) in electrophysiological experiments, the pipette was filled with high Cs+ solution of the following composition: (in mM) 130 CsCl, 2.5 MgCl2, 10 HEPES, 10 EGTA, and 2 Na2ATP (pH was adjusted to 7.2 with CsOH). The bath solution contained (in mM) 140 NaCl, 2.7 KCl, 10 BaCl2, and 10 HEPES (pH was adjusted to 7.4 with NaOH). All experiments were performed in the presence of 200 nM tetrodotoxin (TTX) to block the Na+ currents previously described in these cells (2). The osmolarity of all internal and external solutions was maintained at 271 and 296 ± 1 mosmol/l, respectively. Osmolarity was measured with a model 5500 vapor pressure osmometer (Wescor, Logan, UT).
Reagents and sources were as follows: ouabain, phenylepherine, nifedipine, collagenase, elastase, and bovine serum albumin (Sigma-Aldrich, St. Louis, MO); SEA0400 (Taisho Pharmaceutical, Tokyo, Japan); and TTX and Bay K 8644 (Calbiochem, San Diego, CA). Other reagents were reagent grade or the highest grade available. TTX was dissolved in deionized water; nifedipine was dissolved in DMSO.
Data Analysis and Statistics
The data are expressed as means ± SE; n denotes the number of animals, the number of arteries studied (1 artery per animal), or the number of cells studied in electrophysiological experiments. Comparisons of data were made using Student's paired or unpaired t-test, as appropriate; one-way or two-way ANOVA was used where indicated. Differences were considered significant at P < 0.05. Electrophysiological data analysis was performed using pClamp software (version 9.0; Axon Instruments). IBa amplitudes were measured relative to the current level before the pulse.
To characterize the voltage dependence of LVGC activation, we fit G–V curves with a Boltzman equation of the following form: where −Itail(Vm) is the magnitude of Itail following a step to Vm, −Itail,max is the maximum tail current, V0.5 is the voltage at which the conductance (i.e., the −Itail magnitude) is half-maximal, and k is the slope factor.
NCX1 Protein Expression Is Reduced in NCX1SM−/− Mouse Arteries
The expression of Cre recombinase, and thus the excision of NCX1 exon 11 in arterial myocytes of NCX1SM−/− mice, was verified by the simultaneous expression of eGFP. GFP fluorescence was observed in about 75–90% of the myocytes in the NCX1SM−/− mouse mesenteric small arteries employed in physiological experiments (Fig. 1C). There was, however, substantial variability not only from artery to artery but also from region to region in a single artery. The absence of a GFP signal in some myocytes (Fig. 1C) may indicate that Cre was not expressed in these cells or that it was only transiently expressed, in which case, NCX1 exon 11 should already have been excised.
The downregulation of normal NCX1 expression in these arteries was confirmed by immunoblot. In cardiac muscle, NCX1 appears as two bands, at 160 and 120 kDa. The relative expression of these bands varies, however, depending on the redox conditions used for membrane preparation (14); the 120-kDa band predominates under our conditions (Fig. 1D). In cardiac-specific knockout of NCX1, the intensities of both bands are reduced and a new, truncated NCX1 band appears at ∼110 kDa (18). As illustrated in Fig. 1D, comparable results are obtained in the arteries of WT and NCX1SM−/− mice, although some bands appear slightly smaller (∼140, ∼120, and ∼90 kDa, respectively). Only the two larger bands are observed in arteries from WT mice. The intensity of the 120-kDa band, which predominates in WT arteries, is reduced by about half in NCX1Fx/− mouse arteries and by ∼80–90% in NCX1SM−/− arteries; a new, truncated, ∼90-kDa band is then observed. The NCX1 expression is similarly reduced in urinary bladders from NCX1Fx/− and NCX1SM−/− mice (Fig. 1D). Quantification is difficult, however, because of the change in band size and the apparent degradation of the truncated, nonfunctional protein. In contrast, both hearts (Fig. 1D) and brains (not shown) from NCX1SM−/− as well as NCX1Fx/− mice express about half the normal (WT) level of NCX1, because both genotypes have one global, null-mutant NCX1 allele, but the SM-specific Cre recombinase is not activated in these tissues. In sum, these data demonstrate that the marked knockdown of NCX1 expression in NCX1SM−/− mice is SM specific; moreover, homozygous global knockout is embryonic lethal (39).
BP is Modestly Reduced in NCX1SM−/− Mice
NCX1SM−/− mice appear normal and grow to adulthood, and there are no obvious behavioral or gross anatomical differences between these mice and either the WT or NCX1Fx/− controls. Nevertheless, the fact that mice which overexpress NCX1 in SM have elevated BP (21) indicates that NCX1 plays a role in BP regulation and suggests that NXC1SM−/− mice might also have a vascular phenotype. To test this, we compared BP in WT, NCX1Fx/−, and NCX1SM−/− mice with two invasive methods (Fig. 2). First, the mean arterial BP (MBP) was measured with an intrafemoral catheter in mice under light isoflurane anesthesia. The results revealed that MBP in NCX1Fx/− mice was comparable with WT controls and was significantly reduced in NCX1SM−/− mice (Fig. 2A). Similar results were obtained with telemetric BP measurements on awake, free-moving mice: MBP was lower in NCX1SM−/− mice than in NCX1Fx/− or WT mice (Fig. 2B,a). Interestingly, the heart rate was significantly slower in both NCX1Fx/− and NCX1SM−/− mice than in WT mice (Fig. 2B,b). The similarity of the heart rate in NCX1Fx/− and NCX1SM−/− mice, despite the difference in BP, implies that the slow heart rate does not account for the low BP in the NCX1SM−/− mice.
MT and Reactivity Are Attenuated by Reduced NCX1 Activity
To determine the contractile properties of the arteries from NCX1Fx/− and NCX1SM−/− mice, the diameter changes in isolated, pressurized mesenteric small arteries were measured. These arteries constrict spontaneously (=myogenic reactivity) when intralumenal pressure is increased to more than 40 or 50 mmHg during superfusion with PSS at 35–37°C (48). Arteries from NCX1SM−/− mice exhibited significantly reduced myogenic reactivity (Fig. 3A) and MT (Fig. 3B) than did WT or NCX1Fx/− arteries.
Figure 3B illustrates the effects of the specific NCX inhibitor, SEA0400 (25), on MT in these arteries. SEA0400 (1 μM) reduced MT in WT mouse arteries to about the level of MT observed in NCX1SM−/− arteries in the absence of SEA0400 but had no effect on MT in NCX1SM−/− arteries. Thus the effect of SEA0400 on WT arteries is comparable with the effect of genetic NCX1 ablation. This lack of effect on arteries with markedly reduced NCX1 expression and the attenuated effect on MT in NCX1Fx/− arteries (Fig. 3B) are evidence for the specificity of 1 μM SEA0400.
Data on the mechanism by which SEA0400 reduces MT were obtained by simultaneous measurements of [Ca2+]Cyt and diameter in WT arteries (Fig. 3C). Previously, we demonstrated that the generation of MT is associated with a sustained rise in [Ca2+]Cyt in myocytes within the walls of pressurized small arteries (48). The confocal images of a fluo-4-loaded WT artery wall in Fig. 3C,a show that [Ca2+]Cyt (i.e., fluo-4 fluorescence) declines and the artery dilates when 1 μM SEA0400 is applied (Fig. 3, C,a, i to ii, and C,b). [Ca2+]Cyt rises and the artery constricts when the SEA0400 is washed out (Fig. 3, C,a, ii to iii, and C,b). The incomplete recovery of both fluo-4 fluorescence and MT were likely primarily due to the slow and incomplete washout of SEA0400, which is very hydrophobic. A modest bleaching of fluo-4, however, probably also contributed to the incomplete recovery of the initial fluorescent signal.
A supplemental video clip of the fluo-4 fluorescence data shown in Fig. 3C is available (note: supplemental material may be found posted with the online version of this article). These data imply that Ca2+ entry via NCX1 contributes to the maintenance of the elevated [Ca2+]Cyt and MT in pressurized small arteries.
NCX1 Function Is Markedly Reduced in NCX1SM−/− Arteries
Additional evidence that arterial myocyte NCX1 activity is markedly reduced in NCX1SM−/− mice was obtained by exposing arteries to low (25.7 mM) [Na+]o solution for 7–10 min before restoring normal (137.7 mM) [Na+]o. In this common test of NCX function (1), the exposure of WT arteries to a sudden reduction in ENa induces an initial, transient vasodilation, followed by a sustained vasoconstriction (Fig. 4A). A preincubation of WT arteries with 1 μM SEA0400 for 10 min to block NCX reduces the dilation by about 50% and the constriction by about 90% (Fig. 4B). Importantly, the magnitudes of both the transient vasodilation and the sustained vasoconstriction are similarly reduced in NCX1Fx/− and NCX1SM−/− arteries (Fig. 4, A and C).
We have reported that nanomolar ouabain raises [Ca2+]Cyt and augments MT in WT arteries; these effects are markedly inhibited by 1 μM SEA0400, suggesting that they result from increased NCX-mediated Ca2+ entry (21, 48). To explore this role of NCX further, we compared the effects of ouabain on MT in arteries from WT, NCX1Fx/−, and NCX1SM−/− mice. As illustrated in Fig. 5, A and B, the ability of 100 nM ouabain to augment MT is significantly impaired in NCX1Fx/− and, to a greater extent, in NCX1SM−/− arteries.
Agonist-Induced Vasoconstriction Is Impaired in NCX1SM−/− Arteries
The effect of reduced NCX1 expression on high [K+]o- and phenylephrine (PE)-evoked vasoconstriction in small arteries are illustrated in Fig. 6. The maximal vasoconstriction induced by elevating [K+]o from 5 to 75 mM in NCX1Fx/− arteries, ∼60% of PD (which nearly obliterated the lumen), is similar to that in WT arteries (Fig. 6A,a). In contrast, the 75 mM [K+]o-induced vasoconstriction is significantly reduced in NCX1SM−/− arteries (Fig. 6A, a and b). This may indicate that the maximal Ca2+ entry through LVGCs is reduced in NCX1SM−/− arteries because the 75 mM [K+]o-evoked vasoconstriction is abolished by the LVGC blocker, nifedipine (47).
The maximal constriction to high-dose (3–100 μM) PE also is significantly smaller in NCX1SM−/− than in WT arteries; the constriction to 100 μM PE is slightly, but not significantly, reduced in NCX1Fx/− arteries (Fig. 6B, a and b). Furthermore, both the high [K+]o (Fig. 6A,b) and PE (Fig. 6B,b) dose-response curves are significantly depressed in NCX1SM−/− compared with in NCX1Fx/− arteries. Thus, for example, the PE constriction is reduced at all PE concentrations, but the concentration required for half-maximal constriction, ∼0.35 μM, is not significantly different in the NCX1SM−/− and WT mouse arteries (Fig. 6B,b). The differences between the NCX1SM−/− and NCX1Fx/− arteries at the low-dose ends of the respective curves can be attributed, at least in part, to the lower MT in the NCX1SM−/− arteries (i.e., at 5 mM [K+]o and at 0 mM PE). Some possible mechanisms for the reduced constriction to high [K+]o and high concentrations of PE are described in discussion.
LVGC Current Is Reduced in NCX1SM−/− Myocytes
The aforementioned studies on high [K+]o-induced vasoconstriction raise the possibility that LVGC activity might be reduced in NCX1SM−/− myocytes. Therefore, LVGC currents were measured in isolated mesenteric artery myocytes, using Ba2+ as the current carrier (47). Indeed, the data reveal that LVGC currents in NCX1SM−/− mouse myocytes are smaller than the currents in myocytes from WT and NCX1Fx/− mice (Fig. 7A, a–c). This was confirmed by measuring both the current-voltage and G-V curves in myocytes from WT, NCX1Fx/−, and NCX1SM−/− mice. The amplitude of the LVGC, −Itail, was used as a measure of the conductance activated during the preceding depolarizing pulse (Fig. 7A,d). Both the LVGC current and conductance are significantly smaller in NCX1SM−/− myocytes compared with WT myocytes (Fig. 7B, a and b). LVGC currents and conductances are, however, nearly identical in WT and NCX1Fx/− myocytes (Fig. 7B, a and b).
Block of NCX1 with SEA0400 Does Not Affect LVGC Current
Because the LVGC current is reduced in NCX1SM−/− mice, we examined the effect of acute block of NCX1 with SEA0400 on LVGC current in dialyzed, patch-clamped WT myocytes. In the experiment illustrated in Fig. 7C, IBa at +10 mV is plotted as a function of time to control for the rundown of the current. The addition of 0.01% DMSO or 1 μM SEA0400 in 0.01% DMSO had no detectable effect on IBa. In another type of control experiment performed in a similar manner, ∼85–90% of IBa was blocked by 10 μM nifedipine within 30 s (data not shown) (47). These experiments demonstrate that an acute block of NCX1 by SEA0400 in WT myocytes does not reduce the nifedipine-sensitive LVGC current.
Voltage-Dependent Inactivation of LVGC Channels Is Unchanged in NCX1SM−/− Myocytes
In cardiac-specific NCX1 knockout mice, too, LVGC current is decreased. In those myocytes, the current declines as a result of an increase in the rate of Ca2+-induced inactivation and increased Ca2+-dependent tonic inactivation of LVGCs, presumably because of the elevation of subsarcolemmal [Ca2+] (32). Such a mechanism cannot explain our results because Ba2+ was used as the current carrier in the arterial myocytes and because Ba2+ does not support Ca2+-dependent inactivation (15). Because LVGCs also exhibit voltage-dependent inactivation, however, we tested whether a shift in voltage dependence or an increase in inactivation could explain the reduction of LVGC current in NCX1SM−/− mice. The results reveal that voltage-dependent inactivation is nearly identical in WT, NCX1Fx/−, and NCX1SM−/− myocytes (Fig. 8A). Thus a change in inactivation cannot explain the reduction of LVGC current in NCX1SM−/− myocytes.
Bay K 8644 Eliminates the Difference in LVGC Current Between Control and NCX1SM−/− Myocytes
Another possible explanation for the reduced LVGC current in NCX1SM−/− myocytes is that the expression of the channels is reduced. To get an indication of LVGC expression, we applied Bay K 8644, a potent activator of LVGCs (19). In both WT and NCX1SM−/− myocytes, 1 μM Bay K 8644 increased LVGC current and maximum conductance and shifted the G–V curve to more negative potentials (Fig. 8B). Interestingly, the current and conductance increases were greater in NCX1SM−/− myocytes than in WT myocytes. In fact, Bay K 8644 eliminated the difference between the current, as well as the conductance, in WT and NCX1SM−/− myocytes (Fig. 8B). This implies that LVGC channel expression is comparable in WT and NCX1SM−/− myocytes.
Bay K 8644 Eliminates the Difference in Ca2+ Sparklet Activity Between Control and NCX1SM−/− Myocytes
Ca2+ sparklets are local Ca2+ signals produced by the opening of single or small clusters of LVGCs in arterial myocytes (28, 29). Accordingly, Ca2+ sparklets are the elementary events underlying dihydropyridine-sensitive Ca2+ influx in these cells. Therefore, in view of the smaller LVGC currents in NCX1SM−/− than in WT or NXC1Fx/− myocytes, we examined Ca2+ sparklet activity in NCX1SM−/− and sibling NXC1Fx/− mouse myocytes to test the hypothesis that sparklet activity is reduced in the NCX1SM−/− myocytes.
The images in Fig. 9A show a representative Ca2+ sparklet recorded from an NCX1SM−/− myocyte and one from an NXC1Fx/− myocyte. Summarized data indicate that the mean peak amplitude of the sparklets, as well as the width at 50% of peak amplitude, is comparable in NXC1Fx/− and NCX1SM−/− myocytes: 41 ± 1 vs. 40 ± 1 nM, and 0.73 ± 0.03 vs. 0.82 ± 0.05 μm (n = 7 and 9 cells, respectively; P > 0.05 for both comparisons). These results suggest that NCX1-mediated Ca2+ transport does not significantly alter the amplitude or spatial spread of quantal Ca2+ sparklets.
Despite these similarities in the quantal events, Ca2+ sparklet density (in sites/mm2) and the overall level of sparklet activity (in nPs, where n is the number of quantal levels and Ps is the probability that a sparklet site is active) (29) are significantly reduced in NCX1SM−/− myocytes compared with NXC1Fx/− myocytes (Fig. 9, A and B). Bay K 8644 (500 nM), however, abolishes this difference (Fig. 9, B and C). These results are consistent with the effect of Bay K 8644 on LVGC currents in NCX1SM−/− and control (WT) myocytes (Fig. 8B).
Bay K 8644 Does Not Eliminate the Difference in Myogenic Reactivity Between WT and NCX1SM−/− Arteries
Because LVGCs play a major role in myogenic constriction (8, 20), one possibility is that the reduced myogenic reactivity observed in NCX1SM−/− arteries (Fig. 3A) might be the result of the reduced LVGC activity in these arteries. Bay K 8644 normalizes LVGC current (Fig. 8) and Ca2+ sparklet activity (Fig. 9) in NCX1SM−/− arteries. Therefore, we reexamined the myogenic reactivity curves in WT and NCX1SM−/− arteries in the presence of 1 μM Bay K 8644. Figure 10 shows that Bay K 8644 constricts WT and NCX1SM−/− arteries at 20 mmHg. The WT arteries then exhibit slight additional constriction when the intralumenal pressure is increased to 40 mmHg or more. In contrast, raising intralumenal pressure from 20 to 40 mmHg dilates the Bay K 8644-treated NCX1SM−/− arteries, albeit, not as much as in the absence of Bay K 8644, increasing intralumental pressure further, and then causes only slight constriction. Importantly, Bay K 8644 did not affect the PD in either WT or NCX1SM−/− arteries (not shown). Clearly, the NCX1SM−/− arteries exhibit significantly less myogenic constriction than WT arteries at intralumenal pressures of 40–120 mmHg, even in the presence of Bay K 8644. This difference apparently cannot be attributed to the reduced LVGC activity in NCX1SM−/− arterial myocytes.
This study on the functional consequences of SM-specific knockout of NCX1 complements our earlier report that SM-specific overexpression of NCX1 elevates BP in mice (21). The results we describe here strengthen the view that NCX1 plays an important role in vascular function.
NCX1Fx/− Mice vs. WT Mice
A complication encountered in this study was the discovery that the Cre recombinase gene, under the control of the SM-MHC promoter, is activated during gametogenesis. Thus both the CreSM+ and Cre− mice used in this study all carried one floxed NCX1 allele and one null-mutant allele, in which NCX1 exon 11 was permanently deleted in all cells (methods). In other words, both Cre− and CreSM+ mice are, effectively, NCX1 heterozygotes; then, upon activation of the Cre gene in the SM cells of CreSM+ mice, exon 11 in the remaining floxed NCX1 allele is excised in these cells. Therefore, in many experiments, we employed two controls for the NCX1SM−/− mice: WT mice and NCX1Fx/− mice. It is noteworthy that the homozygous global null mutation of NCX1 is embryonic lethal (39).
Relative to WT controls, the NCX1Fx/− mice have a reduced expression of full-length NCX1 in all tissues tested (see Fig. 1D). As a result, the NCX1Fx/− arteries exhibit a reduced vasodilator response to SEA0400 (Fig. 3B), a reduced low [Na+]o-induced vasodilation and vasoconstriction (Fig. 4B), and a reduced vasoconstrictor response to low-dose ouabain (Fig. 5). In many other respects, however, the function of NCX1Fx/− arteries is comparable with that of WT controls. For example, MT, vasoconstrictor responses to high [K+]o, and the function of LVGCs are all normal; moreover, the NCX1Fx/− mice have normal BP. The implication is that approximately half of the normal level of NCX1 in ASMC is sufficient to maintain normal arterial function.
Similarity of Effects of Genetic and Pharmacological Knockout of NCX1 on Arterial Function
In contrast to the situation in NCX1Fx/− mice, vascular function is substantially impaired in NCX1SM−/− mice. This is exemplified by the reduced myogenic reactivity and MT (Fig. 3), attenuated vasoconstrictor responses to PE and high [K+]o (Fig. 6), and reduced LVGC activity (Fig. 7). Moreover, SM-specific knockout of NCX1 and an acute SEA0400 application to WT mouse arteries, i.e., a genetic and pharmacological reduction of NCX activity, respectively, have comparable effects on MT (Fig. 3) and on the responses to low [Na+]o (Fig. 4) and low-dose ouabain (Fig. 5 and Ref. 48). In rat small arteries, too, pharmacological blockade or antisense oligonucleotide knockdown of NCX1 reduces myogenic reactivity and tone (36). As expected for a NCX-specific blocker [albeit somewhat controversial (38)], SEA0400 has no effect on MT in NCX1SM−/− mouse arteries (Fig. 3) or on LVGC current in WT artery myocytes (Fig. 7C). These findings emphasize the importance of NCX1-mediated Ca2+ entry in arterial function. Clearly, this mode of NCX operation is a normal mode and, perhaps, the dominant mode in ASMCs in arteries with tone.
The NCX1-mediated, low [Na+]o-induced initial vasodilation and, later, sustained vasoconstriction (Fig. 4A) raise the interesting question of mechanism(s). The vasoconstriction appears to be explicable by the decline in the inwardly directed ENa; this is expected to promote net Ca2+ gain via NCX1 (Introduction) and direct activation of myosin light chain kinase and, therefore, ASMC contraction. The explanation for the early vasodilation is less clear. About half of the dilation is blocked by bath-applied SEA0400 or by SM-specific NCX1 knockout and therefore appears to be attributable to SM NCX1 (Fig. 4C). Two possible mechanisms that could also be triggered by NCX-mediated Ca2+ entry and a rise in [Ca2+]Cyt are 1) the rapid activation of large-conductance Ca2+-activated K+ channels in ASMCs (7) and 2) the Ca2+-induced activation of nitric oxide synthase in endothelial cells (43). The latter possibility is consistent with the view that NCX1 activity in ASMCs and endothelial cells may have opposing effects on arterial constriction.
Reduced LVGC Activation: Cause of Reduced MT or Consequence of Reduced NCX1 Activity?
An unexpected observation was that LVGC activity is reduced in arterial myocytes from NCX1SM−/− mice (Fig. 7). Three possible explanations are 1) a reduction in the number of expressed LVGCs in NCX1SM−/− myocytes or 2) a decrease in the magnitude of the LVGC-mediated elementary Ca2+ influx events (Ca2+ sparklets) in NCX1SM−/− myocytes (i.e., a decrease in quantal size), or 3) a reduction in the number of open channels under the physiological conditions that prevail in NCX1SM−/− myocytes. The maximal activation of LVGCs (both whole cell currents and Ca2+ sparklets) by Bay K 8644 reveals that the reduced channel activity is functional and is not due to reduced LVGC expression.
To distinguish between the second and third possibilities, we employed sparklet analysis. The evidence that the magnitude and spatial spread of individual sparklets do not differ in NCX1SM−/− and NCX1Fx/− myocytes indicates that the individual quanta are similar; this also implies that the single-channel conductance of the LVGCs is not altered. Thus the reduced LVGC current must be due to a decreased open-channel probability. This fits with the observed reduction in sparklet activity (number of active sites and probability of activation) in NCX1SM−/− myocytes (Fig. 9).
LVGC activity plays a critical role in the maintenance of MT (8, 20, 47), and the reduced LVGC activity might be the cause of the reduced MT and low BP in NCX1SM−/− mice. Figure 10 shows, however, that myogenic constriction is reduced in NCX1SM−/− arteries, compared with WT arteries, even in the presence of 1 μM Bay K 8644, which abolishes the difference between LVGC activity in these arteries (Fig. 8). Furthermore, evidence presented in the accompanying article (37) implies that the depressed LVGC activity is a consequence of the low [Ca2+]Cyt and reduced Ca2+-dependent protein kinase C activity that likely results from a marked knockdown of NCX1 expression. Indeed, an acute inhibition of NCX by SEA0400, which does not block LVGCs (Fig. 7C), lowers [Ca2+]Cyt and reduces MT in WT arteries (Fig. 3, B and C). The fact that NCX1SM−/− mouse arteries have significantly reduced MT therefore implies that [Ca2+]Cyt may also be low in these ASMCs. Thus the attenuation of MT (Fig. 3A), LVGC activity (Fig. 7), and high [K+]o-induced vasoconstriction (Fig. 6A) in NCX1SM−/− arteries may all be the consequence of a low [Ca2+]Cyt as a result of reduced NCX1-mediated Ca2+ entry.
The mechanism(s) responsible for the attenuated high-dose PE-induced vasoconstriction (Fig. 6B) is (are) uncertain. Preliminary data imply that the mobilization of stored Ca2+ (42) is not impaired in arteries from NCX1SM−/− mice because high PE-induced vasoconstriction in Ca2+-free media is not attenuated (data not shown). A likely possibility is that receptor-operated channels (ROCs), which are an important source of Ca2+ during receptor activation (44), especially at high catecholamine concentrations (30), may be downregulated in NCX1SM−/− arteries. In primary cultured arterial myocytes, a knockout of NCX1 markedly decreases the expression of transient receptor potential canonical 6 channel protein (a component of ROCs) and reduces ROC activity (33).
Correlation Between BP and In Vitro MT
Davis and Hill (8) observed that the level of tone in isolated arteries "is often comparable to that observed in the same vessels in vivo." This implies that MT in isolated arteries predicts not only in vivo vascular tone but BP as well, as exemplified by the increased MT and elevated BP in α2 Na+-pump heterozygous null mutant mice (48). The present report extends this concept by showing that, at the other end of the spectrum, SM-specific knockout of NCX1 not only reduces MT but lowers BP as well. Conversely, SM-specific overexpression of NCX1 is associated with elevated BP (21); an increase in MT is anticipated, but this has not yet been tested in arteries from these mice.
Most important, the present report adds to the growing body of evidence that α2 Na+ pumps and adjacent NCX1 are critical mechanisms that link Na+ metabolism to BP regulation (6). Recently, Wirth and colleagues (45) suggested that salt-dependent hypertension is specifically mediated by a G12-G13 protein-coupled receptor-mediated pathway, but our findings do not support their conclusion. On the contrary, our results suggest that the modulation of NCX1 activity directly affects Ca2+ metabolism and the level of [Ca2+]Cyt and thus likely acts via myosin light chain kinase to modulate vasoconstriction. In conjunction with earlier reports (12, 13, 21, 48), the present study is consistent with the view that retained salt can act via the endogenous ouabain-α2 Na+ pump-NCX1 pathway to elevate BP without directly involving G protein-coupled receptor pathways (6). Of course, the downstream effects are likely to be modulated by the G protein-coupled receptor pathways that regulate the Ca2+ sensitivity of the contractile apparatus (24).
We conclude that the arterial SM NCX1 is needed to maintain the normal [Ca2+]Cyt, MT, and BP. This direct link to NaCl metabolism is often overlooked in studies of the mechanisms that influence the BP in salt-dependent hypertension.
This work was supported by a National Scientist Development grant from the American Heart Association (to J. Zhang); an International Society of Hypertension-Pfizer award (to J. Zhang); an National Institute of Arthritis and Musculoskeletal and Skin Diseases training grant (to L. K. Antos); and National Heart, Lung, and Blood Institute research grants (to M. P. Blaustein, K. D. Philipson, M. I. Kotlikoff, L. F. Santana, and W. G. Wier).
No conflicts of interest are declared by the author(s).
We thank Michelle Izuka for technical support and transgenic mouse genotyping and Katherine Frankel for assistance with the manuscript. We also thank Taisho Pharmaceutical (Tokyo, Japan) for a gift of SEA0400.
Present address of C. Ren: Neuroscience program, Otolaryngology/Department of Surgery, University of Utah, Salt Lake City, UT.
- Copyright © 2010 the American Physiological Society


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Maid of the Mist launches brand campaign
A statue of a seated President Abraham Lincoln wears the same blue poncho used by millions every year to keep away the mist of Niagara Falls.
So do Lady Liberty and a few other important figures in American history.
Those are the images of a new brand campaign being undertaken by the Maid of the Mist Corp., which will use the Statue of Liberty, the Lincoln Memorial and Mount Rushmore in national and international advertising over the coming year.
"Our research uncovered that many people view the Maid of the Mist as an American treasure and beloved tourist attraction," said Tim Ruddy, Maid of the Mist vice president of marketing. "We wanted our brand to highlight that viewpoint and better position us as an authentic, homegrown destination that, much like our nation's monuments, is true to its American roots."
The Maid of the Mist, which up until this year had been operating boat rides below Niagara Falls on both sides of the border uninterrupted since 1885, hired Eric Mower + Associates, which has an office in Buffalo, for the marketing campaign.
The campaign's tagline: America's Most Monumental Experience since 1846.
"Taking a ride on the Maid of the Mist is a time-honored event that is enjoyed by people all around the world," said Tom Merrick, executive creative director for Eric Mower. "It was important that the brand celebrate that and communicate the thrill of an experience that can only be found in the United States."
The campaign will include outdoor advertising, as well as consumer and trade print and online ads.
The ad with the Lincoln Memorial has already started running in the Northeast.
Maid of the Mist Corp. is finalizing work on its $32 million boat storage facility, built on the site of the former Schoellkopf Power Station in the Niagara Gorge.
Starting this year, Hornblower Cruises has taken over operating the boat tours on the Canadian side of the border.
For more Niagara Falls news, follow @The_Brink_Blog on Twitter email: firstname.lastname@example.com

subset: commoncrawl


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Selinger Marsh
Open to the Public
Plan Your Visit
Get Directions
Why You Should Visit
Selinger Marsh lies in the rain shadow of the Appalachian Mountains — this area receives the least rainfall in Maryland. Springs on the surrounding slopes, however, provide the water for the Marsh, making it an unusual wetland in a otherwise very dry region. Selinger Marsh is open year round to the public for birdwatching and nature walks. Admission is free.
Before You Go
- View Preserve Guidelines. Please note: dogs are not allowed at any Conservancy preserve.
- Download the Selinger Marsh Preserve Visitors Guide (pdf)
Two hour drive from Baltimore and Washington, D.C., near Flintstone
Why the Conservancy Selected This Site
Selinger Marsh is a rare wetland in this dry region of the state; its importance as a breeding ground for amphibians makes the site a priority in order to maintain viable populations of these species in this part of Maryland.
What the Conservancy Has Done/Is Doing Here
Protected by The Nature Conservancy since 1985, Selinger Marsh's 85 acres continue to be areas for research and monitoring, and the Conservancy acts as a steward to ensure that the site maintains its ecological viability.
What to See: Plants
- State-rare fringe-tipped gentian
- Arrowhead, monkey-flower, cardinal flower, New England aster, great blue lobelia, water plantain, blue flag, silky dogwood, alder, and elderberry
What to See: Animals
- Selinger Marsh is an important haven in this otherwise dry region for several amphibians, including the upland chorus frog and the Jefferson salamander
Get the Most from Your Visit
- Please do not remove any plants, animals, or rocks
- There are no trails, other than deer trails, so a compass may help you keep your bearings
- Please help us maintain this unique natural environment by taking home everything that you bring, including biodegradable materials
- For more information, please contact the Maryland/DC Chapter office at 301-897-8570 or firstname.lastname@example.com
From Washington
- Take the Capital Beltway (I-495) to I-270 North
- From I-270 North take I-70 West
- Near Hancock, take I-68 West / US-40 West. Take the Flintstone exit
- From the center of Flintstone continue about .5 mile east to intersection at Town Creek Road
- Turn right across metal bridge onto Town Creek Road
- In about 1.5 miles bear left (stay on pavement) and cross a second metal bridge
- The preserve is 2 miles on the right
- There is no parking lot; please park carefully on the roadside
Have you been to this preserve? Are you thinking of visiting? See what others are saying about their experiences and add your comments below.
Add Your Comments
Time for you to join the discussion. Tell us about your experience at this preserve. What plants and animals did you see? When did you go? You can help others plan their visit when you share your thoughts. And thank you for visiting one of our nature preserves!comments powered by Disqus
Read our guidelines on posting comments


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Macally's BTKEYPRO looks like a nice do-everything keyboard, for all your devices. The main selling point is that it can pair with and switch between up to five devices, letting you use it with your iMac, MacBook, iPad, iPad Mini and iPhone, all at the touch of a key.
But it does more than just that.
Without getting my fingers on this keyboard, It's impossible to tell whether it's good or not. On paper, though, it looks great. Bluetooth, AA batteries for long life and easy switching (batteries are included, too!) and rubber feet join the familiar scissor keys (like those on your MacBook). You even get to use the keyboard as a USB peripheral if you like.
There are also a whole lot of special function keys baked into the 108-key layout (108 keys means there's a number pad). You get six dedicated device switching keys, all of which are labelled. You get the regular media keys, plus buttons for Spotlight, Home and lock/power (for the iDevices).
So feature-wise it looks amazing. The only thing to be determined is the quality of the keys, which is arguably the most important part.
Available now for a reassuring $70.
Source: Macally


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Grand Rapids Police Say No Gun Was In Building
Edit: (2:50pm)
Police have cleared an office building in the 3500 Block of Lake Eastbrook after receiving reports of a possible gunman in the building.
Officers searched the East Lake Office Center and say that they didn't find anyone with a gun.
Authorities responded to the scene after receiving a call from someone claiming to have seen a man going inside with a weapon.
Edit: (2:13pm)
According to WZZM, police are responding to a report of a gunman who has barricaded himself near Centerpointe Mall in a business complex at 3501 Lake Eastbrook SE.
Authorities are evacuating nearby businesses and residences. Motorists near the scene should expect delays and seek an alternate route if possible.
It is not known at this time if anyone else is in the building with the gunman.


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SPORTS: Port Angeles softball wins on walk-off hit; other prep reports
Peninsula Daily News
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Two of the Roughriders' three hits came in the bottom of the seventh, including Politika's two-out hit that scored Ashlee Reid.
The rest of the game between second-place Port Angeles (12-2, 13-2) and third-place Kingston (9-4) was dominated by the pitching of Sarah Steinman and Allison Hilse.
Steinman pitched a complete-game shutout for the Riders, striking out eight and giving up just two hits.
Hilse had a one-hitter going into the seventh inning. She struck out one and walked one.
Hilse also had one of the Buccaneers' two hits.
Along with the game-winning run, Reid had two hits for Port Angeles.
With two games remaining, the Riders are one win away from clinching second place in the Olympic League. Their first chance to clinch comes today at home against North Kitsap (5-8, 5-9).
Port Angeles 1, Kingston 0
Kingston 0 0 0 0 0 0 0 — 0 2 2
Port Angeles 0 0 0 0 0 0 1 — 1 3 2
WP- Steinman; LP- Hilse
Pitching Statistics
Kingston: Hilse 6 2/3IP, 3H, R, BB, K.
Port Angeles: Steinman 7IP, 2H, 0R, 8K, 8BB.
Hitting Statistics
Kingston: Hilse 1-2; Reitan 1-4.
Port Angeles: Reed 2-3, R; Politika 1-3, RBI.
Sequim 9,
North Mason 1
BELFAIR — The Wolves were held to three runs over the first five innings, but exploded with a six-run sixth to take a commanding 9-0 lead.
Alexas Besand doubled and drove in three runs for Sequim (13-0, 14-0), which leads the Olympic League by two games with three games left.
Columbia Haupt contributed two RBI and Rylleigh Zbaraschuk doubled twice, walked twice and scored twice.
On the mound, Makayla Bentz tossed a complete game and striking out eight. The only run she allowed was a homer by North Mason's Kimber Bolin.
Sequim plays its third road game in three days today at Olympic (10-4, 10-7), which is tied for third place in the Olympic League. The Wolves pounded the Trojans 28-2 on Friday.
Sequim 9, North Mason 1
Sequim 0 0 2 0 1 6 0 — 9 11 2
North Mason 0 0 0 0 0 1 0 — 1 5 3
WP- Bentz; LP- Bolin
Pitching Statistics
Sequim: Makayla Bentz 7IP, 5H, R, 4BB, 8K.
North Mason: Bolin 5 1/3IP, 8H, 7R, 2BB, 5K; Crover 1 2/3IP, 3H, 2R, 2BB, K.
Hitting Statistics
Sequim: Besand 2-3, BB, 2B, 3RBI; Grubb 3-4, 2B, 2R, SB; Zbaraschuk 2-2, 2 2B, 2R, RBI; Haupt 1-3, R, 2RBI, SB.
North Mason: Bolin 1-2, HR; Pesk 2-3; Gascoyne 2-3.
Chimacum 17,
Vashon 0, 5 innings
VASHON — Kristen Castillo drove in four runs to lead the Cowboys' onslaught against the winless Pirates (0-13).
As a team, Chimacum pounded out 16 hits, including four by Mallori Cossell, three by Castillo and two each from Cydney Nelson, Krista Hathaway and Megan Dukek.
Cowboys pitchers Nelson and Ryley Eldridge combined for the shutout.
Eldridge started and earned the win with two strikeouts in three innings, while Nelson fanned three in two innings. Both pitchers allowed one hit apiece.
Chimacum (6-4, 7-5), which sits in third place in the Nisqually League, hosts Cascade Christian (4-6) today.
Chimacum 17, Vashon 0 (5 innings)
Chimacum 3 0 4 7 3 — 17 16
Vashon 0 0 0 0 0 — 0 2
WP- Eldridge; LP- Frazier
Pitching Statistics
Chimacum: Eldridge 3IP, 2K, H, 2BB; Nelson 2IP, 3K, H.
Vashon: Frazier 3IP, 2K, 7H, 2BB; Baker 2IP, K, 9H, 6BB.
Hitting Statistics
Chimacum: Castillo 3-3, 2B, 4RBI; Cossell 4-5, 2B; Hathaway 2-4, 2RBI; Dukek 2-3, 2RBI; Nelson 2-4, 2B; Snyder, 2B, RBI.
Vashon: Frazier 1-2; Baker 1-1.
Port Angeles 5,
Kingston 3
PORT ANGELES — By beating the Buccaneers, the Roughriders clinched the fourth spot in the Olympic League tournament.
Port Angeles (9-7) will play Klahowya today at the Kitsap County Fairgrounds.
The winner will face Olympic on Friday for the league's third seed in the West Central District tournament, and the loser will be relegated to the fifth seed.
Wes Giddings started against Kingston and allowed only three hits and held the Buccaneers scoreless in his six innings on the mound.
"We did what we needed to do today and took care of business," Port Angeles coach Chad Wagner said.
"Wes Giddings took hold of this game and forced us to win. Great to see a senior who deserves it step up on senior night."
At the plate, Giddings was 2 for 2 with a walk and a run.
Larsson Chapman tripled, scored and drove in a run, and Zach Lovik had a hit and an RBI.
Sequim 7,
North Mason 2
BELFAIR — The Wolves ended the Bulldogs' quest for postseason by scoring seven unanswered runs.
North Mason (7-9, 10-9) plated two runs in the bottom of the first inning, but Sequim pitcher Austin Clement held them in check the rest of the way.
Clement pitched a complete game, fanning six and allowing just four hits.
The Wolves (4-11, 6-11) trailed 2-0 going into the fourth inning before their bats started bringing in runs.
Sequim scored three in the fourth, and added one in the fifth and three more in the top of the seventh inning.
Nick Johnston was 3 for 4 with a double and two RBI. Fred Serrano added two hits and an RBI, and Brett Wright had two hits and an RBI.
Sequim 7, North Mason 2
Sequim 0 0 0 3 1 0 3 — 7 11 0
North Mason 2 0 0 0 0 0 0 — 2 4 0
WP- Clement; LP- Sandell
Pitching Statistics
Sequim: Clement 7IP, 2R, 6K, 4H, 3BB.
North Mason: McKean 3IP, 0R, 5K, H; Sandell 4IP, 6ER, 3BB.
Hitting Statistics
Sequim: Johnston 3-4, 2B, BB, R, 2RBI, SB; F. Serrano 2-4, 2B, R, RBI, SB; Wright 2-4, R, RBI, SB; Donahue 0-3, R, 3SB; Koonz 1-2, R.
North Mason: Stevens 2-3, 2B; Burrgraaf 1-4, R.
Vashon 7, Chimacum 5
VASHON — The Pirates big start was too much for the Cowboys to overcome in this Nisqually League matchup.
Vashon began the game with a six-spot in the first inning, and added another run in the second to jump out to a 7-0 lead.
Chimacum held the Pirates (9-6, 9-8) scoreless the remainder of the game and countered with five runs of its own — two in the third inning and three in the fifth.
Colton Shaw pitched four solid innings of relief for the Cowboys, allowing just one hit and striking out one.
Drew Yackulic had a pair of hits for Chimacum (3-10, 3-12), which plays at Cascade Christian (10-3) today. The Cougars are currently in second place in the Nisqually League.
Vashon 7, Chimacum 5
Chimacum 0 0 2 0 3 0 0 — 5 5 0
Vashon 6 1 0 0 0 0 x — 7 4 2
WP- Mathena; LP- Schreier
Pitching Statistics
Chimacum: Shaw 4IP, H, K.
Hitting Statistics
Chimacum: Yackulic 2-4.
Vashon: Bernheisel 1-2, 2RBI.
Girls Tennis
Sequim 4, Kingston 3
KINGSTON — The Wolves won the Olympic League title by going on the road and winning the battle of 12-0 teams.
Sequim swept the singles matches, with No. 1 Hannah Gauthun defeating Miriam Robertson 6-2, 6-2; No. 2 Tenisha Powless beating Sarah Zischke 6-2, 6-3; and Andrea Tjemsland besting Sara Beckstrom 7-6(7-5), 6-4.
Kingston won three of the four doubles matches.
The Wolves' only doubles winner was the No. 2 team of Hillary Smith and Anna Prorok, who defeated Kignston's Paige Heaps and Nicole Westendorp 7-5, 3-6, 6-4.
Sequim next plays at the Olympic League tournament.
Last modified: April 30. 2013 5:42PM


text:
On May 28th, Microsoft released the initial version of this bulletin, rating the severity of the vulnerability as Moderate. Subsequent to that release we have determined that the actions an attacker could take as a result of exploiting this vulnerability could include the ability to execute arbitrary code. As a result Microsoft has reissued this bulletin and changed the severity rating to Important. The original corrects the vulnerability and is not being re-released.
Originally posted: May 28, 2003
Updated: May 30, 2003
Impact of vulnerability: Allow an attacker to execute code of their choice
Maximum Severity Rating: Important
Affected Software:
View: Microsoft TechNet - Security Bulletin ID MS03-019
View: Neowin - Windows Security Patch: Flaw in ISAPI Extension


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Legal Question in Civil Litigation in Florida
I was dating a very controlling man and we recently broke up. He took me on a cruise during the relationship as well as gave me and my children expensive gifts. Most recently a hot tub. He was mad when I ended things (found out he is still married) and he asked for me to drop off his clothes and personal belongings at his work. I did. Now he is texting me demanding the hot tub back and said he will take me to small claims court for the price of the hot tub and my half of the cruise. The hot tub was purchased used, he paid cash $500. What do I do?
Asked on 6/15/13, 6:31 am
1 Answer from Attorneys
Barry Stein De Cardenas, Freixas, Stein & Zachary
Probably best to ignore him. It sounds like a gift and he wont be getting it back in Court if he decides to waste his time. I would block his email and phone and move on.
Answered on 6/15/13, 8:02 am


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It's an interesting world when online entities strike a formal claim on the land. Go Figurative, an online gallery dedicated to figurative art, will host "Real" from March 19 to 21, 2010. The show will provide an opportunity to snatch up some 130 works from 42 different artists at "reasonable prices." In keeping with the Go Figurative site, the exhibited paintings and sculptures are realist and (obviously) figurative.
Hosted by Heatherley's School of Fine Art, 75 Lots Road, London Sw10.
The Catalog is worth a gander, some highlights after the jump.


text:
"The Pub Sofas are terrific. Even better than the pictures and descriptions. However, we did have some confusion and issues about the delivery. Wrong phone number & work order did not reflect "White Glove" delivery. Everything was cleared up but it was very time consuming."
Home Theater Seating
- Media Room
- Movie, VIP & Cinemas
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My avatar is the third quilt I ever made, and the first full sized one. I made it for my grandfather's 90th birthday. The photos are of family, and the top row shows him and Grandma, their wedding photo and their 50th anniversary photo. The lattice blocks are autograph blocks that family and friends signed. Grandpa was from Ohio, hence the Ohio star with the heart in the middle. The photos were done by a special process that guaranteed they would last for at least 50 washings, and since this quilt has been washed at least 10 times, they were worth the price.
The little pillow is one I made for Grandpa after Gram died. He kept it with him always.
Virgil & Clara Engagement photos, then Wedding Photo, then 50th Anniversary Photo


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The Georgetown University law student labeled a "slut" by conservative radio host Rush Limbaugh says his apology changes nothing and that Americans have to decide whether to support companies that continue to advertise on his program.
Sandra Fluke told ABC's "The View" on Monday that she hasn't heard from Limbaugh since he issued a written apology late Saturday. But she says his comments about her testimony before members of Congress have been so personal, she'd rather not have him call. Fluke testified to Democratic members of Congress in support of a requirement that health care companies provide coverage for contraception.
Seven companies have now pulled advertising from Limbaugh's three-hour radio show. Fluke says his comments were an "attempt to silence me" and an attack on women's health.


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London, July 31 (IANS) Zimbabwe has sought from the US extradition of the dentist who killed Cecil the lion, the most famous creature in the country's national park.
Zimbabwe's Environment Minister Oppah Muchinguri said at a press conference that Walter Palmer, 55, was a "foreign poacher" and that Zimbabwe's prosecutor general had already started the process to have him extradited, BBC reported on Friday.
A bilateral extradition treaty between the US and Zimbabwe has been in effect since April 2000 in cases where an individual is charged with what would be a criminal offence in both countries.
Palmer, a keen big game hunter who is said to have paid around $50,000 for a chance to kill Cecil, will be facing trial for financing the illegal hunt if he is extradited to Zimbabwe.
Cecil was a male African lion who lived in the Hwange National Park in Zimbabwe. The animal was shot on July 1 in the park.
The big lion – a major attraction at the park – was recognisable because of his large size and distinctive black mane and who seemingly enjoyed human company. He was being studied and tracked by Oxford University as part of a larger study.
In further infringement of the park rules, the hunters then removed his collar.
During the hunt, it was alleged that Cecil was lured at night about half a mile out of the national park using bait, and then shot with a bow and arrow. The next day he was found wounded by the hunters and killed, before being beheaded and skinned.
Hunting is legal in Zimbabwe and other neighbouring countries, but only with permits granted by the Zimbabwe Parks and Wildlife Management Authority.
Only animals that have been put on an annual hunting quota by the Zimbabwe Parks and Wildlife Management can be killed within a private hunting concession and government-controlled safari areas — but never in a national park.
Cecil's death sparked outrage among animal conservationists across the world and prompted responses from top politicians.


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Does it work with Airplay?
Well, does it?
- Asked by Joel C S from Viola
Marshall Stanmore Bluetooth Speaker
Product No Longer Available
4 Answers from the Community
No. Bluetooth is the only wireless standard supported by this speaker. Pair it with an airport express if you would like AirPlay capability. I would recommend an optical cable between the two.
- Answered by Kyle B from Franklin
Best Answer:
It will indeed work with your airplay network provided that you: a) have an airport express or extreme to plug into, and b) you use the 1/8" jack to hardware the Marshall speaker into the airport express so that you receive the signal via your airport network setup. The Marshall speaker could also be used with the Bluetooth capabilities it has, however you would then eliminate the airplay capabilities as most Bluetooth capable electronics are only able to source signal to one type of connection at a time.
- Answered by David C from Osprey
No. You can use bluetooth (wireless), RCA, optical (TOSLINK) or 3.5mm (1/8in) audio cable.
- Answered by Kutter C from Durham
Yes it works with air play. Works best with toslink optical audio cable which comes with 3.5mm adapter. Then use airplay, works flawlessly, sounds great, can control volume through iPad ( no remote)
- Answered by Adam J from Fiskdale


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Pattern writing using nouns and adjectives
In this lesson plan, students will think and write creatively using nouns and adjectives. They will experience all phases of the writing process.
A lesson plan for grades 3 and 6 English Language Arts
Learning outcomes
Students will:
- demonstrate a well-developed knowledge of adjectives and nouns.
- use adjectives and nouns in the development of poetry.
- think and write creatively.
Teacher planning
Time required for lesson
1 hour
- The poem "Beans, Beans, Beans" from Hooray for Chocolate by Lucia and James Hymes
- The outlined pattern:
noun,noun,noun,_______________ noun,_______________ noun,____________ _____________ _____________ ____________ noun,____________ _____________ _____________ ____________ noun- Those are just a few.____________ noun,____________ noun,____________ _____________ _____________ ____________ noun,____________ _____________ _____________ ____________ noun,____________ noun too.____________ noun,____________ noun,Don't forget _____________ ____________ noun.Last of all, best of all.I like _____________ ____________ noun.
Technology resources
word processing software
This lesson needs to be presented to students who have an understanding of nouns and adjectives. The poem "Beans, Beans, Beans," by Lucia and James Hymes in Hooray for Chocolate is read orally to the students:
- The outlined pattern for writing the poem should be done through oral composition with whole class participation or the following poems completed by students of Eileen Hoernlein may be shared with the students:
- "Pickles, Pickles, Pickles. Fat pickles, Skinny pickles, Little, sour, juicy pickles- Those are just a few. Dill pickles, Hamburger pickles, Sandwich and lunch pickles, All-by-themselves pickles Whopper pickles too. Salad pickles, Tuna fish pickles, Don't forget spiced pickles. Last of all, best of all, I like sliced pickles." by Rodney Keys
- "Bugs, Bugs, Bugs. Bad bugs, Spider bugs, Daddy long legs bugs, Dead, smushed up bugs- Those are just a few. Cockroach bugs, Green bugs, Brown, yellow, black bugs, Lightening bugs too. Don't forget ant bugs. Last of all, best of all, I like ladybugs!" by Michael Robinson.
- The students will pick one noun and describe it using the outlined pattern provided.
- The students should be allowed to use words other than adjectives if the words communicate a clearer message.
- The students will use the phases of the writing process from pre-writing to publishing using a word processor.
The completed poems will be the verification of the students' progress. The poems will be assessed using the outlined pattern given for the poem. The phases of the writing process from pre-writing to publishing will be assessed.
Supplemental information
The lesson was adapted from Communication in Action: Teaching the Language Arts (Boston, MA: Houghton Mifflin Company, 1986), 347-349.
- Common Core State Standards
- English Language Arts (2010)
- Grade 3
- 3.L.1 Demonstrate command of the conventions of standard English grammar and usage when writing or speaking. 3.L.1.1 Explain the function of nouns, pronouns, verbs, adjectives, and adverbs in general and their functions in particular sentences. 3.L.1.2 Form...
- Grade 3
- English Language Arts (2010)
North Carolina curriculum alignment
English Language Arts (2004)
Grade 6
- Goal 1: The learner will use language to express individual perspectives drawn from personal or related experience.
- Objective 1.02: Explore expressive materials that are read, heard, and viewed by:
- monitoring comprehension for understanding of what is read, heard, and/or viewed.
- analyzing the characteristics of expressive works.
- determining the effect of literary devices and/or strategies on the reader/viewer/listener.
- making connections between works, self and related topics.
- comparing and/or contrasting information.
- drawing inferences and/or conclusions.
- determining the main idea and/or significance of events.
- generating a learning log or journal.
- creating an artistic interpretation that connects self to the work.
- discussing books/media formally and informally.
- Objective 1.02: Explore expressive materials that are read, heard, and viewed by:
- Goal 6: The learner will apply conventions of grammar and language usage.
- Objective 6.01: Demonstrate an understanding of conventional written and spoken expression by:
- using a variety of sentence types correctly, punctuating them properly, and avoiding fragments and run-ons.
- using appropriate subject-verb agreement and verb tense that are appropriate for the meaning of the sentence.
- demonstrating the different roles of the different parts of speech in sentence construction.
- using pronouns correctly, including clear antecedents and correct case.
- using phrases and clauses correctly (e.g., prepositional phrases, appositives, dependent and independent clauses).
- determining the meaning of unfamiliar vocabulary words by using context clues, a dictionary, a glossary, a thesaurus, and/or structural analysis (roots, prefixes, suffices) of words.
- extending vocabulary knowledge by learning and using new words.
- exploring the role and use of dialects and of standard English to appreciate appropriate usage in different contexts.
- developing an awareness of language conventions and usage during oral presentations.
- Objective 6.01: Demonstrate an understanding of conventional written and spoken expression by:


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The story of the Exodus and the Passover Haggadah, which will be read this weekend, carry perennial relevance and significance. "In every generation a person is obligated to see himself as though he actually left Egypt," the Haggadah tells us. Then we sing the well-known verse Vehi Sheamda: "This is what has stood by our fathers and us. For not just one alone has risen against us to destroy us, but in every generation they rise against us to destroy us; and the Holy One, blessed be He, saves us from their hand." What has stood by our fathers was the promise of the Almighty to save and redeem the Israelites, as the preceding verse of the Haggadah states.
In their book The Haggada of the Sages, Professors Shmuel and Zeev Safrai comment on the verse Vehi Sheamda, explaining that "the promise of the Almighty is a continuing reality that is passed on from generation to generation. It ties in with other Haggadah verses that underscore the religious and contemporary significance of the Exodus."
Yet ever since the Holocaust, to my mind, this verse has become problematic — especially for the believers. It touches a raw nerve pertaining to faith, contending with a most profound and tormenting issue for which no one has the answer. Where was the Almighty that was to "save us from their hands" during the Holocaust? Where was He during the greatest attempt to bring about the extinction of the Jewish people — or in fact that of any people?
Delving into the quintessential issues of our existence, this acute dilemma has shaken the belief of many people in the aftermath of the Holocaust, driving them away from religion. Others who remained steadfast in their faith continued to be tormented for the rest of their lives. God works in mysterious ways and we must not do any reckoning in His stead — that was the explanation provided by religious leaders. In effect, they said that "this is too big for us." (But that did not stop them from explaining to us God's reckoning in connection with other matters, when lesser disasters struck.) Still, that explanation was unsatisfying and could not heal anyone's soul. I know some people who would skip Vehi Sheamda. They may have muttered something, but saw no reason to sing the verse.
Since the very beginning of its founding, the State of Israel has had to combat forces that have sought to destroy it. Like David's victory over Goliath, the Israeli victory in the War of Independence seemed a miracle to some people. They perceived it to be a kind of a heavenly tikkun — mending fences if you will — that compensated for the disappearance of God's saving hand just a few years earlier.
Once the State of Israel became stronger, we wanted to believe that the danger of "every generation rises against us to destroy us" had faded. And if that was the case, Vehi Sheamda had also become obsolete. Threats to throw us to the sea were still being made, but they were primarily blustery bravado. Indeed, there were times of concern and apprehension, such as on the eve of the Six Day War or the Yom Kippur War, yet Israel emerged triumphant. The belief that what had befallen the Jewish people during the Holocaust would not happen again became an axiom, as did the assumption that if the State of Israel had existed at that time, the Holocaust would not have happened. As a premise, that is quite dubious.
And now, on the eve of Israel's sixty-fourth year of independence, we are facing yet another attempt at our extinction — this time by villainous Iranian despots. Once again, this part of Vehi Sheamda has become relevant. Although there is no more room to make any comparisons with the Holocaust, we are once again experiencing concern and apprehension, perhaps even fear, of whether we would be able to rise up to the test. Unfortunately, part of this intimidation hails from our own leaders who talk too much. Instead of letting them alarm us, both the IDF and the home front should be making appropriate preparations. It seems that the preparedness of the home front is deficient, already portending the establishment of a commission of inquiry.
Those who still believe in divine help, despite the trauma of the Holocaust and the religious questions it raises — and all the more so those who utterly do not believe in such a thing — should know that such heavenly help would not be forthcoming without our own more earthly self-rescue.


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"...as lovely as a tree"
"...as lovely as a tree"
The technique of Photoshop collage, as a legitimate art form, has taken a hit over recent years. It's been denigrated as a gimmick that anyone with minimal computer skills and no drawing chops can do. Illustrators complained that designers were doing Photoshop collages themselves, rather than commissioning an illustration. I think the bad rap is unjustified. You still have to deal with composition, color, balance, contrast, etc., while still drawing the viewer's interest. There are plenty of other recognized art media, like photography, that don't require drawing skills, although a strong drawing foundation will always inform and elevate any artistic discipline you choose.
I've always been intrigued by its possibilities, mainly because I love collage. My approach to it involves creating some of the imagery by hand through drawing or painting, and integrating that with photographic elements. Hopefully that gives the work a softer, less technical feel.
Once I had all the elements I needed to work with, these two illustrations took about an hour each on the computer. They are my first tentative exploration of digital collage. I don't foresee this replacing my totally hand-done work, but it was an interesting experiment that I may pursue on the side. These images were done for a folio that was created to accompany a show at Gallery Della-Piana in Wenham, MA on the theme of trees. I also had a painting in the show.
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Fall of Fornost
This article or section needs more/new/more-detailed sources to conform to a higher standard and to provide proof for claims made.
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|Fall of Fornost
|Conflict: War of Angmar
|Date: T.A. 1974
|Place: Fornost Erain
|Outcome: Decisive Angmar victory
Men, Elves, Hobbits
Est. 10-20,000 Men of Arnor, several thousand Elves from, Lindon, Rivendell, and Lothlórien, and 500 Hobbit archers[source?]
The Fall of Fornost was an event that resulted from the defeat of the forces of Arnor, and the taking of their capital city of Fornost by the Witch-king of Angmar and his armies.
By the year T.A. 1974 things were looking very grim for the Dúnedain of Arnor. The Great Plague three hundred years previously had killed most of the people of Cardolan which later fell to the Witch-king. And even with the aid of Elves and Hobbits most of the now diminished and totally demoralized people of Arnor were now waiting to make their final stand at Fornost, capital city of Arthedain.
Opposing Forces
For the forces of Eriador exact numbers are not known, but it can be estimated that at the most ten thousand Men of Arnor were present at the city. Along with likely several thousand Elves of Lindon, Rivendell, Lothlórien, and five hundred Hobbits archers from the Shire.
For the forces of Angmar it can be estimated that there was at least forty thousand or at the most sixty thousand Orcs, Trolls, Evil Men, Wolves, and all other manner of evil creatures.
The Battle
Not much is known of the battle, and it is simply stated that the Witch-king overran Fornost and the King was driven into the North Downs.
It is likely that the Witch-king issued from Angmar and swept aside Arthedain's defences along the Great North South Road and assaulted the city from the south and east. Considering that Arvedui was able to flee into the North Downs and hold out there for a while, and the fact that refugees and fleeing soldiers were able to escape, it is likely that Arthedain repulsed Angmar along the North Downs.[source?]
It is likely that several companies of Orcs were sent down to Bree-Land and possibly the Shire, but considering that those lands were largely unscathed by the war, we can assume that any assaults on them regions were repulsed and the majority of the war was fought in Fornost and the Downs.[source?]
The fighting outside the gates of Fornost was fierce, as the might of Arthedain held the walls against the seemingly endless hordes of the servants of Angmar. Whether by brute strength or dark magic, the gates were breached and the Orcs and Evil Men poured in. The street to street fighting would've been a nightmare for the defenders and attackers, and it is probable that during this stage, Arvedui ordered the military evacuation of the city to the north and west. It is possible that Arvedui was trapped in the city and was forced to fight his way out, or he may have departed the city moments before its fall. Thousands of refugees would've been fleeing from the city at this point and it is likely that the remnants of Arthedain's Army protected these bands of refugees as they made their way to the Lhun.[source?]
Regardless, the North Downs possibly held out for several more weeks with the arrival of Arvedui and his personal guard, giving the King hope that if Gondor arrived soon, Fornost may yet be recovered. However, with most of his enemies dead or fleeing west, the Witch-king was able to concentrate the remainder of his forces on the Downs, and Arvedui was forced to flee, giving the Witch-king supremacy of most of Arthedain.[source?]
With Arthedain in his hands, the Witch-king had conquered all of Arnor. Arvedui perished in the Icebay of Forochel when a ship sent to rescue him sank in a storm. The Witch-king took the throne in the king's palace, until a year later in T.A. 1975 when the forces of Eriador had regrouped with newly arrived reinforcements from Gondor. In the Battle of Fornost his army was destroyed, and he himself fled east over the Misty Mountains.
Fornost was not resettled after the war. It became a deserted place, feared by the men of Bree, who called it Deadman's Dike. Not until a thousand years later after the War of the Ring under King Elessar was it refounded.


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Originally released back in 1991, the VI Infrared has become one of the most sought after Jordans of all time. For collectors and Jordan fans alike, it holds grail status. It was retroed in the Summer of 2000, and will make a return next January. mash rating: 9/10
More after the jump.
pic and info: Gradient Magazine


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jbrock2810 4 years ago
We really need the option to keep track of players salary's and contract years on each team. We have an auction draft but cant put the player's salary on the website. This make me run an excel sheet that i have to send out a least once a week. This would save a lot of time for me each week. Last year I had to change players salary's when they were dropped, added, traded, and pickup each week. We have blind bidding which should stay with the player in case of a trade or drop not just taken off the overall budget. With the contracts we have an option to have keepers but would like to see where we can put the number of years we are keeping them for. Thank you for your time and hard work to keep the site running smoothly.
FleaMod Admin 4 years ago
Agree that a salary sheet is important for dynasty leagues and we hope to have it implemented in the near future. It's on our agenda.
Deschain19 4 years ago


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The stories are great. And this float too. Thanks.
Splendid! King Arthur has always bee a favorite of mine!
Hi Monica and Cathy, it's a nice one and the horse is beautiful. Thanks
Wow! Very nice float. I love the horse!
I love King Arthur! Thanks Ank, this was wonderful fun!...Monica
Often it takes months to build a float.
Beautiful, a lot of work went into this one, Thanks Ank.
You're so welcome Jan
The horse and castle are both gorgeous! Thanks, Ank!
Thanks friends, it's a beauty indeed. I agree, the horse looks very real.
Gorgeous thanks Sis
It's really formidable!! Thanks so very much Ank!!
Another beautiful float and the horse looks so real. Thanks, Ank.
I just can say, girls I agree. Thanks.
It is beautiful, and I love the horse!
Another lovely float Ank. The colours are beautiful. Thank you:-))
Another smile, this is such a BEAUTY, I thank my dear Ank :-))


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Comic Book Art by Eric Guzman
Comic-Con began today in San Diego, and to celebrate we wanted to feature the comic book art of Eric Guzman. We could honestly showcase a different artist each day for a year and come no where close to running out, but that doesn't discount the skill and style that each artist brings to their work. Marvel and DC Comics are the big boys on the scene that most have heard of, so we wanted to highlight an artist who has covered both of their characters fairly equally. Check out Eric's take on The Avengers and The Justice League below:
Eric's work originally caught our attention when WalYou put together a gallery of his DC and Marvel Amalgam collection. In it, Guzman combines together villains from each comic universe to make new characters. We haven't included any of those images here, but instead decided to focus on some of his more traditional offerings. Below are 20 of our favorites, but be sure to check out his Deviant Art profile for all of his creations :


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On this day fifty-four years ago, John F Kennedy, the 35th President of the United States of America, would leave the White House and never return.
You are here
After $1m fire El Pecos to reopen in three months
After suffering an estimated loss of just over $1 million, owner of the El Pecos chain of restaurants is promising that his Ariapita Avenue, Port-of-Spain, branch will reopen in three months. Speaking with the media outside his partially burnt business place yesterday, owner Richard Camacho said there had been $750,000 worth of damage to equipment and the structure and an estimated $350,000 in stocks were burnt. He added: "Thanks to the swift response of the fire officials, the damage is mostly superficial.
"They did a good job, I must say. They responded quickly, no one got hurt and they were able to contain the blaze and from what they told me, prevent damage to the structure. "We will need a new roof, change the plastering on walls, purchase new equipment and re-do electricals but beyond that not much. I expect to be open again within three months." Camacho said in the interim the employees would be placed in his three other branches at Diego Martin, Maraval and Barataria.
Fire Sub-Station Officer (FSSO) Sylvester Reyes said the blaze started around 1.50 pm yesterday and was confined to the kitchen area. The cause of the fire was not known, he said, but the T&T Guardian overheard an employee telling another employee it was a grease fire. One of the 24 members of staff, Keith Gordon, told the media he had just taken up his shift at the restaurant when around 1.30 pm he saw smoke coming from the chimney.
Gordon, a cook, added that within three to four minutes the restaurant had been evacuated and fire officers from the Wrightson Road Fire Station arrived, saving most of the building. Divisional Fire Officer Cleybert Grayson said officers took two minutes to respond to the fire and when they arrived workers had already began to fight the fire, using extinguishers. As a result of the fire, traffic heading west was diverted to Mucurapo Road and traffic headed east to Wrightson Road. Investigations are continuing.


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ROD Davies' flying feet were in a Test backline 18 months ago but the winger is now eager to force his way out of a five-way crush to claim a Queensland starting spot.
Such is the depth on the wing for the Reds that Digby Ioane, Dom Shipperley, Luke Morahan and Davies are all Wallabies while Samoan-bred powerhouse Chris F'Sautia is a teenager waiting to build on his three Super Rugby appearances last year.
Davies is on the bench behind Ioane and Morahan for Saturday night's Super Rugby trial against the Auckland Blues in Toowoomba.
"This is my fifth season at the Reds and it's the most competitive I've seen for wing spots," Davies said.
"Chris is as good a young kid as I've seen talent-wise so it's not just the more experienced guys going for the spots.
"I'm the fittest I've been for a while so I'm keen to get on in Toowoomba, rediscover some form and hopefully impress.
"You've got to be 'on' at every training session. That's the environment at the Reds.
"The competition is fierce but I'm still confident in my ability."
Davies hit the heights against the Blues in the 2011 Super Rugby semi-final with a breathtaking hat-trick of tries which showcased his pace, his timing and confidence.
He earned a Test jersey against Samoa with that display but 2012 was a tame follow-up that was cut short by visits to the surgeon for a shoulder reconstruction and a knee operation.
The popular Davies is looking well-conditioned and is showing the benefits of a full pre-season, two factors that will hopefully feed into "Rocket" making a successful relaunch against the Blues.
Senior Blues Ali Williams, Piri Weepu and Rene Ranger will not make the trip for the trial but it will be an energised Blues touring party eager to impress new coach John Kirwan.
Young lock Blake Enever, 21, gets a starting chance to show his improvement while Albert Anae's growth as a prop will also be measured against a top opponent.
Livewire halfback Nick Frisby will make his return from a shoulder reconstruction off the bench.
Ben Lucas and Frisby have to find a dynamic and resourceful 80 minutes to fill as much of the void as possible while Wallaby Will Genia spends the opening month of the season on the sidelines building more strength into his knee.
Originally published as Five-way crush for Reds wing spot


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CARSON, Calif. – The LA Galaxy are in the midst of yet another bye week that the team must savor because soon the grind of the MLS Regular Season will truly begin.
This weekend will serve as the Galaxy's third off week of the 2014 campaign which was set in place—in large part—due to LA's participation in the CONCACAF Champions League. Following this weekend's bye, the Galaxy will not experience another weekend off until MLS action is halted due to the 2014 FIFA World Cup. Once MLS action resumes, the Galaxy's campaign will truly begin as the team will play at least once each week until the season's end.
Although the schedule will pick up at a rapid pace after this weekend's respite, the Galaxy actually believe that 2014 will bring a smaller work-load than past seasons.
"I think we actually see this as a lighter year, because we're not in the [next edition of] the Champions League. We're not playing extra games [down the stretch], so for us to play every week is fine. I personally would prefer to be playing regularly," Landon Donovan told reporters earlier this week. "At different points of the season, which you're nicked up, you accept a bye week happily, but to have them so early is a little unnecessary, I guess. But it is what it is. I would think preferably we'd have them more late in the season, but that's the way it goes."
But midfielder Stefan Ishizaki thinks that the bye week may turn out to be advantageous for LA ahead of their match against the Colorado Rapids next Saturday.
"Playing on hard turf in Vancouver made guys a bit sore and I think that it's a good thing that we got that out," said Ishizaki. "We can train hard during the bye week and then ease up our preparations as we get closer to the Colorado match."


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by Christopher Freeburn | July 23, 2013 10:23 am
[1]British Prime Minster David Cameron has announced new restrictions on online pornography in England and Wales[2].
Under an agreement with the four largest Internet service providers (ISPs) in the U.K., new and existing customers will have filters applied to their Internet connections to block pornography. The filters will only be removed if customers request it, the BBC notes.
The prime minister also signaled that search engines will be expected to begin banning search terms that could generate links relating to child pornography. If search engines don't implement the restrictions voluntarily, the British government could impose the bans by law.
Any online pornography that depicts rape will be also banned.
Cameron called online pornography corrosive to the innocence of children. He noted Internet companies had a responsibility to do more to police online pornography.
The new proposals would bring England and Scotland closer to Scotland's more restrictive pornography laws.
Source URL: https://investorplace.com/2013/07/all-porn-will-be-blocked-in-uk-unless/
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