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【关键词】 calcium
Vascular Biology Center and Departments of Surgery and Physiology, Medical College of Georgia, Augusta, Georgia
ABSTRACT
The signaling pathways of endothelin (ET)-1-mediated vasoconstriction in the renal circulation have not been elucidated but appear to be distinct between ETA and ETB receptors. The purpose of this study was to determine the role of L-type Ca2+ channels in the vasoconstrictor response to ET-1 and the ETB receptor agonist sarafotoxin 6c (S6c) in the rat kidney. Renal blood flow (RBF) was measured with an ultrasonic flow probe in anesthetized rats, and a microcatheter was inserted into the renal artery for drug infusion. All rats were given vehicle (0.9% NaCl) or three successive bolus injections (1, 10, and 100 pmol) of ET-1 or S6c at 30-min intervals (n = 6 in each group). ET-1 and S6c produced dose-dependent decreases in RBF. The Ca2+ channel blocker nifedipine (1.5 μg) significantly attenuated the RBF response only at the highest doses of ET-1 and S6c. In the isolated blood-perfused juxtamedullary nephron preparation, Ca2+ channel blockade with diltiazem had a very small inhibitory effect on ET-1-induced decreases in afferent arteriolar diameter only at the lowest concentrations of ET-1. In vascular smooth muscle cells isolated from preglomerular vessels, ET-1 produced a typical biphasic Ca2+ response, whereas S6c had no effect on cytosolic Ca2+. Furthermore, Ca2+ channel blockade (diltiazem or Ni2+) had no effect on the peak or sustained increase in cytosolic Ca2+ produced by ET-1. These results support the hypothesis that L-type Ca2+ channels play only a minor role in the constrictor responses to ET-1 in the renal microcirculation.
renal blood flow; endothelin receptors; afferent arteriole; calcium channel blockers
THE RENAL CIRCULATION CONTAINS endothelin (ET) type A and B (ETA and ETB) receptors, which can mediate vasoconstriction. Although it is generally accepted that ETA receptor activation stimulates mobilization of Ca2+ from extracellular and intracellular sources (28), there is very little information about ETB receptor subtype signaling pathways in vascular smooth muscle. Both receptor subtypes stimulate G protein-dependent pathways. Although a number of mediators have been identified for their involvement in the Ca2+ mobilization response to ET-1, the precise function and coordination of these messengers is not fully resolved. In the vasculature, the assumption that the ETA receptor is the predominant subtype underlies a number of studies investigating the nature of the vasoconstrictor actions of ET-1 (28). However, ETA and ETB receptors contribute to the renal vasconstrictor actions of ET-1, and the extent to which the signal transduction mechanisms may be receptor subtype specific is unknown.
Cao and Banks (1) reported that Ca2+ channel antagonists prevent the systemic hemodynamic actions of ET-1 but have no effect on ET-1-induced renal vasoconstriction. Their results are consistent with the hypothesis that ETB receptor-mediated vasoconstriction does not depend on extracellular Ca2+, because they used high doses of ET-1, which primarily activate ETB receptors in the kidney. On the other hand, Loutzenhiser et al. (22) reported that Ca2+ channel blockade completely reversed ET-1-induced vasoconstriction in the isolated perfused hydronephrotic kidney. In isolated rabbit arterioles, Edwards et al. (5) observed that ET-1-induced contraction of afferent, but not efferent, arterioles was sensitive to Ca2+ channel antagonists. In isolated smooth muscle cells obtained from microdissected interlobar and arcuate arteries of the rat, Gordienko et al. (10) observed that ET-1 increased cytosolic Ca2+ via intracellular and extracellular sources. They also provided evidence that ET-1 activates T- and L-type channels as a means of stimulating extracellular Ca2+ influx.
Touyz et al. (35) reported that ETB receptor-mediated contraction in small mesenteric arteries is mediated by increases in intracellular Ca2+. However, ETB receptor signaling systems may not be ubiquitous among cell types. As an example, Wu-Wong et al. (36, 38) reported that ETB receptors do not stimulate Ca2+ mobilization in astrocytoma cells. Although a large number of ETB receptors are present in the kidney, primarily in tubular epithelium, there is only limited information regarding the Ca2+ signaling pathways during ETB receptor-mediated renal vasoconstriction. Our laboratory has reported that, in contrast to ET-1, ET-3, the natural ligand for the ETB receptor, has no effect on intracellular Ca2+ in isolated preglomerular vascular smooth muscle cells (32). In contrast, using a similar preparation, Fellner and Arendshorst (8) recently reported that the ETB receptor agonist IRL-1620 increases cytosolic Ca2+ in a fashion nearly identical to ET-1.
The purpose of the present study was to determine the role of L-type Ca2+ channels in the renal hemodynamic response to ET-1 and specific ETB receptor activation by in vivo and in vitro approaches. The effect of Ca2+ channel blockade on renal vasoconstrictor responses to direct intra-arterial administration of ET-1 and the ETB receptor agonist sarafotoxin 6c (S6c) were determined in anesthetized rats. In addition, the role of L-type Ca2+ channels in response to ET-1 was determined in the isolated blood-perfused juxtamedullary nephron preparation and in isolated preglomerular microvascular smooth muscle cells.
METHODS
The Institutional Animal Care and Use Committee at the Medical College of Georgia approved these studies.
In vivo hemodynamic experiments. Male Sprague-Dawley rats (225250 g body wt; Harlan Laboratories, Indianapolis, IN) were anesthetized with thiobutabarbital (Inactin, 50 mg/kg) and placed on a servo-controlled heating table to maintain a constant temperature of 37°C. A tracheotomy was performed to facilitate unobstructed breathing. The left jugular vein was cannulated for infusion of BSA (6.2%) in saline (0.9% NaCl) and the ETB receptor antagonist A-192621, while the right femoral artery was cannulated to monitor mean arterial pressure (MAP) with a MacLab data acquisition system. After cannulation of the right femoral artery, a microcatheter was advanced 3 mm into the left renal artery for intrarenal infusion of nifedipine, ET-1, or S6c. An ultrasonic flow probe (Transonic Systems) was placed on the left renal artery to measure renal blood flow (RBF). After a 60-min equilibration period, rats were given a bolus of 0.9% NaCl or A-192621 (30 mg/kg) via the jugular vein. Three 30-min periods followed in which ET-1 or S6c (1, 10, and 100 pmol) was given intra-arterially in the presence or absence of nifedipine (1.5 μg). This dose and method of nifedipine administration have been previously shown to block 90% of the maximum decrease in RBF produced by the voltage-gated Ca2+ channel activator BAY K 8644 (30).
In vitro blood-perfused juxtamedullary nephron experiments. Experiments were conducted, in vitro, using the blood-perfused juxtamedullary nephron technique, as previously described (3, 13, 14). Two male Sprague-Dawley rats (350400 g body wt) were used for each experiment. Rats were anesthetized with pentobarbital sodium (40 mg/kg ip), and perfusate blood was collected and prepared as previously described (13, 14). Briefly, blood was collected from the nephrectomized blood donor rat into a heparinized (500 U) syringe. The plasma and erythrocyte fractions were separated, and the leukocyte fraction was discarded. Washed erythrocytes were combined with the filtered (0.2-μm exclusion) plasma to yield a hematocrit of 33%. The reconstituted blood was filtered through a 5-μm nylon mesh and saved for later use.
The right renal artery of the kidney donor was cannulated and perfused with a Tyrode buffer solution containing 5.2% BSA and a complement of L-amino acids (18). The perfused kidney was removed and sectioned along the longitudinal axis, with care taken to leave the papilla intact on the dorsal two-thirds of the kidney (3). The papilla was reflected and the pelvic mucosa was removed to expose the main arterial branches, renal tubules, glomeruli, and related microvasculature of juxtamedullary nephrons. The terminal ends of the large arteries were ligated to restore intravascular pressure to the perfused cortical and papillary tissue.
After completion of the microdissection procedures, the cell-free perfusate was replaced with the reconstituted blood. The blood perfusate was stirred continuously in a closed reservoir during oxygenation with 95% O2-5% CO2. Perfusion pressure was set at 110 mmHg and monitored continuously. The inner cortical surface of the kidney was superfused with warmed (37°C) Tyrode buffer containing 1% BSA.
The perfusion chamber, containing the prepared kidney, was attached to the stage of a Nikon Optiphot-2UD microscope equipped with a Zeiss water-immersion objective (x40). The tissue was transilluminated, and the kidney surface was visualized using a Newvicon camera, an image processor, and a video monitor while being simultaneously recorded on videotape for later analysis. Vascular inside diameters were measured using an image shearing monitor.
Experimental protocols consisted of consecutive 5-min treatment periods. ET was administered in the superfusate solution. After an initial control period, the tissue was exposed to increasing concentrations of ET-1 (1 pM-10 nM). Two groups of arterioles were examined. Untreated kidneys were used to establish the control response to ET. A second set of kidneys were treated with 10 μM diltiazem in the superfusate to determine the effect of Ca2+ channel blockade on ET-mediated responses over the same concentration range. Arteriolar diameter was monitored continuously throughout the protocol, while measurements of vascular inside diameter were obtained at 12-s intervals. Steady-state diameter was calculated from the average diameter during the final 2 min of each 5-min treatment period.
Isolation of microvascular smooth muscle cells. Freshly isolated preglomerular smooth muscle cells were prepared as previously described (15, 16, 19). Male Sprague-Dawley rats (250375 g body wt; Charles River Laboratories) were anesthetized with pentobarbital sodium (40 mg/kg ip), and the abdominal cavity was exposed to allow perfusion of the kidneys. An ice-cold, low-Ca2+ (0.1 mM Ca2+) physiological salt solution was perfused through the kidneys. Kidneys were removed and decapsulated, and the renal medullary tissue was separated. The renal cortex was pressed through a sieve (180 μm mesh) and washed with ice-cold physiological salt solution. The remaining tissue was transferred to an enzyme solution containing 0.075% collagenase, 0.02% dithiothreitol, 0.2% soybean trypsin inhibitor, and 0.1% BSA in physiological salt solution. After 30 min of incubation at 37°C, the vascular tissue was removed and transferred to a 70-μm nylon mesh, where it was rinsed with ice-cold physiological salt solution, and segments of interlobular artery with attached afferent arterioles were collected by microdissection. Collected tissue was placed in a solution of 0.075% papain and 0.02% dithiothreitol in physiological salt solution for 15 min at 37°C before being centrifuged at 2,000 g for 50 s. The tissue pellet was transferred to an enzyme solution containing 0.3% collagenase and 0.2% soybean trypsin inhibitor in physiological salt solution at 37°C. After a 15-min incubation period, the mixture was gently triturated and then centrifuged at 500 g for 5 min. The supernatant was discarded, and the dispersed cells were gently resuspended in 1.0 ml of DMEM supplemented with 20% fetal calf serum, 100 U/ml penicillin, and 200 μg/ml streptomycin. Cell suspensions were stored on ice until used.
Fluorescence measurements in single microvascular smooth muscle cells. Experiments were performed using standard microscope-based fluorescence spectrophotometry techniques as previously described (15, 16). The excitation wavelengths were set at 340 and 380 nm, and the emitted light was collected at 510 ± 20 nm. Fluorescence intensity was collected (5 data points/s) and analyzed with the aid of Photon Technology International software. Fluorescence data were calibrated in vitro according to the method used by Grynkiewicz et al. (12).
Intracellular Ca2+ concentration ([Ca2+]i) in single preglomerular smooth muscle cells was measured as described previously (15, 16, 19). Suspensions of microvascular smooth muscle cells were loaded with 10 μM fura 2-AM (Molecular Probes, Eugene, OR), and an aliquot of cell suspension was transferred to the perfusion chamber (Warner Instrument, Hamden, CT) and mounted on the stage of a Nikon Diaphot inverted microscope. The cells were superfused at 37°C with a control physiological salt solution of the following composition: 125 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 20 mM HEPES, 1.8 mM CaCl2, and 0.1 g/l BSA. For each experiment, fluorescence data were collected from a single cell after background subtraction. A new coverslip was used for each experiment.
The effect of ET on [Ca2+]i was determined by exposure of single cells to normal-Ca2+ physiological salt solutions containing 100 nM ET-1, 1 μM S6c, or another ETB receptor agonist, IRL-1620, at 10 μM. ET-1 was tested in the presence and absence of 10 μM diltiazem. Diltiazem, instead of nifedipine, was used to avoid nifedipine-related autofluorescence at the excitation wavelengths for fura 2. Previous studies establish that 10 μM diltiazem effectively inhibits the Ca2+ influx response evoked by exposure of preglomerular smooth muscle cells to 90 mM KCl (16).
Cells were also superfused with a nominally Ca2+-free solution (Ca2+-free physiological salt solution), which resembled the physiological salt solutions, except no CaCl2 was added. No EGTA was added to the solution. Previous studies have shown that exposure of preglomerular microvascular smooth muscle cells to 90 mM KCl in a similar nominally Ca2+-free solution resulted in no detectable increase in [Ca2+]i (16).
S6c, IRL-1620, and ET-1 were obtained from American Peptide, collagenase from Boehringer Ingelheim Chemicals (Petersburg, VA), and BSA from Calbiochem (La Jolla, CA). All other reagents were obtained from Sigma (St. Louis, MO). Values are means ± SE or representative traces. Within-group comparisons were assessed by ANOVA for repeated measures; differences between groups were analyzed with Fisher's protected least square difference post hoc tests. Significant differences were noted when P < 0.05.
RESULTS
In vivo hemodynamic experiments. Intrarenal injection of ET-1 produced dose-dependent decreases in RBF (Fig. 1A). Coadministration of nifedipine had no effect on the response to ET-1 at the two lower doses; yet the decrease in RBF produced by ET-1 was significantly attenuated at the highest dose by 50%. Intrarenal injection of S6c also produced a dose-dependent decrease in RBF (Fig. 1B). In contrast to ET-1, the highest dose of S6c produced a biphasic vasoconstriction typified by a rapid, pronounced vasoconstriction followed by a partial recovery to an intermediate plateau level. Previous studies demonstrated a net vasoconstrictor effect of ETB receptor agonists, despite evidence for vasodilator and vasoconstrictor actions within the renal circulation (26). Coadministration with the Ca2+ channel blocker nifedipine had no significant effect on the renal vasoconstrictor response to the lower doses of S6c. However, nifedipine significantly attenuated the decrease in RBF produced by the highest dose of S6c. To verify that S6c was producing the vasoconstriction through the ETB receptor, A-192621 was administered 15 min before injection of S6c in a separate group of rats (Fig. 1C). A-192621 alone produced a slowly developing decrease in RBF consistent with a tonic influence of ETB receptors to maintain an endothelium-dependent vasodilatory influence within the renal circulation and increased ETA receptor activation associated with reduced clearance of endogenous ET-1. S6c had no effect on RBF after A-192621 treatment.
Intrarenal ET-1 infusion had no significant effect on MAP (Fig. 2A). MAP did not significantly change after intrarenal infusion of S6c at the two lower doses (Fig. 2B). However, the higher dose produced a small, transient decrease in MAP followed by a more prolonged elevation in blood pressure. The hypotensive response was exaggerated in rats given nifedipine, although the increase in arterial pressure was unaffected. A-192621 increased MAP and prevented any change in MAP after S6c injection at all doses (Fig. 2C).
In vitro blood-perfused juxtamedullary nephron experiments. The effect of Ca2+ channel blockade on the afferent arteriolar response to ET-1 was directly assessed, in vitro, using the blood-perfused juxtamedullary nephron technique. Two groups of arterioles were studied. The baseline diameters of the control and diltiazem-treated arterioles were similar and averaged 16.8 ± 0.7 and 18.8 ± 1.1 μm, respectively. Diltiazem increased afferent diameter significantly by 42% to 26.5 ± 1.3 μm. Exposure of afferent arterioles to increasing concentrations of ET-1 (1 pM-10 nM) produced concentration-dependent vasoconstriction under control conditions (Fig. 3). Diameter decreased by 12 ± 2, 25 ± 3, 40 ± 4, 65 ± 4, and 78 ± 2%, respectively, with each successive increase in ET-1 concentration. Diltiazem treatment significantly attenuated the vasoconstrictor response evoked by ET-1 at 1 and 10 pM but had no significant effect on vasoconstrictor responses to higher concentrations.
Isolated microvascular smooth muscle cell experiments. The effect of Ca2+ channel blockade on Ca2+ signaling responses elicited by ET-1 was directly assessed using freshly isolated preglomerular smooth muscle cells. A total of 84 individual cells prepared from 18 tissue dispersions were examined. Baseline [Ca2+]i averaged 104 ± 3 nM, which is similar to values reported previously (15, 16, 19). S6c at 1 μM had no significant effect on [Ca2+]i (Fig. 4A), but ET-1 treatment at 100 nM in the same cell resulted in a marked increase in Ca2+. [Ca2+]i increased an average of 34 ± 19 nM during exposure to 1 μM S6c (n = 9 cells from 4 dispersions). In separate experiments, we examined the effect of IRL-1620 on [Ca2+]i in preglomerular smooth muscle cells. IRL-1620 at 10 μM evoked a minimal increase in [Ca2+]i, whereas 100 nM ET-1 produced a robust and significantly greater Ca2+ response (Fig. 4B). Peak increases in [Ca2+]i induced by IRL-1620 averaged 26 ± 5 nM (n = 8) under control conditions and 39 ± 4 nM (n = 25) in the presence of diltiazem.
Similarly prepared cells responded to ET-1 with a marked increase in [Ca2+]i. ET-1 at 100 nM increased [Ca2+]i from a baseline of 120 nM to a peak value of 895 nM before stabilization at a sustained level of 127 nM (Fig. 5A). Group analysis revealed that ET-1 increased [Ca2+]i from a baseline of 94 ± 5 nM to a maximum of 577 ± 70 nM (P < 0.001 vs. baseline, n = 23 cells from 9 dispersions). This response profile was unaltered by pretreatment with the Ca2+ channel blocker diltiazem (10 μM). Consistent with responses from untreated control cells and with the example trace shown in Fig. 5B, 100 nM ET-1 increased [Ca2+]i from a baseline of 104 ± 4 to a peak of 592 ± 95 nM. Similar results were obtained when more general blockade of Ca2+ channels was imposed by the introduction of 5 mM Ni2+ to the bathing medium. These responses, along with the effects of diltiazem, are summarized in Fig. 6. Neither diltiazem nor Ni2+ had an effect on the peak or sustained change in [Ca2+]i evoked by ET-1 or on baseline Ca2+ levels. However, 5 mM Ni2+ completely blocked the Ca2+ response to KCl. Under control conditions, 90 mM KCl increased Ca2+ from 109 ± 12 to 203 ± 42 nM (P < 0.05), whereas [Ca2+]i averaged 100 ± 15, 99 ± 14, and 94 ± 12 nM during the control, NiCl, and NiCl + KCl periods, respectively. We previously established the ability of 10 μM diltiazem to block the Ca2+ response to 90 mM KCl (15, 16).
DISCUSSION
The present study utilized three model systems to determine the role of L-type Ca2+ channels in the renal microvascular response to ET-1 and ETB receptor activation. In whole animal experiments, Ca2+ channel blockade inhibited the vasoconstrictor responses to the nonselective ligand ET-1 and the ETB receptor agonist S6c only at the highest dose. These findings are consistent with a relatively minor role for L-type Ca2+ channels in the response to ET receptor activation by ETA or ETB receptors. In vitro responses of blood-perfused juxtamedullary afferent arterioles were slightly attenuated by Ca2+ channel blockade only at very low concentrations of ET-1. These results are in general agreement with findings in isolated renal vascular smooth muscle cells that neither diltiazem nor Ni2+ had an effect on the changes in [Ca2+]i produced by ET-1. Selective activation of ETB receptors with S6c had no effect on [Ca2+]i. Collectively, these results clearly indicate that ET-1 and ETB receptor-dependent renal vasoconstriction only has a minor dependence on L-type Ca2+ channels in the renal circulation. It is not clear why the minor influence of Ca2+ channel blockade was observed only at the low concentrations in the juxtamedullary nephron preparation but only at high doses in the whole kidney preparation. The small differences in the contribution of L-type Ca2+ channels in the different model systems may be related to contributions of other cell types or mediators that could modulate the response to ET-1 in the different preparations.
ETA receptor activation results in a biphasic increase in [Ca2+]i. In the first phase, ET-1 binds to a specific receptor at the cell surface, activating a pertussis toxin-insensitive G protein, which in turn stimulates phospholipase C. Hydrolysis of phosphatidylinositol by phospholipase C generates inositol triphosphate (IP3) and diacylglycerol, both of which are known to function as second messengers, transducing information from the surface of the cell to the interior (28). IP3 is a fast-acting mediator that facilitates the first phase of the response by binding to a receptor on the sarcoplasmic reticulum and triggering release of Ca2+ from intracellular stores. In conjunction with the release of Ca2+ from intracellular stores, a second phase is initiated, in which Ca2+ moves into the cell from the extracellular space. The sustained elevation in intracellular Ca2+ that results from Ca2+ influx is thought to be responsible for increases in contractility. Although a number of studies have attributed the activation of voltage-dependent, L-type Ca2+ channels to the preglomerular vasoconstriction evoked by numerous hormones, agonists, and autacoids (2, 7, 10, 16, 17, 19, 23, 24, 34), the precise mechanism by which ET activates this channel is largely unresolved. Furthermore, it has been shown that L-type Ca2+ channels account for only a portion of the Ca2+ response and that other channels may be involved (20, 33). In contrast to the IP3-mediated pathway by which intracellular Ca2+ is released, the purported processes by which extracellular Ca2+ enters vascular smooth muscle cells are less clearly defined.
The in vivo approach we used to study L-type Ca2+ channel involvement in renal vasoconstrictor responses to ET-1 has been effectively utilized to demonstrate the dependence of angiotensin II and other vasoconstrictors on L-type Ca2+ channels (9, 30, 31). These investigators were able to observe a significant inhibition of vasoconstrictor responses to angiotensin II, norepinephrine, and vasopressin when the Ca2+ channel blocker was injected along with the active peptide. In contrast to angiotensin II, ET-1 does not appear to be as dependent on L-type Ca2+ channels to produce renal vasoconstriction. This is somewhat surprising, because a number of reports indicate that the vasoconstrictor actions of angiotensin II can be inhibited by ETA receptor blockade (4, 29). However, these findings are consistent with reports that Ca2+ entry from extracellular sources may involve mechanisms other than L-type Ca2+ channels (20, 33). We previously reported that the sustained phase of ET-1-induced increases in intracellular Ca2+ is eliminated in a Ca2+-free medium, indicating the requirement for extracellular Ca2+ (32). Because the present study demonstrates that this does not involve L-type Ca2+ channels, these data support an alternative mechanism for Ca2+ entry. These mechanisms appear to be unique to the renal vasculature, because dihydropyridine-sensitive Ca2+ channels are activated by ET-1 in other vascular beds (11, 21).
We previously reported that ET-3, a selective ETB receptor agonist at low doses, had no effect on intracellular Ca2+ levels in preglomerular smooth muscle cells, whereas ET-1, a nonselective ligand, produced a transient peak increase in intracellular Ca2+ consistent with IP3-mediated release of Ca2+ from intracellular stores (32). This was followed by slightly sustained increase that was more prolonged. Many vasoconstrictors can sustain this increase in intracellular Ca2+ by activating L-type Ca2+ channels to allow entry of extracellular Ca2+ (2, 7, 10, 16, 17, 19, 23, 24, 34). We also previously reported that the sustained, but not the peak, increase in intracellular Ca2+ is eliminated in the absence of extracellular Ca2+ (32). In the present study, we report that blockade of L-type Ca2+ channels had no effect on the peak or sustained rise in intracellular Ca2+, suggesting an alternate pathway for Ca2+ entry in preglomerular vascular smooth muscle in response to ET-1.
Similar to the endogenous ligand ET-3, we observed that S6c, another selective ETB receptor agonist, had no effect on intracellular Ca2+. These data indicate that ETB receptor-mediated constriction is through a Ca2+-independent mechanism or occurs at sites other than interlobular arteries and afferent arterioles from which these cells are isolated. Endlich et al. (6) reported in the hydronephrotic kidney that ETB receptor-mediated vasoconstriction occurs primarily in efferent arterioles or sites in the afferent arteriole immediately adjacent to the glomerulus. Our observations that ET-3, IRL-1620, and S6c have no marked effect on intracellular Ca2+ contrast with a recent report of Fellner and Arendshorst (8) using the ETB receptor agonist IRL-1620 in a similar isolated cell preparation. In their report, IRL-1620 was the only ETB receptor agonist examined, and it produced a peak increase in intracellular Ca2+ of 106 nM compared with the small but significant 26 nM change we observed. In contrast, our response to ET-1 was about twofold larger. The difference in results is not clear, but differences between IRL-1620 and other ETB receptor ligands have been reported in terms of binding characteristics to subpopulations of ETB receptors (25, 37). Interestingly, we previously reported considerably fewer IRL-1620 than ET-3 binding sites in the kidney (27).
In summary, using three different experimental approaches, our studies indicate a relatively minor role for L-type Ca2+ channel activation in the renal vasoconstrictor response to ET-1 and the selective ETB receptor agonist S6c. These data indicate that mechanisms unrelated to L-type Ca2+ channels contribute to ET-1-mediated vasoconstriction.
GRANTS
These studies were supported by National Institutes of Health Grants HL-64776 (D. M. Pollock), DK-38226 (J. D. Imig), and DK-44628 (E. W. Inscho) and Established Investigator Awards (D. M. Pollock and J. D. Imig) and a Grant-In-Aid from the American Heart Association (E. W. Inscho).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Cao LQ and Banks RO. Cardiovascular and renal actions of endothelin: effects of calcium-channel blockers. Am J Physiol Renal Fluid Electrolyte Physiol 258: F254F258, 1990.
Carmines PK and Navar LG. Disparate effects of Ca channel blockade on afferent and efferent arteriolar responses to ANG II. Am J Physiol Renal Fluid Electrolyte Physiol 256: F1015F1020, 1989.
Casellas D and Navar LG. In vitro perfusion of juxtamedullary nephrons in rats. Am J Physiol Renal Fluid Electrolyte Physiol 246: F349F358, 1984.
d'Uscio LV, Moreau P, Shaw S, Takase H, Barton M, and Luscher TF. Effects of chronic ETA-receptor blockade in angiotensin II-induced hypertension. Hypertension 29: 435441, 1997.
Edwards RM, Trizna W, and Ohlstein EH. Renal microvascular effects of endothelin. Am J Physiol Renal Fluid Electrolyte Physiol 259: F217F221, 1990.
Endlich K, Hoffend J, and Steinhausen M. Localization of endothelin ETA and ETB receptor-mediated constriction in the renal microcirculation of rats. J Physiol 497: 211218, 1996.
Epstein M and Loutzenhiser RD. Effects of calcium antagonists on renal hemodynamics. Am J Kidney Dis 16: 1014, 1990.
Fellner SK and Arendshorst WJ. Endothelin A and B receptors of preglomerular vascular smooth muscle cells. Kidney Int 65: 18101817, 2004.
Feng JJ and Arendshorst WJ. Calcium signaling mechanisms in renal vascular responses to vasopressin in genetic hypertension. Hypertension 30: 12231231, 1997.
Gordienko DV, Clausen C, and Goligorsky MS. Ionic currents and endothelin signaling in smooth muscle cells from rat renal resistance arteries. Am J Physiol Renal Fluid Electrolyte Physiol 266: F325F341, 1994.
Goto K, Kasuya Y, Matsuki N, Takuwa Y, Kurihara H, Ishikawa T, Kimura S, Yanagisawa M, and Masaki T. Endothelin activates the dihydropyridine-sensitive, voltage-dependent Ca2+ channel in vascular smooth muscle. Proc Natl Acad Sci USA 86: 39153918, 1989.
Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 34403450, 1985.
Inscho EW, Cook AK, Mui V, and Imig JD. Calcium mobilization contributes to pressure-mediated afferent arteriolar vasoconstriction. Hypertension 31: 421428, 1998.
Inscho EW, Cook AK, Mui V, and Miller J. Direct assessment of renal microvascular responses to P2-purinoceptor agonists. Am J Physiol Renal Physiol 274: F718F727, 1998.
Inscho EW, LeBlanc EA, Pham BT, White SM, and Imig JD. Purinoceptor-mediated calcium signaling in preglomerular smooth muscle cells. Hypertension 33: 195200, 1999.
Inscho EW, Mason MJ, Schroeder AC, Deichmann PC, Stiegler KD, and Imig JD. Agonist-induced calcium regulation in freshly isolated renal microvascular smooth muscle cells. J Am Soc Nephrol 8: 569579, 1997.
Inscho EW, Ohishi K, Cook AK, Belott TP, and Navar LG. Calcium activation mechanisms in the renal microvascular response to extracellular ATP. Am J Physiol Renal Fluid Electrolyte Physiol 268: F876F884, 1995.
Inscho EW, Ohishi K, and Navar LG. Effects of ATP on pre- and postglomerular juxtamedullary microvasculature. Am J Physiol Renal Fluid Electrolyte Physiol 263: F886F893, 1992.
Inscho EW, Schroeder AC, Deichmann PC, and Imig JD. ATP-mediated Ca2+ signaling in preglomerular smooth muscle cells. Am J Physiol Renal Physiol 276: F450F456, 1999.
Iwasawa K, Nakajima T, Hazama H, Goto A, Shin WS, Toyo-oka T, and Omata M. Effects of extracellular pH on receptor-mediated Ca2+ influx in A7r5 rat smooth muscle cells: involvement of two different types of channel. J Physiol 503: 237251, 1997.
Kasuya Y, Takuwa Y, Yanagisawa M, Masaki T, and Goto K. A pertussis toxin-sensitive mechanism of endothelin action in porcine coronary artery smooth muscle. Br J Pharmacol 107: 456462, 1992.
Loutzenhiser R, Epstein M, Hayashi K, and Horton C. Direct visualization of effects of endothelin on the renal microvasculature. Am J Physiol Renal Fluid Electrolyte Physiol 258: F61F68, 1990.
Mitchell KD and Navar LG. Tubuloglomerular feedback responses during peritubular infusions of calcium channel blockers. Am J Physiol Renal Fluid Electrolyte Physiol 258: F537F544, 1990.
Navar LG, Inscho EW, Majid SA, Imig JD, Harrison-Bernard LM, and Mitchell KD. Paracrine regulation of the renal microcirculation. Physiol Rev 76: 425536, 1996.
Nishiyama M, Takahara Y, Masaki T, Nakajima N, and Kimura S. Pharmacological heterogeneity of both endothelin ETA- and ETB-receptors in the human saphenous vein. Jpn J Pharmacol 69: 391398, 1995.
Pollock DM. Renal endothelin in hypertension. Curr Opin Nephrol Hypertens 9: 157164, 2000.
Pollock DM, Allcock GH, Krishnan A, Dayton BD, and Pollock JS. Upregulation of endothelin B receptors in kidneys of DOCA-salt hypertensive rats. Am J Physiol Renal Physiol 278: F279F286, 2000.
Pollock DM, Keith TL, and Highsmith RF. Endothelin receptors and calcium signaling. FASEB J 9: 11961204, 1995.
Rajagopalan S, Laursen JB, Borthayre A, Kurz S, Keiser J, Haleen S, Giaid A, and Harrison DG. Role for endothelin-1 in angiotensin II-mediated hypertension. Hypertension 30: 2934, 1997.
Ruan X and Arendshorst WJ. Calcium entry and mobilization signaling pathways in ANG II-induced renal vasoconstriction in vivo. Am J Physiol Renal Fluid Electrolyte Physiol 270: F398F405, 1996.
Salomonsson M and Arendshorst WJ. Calcium recruitment in renal vasculature: NE effects on blood flow and cytosolic calcium concentration. Am J Physiol Renal Physiol 276: F700F710, 1999.
Schroeder AC, Imig JD, LeBlanc EA, Pham BT, Pollock DM, and Inscho EW. Endothelin-mediated calcium signaling in preglomerular smooth muscle cells. Hypertension 35: 280286, 2000.
Takenaka T, Epstein M, Forster H, Landry DW, Iijima K, and Goligorsky MS. Attenuation of endothelin effects by a chloride channel inhibitor, indanyloxyacetic acid. Am J Physiol Renal Fluid Electrolyte Physiol 262: F799F806, 1992.
Takenaka T, Forster H, and Epstein M. Protein kinase C and calcium channel activation as determinants of renal vasoconstriction by angiotensin II and endothelin. Circ Res 73: 743750, 1993.
Touyz RM, Deng LY, and Schiffrin EL. Ca2+ and contractile responses of resistance vessels of WKY rats and SHR to endothelin-1. J Cardiovasc Pharmacol 26 Suppl 3: S193S196, 1995.
Wu-Wong JR, Chiou W, Magnuson SR, Bianchi BR, and Lin CW. Human astrocytoma U138MG cells express predominantly type-A endothelin receptor. Biochim Biophys Acta 1311: 155163, 1996.
Wu-Wong JR, Chiou WJ, Magnuson SR, and Opgenorth TJ. Endothelin receptor agonists and antagonists exhibit different dissociation characteristics. Biochim Biophys Acta 1224: 288294, 1994.
Wu-Wong JR, Dayton BD, and Opgenorth TJ. Endothelin-1-evoked arachidonic acid release: a Ca2+-dependent pathway. Am J Physiol Cell Physiol 271: C869C877, 1996.