Segment-specific effect of chloride channel blockade on rat renal arteriolar contractile responses to angiotensin II

A]H

1995; 8:90-94

Segment-Specific Effect of Chloride Channel Blockade on Rat Renal Arteriolar Contractile Responses to Angiotensin II Pamela K. Carmines

Experiments were performed to determine the effect of a chloride channel blocker (indanyloxyacetic acid, IAA-94) on renal arteriolar vasoconstrictor responses to angiotensin II (AngII). The in vitro blood-perfused juxtamedullary nephron technique was exploited to provide access to the renal microvasculature of enalaprilat-treated rats. Under control conditions, 1 to 100 nmol/L AngII evoked concentration-dependent afferent arteriolar vasoconstriction. Baseline diameter of afferent arterioles was not altered by 30 ~mol/L IAA-94; however, AngII responsiveness was markedly attenuated. The afferent response to K-induced

depolarization was sustained in the presence of IAA-94. In efferent arterioles, neither baseline diameter nor AngII responsiveness was altered by IAA-94. These results suggest that full expression AngII-induced afferent (but not efferent) arteriolar vasoconstriction requires participation of chloride channels, which likely engender depolarization and subsequent opening of voltage-gated calcium channels. Am J Hypertens 1995;8:90-94

ccumulating evidence suggests that angiotensin II (AnglI) utilizes disparate intracellular signaling processes to evoke va• soconstriction of renal afferent and efferent arterioles. Most strikingly, pharmacologic agents which impede opening of voltage-gated calcium channels block the afferent arteriolar response to AnglI, but have little or no impact on the efferent arteriolar response.l"2 Thus, the afferent arteriolar response to AngII must include membrane depolariza-

tion sufficient to engender opening of the voltagegated calcium channels. Indeed, AngII-induced depolarization has been documented in mouse afferent arterioles; 3 however, the mechanism through which AngII evokes afferent arteriolar membrane depolarization remains undefined. In mesangial cells and vascular smooth muscle cells, agonist-induced depolarization responses can result from the opening of chloride channels. 4-9 Based on these reports, experiments were performed to test the hypothesis that the afferent arteriolar response to AngII involves activation of chloride channels. The strategy employed in this study was to assess the influence of indanyloxyacetic acid (IAA-94, a chloride channel blocker 1°) on vasoconstrictor responses to exogenous AngII.

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Received June 6, 1994. Accepted September 12, 1994. From the Department of Physiology & Biophysics, University of Nebraska Medical Center, Omaha, Nebraska. This work was supported by grants from the American Heart Association (910709) and the National Institutes of Health (DK39202). Dr. Carmines is an Established Investigator of the American Heart Association. Address correspondence and reprint requests to Pamela K. Carmines, Department of Physiology & Biophysics, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 681984575.

© 1995 by the American Journal of Hypertension, Ltd.

KEY WORDS: Angiotensin II, arterioles, chloride channels, indanyloxyacetic acid, renal circulation.

METHODS The procedures used in this study were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee. Twenty-four 0895-7061/95/$9.50 0895-7061(94)00170-G

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male Sprague-Dawley rats were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneally) and treated acutely with enalaprilat (2 rag, intraarterially) to reduce the influence of endogenous AngII on the microvasculature. Each experiment required two rats, which served as tissue donors for study of the renal microvasculature using the in vitro bloodperfused juxtamedullary nephron technique. 11 One rat was nephrectomized bilaterally and exsanguinated into a heparinized syringe. Blood collected from this rat was processed as previously described. 7 The right kidney of the second rat was perfused in situ with Tyrode's solution containing 51 g/L bovine serum albumin. While maintaining the perfusion, this kidney was excised and hemisected, retaining the papilla in the perfused portion of the organ. The papilla was lifted from the pelvic cavity and retained in that position by insect pins. The pelvic mucosa and associated adipose tissue were removed, and the walls of the large veins were opened, thus revealing the surface of the cortex which normally apposes the pelvic cavity. Placement of tight ligatures on distal aspects of the large arteries allowed perfusion of a small portion of the kidney, including juxtamedullary glomeruli and associated arterioles located on the tissue surface. After completing the dissection procedure, the Tyrode's perfusate was replaced with blood. Renal arterial pressure was maintained at 110 mm Hg, and the tissue surface was continuously bathed with Tyrode's solution containing 10 g/L bovine serum albumin at 37°C. The microvasculature was visualized by videomicroscopy and a single afferent or efferent arteriole was chosen for study. The arteriolar image was recorded continuously on videotape for later analysis. After a 5-rain control (baseline) period, the tissue was exposed to increasing concentrations (1 to 100 nmol/ L) of AngII via the bathing solution. A recovery period was followed by the addition of IAA-94 to the bathing solution at a final concentration of 30 ~mol/L, and the AngII exposure protocol was repeated. In some instances, the tissue was subsequently exposed to Tyrode's solution containing 40 mmol/L K (prepared by isosmotic substitution for Na). Luminal diameter was measured from videotaped images at 12-sec intervals using a digital imageshearing monitor. Preliminary studies indicated that arteriolar diameter responses to two sequential AnglI exposure sequences were indistinguishable, and that the response to each concentration of the peptide achieved a stable plateau within 2 min. Accordingly, responses are reported as the average diameter over the final minute of each 3-min treatment period. Statistical comparisons were made by ANOVA for repeated measures and Newman-Keuls tests. P val-

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91

ues less than .05 were considered significant. Data are expressed as mean + SEM (n = n u m b e r of arterioles). RESULTS Afferent arteriolar responses to exogenous AnglI are depicted in the upper panel of Figure 1. Under control conditions (before IAA-94 treatment), baseline afferent diameter averaged 18.1 ___ 1.4 ~xm (n = 7) and AnglI evoked a concentration-dependent vasoconstrictor response. One nanomolar AngII reduced afferent arteriolar diameter to 72 +- 6% of baseline, and 100 nmol/L AnglI completely closed six of the seven vessels studied. After a 10-min recovery period, afferent arteriolar diameter was restored to 18.8 +- 1.3 ~xm (P > .05 v baseline). Although IAA-94 did not alter baseline afferent arteriolar diameter (18.7 --- 1.4 ixm), AnglI responsiveness was markedly attenuated. During IAA-94 exposure, the afferent arteriolar diameter response to each Angll concentration was significantly (P < .05) reduced compared to the response observed in the absence of IAA-94. Although

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AngII Concentration (nM) FIGURE 1. Effects of IAA-94 on renal arteriolar diameter responses to exogenous AnglI. Top: Afferent arterioles (n = 7). Bottom: Efferent arterioles (n = 5). Untreated (C)); 30 ~moI/L IAA-94 (0). Values are mean +-- SEM. *P < .05 v baseline (0 nmol/L AnglI), f P < .05 v untreated.

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100 nmol/L AngII only decreased afferent arteriolar diameter to 68 + 8% of baseline during IAA-94 treatment, afferent arteriolar blood flow was arrested, indicating that another renal vascular segment remained highly responsive to AngII under these conditions. After recovery from the second AngII treatment sequence, and during continued exposure to IAA-94, five of the afferent arterioles were exposed to a bathing solution containing 40 mmol/L K. In contrast with the weak AngII responsiveness observed during IAA-94 treatment, K-induced depolarization under the same conditions markedly reduced afferent arteriolar diameter from 18.0 + 1.6 ~m to 4.7 _+ 2.2 p~m (P < .05). The influence of IAA-94 on efferent arteriolar AngII responsiveness is depicted in the lower panel of Figure 1. Under control conditions (untreated), baseline efferent arteriolar diameter averaged 17.2 + 1.4 p,m (n = 5), and AngII evoked a concentration-dependent vasoconstriction which was similar in magnitude to that observed in afferent arterioles. One nanomolar AngII reduced efferent arteriolar diameter to 90 + 3% of baseline, and 100 nmol/L AngII closed three of the five efferent arterioles studied. After recovery from the AngII exposure sequence (diameter restored to 16.2 + 0.6 ~m), addition of IAA-94 to the bathing solution did not alter either baseline efferent arteriolar diameter (16.6 + 0.9 p~m) or efferent arteriolar AngII responsiveness. One nanomolar AnglI reduced efferent arteriolar diameter to 89 + 8% of baseline during IAA-94 treatment, and 100 nmol/L AngII completely closed three of the five vessels studied (diameter reduced to 16 -+ 14% of baseline). Thus, efferent arteriolar AngII responsiveness was sustained during treatment with IAA-94. DISCUSSION IAA-94 is a potent and specific blocker of epithelial chloride channels, having extremely weak inhibitory effects on the CI:HCO 3 exchanger and other erythrocyte anion transport processes. 1° The concentration of IAA-94 used in the present study virtually abolishes 36C1flUXacross renal cortical membrane vesicles enriched in epithelial chloride channels. 1° IAA-94 is also effective in cultured vascular smooth muscle cells, blunting the endothelin-induced decrease in intracellular chloride concentration as well as the attendant depolarization and sustained elevation of cytosolic calcium concentration. 7-9 Moreover, afferent arteriolar contractile r e s p o n s e s to e n d o t h e l i n are blocked by IAA-94. 7 Results of the present study confirm and extend this observation, revealing that IAA-94 blocks the afferent (but not efferent) arteriolar vasoconstrictor response to AnglI. The seg-

ment-selective renal arteriolar effects of IAA-94 implicate opening of chloride channels as a pivotal event in agonist-induced regulation of the renal microvasculature. The in vitro blood-perfused juxtamedullary nephton technique allows direct study of the renal microvasculature while retaining glomerular and tubular function. 11 Thus, it is possible that the effects of IAA-94 on arteriolar function in this experimental setting developed secondary to a direct effect of the agent on epithelial chloride channels. Indeed, IAA-94 inhibits the opening of renal tubular chloride channels, 1° possibly including those on the basolateral aspect of the thick ascending limb. An alteration in the gating of these channels can be expected to impact on solute delivery to the macula densa and, hence, on afferent arteriolar function through the tubuloglomerular feedback mechanism. Inhibition of chloride channels in the thick ascending limb should impair the diluting capacity of this nephron segment, resulting in increased solute delivery to the macula densa and tubuloglomerular feedback-mediated afferent vasoconstriction. Since baseline diameter was unaffected and vasoconstrictor responsiveness was reduced during IAA-94 treatment, it is unlikely that these events developed secondary to a tubular effect of the agent. In agreement with the observations of Takenaka and coworkers, 7 IAA-94 did not alter baseline renal arteriolar diameter in the present study. This observation suggests that chloride channels are minimally involved in setting baseline or myogenic tone within the renal microvasculature. This contention is c o n s i s t e n t with a c c u m u l a t i n g e v i d e n c e t h a t gating of stretch-activated ion channels or the Kconductance properties of the sarcolemma are the primary determinants of membrane potential events which e n g e n d e r baseline m y o g e n i c tone in the microvasculature. 12 Voltage-gated calcium channels are prominent in afferent arterioles, but functionally suppressed or absent in efferent arterioles. 2'13'14 Moreover, pharmacologic antagonists of voltage-gated calcium channels block afferent arteriolar responses to AngII, having little or no influence on the efferent arteriolar response to this peptide. 12 Thus, the segment-specific effect of IAA-94 on renal arteriolar AngII responsiveness is similar to the action of calcium channel blockers. This situation could result from either an effect of IAA-94 to interfere with cellular mechanisms which ultimately result in the opening of voltage-gated channels (ie, membrane depolarization) or a direct effect of IAA-94 to block voltage-gated calcium channels. To assess the latter possibility, IAA-94-treated afferent arterioles were exposed to a bathing solution

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containing 40 mmol/L potassium. This manipulation induces membrane depolarization through a mechanism which is independent of chloride channels. In accord with previous reports, K-induced depolarization evoked a strong afferent arteriolar vasoconstrictor response during IAA-94 treatment, indicating that this agent does not produce a nonspecific inhibition of contractile responses to depolarizing stimuli. Rather, the similarity between the selective afferent arteriolar effects of IAA-94 and calcium entry antagonists indicates that IAA-94 interferes with agonistinduced mechanisms which ultimately engender membrane depolarization. Chloride is a c c u m u l a t e d in the cytoplasm of smooth muscle cells, achieving concentrations which exceed those predicted by electrochemical equilibrium. 15 Indeed, membrane potential in afferent arteriolar vascular smooth muscle averages - 5 8 mV, 3 which is 30 mV more negative than the approximate equilibrium potential for chloride in smooth muscle cells. Thus, the AngII-induced opening of afferent arteriolar chloride channels should allow chloride efflux, a resulting membrane depolarization, and subsequent opening of voltage-gated calcium channels. This process would increase cytosolic calcium concentration and lead to contraction. Similar agonistinduced signaling events have been described in cultured mesangial cells and in vascular myocytes from nonrenal s o u r c e s . 4 - 7 ' 9 Moreover, recent patch clamp studies have revealed the involvement of a calciumactivated chloride current in the signaling events evoked by endothelin in isolated smooth muscle cells from interlobar and arcuate arteries, s Although the results of the present study are consistent with the postulate that the afferent arteriolar response to AngII involves similar processes, verification of this hypothesis awaits direct assessment of transmembrane ion fluxes and intracellular ion concentration responses evoked by AngII in smooth muscle cells from this vascular segment. As we have reported previously, 1 the AngII responsiveness of efferent arterioles was similar to that of afferent arterioles when studied using the in vitro blood-perfused juxtamedullary nephron technique. However, in contrast with the behavior of afferent arterioles, the efferent arteriolar vasoconstrictor response to AngII was sustained during IAA-94 treatment and likely caused the interruption of afferent arteriolar blood flow observed under these conditions. This observation suggests that the opening of chloride channels is not involved in the development of AngII-induced efferent arteriolar vasoconstriction and further supports the contention that AngII evokes vasoconstriction through disparate signaling mechanisms at pre- and postglomerular sites. 1'2 In summary, the chloride channel blocker IAA-94

CHLORIDECHANNELSAND RENALANGII RESPONSES 93

did not alter afferent or efferent arteriolar diameter, suggesting that baseline tone is sustained during relative inactivation of chloride channels. Afferent arteriolar contractile responses to exogenous AngII were substantially attenuated by IAA-94, while responses to K-induced depolarization were sustained. In contrast, efferent arteriolar responses to AngII were not influenced by IAA-94. These observations indicate that full expression of AngII-induced afferent arteriolar vasoconstriction requires participation of chloride channels which are sensitive to IAA-94. A potential mechanism which might underlie this phenomenon involves AngII-stimulated opening of afferent arteriolar chloride channels, resulting in chloride efflux and consequent membrane depolarization. This process would drive the opening of voltage-gated calcium channels, calcium influx, and vasoconstriction. Efferent arteriolar AnglI responses, which arise independently of voltage-gated calcium channels, apparently do not require the opening of chloride channels. ACKNOWLEDGMENTS Excellent technical assistance was provided by Rachel W. Fallet. REFERENCES 1. Carmines PK, Navar LG: Disparate effects of Ca channel blockade on afferent and efferent arteriolar responses to ANG II. Am J Physiol 1989;256:F1015F1020. 2. Conger JD, Falk SA, Robinette JB: Angiotensin IIinduced changes in smooth muscle calcium in rat renal arterioles. J Am Soc Nephrol 1993;3:1792-1803. 3. Biihrle CP, Nobiling R, Taugner R: Intracellular recordings from renin-positive cells of the afferent glomerular arteriole. Am J Physiol 1985;249:F272-F281. 4. Kremer SG, Breuer WV, Skorecki KL: Vasoconstrictor hormones depolarize glomerular mesangial cells by activating chloride channels. J Cell Physiol 1989;138: 97-105. 5. Okuda T, Yamashita N, Kurokawa K: Angiotensin II and vasopressin stimulate calcium-activated chloride conductance in rat mesangial cells. J Clin Invest 1986; 78:1443-1448. 6. Pacaud P, Loirand G, Baron A, et al: C a 2 ÷ channel activation and membrane depolarization mediated by C1- channels in response to noradrenaline in vascular myocytes. Br J Pharmacol 1991;104:1000--1006. 7. Takenaka T, Epstein M, Forster H, et al: Attenuation of endothelin effects by a chloride channel inhibitor, indanyloxyacetic acid. Am J Physiol 1992;262:F799F806. 8. Gordienko DV, Clausen C, Goligorsky MS: Ionic currents and endothelin signaling in smooth muscle cells from rat renal resistance arteries. Am J Physiol 1994; 266:F325-F341. 9. Iijima K, Lin L, Nasjletti A, Goligorsky MS: Intracellular ramification of endothelin signal. Am J Physiol 1991;260:C982-C992.

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