On the natriuretic effect of verapamil: inhibition of ENaC and transepithelial sodium transport

Alan S. Segal, John P. Hayslett, and Gary V. Desir

University of Vermont, Burlington, Vermont 05405; and Yale University School of Medicine and West Haven Veterans Affairs Medical Center, New Haven, Connecticut 06510


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

The natriuretic effect of Ca2+ channel blockers has been attributed to hemodynamic changes and to poorly defined direct tubular effects. To test the possibility that verapamil may inhibit Na+ reabsorption at the distal tubule, its effect on transepithelial Na+ transport in aldosterone-stimulated A6 cells was determined. Cells were grown on permeable supports, and short-circuit current (Isc) measured in an Ussing chamber was used as a surrogate marker for transepithelial Na+ transport. Application of 300 µM verapamil to the apical side inhibited Isc by 77% and was nearly as potent as 100 µM amiloride, which inhibited Isc by 87%. Verapamil-induced inhibition of Isc was accompanied by a significant increase in transepithelial resistance, suggesting blockade of an apical conductance. Its action on transepithelial Na+ transport does not appear to occur through inhibition of L-type Ca2+ channels, since Isc was unaffected by removal of extracellular Ca2+. Verapamil also does not appear to inhibit Isc by modulating intracellular Ca2+ stores, since it fails to inhibit transepithelial Na+ transport when added to the basolateral side. The effect on Na+ transport is specific for verapamil, since nifedipine, Ba2+, 4-aminopyridine, and charybdotoxin do not significantly affect Isc. A direct effect of verapamil on the epithelial Na+ channel (ENaC) was tested using oocytes injected with the alpha -, beta -, and gamma -subunits. We conclude that verapamil inhibits transepithelial Na+ transport in A6 cells by blocking ENaC and that the natriuresis observed with administration of verapamil may be due in part to its action on ENaC.

verapamil; epithelial sodium channel; Xenopus laevis oocyte; sodium excretion and regulation; diuretic; natriuresis; A6 cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

CALCIUM CHANNEL BLOCKERS are used extensively in the treatment of hypertension. They lower blood pressure by relaxing vascular smooth muscle and decreasing peripheral vascular resistance. Vasorelaxation is a result of blocking Ca2+ entry through voltage-gated Ca2+ channels. Ca2+ channel blockers also have significant effects on heart rate and renal function. They increase renal blood flow and glomerular filtration rate and decrease the activity of the renin-angiotensin-aldosterone system and the reabsorption of salt and water (4, 6, 10).

Although the natriuretic and diuretic effect of Ca2+ channel blockers is in part due to changes in renal hemodynamics, direct tubular actions have also been well documented. The dihydropyridine class of Ca2+ channel blockers has been studied most extensively. Intrarenal infusion of nifedipine significantly increased Na+ excretion without any detectable change in renal blood flow, creatinine clearance, and glomerular filtration rate (7). These effects are thought to result from a decrease in Na+ reabsorption in the proximal tubule. On the other hand, felodipine, another dihydropyridine, appears to increase Na+ excretion by blocking reabsorption at a distal tubular site (6). Verapamil, a phenylalkylamine, causes natriuresis when injected into the renal artery of the dog (1). Renal Na+ excretion is also significantly enhanced in hypertensive patients treated with verapamil (5, 10).

The molecular mechanisms underlying the tubular effects of Ca2+ channel blockers are unclear. Na+ reabsorption occurs all along the nephron, and inhibition at any site could account for the modest degree of natriuresis observed with these agents. The collecting duct is an important site for Na+ homeostasis. In this nephron segment, principal cells are responsible for Na+ reabsorption. The rate-limiting step is the apical entry of Na+ through the epithelial Na+ channel (ENaC) (2). This process is regulated by mineralocorticoids and plays a critical role in overall Na+ balance. The A6 cell line, derived from the Xenopus laevis kidney, possesses many of the properties of principal cells and has been used extensively as a model for the study of electrogenic transepithelial Na+ transport. When grown on permeable supports, these cells develop a high transepithelial resistance (TER), express the ENaC, and engage in transepithelial electrogenic Na+ transport that is regulated by insulin, aldosterone, and antidiuretic hormone. A6 cells and X. laevis oocytes expressing ENaC were used in the present study to examine the possibility that Ca2+ channel blockers exert their natriuretic effect by inhibiting Na+ transport in the cortical collecting duct (CCD).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Cell culture. A6 cells were plated and maintained in culture as previously described (9). Briefly, cells were seeded at a density of 1 × 106 cells/cm2 on permeable supports in DMEM modified for amphibian culture and supplemented with 10% fetal bovine serum. They were grown in a humidified atmosphere of 2% CO2 at 28°C. Aldosterone (1.5 µM) was added for 2 days after the initial seeding and then for 18 h before measurements.

Solution and drugs. Unless stated otherwise, the apical and basolateral solutions were identical and consisted of 97 mM NaCl, 1 mM CaCl2, 1 mM KCl, 0.5 mM MgCl2, and 5 mM HEPES (pH 7.2). For Ca2+-free solutions, CaCl2 was omitted and 1 mM EGTA was added. Where indicated, Ba2+ was added as BaCl2. Verapamil and nifedipine were dissolved in 100% DMSO and added from 100 mM stock. The final DMSO concentration was <0.1%. In control studies, we ascertained that 0.1% DMSO added for up to 1 h to the apical membrane of A6 cells had no effect on short-circuit current (Isc = 15.7 ± 1.3 µA, n = 4). Similarly, 0.1% DMSO did not affect ENaC current measured in oocytes at -80 mV (current = -2.1 ± 0.2 µA, n = 4). For dose-response experiments, drugs were added to the apical or basolateral compartment in increasing concentrations. The inhibition constant (Ki) for verapamil was calculated from the best logistic fit of the dose response. Values are means ± SE.

Electrical measurements in A6 cells. Falcon inserts were mounted on plastic rings (effective surface area = 0.64 cm2) and placed in an Ussing chamber modified to allow continuous independent perfusion of apical and basolateral compartments. The compartments were connected by 1 M KCl-2% agar bridges. Current and transepithelial potential were measured using Ag-AgCl half-cells and a current-voltage clamp (model DVC-1000, WPI). Current flowing from the apical to the basolateral side was measured as positive by convention. The solution resistance was measured and compensated for before recordings began. Isc was measured by clamping the transepithelial voltage to 0 mV for 5 s. TER was calculated from the difference in current measured when the cells were voltage clamped to 0 or 60 mV, as follows: TER (Omega  · cm2) = 60 mV/[Isc(60 mV) - Isc(0 mV)] µA/cm2.

Expression and measurement of ENaC current in X. laevis oocytes. Stage V-VI X. laevis oocytes were dissected from ovarian lobes and stored in modified Barth's solution, as previously described. Oocytes were injected with 50 nl of solution containing either a mixture of in vitro-transcribed, 5'-capped alpha -, beta -, and gamma -ENaC RNA or water as a negative control.

Whole cell currents were recorded using a standard two-microelectrode voltage clamp (model OC-725, Warner Instruments) 1-8 days after injection. Oocytes were impaled with microelectrodes filled with 1 M KCl (resistance = 1-4 MOmega ). The bath contained Na+ (or Li+)-ND-96: 96 mM NaCl (or LiCl), 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES (pH 7.4). Voltage-clamp protocols were controlled by PULSE (HEKA Lambretch), and amplified currents were filtered at 1-2 kHz and then recorded and analyzed using PULSE-FIT (HEKA Lambretch) and Igor-Pro (Wavemetrics).


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Effect of verapamil on transepithelial Na+ transport (Isc). Isc is used as a surrogate marker for transepithelial Na+ transport in A6 cells, because ~90% of the current is amiloride sensitive. We confirmed this for A6 cells grown on permeable supports and treated with aldosterone for 18 h before measurements. Under these conditions, 100 µM amiloride inhibited Isc by 87.3 ± 2% (n = 4). Therefore, the amiloride-sensitive Isc correlates well with Na+ transport from the apical to the basolateral side through the ENaC.

The effect of verapamil on Isc (and, therefore, on Na+ transport) was tested on A6 cells mounted in an Ussing chamber. As shown in Fig. 1, 0.2 mM verapamil added to the apical side significantly inhibited Isc compared with control conditions (16.0 ± 2.6 to 5.3 ± 1.1 µA/cm2, n = 8, P < 0.05). Because verapamil is a known inhibitor of L-type Ca2+ channels, it might exert its effect on Isc by decreasing apical Ca2+ entry. To test the dependence of Isc on extracellular Ca2+, the effect of Ca2+ removal on Isc was measured. Figure 1 shows that removal of apical Ca2+ had no effect on Isc under control conditions and TER was unaffected. Furthermore, verapamil inhibited Isc to a similar degree in the absence and presence of Ca2+ (Fig. 1). Verapamil is lipophilic, so it penetrates the cell membrane and accumulates inside the cell. Therefore, it could inhibit Ca2+ release from intracellular stores, which could in turn modulate Isc. To further rule out the possibility that verapamil's action on Isc might be Ca2+ dependent, its inhibitory effect on Isc was compared with that of nifiedipine, a structurally unrelated, potent L-type Ca2+ channel blocker. Although nifedipine is a significantly more potent blocker of L-type Ca2+ channels than verapamil, it decreased Isc by only 7.3 ± 0.3% when applied to the apical side at the highest concentration (200 µM) achievable in aqueous media. Because, as shown in Fig. 1, 0.2 mM verapamil added to the basolateral side has no significant effect on Isc, we conclude that the drug inhibits Isc and Na+ transport only when applied to the apical side and through a mechanism unrelated to its action on plasma membrane L-type Ca2+ channels and intracellular Ca2+ stores.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Verapamil inhibits transepithelial Na+ transport in A6 cells grown on permeable supports and treated with 1 µM aldosterone 18 h before study. Monolayers were mounted in Ussing chambers, and short-circuit current (Isc) was measured by clamping to 0 mV for 5 s. Apical Ca2+ concentration was 0 or 1 mM. Verapamil (Vera) was added to apical or basolateral perfusate.

Effect of verapamil on TER. It is possible that verapamil could inhibit Isc and yet cause no change in net Na+ transport. Indeed, if addition of the drug leads to a nonspecific decrease in TER, through interaction with the paracellular junction or by damaging the plasma membrane, transepithelial voltage and Isc would be expected to decrease independently of a net decrease in Na+ transport. If that were the case, the inhibition of Isc would be accompanied by a fall in membrane resistance. For instance, diltiazem is known to increase the permeabilities of anions and cations in photoreceptor rod outer segments and in intact red blood cells (3). To exclude this possibility, the effect of verapamil on membrane resistance was determined from the change in monolayer current resulting from a 60-mV voltage step. As shown in Fig. 2, control cells had a TER of 2,575 ± 350 Omega  · cm2 (n = 11). Increasing concentrations of verapamil from 100 to 400 µM led to progressive increases in TER (5,002 ± 406 Omega  · cm2, n = 5). These results indicate that the fall in Isc observed with the addition of verapamil does not occur because of nonspecific changes in the paracellular junction or damage to the cell membranes. Instead, verapamil appears to have a direct inhibitory effect on transepithelial Na+ transport.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2.   Verapamil increases transepithelial resistance. Transepithelial resistance of A6 cells was obtained by measuring change in current when voltage was stepped from 0 to 60 mV. Increasing concentrations of verapamil were added to apical perfusate. Inhibition of Isc is accompanied by an increase in transepithelial resistance.

Dose-dependent inhibition of Na+ transport by verapamil. The dose-response curve for verapamil with respect to inhibition of Isc in A6 cells was determined by adding increasing concentrations of the drug to the apical side. The amiloride-sensitive component of Isc was determined after each experiment by the addition of 100 µM amiloride. As shown in Fig. 3, the Ki of Isc for verapamil is 104.5 µM.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 3.   Dose-response curve for inhibition of Isc by apical verapamil. Amiloride-sensitive component was determined by addition of 100 µM amiloride at the end of each experiment. Percent inhibition of amiloride-sensitive current was determined at each concentration (n = 4 for each data point). Data were fitted using Origin 6.0. Ki, concentration for 50% inhibition.

Verapamil contains a nitrogen at position 9 that undergoes pH-dependent protonation (pKa = 8.5). The nonprotonated form of verapamil is lipophilic and readily penetrates biological membranes. Figure 4 shows that the effect of verapamil is strongly influenced by the pH of the apical perfusate. As pH increases from 5 to 8, verapamil becomes deprotonated and its potency to block Isc increases. Indeed, verapamil predominantly blocks L-type Ca2+ and K+ channels from the cytoplasmic side (13). It is likely that verapamil also inhibits Isc from the cytoplasmic side, since, as observed for inhibition of Ca2+ and K+ channels, its onset of action on Isc is delayed (3-4 min) and slowly reaches a plateau within 10-15 min. Therefore, the true Ki for verapamil-induced inhibition of Isc cannot be determined without measuring the intracellular concentration of the drug.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 4.   Alkaline pH potentiates inhibitory action of verapamil. Apical pH was varied from 5 to 8, and inhibition of Isc by 100 µM verapamil was determined. Verapamil was 4.5 times more potent at pH 8 than at pH 5.

Effect of nifedipine and K+ channel blockers on Na+ transport. Verapamil is also known to inhibit voltage-gated K+ channels, such as Kv1.3 and KCNA10 (Ki = 50 µM). Therefore, it is possible that it could mediate its effect on transepithelial Na+ transport by blocking K+ channels. This possibility was tested by examining the effect of K+ channel blockers applied to the apical side of A6 cells. Ba2+ (10 mM), which inhibits a variety of K+ channels, caused only a small decrease in Isc (Fig. 5) and TER (from 2,520 ± 223 to 2,001 ± 216 Omega  · cm2, n = 4). This confirms the results of Thomas and Mintz (11), who showed that Ba2+ depolarizes the apical membrane of A6 cells in culture and also reduces TER, probably by opening a paracellular conductive pathway. Inhibitors of voltage-gated and Ca2+-activated K+ channels (charybdotoxin and 4-aminopyridine, respectively) had no detectable effect on Isc and Na+ transport. Nifedipine, a dihydropyridine Ca2+ channel blocker chemically unrelated to verapamil, blocks Na+ channels in rat cardiac myocytes with a Ki of 3.0 µM (12). Its effect on Isc in A6 cells was tested, and, as shown in Fig. 5, it was ineffective at blocking Isc at the highest concentration achievable in aqueous media (0.2 mM). The inability of nifedipine to block Isc supports the conclusion that the effect of verapamil on Isc is independent of its action on L-type Ca2+ channels. These data also indicate that verapamil is a specific inhibitor of transepithelial Na+ transport in A6 cells and that its mechanism of action does not depend on the inhibition of K+ channels.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 5.   Verapamil is a specific inhibitor of Na+ transport in A6 cells. Drugs were added to apical perfusate. Amiloride and verapamil specifically block Isc, while nifedipine (a dihydropyridine) was ineffective. Classic K+ channel blockers such as Ba2+, 4-aminopyridine (4-AP), and charybdotoxin (CTX) were also without significant effect.

Effect of verapamil on ENaC in X. laevis oocytes. We considered the possibility that verapamil directly blocks ENaC. Indeed, its chemical structure resembles that of two inhibitors of ENaC, namely, trimethoprim and benzamil. To test that hypothesis, cRNA for alpha -, beta -, and gamma -ENaC were coinjected into X. laevis oocytes, and the effect of verapamil on amiloride-sensitive inward current was examined using the two-microelectrode voltage-clamp method. Figure 6 depicts a representative experiment in which membrane voltage is clamped from -80 to +40 mV in 10-mV increments and the resulting current is measured. In oocytes expressing ENaC, 85.1 ± 3% (n = 4) of the current measured at -80 mV was amiloride sensitive and mediated by ENaC. Verapamil inhibited amiloride-sensitive current with a Ki of 34 ± 5.2 µM (n = 4). The Ki observed in oocytes is similar to that measured in A6 cells, supporting the notion that ENaC inhibition underlies the effect of verapamil on Isc.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6.   Verapamil inhibits epithelial Na+ channel (ENaC) currents expressed in Xenopus laevis oocytes. A: 2-microelectrode voltage clamp. Whole cell currents were recorded from oocytes in ND-96 injected with alpha -, beta -, and gamma -ENaC with no drug (left) or in the presence of 100 µM verapamil (middle) or amiloride (right). B: current-voltage relation. Verapamil blocks 93 ± 3% (n = 6) of amiloride-sensitive current detected in oocytes expressing alpha -, beta -, and gamma -ENaC.

The data presented above confirm the hypothesis that verapamil inhibits ENaC. It is not clear whether the drug inhibits from the cytoplasmic side, since, although the slow time course of inhibition and its dependence on external pH suggest that it does, its inability to block when added to the basal side argues otherwise. The latter observation does not completely rule out the possibility of blocks from the inside, since it is not known whether verapamil that enters from the basolateral side freely diffuses to the apical side.

Physiological relevance. On the basis of studies examining the urinary concentration of verapamil and its metabolites, ~5% of the dose of verapamil administered is excreted unchanged in the urine over 24 h (8). With a maximum daily dose of 500 mg, distal tubular fluid may contain 25 mg/l verapamil or ~50 µM. The data in the present study suggest that this concentration would be sufficient to exert a significant effect on Na+ transport through inhibition of ENaC. It should be noted, however, that inhibition of Isc by verapamil was greater at alkaline than acidic pH. The pH at the distal tubule can be as low as 5.5 and would predict a significant decrease in the inhibitory potency of verapamil. On the other hand, verapamil tends to accumulate in cells, and the steady-state intracellular concentration at distal tubular cells might be even higher than expected from a luminal concentration of 50 µM.

Conclusion. The present study shows that verapamil, unlike nifedipine, inhibits Na+ reabsorption in A6 cells, a cell model for the principal cells of the CCD. The concentration of verapamil thought to be present in distal tubular fluid is more than sufficient to cause significant inhibition of Na+ reabsorption through ENaC. Therefore, the natriuresis observed with the administration of verapamil is likely mediated by a combination of hemodynamic factors and the direct tubular effect demonstrated in this study. The present study documents a previously unrecognized amiloride-like effect of verapamil on Na+ transport in A6 cells and a direct inhibitory effect of the drug on ENaC. We speculate that the antihypertensive action of verapamil results from a combination of a direct effect on vascular smooth muscle and a small but significant decrease in total body Na+ stores through its action on Na+ reabsorption in the CCD. It may be possible to improve the efficacy and safety profile of verapamil and develop analogs with enhanced natriuretic properties.


    ACKNOWLEDGEMENTS

We thank Cecilia Canessa for providing the ENaC subunit clones and Larry J. Macala for technical expertise.


    FOOTNOTES

G. V. Desir and J. P. Hayslett are supported by a Merit Review Award from the Department of Veterans Affairs. G. V. Desir was also supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48105B and is an Established Investigator of the American Heart Association.

Address for reprint requests and other correspondence: G. V. Desir, Dept. of Medicine, Sect. of Nephrology, Yale University School of Medicine, 2073 LMP, 333 Cedar St., New Haven, CT 06510 (E-mail: gary.desir{at}yale.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

May 22, 2002;10.1152/ajprenal.00253.2001

Received 15 August 2001; accepted in final form 22 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

1.   Bell, AJ, and Lindner A. Effects of verapamil and nifedipine on renal function and hemodynamics in the dog. Renal Physiol 7: 329-343, 1984[ISI][Medline].

2.   Canessa, CM, Schild L, Buell G, Thorens G, Gautschi I, Horisberger JD, and Rossier BC. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367: 463-467, 1994[ISI][Medline].

3.   Caretta, A, Sorbi RT, Stein PJ, and Tirindelli R. Diltiazem at high concentration increases the ionic permeability of biological membranes. J Membr Biol 122: 203-213, 1991[ISI][Medline].

4.   Chellingsworth, MC, and Kendall MJ. Effects of nifedipine, verapamil and diltiazem on renal function. Br J Clin Pharmacol 25: 599-602, 1988[ISI][Medline].

5.   De Leeuw, PW, and Birkenhager WH. Effects of verapamil in hypertensive patients. Acta Med Scand Suppl 681: 125-128, 1984[Medline].

6.   Dibona, GF, and Sawin LL. Renal tubular site of action of felodipine. J Pharmacol Exp Ther 228: 420-424, 1984[Abstract].

7.   Dietz, JR, Davis JO, Freeman RH, Villarreal D, and Echtenkamp SF. Effects of intrarenal infusion of calcium entry blockers in anesthetized dogs. Hypertension 5: 482-488, 1983[Abstract].

8.   Hamann, SR, Blouin RA, and McAllister RG, Jr. Clinical pharmacokinetics of verapamil. Clin Pharmacokinet 9: 26-41, 1984[ISI][Medline].

9.   Hayslett, JP, Macala LJ, Smallwood JI, Kalghatgi L, Gassala-Herraiz J, and Isales C. Adenosine stimulation of Na+ transport is mediated by an A1 receptor and a [Ca2+]i-dependent mechanism. Kidney Int 47: 1576-1584, 1995[ISI][Medline].

10.   Leonetti, G, and Zanchetti A. Renal effects of calcium antagonists in hypertensive patients. J Hypertens Suppl 3: S535-S539, 1985[Medline].

11.   Thomas, SR, and Mintz E. Time-dependent apical membrane K+ and Na+ selectivity in cultured kidney cells. Am J Physiol Cell Physiol 253: C1-C6, 1987[Abstract/Free Full Text].

12.   Yatani, A, and Brown AM. The calcium channel blocker nitrendipine blocks sodium channels in neonatal rat cardiac myocytes. Circ Res 56: 868-875, 1985[Abstract].

13.   Zhang, S, Zhou Z, Gong Q, Makielski JC, and January CT. Mechanism of block and identification of the verapamil binding domain to HERG potassium channels. Circ Res 84: 989-998, 1999[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 283(4):F765-F770




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
283/4/F765    most recent
00253.2001v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Google Scholar
Articles by Segal, A. S.
Articles by Desir, G. V.
Articles citing this Article
PubMed
PubMed Citation
Articles by Segal, A. S.
Articles by Desir, G. V.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online