University of Vermont, Burlington, Vermont 05405; and Yale University School of Medicine and West Haven Veterans Affairs Medical Center, New Haven, Connecticut 06510
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ABSTRACT |
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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 -,
-, and
-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
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INTRODUCTION |
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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).
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MATERIALS AND METHODS |
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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 ( · 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 -,
-,
and
-ENaC RNA or water as a negative control.
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RESULTS AND DISCUSSION |
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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.
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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 · cm2
(n = 11). Increasing concentrations of verapamil from
100 to 400 µM led to progressive increases in TER (5,002 ± 406
· 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.
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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.
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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 · 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.
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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 -,
-, and
-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.
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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.
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ACKNOWLEDGEMENTS |
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We thank Cecilia Canessa for providing the ENaC subunit clones and Larry J. Macala for technical expertise.
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FOOTNOTES |
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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.
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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
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
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