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 (I_sc = 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 (K_i) 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. I_sc 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/[I_sc(60 mV)  I_sc(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.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Effect of verapamil on transepithelial Na+ transport (I_sc). I_sc 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 I_sc by 87.3 ± 2% (n = 4). Therefore, the amiloride-sensitive I_sc correlates well with Na⁺ transport from the apical to the basolateral side through the ENaC.

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 I_sc 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 I_sc would be expected to decrease independently of a net decrease in Na⁺ transport. If that were the case, the inhibition of I_sc 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 I_sc 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 I_sc in A6 cells was determined by adding increasing concentrations of the drug to the apical side. The amiloride-sensitive component of I_sc was determined after each experiment by the addition of 100 µM amiloride. As shown in Fig. 3, the K_i of I_sc 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.

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 (K_i = 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 I_sc (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 I_sc and Na⁺ transport. Nifedipine, a dihydropyridine Ca²⁺ channel blocker chemically unrelated to verapamil, blocks Na⁺ channels in rat cardiac myocytes with a K_i of 3.0 µM (12). Its effect on I_sc in A6 cells was tested, and, as shown in Fig. 5, it was ineffective at blocking I_sc at the highest concentration achievable in aqueous media (0.2 mM). The inability of nifedipine to block I_sc supports the conclusion that the effect of verapamil on I_sc 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 -, -, 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 K_i of 34 ± 5.2 µM (n = 4). The K_i observed in oocytes is similar to that measured in A6 cells, supporting the notion that ENaC inhibition underlies the effect of verapamil on I_sc.

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 -, -, and -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 -, -, and -ENaC.

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 I_sc 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.