Effects of amiloride, benzamil, and alterations in extracellular Na+ on the rat afferent arteriole and its myogenic response

Abstract

Recent studies have implicated epithelial Na+ channels (ENaC) in myogenic signaling. The present study was undertaken to determine if ENaC and/or Na+ entry are involved in the myogenic response of the rat afferent arteriole. Myogenic responses were assessed in the in vitro hydronephrotic kidney model. ENaC expression and membrane potential responses were evaluated with afferent arterioles isolated from normal rat kidneys. Our findings do not support a role of ENaC, in that ENaC channel blockers did not reduce myogenic responses and ENaC expression could not be demonstrated in this vessel. Reducing extracellular Na+ concentration ([Na+]o; 100 mmol/l) did not attenuate myogenic responses, and amiloride had no effect on membrane potential. Benzamil, an inhibitor of ENaC that also blocks Na+/Ca2+ exchange (NCX), potentiated myogenic vasoconstriction. Benzamil and low [Na+]o elicited vasoconstriction; however, these responses were attenuated by diltiazem and were associated with significant membrane depolarization, suggesting a contribution of mechanisms other than a reduction in NCX. Na+ repletion induced a vasodilation in pressurized afferent arterioles preequilibrated in low [Na+]o, a hallmark of NCX, and this response was reduced by 10 micromol/l benzamil. The dilation was eliminated, however, by a combination of benzamil plus ouabain, suggesting an involvement of the electrogenic Na+-K+-ATPase. In concert, these findings refute the premise that ENaC plays a significant role in the rat afferent arteriole and instead suggest that reducing [Na+](o) and/or Na+ entry is coupled to membrane depolarization. The mechanisms underlying these unexpected and paradoxical effects of Na+ are not resolved at the present time.

Fig. 1.

Original tracing (A) and mean data (B, n = 6) showing the effects of benzamil on myogenic responses of the afferent arteriole. Note that rather than blocking myogenic reactivity, 1.0 μmol/l benzamil actually enhanced the vasoconstriction (*P < 0.05 vs. control response).

Fig. 2.

Amiloride had no significant effect on the myogenic response of the afferent arteriole to changes in renal arterial pressure (n = 5).

Fig. 3.

A: representative RT-PCR assays of afferent arterioles using the α, β, and γ₁ nested primers (Table 1, 35 + 35 cycles) and single (300 μm) segment of the cortical collecting duct (CCD). Nos. in parentheses indicate frequency of observation in all 20 kidney preparations. B: with the use of the α, β, and γ₂ primers (Table 1), mRNA for all three epithelial Na⁺ channel (ENaC) subunits could be detected in 10 CCD epithelial cells but not in 10 afferent arterioles (∼1,000 cells). C: same assay could detect ENaC subunit expression in an intact interlobar artery but not in isolated interlobar myocytes (see text for description).

Fig. 4.

As shown in top, the ENaC α-subunit assay produced multiple PCR products in 4/20 assays (labeled 1–3 in top left). These three products were sequenced and found to correspond to the predicted processed, partially processed, and unprocessed mRNA sequences shown in bottom. These products were not seen in −RT control (data not shown). Nos. in parentheses represent the frequency of these observations in the 20 RT-PCR assays.

Fig. 5.

Original tracings (left) and mean data showing effects of 10 μmol/l benzamil (A and B, n = 4) and reducing extracellular Na⁺ concentration ([Na⁺]_o) from 140 to 100 mmol/l (C and D, n = 5) on the afferent arteriole. Kidneys were perfused at an arterial pressure of 80 mmHg. Benzamil and reduced [Na⁺]_o each elicited a marked vasoconstriction that was partially reversed by diltiazem (10 μmol/l). Note that, in regard to the latter, restoration of normal [Na⁺]_o reversed the diltiazem-insensitive component of this response (C and D).

Fig. 6.

Original tracings (left) and mean data showing effects of 10 μmol/l benzamil (n = 4), 3 μmol/l amiloride (A and B, n = 3), and reducing [Na⁺]_o from 140 to 100 mmol/l (C and D, n = 4) on the membrane potentials of afferent arterioles isolated from normal rat kidneys. Benzamil and low [Na⁺]_o caused a significant and reversible membrane depolarization. Amiloride had no effect.

Fig. 7.

Myogenic responses in normal (140 mmol/l) and low [Na⁺]_o (100 mmol/l). Note that following the low [Na⁺]_o-induced vasoconstriction, diameters could be partially restored by reducing perfusion pressure from 80 to 60 mmHg (tracing A, mean data, B). Myogenic responses to elevating renal arterial pressure to 140 mmHg could then be elicited (n = 9), and the level of myogenic tone attained was similar to that elicited in normal [Na⁺]_o (n = 6). Statistical analyses are described in the text.

Fig. 8.

Na⁺-induced vasodilation seen when [Na⁺]_o was increased from 100 to 140 mmol/l. Afferent arterioles were preconstricted by elevating renal perfusion pressure from 60 to 140 mmHg. As shown in A and B, restoration of normal [Na⁺]_o resulted in an initial vasodilation that restored diameters to values greater than that seen at 60 mmHg (n = 9). The initial vasoconstriction was restored upon returning [Na⁺]_o to 100 mmol/l. C and D show that partially attenuated vasodilatory responses could be elicited in the presence of 10 μmol/l benzamil (n = 6). Statistical analyses are described in the text.

Fig. 9.

Na⁺-induced vasodilation could also be elicited in the presence of ouabain. Ouabain (3 mmol/l) produced a marked vasoconstriction of the afferent arteriole (renal arterial pressure held constant at 60 mmHg) in the low [Na⁺]_o media. Restoring [Na⁺]_o to 140 mmol/l resulted in a more slowly developing vasodilation. Mean data (n = 5) shown in B. Statistical analysis is described in the text. Time courses for the vasodilatory responses in controls, benzamil, and ouabain are compared in C.

Fig. 10.

Original tracing (A) and mean data (B, n = 6) illustrating that the combined treatment with benzamil (10 μmol/l) and ouabain (3 mmol/l) completely eliminated Na⁺-induced vasodilation. Note that diltiazem (10 μmol/l) was able to fully reverse the vasoconstriction while Na⁺ replacement had no effect. Statistical analyses are described in the text. C compares the magnitude of responses observed in each setting.