Effects of mibefradil and nifedipine on arteriolar myogenic responsiveness and intracellular Ca(2+)

Abstract

1. Ca(2+) entry mechanisms underlying spontaneous arteriolar tone and acute myogenic reactivity remain uncertain. These studies aimed to compare the effects of nifedipine and the putative T-channel blocker, mibefradil, on arteriolar myogenic responsiveness and intracellular Ca(2+) (Ca(2+)(i)). 2. First order cremaster muscle arterioles (1A) were isolated from rats, cannulated, pressurized to 70 mmHg in the absence of intraluminal flow, and mechanical responses studied by video microscopy. The Ca(2+)(i) was measured using fluorescence imaging of Fura 2 loaded arterioles. 3. Both nifedipine and mibefradil showed dose-dependent inhibition of spontaneous myogenic tone (at 70 mmHg; pEC(50) 7.04+/-0.17 vs 6.65+/-0.20 respectively, n=6 for both, n.s.) and KCl-induced vasoconstriction (at 70 mmHg; pEC(50) 6.93+/-0. 38 vs 6.45+/-0.27 respectively, n=6 for both, n.s.). 4. In arterioles maintained at 50 mmHg, nifedipine (10(-7) and 10(-5) M) caused a concentration dependent reduction in Ca(2+)(i), however, mibefradil (10(-7) and 10(-5) M) had no effect. Furthermore nifedipine significantly attenuated the increase in Ca(2+)(i) associated with an acute pressure step (50 - 120 mmHg) whereas mibefradil was considerably less effective. 5. Mibefradil (10(-7) M) significantly attenuated contractile responses to 60 mM KCl without altering the KCl-induced increase in Ca(2+)(i), in contrast to nifedipine (10(-7) M) which reduced both Ca(2+)(i) and contraction. 6. Membrane potential of arterioles with spontaneous myogenic tone (70 mmHg) was -41.5+/-1. 0 mV. Nifedipine (10(-7) or 10(-5) M) had no effect on membrane potential, however mibefradil (10(-5) M) caused significant depolarization. 7. In summary, both mibefradil and nifedipine inhibit arteriolar spontaneous tone and acute myogenic reactivity. While there may be overlap in the mechanisms by which these agents inhibit tone, differences in effects on membrane potential and intracellular Ca(2+) levels suggest mibefradil exhibits actions other than blockade of Ca(2+) entry in skeletal muscle arterioles.

Figure 1

Concentration-dependent effects of mibefradil (n=6) and nifedipine (n=6) on spontaneous arteriolar tone (A) and KCl (60 mM)–induced vasoconstriction (B). Vessel diameters are plotted relative to the passive diameter at an intraluminal pressure of 70 mmHg. Basal indicates the level of spontaneous tone at 70 mmHg in the absence of Ca²⁺ channel antagonists (A); in (B) responses to 60 mM KCl in the absence of antagonist are shown to the left of the figure. (A) includes studies of the effect of increasing concentrations of mibefradil on myogenic tone in the presence of a maximally effective concentration of nifedipine (n=3). (B) includes a time control for KCl-induced vasoconstriction performed in the absence of Ca²⁺ channel antagonists (n=3). Results are presented as mean±s.e.mean.

Figure 2

Concentration-dependent effects of mibefradil (A; n=6) and nifedipine (B; n=5) on Ca²⁺_i at steady-state intraluminal pressures of 50, 70 and 120 mmHg. Fluorescence ratios (340 : 380 nm) are normalized to the ratio obtained under passive conditions at 70 mmHg. Results are presented as mean±s.e.mean; ^*P<0.05.

Figure 3

Group data from experiments summarizing the effects of mibefradil (n=6) and nifedipine (n=5) on basal, minimum and steady-state diameter (A and B), and basal, maximum and steady-state Ca²⁺_i (C and D) responses occurring during a pressure step from 50 to 120 mmHg. See text for methods of determining maximum and steady-state values. Diameters are expressed as per cent of the basal diameter (50 mmHg) in the absence of Ca²⁺ channel antagonists. Changes in intracellular Ca²⁺ are presented as a percent of the pre-stimulation ratio value independent of the effect of the Ca²⁺ channel antagonists (C and D). Results are presented as mean±s.e.mean; ^*P<0.05 compared to corresponding value in the absence of antagonist; ΔP<0.05 compared to basal value at a given antagonist concentration.

Figure 4

Group data from experiments summarizing the effects of mibefradil (n=5) and nifedipine (n=5) on diameter (A and B) and Ca²⁺_i (C and D) responses to 60 mM KPSS. Diameters are expressed as per cent of the basal diameter (70 mmHg) in the absence of Ca²⁺ channel antagonists. Changes in intracellular Ca²⁺ are presented as a per cent of the basal ratio value. Results are presented as mean±s.e.mean; ^*P<0.05 compared to corresponding value in the absence of antagonist; ΔP<0.05 compared to basal value at a given antagonist concentration.

Figure 5

Comparison of the effects of mibefradil and nifedipine on arteriolar membrane potential (n=4 per group). At 10 mmHg vessels lack myogenic tone due to insufficient distending pressure while 70 mmHg approximates the in vivo pressure within the cremaster muscle 1A (Hill et al., 1992). Effects in the presence of mibefradil are shown on the left and in the presence of nifedipine in panels on the right. Results are presented as mean±s.e.mean; ^*P<0.05 compared to corresponding value in the absence of antagonist; ΔP<0.05 compared to value at 10 mmHg at a given antagonist concentration.