Mechanisms of blockade of two types of Ca2+ channels by the organic Ca2+ antagonists, nicardipine, diltiazem, verapamil, and flunarizine, were examined in rat aorta smooth muscle cells in primary culture by using the whole-cell voltage-clamp method. T-type Ca2+ current (T-type I(Ca)) was isolated by an internal perfusion of 5 mM F-, which irreversibly suppressed the L-type I(Ca), without affecting T-type I(Ca). L-type I(Ca) was isolated by setting a holding potential at -60 mV, at which most of the T-type Ca2+ channels were inactivated. L-type I(Ca) is halved by 0.1 μM nicardipine, 3.0 μM diltiazem, 0.6 μM verapamil, and 0.1 μM flunarizine, whereas T-type I(Ca) is halved by the same drugs at 0.6, 30, 30, and 0.1 μM, respectively. Diltiazem and verapamil accelerated the decay of L-type I(Ca) and cumulatively blocked L-type I(Ca) during repetitive step depolarizations elicited every 30 seconds ('use-dependent block'). Diltiazem and verapamil neither changed the decay of T-type I(Ca) nor showed a use-dependent block of T-type I(Ca). Nicardipine and flunarizine blocked both L- and T-type I(Ca) from the first depolarization step after drug treatment ('tonic block') and shifted their steady-state inactivation curves to the left. The estimated binding constants of nicardipine and flunarizine for the inactivated state of T-type Ca2+ channels (48 and 19 nM, respectively), were smaller than those for the resting states of L-type Ca2+ channels (160 and 90 nM, respectively). A low concentration (0.1 μM) of nicardipine initially potentiated T-type I(Ca) and then reduced it. We conclude from these results that 1) nicardipine and flunarizine block not only the resting state but, more preferentially, the inactivated state of both the L- and T-type Ca2+ channels; 2) verapamil and diltiazem preferentially act on the open state of the L-type Ca2+ channel and on the resting and inactivated state of the T-type Ca2+ channel; and 3) the T-type Ca2+ channel of the rat aorta smooth muscle cells appears to be more sensitive to nicardipine and flunarizine than does the L-type Ca2+ channel at around the resting membrane potential.
All Science Journal Classification (ASJC) codes
- Cardiology and Cardiovascular Medicine
- Calcium antagonist
- Calcium channel
- Vascular smooth muscle cell
- Whole-cell clamp method