Separate activation of intracellular Ca2+ release, voltage-dependent and receptor-operated Ca2+ channels in the rat aorta.

Both intracellular Ca2+ release and Ca2+ influx through plasma membrane channels contribute jointly to the elevation of cytoplasmic Ca2+ levels which regulate the contractility of vascular smooth muscle in response to vasoconstrictors. The objective of the present study was to design new protocols to experimentally separate three Ca(2+)-elevating pathways involved in the noradrenaline-induced contractile response, intracellular Ca2+ release, Ca2+ influx through the voltage-dependent (VDCCs) and the receptor-operated (ROCCs) Ca2+ channels with Ca2+ channel blockers in the isolated rat aortic rings. VDCCs were simply activated by membrane depolarization with 60 mM K+ solution and Ca2+ release from internal stores was reflected by the noradrenaline-induced contraction in Ca(2+)-free, high K4 solution. Nifedipine, diltiazem and verapamil concentration-dependently inhibited VDCCs and intracellular Ca2+ release with high affinity. ROCCs were activated by the following procedures: the noradrenaline-induced contractile response due to intracellular Ca2+ release was fully reversed by nifedipine at 100 nM, diltiazem at 3 microM and verapamil at 10 microM, the concentration of each drug was 10-fold greater than that causing a complete inhibition of VDCCs. Under this condition, addition of 1 mM Ca2+ induced a second sustained contraction which was fully reversed by 0.3 microM prazosin. The second contractile response was most likely caused by Ca2+ influx through the noradrenaline-activated ROCCs. Higher concentrations of diltiazem and verapamil were needed to inhibit ROCCs, and nifedipine was without effect. The sensitivity order of Ca2+ channel blockers for three Ca(2+)-elevating pathways was the same: VDCCs > intracellular Ca2+ release > ROCCs. The present results indicate that VDCCs can be functionally separated from ROCCs with the experimental designs used in this study and noradrenaline evoked contractions of rat aorta by activating intracellular Ca2+ release and Ca2+ influx through VDCCs and ROCCs. However, Ca2+ influx via ROCCs makes a small contribution to the noradrenaline-induced contractile response.