Modulation of dihydropyridine receptors in vascular smooth muscle cells by membrane potential and cell proliferation

We have studied binding of isradipine to A7r5 vascular smooth muscle cells as a function of membrane potential and cell proliferation. Consistent with a voltage-modulated receptor model, two classes of binding sites were detected in confluent cultures: high-affinity sites under depolarizing (50 mM K⁺) conditions (K_d = 45 ± 3 pM), and lower affinity sites under resting (5 mM K⁺) conditions (K_d = 181 ± 20 pM). However, proliferating cells also displayed the high-affinity state at rest (K_d = 29 ± 9 pM) in addition to a low-affinity site (K_d = 869 ± 383 pM). Analysis of dissociation rates also revealed two receptor classes during proliferation. Proliferating cells showed a single class of high-affinity sites (K_d = 39 ± 6 pM) when depolarized, similar to confluent cells. Receptor density in confluent monolayers increased from 15 ± 3 fmol/10⁶ cells at 5 days to 72 ± 6 fmol/10⁶ cells after 10 days. These results suggest (i) that some L-type Ca²⁺ channels are spontaneously active in proliferating vascular smooth muscle cells, but require depolarization to activate in a confluent monolayer, and (ii) that the density of dihydropyridine receptors increases after a monolayer becomes confluent.