MICROMOLAR CALCIUM DECREASES AFFINITY OF INOSITOL TRISPHOSPHATE RECEPTOR IN VASCULAR SMOOTH-MUSCLE

The mechanism by which Ca2+ inhibits InsP(3)-induced Ca2+ release from sarcoplasmic reticulum of vascular smooth muscle was investigated. In sP(3) binding to sarcoplasmic-reticulum vesicles from dog aortic smoot h muscle was inhibited by 51 +/- 6% by 2 mu M Ca2+ in the presence of 10 nM [H-3]InsP(3). Scatchard analysis indicated the presence of two I nsP(3)-binding sites in the absence of Ca2+ (K-d = 2.5 +/- 0.9 and 49 +/- 8 nM InsP(3)), though the low-affinity site was more prevalent (re presenting 92 +/- 3% of the total number of binding sites). Ca2+ (2 mu M) did not alter InsP(3) binding to the high-affinity site (P > 0.05) , but increased the K-d of the low-affinity site 3-fold (K-d = 155 +/- 4 nM InsP(3); P < 0.001). The possibility that the apparent decrease in InsP(3) affinity was caused by Ca2+-dependent activation of an endo genous phospholipase C could be excluded, because the Ca2+-dependent i nhibition of InsP(3) binding was completely reversible and insensitive to an inhibitor of phospholipase C. Moreover, Ca2+ did not inhibit In sP(3) binding to InsP(3) receptor partially purified by heparin-Sephar ose chromatography, though another fraction (devoid of InsP(3) recepto r) restored Ca2+-sensitivity of the partially purified InsP(3) recepto r. Thus Ca2+ binding to a Ca2+-sensitizing factor associated with the InsP(3) receptor decreases the affinity of the receptor complex for In sP(3). This Ca2+-sensitizing factor may provide a negative-feedback me chanism for regulating the rise in cytosolic Ca2+ concentration in vas cular smooth muscle after hormone activation of the phosphoinositide c ascade.