COMPARATIVE LOCALIZATION OI INOSITOL 1,4,5-TRISPHOSPHATE AND RYANODINE RECEPTORS IN INTESTINAL SMOOTH-MUSCLE - AN ANALYTICAL SUBFRACTIONATION STUDY

[H-3]Ins(1,4,5)P-3- and [H-3]ryanodine-binding sites were characterized in membrane fractions from guinea-pig intestinal smooth muscle (longitudinal layer) and their subcellular localization was investigated by analytical cell-fractionation techniques. Fractions collected at low centrifugal fields (N and M fractions) contained predominantly low-affinity [H-3]Ins(1,4,5)P-3-binding sites (K-D 80 nM), whereas microsomal (P) fractions contained only high-affinity binding sites (K-D 5 nM). Total sedimentable high-affinity binding sites of [H-3]Ins(1,4,5)P-3 were 9-10-fold more numerous than those of [H-3]ryanodine. Both high-affinity binding sites were purified in microsomal fractions, and their sub-microsomal distribution patterns after isopycnic density-gradient centrifugation were similar to those of presumed endoplasmic reticulum (ER) constituents, indicating that Ins(1,4,5)P-3 and ryanodine receptors were localized primarily in ER and probably associated with rough as well as smooth ER. However, the stoichiometric ratio of Ins(1,4,5)P-3 to ryanodine receptors was distinctly higher in high-density RNA-rich subfractions than in low-density RNA-poor subfractions, suggesting that Ins(1,4,5)P-3 receptors were somewhat concentrated in the ribosome-coated portions of ER. The low overall stoichiometric ratio of ryanodine to Ins(1,4,5)P-3 receptors in intestinal smooth muscle (1:9-10) might explain, at least partly, the existence of a Ca2+-storage compartment devoid of ryanodine-sensitive Ca2+ channels, but equipped with Ins(1,4, S)P-3-sensitive channels, in saponin-permeabilized smooth-muscle cells [Iino, Kobayashi and Endo (1988) Biochem. Biophys. Res. Commun. 152, 417-422].