Comparative localization of inositol 1,4,5-trisphosphate and ryanodine receptors in intestinal smooth muscle: an analytical subfractionation study.
[3H]Ins(1,4,5)P3- and [3H]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 [3H]Ins(1,4,5)P3-binding sites (KD 80 nM), whereas microsomal (P) fractions contained only high-affinity binding sites (KD 5 nM). Total sedimentable high-affinity binding sites of [3H]Ins(1,4,5)P3 were 9-10-fold more numerous than those of [3H]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)P3 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)P3 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)P3 receptors were somewhat concentrated in the ribosome-coated portions of ER. The low overall stoichiometric ratio of ryanodine to Ins(1,4,5)P3 receptors in intestinal smooth muscle (1:9-10) might explain, at least partly, the existence of a Ca(2+)-storage compartment devoid of ryanodine-sensitive Ca2+ channels, but equipped with Ins(1,4,5)P3-sensitive channels, in saponin-permeabilized smooth-muscle cells [Iino, Kobayashi and Endo (1988) Biochem. Biophys. Res. Commun. 152, 417-422].
Wibo
M
M
Laboratoire de Pharmacologie, Université Catholique de Louvain, Brussels, Belgium.
Godfraind
T
T
D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D002500:Centrifugation, Isopycnic; D004072:Digitonin / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D006168:Guinea Pigs; D006493:Heparin / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D007425:Intracellular Membranes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel; D013347:Subcellular Fractions / Q000201:enzymology
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009124:Muscle Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D019837:Ryanodine Receptor Calcium Release Channel; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D004072:Digitonin