IMMUNOGOLD LOCALIZATION OF INOSITOL 1,4,5-TRISPHOSPHATE RECEPTORS ANDCHARACTERIZATION OF ULTRASTRUCTURAL FEATURES OF THE SARCOPLASMIC-RETICULUM IN PHASIC AND TONIC SMOOTH-MUSCLE

Although agonist stimulation leads to an increase in inositol 1,4,5-tr isphosphate (InsP(3)) and decreased calcium in peripherally and centra lly located sarcoplasmic reticulum in smooth muscle, the distribution of InsP(3) receptors is unknown. InsP(3) receptor and the calcium bind ing protein, calsequestrin were localized by immunolabelling in a toni c and a phasic smooth muscle. InsP(3) receptor labelling was predomina ntly localized at the cell periphery, where most of the sarcoplasmic r eticulum is localized in vas deferens (phasic muscle). Elements of cen tral sarcoplasmic reticulum, where present, were also labelled. Distri bution of calsequestrin in vas deferens was similar to that of the Ins P(3) receptor. In aorta (tonic muscle) the InsP(3) receptor labelling was proportional to sarcoplasmic reticulum distribution: predominantly central. No labelling of sections or immunoblots was observed with th e anti-calsequestrin antibody in aorta. InsP(3) and caffeine, but not cyclic ADP-ribose, released intracellular Ca2+ in permeabilized vas de ferens and aorta. The ultrastructure of the sarcoplasmic reticulum, in vestigated in stereo views of semi-thick and thin sections of osmium f erricyanide stained tissue, is shown to have several distinctive featu res, such as fenestrated sheets (single or in stacks), as well as nume rous regions of continuity between central and peripheral sarcoplasmic reticulum, suggesting a single compartment within the smooth muscle c ell. Regions of the sarcoplasmic reticulum were closely apposed to and often ensheathed mitochondria. We conclude that InsP(3) receptors are present in both the central and the peripheral sarcoplasmic reticulum of tonic and phasic smooth muscle, consistent with electron probe ana lysis results showing calcium release from both regions.