Distinct ryanodine- and inositol 1,4,5-trisphosphate-binding sites in hepatic microsomes

Abstract

A light hepatic microsomal preparation was fractionated by sucrose-density centrifugation into one rough, one intermediate and two smooth fractions. The four fractions were characterized with respect to parameters relevant to Ca2+ sequestration. Ca2(+)-ATPase activity was similar in the rough, intermediate and smooth I fractions, but lower in the smooth II fraction. Ca2+ accumulation was the highest in the smooth I and intermediate fractions. On the other hand, Ca2+ efflux from the rough fraction was several-fold faster than from the smooth I fraction. All four subfractions exhibited specific binding sites for inositol 1,4,5-trisphosphate (IP3) and ryanodine; however, the receptors were especially enriched in the smooth I fraction. The total binding sites for ryanodine in that fraction exceeded the number of binding sites for IP3 by about 10-fold. The two receptors responded differently to pharmacological agents; caffeine and dantrolene strongly inhibited ryanodine binding but not IP3 binding, whereas heparin inhibited IP3 binding only. Thus the two receptors are distinct entities. The four fractions also showed distinct gel electrophoretic patterns. The use of two different SDS/polyacrylamide-gel gradients and two protein-staining methods revealed major differences in the distribution of the bands corresponding to Mr values of (x 10(-3) 380, 320, 260, 170, 90, 29 and 21. These proteins were enriched in the smooth fraction. The results indicate that the smooth I fraction might have special importance in stimulus-evoked Ca2(+)-release processes.

Expression and subcellular localization of the ryanodine receptor in rat pancreatic acinar cells

“…However, the polarity of the distribution of the fluorescent tag was not examined. In addition, ryanodine-binding sites do not always reflect the location of RyR itself, as evidenced by the fact that hepatocyte microsomes contain ryanodine-binding sites [30,31], yet hepatocytes do not express RyR [15]. In another recent study, immunochemistry was used to examine RyR expression in pancreatic acini, but no labelling was detected [8].…”

Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin

“…The gradual increase in lCa2+Ii induced by exposure to thapsigargin in glial cells is similar to that seen in other cell types (Law et al, 1990;Thastrup et al, 1990), and is consistent with the inhibition of an endoplasmic reticulum Ca' ' -ATPase and subsequent leakage of Ca2 ' from intracellular stores into the cytoplasm. In contrast, studies of brain, hepatic, and skeletal muscle microsomal preparations provide evidence that dantrolene inhibits Ca' ' -induced Ca2+ release but does not affect IPS-mediated Ca2-release (Ohta et al, 1990;Palade et al, 1989;Shoshan-Barmatz et al, 1990). The effect of dantrolene on intracellular Ca" ' release in sympathetic neurons has been reported to require illumination with ultraviolet light in the range of 335-385 nm (Nohmi et al, 1991).…”

“…Dantrolene has also been shown to have effects on brain cell function in vitro, including inhibition of long-term potentiation in hippocampal slices (Obenhaus et al, 1989) and protection against glutamate toxicity in neurons (Frandsen and Schousboe, 1991). Studies using microsomal preparations have shown that dantrolene specifically inhibits Ca'+-induced Ca" release without affecting IP,-mediated Ca2+ release (Ohta et al, 1990;Palade et al, 1989) and inhibits binding of ryanodine but not IP, to intracellular membrane binding sites (Shoshan-Barmatz et al, 1990). Thapsigargin is a specific inhibitor of endoplasmic reticulum CaZt -ATPases, which causes a leakage of Ca'+ from intracellular IP,-sensitive stores (Lytton et al, 1991;Thastrup et al, 1990).…”

Peptide Probe of Ryanodine Receptor Function IMPERATOXIN A, A PEPTIDE FROM THE VENOM OF THE SCORPION PANDINUS IMPERATOR, SELECTIVELY ACTIVATES SKELETAL-TYPE RYANODINE RECEPTOR ISOFORMS

“…RyRs are also found in neurons (2,3), exocrine cells (4), smooth muscle cells (5,6), epithelial cells (7), lymphocytes (8), and sea urchin eggs (9). In all of these cells, RyRs play a central role in the regulation of the intracellular free Ca 2ϩ concentration, whose elevation triggers a cascade of events that culminates in muscle contraction, hormone secretion, lymphocyte activation, egg fertilization, etc.…”