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3':5'-cyclic guanosine monophosphate (cGMP) potentiates the inositol 1,4,5-trisphosphate-evoked Ca2+ release in guinea-pig hepatocytes.

ABSTRACT: The effect of cGMP on noradrenaline-induced intracellular Ca2+ mobilization was investigated in whole-cell voltage-clamped guinea-pig hepatocytes. Treatment of the cells with 8-Br-cGMP (1-500 microM) resulted in an increase in the sensitivity of the cells to noradrenaline and to inositol 1,4,5-trisphosphate (InsP3) photo-released from caged InsP3. The positive effect of 8-Br-cGMP on the Ca2+ release evoked by Ca(2+)-mobilizing agonists or InsP3 was blocked by a protein kinase G (PKG; cGMP-dependent protein kinase) inhibitor, the RP-8-(4-chlorophenylthio)guanosine 3':5'-monophosphorothioate. 8-Br-cGMP affected neither the basal InsP3 concentration nor the noradrenaline-induced production of InsP3. In permeabilized hepatocytes, the dose-response curve for InsP3-induced Ca2+ release was shifted to the left in the presence of 8-Br-cGMP. Furthermore, the treatment with 8-Br-cGMP did not affect the Ca2+ content of the InsP3-sensitive Ca2+ stores. These results indicate that intracellular cGMP potentiates the noradrenaline-induced Ca2+ response by enhancing Ca2+ release from the intracellular Ca2+ stores. We suggest that cGMP increases the apparent affinity of InsP3 receptors for InsP3 in guinea-pig hepatocytes probably by phosphorylation via the activation of PKG.

SUBMITTER: Guihard G

PROVIDER: S-EPMC1217695 | biostudies-other | 1996 Sep

REPOSITORIES: biostudies-other

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Project description:Osteoclast activity is central to balanced bone turnover to maintain normal bone mass. A specialized osteoclast attachment to bone localizes acid secretion to remove bone mineral; in some cases, attachment is functionally impaired despite normal attachment proteins. The inositol-1,4,5-trisphosphate receptor-1 (IP3R1) is an intracellular calcium channel required for regulation of reversible osteoclast attachment by nitric oxide (NO), an important regulator of both normal and pathological bone degradation. In studies using human osteoclasts produced in vitro, we found that IP3R1 binds an endosomal isoform of the IP3R-associated cGMP-dependent kinase substrate (IRAG). IRAG is a substrate of cGMP-dependent kinase-1 (PKG1) and binds the PKG1 isoform PKG1β, which was the predominant form of PKG1 in human osteoclasts. Western blots of IRAG were consistent with NO-dependent serine phosphorylation of IRAG. An additional effect of PKG1β activity in osteoclasts was disassociation of IP3R1-IRAG complexes, as shown by analysis of IP3R1 complexes and by localization of the proteins within cells. IP3R1-IRAG complexes were stabilized by PKG or Src antagonists, Src activity being a requirement for IP3R1 calcium release downstream of PKG. IP3R1-mediated calcium release regulates cellular detachment in part through the calcium-dependent proteinase μ-calpain. In osteoclasts with IRAG suppressed by siRNA, activity of μ-calpain was increased relative to cells with normal IRAG, and regulation of μ-calpain by NO was lost. Furthermore, cells deficient in IRAG detached easily from substrate and had smaller attached diameters and randomly distributed podosomes, although IRAG knockdown did not affect cell viability. Our results indicate that IRAG is required for PKG1β-regulated cyclic calcium release during motility, and that disruption of the IP3R1-IRAG calcium regulation system is a novel cause of dysfunctional osteoclasts unrelated to defects in attachment proteins or acid secretion.

Project description:Metabolism of inositol 1,4,5-trisphosphate was investigated in permeabilized guinea-pig hepatocytes. The conversion of [3H]inositol 1,4,5-trisphosphate to a more polar 3H-labelled compound occurred rapidly and was detected as early as 5 s. This material co-eluted from h.p.l.c. with inositol 1,3,4,5 tetrakis[32P]phosphate and is presumably an inositol tetrakisphosphate. A significant increase in the 3H-labelled material co-eluting from h.p.l.c. with inositol 1,3,4-trisphosphate occurred only after a definite lag period. Incubation of permeabilized hepatocytes with inositol 1,3,4,5-tetrakis[32P]phosphate resulted in the formation of 32P-labelled material that co-eluted with inositol 1,3,4-trisphosphate; no inositol 1,4,5-tris[32P]phosphate was produced, suggesting the action of a 5-phosphomonoesterase. The half-time of hydrolysis of inositol 1,3,4,5-tetrakis[32P]phosphate of approx. 1 min was increased to 3 min by 2,3-bisphosphoglyceric acid. Similarly, the rate of production of material tentatively designed as inositol 1,3,4-tris[32P]phosphate from the tetrakisphosphate was reduced by 10 mM-2,3-bisphosphoglyceric acid. In the absence of ATP there was no conversion of [3H]inositol 1,4,5-trisphosphate to [3H]inositol tetrakisphosphate or to [3H]inositol 1,3,4-trisphosphate, which suggests that the 1,3,4 isomer does not result from isomerization of inositol 1,4,5-trisphosphate. The results of this study suggest that the origin of the 1,3,4 isomer of inositol trisphosphate in isolated hepatocytes is inositol 1,3,4,5-tetrakisphosphate and that inositol 1,4,5-trisphosphate is rapidly converted to this tetrakisphosphate. The ability of 2,3-bisphosphoglyceric acid, an inhibitor of 5-phosphomonoesterase of red blood cell membrane, to inhibit the breakdown of the tetrakisphosphate suggests that the enzyme which removes the 5-phosphate from inositol 1,4,5-trisphosphate may also act to convert the tetrakisphosphate to inositol 1,3,4-trisphosphate. It is not known if the role of inositol 1,4,5-trisphosphate kinase is to inactivate inositol 1,4,5-trisphosphate or whether the tetrakisphosphate product may have a messenger function in the cell.

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3':5'-cyclic guanosine monophosphate (cGMP) potentiates the inositol 1,4,5-trisphosphate-evoked Ca2+ release in guinea-pig hepatocytes.

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