Investigation of the role of sigma1-receptors in inositol 1,4,5-trisphosphate dependent calcium signaling in hepatocytes.

In hepatocytes, as in other cell types, Ca(2+) signaling is subject to complex regulations, which result largely from the intrinsic characteristics of the different inositol 1,4,5-trisphosphate receptor (InsP(3)R) isoforms and from their interactions with other proteins. Although sigma1 receptors (Sig-1Rs) are widely expressed in the liver, their involvement in hepatic Ca(2+) signaling remains unknown. We here report that in this cell type Sig-1R interact with type 1 isoforms of the InsP(3) receptors (InsP(3)R-1). These results obtained by immunoprecipitation experiments are confirmed by the observation that Sig-1R proteins and InsP(3)R-1 colocalize in hepatocytes. However, Sig-1R ligands have no effect on InsP(3)-induced Ca(2+) release in hepatocytes. This can be explained by the rather low expression level expression of InsP(3)R-1. In contrast, we find that Sig-1R ligands can inhibit agonist-induced Ca(2+) signaling via an inhibitory effect on InsP(3) synthesis. We show that this inhibition is due to the stimulation of PKC activity by Sig-1R, resulting in the well-known down-regulation of the signaling pathway responsible for the transduction of the extracellular stimulus into InsP(3) synthesis. The PKC sensitive to Sig-1R activity belongs to the family of conventional PKC, but the precise molecular mechanism of this regulation remains to be elucidated.

Effects of the prostaglandins PGF2alpha and PGE2 on calcium signaling in rat hepatocyte doublets.

Coordination of intercellular Ca2+ signals is important for certain hepatic functions including biliary flow and glucose output. Prostaglandins, such as PGF2alpha and PGE2, may modify these hepatocyte functions by inducing Ca2+ increase, but very little is known about the organization of the Ca2+ signals induced by these agonists. We studied Ca2+ signals induced by PGF2alpha and PGE2 in fura-2 AM-loaded hepatocyte doublets. Even though both prostaglandins induced Ca2+ oscillations, neither PGF2alpha nor PGE2 induced coordinated Ca2+ oscillations in hepatocyte doublets. Gap junction permeability (GJP), assessed by fluorescence recovery after photobleaching, showed that this absence of coordination was not related to a defect in GJP. Inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] assays and the increase in Ins(1,4,5)P3 receptor sensitivity to Ins(1,4,5)P3 observed in response to thimerosal suggested that the absence of coordination was a consequence of the very small quantity of Ins(1,4,5)P3 formed by these prostaglandins. Furthermore, when PGE2 and PGF2alpha were added just before norepinephrine, they favored the coordination of Ca2+ signals induced by norepinephrine. However, GJP between hepatocyte doublets was strongly inhibited by prolonged (>or=2 h) treatment with PGF2alpha, thereby preventing the coordination of Ca2+ oscillations induced by norepinephrine in these cells. Thus, depending on the time window, prostaglandins, specially PGF2alpha, may enhance or diminish the propagation of Ca2+ signals. They may therefore contribute to the fine tuning of Ca2+ wave-dependent functions, such as nerve stimulation, hormonal regulation of liver metabolism, or bile secretion, in both normal and pathogenic conditions.

Ca2+ oscillations in hepatocytes do not require the modulation of InsP3 3-kinase activity by Ca2+.

Receptor-mediated production of inositol 1,4,5-trisphosphate (InsP(3)) initiates Ca(2+) release and is responsible for cytosolic Ca(2+) oscillations. InsP(3) oscillations have also been observed in some cells. One of the enzymes controlling InsP(3) catabolism, the InsP(3) 3-kinase, is stimulated by Ca(2+); this regulation is presumably part of the reason for InsP(3) oscillations that have been observed in some cells. Here, we investigate the possible role of Ca(2+)-activated InsP(3) catabolism on the characteristics of the InsP(3)-induced Ca(2+) oscillations. Numerical simulations show that if it is assumed that the Ca(2+)-independent InsP(3) catabolism is predominant, Ca(2+) oscillations remain qualitatively unchanged although the relative amplitude of the oscillations in InsP(3) concentrations becomes minimal. We tested this prediction in hepatocytes by masking the Ca(2+)-dependent InsP(3) catabolism by 3-kinase through the injection of massive amounts of InsP(3) 5-phosphatase, which is not stimulated by Ca(2+). We find that in such injected hepatocytes, Ca(2+) oscillations generated by modest agonist levels are suppressed, presumably because of the decreased dose in InsP(3), but that at higher doses of agonist, oscillations reappear, with characteristics similar to those of untreated cells at low agonist doses. Altogether, these results suggest that oscillations in InsP(3) concentration due to Ca(2+)-stimulated InsP(3) catabolism do not play a major role for the oscillations in Ca(2+) concentration.