PubMed · 4300737
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J T Fitzsimons. 1968. [Extracellular dehydration].. https://pubmed.ncbi.nlm.nih.gov/4300737/
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Expression of the antigen-regulated, cyclosporin A-sensitive nuclear factor of activated T cells (NFAT) is not restricted to lymphoid cells, as thought initially, but the physiological inducers of NFAT-mediated transcription in non-lymphoid cells are unknown. Here, cultured vascular smooth muscle cells (VSMC) are shown to express two isoforms of the NFAT family endogenously, which are localized differentially in cells under resting conditions. Using a retroviral NFAT-specific luciferase reporter, we show that VSMC support previously unrecognized complexities in NFAT-mediated transcription, including evidence for negative regulation by Ca2+ signaling and positive regulation through co-activation of adenylyl cyclase and Ca2+ mobilization. The VSMC mitogen platelet derived growth factor-BB (PDGF-BB) induces NFAT-mediated transcription in VSMC. Thrombin and angiotensin II, which activate Galphaq-coupled receptors, are significantly weaker inducers of NFAT-mediated luciferase expression than is PDGF-BB. However, co-stimulation studies show that Galphaq receptor agonists augment the NFAT-mediated transcriptional response to PDGF-BB. This synergy can be explained in part by augmented intracellular Ca2+ transients elicited by multiple agonist challenges. These data indicate that agonists for phospholipase C-coupled receptors stimulate NFAT-mediated transcription in VSMC differentially, and that NFAT can function to integrate co-activating signals in the extracellular environment.
Activation of phospholipase C (PLC) is one of the earliest events in angiotensin II (Ang II) type 1 (AT1) receptor (R)-mediated signal transduction in vascular smooth muscle cells (VSMCs). The coupling mechanisms of AT1 Rs to PLC, however, are controversial, because both tyrosine phosphorylation of PLC-gamma and G protein-dependent PLC-beta activation pathways have been reported. The expression of PLC-beta1, furthermore, has not been consistently demonstrated in VSMCs. Here we identified the PLC subtypes and subunits of heterotrimeric G proteins involved in AT1 R-PLC coupling using cultured rat VSMCs. Western analysis revealed the expression of PLC-beta1, -gamma1, and -delta1 in VSMCs. Ang II-stimulated inositol trisphosphate (IP3) formation measured at 15 s, which corresponds to the peak response, was significantly inhibited by electroporation of antibodies against PLC-beta1, but not by anti-PLC-gamma and -delta antibodies. Electroporation of anti-Galphaq/11 and -Galpha12 antibodies also showed significant inhibition of the Ang II-induced IP3 generation at 15 s, while anti-Galphai and Galpha13 antibodies were ineffective. Furthermore, in VSMCs electroporated with anti-Gbeta antibody and cells stably transfected with the plasmid encoding the Gbetagamma-binding region of the carboxyl terminus of beta-adrenergic receptor kinase1, the peak Ang II-stimulated PLC activity (at 15 s) was significantly inhibited. The tyrosine kinase inhibitor, genistein, had no effect on the peak response to Ang II stimulation, but significantly inhibited IP3 production after 30 s, a time period which temporally correlated with PLC-gamma tyrosine phosphorylation in response to Ang II. Moreover, electropor-ation of anti-PLC-gamma antibody markedly inhibited the IP3 production measured at 30 s, indicating that tyrosine phosphorylation of PLC-gamma contributes mainly to the later phase of PLC activation. Thus, these results suggest that: 1) AT1 receptors sequentially couple to PLC-beta1 via a heterotrimeric G protein and to PLC-gamma via a downstream tyrosine kinase; 2) the initial AT1 receptor-PLC-beta1 coupling is mediated by Galphaq/11beta gamma and Galpha12 beta gamma; 3) Gbeta gamma acts as a signal transducer for activation of PLC in VSMCs. The sequential coupling of AT1 receptors to PLC-beta1 and PLC-gamma, as well as dual coupling of AT1 receptors to distinct Galpha proteins, suggests a novel mechanism for a temporally controlled, highly organized and convergent Ang II-signaling network in VSMCs.
Divalent cations are known to affect the activity of the cAMP-generating system. By observing the effects of the addition of cobalt (Co2+) and the depletion of calcium (Ca2+), this study tried to determine the relative contribution of Ca2+-dependent mechanism in glucagon-induced glucose output from the isolated perfused rat liver. Co2+ (1 mM) completely suppressed glucose and cAMP output induced by 0.1 nM glucagon and partly suppressed those induced by 1 to 10 nM glucagon. Co2+ (1-5 mM) did not inhibit 125I-labeled glucagon binding to hepatic cell membrane. Phenylephrine- or angiotensin II-induced glucose output was not affected by 1 mM Co2+. Co2+ (1 mM) inhibited a glucagon-induced increase in [Ca2+]i in isolated rat hepatocytes but did not inhibit a phenylephrine-induced increase in [Ca2+]i. The removal of Ca2+ from the perfusion medium impaired phenylephrine- or angiotensin II-induced glucose output, but did not impair glucagon-induced glucose output. When glucagon-induced cAMP production was inhibited by Co2+, the glucose output produced by 1 to 10 nM glucagon was impaired further in the Ca2+-free perfusion. Addition of 0.1 mM IBMX increased the glucose output produced by 1 nM glucagon but did not increase that produced by 10 nM glucagon in the Co2+-containing Ca2+-free perfusion. These results suggest that Co2+ inhibits the glucagon-responsive adenylyl cyclase system directly, resulting in impaired glucose output. Glucagon increases [Ca2+]i through a mechanism different from that of phenylephrine. Glucagon (0.01-10 nM)-induced glucose output from the liver is derived mainly through a cAMP-dependent mechanism. Only when glucagon-induced cAMP production was inhibited by Co2+ was the Ca2+ dependency observed in high concentrations (>/=1 nM) of glucagon-induced glucose output, and it approximated 30% of the glucose output produced by 10 nM glucagon.