Insulin and vasopressin elicit inhibition of cholera-toxin-stimulated adenylate cyclase activity in both hepatocytes and the P9 immortalized hepatocyte cell line through an action involving protein kinase C

Abstract

Incubation of hepatocytes or the SV40-DNA-immortalized hepatocyte P9 cell line with cholera toxin led to a time-dependent activation of adenylate cyclase activity, which occurred after a defined lag period. When added together with cholera toxin, each of the hormones insulin and vasopressin was capable of attenuating the maximum stimulatory effect achieved by cholera toxin over a period of 60 min through a process which could be blocked by the compounds staurosporine and chelerythrine. Attenuating effects on cholera-toxin-stimulated adenylate cyclase activity could also be elicited by using either the protein kinase C (PKC)-stimulating phorbol ester PMA (phorbol 12-myristate 13-acetate) or the protein phosphatase inhibitor okadaic acid. Alkaline phosphatase treatment of membranes reversed the inhibitory effect of PMA. Cholera toxin also stimulated the adenylate cyclase activity of intact CHO (Chinese-hamster ovary) and NIH-3T3 cells, but this activity was insensitive to the addition of PMA. Overexpression of various PKC isoforms in CHO cell lines did not confer sensitivity to inhibition by PMA upon cholera-toxin-stimulated adenylate cyclase activity. Rather, overexpression of the gamma isoform of PKC allowed PMA to stimulate adenylate cyclase activity in CHO cells. It is suggested that the PKC-mediated phosphorylation of a membrane protein attenuates cholera-toxin-stimulated adenylate cyclase activity in hepatocytes and P9 cells. The cellular selectivity of such an action may be due to the target for this inhibitory action of PKC being a particular isoform of adenylate cyclase which provides the major activity in hepatocytes and P9 cells, but not in either CHO or NIH-3T3 cells.

Protein Kinase C Inhibits Adenylyl Cyclase Type VI Activity during Desensitization of the A2a-Adenosine Receptor-mediated cAMP Response

“…Most intriguingly, PKC-evoked phosphorylation allows AC2 to integrate multiple signals produced by G q␣ -and G i␣ -coupled receptors in a G s␣ -independent manner (29). An inhibitory effect of PKC on a specific, unidentified AC subtype which is expressed mainly in hepatocytes has also been reported (30). We have shown in the present study that PKC specifically inhibits AC6 activity through protein phosphorylation.…”

Proteolytic activation of internalized cholera toxin within hepatic endosomes by cathepsin D

“…These competition studies correlated with results obtained from degradation studies using endosomal fractions or pure bovine cathepsin D in which the rate of peptide hydrolysis was found to be low with the A‐subunit and high with the B‐subunit. Interestingly, insulin elicits inhibition of CT‐stimulated adenylate cyclase activity in both hepatocytes and the P9 immortalized hepatocyte cell line [22]. Attenuating effects on CT action may well originate from the ability of both peptides to accumulate into hepatic endocytic vesicles and to interact with endosomal cathepsin D (this study and [12]).…”

Specific features of glycogen metabolism in the liver

“…Other insulin effects that decrease glycogenolysis and promote glycogen synthesis in the liver are not yet understood at the molecular level [64,74,114,115]. These include an inhibition of adenylate cyclase, apparently mediated by protein kinase C, an inactivation of phosphorylase kinase that is independent of changes in the activity of protein kinase A, an increase of the phosphorylase phosphatase activity and an inhibition of αadrenergic signalling.…”