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Regulation by forskolin of cyclic AMP phosphodiesterase and protein kinase C activity in LLC-PK1 cells.

ABSTRACT: Forskolin, a naturally occurring activator of adenylate cyclase, inhibits total and high-affinity cyclic AMP phosphodiesterase activity in soluble and particulate fractions of cultured LLC-PK1 renal epithelial cells. The naturally occurring forskolin analogue 1,9-dideoxyforskolin, which does not stimulate adenylate cyclase activity, is a more potent inhibitor of cyclic AMP phosphodiesterase activity than forskolin. To clarify the structural feature of the forskolin molecule responsible for inhibition of cyclic AMP phosphodiesterase activity, the effects of two agents which share structural identity with portions of the forskolin ring were tested. The steroid 5-pregnenolone, but not the hexose alpha-D-galactose, inhibited cyclic AMP phosphodiesterase activity in LLC-PK1 cells. Forskolin and 1,9-dideoxyforskolin both stimulate protein kinase C activity in LLC-PK1 cells. The effect of 1,9-dideoxyforskolin in stimulating LLC-PK1 protein kinase C activity can be attenuated by staurosporine. Both 5-pregnenolone and alpha-D-galactose also stimulate protein kinase C activity in LLC-PK1 cells. 5-Pregnenolone and the phorbol ester phorbol 12-myristate 13-acetate cause translocation of protein kinase C from a soluble to a particulate fraction, while both 1,9-dideoxyforskolin and alpha-D-galactose increase protein kinase C activity in both soluble and particulate fractions. Our results demonstrate that forskolin exerts diverse enzymic effects in cultured LLC-PK1 cells.

SUBMITTER: Anderson RJ

PROVIDER: S-EPMC1151541 | biostudies-other | 1991 Oct

REPOSITORIES: biostudies-other

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Project description:To investigate substrates for cyclic AMP-dependent protein kinase in intact islets of Langerhans, batches of islets were incubated with [32P]Pi for 1 h in the presence of 10 mM-glucose; the adenylate cyclase activator forskolin, which in parallel experiments was shown to increase islet cyclic AMP content and insulin release, was then added. Islets were homogenized and subcellular fractions prepared by differential centrifugation. Phosphopeptides were electrophoresed on sodium dodecyl sulphate/polyacrylamide gels and quantified by autoradiography and densitometry. Within 5 min forskolin caused increased labelling of Mr-25 000 and -30 000 cytosolic and Mr-23 000 and -32 000 particulate peptides; a rapid decrease in phosphorylation of Mr-18 000 and -34 000 cytosolic peptides was also observed. In addition, rather slower phosphorylation occurred of the Mr-15 000 peptide previously identified as histone H3 [Christie & Ashcroft (1984) Biochem. J. 218, 87-99]. When similar subcellular fractions were incubated with [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase, peptides phosphorylated included cytosolic species of Mr 25 000 and 30 000 and particulate species of Mr 23 000 and 32 000. The distribution of RNA in the subcellular fractions suggested that the Mr-32 000 species could be a ribosomal protein. The 24 000 g pellet was heterogeneous, as judged by marker assays, and was therefore fractionated further by Percoll-density-gradient centrifugation. The peak containing the Mr-23 000 peptide was resolved from marker enzymes for plasma membranes, mitochondria and endoplasmic reticulum and coincided with a peak for insulin: hence the Mr-23 000 peptide is likely to be a secretory-granule component. The study demonstrates that the potentiation of insulin release that occurs when islet cyclic AMP is increased is accompanied by rapid phosphorylation of specific islet substrates for cyclic AMP-dependent protein kinase. The data are consistent with the hypothesis that protein phosphorylation is involved in the regulation of insulin secretion.

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Regulation by forskolin of cyclic AMP phosphodiesterase and protein kinase C activity in LLC-PK1 cells.

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