Inhibition of 25-hydroxyvitamin D3-24-hydroxylase by forskolin: evidence for a 3',5'-cyclic adenosine monophosphate-independent mechanism.
Forskolin has long been used to demonstrate the involvement of cAMP in the regulation of cellular function, by virtue of its ability to stimulate adenylate cyclase directly. Recently, however, forskolin has been shown to affect plasma membrane transporter and channel function in a manner unrelated to cAMP. The present study examines whether forskolin-mediated inhibition of a mitochondrial membrane-associated enzyme, 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), also occurs by a cAMP-independent mechanism. Both forskolin and PTH stimulated cAMP accumulation and inhibited 24-hydroxylase activity in a dose-dependent manner in fresh mouse renal tubules. However, the level of inhibition of 24-hydroxylase achieved with forskolin was consistently greater than that obtained with PTH, at comparable levels of cAMP. 1',9'-Dideoxyforskolin, a cyclase-inactive analog of forskolin, also inhibited 24-hydroxylase activity, without stimulating cAMP production. Moreover, both forskolin and 1',9'-dideoxyforskolin directly inhibited 24-hydroxylase in isolated renal mitochondria. Kinetic analysis revealed a competitive mode of inhibition for both agents; however, 1',9'-dideoxyforskolin proved to be a more potent inhibitor of 24-hydroxylase than forskolin (inhibitory constant, 0.25 vs. 22 microM, respectively). Finally, both forskolin and 1',9'-dideoxyforskolin also inhibited inducible 24-hydroxylase in renal tubules prepared from 1,25-(OH)2D3-treated mice. However, inducible 24-hydroxylase activity was less susceptible to inhibition by the diterpenes than the basal enzyme activity. The present study provides evidence for cAMP-independent inhibition of 24-hydroxylase by forskolin and represents the first demonstration of a cAMP-independent effect of forskolin on a protein that is not a plasma membrane-associated transporter or channel. Our data advocate caution in the interpretation of studies using forskolin to assess the role of cAMP in cellular processes.
Mandla
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Department of Pediatrics, McGill University, McGill University-Montreal Children's Hospital Research Institute, Quebec, Canada.
Tenenhouse
H S
HS
D000818:Animals; D002117:Calcitriol / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000502:physiology*; D065607:Cytochrome P-450 Enzyme Inhibitors; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010281:Parathyroid Hormone / Q000494:pharmacology; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors*; D065668:Vitamin D3 24-Hydroxylase
D065607:Cytochrome P-450 Enzyme Inhibitors; D010281:Parathyroid Hormone; D005576:Colforsin; D000242:Cyclic AMP; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol