Kenneth B. Seamon

The diterpene, forskolin [half-maximal effective concentration (EC50), 5-10 microM] activates adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in rat cerebral cortical membranes in a rapid and reversible manner. Activation is not dependent on exogenous guanyl nucleotides and is not inhibited by guanosine 5'-O-(2-thiodiphosphate) when assayed with adenosine 5'-[beta, gamma-imido]triphosphate as substrate. GTP and GDP potentiate responses to forskolin. The activations of adenylate cyclase by forskolin and guanosine 5'-[beta, gamma-imido]triphosphate p[NH]ppG are not additive, whereas activations by forskolin and fluoride are additive or partially additive. The responses of adenylate cyclase to forskolin or fluoride are not inhibited by manganese ions, whereas the response to p[NH]ppG is completely blocked. Activation of adenylate cyclase by forskolin is considerably greater than the activation by fluoride in membranes from rat cerebellum, striatum, heart, and liver, while being about equal or less than the activation by fluoride in other tissues. Forskolin (EC50, 25 microM) causes a rapid and readily reversible 35-fold elevation of cyclic AMP in rat cerebral cortical slices that is not blocked by a variety of neurotransmitter antagonists. Low concentrations of forskolin (1 microM) augment the response of cyclic AMP-generating systems in brain slices to norepinephrine, isoproterenol, histamine, adenosine, prostaglandin E2, and vasoactive intestinal peptide. Forskolin would appear to activate adenylate cyclase through a unique mechanism involving both direct activation of the enzyme and facilitation or potentiation of the modulation of enzyme activity by receptors or the guanyl nucleotide-binding subunit, or both.

Forskolin, a novel diterpene activator of adenylate cyclase in membranes and intact cells, activates the enzyme in membranes from mutant cyc-S49 murine lymphoma cells and the soluble enzyme from rat testes. Each of these enzymes consists only of the catalytic subunit and does not have a functional guanine nucleotide-binding protein. In both cases forskolin converts the manganese-dependent enzymes to a form which does not require manganese for activity. Forskolin can also stimulate a detergent-solubilized preparation of adenylate cyclase from rat cerebral cortex. Activation of adenylate cyclase by forskolin is therefore not dependent on a perturbation of membrane structure nor does it require a functional guanine nucleotide-binding subunit.

Forskolin, a diterpene of the labdane family, activates adenylate cyclase in broken cell preparations as well as in intact tissues. This activation does not require the guanine nucleotide regulatory subunit of the enzyme and probably occurs via an interaction with the catalytic subunit of adenylate cyclase. Activation of adenylate cyclase by forskolin results in marked increases in levels of intracellular cyclic AMP in a variety of eukaryotic cells. Low concentrations of forskolin which alone elicit small increases in intracellular cyclic AMP greatly potentiate hormonal activation of adenylate cyclase in a number of intact cells. Forskolin elicits cellular responses which have been proposed to be dependent o cyclic AMP as a second messenger. Forskolin, thus provides an invaluable tool for the investigation of the role of cyclic AMP in physiological responses to hormones, both through it direct activation of adenylate cyclase and through its ability to potentiate hormonal activation of adenylate cyclase.