Conditional inhibition of forskolin-activated adenylate cyclase by guanosine diphosphate and its analog.

Forskolin-activated adenylate cyclases (AC) in intact membranes, solubilized with Lubrol or eluted following adsorption on a forskolin-Sepharose column, were examined for inhibition by GDP and GDP beta S. AC in intact membranes of rat or rabbit adipocytes was activated by 100 microM forskolin and further potentiated by 10 microM Gpp(NH)p in combination with either 230 microM epinephrine or 50 mU X ml-1 ACTH. GDP (0-1 mM) or GDP beta S (0-500 microM) inhibited activation in a dose-dependent manner to a level similar to or slightly below that produced by 100 microM forskolin alone. Forskolin at 100 microM stimulated solubilized AC of rabbit adipocytes and rat liver membranes for 10 +/- 4 to 160 +/- 10 and from 26 +/- 2 to 274 +/- 21 pmol(mg X min)-1, respectively, in the absence of GDP beta S; forskolin-activated activity decreased from 160 +/- 10 to 157 +/- 6 and from 274 +/- 21 to 238 +/- 14 pmol(mg X min)-1 in the presence of 500 microM GDP beta S. Forskolin-activated solubilized enzyme was further potentiated by 10 microM Gpp(NH)p from 160 +/- 10 to 289 +/- 52 and from 274 +/- 21 to 702 +/- 50 pmol(mg X min)-1. GDP beta S at 500 microM inhibited 93 and 103% of the Gpp(NH)p-potentiated activity. AC of rat adipocytes eluted from forskolin-Sepharose affinity column with 500 mM NaCl and 100 microM forskolin was not significantly activated by Gpp(NH)p nor inhibited by GDP beta S. However, it was activated by forskolin. The lack of inhibition of unmodified forskolin-activated activity by GDP or GDP beta S in contrast to the inhibition of Gpp(NH)p-activated enzyme or Gpp(NH)p-potentiated forskolin-activated enzyme may be a general phenomenon descriptive of the action of forskolin on AC. Furthermore, inhibition of forskolin-activated AC by GDP and its analog may be a useful index in analyzing the degree of guanine nucleotide potentiation of this enzyme.