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The issue of whether the adenosine 3',5'-monophosphate (cAMP)-generating system contributes to luteinizing hormone (LH) release was addressed by using several complementary probes in vitro. Pertussis toxin is considered to modify covalently an inhibitory adenylate cyclase regulatory protein. Treatment of gonadotrophs with this toxin increased both basal LH release and the efficacy of gonadotropin-releasing hormone (GnRH)-stimulated LH release with no apparent effect on GnRH potency. Cholera toxin, which probably activates adenylate cyclase by covalently altering another regulatory protein, forskolin, which directly stimulates the catalytic subunit of adenylate cyclase, and the cAMP analogue 8-Br-cAMP amplified both basal LH release (in a dose-dependent manner) and GnRH-stimulated LH release after a lag of 1 (cholera toxin and 8-Br-cAmP) and 4 (forskolin) h. It is noteworthy that these belated effects occurred in spite of the fact that cellular cAMP accumulation was markedly increased within 30 min after cholera toxin and at 1 min after forskolin addition. There was no change in total radioimmunoassayable LH (cellular + released) in either the basal or GnRH-treated cells after cholera toxin and forskolin for up to 24 h. Finally, the forskolin-amplified LH release was reversible and calcium dependent because D-600, EDTA, and calcium-free medium inhibited this effect. These results, generated with three complementary probes that affect integral proteins of the adenylate cyclase complex, suggest a function for cAMP in modulating LH release.

Bordetella pertussis synthesizes a variety of virulence factors including a calmodulin-dependent adenylate cyclase (AC) toxin. Treatment of anterior pituitary cells with this AC toxin resulted in an increase in cellular cAMP levels that was associated with accelerated exocytosis of growth hormone (GH), prolactin, adrenocorticotropic hormone (ACTH), and luteinizing hormone (LH). The kinetics of release of these hormones, however, were markedly different; GH and prolactin were rapidly released, while LH and ACTH secretion was more gradually elevated. Neither dopamine agonists nor somatostatin changed the ability of AC toxin to generate cAMP (up to 2 h). Low concentrations of AC toxin amplified the secretory response to hypophysiotrophic hormones. We conclude that bacterial AC toxin can rapidly elevate cAMP levels in anterior pituitary cells and that it is this response that explains the subsequent acceleration of hormone release.

Pertussis toxin, a protein exotoxin produced by Bordetella pertussis, markedly reduced or eliminated the ability of dopamine or the dopamine agonist bromocriptine to inhibit prolactin release from anterior pituitary cells in vitro. Toxin-mediated reversal of the effect on dopamine agonist inhibition of prolactin release occurred with a lag of greater than 6 h, was maximal by 24 h, and persisted for at least 6 days after removal of the toxin from the medium. The toxin reduced dopamine agonist efficacy without altering potency or directly modifying the dopamine receptor (as measured by [3H]spiperone binding). The ability of dopamine to reduce cellular cyclic AMP content was also antagonized by pertussis toxin, supporting the hypothesis that reduction of cellular cyclic AMP content and inhibition of prolactin secretion may be causally related. These data demonstrated that pertussis toxin can prevent the typical inhibitory action of dopamine agonists on anterior pituitary prolactin release and suggest that this receptor-mediated inhibitory hormone system is analogous to other inhibitory receptors coupled to adenylate cyclase.

The effects of forskolin, an agent which increases intracellular levels of cAMP, on basal luteinizing hormone (LH) and growth hormone (GH) release and on gonadotropin-releasing hormone (GnRH)-stimulated LH release were documented. Continuously perifused dispersed anterior pituitary cells from female rats at random stages of the estrous cycle were used. Secretory rates of both LH and GH increased in a concentration-dependent manner in response to a 1-h challenge with 0.03, 0.1, 0.3, 1, or 3 microM forskolin. In response to 0.3 microM forskolin, maximum GH release was achieved within 15-20 min, after which secretion decreased. In contrast, LH release increased gradually, became maximal at 1.5-2 h, and remained constant until the forskolin was withdrawn. Cells exposed to 10 nM GnRH for 4 h exhibited a biphasic release of LH with the interphase nadir occurring at 30 min. The second phase of LH release was enhanced by simultaneous addition of forskolin with the GnRH. Whereas second phase release did not increase further, exposure of the cells to forskolin for 60 or 120 min before GnRH resulted in increased first-phase LH release. We suggest that, whereas our data are consistent with a role for cAMP in mediating the acute release of GH, cAMP may be involved in the process through which nonimmediately releasable LH becomes available for release.