The Use of Flufenamate and Forskolin to Evaluate the Role of cAMP in Gonadotropin‐Releasing Hormone‐Stimulated Luteinizing Hormone Secretion from Pituitaries of Ovariectomized Rats

Abstract

Flufenamate which inhibited gonadotropin-releasing hormone (GnRH)-stimulated cAMP production in pituitaries from ovariectomized (72 hr) rats, was used to determine whether ovariectomy induces a change in the role of cAMP as a mediator of luteinizing hormone (LH) secretion. Additionally, the study evaluated the practicability of utilizing forskolin to restore intracellular cAMP concentrations in the presence of flufenamate. Infusions of flufenamate to perifused pituitary tissue blocks did not affect the protein synthesis-independent component of GnRH-stimulated LH secretion, but completely inhibited the protein synthesis-dependent component. Dibutyryl cAMP (dbcAMP) and forskolin potentiated the GnRH-stimulated responses, and restored the LH secretion inhibited by flufenamate, even though these agents were ineffective secretagogues when administered singly. The LH responses affected by forskolin were dependent on protein synthesis, while dbcAMP affected both the protein synthesis-dependent and -independent components of GnRH-stimulated LH secretion. Since the effects of dbcAMP on the protein synthesis-independent component of LH secretion might be due to interactions with GnRH receptors, the results suggest that forskolin might be a better choice for restoring intracellular cAMP levels in the presence of flufenamate when assessing the role of cAMP in gonadotropes. The study also indicates that ovariectomy does not result in a change in the role of cAMP, which appears to be a pivotal, but indirect mediator of the protein synthesis-dependent component of GnRH-stimulated LH secretion.

Cellular Mechanism of Melatonin Action in Neonatal Rat Pituitary

“…Melatonin thus does not seem to act via diacylglycerol and protein kinase C. cAMP may be involved in regulation of LH release from neonatal gonadotropes as the cAMP derivative po tentiated the GnRH-induced LH release. This is in agree ment with previously published data showing the stimula tory effect of cAMP on LH release in adult gonadotropes [ 19,20], Alternatively, the potentiation of LH release may be secondary to the stimulation of LH synthesis, which seems to be regulated by cAMP [21], In amphibian melanophores, the melatonin-induced decrease of cAMP is the signal transducing the blanching effect of melatonin [4]. In neonatal rat gonadotropes, however, the melatonininduced decrease of cAMP is not the critical signal for inhibition of LH release.…”

Forskolin but Not Cholera Toxin Bypasses Flufenamate‐Induced Inhibition of cAMP Production in Anterior Pituitaries

“…If this likelihood is indeed true then obviously the use of flufenamate to investigate the role of cAMP in various cellular processes would be severely limited. However, since phospholipase C activation is involved in the mechanism of action of GnRH (Conn et al 1987), the results from the following study (Das & Bourne 1992) tend to negate this possibility.…”

“…The results of this study clearly demonstrate that flufenamate does not affect forskolin-stimulated increases in cAMP concentrations. As a result, forskolin can be used to restore endogenous cAMP concentrations in the presence of GnRH and flufenamate, thereby removing the compounding factors which arise when exogenous cAMP analogues are used (Das & Bourne 1992).…”

“…The observation that the actions of forskolin which directly stimulates the catalytic component of adenylyl cyclase (Downs & Aurbach 1982;Berridge 1985) were not affected by flufenamate, suggest that flufenamate does not significantly affect the activity of phosphodiesterases. Infact, flufenamate exerts its inhibitory effect on cAMP production in the presence of a phosphodiesterase inhibitor (Bourne 1988;Das & Bourne 1992).…”