The involvement of dihydropyridine-sensitive calcium channels in phorbol ester-induced luteinizing hormone and growth hormone release

We examined the role of voltage-activated, L-type, Ca2+ channels in phorbol ester-induced luteinizing hormone (LH) and growth hormone (GH) release from rat anterior pituitary tissue. The L-type Ca2+ channel inhibitor, nimodipine (NMD), inhibited phorbol 12,13-dibutyrate (PDBu)-induced GH release but had no significant effect on LH release. The L-type Ca2+ channel activator BAY K 8644 had no effect on PDBu-induced GH release but potentiated PDBu-induced LH release. In contrast, 60 mM K+-induced LH and GH release were inhibited by NMD, whereas BAY K 8644 had no effect. When PDBu and either K+ or BAY K 8644 were used together, they acted synergistically to evoke levels of LH release greater than addition of release caused by each secretagogue alone. However, the release of GH was additive with PDBu and either K+, BAY K 8644. The protein kinase C (PKC) inhibitor staurosporine inhibited both PDBu-induced LH release and GH release. A structurally different PKC inhibitor, H7, significantly inhibited PDBu-induced LH release but had no effect on PDBu-induced GH release. Both staurosporine and H7 inhibited LH release induced by PDBu and BAY K 8644 together. In contrast, although staurosporine inhibited GH release induced by PDBu and BAY K 8644, H7 significantly potentiated this response. A difference in the action of these two inhibitors was also apparent on K+-induced hormone release where staurosporine partially blocked K+-induced LH and GH release but H7 had no effect on the release of either hormone. Data obtained in 45Ca2+ influx experiments further suggested that a staurosporine-sensitive, but H7-resistant, PKC-like kinase may tonically maintain L-channels in a voltage-sensitive state, as down-regulation of PKC in dispersed anterior pituitary cells by long term PDBu treatment caused a significant reduction in K+-induced 45Ca2+ influx. We conclude that phorbol ester-induced GH release, but not LH release, is a result of L-type Ca2+ channel activation which may occur by means of alterations in the channel itself to increase its responsiveness to a given depolarisation

Oestradiol-17 modulates the actions of pharmacologically distinct forms of protein kinase C in rat anterior pituitary cells

Phorbol ester-induced release of LH and GH from rat anterior pituitary tissue in vitro is differentially inhibited by some, but not other, inhibitors of protein kinase C (PKC), suggesting that pharmacologically distinct species of PKC may have different functional roles in these cells. Since stimulus-induced anterior pituitary hormone release can be enhanced by oestradiol-17β (OE2) pretreatment, we investigated the effect of OE2 treatment of long-term (4 weeks) ovariectomized rats on the amount, activity and cellular actions of pharmacologically distinct PKC species in rat anterior pituitary tissue. Here we report that OE2 treatment enhanced phorbol 12,13-dibutyrate (PDBu)-induced LH but not GH release measured in vitro. This effect of OE2 on LH release may involve synthesis of additional PKCs that are not targeted by the synthetic diacylglycerol, 1,2-dioctanoyl-snglycerol (DOG). Measurements of anterior pituitary PKC activity and [3H] phorbol ester-binding studies suggested that the facilitatory action of OE2 on LH release may occur, at least in part, by altering the quantity and activity of PKC(s). Our results also demonstrate that the OE2-induced PKC(s) which facilitate LH release may be of the type that are not dependent upon raised intracellular Ca2+ for their activation and display distinct pharmacological properties (being readily activated by PDBu, but not by DOG, and are staurosporine-sensitive but H7-insensitive). This facilitatory action of OE2 on PKC-induced LH release does not appear to involve OE2-induced changes in the affinity of existing PKC(s) for PDBu, or changes in the amount of releasable LH in the pituitary prior to the stimulus