New stimulators and a new mechanism of regulated secretion in pituitary gonadotropes

Abstract

The major regulator of gonadotropin (luteinizing hormone, LH; and follicle-stimulating hormone, FSH) secretion is the peptide gonadotropin-releasing hormone (GnRH). It acts via a rise in intracellular Ca$\sp{2+}$ concentration ( (Ca$\sp{2+}\rbrack\sb{\rm i}$). I used cultured pituitary gonadotropes of male rats to seek other possible agents and mechanisms that might regulate gonadotropin secretion. The GnRH-evoked (Ca$\sp{2+}\rbrack\sb{\rm i}$ rise is oscillatory and accompanied by oscillations of outward K$\sp{+}$ current that is activated by Ca$\sp{2+}$. Substances that caused current responses similar to those with GnRH were hypothesized to evoke gonadotropin secretion via the Ca$\sp{2+}$-dependent mechanism. Endothelin-1, oxytocin, neurotensin, pituitary adenylate cyclase-activating polypeptide, and serotonin raised (Ca$\sp{2+}\rbrack\sb{\rm i}$ and evoked LH release as assayed by the reverse hemolytic plaque assay (RHPA). These agents affected only subpopulations of gonadotropes establishing functional heterogeneity of pituitary gonadotropes. On the other hand, a protein kinase C (PKC) activator--phorbol-12-myristate-13-acetate (PMA)--caused LH release from pituitary gonadotropes without evoking changes in (Ca$\sp{2+}\rbrack\sb{i}$. After 1 hour incubation in RHPA, PMA elicited an increase in LH secretion comparable to that caused by GnRH. A PKC inhibitor did not alter GnRH-induced LH release indicating that PKC activation is not important for GnRH-induced secretion in a 1 hour application. Buffering of intracellular Ca$\sp{2+}$ by AM-loaded BAPTA reduced GnRH-induced LH secretion almost to control levels without significantly affecting PMA-induced secretion. Single-cell photometry on gonadotropes previously identified by incubation with GnRH in RHPA, revealed no changes in (Ca$\sp{2+}\rbrack\sb{\rm i}$ during a one hour application of PMA. Hence, there appears to be a Ca$\sp{2+}$-independent pathway of regulated secretion in gonadotropes. Its nature and physiological ligands remain to be elucidated. Finally, I attempted to develop a technique to study differential regulation of LH and FSH release. This attempt failed due to methodological problems but the method may allow study of secretion of two hormones from cell systems other than pituitary gonadotropes. Thus, gonadotropes possess several mechanisms of regulated secretion and can be regulated by a variety of neuromodulators, suggesting possible ways of differential regulation of LH and FSH secretion.