Heterogeneity of responses to LH-releasing hormone and phorbol ester among rat gonadotrophs: a study using a reverse haemolytic plaque assay for LH.

Protein kinase C (PK-C) has been implicated in the action of LHRH because LHRH-induced release of LH is partially mimicked by phorbol esters which activate PK-C, and reduced in magnitude by inhibitors of PK-C. We have used a reverse haemolytic plaque assay for LH to visualize and compare the direct effects on individual rat gonadotrophs of (1) a 2-h exposure to a range of concentrations of LHRH and phorbol 12-myristate 13-acetate (PMA) and (2) a single maximally stimulatory dose (10 nM) of LHRH or PMA, or LHRH in the presence of inhibitors of PK-C (retinal and isoquinolone sulphonamide; H7) over six consecutive 30-min intervals. Quantitative analysis of the size and number of haemolytic plaques indicated that LHRH induced a dose- and time-dependent increase in the amount of LH release by individual gonadotrophs, with no evidence of the priming effect of LHRH. Stimulation by 10nM LHRH induced recruitment of actively secreting gonadotrophs which reached maximum levels by 90 min. There was a delay of 90-120 min before 10 nM PMA caused a significant release of LH, as assessed by both the size and number of plaques. During the first 30 min of exposure, the presence of 10 microM retinal or H7 augmented LHRH-induced secretion of LH, with the absence of any inhibition of the effects of LHRH until 90-120 min, when both the size and number of plaques were reduced compared with those formed in the presence of LHRH alone.(ABSTRACT TRUNCATED AT 250 WORDS)