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 released by individual gonadotrophs, with no evidence of the priming effect of LHRH. Stimulation by 10 nM 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 µM 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.
Heterogeneity of responsiveness to LHRH among gonadotrophs was evident from the bimodal distribution of plaque sizes formed after 60–180 min of exposure to LHRH. The size of gonadotrophs was correlated with that of plaques by 60 min of exposure to LHRH, with the larger cells secreting more LH. By contrast, not until 180 min did PMA induce such heterogeneity in secretion. PMA or LHRH in the presence of retinal or H7 resulted in only a unimodal distribution of plaque sizes, with the absence of a discrete population of cells producing the larger plaques.
These findings indicate that PK-C is not crucial for the immediate LH-releasing action of LHRH but could well be involved in other, slower LHRH-induced effects such as post-translational modification of LH, up-regulation of LHRH receptors and/or gonadotroph multiplication.
