The possible involvement of polyphosphoinositides in the stimulation of LH release was investigated. Dispersed sheep pituitary cells were incubated in test-tubes, or perifusedns in columns, with gonadotrophin-releasing hormone (GnRH) and Li+, or with a phorbol ester, and the amounts and patterns of LH release over time compared.
Treatment with Li+ (10 mmol/l), which is known to increase levels of inositol phosphates in gonadotrophs, was shown to have effects only on the responses of desensitized cells, significantly decreasing the rate at which the cells desensitize (P<0·005) and decreasing the response to supramaximal levels of GnRH stimulus (P<0·01). It is suggested that these effects could be due to increased levels of inositol monophosphate, inositol bisphosphate inositol 1,3,4-trisphosphate. Responses to single or repeated pulses of GnRH at 18-, 30- and 60-min intervals were not significantly altered.
Phorbol 12-myristate 13-acetate (PMA), an activator of the calcium and phospholipid-dependent protein kinase (protein kinase C), was specifically active in releasing LH with a half-maximal stimulating dose of approximately 3 nmol/l. Phorbol 12,13-diacetate, which is structurally similar to PMA but does not activate protein kinase C, did not release LH, except at high levels in freshly dispersed cells. The timing of PMA-stimulated LH release was similar to that for GnRH-stimulated release, and PMA was able to release greater amounts of LH than could GnRH. This suggests that activation of protein kinase C is likely to be important in the GnRH-stimulated release of LH from gonadotrophs. It also shows that the desensitization to GnRH stimulation observed after 10 min is unlikely to be caused by lack of releasable LH. Cells desensitized to maximally stimulating levels of GnRH still responded strongly to PMA stimulation, indicating that the desensitization to GnRH stimulation involves a step in the transduction mechanism before activation of protein kinase C.