Evidence for a role of protein kinase-C in His-D-Trp-Ala-Trp-D-Phe-Lys-NH2-induced growth hormone release from rat primary pituitary cells.
We have recently reported that His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6) synergizes with GH-releasing factor (GRF) to increase GH release and cAMP accumulation in rat pituitary cells in vitro. This study was undertaken to further investigate the mechanism of action of GHRP-6 on GH release, particularly the involvement of protein kinase-C. Forskolin (10(-5) M), A23187 (10(-6) M), and phorbol 12-myristate 13-acetate (PMA; 10(-7) M) all stimulated GH release. However, only PMA can mimic the synergistic effects of GHRP-6 on GRF-stimulated GH release and intracellular cAMP accumulation. 4 alpha-Phorbol 12,13-didecanoate, an inactive phorbol ester, was unable to stimulate GH release or potentiate the effect of GRF. Extracellularly added phospholipase-C not only stimulated GH release in a dose-dependent manner, but also potentiated GRF-induced GH release. Phloretin, a protein kinase-C inhibitor, in a concentration range of 10-250 microM had very little or no effect on basal and GRF-stimulated GH release, but markedly inhibited the stimulatory effects induced by either PMA or GHRP-6. Incubation of rat pituitary cells with 10(-6) M PMA for 24 h completely down-regulated protein kinase-C, since such PMA-pretreated cells did not release GH in response to a second dose of PMA. The protein kinase-C-depleted cells had an attenuated GHRP-6 response, but they responded normally to GRF. Moreover, the synergistic effects of GHRP-6 and GRF on GH release and cAMP accumulation were also greatly inhibited by protein kinase-C down-regulation. These data suggest that the effects of GHRP-6 on GH release, either alone or together with GRF, are at least partially mediated via the activation of protein kinase-C.
Cheng
K
K
Department of Growth Biochemistry and Physiology, Merck, Sharp, and Dohme Research Laboratories, Rahway, New Jersey 07065.
Chan
W W
WW
Butler
B
B
Barreto
A
A
Smith
R G
RG
D000595:Amino Acid Sequence; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004357:Drug Synergism; D013006:Growth Hormone / Q000378:metabolism*; D013007:Growth Hormone-Releasing Hormone / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology*; D010693:Phloretin / Q000494:pharmacology; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D009842:Oligopeptides; D005576:Colforsin; D000001:Calcimycin; C041048:growth hormone releasing hexapeptide; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone; D000242:Cyclic AMP; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D010693:Phloretin