Mechanisms of action of a second generation growth hormone-releasing peptide (Ala-His-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2) in rat anterior pituitary cells.
The mechanism by which GH-releasing peptides elicit GH secretion has remained largely unknown. In this study, the effects of a second generation GH-releasing peptide, Ala-His-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2(GHRP-1), on cAMP, intracellular Ca2+ ([Ca2+]i), and GH release were examined using rat pituitary gland static monolayer cell cultures. It was found that GHRP-1 increased GH release in a dose-dependent manner up to 3-fold, while having no effect on cAMP levels. In contrast, simultaneous elevations of cAMP and GH were observed after treatment with GHRH. To further define the underlying mechanism of GHRP-1-mediated GH release, its effect on [Ca2+]i was determined using a fluorescent Ca2+ indicator, fura-2. GHRP-1 dose dependently increased [Ca2+]i up to 45.5 nM +/- 5.6 nM. A similar elevation of [Ca2+]i was observed after GHRH treatment. Similar to GHRH, GHRP-1-induced increases in [Ca2+]i and GH release were inhibited by somatostatin. Furthermore, the GHRP-1-induced increases in [Ca2+]i and GH were also suppressed by nifedipine. The interaction between the voltage-dependent Ca2+ channels and GHRP-1 was investigated in cells maximally stimulated by KCl. The addition of GHRP-1 had no effect on the KCl-stimulated GH release. To investigate the possible interaction between the adenylyl cyclase pathway and GHRP-1, cells were maximally stimulated with forskolin or (Bu)2cAMP. Addition of GHRP-1 stimulated GH release beyond that observed using cAMP elevating agents. Similar results were obtained in the presence of a protein kinase C, 4 beta-phorbol 12-myristate 13-acetate (PMA). The GHRP-1-stimulated GH release was additive to that observed with PMA stimulation. Based on these findings, it was concluded that 1) GHRP-1 treatment leads to an increase in [Ca2+]i; 2) unlike GHRH, GHRP-1 releases GH via a Ca(2+)-dependent, cAMP-independent mechanism; 3) GHRP-1-induced increases in [Ca2+]i and GH release are sensitive to somatostatin inhibition; and 4) cAMP-elevating agents and PMA have an additive effect on the GHRP-1-stimulated GH release, indicating these agents stimulate GH release via a mechanism separate from that of GHRP-1.
Akman
M S
MS
Department of Medicine, Faculty of Medicine, University of Alberta, Edmonton, Canada.
Girard
M
M
O'Brien
L F
LF
Ho
A K
AK
Chik
C L
CL
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D013006:Growth Hormone / Q000378:metabolism*; D013007:Growth Hormone-Releasing Hormone / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009543:Nifedipine / Q000494:pharmacology; D009842:Oligopeptides; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013004:Somatostatin / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D009842:Oligopeptides; C079956:alanyl-histidyl-(2-naphthyl)alanyl-tryptophyl-phenylalanyl-lysinamide; D005576:Colforsin; D013004:Somatostatin; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone; D000242:Cyclic AMP; D009543:Nifedipine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium