title: Inhibitory effects of antagonistic analogs of GHRH on GH3 pituitary cells overexpressing the human GHRH receptor.
abstract: GH3 rat pituitary tumor cells produce GH and prolactin (PRL), but lack the GHRH receptor (GHRH-R). We expressed human GHRH-R (hGHRH-R) in GH3 cells using recombinant adenoviral vectors and studied the effects of GHRH antagonists. The mRNA expression of the GHRH-R gene in the cells was demonstrated by RT-PCR. An exposure of the GH3 cells infected with hGHRH-R to 10(-10), 10(-9) and 10(-8) m hGHRH for 1 or 2 h in culture caused a dose-dependent elevation of the intracellular cAMP concentration and the cAMP efflux. Exposure to hGHRH also elicited dose-dependent increases in GH and PRL secretion from these cells. Neither the uninfected nor the antisense hGHRH-R-infected control cells exhibited cAMP, GH and PRL responses to GHRH stimulation. GHRH antagonists JV-1-38 and jv-1-36 applied at 3x10(-8) m for 3 h, together with 10(-9) m GHRH, significantly inhibited the GHRH-stimulated cAMP efflux from the hGHRH-R-infected cells by 36 and 80% respectively. The more potent antagonist JV-1-36 also decreased the intracellular cAMP levels in these cells by 55%. Exposure to JV-1-36 for 1 h nullified the stimulatory effect of GHRH on GH secretion and significantly inhibited it by 64 and 77% after 2 and 3 h respectively. In a superfusion system, GHRH at 10(-10), 10(-9) and 10(-8) m concentrations induced prompt and dose-related high cAMP responses and smaller increases in the spontaneous GH secretion of the hGHRH-R-infected cells. Antagonists JV-1-36 and JV-1-38 applied at 3x10(-8) m for 15 min, together with 10(-9) m GHRH, inhibited the GHRH-stimulated cAMP response by 59 and 35% respectively. This work demonstrates that GHRH antagonists can effectively inhibit the actions of GHRH on the hGHRH-R. Our results support the view that this class of compounds would be active clinically.
authors.affiliation: Endocrine, Polypeptide, and Cancer Institute, Veterans Affairs Medical Center, New Orleans, Louisiana 70112, USA.
authors.forename: M
authors.initials: M
authors.lastname: Kovacs
authors.forename: A V
authors.initials: AV
authors.lastname: Schally
authors.forename: E-J
authors.initials: EJ
authors.lastname: Lee
authors.forename: R
authors.initials: R
authors.lastname: Busto
authors.forename: P
authors.initials: P
authors.lastname: Armatis
authors.forename: K
authors.initials: K
authors.lastname: Groot
authors.forename: J L
authors.initials: JL
authors.lastname: Varga
mesh_terms: D000818:Animals; D000242:Cyclic AMP / Q000032:analysis / Q000096:biosynthesis; D015870:Gene Expression / Q000235:genetics; D013006:Growth Hormone / Q000032:analysis / Q000378:metabolism; D013007:Growth Hormone-Releasing Hormone / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000235:genetics; D006801:Humans; D010902:Pituitary Gland / Q000166:cytology / Q000502:physiology*; D011388:Prolactin / Q000032:analysis; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011986:Receptors, Somatotropin / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: D012333:RNA, Messenger; D011986:Receptors, Somatotropin; D011388:Prolactin; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone; D000242:Cyclic AMP