title: Bradykinin receptor localization and cell signaling pathways used by bradykinin in the regulation of gonadotropin-releasing hormone secretion.
abstract: In a previous publication we provided evidence of a novel neuronal pathway for the control of GnRH secretion by bradykinin. The action of bradykinin appeared to be exerted through the bradykinin B2 receptor. In this study we demonstrated that the bradykinin B2 receptor is densely localized in the arcuate nucleus, median eminence, organum vasculosum of the lamina terminalis, and preoptic area, regions known to be critical for the control of GnRH secretion. To determine the mechanism of action of bradykinin in stimulating GnRH release, we used immortalized GnRH (GT1-7) cells in vitro. Bradykinin stimulation of GnRH secretion from GT1-7 cells appears to involve activation of the phospholipase C signaling pathway and mobilization of extracellular and intracellular calcium stores. Evidence to support this contention was derived from the observations that incubation of the phospholipase C inhibitor, U-73122 with bradykinin, blocked the ability of bradykinin to stimulate release from GT1-7 cells. This effect was specific, as a nitric oxide synthase inhibitor and a cyclooxygenase inhibitor were found to have no effect on bradykinin-induced GnRH secretion, suggesting that nitric oxide and PGs do not mediate bradykinin effects. Pertussis toxin also had no effect on bradykinin action. This suggests that the bradykinin B2 receptor may be coupled to a pertussis toxin-insensitive G protein in GT1-7 cells. With respect to calcium involvement in bradykinin action, fura-2 calcium indicator studies revealed that bradykinin can rapidly increase intracellular Ca2+ levels in GT1-7 cells. A role for intracellular Ca2+ in bradykinin action was further suggested by the finding that an intracellular calcium chelator, 1,2-bis(O-aminophenoxy)]ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, significantly attenuated the effects of bradykinin on GnRH release. The elevation of intracellular calcium by bradykinin appears to be due to mobilization of calcium from the endoplasmic reticulum, as incubation of the Ca2+-adenosine triphosphatase inhibitor thapsigarin, which depletes endoplasmic reticulum Ca2+ stores, significantly attenuated bradykinin action on GnRH release. Extracellular calcium may also be involved in bradykinin action, as the L-type Ca2+ channel blockers verapamil and nifedipine had no effect on bradykinin-induced GnRH release, whereas the nonselective Ca2+ channel blocker, nickel chloride, attenuated bradykinin-induced GnRH release. Taken as a whole, these studies demonstrate that the bradykinin B2 receptor is densely localized in key hypothalamic nuclei responsible for regulation of GnRH release, and that the mechanism of bradykinin stimulation of GnRH secretion involves activation of the phospholipase C signaling pathway, with a critical role implicated for calcium in bradykinin action in GT1-7 cells.
authors.affiliation: Department of Physiology and Endocrinology, Medical College of Georgia, Augusta 30912, USA.
authors.forename: B
authors.initials: B
authors.lastname: Shi
authors.forename: G
authors.initials: G
authors.lastname: Bhat
authors.forename: V B
authors.initials: VB
authors.lastname: Mahesh
authors.forename: M
authors.initials: M
authors.lastname: Brotto
authors.forename: T M
authors.initials: TM
authors.lastname: Nosek
authors.forename: D W
authors.initials: DW
authors.lastname: Brann
mesh_terms: D000818:Animals; D001920:Bradykinin / Q000494:pharmacology / Q000502:physiology*; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000502:physiology; D002460:Cell Line; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D007150:Immunohistochemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D018002:Receptors, Bradykinin / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D018002:Receptors, Bradykinin; D007987:Gonadotropin-Releasing Hormone; D010738:Type C Phospholipases; D001920:Bradykinin; D002118:Calcium