Pituitary adenylate cyclase-activating polypeptide increases [Ca2]i in rat gonadotrophs through an inositol trisphosphate-dependent mechanism.

S.R Rawlings,N Demaurex,W Schlegel

doi:10.1016/s0021-9258(17)37514-2

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases cAMP production and stimulates hormone release from a variety of anterior pituitary cells. However, in anterior pituitary gonadotrophs PACAP stimulates oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) that appear to be independent of cAMP. To study the mechanisms involved in this response, we used the patch-clamp technique to microperfuse various agents into single rat gonadotrophs while monitoring [Ca2+]i with microfluorometry. Extracellular application of PACAP to single gonadotrophs stimulated high amplitude (> 1 microM) oscillations in [Ca2+]i, which were blocked by intracellular application of GDP beta S (guanosine 5'-O-2-thiodiphosphate), indicating the involvement of a G-protein. To identify the intracellular messenger(s) involved, we microperfused gonadotrophs with cAMP, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), or heparin, an antagonist of the Ins(1,4,5)P3 receptor. A high concentration of cAMP (100 microM) had no significant effect on basal [Ca2+]i and did not alter the PACAP-stimulated Ca2+ response. Heparin, but not its inactive isoform, completely blocked the PACAP-stimulated increase in [Ca2+]i, while Ins(1,4,5)P3 stimulated oscillations in [Ca2+]i very similar to those observed in response to PACAP. These results strongly suggest that PACAP mobilizes Ca2+ through an Ins(1,4,5)P3-dependent mechanism. The fact that PACAP stimulates two signaling pathways in pituitary cells could substantially enhance the signaling potential of this hypothalamic peptide.

Highlights

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases cAMP production and stimulates hormone release from a variety of anterior pituitary cells
A number of its actions in theregulation of pituitary cell function appear tobe through theCAMP system, including the stimulationof changes in Ca2+ influxin somatotrophs (Rawlings et al, 1993), the release of interleukin-6 from folliculo-stellate cells (Tatsunoet al., 19911, and the release of a-melanocyte-stimulatinghormone from melanotrophs (Koch and Lutz-Bucher, 1992).The present study has directly demonstrated at the single cell level that PACAP can have a specific cellular action through the activation of the Ins(1,4,5)P3 system
PACAP Stimulation of Ca2+ Mobilization-The effect of PACAP to mobilize Ca2+ from intracellular Ca2+ stores was blocked by the Ins(1,4,5)P3 receptor antagonist heparin

Summary

EXPERIMENTAL PROCEDURES

Activating polypeptide; PACAP38, the 38-amino acid form of PACAP; PACAP27, the 27-amino acid form of PACAP;VIP, vasoactive intestinal polypeptide; LH, luteinizing hormone; LHRH, LH-releasing hormone;. Pipette and the insideof the cell significantly reducesthe ability of the [Ca'+], Measurements-Anterior pituitarycellswere loaded with pipette contents to diffuse into the cell (Marty and Neher, 1983),an indo-1/AM (2.5 PM) in RPMI for 30 min a t room temperature, washed effect that is profound for large molecular weight comwith RPMI to remove any remaining ester, and kept at room pounds like heparin. For this reason, when blockers were introduced temperature until use. Pipette-cell membrane seal resistances were between 20 and 50 gigohms, and access resis-

RESULTS

DISCUSSION