Abstract
High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and a high density of PACAP binding sites have been detected in the developing rat cerebellum. In particular, PACAP receptors are actively expressed in immature granule cells, where they activate both adenylyl cyclase and phospholipase C. The aim of the present study was to investigate the ability of PACAP to induce calcium mobilization in cerebellar granule neurons. Administration of PACAP-induced a transient, rapid, and monophasic rise of the cytosolic calcium concentration ([Ca2+]i), while vasoactive intestinal peptide was devoid of effect, indicating the involvement of the PAC1 receptor in the Ca2+ response. Preincubation of granule cells with the Ca2+ ATPase inhibitor, thapsigargin, or the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor antagonist, 2-aminoethoxydiphenyl borate, markedly reduced the stimulatory effect of PACAP on [Ca2+]i. Furthermore, addition of the calcium chelator, EGTA, or exposure of cells to the non-selective Ca2+ channel blocker, NiCl2, significantly attenuated the PACAP-evoked [Ca2+]i increase. Preincubation of granule neurons with the N-type Ca2+ channel blocker, ω-conotoxin GVIA, decreased the PACAP-induced [Ca2+]i response, whereas the L-type Ca2+ channel blocker, nifedipine, and the P- and Q-type Ca2+ channel blocker, ω-conotoxin MVIIC, had no effect. Altogether, these findings indicate that PACAP, acting through PAC1 receptors, provokes an increase in [Ca2+]i in granule neurons, which is mediated by both mobilization of calcium from IP3-sensitive intracellular stores and activation of N-type Ca2+ channel. Some of the activities of PACAP on proliferation, survival, migration, and differentiation of cerebellar granule cells could thus be mediated, at least in part, through these intracellular and/or extracellular calcium fluxes.
Highlights
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38- or 27-amino acid peptide that was isolated from hypothalamic extracts for its ability to stimulate cAMP formation in anterior pituitary cells (Miyata et al, 1989)
The response profile determined as the ratio between the fluo-3 fluorescence intensity under resting conditions and after exposure to test substances, revealed that both 38-amino acid form of PACAP (PACAP38) and 27-amino acid form of PACAP (PACAP27) (10−6 M) induced a transient, rapid, and monophasic increase in [intracellular calcium concentration (Ca2+]i) followed by gradual return to baseline (Figures 1B,C) while application of vehicle had no effect
Because primary cultures of rat cerebellar granule cells are mainly composed of a single population of cells, the effect of PACAP on the [Ca2+]i response could be monitored on a large number of neurons
Summary
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38- or 27-amino acid peptide that was isolated from hypothalamic extracts for its ability to stimulate cAMP formation in anterior pituitary cells (Miyata et al, 1989). Three PACAP/VIP receptors, which belong to the class B family of seven-transmembrane G protein-coupled receptors, have been cloned and termed respectively PAC1,VPAC1, and VPAC2, according to their relative affinity for PACAP and VIP (Harmar et al, 2012). All three receptors can activate a variety of second messengers including cAMP, cGMP, IP3, or calcium, depending on the receptor types, splice variants, G proteins, and other intracellular components expressed by each cell type (Vaudry et al, 2009). This cellular context plays a crucial role in determining the effects of PACAP, as differential expression of PAC1 splice variants is sufficient to www.frontiersin.org
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