Activation of PAC1 Receptors in Rat Cerebellar Granule Cells Stimulates Both Calcium Mobilization from Intracellular Stores and Calcium Influx through N-Type Calcium Channels

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 ([Ca(2+)]i), while vasoactive intestinal peptide was devoid of effect, indicating the involvement of the PAC1 receptor in the Ca(2+) response. Preincubation of granule cells with the Ca(2+) 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 [Ca(2+)]i. Furthermore, addition of the calcium chelator, EGTA, or exposure of cells to the non-selective Ca(2+) channel blocker, NiCl2, significantly attenuated the PACAP-evoked [Ca(2+)]i increase. Preincubation of granule neurons with the N-type Ca(2+) channel blocker, ω-conotoxin GVIA, decreased the PACAP-induced [Ca(2+)]i response, whereas the L-type Ca(2+) channel blocker, nifedipine, and the P- and Q-type Ca(2+) channel blocker, ω-conotoxin MVIIC, had no effect. Altogether, these findings indicate that PACAP, acting through PAC1 receptors, provokes an increase in [Ca(2+)]i in granule neurons, which is mediated by both mobilization of calcium from IP3-sensitive intracellular stores and activation of N-type Ca(2+) 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.

Keywords: autoradiography; calcium; calcium channels; cerebellum; cytoplasmic calcium stores; granule cells; pituitary adenylate cyclase-activating polypeptide.

Figure 1

Effect of PACAP38, PACAP27, or VIP on [Ca²⁺]_i in cultured cerebellar granule cells. (A) Time series of pseudocolor images illustrating the [Ca²⁺]_i changes in cultured granule cells loaded with the Ca²⁺-sensitive dye fluo-3/AM after infusion of 10⁻⁶ M PACAP38 at the vicinity of the cells. The pseudocolor scale indicates the corresponding [Ca²⁺]_i changes expressed in arbitrary units. (B–D) Typical profiles illustrating the time-course of the variation of the fluorescence ratio evoked by a single application of 10⁻⁶ M PACAP38 (B), PACAP27 (C), or VIP (D) on [Ca²⁺]_i in cultured granule cells. The arrows indicate the onset of peptide application.

Figure 2

Involvement of intracellular calcium stores in the mechanism of action of PACAP38 in cerebellar granule cells. (A–D) Effect of a single application of 10⁻⁶ M PACAP38 on the amplitude (A,C) and the area under the curve (B,D) of the [Ca²⁺]_i response in the absence (black bars) or presence (white bars) of 10⁻⁶ M thapsigargin, a Ca²⁺ ATPase inhibitor, or 10⁻⁵ M 2APB, a membrane permeable d-myo-inositol 1,4,5-trisphosphate receptor antagonist. Thapsigargin and 2APB were added 15 and 30 min before application of the pulse of PACAP, respectively. (A,C) Histograms showing the distribution of granule cells according to the amplitude of their response to PACAP. Each graph represents the analysis of a least 50 cells from 3 different cultures. 2APB, 2-aminoethoxydiphenyl borate; AUC, area under the curve. ***p < 0.001.

Figure 3

Involvement of calcium influx in the mechanism of action of PACAP38 in cerebellar granule cells. (A–D) Effect of a single application of 10⁻⁶ M PACAP38 on the amplitude (A,C) and the area under the curve (B,D) of the [Ca²⁺]_i response in the absence (black bars) or presence (white bars) of 6 mM EGTA, a Ca²⁺ chelator, or 3 mM NiCl₂, a blocker of voltage-operated calcium channels. EGTA and NiCl₂ were added to the incubation medium 10 min before application of the pulse of PACAP. (A,C) Histograms showing the distribution of granule cells according to the amplitude of their response to PACAP. Each graph represents the analysis of a least 50 cells from 3 different cultures. AUC, area under the curve; EGTA, ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid; NiCl₂, nickel chloride. **p < 0.01, ***p < 0.001.

Figure 4

Effect of the Ca²⁺ chelator EGTA (6 mM) on [¹²⁵I]PACAP27 binding to cerebellar tissue slices or cerebellar granule cells. (A–D) Autoradiographic visualization of [¹²⁵I]PACAP27 binding sites in the absence (A,C) or presence of 10⁻⁶ M PACAP38 (B,D) in consecutive sections of P8 rat cerebellum. Scale bar = 1.7 mm. (E) Histograms showing specific binding of [¹²⁵I]PACAP27 in the absence (black bars) or presence (white bars) of 6 mM EGTA on cultured cerebellar granule cells. EGTA, ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid. NS, not significantly different from Hepes buffer.

Figure 5

Effect of selective blockers of voltage-operated calcium channels on calcium influx induced by PACAP38 in cerebellar granule cells. (A–F) Histograms showing the effect of a single application of 10⁻⁶ M PACAP38 on the amplitude (A,C,E) and the area under the curve (B,D,F) of the [Ca²⁺]_i response in the absence (black bars) or presence (white bars) of 10⁻⁵ M of the L-type Ca²⁺ channel blocker nifedipine (A,B), 10⁻⁶ M of the P- and Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC (C,D), or 10⁻⁶ M of the N-type Ca²⁺ channel blocker ω-conotoxin GVIA (E,F). Nifedipine, ω-conotoxin MVIIC, and ω-conotoxin GVIA were added to the incubation medium 30 min before application of the pulse of PACAP38. (A,C,E) Histograms showing the distribution of granule cells according to the amplitude of their response to PACAP. Each graph represents the analysis of a least 50 cells from 3 different cultures. AUC, area under the curve; NS, not significantly different from the PACAP38; ***p < 0.001.