Pituitary adenylate cyclase-activating peptide (PACAP) recruits low voltage-activated T-type calcium influx under acute sympathetic stimulation in mouse adrenal chromaffin cells

Abstract

Low voltage-activated T-type Ca(v)3.2 calcium channels are expressed in neurosecretory chromaffin cells of the adrenal medulla. Previous studies have shown that naïve adrenal chromaffin cells express a nominal Ca(v)3.2-dependent conductance. However, Ca(v)3.2 conductance is up-regulated following chronic hypoxia or long term exposure to cAMP analogs. Thus, although a link between chronic stressors and up-regulation of Ca(v)3.2 exists, there are no reports testing the specific role of Ca(v)3.2 channels in the acute sympathoadrenal stress response. In this study, we examined the effects of acute sympathetic stress on T-type Ca(v)3.2 calcium influx in mouse chromaffin cells in situ. Pituitary adenylate cyclase-activating peptide (PACAP) is an excitatory neuroactive peptide transmitter released by the splanchnic nerve under elevated sympathetic activity to stimulate the adrenal medulla. PACAP stimulation did not evoke action potential firing in chromaffin cells but did cause a persistent subthreshold membrane depolarization that resulted in an immediate and robust Ca(2+)-dependent catecholamine secretion. Moreover, PACAP-evoked secretion was sensitive to block by nickel chloride and was acutely inhibited by protein kinase C blockers. We utilized perforated patch electrophysiological recordings conducted in adrenal tissue slices to investigate the mechanism of PACAP-evoked calcium entry. We provide evidence that stimulation with exogenous PACAP and native neuronal stress stimulation both lead to a protein kinase C-mediated phosphodependent recruitment of a T-type Ca(v)3.2 Ca(2+) influx. This in turn evokes catecholamine release during the acute sympathetic stress response.

FIGURE 1.

PACAP evokes membrane depolarization and nickel-sensitive catecholamine release. A, panel i, a chromaffin cell was held in perforated patch configuration, and membrane potential was measured under current clamp. Focal perfusion of PACAP (1 μm) is indicated by the bar. The inset icon indicates the patch clamp recording condition. A, panel ii, representative current recorded during perforated patch voltage clamp recordings of chromaffin cells stimulated with an action potential voltage template in control cells (dash line) or PACAP-treated cells (solid line). B, data quantified from pooled experiments such as in A show that PACAP stimulation does not significantly alter peak Ca²⁺ influx during the action potential but does cause a significant membrane depolarization to approximately −53 mV (P_Vm). Data are plotted as mean value, with error bars representing S.E. * indicates significance at p < 0.02 by Student's t test; sample size is indicated by numbers at the base of each category bar. C, carbon fiber amperometry was utilized to measure catecholamine release under PACAP stimulation. Total catecholamine release for each condition was determined by integrating the time-resolved amperometric traces. The inset icon indicates the amperometric recording condition. Panel i, in untreated “Control” cells, focal perfusion of 1 μm PACAP elicits a robust catecholamine release indicated by a rapidly rising integrated amperometric current. Panel ii, perfusion in Ringer's solution supplemented with 50 μm NiCl₂ effectively blocks catecholamine release under PACAP stimulation. pF, picofarads; memb., membrane; pC, picocoulombs.

FIGURE 2.

PACAP facilitates nickel-sensitive current at negative potentials. A, immunostaining for tyrosine hydroxylase (TH; green panel), a marker for catecholamine-secreting adrenal chromaffin cells, and Ca_v3.2 T-type calcium channels (red panel) are shown. The merge overlay (“Merge”) indicates that both signals are present in the same cells of the adrenal medulla (scale bar, 50 μm). The panel to the right shows staining for PACAP and Ca_v3.2 at a higher resolution (scale bar, 10 μm) and shows a peripheral staining for PACAP surrounding the Ca_v3.2-positive chromaffin cell. B, a chromaffin cell was held at −53-mV command potential to match P_Vm, and the membrane current was measured. Puffing of exogenous PACAP (1 μm) elicited a sustained inward current. Vertical scale bar, 6 pA/picofarad; horizontal scale bar, 30 s. The PACAP-evoked current was blocked by the additional perfusion with 50 μm NiCl₂. C, a protocol was designed to assess PACAP-dependent effects on Ca²⁺ tail currents. Representative records are provided, demonstrating that a depolarization to 20 mV and return to P_Vm resulted in a specific enhancement of a non-inactivating window current in PACAP-treated cells. Vertical scale bar, 6 pA/picofarad; horizontal scale bar, 10 ms. D, voltage clamp depolarization from −80-mV holding potential to P_Vm (−53 mV) elicits both a specific augmentation of inward current and a slower deactivating tail current specifically after stimulation with exogenous PACAP (10 μm). Depolarization to −20 mV (“Peak”) to maximally activate all voltage-gated calcium channels elicits a specific emergence of a rapidly activating component as well as a slowly deactivating tail current after PACAP stimulation. Horizontal scale bar, 15 ms; vertical scale bar: −53 mV, 4 pA/picofarad; −20 mV, 5 pA/picofarad.

FIGURE 3.

PACAP-evoked facilitation of nickel-sensitive current is reversed by PKC inhibition. A, a cell was held in perforated voltage clamp in situ and stimulated with an I-V protocol as described in the text. Steady state current was measured (panel i, vertical dotted line) and plotted against step potential in control Ringer's solution conditions (panel ii, ●) and after perfusion with Ringer's solution supplemented with 50 μm NiCl₂ (panel ii, ○). B, top left plot, a subtraction current of the data presented in A demonstrates the Ni²⁺-sensitive current component. The same protocol as summarized in A was repeated in cells either treated with 1 μm exogenous PACAP (bottom left plot, ●) or PACAP and 100 nm Gö6983 (bottom left plot, ○). Ni²⁺-sensitive (sens.) subtraction currents are plotted for each condition. For comparison, the nickel-sensitive current measured in control cells (top left plot) is replotted as a dotted line. The same protocol as summarized in A was repeated in 100 nm PMA (top right plot, ●) and Gö6983 (top right plot, ○)-treated cells (top right plot). Conditions are the same as in the other plots except the pharmacological conditions are 100 μm 8-pCPT-2′-O-Me-cAMP (8-cPCT; bottom right plot, ●) or 8-pCPT-2′-O-Me-cAMP and Gö6983 (bottom right plot, ○).

FIGURE 4.

PACAP, PMA, and 8-pCPT-2′-O-Me-cAMP facilitate nickel-sensitive current. A, inset icons indicate the point in the I-V curve where current magnitudes were measured. Category plots on the left (panel i) represent peak inward values, whereas those on the right (panel ii) represent P_Vm values. Data were pooled from experimental conditions as represented in Fig. 3 and are plotted as mean value with error bars representing S.E. Upper category plot, no significant difference was found in total peak current for any condition versus control. No significant difference was found in any pharmacological condition with respect to control peak Ca²⁺ current. Lower category plot, nickel-sensitive Ca²⁺ influx was determined as the difference between currents measured in normal Ringer's and Ni²⁺-containing Ringer's solutions. * indicates p ≤ 0.05 determined by one-way analysis of variance. Sample size is indicated by the number at the base of each category bar. 8-cPCT, 8-pCPT-2′-O-Me-cAMP. B, the inset icon indicates a combined voltage clamp and amperometry experimental configuration. A voltage protocol was designed to test catecholamine secretion at P_Vm. A cell was held in perforated patch configuration and stepped from the holding potential of −80 to −53 mV. The resulting integrated amperometric current is shown for a control, naïve cell (panel i, Control) and for a cell that had been treated for 5 min with 1 μm exogenous PACAP by focal perfusion (panel ii, +PACAP). The increased integrated amperometric current in the lower trace indicates a sustained and robust catecholamine secretion in the PACAP-treated cell. pF, picofarad; sens., sensitive; pC, picocoulombs.

FIGURE 5.

Elevated neuronal stimulation facilitates nickel-sensitive calcium influx. A, left, a diagram illustrates the nerve stimulation and chromaffin cell recording condition. Right, evoked catecholamine release was measured by carbon fiber amperometry. Total catecholamine release was determined by integrating the amperometric record as in Figs. 1 and 4. Representative traces are plotted in B for Low Hz stimulation (stim.) and High Hz stimulation in a normal Ringer's bath solution and High Hz stimulation in the presence of Ni²⁺ or Gö6983-containing Ringer's bath solution. B, cells determined to be excitable by neuronal stimulation were patch-clamped in the perforated voltage clamp configuration after bipolar stimulation, and I-V protocols were conducted. Data are plotted as mean value, with error bars representing S.E. Panel i, peak inward (left axis) and the Ni²⁺-sensitive (sens.) inward component (right axis) currents were measured as in Fig. 3 after bipolar neuronal stimulation with Low Hz- and High Hz-stimulated firing patterns. Panel ii, mean inward current at P_Vm and the Ni²⁺-sensitive component were measured in each condition as described in Fig. 4, pooled, and plotted. * indicates p ≤ 0.05 with respect to control values determined by one-way analysis of variance. Sample size is indicated by the number at the base of each category bar. pF, picofarad; Gö, Gö6983.