Effects of VPAC1 activation in nucleus ambiguus neurons

Abstract

The pituitary adenylyl cyclase-activating polypeptide (PACAP) and its G protein-coupled receptors, PAC1, VPAC1 and VPAC2 form a system involved in a variety of biological processes. Although some sympathetic stimulatory effects of this system have been reported, its central cardiovascular regulatory properties are poorly characterized. VPAC1 receptors are expressed in the nucleus ambiguus (nAmb), a key center controlling cardiac parasympathetic tone. In this study, we report that selective VPAC1 activation in rhodamine-labeled cardiac vagal preganglionic neurons of the rat nAmb produces inositol 1,4,5-trisphosphate receptor-mediated Ca²⁺ mobilization, membrane depolarization and activation of P/Q-type Ca²⁺ channels. In vivo, this pathway converges onto transient reduction in heart rate of conscious rats. Therefore we demonstrate a VPAC1-dependent mechanism in the central parasympathetic regulation of the heart rate, adding to the complexity of PACAP-mediated cardiovascular modulation.

Keywords: Autonomic cardiac tone; Bradycardia; Calcium signaling; PACAP.

Figure 1. Concentration-dependent [Ca²⁺]_i elevations induced by VPAC1 activation in cardiac preganglionic vagal nucleus ambiguus neurons

A, Changes in Fura-2 fluorescence ratio (340 nm/380 nm) of rhodamine-labeled neurons upon administration of 100 nM VPAC1 agonist alone (top) and in presence of antagonist pretreatment (bottom). B, Typical tracings of Ca²⁺ responses induced by administration of VPAC1 agonist alone (left) or in presence of VPAC1 antagonist. C, Concentration-dependent effect of VPAC1 agonist (10–1000 nM) on [Ca²⁺]_i of cardiac vagal nAmb neurons and lack of effect of 100 nM agonist in neurons pretreated with VPAC1 antagonist (10 μM, 20 min); *P < 0.05 compared to basal Ca²⁺ levels and within the group; ^#P < 0.05 compared to 100 nM agonist.

Figure 2. VPAC1 activation triggers Ca²⁺ influx via P/Q-type Ca²⁺ channels

A, Representative examples of Ca²⁺ responses triggered by VPAC1 agonist in the absence and presence of blockers of voltage-activated Ca²⁺ channels (N-type, ω-conotoxin GVIA; P/Q-type, ω-conotoxin MVIIC). B, Quantification of the increase in [Ca²⁺]_i produced by VPAC1 stimulation in the conditions mentioned in A; *P < 0.05 compared with VPAC1 agonist alone.

Figure 3. VPAC1 activation releases Ca²⁺ from IP₃-sensitive Ca²⁺ stores in cardiac vagal neurons of nucleus ambiguus

A, Characteristic increases in [Ca²⁺]_i produced by VPAC1 agonist (100 nM) in Ca²⁺-free saline, in the absence and presence of lysosomal disruptor bafilomycin A1 (Baf), ryanodine receptor blocker ryanodine (Ry), IP₃R inhibitors xestospongin C (XeC) and 2-aminoethoxydiphenyl borate (2-APB). B, Comparison of the effects on [Ca²⁺]_i induced by treatments indicated in A, in cultured cardiac vagal neurons; *P < 0.05 as compared with VPAC1 agonist alone.

Figure 4. VPAC1 activation depolarizes cardiac vagal neurons of nucleus ambiguus

A, Representative changes in membrane potential elicited by VPAC1 agonist (100 nM) alone or after antagonist (10 μM) pretreatment in rhodamine-labeled cardiac vagal nAmb neurons. B, Response quantifications reveal concentration-dependent depolarizing effect of VPAC1 agonist (10–1000 nM) on the membrane potential of cardiac preganglionic neurons; *P < 0.05 compared with resting membrane potential and within the group, ^#P < 0.05 compared to the effect of 100 nM VPAC1 agonist.

Figure 5. Bradycardic effects of microinjection of VPAC1 agonist in the nucleus ambiguus of conscious rats

A, Characteristic heart rate and blood pressure recordings after microinjection of saline, L-glutamate (L-Glu, 5 mM, 50 nL) and VPAC1 agonist (100 nM, 50 nL). B, Microinjection of VPAC1 agonist (10, 100 and 1000 nM) induces dose-dependent bradycardic responses. *P < 0.05 as compared to basal heart rate and within the group.

Figure 6. Microinjection of VPAC1 antagonist into the nucleus ambiguus prevents the bradycardic effect of the VPAC1 agonist in conscious rats

A, Representative examples of heart rate and blood pressure recordings after microinjection of saline, L-glutamate (L-Glu, 5 mM, 50 nL), VPAC1 antagonist (500 nM, 50 nL) alone, and co-administration of VPAC1 antagonist (500 nM, 50 nL) and VPAC1 agonist (100 nM, 50 nL). The bradycardic effect of VPAC1 agonist (100 nM) is blocked by co-administration of VPAC1 antagonist; *P < 0.05 compared with the effect of the antagonist alone or of the co-administration of the antagonist and agonist.

Figure 7. Diagram summarizing the effects of VPAC1 receptor activation in the nucleus ambiguus neurons

VPAC1 agonists such as PACAP or VIP activate VPAC1 receptor and release Ca²⁺ from endoplasmic reticulum (ER) via inositol 1,4,5-trisphosphate receptor (IP₃R). VPAC1 agonists also produce membrane depolarization, facilitating activation of P/Q-type voltage-gated Ca²⁺ channels (VGCC) and subsequent Ca²⁺ influx. The depolarization is transmitted along the axon and leads to the release of acetylcholine (ACh) in cardiac ganglia followed by bradycardia.