Cardiac output is regulated by the coordinate interactions of stimulatory sympathetic and inhibitory parasympathetic signals. Intracardiac parasympathetic ganglia are integrative centers of cardiac regulation, and modulation of the parasympathetic drive on the heart is accomplished by altering intrinsic cardiac ganglion neuron excitability. The pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) family of peptides modulates cardiac function, and in guinea pig heart, PACAP appears to act directly on intrinsic parasympathetic cardiac ganglia neurons through PACAP-selective receptors. A multidisciplinary project tested whether cardiac PACAP peptides act through PACAP-selective receptors as excitatory neuromodulators amplifying the parasympathetic inhibition from guinea pig cardiac ganglia. The in vivo sources of regulatory PACAP peptides were localized immunocytochemically to neuronal fibers and a subpopulation of intrinsic postganglionic cardiac neurons. RT-PCR confirmed that cardiac ganglia expressed proPACAP transcripts and have PACAP peptide biosynthetic capabilities. Messenger RNA encoding PACAP-selective PAC1 receptor isoforms were also present in cardiac ganglia. Alternative splicing of PAC1 receptor transcripts produced predominant expression of the very short variant with neither HIP nor HOP cassettes; lower levels of the PAC1HOP2 receptor mRNA were present. Almost all of the parasympathetic neurons expressed membrane-associated PAC1 receptor proteins, localized immunocytochemically, which correlated with the population of cells that responded physiologically to PACAP peptides. PACAP depolarized cardiac ganglia neurons and increased neuronal membrane excitability. The rank order of peptide potency on membrane excitability in response to depolarizing currents was PACAP27>PACAP38>VIP. The PACAP-induced increase in excitability was not a function of membrane depolarization nor was it caused by alterations in action potential configuration. These results support roles for PACAP peptides as integrative modulators amplifying, through PACAP-selective receptors, the parasympathetic cardiac ganglia inhibition of cardiac output.

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Following G protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex often is rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. Formation of signaling endosomes is recognized to be a mechanism to produce sustained intracellular signals, which may be distinct from those generated at the cell surface, for cellular responses including growth, differentiation and survival. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) is a potent neurotransmitter/neurotrophic peptide and mediates its diverse cellular functions in part through internalization of its cognate G protein-coupled PAC1 receptor (Adcyap1r1). In the current studies, we examined whether PAC1 receptor endocytosis participates in regulation of neuronal excitability. While PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynaminI/II, respectively, suppressed the PACAP effect. Subsequent addition of inhibitor, after the PACAP- induced increase in excitability developed, gradually attenuated excitability with no changes in action potential properties. Likewise, the PACAP-induced increase in excitability was markedly decreased at ambient temperature. Receptor trafficking studies with GFP-PAC1 cell lines demonstrated the efficacy of Pitstop 2 and dynasore and low temperature to suppress PAC1 receptor endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1 receptor signaling still increased neuronal cAMP production even with endocytic blockade. In aggregate, these studies demonstrate that PACAP/PAC1 receptor complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

Whole mount preparations from guinea‐pig hearts were used to characterize the receptors and ionic mechanisms mediating the substance P (SP)‐induced depolarization of para‐sympathetic postganglionic neurones of the cardiac ganglion. Measurement of the amplitude of depolarization in response to superfusion of different tachykinin agonists (neurokinins A (NKA) and B (NKB), SP, and senktide) gave a rank‐order potency of NKB=senktide > NKA > SP, indicating involvement of an NK3 receptor. The use of the selective tachykinin receptor antagonists SR 140333, SR 48986, and SR 142801 demonstrated that only the NK3 receptor antagonist SR 142801 inhibited the SP‐induced depolarization. The SP‐induced depolarization was not inhibited by Ba2+, TEA, or niflumic acid, or altered by reduced Cl− solutions, but was attenuated in reduced Na+ solutions. Single electrode voltage clamp studies demonstrated that the SP‐induced inward current increased in amplitude at more negative potentials, had a reversal potential of approximately 0 mV, and was reduced in amplitude in reduced Na+ solutions. We conclude that the SP‐induced depolarization in guinea‐pig postganglionic parasympathetic neurones of the cardiac ganglion is due to NK3‐mediated activation of a non‐selective cation conductance.

This paper aims to determine whether chronic vagus nerve stimulation (VNS) mitigates myocardial infarction (MI)-induced remodeling of the intrinsic cardiac nervous system (ICNS), along with the cardiac tissue it regulates. Guinea pigs underwent VNS implantation on the right cervical vagus. Two weeks later, MI was produced by ligating the ventral descending coronary artery. VNS stimulation started 7 days post-MI (20 Hz, 0.9 ± 0.2 mA, 14 s on, 48 s off; VNS-MI, n = 7) and was compared with time-matched MI animals with sham VNS (MI n = 7) vs. untreated controls (n = 8). Echocardiograms were performed before and at 90 days post-MI. At termination, IC neuronal intracellular voltage recordings were obtained from whole-mount neuronal plexuses. MI increased left ventricular end systolic volume (LVESV) 30% (P = 0.027) and reduced LV ejection fraction (LVEF) 6.5% (P < 0.001) at 90 days post-MI compared with baseline. In the VNS-MI group, LVESV and LVEF did not differ from baseline. IC neurons showed depolarization of resting membrane potentials and increased input resistance in MI compared with VNS-MI and sham controls (P < 0.05). Neuronal excitability and sensitivity to norepinephrine increased in MI and VNS-MI groups compared with controls (P < 0.05). Synaptic efficacy, as determined by evoked responses to stimulating input axons, was reduced in VNS-MI compared with MI or controls (P < 0.05). VNS induced changes in myocytes, consistent with enhanced glycogenolysis, and blunted the MI-induced increase in the proapoptotic Bcl-2-associated X protein (P < 0.05). VNS mitigates MI-induced remodeling of the ICNS, correspondingly preserving ventricular function via both neural and cardiomyocyte-dependent actions.