Jean C. Hardwick

Evidence for afferent fiber innervation of parasympathetic neurons of the guinea-pig cardiac ganglion

The present study was done to establish whether peptidergic afferent inputs can modulate parasympathetic neurons of the guinea-pig cardiac ganglion. Whole mount preparations from the guinea-pig heart were utilized to localize afferent terminals by immunohistochemistry and for intracellular recordings from individual neurons in situ. Action potentials could be elicited by both intracellular current injection and stimulation of interganglionic fiber bundles. Two types of neuron, phasic (95%) and tonic (5%) as defined by their firing properties, were observed. High frequency (5-10 Hz) interganglionic fiber stimulation produced a calcium-dependent, slow depolarization in many cells which was not blocked by 100 microM hexamethonium or 1 microM atropine. A prolonged depolarization was also produced by local application of capsaicin (1 mM), which releases substance P and CGRP from afferent nerve terminals. Microinjection of the mammalian tachykinins substance P, neurokinin A and neurokinin B (all at 100 microM), also produced a slow depolarization. Application of specific agonists for the tachykinin receptor subtypes indicated that these neurons express both NK2 and NK3 receptors. Individual cells were filled with neurobiotin to examine their morphology and the preparations were counter-stained for SP-like immunoreactivity. The results demonstrated that SP-positive fibers are found in close apposition to both phasic and tonic neurons. From these results, we suggest that the parasympathetic neurons of the guinea-pig cardiac ganglion receive inputs from peptidergic, afferent fibers and that this input provides a pathway for potential local reflex control of cardiac function.

Tachykinin-induced activation of non-specific cation conductance via nk3 neurokinin receptors in guinea-pig intracardiac neurones

1 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. 2 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. 3 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. 4 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.

Pituitary Adenylate Cyclase 1 Receptor Internalization and Endosomal Signaling Mediate the Pituitary Adenylate Cyclase Activating Polypeptide-Induced Increase in Guinea Pig Cardiac Neuron Excitability

After G-protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex is often rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. The formation of signaling endosomes is recognized as a mechanism that produces sustained intracellular signals that 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 (PAC1R; Adcyap1r1). In the present study, we examined whether PAC1R endocytosis participates in the regulation of neuronal excitability. Although PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynamin I/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, dynasore, and low temperatures at suppressing PAC1R endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1R signaling still increased neuronal cAMP production even with endocytic blockade. Our results demonstrate that PACAP/PAC1R complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

Chronic myocardial infarction induces phenotypic and functional remodeling in the guinea pig cardiac plexus

Chronic myocardial infarction (CMI) is associated with remodeling of the ventricle and evokes adaption in the cardiac neurohumoral control systems. To evaluate the remodeling of the intrinsic cardiac nervous system following myocardial infarction, the dorsal descending coronary artery was ligated in the guinea pig heart and the animals were allowed to recover for 7-9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential configuration compared with age-matched controls and sham-operated animals. The intrinsic cardiac neurons from chronic infarcted hearts did demonstrate an increase in evoked action potential (AP) frequency (as determined by the number of APs produced with depolarizing stimuli) and an increase in responses to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide (PACAP)-induced increases in intrinsic cardiac neuron-evoked AP frequency were similar between control and CMI animals. Immunohistochemical analysis demonstrated a threefold increase in percentage of neurons immunoreactive for neuronal nitric oxide synthase (NOS) in CMI animals compared with control and the additional expression of inducible NOS by some neurons, which was not evident in control animals. Finally, the density of mast cells within the intrinsic cardiac plexus was increased threefold in preparations from CMI animals. These results indicate that CMI induces a differential remodeling of intrinsic cardiac neurons and functional upregulation of neuronal responsiveness to specific neuromodulators.

Remodeling of the guinea pig intrinsic cardiac plexus with chronic pressure overload

Chronic pressure overload (PO) is associated with cardiac hypertrophy and altered autonomic control of cardiac function, in which the latter may involve adaptations in central and/or peripheral cardiac neural control mechanisms. To evaluate the specific remodeling of the intrinsic cardiac nervous system following pressure overload, the descending thoracic aorta artery of the guinea pig was constricted approximately 20%, and the animals recovered for 9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential depolarization compared with age-matched controls and sham-operated animals, but afterhyperpolarization duration was increased in PO animals. Neuronal excitability, as determined by the number of action potentials produced with depolarizing stimuli, was differentially increased in phasic neurons derived from PO animals in response to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide-induced increases in intrinsic cardiac neuron evoked AP frequency were similar between control and PO animals. Immunohistochemical analysis demonstrated a twofold increase in the percentage of neurons immunoreactive for neuronal nitric oxide synthase in PO animals compared with control. The density of mast cells within the intrinsic cardiac plexus from PO animals was also increased twofold compared with preparations from control animals. These results indicate that congestive heart failure associated with chronic pressure overload induces a differential remodeling of intrinsic cardiac neurons and upregulation of neuronal responsiveness to specific neuromodulators.

Mechanisms of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP)-Induced Depolarization of Sympathetic Superior Cervical Ganglion (SCG) Neuronsa

Our understanding of PACAP expression and regulation of sympathetic neuronal function has been augmented considerably over the last few years. Among the three major VIP/PACAP receptor subtypes, the SCG appears to express preferentially one particular variant of the PACAP-selective PACAP1 receptor coupled to multiple intracellular signaling cascades. The in situ histochemical hybridization and immunocytochemical studies of PACAP1 receptor mRNA and protein are in good agreement; nearly all of the SCG neurons express the PACAP-selective receptor, suggesting that most of the sympathetic neurons are under PACAP neuromodulation. In accord with that possibility, several independent studies have now demonstrated PACAP peptide expression in the IML sympathetic preganglionic neurons and fibers, including those projecting to the SCG, further emphasizing the significance of PACAP peptides as a preganglionic noncholinergic mediator of sympathetic function. Given the high potency of PACAP on any of a number of cellular responses, the functional relevance of PACAP peptides on SCG neurons is considerable. We have previously demonstrated the potency and efficacy of both PACAP27 and PACAP38 on sympathetic neuron neurotransmitter/neuropeptide production and secretion; the ability of these peptides to stimulate neuronal second messenger activation was also in the nanomolar range. These results are congruous with our current electrophysiological studies, which were driven to further define the dynamic sympathetic responses to PACAP. In line with the morphological studies, for example, more than 90% of the sympathetic neurons responded to PACAP. In agreement with previous neuropharmacological data, the PACAP-induced depolarizations were elicited at physiologically relevant peptide concentrations at high affinity PACAP-selective receptors. The effects were direct and the alterations in postganglionic neuronal membrane properties appeared to be mediated by several ionic mechanisms. If these studies were analogous to pieces in a puzzle to understand the effects of PACAP in sympathetic development and function, the picture of late has been more completely assembled. But several important challenges still remain. What are the signal transduction mechanisms that mediate the PACAP-induced changes in sympathetic membrane properties? How do the resulting alterations impact the acute and more long-term responses of sympathetic neurons? Does the coupling of PACAP1 receptors to intracellular signaling pathways differ during development, resulting in a transition from the neurotrophic properties of PACAP in neuroblasts to neuromodulatory roles of the peptides in postmitotic neurons? By looking at these issues in one distinct neuronal system, we enlarge our understanding and appreciation of peptides, and PACAP in particular, in the molecular and cellular events guiding neuronal development, function, and plasticity.

Extracellular ATP Stimulates Norepinephrine Uptake in PC12 Cells

Abstract: This study examined the effects of extracellular ATP on norepinephrine (NE) uptake, using PC12 cells as a model of noradrenergic neurons. Previous experiments with syn‐aptosomes led to the hypothesis that extracellular ATP can regulate NE uptake via an ecto‐protein kinase. In the present study, we examined the high‐affinity uptake of NE (referred to as uptake 1) in PC12 cells in the presence of varying concentrations of extracellular ATP. In the presence of Ca2+, low concentrations of ATP (0.1 μM) increased uptake 1 by approximately 36%. This increase could be mimicked by aden‐osine‐5′‐O‐(3‐thiotriphosphate) tetralithium salt (ATPγS), an analogue of ATP which can be utilized by protein kinases, and not by 5′‐adenylylimidodiphosphate tetralithium salt, a nonhydrolyzable analogue of ATP. GTP, ADP, and adenosine also had no effect on uptake 1. Preincubation of the cells with NE and ATPγS, followed by washing and assaying NE uptake 30 min later, resulted in a persistent increase in uptake 1. Similar pretreatment with ATP did not show this increase; however, simultaneous pretreatment with ATP and ATPγS blocked the activation produced by ATPyS alone. Kinetic analysis showed that ATPγS pretreatment produces an increase in the Vmax of uptake 1 without altering the apparent Km for NE. These results support the hypothesis that extracellular ATP can regulate NE uptake via an ecto‐protein kinase.

Expression and physiological actions of neuropeptide Y in guinea pig parasympathetic cardiac ganglia.

Guinea pig atrial whole mount preparations containing the parasympathetic cardiac ganglia were used to establish the expression, distribution and actions of neuropeptide Y (NPY) in atrial tissues. NPY-immunoreactive fibers densely innervated the atrial myocardium and blood vessels. Fibers containing NPY also innervated intrinsic parasympathetic cardiac neurons. Four percent of the cardiac neurons, identified using microtubule associated protein-2 antiserum, were NPY-positive. An endogenous source of NPY was confirmed with reverse transcription PCR which demonstrated the presence of proNPY mRNA. Sixty percent of the parasympathetic cardiac neurons were hyperpolarized by local application of NPY. NPY also decreased the amplitude and duration of the action potential after hyperpolarization in 60% of the neurons and decreased the fast excitatory postsynaptic potential in about 50% of the cells. These observations indicate that NPY is anatomically positioned to directly alter the output of the parasympathetic cardiac ganglia either by hyperpolarizing the cardiac neurons or by decreasing the fast synaptic input which drives individual neurons.

Vagus nerve stimulation mitigates intrinsic cardiac neuronal and adverse myocyte remodeling postmyocardial infarction

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.

Dynamic remodeling of the guinea pig intrinsic cardiac plexus induced by chronic myocardial infarction

Myocardial infarction (MI) is associated with remodeling of the heart and neurohumoral control systems. The objective of this study was to define time-dependent changes in intrinsic cardiac (IC) neuronal excitability, synaptic efficacy, and neurochemical modulation following MI. MI was produced in guinea pigs by ligation of the coronary artery and associated vein on the dorsal surface of the heart. Animals were recovered for 4, 7, 14, or 50 days. Intracellular voltage recordings were obtained in whole mounts of the cardiac neuronal plexus to determine passive and active neuronal properties of IC neurons. Immunohistochemical analysis demonstrated an immediate and persistent increase in the percentage of IC neurons immunoreactive for neuronal nitric oxide synthase. Examination of individual neuronal properties demonstrated that after hyperpolarizing potentials were significantly decreased in both amplitude and time course of recovery at 7 days post-MI. These parameters returned to control values by 50 days post-MI. Synaptic efficacy, as determined by the stimulation of axonal inputs, was enhanced at 7 days post-MI only. Neuronal excitability in absence of agonist challenge was unchanged following MI. Norepinephrine increased IC excitability to intracellular current injections, a response that was augmented post-MI. Angiotensin II potentiation of norepinephrine and bethanechol-induced excitability, evident in controls, was abolished post-MI. This study demonstrates that MI induces both persistent and transient changes in IC neuronal functions immediately following injury. Alterations in the IC neuronal network, which persist for weeks after the initial insult, may lead to alterations in autonomic signaling and cardiac control.