IONIC BASIS OF EXCITABILITY IN INTRINSIC CARDIAC NEURONS

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The ionic mechanisms underlying excitability in neurons of parasympathetic ganglia of the mammalian heart and the action of neurotransmitters on their function will be studied. Neurotransmitters influence the function of a variety of ion channels and intracellular proteins in excitable cells to alter resting and active electrical properties and, hence, the physiological responses of the cell. Voltage-dependent and receptor- operated ion channels will be studied in dissociated ganglion cells prepared from explants of intracardiac ganglia dissected from the atrial myocardium of newborn rats and grown in tissue culture. The primary objective is to study the mechanisms of action of neurotransmitters on receptor-operated and voltage-gated ion channels in cardiac neurons and subsequent changes in cytoplasmic free calcium concentration ([Ca2+]i) on membrane excitability. Experiments will focus on the molecular mechanisms of adrenergic and peptidergic modulation of neuronal voltage-dependent channels and on the permeability of the ion channels activated by the neurotransmitters, acetylcholine and adenosine triphosphate (ATP). Receptor-operated cation channels which function to permit Ca2+ entry during activation by these agonists will be examined by single channel recording to determine the ionic selectivity, gating kinetics and role of intracellular second messengers in receptor-channel function. The influence of receptor activation on ion channel activity and [Ca2+]i will be evaluated in voltage clamped neurons loaded with the fluorescent Ca2+ indicator dye, Indo-1, to simultaneously monitor [Ca2+]i fluorescence and membrane currents. Specifically, the relative contributions of receptor- mediated Ca2+ entry and Ca2+ release from intracellular stores by biochemical second messengers will be assessed in experiments in which various candidates for the second messenger (e.g., Ca2+, cyclic nucleotides, arachidonate metabolites, inositol phosphates, guanine nucleotides) are manipulated by direct application to the cytoplasm with the patch clamp method. The goal of the research is to elucidate mechanisms that underlie ion channel function in cardiac neurons and the role of neurotransmitters in regulating neuronal excitability in mammalian parasympathetic cardiac ganglia.