Abstract 1623: Reactive Oxygen Species increases Intracellular Calcium, alters the Electrophysiological Properties and Synaptic Transmission in Intrinsic Cardiac Ganglion Neurons: in vitro

Abstract

Anerobic metabolism generates reactive oxygen species (ROS) as by-products and this is increased during ischemia. ROS have been shown to interact with and impair the functioning of membrane proteins such as ion channels and transporters and cause disturbances in intracellular Ca 2+ homeostasis. We have investigated the effects of ROS upon [Ca 2+ ] i , the intrinsic electrophysiological characteristics and synaptic transmission in neurons of the ICG regulating the sinoatrial node. The hypothesis is that ROS-induced damage increases [Ca 2+ ] i and attenuates synaptic transmission in ICG. used a whole-mount adult rat ICG preparation, in vitro (rats were killed by stunning and cervical dislocation, according to current UK Home Office guidelines). Intracellular recordings were made using sharp glass microelectrodes filled with Oregon Green 488 BAPTA-1, allowing simultaneous recording of electrical properties and measurement of [Ca 2+ ] i . Signals resulting from [Ca 2+ ] i changes were expressed as the ratio of fluorescence changes over baseline fluorescence, (f-fo)/fo . The ROS-donors hydrogen peroxide (H 2 O 2 , 1 mM) and tert-butyl hydroperoxide ( t -BHP, 1mM) had broadly similar actions on postganglionic somata, both producing membrane potential hyperpolarization (from −48.4 mV ± 6.5 S.D control to −69.9 mV ± 7.9 H 2 O 2 , n=7; and from −51.9 mV ± 7.1 control to −59.2 mV ± 7.9 t -BHP, n=17, p<0.001). Considering the somatic action potential (AP), t -BHP decreased the rate of rise (from 159 V/sec ± 79 control to 117 V/sec ± 53, n=6, p<0.05), but did not alter the rate of fall and increased AP duration (measured at 0 mV) from 0.7ms (± 0.2) control to 1.1ms (± 0.3, n=5, p<0.05). In addition, t -BHP reduced the AP afterhyper-polarization (AHP) amplitude (from 15.5 mV ± 5.2 control to 9.9 mV ± 4.5, n=10, p<0.001) but had no impact on AP overshoot or AHP duration. t -BHP and H 2 O 2 markedly increased resting [Ca 2+ ] i to 1.59 (± 0.03, n=8,p<0.001) and 2.36 (± 0.04, n=3, p<0.05) respectively, of control values (~ 60 nM ). H 2 O 2 blocked synaptic transmission in 3 /4 neurons. In contrast, t -BHP had no significant action on synaptic transmission. Together, these data demonstrate that ROS alters the excitability of ICG neurons attenuating parasympathetic control of the heart during ischemia/ reperfusion.