Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC172

Poster Communications

Targeted neuronal nitric oxide synthase transgene delivery into cardiac sympathetic neurons reverses abnormal calcium handling in prehypertensive spontaneously hypertensive rats

D. Li1, N. Nikiforova1, D. J. Paterson1

1. BHF Centre of Research Excellence, Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford, United Kingdom.

Hypertension is associated with the early onset of cardiac sympathetic hyper-responsiveness. The NO-cGMP pathway has an inhibitory action on noradrenergic neurotransmission, and therefore we hypothesised that calcium handling in postganglionic cardiac sympathetic neurons is enhanced in young (pre-hypertensive) spontaneously hypertensive rats (SHR) because of impaired NO-cGMP signalling. Rats were humanely killed by an approved Home Office schedule 1 method. Cardiac stellate ganglia were enzymatically isolated from 4-5 weeks old prehypertensive SHR and age matched normotensive Wistar-Kyoto (WKY) rats. A novel noradrenergic cell specific vector marked by red fluorescent protein mCherry (Ad.PRS-mCherry/nNOS) or empty vector (Ad.PRS-mCherry, control virus) was transferred to cultured sympathetic neurons for 3-4 days. Intracellular free Ca2+ concentration ([Ca2+]i) was measured by ratiometric fluorescence imaging using fura-4AM in neurons that had been infected with mCherry. The sympathetic neurons from the prehypertensive SHR (n=10) had a significantly higher depolarization evoked [Ca2+]i increase when compared with the age matched normotensive WKY rats (n=8) (F/F0: SHR: 2.33 ± 0.14; WKY: 1.93 ± 0.15, p<0.03). Ad.PRS-nNOS/mCherry significantly reduced the depolarization induced [Ca2+]i in the SHR (F/F0: 1.54 ± 0.11, n=12) when compared with the empty vector transduced neurons (F/F0: 2.03 ± 0.08, n=5, p<0.05)). nNOS specific inhibition by N-[(4S)-4-Amino-5-[(2-aminoetyl](amino] pentyl]-N'-nitroguanidine (AAAN) reversed this response in transfected SHR with Ad.PRS-nNOS/mCherry (F/F0 from 1.54 ± 0.11 to 2.14 ± 0.17, n=12, p<0.01). These results show that cardiac sympathetic Ca2+ handling is impaired in young SHR before the actual onset of hypertension. Moreover, artificial up-regulation of cardiac sympathetic nNOS via gene transfer can directly attenuate intracellular Ca2+, and may provide a novel method for modulation of cardiac sympathetic neurotransmission.

Where applicable, experiments conform with Society ethical requirements