Targeted Neuronal Nitric Oxide Synthase Transgene Delivery Into Stellate Neurons Reverses Impaired Intracellular Calcium Transients in Prehypertensive Rats

Abstract

Hypertension is associated with the early onset of cardiac sympathetic hyperresponsiveness and enhanced intracellular Ca 2+ concentration [Ca 2+ ] i in sympathetic neurons from both prehypertensive and hypertensive, spontaneously hypertensive rats (SHRs). Oxidative stress is a hallmark of hypertension, therefore, we tested the hypothesis that the inhibitory action of the nitric oxide-cGMP pathway on [Ca 2+ ] i transients is impaired in cardiac sympathetic neurons from the SHR. Stellate ganglia were isolated from young prehypertensive SHRs and age-matched normotensive Wistar-Kyoto rats. [Ca 2+ ] i was measured by ratiometric fluorescence imaging. Neurons from the prehypertensive SHR ganglia had a significantly higher depolarization evoked [Ca 2+ ] i transient that was also associated with decreased expression of neuronal nitric oxide synthase (nNOS), β1 subunit of soluble guanylate cyclase and cGMP when compared with the Wistar-Kyoto rat ganglia. Soluble guanylate cyclase inhibition or nNOS inhibition increased [Ca 2+ ] i in the Wistar-Kyoto rats but had no effect in SHR neurons. A nitric oxide donor decreased [Ca 2+ ] i in both sets of neurons, although this was markedly less in the SHR. A novel noradrenergic cell specific vector (Ad.PRSx8-nNOS/Cherry) or its control vector (Ad.PRSx8-Cherry) was expressed in sympathetic neurons. In the SHR, Ad.PRSx8-nNOS/Cherry-treated neurons had a significantly reduced peak [Ca 2+ ] i transient that was associated with increased tissue levels of nNOS protein and cGMP concentration compared with gene transfer of Ad.PRSx8-Cherry alone. nNOS inhibition significantly increased [Ca 2+ ] i after Ad.PRSx8-nNOS/Cherry expression. We conclude that artificial upregulation of stellate sympathetic nNOS via targeted gene transfer can directly attenuate intracellular Ca 2+ and may provide a novel method for decreasing enhanced cardiac sympathetic neurotransmission.