Nitric oxide (NO) generated from neuronal nitric oxide synthase (NOS-1) in intrinsic cardiac ganglia has been implicated in parasympathetic-induced bradycardia. We provide direct evidence that NOS-1 acts in a site-specific manner to promote cardiac vagal neurotransmission and bradycardia. NOS-1 gene transfer to the guinea pig right atrium increased protein expression and NOS-1 immunolocalization in cholinergic ganglia. It also increased the release of acetylcholine and enhanced the heart rate (HR) response to vagal nerve stimulation (VNS) in vitro and in vivo. NOS inhibition normalized the HR response to VNS in the NOS-1-treated group compared with the control groups (enhanced green fluorescent protein and sham) in vitro. In contrast, an acetylcholine analogue reduced HR to the same extent in all groups before and during NOS inhibition. These results demonstrate that NOS-1-derived NO acts presynaptically to facilitate vagally induced bradycardia and that upregulation of NOS-1 via gene transfer may provide a novel method for increasing cardiac vagal function.T he biological messenger nitric oxide (NO) is thought to be a fundamental signaling molecule in the regulation of cardiac cholinergic function. [1][2][3][4] Neuronal nitric oxide synthase (NOS-1), the enzyme responsible for NO synthesis, colocalizes with choline acetyltransferase in the intracardiac ganglia. 5 Functionally, pharmacological evidence suggests that NO generated from NOS-1 directly enhances the negative chronotropic effect of cholinergic stimulation 6,7 by activating the guanylate cyclase/ cGMP pathway 7,8 to facilitate the release of acetylcholine (ACh), 9 and indirectly via endothelial NOS-3 M 2 receptor coupled inhibition of I Ca-L in pacemaking cells, 10 although this latter point is disputed. 11 Moreover, the vagal heart rate (HR) response to modulators of the NO-cGMP pathway is not mimicked by the stable analogue of ACh, carbachol, suggesting that the dominant functional role of this pathway is presynaptic to the neuroeffector junction. 7,8 We tested the hypothesis that NOS-1 gene transfer into the right atrium would enhance vagal-induced neurotransmission and bradycardia but would be ineffective when HR was decreased by carbachol.
Materials and MethodsDetailed methods for gene transfer, 12 immunohistochemistry, 13 fluorescence microscopy, 14 confocal imaging, 5 immunoblotting, 7 measurements of ACh release, 9 and in vivo or in vitro autonomic phenotyping 7,8 can be found in the online data supplement, available at http://www.circresaha.org.
Results and DiscussionQualitative examination using NADPH-diaphorase staining of tissue cryosections showed greater expression in atrial tissue after replication-deficient adenoviral vector transfection with neuronal NOS (Ad.NOS-1) ( Figure 1A). This was confirmed by Western blotting in which Ad.NOS-1-treated atria (nϭ8) showed significantly greater expression of NOS-1 protein compared with atria infected with an adenoviral vector encoding recombinant enhanced green fluorescent protein (Ad.eGFP) (nϭ8, ...
Mechanisms underlying the circadian rhythm in lung ventilation were investigated. Ten healthy male subjects were studied for 36 h using a constant routine protocol to minimize potentially confounding variables. Laboratory light, humidity, and temperature remained constant, subjects did not sleep, and their meals and activities were held to a strict schedule. Respiratory chemoreflex responses were measured every 3 h using an iso-oxic rebreathing technique incorporating prior hyperventilation. Subjects exhibited circadian rhythms in oral temperature and respiratory chemoreflex responses, but not in metabolic rate. Basal ventilation [i.e., at subthreshold end-tidal carbon dioxide partial pressure (PET(CO(2)))] did not vary with time of day, but the ventilatory response to suprathreshold PET(CO(2)) exhibited a rhythm amplitude of approximately 25%, mediated mainly by circadian variations in the CO(2) threshold for tidal volume. We conclude that the circadian rhythm in lung ventilation is not a simple consequence of circadian variations in arousal state and metabolic rate. By raising the chemoreflex threshold, the circadian timing system may increase the propensity for respiratory instability at night.
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