Our long term goal is to understand the role of neurotransmitters in carotid body function. Our preliminary data have shown that carotid body can synthesize nitric oxide (NO) and it may function as a transmitter. The current proposal focuses ont he physiological significance of NO in carotid body chemoreception. Our hypothesis is that: NO exerts a tonic inhibitor influence on the chemo-sensory activity and the actions of NO are associated with changes in [Ca2+]i, mediated either by a cGMP- dependent and/or independent pathway involving mitochondria. We propose to test this hypothesis using a combination of physiological, histochemical, biochemical and biophysical techniques. Proposed experiments will be performed on anaesthetized cats. Experiments outlined in Specific Aim 1 will determine the source of NO production in the carotid body. This will be accomplished by examining the distribution of nitric oxide synthase (NOS), the enzyme the catalyzes the formation of NO by histochemistry. Enzyme distribution will examined after unilateral chronic sectioning of carotid sinus nerve of sympathectomy of nodose ganglionectomy of combination of all. The results will be compared with contralateral controls. In parallel experiments the enzyme activity will be quantified biochemically. Experiments in Aim 2 will characterize the effects of endogenous NO on the sensory activity of the carotid body. The influence of endogenous NO will be examined by (a) inhibitors of NO synthesis and (b) hemoglobin which traps endogenous NO. Experiments will be performed on carotid bodies in vitro. Chemoreceptor activity will be recorded. No levels in the carotid body will be measured with a chemical microsensor. Experiments in Aims 3 will test the hypothesis that NO mediates inhibition of afferent carotid body activity by stimulation of the sinus nerve. To this end, chemosensory response to electrical stimulation of the sinus nerve will be examined (a) after blockade of NO synthesis, (b) in presence of hemoglobin, and (c) after blockade of soluble guanylate cyclase, a purported target for the actions of NO. Chemosensory activity will be recorded from the isolated carotid bodies. Tissue levels of NO will be monitored with a chemical sensor. Studies proposed in Aim 4 will elucidate the cellular mechanisms associated with the effects of NO on the carotid body. We propose that the effects of NO on chemosensory activity are associated with alterations in [Ca2+]i of the carotid body cells. The changes in calcium are coupled to cGMP- dependent and/or an independent pathway involving mitochondria. The role of cGMP will be examined by immunocytochemical and biochemical techniques. The changes in [Ca2+]i and mitochondrial membrane potential in carotid body cells will be determined by fluorescence imaging. It has long been postulated that heme pigment/s in chemoreceptor tissue are important for O2 sensing mechanisms. Certain features of NO are relevant to this postulation. Molecular O2 is necessary for NO synthesis and the NOS is an heme containing enzyme. The receptor for its cellular actions is a heme-containing enzyme, guanylate cyclase. Examining the significance of NO may yield potential clues as to the O2 sensing mechanisms at the carotid body. This proposal may also yield information on therapeutic uses of NO or its blockers in treatment of hypoxemia, a problem associated with many pulmonary deceases.
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