The carotid bodies are peripheral chemoreceptor organs. They transduce changes in the levels of arterial oxygen, carbon dioxide, and hydrogen ion concentrations into action potential encoded nerve signals. This transduction process is believed to occur through a complex mechanism, mediated by a variety of neurochemicals such as biogenic amines, neuropeptides and gas molecules like nitric oxide. Our studies on the metabolism of neuropeptides in the carotid body have shown that neutral endopeptidase (NEP) plays a major role in the regulation of neuropeptides like tachykinins and enkephalins in the carotid body. Also, we have found that NEP in the carotid body occurs in two active forms viz., a less common cytosolic and conventional membrane-bound form in near equal proportions. Furthermore, inhibition of the NEP activity in vivo appears to potentiate the response of the carotid body to hypoxia suggesting that the two NEP forms may play hitherto undefined important functional roles in the carotid body. This investigation, specifically, addresses the molecular characterization of the two NEP forms of the carotid body and their importance in the function(s) of the carotid body. Our hypothesis is that physiological stimulus such as hypoxia alters the activity and/or synthesis of NEP. These changes in NEP, in turn, regulate the concentrations and modulate the actions of both the excitatory and inhibitory neuropeptides and thus determine the magnitude of chemosensory responses of the carotid body to either acute and/or chronic hypoxia. This hypothesis will be tested using biochemical and cellular biological techniques in combination with immunological and immunocytochemical studies on the two forms of NEP of the carotid body. Most of the experiments will use carotid bodies obtained from cats and rats exposed to defined gas challenges. Experiments outlined in Specific Aim 1 will address the physico-chemical characterization of the soluble and membrane- bound forms of NEP of the carotid body and evaluate the structural relationship among the two NEP forms. The kinetic experiments proposed in Specific Aim 2 will examine the possibility that the NEP forms of the carotid body have different enzymological properties.
In Specific Aim 3, using immunocytochemical approaches, we will determine the cellular location of NEP in relation to the location of tachykinins, enkephalins and atrial natriuretic peptides in the carotid body. The potential alterations in NEP forms of the carotid body in response to different gas challenges and possible mechanisms involved in these alterations will be addressed in Specific Aim 4. It is anticipated that the proposed studies will provide clues as to the role of NEP in the functions of the carotid body. These studies may also yield information in the development of enzyme and its inhibitor(s) based therapeutic agents in the treatment of hypoxemia, a major problem associated with chronic obstructive lung diseases and high altitude adaptation.
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