Two central hypotheses will be explored: 1) Enhanced peripheral chemoreflex function contributes to the development and maintenance of impaired baroreflex function and elevated resting sympathetic function in the rabbit model of pacing-induced heart failure (HF); and 2) Down-regulation of nitric oxide synthase (NOS) in the carotid body (CB) during the development of HF leads to altered ion channel function of type I cells and enhanced CB chemoreceptor sensitivity. To test the first hypothesis, we will determine the time-course of changes in peripheral chemoreflex and baroreflex control of renal sympathetic nerve activity and ventilation during the development of HF in conscious rabbits; and to determine whether the altered peripheral chemoreflex function contributes to impaired baroreflex function and the elevation in resting sympathetic activity in the HF state. To test the second hypothesis, we will determine whether NOS contributes to the altered discharge characteristics or CB chemoreceptor afferents in HF rabbits in response to graded levels of hypoxia, hypercapnia, and chemical stimuli. Afferent responses to these stimuli will be assessed before and after NOS inhibition, exposure to an NO donor to 1 arginine. In other experiments, afferent responses to stimuli will be assessed before and after guanylate cyclase inhibition, phosphodiesterase inhibition, or exposure to cGMP analogues to determine the extent to which NO effects can be attributed to cGMP activation. Using a whole cell patch-clamp, we will determine whether the current density of O2-sensitive K+ channels is altered in CB type 1 (glomus) cells of HF rabbits, and determine whether NOS and cGMP pathways contribute to the altered K+ channel characteristics of these type I cells as in the afferent experiments. We will determine the extent to which NOS activity and the expression and distribution of endothelial (ec) and neural (nc) NOS in cells within the CB is altered in HF rabbits. We will: measure expression of ec- and nc-NOS mRNA in the CB using RT-PCR methods; measure the distribution of NOS isoforms in the CB using immunocytochemical methods; and measure NO production from Cbs under normoxic and graded hypoxic conditions using enzymatic and chemiluminescence methods.
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