(Copied from author abstract): The importance of impaired HC03 secretion in the pathophysiology of the pancreas of cystic fibrosis (CF) patients has been well documented for many years. Perhaps, because CF secretion has been assumed to be of higher importance and thus received the greater attention, the transcellular mechanisms of HC03 secretion in the airways remain poorly understood. Studies from our laboratory and others have now established that the human airway serous cell line, Calu-3 cells, secretes HC03 and not CF. in response to cAMP-mediated agonists. Serous cells are the most abundant cell type of the submucosal glands and are the predominate site of CFTR expression in the airways. Thus, the focus of this proposal is to obtain a better understanding of the transport mechanisms of serous cells. Specifically, we will investigate the mechanisms of HC03 secretion in Calu-3 cells and native submucosal gland serous cells. We will test several hypotheses predicated on our recently proposed model of HC03 secretion across Calu-3 cells. Our proposed studies are divided into three specific aims: 1) We will use double-barreled microelectrodes to test several predictions of our model in Calu-3 cells. These studies will establish the electrochemical driving forces involved in Calu-3 cells HC03 secretion. 2) Isolated submucosal gland obtained by microdissection will be used to investigate, by microelectrode and fluorescence ratio imaging methods whether native serous cells secrete an alkaline solution by the mechanism delineated in Calu-3 cells. These studies will be conducted on simian and human derived submucosal glands, the latter from patients expressing wild type and mutant CFTR. 3) We propose that the uptake of HC03 across the basolateral membrane of serous cells is mediated by the recently cloned pancreatic isoform of the Na+:HCO3 cotransport (pNBC) and that the activity of the pNBC is subject to voltage- and phosphorylation-dependent regulation. Thus, we will investigate which of the different NBC isoforms are expressed in Calu-3 cells and native submucosal gland serous cells by molecular and immunological methods using isoform specific probes and antibodies. Immunofluorescence studies will be performed to ascertain the subcellular localization of the NBC. In vivo phosphorylation studies will be performed under different states of activation of the NBC to ascertain the phosphorylation status of the NBC protein. The successful completion of our proposed studies will firmly establish the mechanism of HC03 secretion in not only Calu-3 cells but also in native serous cells of submucosal glands. The use of isolated, microdissected submucosal glands and the implementation of the microelectrode and imaging studies we propose is certain to provide new knowledge of submucosal gland transport physiology. The results with the human derived submucosal glands will further define the role of CFTR in HC03 secretion, and therefore, have important implications in our understanding of the submucosal gland contribution to the pathophysiology in CF and perhaps other more prevalent airway diseases. Finally, our proposed studies on the recently cloned NBC will provide new information on the expression localization and regulation of a key protein involved in the transcellular secretion of HCO3
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