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dc.contributor.advisorForsyth, George W.en_US
dc.creatorLoewen, Matthew Ericen_US
dc.date.accessioned2004-04-14T14:27:23Zen_US
dc.date.accessioned2013-01-04T04:29:07Z
dc.date.available2005-04-14T08:00:00Zen_US
dc.date.available2013-01-04T04:29:07Z
dc.date.created2004-04en_US
dc.date.issued2004-04-12en_US
dc.date.submittedApril 2004en_US
dc.identifier.urihttp://hdl.handle.net/10388/etd-04142004-142723en_US
dc.description.abstractA CLCA protein (CL for chloride channel and CA for calcium) cloned from porcine ileum was expressed and characterized. The regulatory behavior, inhibitor sensitivity, and functional properties of chloride conductance associated with the expression of pCLCA1 cDNA were investigated in non-epithelial NIH/3T3 fibroblasts and in an epithelial Caco-2 cell line. These properties were also investigated in freshly isolated retinal pigment epithelial (RPE) cells and in primary cultures of these cells which express an endogenous cCLCA1. In NIH/3T3 fibroblasts, the chloride efflux induced by pCLCA1 was directly activated by calcium. A and C kinase agonists were without effect. The electrogenic nature of chloride efflux was confirmed by detection of outwardly rectified chloride currents. Selected anion channel blockers inhibited both the pCLCA1 agonist-induced current and chloride efflux. The inhibitors also reduced Ussing chamber short circuit current and chloride efflux from primary RPE cultures. However, these same agents did not inhibit chloride efflux in fibroblasts expressing the cystic fibrosis transmembrane regulator (CFTR) conductive chloride channel. The expression of pCLCA1 increased cAMP/A kinase-dependent chloride ion release from fibroblasts and Caco-2 cells expressing CFTR. These pleiotropic effects of CLCA protein expression suggested that the protein may regulate the activity of chloride conductance, rather than functioning as a primary ion transporter. This putative regulatory behavior was further investigated in Caco-2 cells. The rate of 36Cl efflux and the amplitude of currents in patch clamp studies after activation of A kinase or intracellular Ca2+ mobilization was significantly increased in freshly passaged Caco-2 cells expressing pCLCA1. However, 36Cl efflux and short circuit Ussing chamber studies in polarized Caco-2 cells provided evidence that both endogenous and pCLCA1-dependent Ca2+-sensitive chloride conductance were lost from 14 day post-passage cells. cAMP-dependent chloride conductance continued to be modulated by pCLCA1 expression in differentiated 14 day post-passage Caco-2 cells, demonstrating the retention of pCLCA1 effects in these mature cells. We conclude that pCLCA1 expression enhances the sensitivity of endogenous chloride channels to both natural agonists, Ca2+and cAMP, but that it lacks inherent Ca2+-dependent chloride channel activity.en_US
dc.language.isoen_USen_US
dc.subjectsecretory diarrheaen_US
dc.subjectbestrophinen_US
dc.subjectanion transporten_US
dc.subjectendothelial adhesion moleculeen_US
dc.subjectmCLCA3en_US
dc.subjecthCLCA1en_US
dc.subjectCLCAen_US
dc.titleCLCA : chloride channel or modulator?en_US
thesis.degree.departmentVeterinary Biomedical Sciencesen_US
thesis.degree.disciplineVeterinary Biomedical Sciencesen_US
thesis.degree.grantorUniversity of Saskatchewanen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophy (Ph.D.)en_US
dc.type.materialtexten_US
dc.type.genreThesisen_US
dc.contributor.committeeMemberJanz, David M.en_US
dc.contributor.committeeMemberHamilton, Donald L.en_US
dc.contributor.committeeMemberCotton, Calvin U.en_US
dc.contributor.committeeMemberAppleyard, Greg D.en_US
dc.contributor.committeeMemberMuir, Gillian D.en_US


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