SNARE-Mediated Exocytosis of Atrial Natriuretic Peptide from Atrial Cardiac Myocytes
The heart, often overlooked as an endocrine gland, is responsible for secreting the vasoactive hormone atrial natriuretic peptide (ANP). Physical stretch of the atrial wall is the conical stimulus; however, ouabain has been shown to efficiently evoke exocytosis. Nevertheless, the Ca2+-dependence and molecular mechanisms that mediate ANP exocytosis remain unclear. In the current study, I identified SNARE proteins and determined their association with ANP-containing secretory granules using primary cultures of neonatal and adult rat atrial cardiac myocytes. Myocytes were screened for mRNA transcripts by RT-PCR and further characterized by Western blot analysis. Functional interactions of identified core SNARE proteins were demonstrated using co-immunoprecipitation methods. Localization of core SNARE proteins were completed using cell fractionation and immunocytochemical methods, revealing that VAMP-1, VAMP-2 and synaptotagmin-1 were localized to subpopulations of ANP-containing secretory granules, suggesting an importance for SNARE proteins. To judge whether secretory activity could be evoked by intracellular Ca2+ elevation and the role of ouabain in this process, we used timeresolved membrane capacitance measurements (Cm) in combination with the flash photolysis of caged Ca2+ to follow the exocytotic activity of individual myocytes. Two sequential flashes at room temperature evoked nearly identical Ca2+ changes that induced exponential Cm rises (78 and 66 granules, respectively). The application of 100 nM ouabain to target the á3-subunit of the Na+/K+-ATPase significantly enhanced the average Cm change to the first stimulus and diminished the response to the second stimulus (138 and 58 granules, espectively) with no effect on Ca2+ levels or size of the readily-releasable pool, suggesting ouabain enhanced the Ca+2-sensitivity of exocytosis. Others have shown binding of ouabain to the Na+/K+-ATPase activates Src and subsequent downstream tyrosine phosphorylation. Using coimmunoprecipitation methods, I demonstrated that synaptotagmin-1 associates with an immune complex comprised of Na+/K+-ATPase á3/Src/syntaxin-4. Moreover, treatment with pervanadate (inhibitor of protein tyrosine phosphatases) or ouabain induced tyrosine phosphorylation of synaptotagmin-1 in a dose dependent manner. Based on these findings, I propose SNARE proteins are required for exocytosis and are assembled with Na+/K+-ATPase á3/Src/syntaxin-4 in a signal complex that upon activation by ouabain induces tyrosine phosphorylation of synaptotagmin-1 to enhance the Ca2+-sensitivity of exocytosis.
School:University of Toledo Health Science Campus
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:atrial natriuretic peptide exocytosis myocytes ca2 imaging membrane capacitance snare proteins
Date of Publication:01/01/2007