The expression and functional study of CNG2 in the role of both cyclic nucleotide response and store independent calcium influx in vascular endothelial cell.

摘要

Ion channels play an indispensable role in endothelial cells, which is a unique signal-transducting surface in the vascular system that is responsible in altering vascular tone. The present study investigated the expression and functional roles of the cyclic nucleotide gated channels (CNG) in regulating the intracellular calcium level of vascular endothelial cells using molecular and calcium measurement techniques.;Cyclic nucleotide-gated (CNG) ion channels are Ca2+ permeable nonselective cation channels that are directly gated by binding of cAMP or cGMP, thus providing a linkage between two important signal molecules, cyclic nucleotides and calcium. They are known to play an important role in sensory transduction and in second-messenger modulation of synaptic neurotransmitter release. Previous studies showed that besides in neuronal cells, CNG were found also in non-neuronal tissues including heart, kidney, blood vessels and spleen, they are reported to be involved in a variety of cell function.;The present study provided evidence that the CNG channels, especially that of CNGA2, were expressed in vascular tissues. I used a variety of different methods, including RT-PCR, northern blot, in-situ hybridization, immunohistochemistry and western blot to study the localization of CNGA2 channels. RT-PCR amplify a CNGA2 fragment of 582bp from RNAs isolated from bovine vascular endothelial cell line, rat vascular smooth muscle cell line and rat aorta. Northern blot analysis detected a 2.3-kilobase (kb) CNGA2 transcript in rat aorta mRNA. The cellular distribution of CNGA2 was further studied by in-situ hybridization, which demonstrated expression of CNGA2 mRNA in human vascular endothelial and vascular smooth muscle cells. Immunohistochemistry data also agreed with those generated from in-situ hybridization. Western blot data also demonstrated proteins of CNG2 was expressed in both human vascular endothelial cells and vascular smooth cells layer. Subcellular localization of CNGA2 inside the vascular endothelial cells was also investigated with the use of a GFP linked CNGA2 channel gene. Taken together, the results showed that CNGA2 proteins were expressed on the plasma membrane of the vascular endothelial cells. (Abstract shortened by UMI.).