| Na,K-ATPase, namely Na pump, is a membrane-bound protein that presents in the plasma membrane of all eukaryotic cells and belongs to a widely distributed class of P-type ATPase, which transports three Na~+ out of the cell and two K~+ in using the energy from the hydrolysis of one molecule of ATP. By establishing and maintaining the normal distribution of these ions across the plasma membrane, Na,K-ATPase plays a central role in the regulation of membrane potential and cell volume. Na,K-ATPase consists of αand βsubunits. The transmemrane αsubunit is responsible for the catalytic and transport properties of the enzyme and contains the binding sites for the cations, ATP, and cardiac glycosides and so the cardiac glycosides sensitivity is conferred by the αsubunit. The βsubunit is essential for the normal activity of the enzyme and regulates the sensitivity of Na,K-ATPase to the cations Na~+ and K~+ because the βsubunit affects the activation of Na,K-ATPase by extracelluar K~+. At present at least 4 isoforms of theαsubunits (α1,α2,α3,α4) and 2 of theβsubunits (β1,β2) that are encoded by different genes are found in mammals and each isoform of the αsubunit has a different sensitivity to cardiac glycosides. The α1 isoform contains the low affinity ouabain binding sites, whereas theα2 andα3 isoform contain the high affinity binding sites. Furthermore, these isoforms exhibit tissue and development specific distrbution. The α1 isoform is expressed ubiquitously among tissues, whereas theα2 isoform has been identified principally in heart, muscle and brain, and the α3 isoform has been identified primarily in neural tissues. In 1998, Shamraj and Lingrel discovered thatα4 isoform is a testis-specific isoform. The α1 isoform is the ubiquitous sodium pump, presumably responsible for maintenamce of intracellular electrolytes in all tissues, whereas α2, α3 and α4 isoforms are expressed in tissue-specific manner suggesting that they may play specific roles. The inhibition of the Na, K-ATPase by cardiac glycosides is belived to be the major mechanism by which these drugs exert their positive cardiac inotropic effect. It has been suggested that the high affinity Na, K-ATPase mediates the positive inotropic effect, whereas the low affinity Na, K-ATPase is responsible for toxic effects of cardiac glycosides. However, in 2003, Lingrel et al clearly demonstrates that the Na, K-ATPase and specifically the α2 Na, K-ATPase isoform mediates oubain-induced cardiac contractivity in mice expressed a oubain-insensitive α2 isoform of the Na, K-ATPase using Cre-Lox technology and subsequently demonstrates thatα1 and α2 coregulate cardiac contractility. In addition, several studies suggest that the decrease in α2/α3 isoform mRNA may underline the lower Na,K-ATPase activity incardiomyopathic hamster heart. These results above clearly suggest that further confirmation of the expression and tissue distribution of each αisoform in various conditions is very important to investigate the physiological and pathological significance of the Na,K-ATPase. Therefore, the present study was to assess which αisoform is expressed in the guinea-pig heart using molecular biology techniques and to provide theoretical and experimental foundations for the reseach of the functional changes of heart failure and other pathological conditions. Objective: To determine which αisoform of cardiac Na, K-ATPase is expressed in normal guinea-pig. Methods: we examined the expression of Na,K-ATPase αisoform by RNase protection assay.(1)the synthesis of radiolabed specific cRNA probes for α1,α2, andα3 isoform: First, Guinea-pig brain mRNA was reverse transcribed, and the subsequent cDNA was used as a template for the PCR reactions. The six primers were used successfully for PCR amplification of the three αsubunit isoforms of Na, K-ATPase from guinea-pig using TAQ polymerase and the PCR products were size-fractionated by electrophoresis through a 1% agarose gel containing 0.5ug/ml ethidium bromide. Ethidium bromide-stained product bands of expec...
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