Abstract
Gap junctions are clusters of cell membrane channels that cross the gap between the adjacent cells and provide pathway for direct intercellular communication. Each channel consists of a pair of connexons, each of which is made of six connexin molecules. The connexins belong to a multigene family. Channels composed of different members of the family possess unique properties. Since exchange of metabolic and electrical signals between adjacent cells depend on gap junction, a variety of biological activities are intimately linked to the junctions, such as maintenance of cardiac and vascular function. The work of this thesis is to investigate the influence of hyperlipidemia and alcoholism on cardiovascular gap junction by using anti-connexin antisera in conjunction with high-resolution confocal laser scanning microscopy. Hypercholesterolemia is well-known to be one key atherogenic factor. Previous reports had shown that hypercholesterolemia affect the gap junctions in the arterial endothelial cells, which contain connexin37, connexin40 and connexin43. However, the effect of hypercholesterolemia on endothelial connexins is not clear. To answer this question, hypercholesterolmia was induced in mice by long-term feeding of cholesterol-enriched diet. In addition, a cholesterol-lowering drug, simvastatin, was given in a short time. The results showed that: i) in hyperlipidemic mice, endothelial connexin37 and connexin40 are downregulated; ii) 7-days use of simvastatin, restores connexin37, not connexin40, nearly to the normal range; iii) connexin43 was not expressed in the aortic endothelia in all animals of different feeding and treatment. On the other hand, excessive intake of alcohol is known harmful to the heart function. However, whether alcohol change the myocardial gap junctions was not clear. I therefore conduct an experiments investigating the change of connexin43, the major connexin expressed in heart, in rats administrated with different concentrations of alcohol in the drinking water for one or two months. The results showed that while the myocardial connexin43 remained stationary in rats drinking 12% alcohol for one month, by those drinking 12% or 36% alcohol for two months had an increase. In addition, the distribution of the connexin43 gap junctions is disorganized in rats drinking 36% alcohol for two months. To investigate the role of gap junction in the cardiovascular system, the first thing is to have antiserum specific to individual connexins. In the thesis, I therefore try to generate anti-connexin polyclonal antibodies in rats followed by affinity purification and characterization by Western blotting plus immunocytochemistry. The results showed the antiserum specific to connexin37, connexin40 and connexin45 are successfully produced. These antisera are useful tools for future gap junction study. These findings demonstrate that cardiovascular diseases are associated with alterations of gap junction distribution and connexin expression. In addition, during the disease process, individual connexins are differentially regulated, suggesting that each member of the connexin family in the cardiovascular system plays an unique role in the pathogenesis of diseases.