Abstract 325: Alternative Splicing in Intron 13 of the Human eNOS Gene: A Potential Mechanism for Regulating eNOS Activity
Nitric oxide, the product of endothelial nitric oxide synthase (eNOS), is a major regulator of vascular homeostasis and a critical factor in preventing cardiovascular diseases. Regarding expression and regulation, nothing is known about alternative splicing of the human eNOS gene. We previously established a positive correlation between the number of variable CA repeats in intron 13 of human eNOS and the risk of coronary artery disease. Furthermore, we had demonstrated that these polymorphic CA repeats function as a length-dependent splicing enhancer. Here we present first evidence for alternative splicing of the human eNOS gene, specifically by use of three novel 3′ splice sites within intron 13. This generates three splice variants - termed eNOS13A, eNOS13B, and eNOS13C, respectively - that share the first 13 exons of human eNOS and the same polyadenylation site at the end of the novel exon. When translated, all these splice variants would result in truncated proteins lacking eNOS activity. Co-expression of full-length eNOS with eNOS13A diminished eNOS enzyme activity in COS-7 cells by formation of heterodimers. The splice variants were expressed in different endothelial cells and various human tissues, with eNOS13A being particularly abundant in testis. Finally, we demonstrate using minigene transfection that the expression of the eNOS13A splice variant is increased with high CA repeat numbers in intron 13. Taken together, these data suggest a new mechanism for the regulation of eNOS activity and NO production in the cardiovascular system by truncated, dominant-negative splice variants of human eNOS.