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Background & Aims: The expression of Cav1.2 channels in colonic circular smooth muscle cells and the contractility of these cells are suppressed in inflammation. Our aim was to investigate whether the activation of p50 and p65 nuclear factor-kappaB subunits mediates these effects., Methods: Primary cultures of human colonic circular smooth muscle cells and muscle strips were used., Results: The messenger RNA and protein expression of the pore-forming alpha1C subunit of Cav1.2 channels decreased time dependently in response to tumor necrosis factor alpha. This effect was blocked by prior transient transfection of the cells with antisense oligonucleotides to p50 or p65. The overexpression of p50 and p65 inhibited the constitutive expression of alpha1C. Three putative kappaB binding motifs were identified on the 5' flanking region of exon 1b of the human L-type calcium channel alpha1C gene. Progressive 5' deletions of the promoter and point mutations of the kappaB binding motifs indicated that the two 5' binding sites, but not the third 3' binding site, were essential for the suppression of alpha1C. Transient transfection of human colonic circular muscle strips with antisense oligonucleotides to p50 and p65 decreased expression of the 2 nuclear factor-kappaB units and reversed the suppression of alpha1C, as well as that of the contractile response to acetylcholine, by 24 hours of treatment with tumor necrosis factor alpha., Conclusions: The activation of p50 and p65 by tumor necrosis factor alpha suppresses the expression of the alpha1C subunit of Cav1.2 channels in human colonic circular smooth muscle cells and their contractile response to acetylcholine. Nuclear factor-kappaB must bind concurrently to the two 5' kappaB motifs on the promoter of alpha1C to produce this effect.

Several different first exons and amino termini have been reported for the cardiac Ca channel known as alpha(1C) or Ca(V)1.2. The aim of this study was to investigate whether the expression of this channel is regulated by different promoters in smooth muscle cells and in heart in humans. Ribonuclease protection assay (RPA) indicates that the longer first exon 1a is found in certain human smooth muscle-containing tissues, notably bladder and fetal aorta, but that it is not expressed to any significant degree in lung or intestine. On the other hand, all four smooth muscle-containing tissues examined strongly express transcripts containing exon 1b, first reported cloned from human fibroblast cells. In addition, primary cultures of human colonic myocytes and coronary artery smooth muscle cells express predominantly transcripts containing exon 1b. The promoter immediately upstream of exon 1b was cloned, and it displays functional promoter activity when luciferase-expressing constructs were transfected into three different cultured smooth muscle cells: primary human coronary artery smooth muscles cells, primary human colonocytes, and the fetal rat aorta-derived A7r5 cell line. These results indicate that expression in smooth muscle is primarily driven by a promoter different from that which drives expression in cardiac myocytes.