Vascular-specific increase in exon 1B-encoded CAV1.2 channels in spontaneously hypertensive rats.

BACKGROUND: The vasculature of the adult spontaneously hypertensive rat (SHR) is known to express more functional L-type Ca channels than the vasculature of normotensive Wistar Kyoto (WKY) rats, but it is not known which Ca(V)1.2 channel isoform is upregulated. METHODS: Western blots and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were used to compare the expression levels of Ca(V)1.2 channel protein and message in selected tissues of adult SHR and WKY rats. RESULTS: The results indicate overexpression in SHR vasculature specifically of the short exon 1b-encoded amino terminus Ca(V)1.2 isoform. Brain and visceral smooth muscle expressing the same isoform were not similarly affected. Differences in message levels are insufficient to account for the differences in isoform-specific protein levels. CONCLUSIONS: We conclude that SHR vasculature must regulate the channel postranscriptionally. Further experiments will be required to determine whether this involves translation of protein from exon 1b-specific transcripts more efficiently, posttranslational chaperoning to the surface membrane more efficiently, or selective degradation of the short amino terminus form of the protein more slowly than in WKY vasculature.

Negative transcriptional regulation of human colonic smooth muscle Cav1.2 channels by p50 and p65 subunits of nuclear factor-kappaB.

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.

Expression of multiple CaV1.2 transcripts in rat tissues mediated by different promoters.

The expression of two different transcripts for Ca(V)1.2 in rat tissues mirrors that which has previously been described for human tissue, in that expression of transcripts expressing exon 1a is predominant only in heart, whereas expression of transcripts expressing exon 1b is greater in smooth muscle rich tissues such as aorta and intestine. Transcripts expressing exon 1b also predominate in brain and in diaphragm. Western blots indicate that the N-terminus coded for by exon 1b is present in much of the protein in all these tissues except heart. The promoter just upstream of exon 1b has been cloned, sequenced and utilized to drive expression of luciferase in smooth muscle A7r5 cells, cardiac HL-1 cells, skeletal muscle L6 cells and neuronal PC12 cells. The nucleotide sequence of the promoter exhibits 80% identity with the equivalent promoter previously identified in humans and 94% identity with the sequence of the equivalent region of the mouse genome. Evidence in favor of still another promoter upstream of exon 2 has been uncovered.

Smooth muscle uses another promoter to express primarily a form of human Cav1.2 L-type calcium channel different from the principal heart form.

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.

A new promoter for alpha1C subunit of human L-type cardiac calcium channel Ca(V)1.2.

The cardiac Ca channel known as alpha1C or Ca(V)1.2 is shown to express a new longer first exon equivalent to that formerly reported in rabbit heart or rat aorta. Ribonuclease protection assay indicates that this exon is found in the majority of Ca(V)1.2 transcripts in human heart RNA. The presence of this exon also suggests that expression of this transcript is driven by a promoter immediately upstream of this exon and its 5' untranslated region. The putative promoter exhibits 69% homology to its rat counterpart and displays functional promoter activity when transfected into heart cells in culture in luciferase-expressing constructs.