Voltage-gated potassium channels of the K_V1 subtype provide negative feedback against voltage-dependent calcium entry in vascular smooth muscle (Cheong et al. 2001a, b). Our aim is to quantify K_V1 mRNA to elucidate mechanisms that regulate expression. Total RNA was isolated from freshly dissected (humanely killed) 8-week-old male mouse aorta after removal of connective tissue and fat, and blood by lumenal perfusion with balanced salt solution. Endothelium was removed by perfusion with Triton-X. Wire myograph recordings demonstrated that the vessels were contractile to 45 mM potassium and phenylephrine. Contractile responses were not diminished after endothelium removal but dilator responses to acetylcholine were abolished. cDNA was synthesized from DNAse I-treated RNA and PCR was performed using gene-specific primers on the Roche Lightcycler combined with SYBR Green detection of dsDNA. Melt-curve analysis and agarose gel electrophoresis were used to determine PCR specificity. Crossing points (C_P) were calculated using Fit Points Method, where C_P corresponds to the PCR cycle at which fluorescence exceeded background signals. Data are given as means ± S.E.M.

Comparison of intact with endothelium-denuded aorta revealed a 10.0 ± 0.1-fold reduction in endothelial nitric oxide synthase (eNOS) mRNA (C_P 27.3 ± 0.1, n = 3 cf. 23.9 ± 0.1, n = 3; unpaired t test, P < 0.01). Levels of β-actin mRNA were similar between intact and denuded aorta (C_P 22.8 ± 0.1, n = 3 cf. 22.5 ± 0.0, n = 3; P = 0.04). K_V1.1-1.6, but not KV1.7, mRNAs were detected in endothelium-denuded aorta. Quantitative analysis revealed no change in K_V1.1 in denuded compared with endothelium-intact aorta (C_P 31.0 ± 0.2, n = 3 cf. 30.9 ± 0.6, n = 3; P > 0.05). K_V1.3 mRNA levels were 5.8 ± 1.0-fold less in denuded compared with endothelium-intact aorta (C_P 32.3 ± 0.7, n = 3 cf. 29.5 ± 0.1, n = 3; P < 0.05). PCR efficiencies were 88 and 94 % for K_V1.1 and K_V1.3 reactions, respectively. K_V1.1-1.6 proteins were detected in smooth muscle of aorta sections by immunostaining with peptide-specific antibodies – K_V1.1 and 1.3 signals were the strongest, and K_V1.4 and K_V1.5 the weakest.

The experiments show the expression of six K_V1 subunits in mouse aorta and demonstrate the potential for using real-time RT-PCR to make quantitative investigations of ion channel mRNA levels in intact and endothelium-denuded arteries. Using this approach we suggest there is K_V1.1 mRNA in smooth muscle but not endothelium, whereas K_V1.3 mRNA is in both.

We thank the MRC and BHF for support.

All procedures accord with current UK legislation.