Endocrine Abstracts (2005) 9 P26

Physiological concentrations of testosterone inhibit extracellular calcium entry via voltage-gated calcium channels in the A7r5 vascular smooth muscle cell line

J Hall1, RD Jones1, P Kang2, KS Channer3,4, C Peers2 & TH Jones1,5


1Hormone and Vascular Biology Group, Academic Unit of Endocrinology, Division of Genomic Medicine, University of Sheffield, Sheffield, UK; 2Institute for Cardiovascular Research, University of Leeds, UK; 3Faculty of Health and Wellbeing, Sheffield Hallam University, Sheffield, UK; 4Department of Cardiology, Royal Hallamshire Hospital, Sheffield, UK; 5Centre for Diabetes and Endocrinology, Barnsley District General Hospital, Barnsley, UK.


Testosterone is a coronary vasodilator and is known to increase ischaemic threshold in men with angina. We have previously demonstrated that testosterone inhibits the pore forming alpha1c subunit of the cardiovascular L-type calcium channel transfected into HEK293 cells. In this study we have investigated the effect of testosterone on potassium-stimulated extracellular calcium entry in A7r5 vascular smooth muscle cells (VSMCs).

A7r5 cells grown on coverslips in 12-well plates in DMEM containing 10% foetal bovine serum were incubated in medium containing the calcium fluorescence probe Fura2-AM (4x10-6M) for 40-min, at 37 degC. Coverslip fragments were placed in a perfusion chamber and changes in calcium were indicated by fluorescence emitted at 510nm after excitation at 340 and 380nm, in the presence of either testosterone, the L-type calcium-channel blocker nifedipine, the T-type calcium-channel blocker pimozide, ethanol-vehicle, or in calcium-free buffer.

High potassium (K+) induced a change in cellular fluorescence of 0.12plus/minus0.02 ratio units. Subsequent recordings are expressed as a percentage of this response. Compared to ethanol (0.1%), 2-min incubation with testosterone (10-10,10-9, 3x10-9, 10-8, 3x10-8 and 10-7M) caused a concentration-dependent inhibition of this response; 94.2plus/minus7.4%K+, 98.3plus/minus4.9%K+, 98.4plus/minus5.1%K+, 67.6plus/minus4.0%K+ (p<0.01), 51.5plus/minus5.8%K+, 36.7plus/minus4.0%K+, 40.9plus/minus2.6%K+all p<0.001 respectively. IC50 = 3.1x10-9nM. Incubation with nifedipine (5x10-6M) also caused similar inhibition of this response; 46.7plus/minus4.1 %K+ (p<0.001), as did pimozide (1x10-6M) 31.3plus/minus4.4%K+ (p<0.001) and calcium-free buffer almost abolished the response 6.5plus/minus1.3 %K+ (p<0.001), as did co-incubation with nifedipine (5x10-6M) and pimozide (1x10-6M) 12.3plus/minus1.3%K+ (p<0.001>. Co-incubation with testosterone 10-8 and nifedipine (5x10-6M) showed no extra inhibition; 44.1plus/minus6.8%K+(p<0.001) compared to nifedipine and testosterone only.

This study shows that physiological concentrations of testosterone inhibit extracellular calcium entry via L-type voltage-gated calcium channels in VSMC's. This effect was similar to that of nifedipine. This provides further evidence that testosterone is an endogenous calcium channel blocker, a mechanism likely to underlie its' vasodilatory action.

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