Endocrine Abstracts (2004) 7 P62

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

J Hall1, RD Jones1, P Kang2, KS Channer1,3, C Peers2 & TH Jones1,4


1Hormone and Vascular Biology Group, Academic Unit of Endocrinology, division of Genomic Medicine, G Floor, The Medical School, Sheffield, UK; 2Institute for Cardiovascular Research, University of Leeds, UK; 3Department of Cardiology, Royal Hallamshire Hospital, Sheffield, UK; 4Centre for Diabetes and Endocrinology, Barnsley District General Hospital, Barnsley, UK.


Testosterone therapy has been shown to be beneficial in men with coronary artery disease (CAD) and chronic heart failure (CHF), effects which are proposed to be due to a direct vasodilatory action. We utilised fluorescence measurements to investigate whether testosterone inhibits extracellular calcium entry induced by 50mM high potassium (K+) buffer, in A7r5 vascular smooth muscle cells.

A7r5 cells were grown on coverslips in 12-well plates in DMEM medium supplemented with 10% foetal bovine serum and left to adhere. Cells were then incubated in medium containing the calcium fluorescence probe Fura2-AM (4x10-6M) for 40-min, at 37 degC. Fragments of coverslip were then placed in a perfusion chamber and changes in calcium were indicated from the fluorescence emitted at 510nm due to alternative excitation at 340 and 380nm, using openlab software, in the presence of either testosterone, nifedipine or ethanol vehicle, or in calcium free buffer.

50mM K+ induced a change in cellular fluorescence of 0.1plus/minus0.007 ratio units. Subsequent recordings are expressed as a percentage of this response. Compared to ethanol (0.1%), 2-min incubation with testosterone (10-9, 10-8, 10-7 10-6M) caused a concentration-dependent inhibition of this response; 90.3plus/minus7.2 %K+, 75.0plus/minus20.7 %K+ (p=0.5), 52.0plus/minus8.2 %K+ (p<0.01), 30.1plus/minus4.6 %K+ (p<0.001), 52.3plus/minus10.4 %K+ (p<0.05) respectively. The IC50 of testosterone was 4.2x10-9nM. Incubation with nifedipine (5x10-6M) also caused statistically similar inhibition of this response; 40.5plus/minus6.7 %K+ (p<0.001), and calcium-free buffer almost abolished the response 6.5plus/minus1.3 %K+ (p<0.001).

These data indicate that physiological concentrations of testosterone inhibit extracellular calcium entry in A7r5 vascular smooth muscle cells, which occurs via voltage-gated calcium channels. Testosterone exhibited a similar efficacy as the L-type calcium channel blocker nifedipine, suggesting a common inhibitory action. This calcium antagonistic effect is likely to underlie the vasodilatory action of testosterone and the clinical benefits associated with testosterone therapy in men with CAD and CHF.

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