Endocrine Abstracts

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 alpha_1c 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.