Testosterone inhibits the prostaglandin F2alpha-mediated increase in intracellular calcium in A7r5 aortic smooth muscle cells: evidence of an antagonistic action upon store-operated calcium channels

Abstract

Testosterone-induced vasodilatation is proposed to contribute to the beneficial effects associated with testosterone replacement therapy in men with cardiovascular disease, and is postulated to occur via either direct calcium channel blockade, or through potassium channel activation via increased production of cyclic nucleotides. We utilised flow cytometry to investigate whether testosterone inhibits the increase in cellular fluorescence induced by prostaglandin F 2 in A7r5 smooth muscle cells loaded with the calcium fluorescent probe indo-1-AM, and to study the cellular mechanisms involved. Two-minute incubation with testosterone (1 µM) significantly inhibited the change in cellular fluorescence in response to prostaglandin F 2 (10 µM) (3·6 0·6 vs 7·6 1·0 arbitrary units, P=0·001). The change in cellular fluorescence in response to prostaglandin F 2 (10 µM) was also significantly attenuated in the absence of extracellular calcium (3·6 0·3 vs 15·6 0·7 arbitrary units, P=0·0000002), and by a 2-min incubation with the store-operated calcium channel blocker SK&F 96365 (50 µM) (4·7 0·8 vs 8·1 0·4 arbitrary units, P=0·003). The response was insensitive to similar incubation with the voltage-operated calcium channel blockers verapamil (10 µM) (12·6 1·2 vs 11·9 0·2 arbitrary units, P=0·7) or nifedipine (10 µM) (13·9 1·3 vs 13·3 0·5 arbitrary units, P=0·7). Forskolin (1 µM) and sodium nitroprusside (100 µM) significantly increased the cellular concentration of cyclic adenosine monophosphate and cyclic guanosine monophosphate respectively, but testosterone (100 nM-100 µM) had no effect. These data indicate that the increase in intracellular calcium in response to prostaglandin F 2 occurs primarily via extracellular calcium entry through store-operated calcium channels. Testosterone inhibits the response, suggesting an antagonistic action upon these channels.

Testosterone-induced coronary vasodilatation occurs via a non-genomic mechanism: evidence of a direct calcium antagonism action

“…Recent evidence suggests that testosterone elicits vasodilatation via a calcium-antagonistic action against VGCCs and SOCCs [7,25,32]. We have investigated the potential calcium-antagonistic action of testosterone in the present study by assessing the influence of testosterone upon coronary arterial vasoconstriction induced by KCl.…”

“…Indeed, a calcium-antagonistic action of testosterone upon SOCCs is reported following exposure to PGF 2α in A7r5 VSMCs [32]. Clearly, there is a need for detailed electrophysiological studies to determine the precise nature of the influence of testosterone upon calcium channel function.…”

The influence of testosterone upon vascular reactivity

“…However, animal studies are inconclusive. Chronic exposure to testosterone via supplemental therapy and acute exposure of isolated vessel preparations to testosterone are reported to exert a positive (21,30,43,52,53) neutral (44) or negative (45) effect (46), which is likely to be a consequence of the diverse models utilised. To date, animal studies have employed varied species (monkey, pig, rabbit, rat and guinea pig), vascular beds (coronary, femoral and aortal), disease states (normal, experimental atherosclerosis and experimental hypertension) and androgen exposures (chronic, acute, physiological and supraphysiological).…”

“…Perusquia and Villalon (52) report that incubation with testosterone significantly attenuates the contraction of isolated rat thoracic aortae induced by KCl and noradrenaline. Similarly, acute exposure to testosterone is reported to inhibit the increase in intracellular calcium elicited by KCl and PGF 2a in isolated smooth muscle cells (21,53), an action which ultimately triggers contraction.…”

“…There is a clear distinction between the concentration at which testosterone can induce vasodilatation in vivo and in vitro, and also between different in vitro methodologies. Testosterone-mediated coronary vasodilatation in vivo is reported at concentrations around 100 nM (7,20), and isolated vessel studies commonly require high (10 -100 mM) micromolar concentrations of testosterone to induce vasodilatation (17), while, in isolated cells, vascularly significant alterations in intracellular calcium are observed at low (1 mM) micromolar concentrations (21). Although the first two values are much higher than the normal physiological range (10 -30 nM), it is premature simply to discard such observations on this basis.…”