Endocrine Abstracts (2002) 3 P267

Testosterone does not increase the production of cyclic AMP (cAMP) in cultured vascular smooth muscle cells

LN Ruban1, RD Jones1, PJ Pugh1,2, KM English2, KS Channer2 & TH Jones1,3


1Endocrine Heart & Pituitary Group, Academic Unit of Endocrinology, Division of Genomic Medicine, The University of Sheffield, Sheffield, UK; 2Department of Cardiology, Royal Hallamshire Hospital, Sheffield Teaching Hospitals NHS Trust, Sheffield, UK; 3Centre for Diabetes & Endocrinology, Barnsley District General Hospital NHS Trust, Barnsley, UK


Background: Testosterone is a coronary vasodilator, and improves myocardial ischaemia in men with coronary artery disease. The dilatory mechanism of testosterone is independent of the classical androgen receptor and endothelial-derived vasodilators. The aim of this study was to determine whether testosterone increases cAMP production in vascular smooth muscle cells (VSMCs) derived from the primary culture of rat thoracic aortae or in a VSMC cell line (A7r5).

Methods: Thoracic aortae were harvested from adult male Wistar rats, and the endothelium removed. The smooth muscle cell layer was cut into pieces and digested with collagenase type I for 16 hours at 37oC. The cells were then transferred to 12-well plates containing supplemented DMEM medium at 37oC, 5% CO2. A7r5 rat aortic smooth muscle cells were grown under identical conditions. Once confluent the VSMCs were collected and re-seeded into 12 well plates. Once attached the cells were washed and exposed to 100uM IBMX for 30min, prior to addition of either 10uM forskolin (positive control), ethanol (negative control) or testosterone (0.1,1,10 or 100uM) for 15 minutes at 37oC. The cells were then fixed and the intracellular cAMP level measured via commercial assay kits (Amersham).

Results. Intracellular cAMP is expressed as the mean (sem) of 3 experiments undertaken in triplicate, in pmol. In primary VSMCs, incubation with forskolin (10uM) increased the production of cAMP from -0.6 (0.2) to 66.0 (15.0) (P<0.0001). Incubation with testosterone (0.1,1,10 or 100uM) had no effect; 0.5 (0.2), -0.9 (0.1), -0.5 (0.3), and -0.6 (0.2) respectively (P>0.1). In A7r5 VSMCs, incubation with forskolin (10uM) increased the production of cAMP from 0.2 (0.03) to 2.1 (0.9) (P<0.05). Incubation with testosterone (0.1,1,10 or 100uM) had no effect; 0.1 (0.03), 0.2 (0.05), 0.2 (0.03), and 0.2 (0.02) respectively (P>0.1).

Conclusion: Testosterone does not increase the production of cAMP in primary or immortalised VSMCs.

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