Membrane testosterone receptors in cultured vascular smooth muscle cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To determine the presence of membrane testosterone receptors in cultured vascular smooth muscle cells (VSMC), and investigate their relationship with classical intracellular androgen receptors (iAR).</p><p><b>METHODS</b>VSMCs were cultured from the thoracic aorta of male Sprague-Dawley rats by the explant method. Subconfluent VSMCs were incubated with serum-free medium for 24 h to obtain quiescent non-dividing cells, and then treated with the indicated agents. The aliquots of VSMCs were labeled with testosterone-BSA-FITC (T-BSA-FITC) and analyzed by flow cytometry. Classical iARs in intact- and permeabilized-cells were detected with anti-iAR antibodies and FITC-labeled secondary antibodies by immunofluorescence, followed by flow cytometry analysis.</p><p><b>RESULTS</b>Incubation of VSMCs with T-BSA-FITC obviously increased their relative fluorescence intensity at 10 sec as compared with the untreated controls (P < 0.01), and so did it at 10 min in comparison with the treatment with BSA-FITC alone or together with free testosterone (P < 0.01). Pretreatment with iAR antagonist flutamide exhibited no significant influence on the relative fluorescence intensity of VSMCs (P = 0.318). Traditional iARs were not detectable on the surface of intact VSMCs, although permeabilized cells contained iARs.</p><p><b>CONCLUSION</b>VSMCs contain testosterone receptors in the plasma membrane, and these membrane receptors are not identical to classical iARs.</p>

Testosterone at physiological level inhibits PGF2alpha-induced increase in intracellular Ca2+ in cultured vascular smooth muscle cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the acute effects of testosterone at the physiological level on PGF2alpha-induced increase in intracellular Ca2+ in cultured vascular smooth muscle cells (VSMCs).</p><p><b>METHODS</b>VSMCs from the thoracic aorta of male Sprague-Dawley rats were cultured using the explant method. The subconfluent VSMCs were incubated with serum-free medium for 24 hours to obtain quiescent non-dividing cells and then treated with the indicated agents. For the measurement of [Ca2+]i, the VSMCs were loaded with fura-2. Changes of [Ca2+]i were determined ratiometrically with a Nikon TE-2000E system.</p><p><b>RESULTS</b>The resting level of [Ca2+]i was around 100 nmol/L in the VSMCs. Exposing cells to perfusate containing 10 micromol/L PGF2alpha triggered an immediate and transient peak in [Ca2+]i, which gradually decreased afterwards. Interference at the peak with the physiological concentration (40 nmol/L) of testosterone significantly decreased the peak-to-baseline time of [Ca2+]i, compared with ethanol vehicle (104.9 +/- 27.0 s vs 153.5 +/- 40.4 s, P < 0.01). Pretreatment with testosterone at 40 nmol/L for 2 minutes also reduced the peak-to-baseline time of [Ca2+]i significantly in comparison with the ethanol control (120.6 +/- 32.0 s vs 151.4 +/- 27.4 s, P < 0.01), but it had no significant effect on the peak level of PGF2alpha-induced intracellular Ca2+ (390.0 +/- 126.0 nmol/L vs 403.4 +/- 160.7 nmol/L, P > 0.05).</p><p><b>CONCLUSION</b>Testosterone at physiological concentration inhibits PGF2alpha-induced Ca2+ fluxes, probably via receptor-operated calcium channels by a non-genomic mechanism in VSMCs, which may be involved in the vasodilatory effect of testosterone.</p>

Inhibition of NHE-1 mRNA expression in penumbra area by human interleukin-10 transfection following cerebral ischemia-reperfusion injury in rats / 中华神经医学杂志

Objective To explore the effect of SA liposome mediated human interleukin-10 (IL-10) gent transfection on NHE-1 mRNA expression in penumbra area following focal cerebral ischemia reperfusion injury in rats. Methods Totally 78 male SD rats were randomly divided into normal control group (n=6), MCAO group (n=24), hIL-10 transfection group (n=24) and empty vector transfection group (n=24). Longa's method was employed to establish MCAO models in the latter 3 groups. The rats in the MCAO group underwent stereotactic operation without drug injection, and the hIL-10 transfection group and empty vector transfection group were injected stereotactieally with pcDNA3.1-IL-10 and pcDNA3.1, respectively, both by SA liposome mediation. After transfection, RT-PCR and ELISA were used to determine the effect of transfection, TTC staining was conducted to detect the infarct volume. Meanwhile, real-time quantitative PCR was performed to examine the expressions of NHE-1 mRNA and NF-κB mRNA in the penumbra area. Results (1) SA liposome effectively mediated the hIL-10 gene to transfect the brain tissue. Also hIL-10 gene transfection played neuroprotective effect by reducing the brain infarct volume. (2) The expression of NF-κB mRNA in different groups was 1.00±0.33, 4.76±0.41, 4.58±0.62 and 2.77±0.43, respectively, hIL-10 gene transfection also inhibited the increase of NF-κB mRNA expression in the penumbra area following the cerebral ischemia reperfusion injury. (3) The expression of NHE-1mRNA was 1.00±0.22, 4.16±0.48, 3.97±0.51 and 2.82±0.47, respectively, hIL-10 gene transfection also inhibited the increase of NHE-1 mRNA expression in the penumbra area following the cerebral ischemia reperfusion injury. Conclusions The hIL-10 transfection can exert the protective effect on the brain against cerebral ischemia-reperfusion injury partly via inhibiting the NHE-1 mRNA expression.