The Mechanisms For Testosterone-induced Mesenteric Artery Relaxation In Hypertension

Abstract: Objective: Hypertension is a chronic disease which often leads to lesion of vital organs such as heart, brain, kidneys. It is important to understand the molecular mechanisms of generation and development of hypertension for its prevention and therapy. The disease is closely related to the pathophysiological mechanisms of tension generation of arterial smooth muscle cells. The regulation of intracellular Ca²⁺ concentration in the vascular smooth muscle cells is a key factor of such mechanisms, and ionic channels, such as Ca²⁺ channels and K⁺ channels are known to be crucially involved in controlling the intracellular Ca²⁺ concentration. Recent research works have demonstrated that large-conductance Ca²⁺ activated K⁺ channels ( BK_Ca ) in vascular smooth muscles play important roles in controlling the vascular tone by determining the level of membrane potential and Ca²⁺ influx through voltage-gated Ca²⁺ channels. Studies on vascular smooth muscles have shown that local Ca²⁺ releases ( Ca²⁺ sparks ) through ryanodine-sensitive channels located in the sarcoplasmic reticulum activate BK_Ca, which can be detected as spontaneous transient outward K⁺ currents ( STOCs ) at the physiological membrane potential under whole cell voltage clamp. It was reported that dysfunction of BK_Ca may be one of the pathological mechanisms of hypertension. In recent years, studies on the relationship between testosterone ( TES ) and hypertension have become a hot spot, but the effects and mechanisms of TES in hypertension is still unclear. There was a study using a hypertension animal model demonstrating that BK_Ca participated the TES induced vasorelaxation on thoracic aorta. However, the results from animal study do not always express the actual relationship between TES and hypertension in humans. The objective of the present study is, therefore, to study the effects of TES on human mensentric artery obtained from patients with hypertension. We used vascular rings to explore the mechanisms in vitro. We also measured the macroscopic currents of BK_Ca and STOCs in isolated human mesenteric artery smooth muscle cells from hypertension patients using whole-cell patch clamp, and investigated changes of sensitivity to TES . Methods: ( 1 ) Vascular rings: The artery was quickly dissected out from a piece of mesenteric tissue in solutionâ… , the free artery of 100²00μm inner diameter was cut to make vascular rings ( 1.4¹.8 mm ). Vascular rings were suspended with two silver wires and fixed in O₂ saturated solutionâ… at 37 oC in a DMT chamber. The vascular rings were linked to an electrophysiolograph through a tension transducer and a biological amplifier. Data was collected and analyzed with the Chart 5 software. KCl was used to test the activity of vascular rings and to cause contraction. We studied the effects of TES on the tension of mesenteric artery vascular rings. ( 2 ) Whole-cell patch clamp: The free mesenteric artery was cut into small segments(2 mm×2 mm)and then transferred to a dissociation solution containing papain andâ…ªtype collagenase for enzymatic digestion. The stereoscopic- and slick-shaped smooth muscle cells were chosen for experiment. The macroscopic currents of BK_Ca and STOCs were recorded under whole-cell, amphotericin-perforated configuration with patch-clamp technique. The currents were amplified and low-pass filtered by an Axopatch 200B amplifier,and the digital data were stored in a Lenovo-compatible personal computer running Clampex 10.1 software and further analyzed by Clampfit 10.1 and MiniAnalysis6.0 software. We studied the changes of the current density of BK_Ca and the amplitude and frequency of STOCs in hypertension, and the sensitivity to TES. Results: ( 1 ) Effects of TES on the tension of mesenteric artery vascular rings:â‘ The relaxation percentages induced by TES in hypertension group ( HT ) and normotension group ( NT ) were 78.92±6.25% and 92.99±3.72%, respectively ( n_HT= 9,n_NT =10, P>0.05 ).â‘¡Before and after 1mM tetraethylammonium ( TEA, a non-specific inhibitor of K⁺ channels ) pretreatment in NT, the relaxation percentages induced by TES were 92.99±3.72% and 75.50±3.29%, respectively(n=10, P<0.05); before and after 1mM TEA pretreatment in HT, the relaxation percentages induced by TES was 78.92±6.25% and 75.50±3.29%, respectively ( n=9, P<0.05 ).â‘¢The relaxation percentage induced by TES in control was 89.48±2.80%; after glibenclamide ( GLI, an inhibitor of K_ATP ), N^ω-nitro-L-arginine methyl ester ( L-NAME, an inhibitor of nitric oxide synthase ) or flutamide ( an inhibitor of androgen receptor ) pretreatment, the percentage of TES-induced relaxation was 88.56±1.08% ( n=4, P>0.05 ), 88.84±3.03% ( n=4, P>0.05 ) or 86.02±1.97% ( n=5, P>0.05 ), respectively. ( 2 ) Effects of TES on BK_Ca and STOCs of mesenteric artery:â‘ The current density of BK_Ca in HT at the voltage of +30⁺60 mV was smaller than that in NT at each voltage tested; at +60 mV test potential, the current density of BK_Ca was 4.14±0.78 pA/pF ( n=6 ) in HT,and was 7.61±1.02 pA/pF in NT( n =12 )( P<0.05).â‘¡At +60 mV test potential, the current densities of BK_Ca in NT before and after adding 100μM TES were 7.61±1.02 pA/pF and 16.21±2.43 pA/pF, respectively ( n=12, P<0.05 ), and those in HT before and after adding 100μM TES were 4.14±0.78 pA/pF and 5.85±1.26 pA/pF, respectively ( n=6, P>0.05 ). ( 3 ) Effects of TES on STOCs in mesenteric artery vascular smooth muscle cells:â‘ At -20 mV test potential, the amplitudes of STOCs in HT and NT were 15.93±3.26 pA and 40.26±3.75 pA, respectively ( n_HT =5, n_NT=6, P<0.05 ); the frequencies of STOCs in HT and NT were 1.52±0.28 Hz and 2.27±0.47Hz, respectively ( n_HT =5, n_NT=6, P<0.050 ).â‘¡Effects of TES on STOCs in HT and NT: We could observe a tendency that both the amplitude and frequency of STOCs between HT and NT were increased after adding TES. Conclusions: ( 1 ) The vasorelaxation effect of TES in HT was smaller than that in NT. ( 2 ) 1 mM TEA pretreatment attenuated the TES-induced relaxation in NT, but not in HT. ( 3 ) The TES-induced relaxation was not inhibited by GLI, L-NAME or flutamide. ( 4 ) Compared with NT, the current density of BK_Ca in HT was smaller, and the amplitude and frequency of STOCs in HT were lower. ( 5 ) BK_Ca in vascular smooth muscle of the mesenteric artery in NT could be activated by TES, but not in HT. In addition, it was observed that the amplitude and frequency of STOCs increased after adding TES. ( 6 ) The effect of TES-induced relaxation in NT was bigger than that in HT, which may be related to the normal sensitivity of BK_Ca in mesenteric artery vascular smooth muscle cells to NT. Due to degradation of the responsibility of BK_Ca to TES in HT, the diastolic function of mesenteric artery is considered to be declined.