| The research on hypertension has an important theoretical significance and potential clinical therapeutic value. Epidemiologic data showed that the incidence of hypertension in plateau inhabitants is lower than residents in plain area, which suggests adaptation of hypoxia in plateau has anti-hypertension effect. It was reported that chronic intermittent hypobaric hypoxia (CIHH) had depression effect on artery pressure inessential hypertension patients and spontaneously hypertensive rats. Recently, our preliminary experiment showed that CIHH decreased artery pressure in renovascular hypertension (RVH) rats. The mechanism, however, underlying the anti-hypertension of CIHH is far from clear.The large conductance calcium-activated potassium channel (BKCa), abundantly expressed in vascular smooth muscle cell (VSMC), plays an important role in regulation of vascular tone and blood pressure. Activation of BKCa channels leads to hyperpolarization of cell membrane, which in turn counteracts vasoconstriction or induces relaxation. It is well known that resistance vessels play a pivotal role in the regulation of generation and maintenance of normal arterial pressure. The peripheral resistance of vessel and blood pressure will increase when resistance vessel contract, and vice versa. The activity of vasoconstriction and vasodilatation depends on the modulation of vasoactive substances and ion-channels on vessels. Our previous study showed that the relaxation of aorta was increased in CIHH-treated rats. So we hypothesize that CIHH can enhance the vasodilatation or/and diminish the vasoconstriction in resistance vessels through activation of BKCa channel in hypertension animals. The present study was designed to investigate the anti-hypertension effect of CIHH and underlying vascular mechanism using microvessel recording, patch-clamp and molecular biology techniques in RVH rats.The study consists of three parts:(1) First part of study aims to confirm the anti-hypertension effect of CIHH in RVH rats using Tail-cuff method, and to determine the effect of CIHH on vasomotion in isolated mesenteric arteries using microvessel recording technique in RVH rats. (2) Second part of study aims to confirm the role of BKCa channel on vasomotion of CIHH in isolated mesenteric arteries of rats using microvessel recording technique and to measure BKCa channel currents in vascular smooth muscle cells (VSMCs) by whole-cell patch technique and protein expression of BKCa channel by western blot analysis. (3) Thrid part of study aims to investigate the molecular mechanism of CIHH on BKCa channel or NO-cGMP-PKG signaling pathway using whole-cell patch technique and Western blots analysis.Part I Effect of CIHH on contraction and relaxation in isolated mesenteric arteries from renal vascular hypertension ratsObjective:To investigate effect of CIHH on contraction and relaxation in rat isolated mesenteric arteries using a microvessel recording technique.Methods:Sprague-Dawley rats were divided into four groups:sham operation group (SHAM), CIHH treatment group (CIHH), renovascular hypertension group (RVH), RVH plus CIHH treatment group (RVH+ CIHH). The CIHH rats were treated with hypobaric hypoxia simulating 5000m altitude for 28-days,6 hours per day in a hypobaric chamber. Renal vascular hypertension was produced in RVH rats through clamping one kidney artery to about half of its original caliber via Q-type silver slip. The RVH+CIHH rats accepted 28-days intermittent hypoxia treatment after production of renovascular hypertension. The SHAM rats accepted abdominal operation without artery clamping and intermittent hypoxia treatment. All rats drank water freely, fed a standard laboratory diet, and were housed in a temperature-controlled room (22±1℃) with a 12 h/12 h light/dark cycle (lights on at 06:00 am). Systolic arterial pressure (SAP) of tail artery was measured in conscious rats with a tail-cuff pressure meter (LE5001, Pressure Meter, Powerlab, ADInstruments Company, AUS) in the same time every week. Microvessel recording technique was used to observe the effect of CIHH on contraction induced by Ang-Ⅱ and PE, endothelium-dependent relaxation induced by ACh and endothelium -independent relaxation induced by SNP in isolated mesenteric artery rings of rats.Results:1 CIHH had no effect on the basis SAP and body weight in rats (P>0.05), but can effectively antagonized increasing of SAP and the reduction of body weight in RVH rats (P＜0.05).2 Compared with SHAM rats, contraction induced by Ang Ⅱ and PE was increased in mesenteric artery rings of RVH rats (P＜0.05), but reduced in mesenteric artery rings of CIHH rats (P<0.05). the increased contraction by Ang Ⅱ and PE in mesenteric artery rings of RVH rats was antagonized by CIHH treatment (P＜0.05).3 Compared with SHAM rats, endothelium-dependent relaxation induced by ACh was reduced in mesenteric artery rings of RVH rats (P<0.05), but increased in mesenteric artery rings of CIHH rats (P<0.05). The reduced endothelium-dependent relaxation by ACh in mesenteric artery rings of RVH rats was antagonized by CIHH treatment (P<0.05).4 Compared with SHAM rats, the blocking effect of L-NAME on endothelium-dependent relaxation by ACh was decreased in mesenteric artery of RVH rats (P＜0.05), but increased in mesenteric artery rings of CIHH rats (P<0.05). The decreased blocking of L-NAME on endothelium-dependent relaxation by ACh in mesenteric artery rings of RVH rats was antagonized by ^IHH treatment (P＜0.05).5 Compared with SHAM rats, endothelium-independent relaxation by SNP was reduced in mesenteric artery rings of RVH rats (P＜0.05), but increased in mesenteric artery rings of CIHH rats (P<0.05). The reduced endothelium-independent relaxation by SNP in mesenteric artery rings of RVH rats was antagonized by CIHH treatment (P＜0.05).Conclusion:1 CIHH treatment can decrease artery blood pressure in renovascular lypertension rats.2 Anti-hypertension effect of CIHH is partly result from decreased contraction by AngⅡand PE as well as increased endothelium-dependent relaxation by ACh and endothelium-independent relaxation by SNP.Part II The involvement of BKCa in modulation of CIHH on vasomotionObjective:To investigate the mechanisms underlying of BKCa involvement in the modulation of CIHH on vasomotion.Methods:Sprague-Dawley rats were divided into four groups:sham operation group (SHAM), CIHH treatment group (CIHH), renovascular hypertension group (RVH), RVH plus CIHH treatment group (RVH+CIHH). The CIHH rats were treated with altitude hypobaric hypoxia simulating 5000m altitude for 28-days,6 hours per day in a hypobaric chamber. Renal vascular hypertension was produced in RVH rats through clamping one kidney artery to about half of its original caliber via Q-type silver slip. The RVH+CIHH rats accepted 28-days intermittent hypoxia treatment after production of renovascular hypertension. The SHAM rats accepted abdominal operation without artery clamping and intermittent hypoxia treatment. All rats drank water freely, fed a standard laboratory diet, and were housed in a temperature-controlled room (22 ± 1 ℃) with a 12 h/12 h light/dark cycle (lights on at 06:00 am). Systolic arterial pressure (SAP) of tail artery was measured in conscious rats with a tail-cuff pressure meter (LE5001, Pressure Meter, Powerlab, ADInstruments Company, AUS) in the same time every week. Microvessel recording technique was used to observe the effect of BKCa opener and blocker on contraction and relaxation in isolated mesenteric artery ring of rats. The VSMCs were isolated from the mesenteric arteries using an enzymatic disassociation. Whole-cell patch clamp technique was used to observe the BKCa currents and western blot analysis was used to examine protein expression of BKCa channel.Results:1 BKCa opener NS1619 suppressed the contraction induced by PE in each group. The suppression of PE-induced contraction was increased in CIHH rats (P＜0.05). The decreased suppression on PE-induced contraction in RVH rats was antagonized after CIHH treatment (P＜0.05).2 BKCa blocker IBTX increased the contraction induced by PE in each group. The increment of PE-induced contraction was increased in CIHH rats (P＜0.05). The decreased increment on PE-induced contraction in RVH rats was antagonized after CIHH treatment (P＜0.05).3 BKCa opener NS1619 induced concentration-dependent relaxation in mesenteric artery rings in each group. The sensitivity of mesenteric artery rings to NS1619 was significantly increased in CIHH rats (P＜0.05). CIHH treatment antagonized the decreased sensitivity of mesenteric artery rings in RVH rats to NS1619 (P＜0.05).4 Endothelium-dependent relaxation induced by ACh was suppressed by BKCa blocker IBTX in each group. The suppression of ACh-induced relaxation was increased in CIHH rats (P＜0.05). The decreased suppression on ACh-induced endothelium-dependent relaxation in RVH rats was antagonized after CIHH tratment (P＜0.05).5 Compared with SHAM rats, the BKCa channel current in VSMCs was decreased in RVH cells, but increased in CIHH cells (P＜0.05). CIHH treatment could antagonize the reduced BKCa channel current in RVH cells (P＜0.05).6 The protein expression of β1 subunits of BKCa channel was decreased in mesenteric ateries of RVH rats, but increased in CIHH rats (P＜0.05). There was no significant difference in protein levels of a subunits of BKCa channel among all rats (P>0.05).Conclusion:1 The BKCa current in VSMCs was decreased and the function of BKCa channels was impaired in RVH cells; the BKCa currents were increased and the function of BKCa channels enhanced in CIHH cells.2 CIHH resulted in decrease of contraction and increase of relaxation through activating BKca.channel current in VSMCs from the mesenteric, arteries.3 The decreased of contraction and increased of relaxation in RVH rats was resulted from the down-regulation of β1 subunits and function of BKCa channel. CIHH would antagonize the increased contraction and reduced relaxation in mesenteric ateries in RVH rats to maintain normal vascular reactivity through upregulation protein expression of β1 subunits and activation of BKCa channel.PartⅢ CIHH modulats BKCa through NO-cGMP-PKG signallingObjective:To explore the role of NO-cGMP-PKG in the modulation of CIHH on BKCa channel and the mechanisms.Methods:Sprague-Dawley rats were divided into four groups:sham operation group (SHAM), CIHH treatment group (CIHH), renovascular hypertension group (RVH), RVH plus CIHH treatment group (RVH+CIHH). The CIHH rat was treated with altitude hypobaric hypoxia simulating 5000m altitude for 28-days,6 hours per day in a hypobaric chamber. Renal vascular hypertension was produced in RVH rats through clamping one kidney artery to about half of its original caliber via Q-type silver slip. The RVH+CIHH rats accepted 28-days intermittent hypoxia treatment after production of renovascular hypertension. The SHAM rats accepted abdominal operation without artery clamping and intermittent hypoxia treatment. All rats drank water freely, fed a standard laboratory diet, and were housed in a temperature-controlled room (22±1℃) with a 12 h/12 h light/dark cycle (lights on at 06:00 am). Systolic arterial pressure (SAP) of tail artery was measured in conscious rats with a tail-cuff pressure meter (LE5001, Pressure Meter, Powerlab, ADInstruments Company, AUS) in the same time every week. A myograph system was used to observe the effect of BKCa opener and blocker on contraction and relaxation in isolated superior mesenteric arteries of rats The VSMCs were isolated from the mesenteric arteries using an enzymatic disassociation. Perforated whole cell patch technique was used to observe the effect of NO-cGMP-PKG on BKCa currents and Western blot analysis to examine protein expression of eNOS. Results:1 L-arg, a precursor of NO, increased BKCa channel current in VSMCs of each group significantly. Compared with SHAM cells, the L-arg-induced enhancement of BKCa channel current was significantly increased in CIHH cells and decreased in RVH cells. The decreased enhancement in RVH cells was antagonized after CIHH treatment (P＜0.5).2 L-NAME, a notric oxide synthase (NOS) inhibitor, could inhibit BKCa channel currents in each group cell significantly. Compared with SHAM cells, the inhibition degree of BKCa channel current was increased in CIHH cells and decreased in RVH cells. The decreased inhibition of BKCa channel current in RVH cells was antagonized after CIHH treatment (P＜0.05).3 SNP, a NO donor, significantly enhanced BKCa channel current in each group cell. Compared with SHAM cells, the enhancement of BKCa channel current was increased in CIHH cells and decreased in RVH cells. The decreased enhancement in RVH cells was antagonized after CIHH treatment (P＜0.05).4 PTIO, a NO scavenger, significantly inhibited in each group cell. Compared with SHAM cells, the inhibition of BKCa channel current was increased in CIHH cells and decreased in RVH cells. The decreased inhibition in RVH cells was antagonized after CIHH treatment (P＜0.05).5 A PKG opener 8-BrcGMP significantly enhanced BKCa channel current in each group cell. Compared with SHAM cells, the enhancement of BKCa channel current was increased in CIHH cells and decreased in RVH cells. The decreased enhancement in RVH cells was antagonized after CIHH treatment (P＜0.05).6 A PKG blocker KT-5823 significantly inhibited BKCa channel current in each group cell. Compared with SHAM cells, the inhibition of BKCa channel current was increased in CIHH cells and decreased in RVH cells. The decreased inhibition in RVH cells was antagonized after CIHH treatment (P＜0.05).7 The protein expression of eNOS in mesenteric ateries was increased in CIHH rats (P＜0.05) and decreased in RVH rats. The decreased eNOS protein levels in mesenteric ateries in RVH rats was antagonized after CIHH treatment (P＜0.05).Conclusion:1 CIHH treatment not only enhanced the sensitivity of BKCa channel current induced by both endogenous and exogenous NO, but also antagonized the decreased sensitivity of BKCa channel current to NO in RVH cells.2 CIHH treatment not only enhanced the function of BKCa channel activated by PKG, but also antagonized the decreased function of BKCa channel in RVH cells.3 CIHH treatment upregulated protein expression, and antagonized the upregulation of eNOS to antagonize the decreased protein expression of eNOS in RVH mesenteric arteries.4 CIHH treatment modulated BKCa channel currents through NO- cGMP-PKG signaling pathway. |
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