The Effect Of Nitric Oxide On Potassium Channel In Human Airway Smooth Muscle Cell Of Passive Sensitization By Serum From Allergic Asthmatic Patients

IntroductionThe changes of cell excitability can induce many diseases. Athma is a chronicinflammatory disease characterized by reversible airway obstruction and bronchialhyperreponsiveness to various stimuli. The increase of airway smooth muscle cells(ASMCs) excitability is the major reason for the bronchial hyperreponsiveness. Of thecell ionic channels potassium (K⁺) channels are important to regulate the cell membranepotential and excitability. ASMCs express three most important K⁺ channels includinglarge Ca²⁺-activated K⁺ channels (BK_Ca), voltage-dependent delayed rectifier K⁺channels (Kv) and ATP-sensitive K⁺ channels (Katp), which play a key role indetermining the resting membrane potential, a relative electrical stability andresponsiveness to both contractile and relaxant agents. Opening K⁺ channels can inducethe increase of outward K⁺, resulting in membrane hyperpolarization thus leading todecline of the cell excitability and Ca²⁺ influx, which may lead to the muscle relaxation.Among these three types of K⁺ channels, the Kv channels can be selectively blocked by4-aminopyridine and are the most important in regulation of airway contractile activityand hyperreactivity. A recent study illustrated that Kv channels contributed significantlyto generation of the resting membrane potential and regulation of the bronchial smoothresting muscle tone in rats. K_ca and K_atp channels had no such effects. In addition,inhibition of Kv channel can increase the proliferation of ASMCs from rats. Though itwas ever reported that the bronchial myocytes from asthmatic rats showed a significantreduction in Kv current density and higher excitability and these mechanisms maycontribute to the development of airway hyperreactivity in asthma, the activity andexpression of K⁺ channels in human bronchial smooth muscle cells (HBSMCs) and howthese channels regulate the cell membrane potential, excitability and muscle tone havenever been studied. Nitric Oxide (NO),the major endothelium-derived relaxing factor(EDRF),canrelax the smooth muscle of airway, vascular and gastrointestinal, In previous study, we found thatNO relaxed the contraction of airway smooth muscle partly via potassium channel opening in rats, Butthere is scarce evidence to illustrate whether NO relaxed the contraction of human airway smoothmuscle (HASM) via potassium channel opening ? So, the aim of this article is to investigate theabove questions and to provide basic experimental data for the further study ofmechanism and treatment of asthma.Part 1 The role of potassium channels in the nitricoxide-induced relaxation of human airway smooth muscle of passively sensitization by serum from allergic asthmatic patients objectiveTo investigate the role of large Ca²⁺-activated, delayed-rectifier, and ATP-sensitive potassium channels in regulating the relaxation induced by nitric oxide (NO) in normal and passively sensitized human airway smooth muscle (HASM) with serum from asthmatic patients.MethodsThe effects of NO or/and potassium channel blockers on the tensions of normal and passively sensitized HASM were measured by using nitric oxide donor and potassium blockers, with the isometric tension recording technique.Results1. In the control group and passively sensitized group, Kv blocker 4-AP (1×10³ mol/L, 5×10³ mol/L, 1×10^-2 mol/L) caused concentration-dependent augmentation in the contraction induced by histamine (p < 0.05), but Glib (1×10^-2 mol/L) and TEA(1× 10^-3 mol/L) had no significant effects on the contraction induced by histamine. The maximum tension induced by histamine in passively sensitized group was higher than that in the control group (p < 0.05).2. NO-donor Sodium Nitroprusside (SNP) brought about significant relaxation in normal and passively sensitized HASM rings (p < 0.05). Relaxations of passively sensitized airway rings [ (29.4±3.3) %] were significant less than those of normal HASM rings[(44.1±10.2)%] (p < 0.05).3. Glib(1×10^-2 mol/L)had no significant effect on the relaxations induced by SNP(1×10^-4 mol/L). 4-AP(1×10^-2 mol/L) inhibited relaxation induced by SNP(1×10^-4 mol/L) (p < 0.01).TEA(1×10-3 mol/L) inhibited relaxation induced by SNP(1×10^-4mol/L) (p < 0.05) , and the inhibiting effect in passively sensitized HASM rings were significant less than in normal HASM (p < 0.05).Conclusions1. Not K_Ca and K_Atp but K_V participated in the regulation of the resting tone of HASM.2. SNP (NO-donor) relaxed the contraction of HASM partly via BKca channel opening.3. In passively sensitized HASM in vitro, the relaxation of SNP decreased compared with control group, which might be associated with the down-regulating activity of BKca in passively sensitized HASM.Part 2 Effect of nitric oxide on apoptosis of asthmatic airway smooth muscle cellsSubject 1 Effect of nitric oxide on apoptosis of airway smooth muscle cells of asthmatic rats in vitroObjectiveTo explore the effect of apoptosis in asthmatic rat airway smooth muscle cells (ASMCs) induced by nitric oxide (NO).Methods1. Wister rats were selected to make the models of asthma.2. The effect of SNP on the survival rate of asthmatic rat airway smooth muscle cells was detected by MTT method.3. The effect of SNP on apoptosis of asthmatic rat airway smooth muscle cells was detected by TUNEL4. The effect of SNP on apoptosis of asthmatic rat airway smooth muscle cells was detected by flow cytometry.Results1. Comparing asthma group, the survival rate of ASMCs decreased in SNP + asthma group by MTT method (n=8, p<0.05) ..2. TUNEL showed that apoptosis index of ASMCs was significantly increased following SNP treatment (n=4, p <0.05).3. Flow cytometry showed that apoptosis rate of ASMCs was significantly increased following SNP treatment (n=4, p <0.05).Conclusions1. NO inhibited the proliferation of asthmatic rat airway smooth muscle cells .2. NO induced apoptosis of asthmatic rat airway smooth muscle cells, which may be related to treatment of airway remodeling in asthma.Subject 2 Effect of nitric oxide on apoptosis of human airway smooth muscle cells of passive sensitization by serum from allergic asthmatic patientsObjectiveTo investigate nitric oxide (NO) induced apoptosis of human airway smooth muscle cells (HASMCs) of passive sensitization by serum from allergic asthmatic patients.Methods:1. The technique of human airway smooth muscle (HASMCs) passively sensitized with serum from allergic asthmatic patients was adopted.2. The effect of SNP on the survival rate of passively sensitized HASMC was detected by MTT method.3. The effect of SNP on apoptosis of passively sensitized HASMC was detected by TUNEL4. The effect of SNP on apoptosis of passively sensitized HASMC by flow cytometry.Results:1. Comparing sensitized group, the survival rate of passively sensitizedHASMCs decreased in SNP + sensitized group (n=5, p <0.05) .2. TUNEL showed that apoptosis index of passively sensitized HASMCs was significantly increased following SNP treatment (n=4, p <0.05).3. Flow cytometry showed that apoptosis rate of passively sensitized HASMCs was significantly increased following SNP treatment (n=4, p <0.05).Conclusion:1. NO inhibited the proliferation of passively sensitized HASMCs2. NO induced apoptosis of passively sensitized HASMCs, which may be related to treatment of airway remodeling in asthma.Part 3 The effect of nitric oxide on potassium channel in human airway smooth muscle cell of passive sensitization by serum from allergic asthmatic patientsobjectiveThe effect of SNP (NO-donor) on potassium channels in cultured HASMCs passively sensitized by serum from asthmatic patients were investigated.MethodsThe technique of HASMCs passively sensitized with serum from allergic asthmatic patients was adopted. HASMCs were randomly divided into three groups: control group (containing 10% serum from nonatopic individuals), sensitized group (containing 10% asthmatic serum) and SNP+sensitized group. The membrane capacitance (Cm), the membrane potential (Em) and potassium current were measured with the whole-cell patch clamp before and after SNP (0.1mmol/L) administration.Results1. The membrane potential in passively sensitized HASMCs were significantly depolarized to — (27.8 + 4.8) mV compared with — (40.1 + 5.8) mV in the controlgroup (n=8,p<0.05) .2. Under Pulse and Ramp protocol, passive sensitization caused significant inhibition of BKca currents in HASMCs compared with control group(n=8,p <0.01), but SNP could significantly increase the BKca currents in passively sensitized HASMCs(n=8,p<0.05) .3. Under Pulse and Ramp protocol, passive sensitization caused significant inhibition of Kv currents in HASMCs compared with control group (n=8,p <0.05) ,but SNP could significantly increase the Kv currents in passively sensitized HASMCs(n=8,p <0.05) .Conclusions1. The Em in passively sensitized HASMCs was depolarized.2. The activity of BKca and Kv in passively sensitized HASMCs was decreased.3. SNP could increase the activity of BKca and Kv and repolarize the Em in passively sensitized HASMCs, which suggest SNP might decrease the excitability of HASMCs via opening potassium channels.SummaryThe tissues and cultured cells of rats and human airway smooth muscle cells (ASMCs) in vitro are used in the series of studies at the level of cell mechanism. We draw the conclusion as follows:1 SNP relaxed the contraction of HASM partly via BKca channel opening.2 NO inhibited the proliferation of airway smooth muscle cells of asthmatic rat and passively sensitized human.3 NO induced apoptosis of airway smooth muscle cells of asthmatic rat and passively sensitized human,which may be related to treatment of airway remodeling inasthma.4 The activity of BKca and Kv in passively sensitized HASMCs was decreased.5 SNP could increase the activity of BKca and Kv and repolarize the Em in passively sensitized HASMCs...