Characteristics of the bradykinin-induced changes in intracellular calcium ion concentration of single bovine tracheal smooth muscle cells.

1. Single bovine tracheal smooth muscle (BTSM) cells were cultured and used to measure bradykinin-induced changes in [Ca2+]i by dynamic video imaging. 2. Bradykinin (10 pM-10 microM)-induced an increase in [Ca2+]i over basal levels (69 +/- 2 nM; n = 353) which was concentration-dependent (log EC50 = -8.7 M) in the presence of extracellular calcium ions (2 mM). The bradykinin B2 receptor antagonist, D-Arg[Hyp3,Thi5,8,D-Phe7]- bradykinin, produced a parallel shift to the right of the bradykinin concentration-response curve (log EC50 = -7.1 M and -5.8 M in the presence of 1 microM and 10 microM antagonist respectively) yielding an apparent KD of 26 nM. 3. In the absence of extracellular calcium ions (with 0.1 mM EGTA), bradykinin (10 pM-10 microM) produced a uniform increase in [Ca2+]i from a basal level of 33 +/- 2 nM (n = 140) to approximately 180 nM in BTSM cells indicating an 'all-or-nothing' release of intracellular calcium ions. In the presence of 10 microM D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin no responses could be induced by bradykinin at concentrations below 100 nM. However, at 100 nM and 1 microM bradykinin there was no change in the uniform increase in [Ca2+]i in these cells previously observed. 4. In both the absence or presence of D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin, there was a concentration-dependent increase in the percentage of cells responding to bradykinin (frequency) under calcium-rich or calcium-free conditions. Individual cells also demonstrated a difference in the sensitivity to any particular concentration of bradykinin.(ABSTRACT TRUNCATED AT 250 WORDS)     

西红花苷对牛主动脉平滑肌细胞内钙离子浓度的影响

目的研究西红花苷对血管平滑肌细胞内钙离子浓度的影响。方法以Fluo-3/AM作为Ca²⁺荧光探针,采用激光扫描共聚焦显微镜观察牛主动脉平滑肌细胞内钙离子浓度的变化。结果无论细胞外有无钙离子,西红花苷(1×10^-8,1×10^-7,1×10^-6 mol·L^-1)均能明显抑制1×10^-2 mol·L^-1 H₂O₂引起的细胞内钙离子浓度的升高,其抑制率含钙条件下分别为34.1%, 57.1%和74.3%(P<0.01),无钙条件下分别为26.2%,32.1%和50.0%(P<0.01);在胞外无钙的条件下,西红花苷 (1×10^-8,1×10^-7,1×10^-6 mol·L^-1)能抑制70 mmol·L^-1 CHCl₃导致雷洛丁敏感钙池的释放,其抑制率分别为27.8%, 27.8%和50.0% (P<0.01)。结论西红花苷能抑制胞外钙离子的内流及内质网上钙离子的释放。     

大鼠肺动脉平滑肌细胞培养及对不同类型激动剂刺激产生的反应

目的分离和培养SD大鼠肺动脉平滑肌细胞（PASMCc）,并检测其功能状态。方法显微分离肺内小动脉,并在含胶原酶（1750 U&#183;mL^-1）和木瓜蛋白酶（9．5U&#183;mL^-1）的低钙HBSS溶液中酶解和培养PASMCs,采用动态细胞荧光成像技术检测PASMCs胞浆游离Ca^2＋浓度（[Ca^2＋]i）的变化。结果在18～24h内可获得PASMCs,并可观察到环匹阿尼酸和5-HT可引起PASMCs[Ca^2＋]i的升高效应。结论大鼠PASMCs一步酶消化法,方法简便实用。所分离的PASMCs细胞形态和功能正常,适用于动态细胞荧光成像技术检测实验及PASMCs信号转导功能的研究。     

Cytosol-free calcium concentration in single bladder smooth muscle cells from normal and diabetic rats.

The effect of diabetes mellitus on intracellular calcium concentration was measured in single rat bladder smooth muscle cells using the fluorescent calcium indicator dye fura-2 AM. Techniques were developed for isolation and short-term culture of rat bladder smooth muscle cells. Cytosol-free calcium concentrations were measured at rest and during carbachol stimulation. Peak intracellular calcium concentration, rate of increase in intracellular calcium concentration and the integral of accumulation of intracellular calcium were determined. The techniques used to isolate and culture bladder smooth muscle cells produced live, physiologically responsive cells. Resting intracellular free calcium levels and were similar in control and diabetic cells. Both control and diabetic cells responded to carbachol stimulation. No significant differences between these cells were noted in peak calcium concentrations, rate of response or integral of response, but standard errors were large. Two patterns of intracellular response to carbachol stimulation were identified, and may explain the large variability in intracellular calcium response to carbachol stimulation.     

糖尿病大鼠膀胱功能及平滑肌细胞内钙离子变化及意义

目的观察糖尿病神经源性膀胱尿动力学及逼尿肌细胞内钙离子变化,探讨其发病机制。方法建立糖尿病大鼠模型,正常大鼠作对照,测定不同糖尿病病程膀胱湿重、最大膀胱容量、膀胱顺应性及单个膀胱平滑肌细胞内钙离子浓度。结果糖尿病组大鼠膀胱湿重增加、最大膀胱容量、膀胱顺应性及平滑肌细胞内钙离子浓度较对照组高。结论糖尿病神经源性膀胱舒缩功能受损,为高顺应性膀胱,且随病程延长损害程度逐渐加重。平滑肌细胞内钙超载可能参与糖尿病后期逼尿肌的病理改变过程。     

Bradykinin B2 receptor-mediated phosphoinositide hydrolysis in bovine cultured tracheal smooth muscle cells.

1. Bovine tracheal smooth muscle cells were established in culture to study agonist-induced phosphoinositide (PI) hydrolysis in this tissue. 2. Bradykinin (0.1 nM-10 microM) evoked a concentration-dependent increase (log EC50 (M) = -9.4 +/- 0.2; n = 8) in the accumulation of total [3H]-inositol phosphates in cultured tracheal smooth muscle cells whereas the selective B1 receptor agonist des-Arg9-bradykinin (10 microM) was significantly less effective (16% of bradykinin maximal response; relative potency = 0.2 with respect to bradykinin = 100). 3. The bradykinin-induced increase in PI hydrolysis was unaffected by the B1 receptor antagonist des-Arg9[Leu8]-bradykinin (1 nM-1 microM) but showed marked attenuation in the presence of the B2 receptor antagonists D-Arg,[Hyp3,D-Phe7]-bradykinin (10 nM-10 microM) or D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin (10 nM-10 microM). The estimated KB values obtained for these two compounds, assuming competitive antagonism, were 40 +/- 14 nM and 8.6 +/- 2.8 nM for D-Arg,[Hyp3,D-Phe7]-bradykinin and D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin respectively. 4. We conclude that bradykinin B2 receptors are expressed in cultured bovine tracheal smooth muscle cells and are coupled to PI hydrolysis mechanisms.     

埃他卡林对兔肺动脉平滑肌内皮细胞钙浓度的影响

目的观察埃他卡林(IPT)对内皮素1(ET-1)诱导培养的兔肺动脉平滑肌细胞(PASMC)增殖的影响,并探讨其作用机制。方法应用细胞培养、氚-胸腺嘧啶核苷([3H]-TdR)参入实验、Fluo-3和激光扫描共聚焦显微镜技术评价IPT对ET-1诱导的兔PASMC增殖及PASMC[Ca2+]i调节的作用。结果ET-1(10-7mol.L-1)使PASMC[3H]-TdR参入量增加146.8%,与对照组比较,差异有显著性(P<0.01);在相同条件下,加入ET-1的同时,分别向培养基中加入IPT10-7、10-6、10-5mol.L-1,细胞[3H]-TdR参入量分别下降(19.8±4.6)%、(41.2±9.5)%、(54.7±10.1)%,与ET-1组比较差异有显著性(P<0.01);对照组PASMC[Ca2+]i荧光强度和荧光光密度值较低;ET-1组中[Ca2+]i荧光光密度值明显增高,从73.7±10.1增加到143.8±28.2,两者比较差异有显著性(P<0.01);而IPT组细胞内荧光光密度值明显降低,仅从74.30±10.2增加到86.03±9.82,与ET-1组比较差异有显著性(P<0.01)。结论IPT可明显抑制ET-1诱导的兔PASMC增殖、DNA合成;减少钙通道的开放时间,抑制细胞内Ca2+浓度增加。     

Modulation of fluoroaluminate-induced inositol phosphate formation by increases in tissue cyclic AMP content in bovine tracheal smooth muscle.

1. The effect of fluoroaluminate complexes (AlCl3 plus NaF) upon smooth muscle tone, [3H]-inositol phosphate accumulation and [3H]-cyclic AMP accumulation has been investigated in slices of bovine tracheal smooth muscle. 2. Fluoroaluminate (10 microM AlCl3 + various concentrations of NaF) elicited concentration-dependent contractions of bovine tracheal smooth muscle strips at concentrations of NaF in the range 1-10 mM. The resultant contractile response was reversed by isoprenaline (50 nM) and was preserved in calcium-free medium. 3. Fluoroaluminate stimulated [3H]-inositol phosphate formation at concentrations of NaF over 1 mM. The response to 20 mM NaF + 10 microM AlCl3 was 164 +/- 29% of the response to 1 mM histamine. Fluoroaluminate also increased the incorporation of [3H]-myo-inositol into membrane phospholipids. 4. Fluoroaluminate produced a small rise in [3H]-cyclic AMP levels (2.1 fold increase over basal with 20 mM NaF). The response to forskolin (1 microM, 8.6 fold over basal) was reduced by fluoroaluminate in a concentration-dependent manner, but still remained significantly (P less than 0.05) elevated over the response to fluoroaluminate alone. 5. The [3H]-inositol phosphate response to fluoroaluminate was inhibited by salbutamol (maximum inhibition 60%, IC50 = 0.08 microM), forskolin (1 microM, 46% inhibition) and isobutylmethylxanthine (1 mM, 73% inhibition). 6. These data suggest that inhibition of agonist-induced inositol phospholipid turnover by cyclic AMP in this tissue can occur at the post-receptor level.     

Des-Arg9-bradykinin increases intracellular Ca2+ in bronchoalveolar eosinophils from ovalbumin-sensitized and -challenged mice

The effects of the selective bradykinin B1 receptor agonist, des-Arg9-bradykinin and the bradykinin B2 receptor agonist, bradykinin were studied on the intracellular free Ca2+ concentration ([Ca2+]i) in murine bronchoalveolar lavage cells from control and ovalbumin-sensitized mice using fura-2 microfluorimetry. The bronchoalveolar lavage cells of control mice, which were predominantly alveolar macrophages, showed an increase in [Ca2+]i in response to bradykinin (1 microM) but not to des-Arg9-bradykinin (1 microM), indicating the presence of functional bradykinin B2 receptors and the absence of B1 receptors. Such elevation in [Ca2+]i induced by bradykinin was totally inhibited by the selective bradykinin B2 receptor antagonist, D-Arg0-Hyp3-Thi5-D-Tic7-Oic8-bradykinin (HOE-140; 10 microM). In contrast, bronchoalveolar lavage cells from ovalbumin-sensitized and -challenged mice significantly responded to both bradykinin and des-Arg9-bradykinin, indicating the presence of both functional bradykinin B1 and B2 receptors. Eosinophils exhibited higher response to des-Arg9-bradykinin (1 microM; 485% increase in [Ca2+]i) compared to bradykinin (1 microM; 163% increase in [Ca2+]i). This des-Arg9-bradykinin-induced [Ca2+]i increase was markedly inhibited by the selective bradykinin B1 receptor antagonist, Ac-Lys-[D-betaNal7, Ile8]des-Arg9-bradykinin (R-715; 10 microM). Des-Arg9-bradykinin neither modified the basal [Ca2+]i in lymphocytes nor in mononuclear cells from ovalbumin-sensitized and challenged mice, while bradykinin produced a [Ca2+]i increase in both cell types. Our results further support the implication of the inducible bradykinin B1 receptors in airway inflammatory response in ovalbumin-sensitized and challenged mice.     

吡那地尔对人肺动脉平滑肌细胞内Ca2+浓度的影响

目的 探讨ATP敏感性钾(KATP)通道开放剂吡那地尔(Pin)对内皮素1(ET-1)诱导的人肺动脉平滑肌细胞(PASMCs)增殖及细胞内[Ca2+]i的影响.方法 体外培养人PASMCs,用ET-1诱导其增殖,应用MTT法、Fluo-3和激光扫描共聚焦显微镜技术评价Pin对ET-1诱导的人PASMCs增殖及PASMCs[Ca2+]i调节的作用.结果 Pin显著抑制ET-1诱导的人PASMCs增殖.呈浓度依赖效应,KATP通道拮抗剂格列本脲呈浓度依赖性阻断Pin的作用;ET-1诱导人PASMCs内[Ca2+]i显著增加,Pin(10 μmol/L)拈抗ET-1诱导的人PASMCs内[Ca2+]i升高.结论 KATP通道开放剂Pin可明显抑制ET-1诱导的人PASMCs增殖作用,抑制细胞内Ca2+浓度增加.     

A patch-clamp study of K(+)-channel activity in bovine isolated tracheal smooth muscle cells.

1. Single smooth muscle cells were isolated from bovine trachealis by enzymic digestion. The properties of large conductance plasmalemmal K(+)-channels in these cells were studied by the patch-clamp recording technique. 2. Recordings were made from inside-out plasmalemmal patches when [K+] was symmetrically high (140 mM) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 10 microM. Large unitary currents of both Ca(2+)-dependent and -independent types were observed. Measured between + 20 and + 40 mV, the slope conductances of the channels carrying these currents were 249 +/- 18 pS and 268 +/- 14 pS respectively. 3. Lowering [K+] on the cytosolic side of the patches from 140 to 6 mM, shifted the reversal potentials of the two types of unitary current from approximately zero to much greater than + 40 mV, suggesting that both currents were carried by K(+)-channels. 4. The Ca(2+)-dependent and -independent K(+)-channels detected in inside-out plasmalemmal patches could also be distinguished on the basis of their sensitivity to inhibitors (tetraethylammonium (TEA), 1-10 mM; Cs+, 10 mM; Ba2+, 1-10 mM; quinidine, 100 microM) applied to the cytosolic surface of the patches. 5. Recordings were made from outside-out plasmalemmal patches when [K+] was symmetrically high (140 mM) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 1 microM. Ca(2+)-dependent unitary currents were observed and the slope conductance of the channel carrying these currents was 229 +/- 5 pS.(ABSTRACT TRUNCATED AT 250 WORDS)     

The relaxant action of nicorandil in bovine tracheal smooth muscle

The relaxant mechanisms of nicorandil were examined by comparing its effects with those of sodium nitroprusside and cromakalim in bovine tracheal smooth muscle. In preparations contracted with methacholine (0.3 μ mol/l) or high K(+)(40 mmol/l), nicorandil and sodium nitroprusside caused concentration-dependent relaxations. Their relaxant effects on high K(+) -contracted preparations were smaller than those on methacholine-contracted muscle. Cromakalim relaxed methacholine-contracted preparations, whereas it had no effect on high K(+) -contracted muscle. The inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 mol/l) completely prevented the relaxation induced by lower concentrations ( <30 μ mol/l) of nicorandil,whereas it partially attenuated relaxation caused by higher concentrations. The ATP-sensitive K(+) (K(ATP)) channel blocker glibenclamide only partially attenuated the relaxant responses to nicorandil (at 100 and 300 μ mol/l). Combination treatment with ODQ and glibenclamide almost completely prevented nicorandil-induced relaxations. The large-conductance Ca2(+) -activated K(+) channel (Maxi K(+) channel) inhibitor iberiotoxin significantly prevented the relaxations induced by lower concentrations (3 and 10 μ mol/l) of nicorandil. The preventive effect of iberiotoxin was markedly enhanced under the blockade of K(ATP) channels with glibenclamide. These results suggest that nicorandil relaxes bovine tracheal smooth muscle through 2 mechanisms: opening of K(ATP) channels and activation of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Nicorandil may also activate Maxi K(+) channels, possibly through the NO-cGMP pathway, and the interaction of K ATP channels and Maxi K(+) channels may affect the relaxant effect of nicorandilin bovine tracheal smooth muscle.     

滨蒿内酯对原代和传代培养豚鼠气道平滑肌细胞内钙的影响

目的探讨滨蒿内酯(scoparone,Scop)对原代及短期传代培养的豚鼠气道平滑肌细胞(airway smooth musclecells,ASMCs)内钙的影响,同时,比较原代与传代ASMCs在形态、生长曲线及内钙释放受Scop及caffeine影响的异同。方法细胞计数法绘制原代及传代培养ASMCs的生长曲线,应用Fluo-3/AM为细胞内Ca2+示踪剂,通过倒置荧光纤维镜观察和记录原代及短期传代培养的ASMCs的细胞形态及其细胞内钙离子浓度([Ca2+]i)的改变。结果原代和传代培养ASMCs的倍增时间分别为(31.89±1.24)h和(22.91±6.82)h,传代培养ASMCs的倍增时间明显缩短(P<0.05),传代培养的ASMCs相对原代培养ASMCs体积增大。在细胞外液无钙条件下,不同浓度的Scop(10-6、10-5、10-4mol.L-1)可降低静息状态下培养的ASMCs的[Ca2+]i,并与给药浓度有关,原代与传代培养的ASMCs比较,对不同浓度的Scop的降钙反应无明显异同(P>0.05);不同浓度咖啡因(caffeine,10-4、10-3、10-2mol.L-1)在10-4mol.L-1Scop存在下,可升高ASMCs的[Ca2+]i,传代培养的ASMCs[Ca2+]i较原代对caffeine的反应下降(P<0.01)。结论Scop可降低培养的ASMCs的[Ca2+]i,并且不受细胞传代影响。短期传代培养的ASMCs相对于原代细胞,形态及内钙释放通道特性发生了改变。     

Histamine-stimulated increases in intracellular calcium in the smooth muscle cell line, DDT1MF-2

Suspensions of undifferentiated cultured vas deferens smooth muscle cells (DDT1MF-2) were loaded with the calcium-sensitive fluorescent dye fura-2. Exposure to histamine elicited a rapid and maintained increase in intracellular free calcium ([Ca2+] i) with an EC50 of 1.3 +/- 0.7 x 10(-5) M. The initial rise is a consequence of calcium release from intracellular stores, whereas the maintained or plateau phase, which is dependent upon the presence of extracellular calcium, is associated with calcium influx. Experiments in nominally Ca(2+)-free buffer attenuated the initial rise in [Ca2+]i (i.e. peak height) and virtually abolished the plateau phase. Re-addition of 2 mM Ca2+ (during experiments performed in nominally Ca(2+)-free buffer) resulted in a return of the plateau phase. Pretreatment with the H1-antagonist mepyramine (100 nM; Kd = 1.0 +/- 0.4 nM, N = 3) completely blocks the response to histamine, whereas tiotidine (2 microM; H2-antagonist) had no effect. In conclusion, the present data would suggest that functional H1-receptors found in hamster vas deferens smooth muscle cells are typical of the "classical" H1-receptor in both its control of intracellular Ca2+ and sensitivity to antagonism by mepyramine.     

醋柳黄酮对血管平滑肌细胞胞内游离钙浓度的影响

目的 :探讨醋柳总黄酮 (TFH)对血管平滑肌细胞胞内游离钙浓度 ([Ca2 ]i)的影响。方法 :采用新一代钙荧光探针Fluo 3 AM检测在高钾、去甲肾上腺素 (NE)、血管紧张素II(AngⅡ )刺激下单层兔主动脉平滑肌细胞内游离钙水平的改变 ,并与传统的钙拮抗剂Verapamil(Ver)进行对照研究。结果 :TFH(10 0mg·L-1)对静息状态的血管平滑肌细胞[Ca2 ]i 无明显影响 ;TFH(6 0～ 10 0mg·L-1)呈剂量依赖性抑制高K 去极化引起的 [Ca2 ]i 升高 ,与Ver作用相似 ,但弱于Ver ;TFH (80、10 0mg·L-1)对NE、AngⅡ通过受体介导引起 [Ca2 ]i 升高均具有明显的抑制作用 ;在无细胞外Ca2 存在下 ,TFH (80、10 0mg·L-1)对NE引起的 [Ca2 ]i 升高也具有一定程度的抑制效应。结论 :醋柳总黄酮通过对电压依赖性钙通道和受体操纵型钙通道双重抑制降低血管平滑肌细胞内游离钙水平 ,这可能是醋柳黄酮产生舒血管降压作用机制之一。     

Beta-adrenoceptor stimulation inhibits histamine-stimulated inositol phospholipid hydrolysis in bovine tracheal smooth muscle.

1. Histamine and carbachol produced concentration-related increases in the accumulation of 3H-inositol phosphates in slices of bovine tracheal smooth muscle. 2. Noradrenaline alone produced a small stimulation of 3H-inositol phosphate accumulation which was inhibited by the alpha-adrenoceptor antagonist phentolamine. In contrast, when noradrenaline (0.1 mM) was added simultaneously with histamine it significantly reduced the inositol phosphate response to high (greater than or equal to 0.1 mM) concentrations of histamine. However, noradrenaline had no inhibitory effect on the carbachol-induced inositol phosphate response. 3. The non-selective beta-agonist isoprenaline (IC50 = 0.08 microM) and the beta 2-selective agonist salbutamol (IC50 = 0.29 microM) both produced a dose-related inhibition of the inositol phosphate response to 0.1 mM histamine. The inhibitory effect of salbutamol was antagonized by propranolol (KA = 2.4 x 10(9) M-1) and the beta 2-selective adrenoceptor antagonist ICI 118551 (KA = 1.7 x 10(9) M-1). 4. The accumulation of 3H-inositol phosphates induced by histamine increased steadily over a 40 min period after an initial lag period of 3-4 min. Following the simultaneous addition of histamine and salbutamol there was a further delay of 3-4 min before the appearance of the inhibitory effect of salbutamol. 5. The effect of histamine on inositol phosphate accumulation was accompanied by a stimulation of [3H]-inositol incorporation into membrane phospholipids which was reduced by the presence of salbutamol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bradykinin increases Na(+)-K(+) pump activity in cultured guinea-pig tracheal smooth muscle cells

1. The effect of bradykinin on the Na+-K+ pump of airway smooth muscle was investigated by measuring ouabain-sensitive (86)Rb(+) uptake in cultured guinea-pig tracheal smooth muscle cells. 2. Bradykinin induced a concentration-dependent increase in ouabain-sensitive (86)Rb(+) uptake, with an EC(50) of 3 nM (pD(2) = 8.50+/-0.10). Stimulation was not affected by indomethacin (1 microM) suggesting that it is not mediated by cycloxygenase products of arachidonic acid. 3. The B(1) receptor agonists Lys-des-Arg(9)-bradykinin and des-Arg(9)-bradykinin had no effect on ouabain-sensitive (86)Rb(+) uptake. In contrast, the B(1) and B(2) receptor agonist Lys-bradykinin induced a concentration-dependent increase in ouabain-sensitive (86)Rb(+) uptake with an EC(50) of 6 nM (pD(2) = 8.21 +/- 0.20). 4. The B(1) receptor antagonist des-Arg(10)-HOE 140 (1 microM) had no effect on bradykinin-stimulated ouabain-sensitive (86)Rb(+) uptake. The B(2) receptor antagonists HOE 140 and WIN 64338 antagonized bradykinin-stimulated ouabain-sensitive (86)Rb(+) uptake with pK(B) values (-log M) of 8.20 +/- 0.08 and 8.11 +/- 0.20 respectively. 5. Reducing extracellular Na+ from 146 mM to 11 mM caused a 53.5% decrease in basal ouabain-sensitive (86)Rb+ uptake and abolished bradykinin-induced uptake. Two inhibitors of the Na(+)-H(+) exchanger, methylisobutyl-amiloride (MIA; 1 - 100 microM) and ethylisopropyl-amiloride (EIPA; 0.1 - 10 microM), inhibited bradykinin-stimulated ouabain-sensitive (86)Rb(+) uptake without affecting basal uptake. 6. These results suggest that bradykinin increases Na+-K+ pump activity of guinea-pig tracheal smooth muscle via stimulation of B(2) receptors and activation of the Na+-H+ exchanger.     

Intracellular calcium in canine cultured tracheal smooth muscle cells is regulated by M3 muscarinic receptors.

1. The regulation of cytosolic Ca2+ concentrations ([Ca2+]i) during exposure to carbachol was measured directly in canine cultured tracheal smooth muscle cells (TSMCs) loaded with fura-2. Stimulation of muscarinic cholinoceptors (muscarinic AChRs) by carbachol produced a dose-dependent rise in [Ca2+]i which was followed by a stable plateau phase. The EC50 values of carbachol for the peak and sustained plateau responses were 0.34 and 0.33 microM, respectively. 2. Atropine (10 microM) prevented all the responses to carbachol, and when added during a response to carbachol, significantly, but not completely decreased [Ca2+]i within 5 s. Therefore, the changes in [Ca2+]i by carbachol were mediated through the muscarinic AChRs. 3. AF-DX 116 (a selective M2 antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, a selective M3 antagonist) inhibited the carbachol-stimulated increase in [Ca2+]i with pKB values of 6.4 and 9.4, respectively, corresponding to low affinity for AF-DX 119 and high affinity for 4-DAMP in antagonizing this response. 4. The plateau elevation of [Ca2+]i was dependent on the presence of external Ca2+. Removal of Ca2+ by the addition of 2 mM EGTA caused the [Ca2+]i to decline rapidly to the resting level. In the absence of external Ca2+, only an initial transient peak of [Ca2+]i was seen which then declined to the resting level; the sustained elevation of [Ca2+]i could then be evoked by the addition of Ca2+ (1.8 mM) in the continued presence of carbachol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bradykinin-stimulated phosphoinositide metabolism in cultured canine tracheal smooth muscle cells.

1. Electrical field stimulation (EFS; 10 V, 10 Hz, 2 ms) of porcine coronary artery strips precontracted with 10 nM endothelin-1 (ET-1) for 5 min caused a biphasic response, consisting of a slight contraction during EFS and a marked and irreversible relaxation just after EFS. This irreversible relaxation after EFS has never been investigated. In the present study, we have investigated the mechanism of the relaxation after EFS. 2. The EFS-induced response was not affected by the presence or absence of endothelium and was insensitive to 10 microM tetrodotoxin (TTX). 3. In the presence of free radical scavengers (40 u ml-1 superoxide dismutase (SOD), 1200 u ml-1 catalase or 80 mM D-mannitol), the relaxation after EFS was significantly inhibited. Moreover, relaxation after EFS was not observed in porcine coronary artery strips precontracted with 20 mM KCl. 4. In a cascade experiment, EFS of Krebs-Ringer solution containing 10 nM ET-1 induced marked suppression of the contractile activity of ET-1 in porcine coronary artery strips, which was in accord with the observed decrease in release of immunoreactive ET-1 (ir-ET-1). This effect of EFS was significantly inhibited by each of the free radical scavengers, 3 mM vitamin C, 40 u ml-1 SOD, 1200 u ml-1 catalase and 80 mM D-mannitol. 5. The exchange of 95% O2/5% CO2 gas for 95% N2/5% CO2 gas significantly inhibited the EFS-induced decrease in release of ir-ET-1.(ABSTRACT TRUNCATED AT 250 WORDS)