Atp-activated ionic permeability in smooth muscle cells isolated from the guinea pig urinary bladder

Smooth muscle cells from the guinea pig urinary bladder were investigated by voltage clamping at the plasma membrane and using an intracellular perfusion technique. Applying adenosine triphosphate (ATP) at a concentration greater than 3 × 10^–8 M and at a membrane potential of –100 to –30 mV produced a rise in fast inward transmembrane current. A similar effect was exerted by adenosine diphosphate (ADP) and -, -, and ,-methylene ATP. Application of guanosine triphosphate, inosine triphosphate, adenosine monophosphate (AMP), and adenosine failed to activate this current. It was found that AMP blocks ATP receptors competitively. No pharmacological differences were found between the latter ATP receptors and those of rat sensory neurons. The ATP receptors were rapidly desensitized and recovered their sensitivity to agonists extremely slowly. Speed of desensitization was reduced by a decrease in ATP concentration.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 95–100, January–February, 1987.     

Differential regulation of Ca2+-activated K+ channels by beta-adrenoceptors in guinea pig urinary bladder smooth muscle

Stimulation of -adrenoceptors contributes to the relaxation of urinary bladder smooth muscle (UBSM) through activation of large-conductance Ca²⁺-activated K⁺ (BK) channels. We examined the mechanisms by which -adrenoceptor stimulation leads to an elevation of the activity of BK channels in UBSM. Depolarization from –70 to +10 mV evokes an inward L-type dihydropyridine-sensitive voltage-dependent Ca²⁺ channel (VDCC) current, followed by outward steady-state and transient BK current. In the presence of ryanodine, which blocks the transient BK currents, isoproterenol, a nonselective -adrenoceptor agonist, increased the VDCC current by 25% and the steady-state BK current by 30%. In the presence of the BK channel inhibitor iberiotoxin, isoproterenol did not cause activation of the remaining steady-state K⁺ current component. Decreasing Ca²⁺ influx through VDCC by nifedipine or depolarization to +80 mV suppressed the isoproterenol-induced activation of the steady-state BK current. Unlike forskolin, isoproterenol did not change significantly the open probability of single BK channels in the absence of Ca²⁺ sparks and with VDCC inhibited by nifedipine. Isoproterenol elevated Ca²⁺ spark (local intracellular Ca²⁺ release through ryanodine receptors of the sarcoplasmic reticulum) frequency and associated transient BK currents by 1.4-fold. The data support the concept that in UBSM -adrenoceptor stimulation activates BK channels by elevating Ca²⁺ influx through VDCC and by increasing Ca²⁺ sparks, but not through a Ca²⁺-independent mechanism. This study reveals key regulatory molecular and cellular mechanisms of -adrenergic regulation of BK channels in UBSM that could provide new targets for drugs in the treatment of bladder dysfunction. Ca²⁺ sparks; voltage-dependent Ca²⁺ channel; ryanodine receptor     

Electrical stimulation of smooth muscle strips from the urinary bladder of the pig

Strips of smooth muscle from pig urinary bladders were electrically stimulated to contract. Stimulation parameters and conditions were optimized so as to obtain a maximum number of isometric contractions with maximal force. It was found that the contractions could be described mathematically by a simple model. In the model there is a constant probability for cells to pass from the non-contractile to the contractile state during stimulation; this leads to a linearly decreasing phase plot (a plot of the rate of rise of a variable as a function of the variable) for the force. ‘Activation’ of the cells is described by a physical step function. Isometric contractions were thus characterized by a set of three parameters: U, the time derivative of the force, extrapolated to zero force, F_iso the value of the isometric force which is approached asymptotically after infinitely long periods of stimulation and t₁, the activation time. The sensitivity of these three parameters to variation of the stimulus parameters was investigated. It was found that the parameter U was consistently correlated with the stimulus parameters, suggesting that this parameter can be used to describe the effectiveness of electrical stimulation of such strips.     

Electrical behavior of guinea pig tracheal smooth muscle

Intracellular recordings were taken from the smooth muscle of the guinea pig trachea, and the effects of intrinsic nerve stimulation were examined. Approximately 50% of the cells had stable resting membrane potentials of -50 +/- 1 mV. The remaining cells displayed spontaneous oscillations in membrane potential, which were abolished either by blocking voltage-dependent Ca(2+) channels with nifedipine or by depleting intracellular Ca(2+) stores with ryanodine. In quiescent cells, stimulation with a single impulse evoked an excitatory junction potential (EJP). In 30% of these cells, trains of stimuli evoked an EJP that was followed by oscillations in membrane potential. Transmural nerve stimulation caused an increase in the frequency of spontaneous oscillations. All responses were abolished by the muscarinic-receptor antagonist hyoscine (1 microM). In quiescent cells, nifedipine (1 microM) reduced EJPs by 30%, whereas ryanodine (10 microM) reduced EJPs by 93%. These results suggest that both the release of Ca(2+) from intracellular stores and the influx of Ca(2+) through voltage-dependent Ca(2+) channels are important determinants of spontaneous and nerve-evoked electrical activity of guinea pig tracheal smooth muscle.     

Reactivity of urinary bladder smooth muscle in guinea pigs to acetylcholine and carbachol--participation of acetylcholinesterase

The authors examined the influence of acetylcholinesterase inhibitor (neostigmine) on the in vitro reactivity of urinary bladder smooth muscle (UBSM) in guinea pigs. The aim of the present study was to determine the participation of pharmacokinetic properties of acetylcholine and carbachol in different UBSM reactivity to these mediators. In vitro method of organ baths was used and reactivity of UBSM strips to cumulative doses of acetylcholine and carbachol was tested before and after the incubation with neostigmine (10(-4) mol.l(-1)). Neostigmine caused a significant increase of UBSM reactivity to acetylcholine. The UBSM reactivity to acetylcholine was significantly higher at concentrations of 10(-5) and 10(-4) mol.l(-1) compared to carbachol at the same concentrations. These findings indicate that in addition to different mediator affinity to muscarinic receptors and to their different intrinsic activity, the pharmacokinetic properties of acetylcholine and carbachol also participate in UBSM reactivity.     

Vagal innervation of guinea pig bronchial smooth muscle

We isolated the guinea pig right bronchus with the vagus nerves intact and evaluated the changes in isometric tension of the smooth muscle in response to nerve stimulation. Brief (10-s) trains of electrical field stimulation or vagus nerve stimulation caused a biphasic contraction: the "first phase" sensitive to atropine and the "second phase" sensitive to capsaicin. The two phases could be dissociated by adjusting the stimulus intensity; greater stimulus intensities (pulse durations or voltage) were required to evoke the capsaicin-sensitive phase. When stimulated at 30-min intervals, the magnitude of both phases of the contractions declined over a 2-h period of repeated stimulation; however, this was prevented by indomethacin. Stimulation of the left vagus nerve resulted in a monophasic contraction of the right bronchus, with little evidence of a capsaicin-sensitive phase. Blocking neurotransmission through the bronchial ganglion, as monitored by intracellular recording techniques, abolished the first-phase contraction but had no effect on the capsaicin-sensitive phase. Selective blockade of muscarinic M1 receptors had no effect on vagus nerve-mediated contractions. The results demonstrate that the left and right vagus nerves carry preganglionic fibers to the right bronchial ganglion. The right but not the left vagus nerve also carries capsaicin-sensitive afferent fibers that, when stimulated, result in a persistent contraction of the right bronchus. Finally, we provide functional and electrophysiological evidence supporting the hypothesis that capsaicin-sensitive afferent neurons communicate with postganglionic motoneurons within the bronchus.     

Cooling-induced contraction of guinea pig tracheal smooth muscle

Cooling of isolated guinea pig tracheal smooth muscle from 38 to 28 degrees C over 2.25 min produced a transient contraction followed by sustained relaxation. The cooling-induced contraction was blocked either by pretreatment with ouabain at concentrations of 10(-5) M or greater or by substitution of normal physiological salt solution with K-free solution. In contrast, the contractile response to cooling was not inhibited by pretreatment with phentolamine (10(-5) M), atropine (10(-5) M), tetrodotoxin (3 X 10(-7) M), diphenhydramine (10(-5) M), cromolyn sodium (10(-3) M), indomethacin (3 X 10(-7) M), nifedipine (10(-7) M), or verapamil (3 X 10(-6) M). Addition of NaHCO3 to the bath during cooling, preventing a change in pH of the physiological salt solution, did not affect the cooling-induced contraction. It is concluded that cooling of isolated guinea pig trachea produces a transient ouabain-sensitive contraction, and that the data suggest the contraction is mediated by inhibition of Na-K-ATPase in the smooth muscle rather than through neuronal stimulation or chemical mediator release.     

豚鼠不同部位微动脉平滑肌细胞电生理学特性的比较

本研究应用电生理技术在豚鼠离体小脑前下动脉(anterior inferior cerebellar artery,AICA)、肠系膜动脉(mesenteric artery,MA)和耳蜗螺旋动脉(spiral modiolar artery,SMA)分支(直径小于100μm)上比较微动脉平滑肌细胞电生理学特性的异同。结果显示:(1)应用细胞内微电极记录技术测得AICA、MA和SMA细胞静息膜电位分别为(-68±1.8)(n=65)、(-71±2.4)(n=80)和(-66±2.9)mV(n=58),各微动脉间无统计学差异。(2)一段血管微动脉标本全细胞膜片钳记录的平滑肌细胞膜电容和膜电导都远大于单个细胞标本,且微动脉间存在统计学差异,大小顺序为MAAICASMA。应用缝隙连接阻断剂2-APB(100μmol/L)后记录一段微动脉平滑肌细胞膜电容和膜电导与单个细胞十分接近。(3)AICA、MA和SMA单个平滑肌细胞膜电流I/V关系呈明显的外向整流特性,都对1mmol/L4-AP和10mmol/LTEA敏感。当指令电压为+40mV时,AICA、MA和SMA血管平滑肌细胞电流密度分别为(26±2.0)、(24±1.7)和(18±1.3)pA/pF,SMA和AICA、MA间存在统计学差异。上述结果提示,豚鼠不同部位微动脉平滑肌细胞在缝隙连接耦联力和电流密度等电生理特性存在差异。     

Calcium waves in intact guinea pig gallbladder smooth muscle cells

Intracellular Ca(2+) waves and spontaneous transient depolarizations were investigated in gallbladder smooth muscle (GBSM) whole mount preparations with intact mucosal layer [full thickness (FT)] by laser confocal imaging of intracellular Ca(2+) and voltage recordings with microelectrodes, respectively. Spontaneous Ca(2+) waves arose most often near the center, but sometimes from the extremities, of GBSM cells. They propagated regeneratively by Ca(2+)-induced Ca(2+) release involving inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptors and were not affected by TTX and atropine (ATS). Spontaneous Ca(2+) waves and spontaneous transient depolarizations were more prevalent in FT than in isolated muscularis layer preparations and occurred with similar pattern in GBSM bundles. Ca(2+) waves were abolished by the Ins(1,4,5)P(3) receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C and by caffeine and cyclopiazonic acid. These events were reduced by voltage-dependent calcium channels (VDCCs) inhibitors diltiazem and nifedipine, by PLC inhibitor U-73122, and by thapsigargin and ryanodine. ACh, CCK, and carbachol augmented Ca(2+) waves and induced Ca(2+) flashes. The actions of these agonists were inhibited by U-73122. These results indicate that in GBSM, discharge and propagation of Ca(2+) waves depend on sarco(endo)plasmic reticulum (SR) Ca(2+) release via Ins(1,4,5)P(3) receptors, PLC activity, Ca(2+) influx via VDCCs, and SR Ca(2+) concentration. Neurohormonal enhancement of GBSM excitability involves PLC-dependent augmentation and synchronization of SR Ca(2+) release via Ins(1,4,5)P(3) receptors. Ca(2+) waves likely reflect the activity of a fundamental unit of spontaneous activity and play an important role in the excitability of GBSM.     

Capacitative calcium entry in guinea pig gallbladder smooth muscle in vitro

This study investigates the involvement of capacitative Ca2+ entry in excitation-contraction coupling in guinea pig gallbladder smooth muscle. Thapsigargin (0.1 nM-1 microM, a sarcoplasmic reticulum Ca(2+)-ATPase inhibitor) produced slowly developing sustained tonic contractions in guinea pig isolated gallbladder strips. All contractions approached 50% of the response to carbachol (10 microM) after 55 min. Contractile responses to thapsigargin (1 microM) were abolished in a Ca(2+)-free medium. Subsequent re-addition of Ca2+ (2.5 mM) produced a sustained tonic contraction (99 +/- 6% of the carbachol response). The contractile response to Ca2+ re-addition following incubation of tissues in a Ca(2+)-free bathing solution in the absence of thapsigargin was significantly less than in its presence (79 +/- 4 % vs 100 +/- 7 % of carbachol; p < 0.05). Contractile responses to Ca2+ re-addition following treatment with thapsigargin were attenuated by (a) the L-type voltage-operated Ca2+ channel antagonist, nifedipine (10 microM) and (b) the general inhibitor of Ca2+ entry channels including store-operated channels, SK&F96365 (50 microM and 100 microM). In separate experiments, responses to Ca2+ re-addition were essentially abolished by the tyrosine kinase inhibitor, genistein (100 microM). These results suggest that capacitative Ca2+ entry provides a source of activator Ca2+ for guinea pig gallbladder smooth muscle contraction. Contractile responses to Ca2+ re-addition following depletion of sarcoplasmic reticulum Ca2+ stores with thapsigargin, are mediated in part by Ca2+ entry through voltage-operated Ca2+ channels and by capacitative Ca2+ entry through store-operated Ca2+ channels which can be blocked by SK&F96365. Furthermore, capacitative Ca2+ entry in this tissue may be modulated by tyrosine kinase.     

Chloride removal and excitation-contraction coupling in guinea pig ileal smooth muscle

The effect of extracellular Cl (Cl-o) removal on contractions evoked by a selective muscarinic agonist, cis-2-methyl-4-dimethylaminomethyl 1,3-dioxolane methiodide (CD), and high K+ depolarizations in the isolated guinea pig ileal longitudinal muscle was studied. The replacement of Cl-o with impermeant anions, such as isethionate (Ise-), was found to selectively inhibit a portion of the initial phasic response to K+ and CD, leaving the secondary and sustained tonic responses unchanged. In Ca2+-free solutions, the loss of contractile responses to high K+ was faster and more pronounced in Cl--free compared with Cl--containing solutions. Furthermore, the uptake of Ca2+, as represented by 45Ca2+, from the saline solution was delayed and reduced in Ise--containing Cl-o-free solutions. Replacement of Cl-o with other impermeant anions, such as gluconate and methylsulphate, had a similar action on contractile activity as for Ise-replacement. Cl-o replacement with permeant anions, such as nitrate, however, did not significantly inhibit the phasic response and sometimes increased the tonic response to K+. These results indicate that there is a Cl-o-dependent Ca2+ pool in the guinea pig ileal longitudinal muscle and we speculate that this Cl-o-dependent Ca2+ pool is associated with membrane structures, such as calveolae, which would thus offer a degree of protection to depletion by removal of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

S-nitrosoglutathione breakdown prevents airway smooth muscle relaxation in the guinea pig

Airway levels of the endogenous bronchodilator S-nitrosoglutathione (GSNO) are low in children with near-fatal asthma. We hypothesized that GSNO could be broken down in the lung and that this catabolism could inhibit airway smooth muscle relaxation. In our experiments, GSNO was broken down by guinea pig lung homogenates, particularly after ovalbumin sensitization (OS). Two lung protein fractions had catabolic activity. One was NADPH dependent and was more active after OS. The other was NADPH independent and was partially inhibited by aurothioglucose. Guinea pig lung tissue protein fractions with GSNO catabolic activity inhibited GSNO-mediated guinea pig tracheal ring relaxation. The relaxant effect of GSNO was partially restored by aurothioglucose. These observations suggest that catabolism of GSNO in the guinea pig 1) is mediated by lung proteins, 2) is partially upregulated after OS, and 3) may contribute to increased airway smooth muscle tone. We speculate that enzymatic breakdown of GSNO in the lung could contribute to asthma pathophysiology by inhibiting the beneficial effects of GSNO, including its effect on airway smooth muscle tone.     

Ultrastructural localization of apamin binding sites in guinea pig intestinal smooth muscle

Binding sites of the neurotoxin apamin were located by immunocytochemical analysis using apamin antibodies. It was found that these bindings sites were located on the plasma membrane of smooth muscle cells in the longitudinal muscle of the cecum and at the nerve terminals in the guinea pig.A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 6, pp. 824–827, November–December, 1985.