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The properties of the outward Ca(2+)-dependent K+ current (KCa) were investigated in single smooth muscle cells (SMCs) isolated from epididymal part of the rat vas deferens (RVD) using amphotericin B perforated patch-clamp technique. The complex kinetic of the net outward current elicited by positive voltage steps from -80 mV to +40 mV suggested the presence of several components of this current. KCa current was separated from the net outward current by removal of Ca2+ from the external solution. KCa was characterized by slow kinetics of current activation and decay. Mycotoxin paxilline, the selective blocker of the large conductance KCa channels, inhibited KCa current in a dose-dependent manner. At the concentration of 70 nM paxilline evoked 50% inhibition of KCa and at 1 mkM complete suppression of KCa current was achieved. The blocking effect of low concentrations of a nonselective KCa channels inhibitor tetraethylammonium (TEA) was compared to that of paxilline. The external application of 0.3 mM TEA inhibited KCa current similarly to 1 mkM of paxilline. Finally, we studied the effect of paxilline on the resting membrane potential ofRVD SMCs. Paxilline (1 mkM) did not affect the membrane potential of SMCs with the resting potential in the range of -60 to -40 mV. However, at potentials more positive than -40 mV application of paxilline significantly (up to 15 mV) depolarized the membrane of SMCs. These results suggest that the large conductance KCa channels in RVD SMCs do not contribute to the resting membrane potential but could serve as a hyperpolarizing mechanism at the significant membrane depolarizations.

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Transfer factor (TF) of immune reactivity (10(-5) - 10(-3) mg/ml) to diphtheria-tetanus anatoxin modulates slow waves and spontaneous contractile activity of non-atropinized smooth muscle stripes (SMS) of guinea-pig taenia coli. TF (10(-4) mg/ml) transforms slow waves into stable depolarization and tonic contraction. After SMS atropinization, the substance acts in the same way. In the presence of methylene blue (10(-5) M), a guanylatecyclase blocker, FT induces transitory increase of SMS muscle tone, which is followed by their stable relaxation. ATP and UTP, purinoceptors agonists, evoke substantial hyperpolarization of smooth muscle cells membrane and their relaxation. FT enhances post-inhibitory excitation in SMS. In the presence of acetylcholine (10(-5) M) FT (10(-4) mg/ml) transforms the inhibitory ATP action on tonic contraction into excitative. This substance (10(-5), 10(-4) mg/ml) enhances Ca2+ mobilization from ryanodine-sensitive calcium store, inhibits the release of these cations from IP3-sensitive calcium store of sarcoplasmic reticulum. TF demolishes the inhibitory actions of sodium nitroprusside (nitric oxide donor), and noradrenaline in taenia coli smooth muscles.

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Immune-active substance of Staphylococcus aureus, cell-bound protein A (CBPA), enhances the acetylcholine- or hyperpotassium (K+) Krebs solution-evoked excitation in Taenia coli smooth muscles. CBPA increases caffeine- and carbachole-evoked Ca2+ signals in smooth muscle cells suspension, loaded with indo-1, and also caffeine- and acetylcholine-evoked contraction in smooth muscles slices. Against a background of CBPA-suppressed action of sodium nitroprusside, ATP evokes the membrane depolarization. CBPA in small concentrations potentiates ATPase (Mg2+,Ca2+-; Mg2+- and Mg2+- in the presence of EGTA) activity of actomyosin in the smooth muscles.

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Cell-bound protein A (CBPA), an immune-active substance of Staphylococcus aureus was ascertained to depolarize membrane of taenia coli smooth muscle (SM) cells, depress ATP inhibiting action (or uridinetriphosphate (UTP)) and sodium nitroprusside (SNP). ATP or UTP-induced membrane hyperpolarization increased during first minutes of CBPA exposure. Bacterial substance enhanced and then inhibited fast component of nicotine-induced relaxation of histamine-activated smooth muscles. This enhancement was inhibited by N(omega)-nitro-Larginine, a NO-syntase blocker. CBPA decreased ATP inhibiting action upon histamine-induced contraction, but enhanced cholinergic SM excitation. All these processes are reversible.

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The G-protein-dependent intracellular signal cascades of excitation in longitudinal and circular intestinal smooth muscles (SM) are compared and summarized in the present review. The key mechanism of excitation in longitudinal SM is an activation of electro-mechanical coupling, in that G-proteins, phospholipase A2, arachidonic acid, membrane-bound cyclic adenosine diphosphoribose and Ca2+ are involved. We observed the role of arachidonic acid-activated chorine and voltage-dependent Ca2+ -channels (L-type) in Ca2+ mobilization in these muscle cells. In contrast to longitudinal, the main mechanism of agonist-induced excitation in circular SM is connected with activation of key methabotropical processes. The role of Rho-kinase in mechanisms of Ca2+ --sensitization of contractile apparatus in SM is also shown in this review. A comparative analysis of involvement of different links of signal cascades in initial and sustained phases of contraction in longitudinal and circular SM are also reviewed.

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The mechanisms of bacterial substances (protein A, peptidoglican Staphylococcus aureus), bacterium toxins (St. aureus, Corynebacterium diphtheriae, Shigella dysenteriae, Clostridium botulinum, Clostridium tetani, Vibrio choleral), transfer factor of immune reactivity to Staphylococcus aureus upon the key link (acetylcholine-, ATP-, inositol-1,4,5-triphosphate-, ryanodin-sensitive receptors, G-proteins, Ca2+, K(+)-transporting systems, second messengers) in the chain of signal conduction of excitatory and inhibitory agonists in excitable cells were examined. The action of these immune-active substances upon contractile proteins ATP-ase activity and protein synthesis was also discussed.

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The carbachol-evoked inward cationic current in guinea-pig ileum smooth muscle cells is comprised of three types nonselective cationic channels (NSCC) with small (10 +/- 2 pS), medium (56 +/- 8pS) and large (135 +/- 14 pS) unitary conductance. All three types of NSCC could be activated by external application of carbachol as well as by internal application of GTPgS. It was found that behavior of carbachol- and GTPgammaS-evoked whole-cell current is mainly determined by the properties of medium conductance channels. The U-shaped I-V relationship of the whole-cell cationic current at negative potentials range arrives from voltage-dependence of its Po of this channel.

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It has been shown that Transfer factor (TF) to Staphilococcus aureus antigens blocked ATP-induced component of the inhibitory junction potential in taenia coli smooth muscle from guinea-pig, and converted an inhibitory action of exogenous ATP into an exciting one (instead of the hyperpolarization of the smooth muscle, TF induced its depolarization). TF at 10-6 mg/ml converted the relaxing effects of sodium nitroprusside (nitric oxide donor) into exciting ones in smooth muscle strips. Higher concentrations (10-5-10-3 mg/ml) of TF slightly amplified the relaxing effect of sodium nitroprusside. Both a- and b-adrenergic activation in taenia coli smooth muscle were not sensitive to that agent.

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Spontaneous transient outward currents (STOCs) were studied in the rat tail artery smooth muscle cells using standard patch-clamp recording techniques in the whole-cell configuration. STOCs evoked by membrane depolarization from -30 to 20 mV varied in size from 50 to 1000 pA, their amplitude increased with membrane depolarization. These currents were inhibited by 1 mM TEA+. Both the frequency and the transferred charge (Q) were decreased in the absence of the extracellular Ca2+ or in the presence of the selective blocker of voltage-gated L-type Ca2+ channels nifedipine. Application of caffeine at 1 mM increased both Q and the frequency of STOCs generation. These results indicate that STOCs are carried by large conductance Ca(2+)-dependent K+ channels and Ca2+ influx plays an important role in their activation.

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K+ current which take part in the controlling of membrane potential in myocytes isolated from rat resistance mesenteric arteries have been investigated using conventional patch clamp method. The mean resting potential of myocytes was--37 mV. Charybdotoxin (200 nM)--selective blocker of large conductance Ca(2+)-activated K+ (KCa) channels--inhibited transmembrane outward K+ current by 60%. 1 mM of tetraethylammonium inhibited outward K+ current same as 200 nM of charybdotoxin, also it inhibited spontaneous spike-like hyperpolarizations and did not affect the membrane potential. Transmembrane current had a 4 aminopyridine (4-AP) sensitive component of delayed rectifier current (KV). Addition of 5 mM of 4-AP evoked membrane depolarization with mean significance of 12.0 +/- 1.5 mV in 5 from 7 single myocytes which had resting potential in the range of -50 ... -35 mV. The obtained results suggest that large conductance KCa channels do not determine the resting potential, but may serve as a negative feedback mechanism at the considerable membrane depolarization. In contrast, 4-AP sensitive KV current take part in the controlling of the resting membrane potential of single myocytes from rat resistance mesenteric arteries.

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The effects of SK&F 96365 on cationic current evoked either by activating muscarinic receptors with carbachol or by intracellularly applied GTPgammaS (in the absence of carbachol) were studied using patch-clamp recording techniques in single guinea-pig ileal smooth muscle cells. SK&F 96365 reversibly inhibited the muscarinic receptor cationic current in a concentration-, time- and voltage-dependent manner producing concomitant alteration of the steady-state I-V relationship shape which could be explained by assuming that increasing membrane positivity increased the affinity of the blocker. The inhibition was similar for both carbachol- and GTPgammaS-evoked currents suggesting that the cationic channel rather than the muscarinic receptor was the primary site of the SK&F 96365 action. Increased membrane positivity induced additional rapid inhibition of the cationic current by SK&F 96365 which was more slowly relieved during membrane repolarization. Both the inhibition and disinhibition time course could be well fitted by a single exponential function with the time constants decreasing with increasing positivity for the inhibition (e-fold per about 12 mV) and approximately linearly decreasing with increasing negativity for the disinhibition. At a constant SK&F 96365 concentration, the degree of cationic current inhibition was a sigmoidal function of the membrane potential with a potential of half-maximal increase positive to about +30 mV and a slope factor of about -13 mV. Increasing the duration of voltage steps at -80 or at 80 mV, increased the percentage inhibition; the degree of inhibition was almost identical at both potentials providing evidence that the same cationic channel was responsible for the cationic current both at negative and at positive potentials. It is concluded that the distinctive and unique mode of SK&F 96365 action on the muscarinic receptor cationic channel is a valuable tool in future molecular biology studies of this channel.

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Whole-cell currents in single smooth muscle cells freshly isolated from the rat tail artery have been studied using patch-clamp recordings techniques. Outward current evoked by depolarizing steps from -70 mV consisted of the initial fast and subsequent sustained components. The former was inhibited by 10 mM nifedipine and Ca(2+)-free solution application whereas the latter decreased by only 16% under these conditions. Caffeine at 4 mM abolished the fast component and only slightly reduced the sustained component. Both component were inhibited by 1 mM TEA+. Adding 10 mM EGTA to the pipette solution abolished the fast outward current. In the presence of 10 mM TEA+ and 4 mM 4-AP an inward current was unmasked. These results suggest that the outward current in these cells is carried mainly via delayed rectifier and Ca(2+)-activated K(+)-channels. 4-AP inhibited the sustained component and had no effect on the initial fast outward current, thus A-current is apparently absent.

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The inward currents in single smooth muscle cells (SMC) isolated from epididymal part of rat vas deferens have been studied using whole-cell patch-clamp method. Depolarising steps from holding potential -90 mV evoked inward current with fast and slow components. The component with slow activation possessed voltage-dependent and pharmacological properties characteristic for Ca(2+) current carried through L-type calcium channels (I(Ca)). The fast component of inward current was activated at around -40 mV, reached its peak at 0 mV, and disappeared upon removal of Na ions from bath solution. This current was blocked in dose-dependent manner by tetrodotoxin (TTX) with an apparent dissociation constant of 6.7 nM. On the basis of voltage-dependent characteristics, TTX sensitivity of fast component of inward current and its disappearance in Na-free solution it is suggested that this current is TTX-sensitive depolarisation activated sodium current (I(Na)). Cell dialysis with a pipette solution containing no macroergic compounds resulted in significant inhibition of I(Ca) (depression of peak I(Ca) by about 81% was observed by 13 min of dialysis), while I(Na) remained unaffected during 50 min of dialysis. These data draw first evidence for the existence of TTX-sensitive Na(+) current in single SMC isolated from rat vas deferens. These Na(+) channels do not appear to be regulated by a phosphorylation process under resting conditions.

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Changes in the muscle tissue stiffness are important and necessary signs of the contractile process. The work is devoted to the study of this parameter of the smooth muscle tissue reaction during the action of phorbol esters, (namely, phorbol 12-myristate 13-acetate (PMA)), synthetic imitators of diacylglycerole. The experiments were carried out on isolated native preparations of aorta thoracic area of guinea pig under isometric conditions. Contraction induced by PMA or by the temperature was not accompanied by the stiffness changes. Potassium contraction (its development, amplitude, relaxation) evoked on the plateau of the PMA-induced contraction was similar in all respects to that in control: the stiffness increased significantly. These findings prove independent development of these two types of contractile processes. At the same time, the stiffness invariability and similar ratios of optical density to mechanical strength changes in the contractions induced by PMA action or by temperature show the likeness of the force generation mechanisms in these mechano-chemical reactions. The results obtained cannot be explained in terms of the generally accepted hypothesis of actomyosin interaction. The conclusion is made that phorbolic esters during the action on the smooth muscle tissue activate the mechanical strength generation by the connective tissue matrix.

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The mechanisms of smooth muscle tissue contractile system functioning are in many respects unexplained. According to the existing hypothesis, the mechanism of smooth muscle contraction is based on the interaction between myosin and actin. The change of muscle tissue stiffness during the contractive process is the important and obligatory feature of this actomyosin interaction. Earlier we have shown that, together with the smooth muscle cells, the connective tissue matrix can also produce the active mechanical strength. This process is not accompanied by changes of stiffness. We suggested that in some cases the induced contraction of smooth muscles is fulfilled, entirely or in part, by the connective tissue matrix. We report here that contractive reaction induced by the phorbol ester--activator of protein kinase C, which is one of the most important enzymes involved in the regulation of the smooth muscle contraction--occurs without any stiffness changes. The results obtained can not be explained in terms of the generally accepted hypothesis of actomyosin interaction. The conclusion is made that phorbol ester during the action on the smooth muscle tissue activates the mechanical strength generation by the connective tissue matrix.

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The matrix of connective tissue was found to take part in generation of the mechanical strength in isolated strips of the v. cava posterior wall under the effect of increased temperature. The finding corroborates the concept of the actomyosin interaction. The vessel tissue response to temperature seems to be formed by three mechanisms, two of them being of a non-actomyosin nature.

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