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M F Shuba

Publications and source records attributed to M F Shuba.

At least 19 recordsLinked to original sources
PubMed2000

Voltage-dependent inhibition of the muscarinic cationic current in guinea-pig ileal cells by SK&F 96365.

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.

Animals↗
PubMed2000

[The role of voltage gated K(+) channels in the modulation of resting membrane potential of myocytes isolated from rat resistance arteries].

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.

Animals↗
PubMed1999

TTX-sensitive Na(+) and nifedipine-sensitive Ca(2+) channels in rat vas deferens smooth muscle cells.

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.

Animals↗
PubMed1994

Activation of the non-actomyosin component of aortic wall contraction by phorbol ester.

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.

Actomyosin↗
PubMed1994

[The "non-actomyosin" component of vascular wall contraction].

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.

Actomyosin↗
PubMed1992

Potential-dependent inward currents in single isolated smooth muscle cells of the rat ileum.

1. Calcium (ICa) and sodium (INa) currents were studied in single smooth muscle cells freshly isolated from both the newborn (1-3 days old) and adult rat ileum, using the patch-clamp technique (whole-cell configuration). 2. Under conditions when INa was blocked, two components of ICa, low-voltage activated or ICa,low and high-voltage activated or ICa,high, were observed in the newborn rat ileal cells. ICa,high and ICa,low have differing voltage ranges of activation and steady-state inactivation and time courses of recovery from inactivation. Potential dependence of ICa,low was much steeper and shifted toward negative membrane potential than that for ICa,high (slope factors and the potential of half-maximal inactivation were 13.6 and -60.6 and 8.8 and -49 mV for ICa,low and ICa,high, correspondingly). 3. Nifedipine at the high concentration of 30 microM exerted no effect on ICa,low and only slightly suppressed ICa,high, decreasing its peak to 0.81 +/- 0.04 (n = 7) at the holding potential of -80 mV and to 0.66 +/- 0.05 (n = 3) at -50 mV. ICa,high was suppressed significantly by Cd2+ ions, while ICa,low was more sensitive to Ni2+ ions. 4. Results presented here suggest that the properties of high-voltage-activated (HVA) Ca2+ channels in the rat small intestine are quite different to those described for L-type Ca2+ channels found in other smooth muscles. It is proposed that HVA Ca2+ channels are similar to N-type Ca2+ channels. 5. Comparison of Ca2+ currents in newborn and adult rat ileal cells showed that the contribution of ICa,low to the net Ca2+ current was negligible in adults, whereas the properties of HVA Ca2+ channels were similar in the neonatal and adult animals. 6. INa, studied in nominally Ca(2+)-free physiological salt solution, activated in the voltage range between -50 and -40 mV and reached its peak at -10 mV. INa was blocked in a dose-dependent manner by TTX with an apparent dissociation constant of 4.5 nM. 7. INa decay was monoexponential in the voltage range studied and its time constant decreased monotonically with membrane depolarization from 4.7 +/- 0.2 ms (n = 6) at -30 mV to 0.51 +/- 0.03 ms (n = 7) at 20 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

A potential-dependent fast outward current in single smooth muscle cells isolated from the newborn rat ileum.

1. Whole-cell outward currents have been studied in single smooth muscle cells isolated from newborn and adult rat ileum, using fire-polished glass micropipettes. 2. Two major outward currents, delayed (I(do)) and fast inactivating potential-dependent (I(fo)), have been observed in the newborn rat ileal cells. I(fo) is activated between -50 and -40 mV from the holding potential of -80 mV, whereas I(do) usually starts to activate at membrane potentials positive to -20 mV. Activation of I(do) was fast, its time-to-peak decreased from 10.8 +/- 0.9 ms (n = 5) at -30 mV to 4.5 +/- 0.7 ms (n = 4) at 20 mV. 3. I(fo) decay was monoexponential and its time constant did not depend on the membrane potential. Dependence of I(fo) inactivation on membrane voltage in normal physiological salt solutions (PSS) could be described by the Boltzmann equation with the following parameters: a half-inactivation potential, V0.5 = -70.8 mV and slope factor, k = 7.7 mV. 4. Recovery of I(fo) from inactivation was fitted by a single exponential and was potential dependent. The average time constant was 28.4 +/- 2.4 ms (n = 11) at -120 mV, 47.7 +/- 3.0 ms (n = 6) at -100 mV and 89.6 +/- 5.3 ms (n = 13) at -80 mV. 5. Removal of Ca2+ ions from the PSS (in the presence of 5 mM-Mg2+) increased I(fo) amplitude by about two times, and shifted its voltage dependence of inactivation towards negative membrane potentials by about 16 mV (V0.5 = -87.2 mV). Removal of Mg2+ from the PSS (in the presence of 2.5 mM-Ca2+) exerted no effects upon either inactivation dependence (V0.5 = -74.2 mV) or I(fo) amplitude. 6. I(do) and I(fo) had different sensitivities to K+ channel blockers. With 10 mM-external TEA+ I(do), was preferentially suppressed, while 5 mM-4-aminopyridine (4-AP) completely blocked I(fo). I(fo) was also partially blocked by a higher TEA+ concentration (30 mM), which suppressed I(fo) to 0.55 +/- 0.02 (n = 9). The blocking effect of 4-AP on I(fo) was potential, use and time dependent. 7. Ileal cells isolated from the adult rat demonstrated the presence of two populations of smooth muscle cells. One has an outward current which seems to be similar to that described in the newborn rat. However, in other cells spontaneous transient outward currents, well described in other single smooth muscle cells, but not found in newborn rat ileal cells, have been observed.

4-Aminopyridine↗
PubMed1992

Some properties of Ca(2+)-induced Ca2+ release mechanism in single visceral smooth muscle cell of the guinea-pig.

1. Late transient outward Ca(2+)-dependent K+ current (ILTO) correlated with Ca(2+)-induced Ca2+ release mechanism was studied in relation to the calcium inward current (ICa) in single isolated smooth muscle cells of the guinea-pig ileum using the whole-cell patch-clamp technique. 2. The voltage dependencies of peak ICa and ILTO were both bell shaped. However, the I-V curve of the outward current was shifted toward more positive potentials by about 60 mV in comparison to that for ICa. 3. Reduction in the external Ca2+ concentration resulted in a decrease of peak amplitude of both ICa and ILTO. However, caffeine-induced outward current was also decreased abruptly suggesting a rapid loss of stored Ca2+ upon lowering the external Ca2+ concentration. 4. Investigation of the relation of ILTO to partially inactivated ICa showed that inactivation of ICa by approximately 65, 80 or 84% of control (produced by prepulse to -20 mV for 2 s, shifting the holding potential to -20 mV for 30 s or by the ramp voltage command from -50 to +10 mV, respectively) was without detectable effect on the ILTO generation. 5. Bath application of the Ca2+ antagonist nifedipine (300 nM) inhibited ICa by 81% without affecting ILTO peak amplitude (92.0 +/- 5.6% of control in six cells). The mean concentration-response curve for ICa inhibition was sigmoidal with the apparent dissociation constant of 86.9 nM, whereas that for the ILTO had a characteristic sharp transition indicating a definite threshold of Ca2+ influx for ILTO generation. 6. Application of Ca(2+)-free external solution during 500 ms of the time when ICa peaked inhibited the current by about 76% whereas the ILTO during such an intervention remained virtually unchanged. 7. In double-pulse experiments, with conditioning and test pulses to +10 mV from -50 mV and an interpulse interval of 600 ms, most of the cells (about 80%) showed larger outward current at the test pulse suggesting continued Ca2+ release triggered by Ca2+ influx during a short (50-200 ms) depolarizing prepulse. The outward current could also be evoked at large positive potentials (presumably near the calcium equilibrium potential) where it did not occur normally by a prepulse to +10 mV for 50 ms. The charge transferred by Ca2+ current necessary to activate Ca2+ release in most of the cells was estimated to be from 6 to 20 pC. 8. The data are interpreted to suggest that the Ca(2+)-induced Ca2+ release mechanism operates in single ileal cells in a regenerative manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Patch-clamp recording in myenteric neurons of guinea pig small intestine.

The results of our research established the feasibility of applying patch-clamp methods in the study of the cellular neurophysiology of myenteric neurons enzymatically dissociated from adult guinea pig small intestine. Recording in current-clamp mode revealed two populations of neurons. One population discharged repetitively during depolarizing current pulses and displayed anodal-break excitation reminiscent of S/type 1 myenteric neurons. In the second population, spike discharge was limited to one or two spikes at the onset of depolarizing pulses and was similar to the behavior of AH/type 2 neurons. Recording in voltage-clamp mode revealed a complex of overlapping inward and outward whole cell currents. Fast and slow components of inward current were interpreted as sodium and calcium currents, respectively. Outward currents were blocked by cesium and consisted of components with properties of delayed rectifier current and A-type potassium current.

Animals↗
PubMed1992

[Effects of the membrane potential level on serotonin-induced contraction of the pulmonary artery smooth muscle in rabbits].

The effects of changes in membrane potential level on the electrical and contractile responses induced by serotonin (10(-6) mol/l) were investigated in muscle strips from rabbit main pulmonary artery using sucrose-gap technique. In spite of the fact that serotonin-induced depolarization did not exceed the threshold level for development of contraction, it was followed by a strong tonic contraction. Nearly a half of this contraction could be relaxed by an electrotonic hyperpolarization of the membrane. A week preliminary depolarization of the muscle cells resulted in an increase while a strong depolarization--in dramatic decrease of serotonin-induced contraction. Nifedipine effectively blocked potassium-induced, but not serotonin induced contraction. We suggest that in addition to voltage-operated and receptor operated Ca channels in vascular smooth muscle cell membrane there is a separate class of nifedipine-insensitive Ca channels operated by both serotonin receptor and membrane potential.

Animals↗
PubMed1991

The inhibitory action of caffeine on calcium currents in isolated intestinal smooth muscle cells.

The patch-clamp method has been used to investigate the action of caffeine on the calcium current (ICa) in single isolated smooth muscle cells of the guinea-pig ileum. Caffeine (10 mM) substantially inhibited ICa. This effect occurred in a biphasic manner and it was not due either to activation of additional ionic currents of opposite direction nor to inhibition of phosphodiesterase activity. It strongly depended upon the ethylenebis-(oxonitrilo)tetraacetate (EGTA) concentration in the pipette solution. When there was K+ in the pipette solution, application of caffeine evoked a transient Ca-dependent K+ current and an abrupt and transient increase in the frequency of channel openings. Such well-known blockers of Ca release as procaine and ruthenium red strongly decreased ICa. Ryanodine had only little effect on ICa, but application of caffeine in the presence of ryanodine led to a complete and irreversible inhibition of ICa. The results of experiments involving different EGTA concentrations and comparison of the time courses of all caffeine-induced phenomena clearly indicated that only the initial, transient component of the ICa inhibition by caffeine was related to a Ca-dependent inactivation of Ca channels, evoked as a result of Ca release from intracellular stores. The tonic component of ICa inhibition was probably due to a direct blocking action of caffeine on Ca channels.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Properties of the late transient outward current in isolated intestinal smooth muscle cells of the guinea-pig.

1. Whole-cell membrane currents in voltage-clamped single isolated cells of longitudinal smooth muscle of guinea-pig ileum were studied at room temperature using patch pipettes filled with either high-K+ solution or high-Cs+ solution, to suppress K+ outward current, and containing 0.3 mM-EGTA. 2. In the presence of high-K+ solution in the pipette, membrane depolarization from the holding potential of -50 mV evoked an initial inward calcium current (ICa) followed by a large initial transient outward current and a sustained outward current with spontaneous oscillations superimposed. Prolonged depolarization above -20 mV produced a late transient outward current which reached a maximum (up to several nanoamps at +10 mV) within approximately 1 s and lasted several seconds. 3. The late outward current (ILTO) was voltage dependent and reversed at the EK (potassium equilibrium potential) in cells exposed to high-K+ external solution. It was blocked by TEA+ (tetraethylammonium) or Ba2+ applied externally (calculated Kd (dissociation constant) values were 0.67 and 4.43 mM, respectively) or by high-Cs+ solution perfusing the cell. The removal of extracellular Ca2+, application of Ca2+ channel blockers (3 mM-Co2+, 0.2 mM-Cd2+ or 1 microM-nifedipine) or perfusion of 5 mM-EGTA inside the cell also abolished the current. Thus, the current seems to be a Ca(2+)-activated K+ current. 4. There is a great discrepancy between the time course of the ICa and that of the late ILTO, which suggests that Ca2+ release from intracellular storage sites may contribute to the generation of the ILTO. 5. Bath application of caffeine (10 mM) during the development of ILTO enhanced the current. However, in the presence of caffeine ILTO was inhibited. Moderate inhibition of ICa by caffeine was also observed. 6. Ryanodine (5 microM) applied to the bathing solution completely inhibited ILTO within 3.5 min; however, it had no or little effect on the ICa. 7. Ruthenium Red (10 microM) completely blocked the ILTO and slightly and more slowly inhibited the ICa. 8. Increasing Mg2+ concentration in the pipette solution from 1 to 6 mM abolished the ILTO. 9. It was concluded that the ILTO was activated mainly by Ca2+ released from the intracellular storage sites following Ca2+ entry, presumably by a Ca(2+)-induced Ca2+ release mechanism.

Animals↗
PubMed1991

Inactivation of calcium channels in single vascular and visceral smooth muscle cells of the guinea-pig.

Inactivation of currents carried through calcium channels by calcium (ICa), barium (IBa) and monovalent cations (In.s.) was studied in single smooth muscle cell (SMC) of the guinea-pig coronary artery and taenia caeci by the whole-cell patch-clamp method. The rate of ICa inactivation in the coronary artery SMC was correlated with ICa amplitude, and acceleration was observed with the increasing ICa peak amplitude. The availability curve of ICa in double-pulse experiments was found to be U-shaped, however, no complete restoration of ICa availability was observed. Inactivation of IBa was considerably slower than that of ICa. These findings may indicate that inactivation of calcium channels in the membrane of coronary artery SMC is, at least partially, a Ca-dependent process. However, some facts observed contradict the validity of this hypothesis for coronary artery SMC in contrast to taenia caeci: 1) elevation of external Ca2+ concentration did not affect the time course of ICa inactivation; 2) inactivation of In.s., i.e. without calcium entry into the cell, was faster than that of ICa. It was concluded that the characteristics of Ca channel inactivation were changed by the removal of divalent cations from extracellular solution. Differences and similarities in Ca channel inactivation between coronary artery and taenia caeci SMC are discussed.

Animals↗