T. Tomita

The effect of sodium and calcium on the action potential of the smooth muscle of the guinea-pig taenia coli

1. Spontaneous spike activity and action potentials evoked by external field stimulation were recorded, intracellularly and with the double sucrose gap method, from the smooth muscle of guinea‐pig taenia coli.

Effects of intracellular pH on calcium-activated potassium channels in rabbit tracheal smooth muscle.

1. The effects of intracellular pH (pHi) on calcium‐activated potassium channels (Ca2(+)‐activated K+ channels) were studied in membrane patches of smooth muscle freshly dispersed from the rabbit trachea. Single‐channel currents were recorded with an ‘inside‐out’ patch clamp technique, mainly at 0 mV, with the external (electrode) medium containing 130 mM‐K+ and the internal (bath) medium 6 mM‐K+. 2. With an internal Ca2+ concentration ([Ca2+]i) of 1 microM, the fraction of time during which the channel was in an open state (the open probability, Po) was more than 0.8 at pHi 7.4. The channel activity nearly disappeared at pHi 7.0. The [Ca2+]i‐Po relationship was shifted to higher [Ca2+]i by acidosis, the shift being approximately an 8‐fold increase for a fall in pHi of 0.5 units. 3. The membrane potential and current intensity (V‐I) relationship of single channels between +30 and ‐50 mV was shifted in a hyperpolarizing direction by intracellular acidosis. The shift was roughly 10 mV for 1 pH unit at 1 microM [Ca2+]i. At pHi 7.4 [Ca2+]i 1 microM, the V‐Po relationship was shifted in a depolarizing direction by acidification. When [Ca2+]i was increased to 10 microM, V‐Po relationship became less sensitive to V as well as pHi changes. 4. When Po was high, the probability density function of open and closed time distributions could be fitted by two exponentials. When Po was decreased to less than 0.3, either by reducing [Ca2+]i or by lowering pHi, another component having long closed times appeared. At similar Po values, the time constant of open time distribution was smaller with lower pHi. 5. It is concluded that the main effect of an increase in intracellular hydrogen ions is to decrease the open probability of the Ca2(+)‐activated K+ channel, by reducing the sensitivity to Ca2+ and also shortening the open state.

Effects of Ca removal on the smooth muscle of the guinea-pig taenia coli

1. The effects of removing the external Ca ions on the spontaneous and evoked activity of the smooth muscle of the guinea‐pig taenia coli were investigated with the double sucrose‐gap method.

Effects of calmodulin antagonists on calcium-activated potassium channels in pregnant rat myometrium.

1 The effects of W‐7, trifluoperazine, and W‐5 on Ca2+‐activated K+‐channels were investigated with the inside‐out patch‐clamp method in smooth muscle cells freshly dispersed from pregnant rat myometrium. These drugs are known to have different potencies as calmodulin antagonists. 2 In the presence of 1 μm Ca2+ on the cytoplasmic side ([Ca2+]i), the fraction of time the channel was open (open probability, Po) was about 0.9 and the calmodulin antagonists (1–30 μm) applied to the cytoplasmic face reduced Po to 0.65‐0.55 dose‐dependently. In the presence of 0.1‐0.16 μm Ca2+, when Po was very low (0.02), calmodulin antagonists increased Po. All antagonists used produced almost identical effects at the same concentration. 3 The probability density function of the open time distribution could be described by the sum of two exponentials. W‐7 decreased the time constant of the slow component of distribution and at 30 μm the slow component disappeared both at 1 and 0.25 μm [Ca2+]i, reflecting the appearance of flickering channel activity. The probability density function of the closed time distribution could be fitted with three exponentials. The time constants of these components were not significantly altered by W‐7. 4 Internally applied calmodulin (1–5 μm) did not produce any significant effect on channel activity. 5 The effects of calmodulin antagonists are considered to be due to a direct action of these compounds on the channel, and suggest that channel activation by Ca2+ is not mediated by calmodulin.

The effect of temperature on the membrane conductance of the smooth muscle of the guinea-pig taenia coli.

1. The effect of temperature on the membrane conductance of the smooth muscle of guinea‐pig taenia coli was investigated electrophysiologically, using the double sucrose gap method, and by ion‐flux determinations.

Inhibitory effects of propiverine on rat and guinea-pig urinary bladder muscle

SummaryIn muscle strips isolated from guinea-pig and rat urinary bladder, propiverine (3–10 μM) inhibited carbachol-induced contractions in the presence of verapamil and Ca2+-induced contractions in excess K+ medium containing atropine, suggesting it has both anticholinergic and Ca2+ channel blocking actions.The Ca2+ channel blocking action was also demonstrated by recording inward Ca 2+ currents in single cells dispersed from both species. The inhibition of inward currents by propiverine was three times stronger in the rat than the guinea-pig, ID50 being 7 μM for rat and 21 μM for guinea-pig. The recovery of the current after washout was faster than that of mechanical inhibition. It is concluded that propiverine blocks not only muscarinic receptors, but also Ca2+ channels at similar concentrations.

Effects of intracellular pH on calcium currents and intracellular calcium ions in the smooth muscle of rabbit portal vein

In smooth muscle cells freshly dispersed from the rabbit portal vein, effects of intracellular pH (pHi) on Ca2+ channel currents were studied with the whole‐cell clamp method using nystatin in the pipette. pHi was modified with ammonium chloride (NH4Cl) and propionate. Changes in intracellular Ca2+ concentration ([Ca2+]i) and pHi were also measured with the fluorescent indicator fura‐2 and a pH‐sensitive dye, respectively, together with the mechanical response in intact tissues. Intracellular alkalinization caused by an application of NH4Cl (20 mM) markedly potentiated and acidification caused by propionate (20 mM) inhibited inward Ca2+ channel currents, without much change in the kinetics. Tension development induced by 60 mM K‐ was inhibited by NH4Cl (20 mM) and potentiated by propionate (20 mM), whereas the peak [Ca2+]i level reached during K+ contracture was reduced in the presence of NH4Cl and increased in the presence of propionate. It was concluded that the modification of Ca2+ channel currents caused by pHi is not directly related to the effects of pHi on the mechanical response to excess K+. The direct effects of pHi on [Ca2+]i and on contractile machinery are considered to be mainly responsible for the mechanical effect of pHi.

Regulation of intracellular free magnesium concentration in the taenia of guinea-pig caecum.

1. In the taenia isolated from the guinea‐pig caecum, changes in free intracellular magnesium concentration ([Mg2+]i) were measured based on the separation of the chemical shift of the alpha‐ and beta‐peaks of ATP obtained with nuclear magnetic resonance (NMR). 2. When external Mg2+ was increased from 1.2 to 12 and 40 mM, [Mg2+]i increased from 0.34 +/‐ 0.05 to 0.39 +/‐ 0.07 and 0.61 +/‐ 0.18 mM (n = 3), respectively, in 125‐150 min. In the absence of Ca2+, the increase was greater, i.e. [Mg2+]i reached 0.92 +/‐ 0.05 (n = 3) and 3.37 mM (n = 1), respectively. 3. In Ca(2+)‐ and Mg(2+)‐free solution, [Mg2+]i decreased to about 10 microM in 100 min. This decrease was not affected by substitution of Na+ with K+, but the recovery of [Mg2+]i on Mg2+ readmission was slower in the absence of Na+. This recovery was prevented by Ca2+ (2.4 mM), but Na+ readmission produced full recovery of [Mg2+]i in the presence of Ca2+. 4. When Na+ was substituted with K+ in the absence of Ca2+, [Mg2+]i increased from 0.38 +/‐ 0.01 mM to 0.56 +/‐ 0.04 mM (n = 4) in 100 min. Substitution of Na+ with N‐methyl‐D‐glucamine produced a much greater increase (to 2.1 mM in 100 min). Removal of the external K+ in the absence of Ca2+ also increased [Mg2+]i to 0.66 +/‐ 0.11 mM (n = 4) in 150 min, but no clear change was observed in the presence of Ca2+. Full recovery of [Mg2+]i could be produced in normal solution even when the intracellular phosphocreatine and ATP concentrations were 70‐80% of the control. 5. It is concluded that in the smooth muscle of the guinea‐pig taenia caeci an Na(+)‐Mg2+ exchange process and possibly also an ATP‐driven Mg2+ pump are playing important roles in maintaining free [Mg2+]i at about 0.3 mM under the physiological condition. In addition to this, Ca2+ inhibits both Mg2+ efflux and influx, probably by reducing the membrane permeability to Mg2+ and by competing with Mg2+ in the Na(+)‐Mg2+ exchange of the plasma membrane.

Impedance components in longitudinal direction in the guinea-pig taenia coli.

1. When the tissue impedance of the guinea‐pig taenia coli was measured across a 2 mm sucrose gap in a longitudinal direction, the impedance locus could be fitted by two different circular arcs. Their characteristic frequencies were about 0‐6 and 240 Hz after 60 min superfusion with sucrose solution. From the effects of changing the width of sucrose gap and of transection of tissue, and also from taking the difference between impedances measured at two distances, it was concluded that the low‐frequency locus corresponds to the transverse impedance of the plasma membrane and the high‐frequency locus to the longitudinal tissue impedance. 2. A change in the longitudinal tissue impedance was measured during superfusion with sucrose solution, using a frequency range between 5 Hz and 10 kHz. The admittance decreased with time of superfusion and this time course could be expressed by the sum of three exponential terms. The fastest component, having a time constant of 1‐3 min at 10 kHz, was interpreted to correspond to a process of wash‐out of extracellular medium. 3. Admittances at zero and infinite frequencies were obtained from the impedance locus. The decrease in these admittances with the time was analysed and the values at the start of washing were obtained by extrapolating the admittance change to zero time. 4. From these values it was estimated that the myoplasmic resistance was 214 omega cm, the junctional resistance 372 omega cm, and the junctional capacity 3‐1 muF/cm at 25 degrees C. In these calculations the equivalent circuit of tissue was assumed to be expressed by two components in series: one for the myoplasmic resistance and the other for the junction which has the junctional resistance and capacity in parallel. 5. After 90 min superfusion with sucrose solution, the total tissue impedance measured at zero frequency was increased from 586 to 3034 omega cm. In the total impedance the myoplasmic resistance was increased from 214 to 914 omega cm and the junctional resistance from 372 to 2120 omega cm. Thus, the change in junctional resistance was greater than that in myoplasmic resistance during superfusion of sucrose solution.

Effects of cromakalim on the electrical slow wave in the circular muscle of guinea-pig gastric antrum.

1 In circular muscle strips of the antrum of guinea‐pig stomach, the effects of cromakalim were studied on mechanical activity and intracellular membrane potential. 2 Cromakalim inhibited mechanical activity at concentrations higher than 1 μm, accompanied by membrane hyperpolarization and a decrease in membrane resistance. The hyperpolarization was markedly potentiated in K+‐free solution and was still observed in the absence of Na+. 3 Slow wave electrical activity was relatively resistant to cromakalim. Changes in its amplitude and frequency were not consistent but blockade of slow waves was never observed. In many preparations cromakalim induced spike‐like potentials at the top of slow waves, or when spike‐like potentials already existed they were potentiated. However, mechanical activity was always inhibited. 4 Inhibition by cromakalim of the phasic contractions associated with the slow waves, could not be reversed by increasing the external K+ concentration (12–30 mm). 5 The results suggest that in guinea‐pig stomach muscle mechanical suppression by cromakalim does not simply result from membrane hyperpolarization or from inhibition of slow waves. A clear dissociation was found between the mechanical and electrical activities. Slow waves, particularly their frequency, are relatively insensitive to membrane hyperpolarization.