Hiroyuki Tokuno

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 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.

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.

Effects of glucose removal and readmission on potassium contracture in the guinea‐pig taenia coli.

The effects of removal and readmission of substrates on the K contracture were investigated in the guinea‐pig taenia coli. When, after exposure to excess K in Ca‐free and glucose‐free medium, the readmission and removal of 2.4 mM‐Ca were repeated at regular intervals, the Ca‐induced contractions decreased progressively. The decrease was more marked in the late than in the early part of the tension response. The rate of O2 consumption decreased when the normal medium was replaced by glucose‐free, Ca‐free, excess‐K solution, but substantially recovered following Ca readmission. ATP and creatine phosphate contents decreased during the Ca‐induced contraction, but recovered partially during the subsequent relaxation in Ca‐free solution. The effects of glucose removal were rapidly reversed when glucose or beta‐hydroxybutyrate (beta‐HB) were readmitted to the bathing solution. In the absence of Ca, readmission and removal of the substrates produced an insignificant change in O2 consumption, but the next Ca contraction was potentiated, the effect being stronger with glucose than beta‐HB. When the tonic contraction evoked by 2.4 mM‐Ca readmission had been abolished in glucose‐free, high‐K solution, a rise of the external Ca concentration to 10 mM, or 5 microM‐carbachol, still produced a transient contraction. This suggests that the tonic contraction has disappeared partially because of diminished Ca influx. In glycogen‐depleted preparations, the depolarization caused by carbachol, or by 20 mM‐K, was increased and spike discharge initiated when glucose was readmitted.(ABSTRACT TRUNCATED AT 250 WORDS)

Collagenase eliminates the electrical responses of smooth muscle to catecholamines.

The effects of collagenase on the membrane response to catecholamines were studied with intracellular microelectrodes in the guinea-pig taenia caeci and main pulmonary artery. In the taenia, the inhibitory actions of adrenaline mediated through alpha- and beta-adrenoceptors were both nearly abolished by two 5-min treatments with 0.005% collagenase. In the pulmonary artery, the depolarizing action of noradrenaline mediated through beta-adrenoceptors was markedly reduced by three 5-min treatments with 0.01% collagenase.

Long‐term potentiation of transmitter exocytosis expressed by Ca2+‐induced Ca2+ release from thapsigargin‐sensitive Ca2+ stores in preganglionic nerve terminals

We have studied whether Ca2+‐induced Ca2+ release (CICR) is involved in the mechanism of long‐term potentiation (LTP) at nicotinic synapses of bullfrog sympathetic ganglia. Fast excitatory postsynaptic potentials (fast EPSPs) were recorded in a low‐Ca2+, high‐Mg2+ solution and quantal analysis was applied. The conditioning stimulation of the B‐type preganglionic nerve at 20 Hz for 4 min consistently enhanced the amplitude and quantal content of fast EPSP for > 2 h, but only sometimes enhanced the quantal size. The LTP of quantal content produced by the conditioning tetanus was blocked by thapsigargin, a blocker of Ca2+ pumps at Ca2+ stores, applied before or after the conditioning tetanus, and by Xestospongin C, a blocker of inositoltrisphosphate (IP3) receptors, applied before the tetanus. It was not, however, blocked by ryanodine, a blocker and/or activator of ryanodine receptors, or by propranolol, a blocker of β‐adrenergic receptors. Thus the long‐lasting activity of the preganglionic nerve at a high frequency causes the LTP of impulse‐evoked transmitter release by the activation of CICR from thapsigargin‐sensitive Ca2+ stores in the nerve terminals. It is likely that a large Ca2+ entry into the nerve terminals during tetanic activity primes ryanodine‐insensitive Ca2+ release channels for activation.

Effects of Ca2+ removal and of tetraethylammonium on membrane currents induced by carbachol in isolated cells from the rat parotid gland

In freshly dispersed rat parotid acinar cells, 10 μM carbachol increased outward currents at 0 mV and also inward currents at −70 mV recorded with the whole-cell clamp method using patch pipettes containing 1 mM EGTA. When EGTA in the pipette was increased to 2.4 mM, carbachol increased only outward currents and a further increase of EGTA to 4 mM blocked the carbachol response. Effects of changes in external K+ and Cl− concentrations suggested that outward currents were carried by K+ and inward by Cl−. Effects of Ca2+ removal from the medium differed between experiments with 0 and 5 mM ATP in the patch pipettes. When pipettes contained no ATP, responses evoked by repeated applications of 10 μM carbachol (0.5–1 min) at 1.5–4 min intervals decreased only slowly after Ca2+ removal, outward currents being reduced to 90±6% and inward currents to 47±11% (n=6) in 10 min. On the other hand, when 5 mM ATP was included in the electrodes, Ca2+ removal abolished the carbachol responses in about 5 min (n=4). It was also found that tetraethylammonium (5 mM) strongly reduced both currents, by blocking muscarinic receptors, while Ba2+ (2.4 mM) inhibited only the outward K+ current.

The physiology of the smooth muscle: an interdisciplinary review—part II

In the guinea pig taenia coli, when glycogen is depleted by repeating Ca-induced contracture in excess K solution containing no glucose, the tension cannot be maintained. The decrease in tension is accompanied by reduction of high energy phosphate compounds and oxygen consumption. When substrate is readmitted to the glycogen-depleted preparation in the presence of 2.4 mM Ca and 20 mM K, the first response is hyperpolarization of the membrane and relaxation, and this is followed by depolarization and development of contracture. The latter response is blocked by verapamil, suggesting that energy supply increases the Ca conductance of the plasma membrane. The early response is considered to be due to activation of electrogenic Ca pump, since this is not affected by ouabain as well as removal of Na and K. ATP produced by substrate readmission is probably preferentially utilized for Ca pump activation to reduce the intracellular Ca. The recovery of tension is likely to be brought about by ATP supply not only to the contractile machinery but also to the plasma membrane to remove inactivation of Ca conductance. It is postulated that as the energy source is depleted, energy consumption is automatically limited by suppressing Ca influx, as a self-defence mechanism. Since beta HB is as effective as glucose in the recovery of these processes, and also in the activation of electrogenic Na pump, the metabolic pathway of oxidative phosphorylation alone can support these functions without a contribution of the glycolytic pathway.In the guinea pig taenia coli, when glycogen is depleted by repeating Ca-induced contracture in excess K solution containing no glucose, the tension cannot be maintained. The decrease in tension is accompanied by reduction of high energy phosphate compounds and oxygen consumption. When substrate is readmitted to the glycogendepleted preparation in the presence of 2.4 mM Ca and 20 mM K, the first response is hyperpolarization of the membrane and relaxation, and this is followed by depolarization and development of contracture. The latter response is blocked by verapamil, suggesting that energy supply increases the Ca conductance of the plasma membrane. The early response is considered to be due to activation of electrogenic Ca pump, since this is not affected by ouabain as well as removal of Na and K. ATP produced by substrate readmission is probably preferentially utilized for Ca pump activation to reduce the intracellular. Ca. The recovery of tension is likely to be brought about by ATP supply not only to the contractile machinery but also to the plasma membrane to remove inactivation of Ca conductance. It is postulated that as the energy source is depleted, energy consumption is automatically limited by suppressing Ca influx, as a selfdefence mechanism. Since βHB is as effective as glucose in the recovery of these processes, and also in the activation of electrogenic Na pump, the matabolic pathway of oxidative phosphorylation alone can support these functions without a contribution of the glycolytic pathway.

Effects of ammonium chloride on membrane currents of acinar cells dispersed from the rat parotid gland

In acinar cells freshly dispersed from rat parotid glands, the effects of ammonium chloride (NH4Cl) on membrane currents were studied using the whole-cell clamp method. When membrane currents were recorded with command pulses to 0 mV, applied at 2-s intervals from a holding potential of −70 mV, NH4Cl (5–20 mM) transiently decreased outward currents and then slowly increased both outward and inward currents. After reaching a peak in about 40–50 s, both outward and inward currents gradually decreased in the presence of NH4Cl and, on its wash-out, the currents returned to the control level. Butyrate (5–20 mM) had little effect on the resting membrane currents, but markedly inhibited the response to NH4Cl. Tetraethylammonium (5 mM) strongly reduced both the resting and NH4Cl-induced outward currents, whereas it slightly potentiated the NH4Cl-induced inward current without affecting the membrane current at the holding potential. Without ATP in the patch pipettes, carbachol-induced membrane currents were relatively resistant to Ca2+ removal from the external medium, but NH4Cl-induced currents were quickly abolished in the absence of Ca2+. We conclude that intracellular alkalinization with NH4Cl increases Ca2+ influx and activates Ca2+-dependent outward K+ and inward Cl− currents.