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Acetylcholine induced an outwardly rectifying current beside Ca2+-activated-Cl− current in swine tracheal smooth muscle cells
WS3-J-2-06
IONIC CURRENTS REGULATING MEMBRANE EXCITABILITY OF TRACHEAL SMOOTH MUSCLE CELLS. Y. Imaizumi, S. Henmi, K. Muraki, M. Watanabe Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan The smooth muscle of canine and bovine trachealis has stable resting membrane potential and low excitability. Neither spontaneous action potentials nor evoked active potential changes have been observed under normal conditions. It has been found, however, that application of K+ channel blockers such as tetraethylammonium (TEA) or 4-aminopyddine (4AP) markedly Increases the membrane excitability. Stimulation of the muscle by a transmitter or an autacoid often causes a small depolarization and a large contraction, suggesting that pharmaco-mechanical coupling via the formation of IP3 and subsequent Ca 2+ release from intracellular storage sites may significantly contribute to the contraction. In guinea-pig trachea, the smooth muscle cells often exhibit spontaneous or evoked electrical slow waves. Analyses of several types of membrane ionic currents, which have been recorded under whole-cell clamp from single.smooth muscle cells dispersed from trachealis of several species by treatment with collagenase, greatly helped our understanding about the ionic current mechanisms regulating membrane excitability of the cells. When outward K currents were blocked by the internal perfusion of Cs+, a substantial inward current through voltagedependent L-type Ca2+ channels (Ica) was observed upon depolarization in canine tracheal smooth muscle cells. The peak amplitude of ICa per unit cell membrane area was comparable to that in other smooth muscle cells which have higher membrane excitability and shows spontaneous action potentials. This may suggest that the density of Ca2+ channels is not too low to elicit action potentials. The outward current upon depeladzaUon consisted of two major K+ currents; Ca2+dependent K + current (IK.Ca) and delayed rectifier type K + current 0K-D) which ware blocked by relatively low concentrations of TEA and 4-AP, respectively. Application of a higher concentration of TEA (>10 mM) suppressed IK. D, either. Application of 1 mM TEA and 4-AP almost abolished the outward currents upon depolarization. Correspondingly, a large outward rectification in response to depolarizing currents injected through a recording pipette under the current clamp was markedly reduced and action potentials were elicited in the presence of these blockers. It can be concluded that the low membrane excitability of canine, bovine and feline tracheal smooth muscle cells may be due to the activation of these large K+ currents upon depolarization which prevents further membrane depolarization and significant activation of Ca2+ channels under physiological conditions. IK.Ca upon depolarization under the voltage-clamp was not simply activated by enterS_ Ca2+ through voltage dependent Ca channels but is gre_aUy amplified by Ca z+ release from storage sites via the CaZ+-induced Caz+ release mechanisms. Moreover, other CaZ+-activated currents such as chloride and non-specific cationic currents were also elicited by the increase in [Ca2+] i_n tracheal smooth muscle cells. Activation of these currents was strongly affected byboth the ammount of releasable Ca z+ and the ability of Ca ;'+ releasing channels in the storage sites, as we l as by Ca2+-influx. Therefore, Ca2+-uptake and -release activities of intracellular storage sites regulate these membrane currents and, indirectly but significantly, affect the excitability of tracheal smooth muscle cells, as has been shown in other smooth muscle cells.
WS3-J-2-07 ACETYLCHOLINE INDUCED AN OUTWARDLY RECTIFYING CURRENT BESIDE Ca2+-ACTIVATED-CI- CURRENT IN SWINE TRACHEAL SMOOTH MUSCLE CFAA_,S T. Yamashita, S. Kokubun Dept. of Physiol. Nihon Univ. School of Medicine, Tokyo, Japan Membrane currents were sntdiedin enzymatically isolated swine and canine tracheal smooth muscle cells (TSMCs) using the whole-cell voltage-clamp technique. In order to suppress nonspecific cationic current andK+ currents, we used TEA+ and Cs + as major monovalent cations in exU'acellularandpipette solutions, respectively With a pipette containing 0.1 mM EGTA, acetylcholine (ACh, 10 uM) and histamine (100 /~M) elicited transient current in canine TSMCs. This current was suggestedto be Ca2+-activated-C1- current 0CI_Ca, Sims-SM, J. Physiol., 1992) by following results: 1) Its reversal potential mensuredwith ramp pulses (-70 to +70 mV, 500 mS) was identical to the equilibrium potential of C1-. 2) In the presence of heparine (5 rag~rut) in the pipette, ACh failed to evoke the current, whereas caffeine(10 raM) elicited it. In swine TSMCs, histamine (100/~M) and caffeine(10 raM) evokedlcl_C a as well as in eanineTSMCs. ACh (10/zM), however, activatedoutwardlyrectifyingcurrent(IoR.ACh) in additionto ICI_Ca. IOR_AChwas activatedeadierandsustainedforlonger time thanlcl_Ca. Fixation of intracellularconcentration of Ca2+ ([Ca2+~) at 50 nM with BAP~-Ca 2+ bufferin the pipette suppressedIca.C a andisolated IOR.ACh. Underthis condition, ACh aetivatedthe currentin a dose-dependentmanner(10/z-1 raM), Voltage steps (500mS) to various potentials revealed that IOR_ACh was activated time-dependently at more positive potentials than +20 mV. Fixation of [Ca2+~ at 0 nM completely abolishedIoR_ACh. ACh failedto activate IOR.ACh with the pipette containing GDP-~S (3 mM) whereas it activatedthe current irreversibly in the presence of GTP-y -S (100pM). These results indicate that the activation meehanism of IOR.ACh involves G.protein(s)and that it requiresat least the resting level of [Ca2+]i.
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IONIC CURRENTS REGULATING MEMBRANE EXCITABILITY OF TRACHEAL SMOOTH MUSCLE CELLS. Y. Imaizumi, S. Henmi, K. Muraki, M. Watanabe Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan The smooth muscle of canine and bovine trachealis has stable resting membrane potential and low excitability. Neither spontaneous action potentials nor evoked active potential changes have been observed under normal conditions. It has been found, however, that application of K+ channel blockers such as tetraethylammonium (TEA) or 4-aminopyddine (4AP) markedly Increases the membrane excitability. Stimulation of the muscle by a transmitter or an autacoid often causes a small depolarization and a large contraction, suggesting that pharmaco-mechanical coupling via the formation of IP3 and subsequent Ca 2+ release from intracellular storage sites may significantly contribute to the contraction. In guinea-pig trachea, the smooth muscle cells often exhibit spontaneous or evoked electrical slow waves. Analyses of several types of membrane ionic currents, which have been recorded under whole-cell clamp from single.smooth muscle cells dispersed from trachealis of several species by treatment with collagenase, greatly helped our understanding about the ionic current mechanisms regulating membrane excitability of the cells. When outward K currents were blocked by the internal perfusion of Cs+, a substantial inward current through voltagedependent L-type Ca2+ channels (Ica) was observed upon depolarization in canine tracheal smooth muscle cells. The peak amplitude of ICa per unit cell membrane area was comparable to that in other smooth muscle cells which have higher membrane excitability and shows spontaneous action potentials. This may suggest that the density of Ca2+ channels is not too low to elicit action potentials. The outward current upon depeladzaUon consisted of two major K+ currents; Ca2+dependent K + current (IK.Ca) and delayed rectifier type K + current 0K-D) which ware blocked by relatively low concentrations of TEA and 4-AP, respectively. Application of a higher concentration of TEA (>10 mM) suppressed IK. D, either. Application of 1 mM TEA and 4-AP almost abolished the outward currents upon depolarization. Correspondingly, a large outward rectification in response to depolarizing currents injected through a recording pipette under the current clamp was markedly reduced and action potentials were elicited in the presence of these blockers. It can be concluded that the low membrane excitability of canine, bovine and feline tracheal smooth muscle cells may be due to the activation of these large K+ currents upon depolarization which prevents further membrane depolarization and significant activation of Ca2+ channels under physiological conditions. IK.Ca upon depolarization under the voltage-clamp was not simply activated by enterS_ Ca2+ through voltage dependent Ca channels but is gre_aUy amplified by Ca z+ release from storage sites via the CaZ+-induced Caz+ release mechanisms. Moreover, other CaZ+-activated currents such as chloride and non-specific cationic currents were also elicited by the increase in [Ca2+] i_n tracheal smooth muscle cells. Activation of these currents was strongly affected byboth the ammount of releasable Ca z+ and the ability of Ca ;'+ releasing channels in the storage sites, as we l as by Ca2+-influx. Therefore, Ca2+-uptake and -release activities of intracellular storage sites regulate these membrane currents and, indirectly but significantly, affect the excitability of tracheal smooth muscle cells, as has been shown in other smooth muscle cells.
WS3-J-2-07 ACETYLCHOLINE INDUCED AN OUTWARDLY RECTIFYING CURRENT BESIDE Ca2+-ACTIVATED-CI- CURRENT IN SWINE TRACHEAL SMOOTH MUSCLE CFAA_,S T. Yamashita, S. Kokubun Dept. of Physiol. Nihon Univ. School of Medicine, Tokyo, Japan Membrane currents were sntdiedin enzymatically isolated swine and canine tracheal smooth muscle cells (TSMCs) using the whole-cell voltage-clamp technique. In order to suppress nonspecific cationic current andK+ currents, we used TEA+ and Cs + as major monovalent cations in exU'acellularandpipette solutions, respectively With a pipette containing 0.1 mM EGTA, acetylcholine (ACh, 10 uM) and histamine (100 /~M) elicited transient current in canine TSMCs. This current was suggestedto be Ca2+-activated-C1- current 0CI_Ca, Sims-SM, J. Physiol., 1992) by following results: 1) Its reversal potential mensuredwith ramp pulses (-70 to +70 mV, 500 mS) was identical to the equilibrium potential of C1-. 2) In the presence of heparine (5 rag~rut) in the pipette, ACh failed to evoke the current, whereas caffeine(10 raM) elicited it. In swine TSMCs, histamine (100/~M) and caffeine(10 raM) evokedlcl_C a as well as in eanineTSMCs. ACh (10/zM), however, activatedoutwardlyrectifyingcurrent(IoR.ACh) in additionto ICI_Ca. IOR_AChwas activatedeadierandsustainedforlonger time thanlcl_Ca. Fixation of intracellularconcentration of Ca2+ ([Ca2+~) at 50 nM with BAP~-Ca 2+ bufferin the pipette suppressedIca.C a andisolated IOR.ACh. Underthis condition, ACh aetivatedthe currentin a dose-dependentmanner(10/z-1 raM), Voltage steps (500mS) to various potentials revealed that IOR_ACh was activated time-dependently at more positive potentials than +20 mV. Fixation of [Ca2+~ at 0 nM completely abolishedIoR_ACh. ACh failedto activate IOR.ACh with the pipette containing GDP-~S (3 mM) whereas it activatedthe current irreversibly in the presence of GTP-y -S (100pM). These results indicate that the activation meehanism of IOR.ACh involves G.protein(s)and that it requiresat least the resting level of [Ca2+]i.
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