Double Sucrose-gap Method Research Articles

Physiological Features of Visceral Smooth Muscle Cells, With Special Reference to Receptors and Ion Channels

Visceral smooth muscle cells (VSMC) play an essential role, through changes in their contraction-relaxation cycle, in the maintenance of homeostasis in biological systems. The features of these cells differ markedly by tissue and by species; moreover, there are often regional differences within a given tissue. The biophysical features used to investigate ion channels in VSMC have progressed from the original extracellular recording methods (large electrode, single or double sucrose gap methods), to the intracellular (microelectrode) recording method, and then to methods for recording from membrane fractions (patch-clamp, including cell-attached patch-clamp, methods). Remarkable advances are now being made thanks to the application of these more modern biophysical procedures and to the development of techniques in molecular biology. Even so, we still have much to learn about the physiological features of these channels and about their contribution to the activity of both cell and tissue. In this review, we take a detailed look at ion channels in VSMC and at receptor-operated ion channels in particular; we look at their interaction with the contraction-relaxation cycle in individual VSMC and especially at the way in which their activity is related to Ca2+ movements and Ca2+ homeostasis in the cell. In sections II and III, we discuss research findings mainly derived from the use of the microelectrode, although we also introduce work done using the patch-clamp procedure. These sections cover work on the electrical activity of VSMC membranes (sect. II) and on neuromuscular transmission (sect. III). In sections IV and V, we discuss work done, using the patch-clamp procedure, on individual ion channels (Na+, Ca2+, K+, and Cl-; sect. IV) and on various types of receptor-operated ion channels (with or without coupled GTP-binding proteins and voltage dependent and independent; sect. V). In sect. VI, we look at work done on the role of Ca2+ in VSMC using the patch-clamp procedure, biochemical procedures, measurements of Ca2+ transients, and Ca2+ sensitivity of contractile proteins of VSMC. We discuss the way in which Ca2+ mobilization occurs after membrane activation (Ca2+ influx and efflux through the surface membrane, Ca2+ release from and uptake into the sarcoplasmic reticulum, and dynamic changes in Ca2+ within the cytosol). In this article, we make only limited reference to vascular smooth muscle research, since we reviewed the features of ion channels in vascular tissues only recently.

Mechanisms of ionic currents involved in suppressive effect of isoprenaline on the action potential of guinea-pig vas deferens in normal Krebs solution.

The effects of beta-adrenoceptor stimulation by isoprenaline (ISO) on action potential and membrane current were studied in the guinea-pig vas deferens in normal Krebs solution by using a double sucrose gap method. In current-clamp experiments, ISO produced the membrane hyperpolarization by reducing the resistance and modified the pattern of multi-spike activity elicited by long depolarizing currents; an initial spike potential was reduced in the amplitude and the rate of rising phases and the slope of diastolic depolarization was slowered, leading to a prolongation of the spike discharge interval. In voltage-clamp experiments, ISO greatly enhanced the late outward K+ current (Ik2) by increasing the conductance, but hardly affected the peak outward K+ current (Ik1). ISO also reduced the inward Ca2+ current (Ica) by decreasing the conductance (ga) as well as by reducing the driving force for Ca2+. While it increased the leakage conductance (g1) due to K+ associated with hyperpolarizing voltage steps. In the preparations treated with Ca2+ channel blocker such as D-600, the enhanced effect of ISO on the Ik was still prominent, suggesting that Ca2+ and K+ channels appear to be independently regulated during beta-adrenoceptor stimulation. These results suggest that ISO exerts depressant actions on the vas deferens muscle by reduction of the Ica as well as by prominent enhancement of the Ik2 via beta-adrenoceptor activation.

Enhancement by dibutyryl cyclic AMP of voltage-dependent Ca2+ and K+ currents in the guinea-pig vas deferens.

This study was designed to investigate a possible role for intracellular cyclic AMP involved in agonist-induced changes in electrical activity of smooth muscle of the guinea-pig vas deferens. The action of dibutyryl adenosine 3', 5'-phosphate (dibutyryl cyclic AMP) (up to 30 microM) was examined in current- and voltage-clamp, using the double sucrose gap method. Under current-clamp, dibutyryl cyclic AMP clearly shortens the duration of action potential by hastening the rates of depolarization and of repolarization and increases the peak amplitude. Under voltage-clamp, dibutyryl cyclic AMP enhances the maximum ICa by increasing the conductance (ga), but without affecting its reversal potential (Ea) and kinetics in preparations in normal Krebs solution as well as in preparations in tetraethylammonium chloride loading solution. In normal Krebs solution, dibutyryl cyclic AMP also enhances the peak (Ib') and late outward K+ currents (Ib) by increasing the conductances (gb') and (gb), respectively. These results indicate that in vas deferens smooth muscle intracellular cyclic AMP may be of functional significance for activation of voltage-dependent peak and late IK channels as well as activation of voltage-dependent ICa channel.

<b>BIPHASIC ACTIONS OF PINACIDIL ON EXCITATORY NEURO-EFFECTOR TRANSMISSION IN DOG </b><b>TRACHEA </b>

Effects of pinacidil were investigated on the resting membrane potential and excitatory neuro-effector transmission in the smooth muscle tissue of dog trachea using tension recording, microelectrode and double sucrose gap methods. Pinacidil (>5×10 -7 M) increased the ionic conductance and hyperpolarized smooth muscle cell membranes in dog trachea, in a dose-dependent manner. This pinacidil-induced hyperpolarization was antagonized by glibenclamide (2×10 -6 M)

Effects of PAF on excitatory neuro-effector transmission in dog airways.

1. Effects of PAF on excitatory neuro-effector transmission in smooth muscle cells of mucosa-free trachea and epithelium-intact bronchiole of the dog were investigated, by isometric tension recording, microelectrode and double sucrose-gap methods. 2. PAF (10(-11)-10(-7) M) dose-dependently enhanced the amplitude of contraction evoked by repetitive field stimulations (10 stimuli at 20 Hz) in both tracheal and bronchiolar tissues. At higher concentrations PAF (> 10(-8) M) increased the amplitude of contraction to a greater extent in the bronchiole than in the trachea. 3. In both muscle tissues, in parallel to the amplitude of contraction, PAF markedly enhanced the amplitude of excitatory junction potentials (e.j.ps) evoked by a single field stimulation in a dose-dependent manner, with no change in the resting membrane potential or input membrane resistance of the smooth muscle cells. PAF (5 x 10(-7) M) enhanced the amplitude of e.j.p. to a greater extent in the bronchiole than in the trachealis. In contrast, lyso-PAF (10(-10)-10(-7) M) showed no effect on e.j.p. amplitude in bronchiolar tissues. At a high concentration (10(-7) M) lyso-PAF slightly enhanced the e.j.p. amplitude in tracheal tissue, however the lyso-PAF induced stimulation of e.j.p. amplitude in the trachea was small compared to that of PAF. 4. PAF (10(-7) M) had no effect on the membrane depolarization induced by acetylcholine (ACh, 10(-9)-10(-5) M) and carbachol (10(-9)-10(-5) M) in tracheal smooth muscle cells. 5. The PAF-antagonists CV3988 (5 x i0-7 M) or WEB2086 (5 x 10-7 M) significantly enhanced the e.j.p. amplitude themselves, PAF (5 x 10-8 M) further enhanced the ej.p. amplitude in the presence of WEB2086 (5 x l0-7 M) but not CV3988 (5 x 10-7 M). In contrast, the new PAF-antagonist, E 6123(5 x l0-8 M), did not affect the ej.p. amplitude itself, and completely inhibited the increase in ej.p. amplitude caused by 5 x 10-8 M PAF. On the other hand, in the presence of the Hi-antagonist,mepyramine, PAF (5 X 10-8 M) further enhanced the ej.p. amplitude.6. The leukotriene synthesis inhibitor AA-861 (10-6 M) or leukotriene antagonist ONO1078 (10-7 M)inhibited the increase in ej.p. amplitude caused by 5 X 10-8 M PAF, respectively.7. In the presence of AA-861 (10-6 M), leukotriene B4 (LTB4, 10-' M) or LTD4 (10-8 M) slightly, and LTC4 (10- M) markedly enhanced the ej.p. amplitude. In contrast, LTE4 (10-8 M) significantly suppressed the e.j.p. amplitude.8. PAF (5 x 10-8 M) attenuated the depression phenomena of ej.ps observed during double stimulus experiments at different time intervals (5-10 s), but had no effect on the summation of ej.ps during repetitive field stimulation at a high frequency (20 Hz) in the trachealis.9. These results indicate that PAF potentiates excitatory neuro-effector transmission mainly through stimulating the release of lipoxygenase products, mainly LTC4 in the dog airway smooth muscle tissues.

Effects of vasoactive intestinal polypeptide antagonists on cholinergic neurotransmission in dog and cat trachea.

1. The effects of vasoactive intestinal polypeptide (VIP) antagonists [AC-Tyr1, D-Phe2]-GRF(1-29)-NH2 and [4-Cl-D-Phe6, Leu17]-VIP on excitatory neuroeffector transmission in the dog and cat trachea were investigated by use of microelectrode, double sucrose-gap and tension recording methods. 2. In the dog trachea, repetitive stimuli at high frequency (20 Hz) markedly enhanced the amplitude of contraction, the amplitude of contractions evoked by 50 stimuli at 20 Hz relative to that evoked by 5 stimuli being 14.2 +/- 3.8 times (n = 7, +/- s.d.). In the cat, the summation was much less marked, the amplitude of contractions evoked by 50 stimuli relative to that evoked by 5 stimuli being only 2.1 +/- 0.6 times (n = 5, +/- s.d.). Neither VIP antagonist had any effect on the relationship between the number of stimuli at 20 Hz and the relative amplitude of contraction in the dog trachea, but did enhance the amplitude of contractions to 1.1-1.5 times control in the cat trachea. 3. VIP antagonists dose-dependently enhanced the amplitude of excitatory junction potentials (e.j.ps) evoked by a single stimulus in the cat trachea, without changing the resting membrane potential or input membrane resistance of the smooth muscle cells. However, neither antagonist had any effect on the amplitude of the e.j.p. in the dog trachea. 4. Neither VIP antagonist had any effect on the post-junctional response of smooth muscle cells to exogenously applied acetylcholine (ACh; 10(-9)-10(-5) M) in the dog or cat trachea.5. In the cat trachea, VIP (10-11 M) suppressed the ej.p. amplitude to 0.74 +/- 0.09 times the control value (n = 6). However, after pretreatment of the tissue with the VIP antagonists [Ac-Tyr', D-Phe2]-GRF(1-29)- H2 (10-8M) and [4-Cl-D-Phe6, Leu17]-VIP (10-8M), VIP (10-11 M) did not suppress the ej.p. amplitude, indicating that VIP antagonists block the presynaptic inhibitory action of exogenous VIP.6. In parallel with the enhancement of contraction, ej.ps showed marked summation when repetitive field stimulations were applied at high frequency (20 Hz) in the dog trachea. The relationship between the relative amplitude of the ej.p. and number of stimuli at 20 Hz was linear and the slope was 2.2 +/- 0.3 mV/stimulation. VIP antagonists did not affect this relationship. However, in the cat trachea, summation of ej.ps was not at all marked and a linear relationship was not observed with the double sucrose-gap method. Incubation of the cat tracheal tissue with either of the VIP antagonists (10-8 or 10-7M) markedly enhanced the summation of ej.ps evoked by repetitive field stimulation at 20 Hz, and after the treatment a linear relationship between the number of stimuli and the amplitude of ej.ps was observed, the slopes being 0.6 +/- 0.1 (n = 8) and 0.55 +/- 0.1 mV/stimulation (n = 5), respectively.7. These results indicate that both VIP antagonists, [Ac-Tyr', D-Phe2]-GRF(1-29)-NH2 and [4-Cl-DPhe6, Leu17]-VIP, have a prejunctional action accelerating the excitatory neuroeffector transmission, presumably by enhancing transmitter release from the vagus nerves in the cat, but not in the dog trachea.

Concentration-related effects of extracellular application of ATP on the action potential and membrane currents of the guinea-pig vas deferens

The ionic mechanisms underlying concentration-related alterations in the action potential configuration caused by ATP were studied using preparations of the guinea-pig vas defercns voltage-clamped by a double sucrose gap method. Under current-clamp conditions, ATP at concentration of 1.6 μM enhanced the rates of rise and of repolarisation of the action potential whereas at concentration of 1.6 mM it reduced both rates. Under voltage-clamp conditions, lower concentrations increased the maximum inward Ca current without altering kinetics or reversal potential. Higher concentrations reduced the maximum inward Ca current with slowing of rates of activations and inactivation, but also caused a negative shift in reversal potential without affecting conductance. These results suggest that a low ATP concentration activates the voltage-dependent Ca current channels and that the action of a high ATP concentration is related to the internal Ca ion concentration.

Effects of azelastine on vagal neuroeffector transmission in canine and human airway smooth muscle

Azelastine is a newly developed antiallergic drug that is reported to antagonize histamine and leukotrienes in addition to its inhibitory action on release of chemical mediators. In the present study, the effects of azelastine on neuroeffector transmission in the airway smooth muscles with double sucrose gap and isometric tension-recording methods were evaluated. Azelastine (10(-8) to 10(-6) mol/L) markedly decreased the contractile response of human bronchial and dog tracheal muscle strips to electrical field stimulation (10 pulses at 20 V, 20 Hz, 800 microseconds) in a dose-dependent manner. In parallel with actions on twitch contractions, azelastine suppressed the amplitude of excitatory junction potentials of dog trachea without changing the resting membrane potential and input resistance of smooth muscle cells. However, azelastine did not alter acetylcholine sensitivity of the smooth muscle cells. These results indicate that azelastine possesses an inhibitory action on the release of acetylcholine by vagal nerve terminals. This inhibitory effect of azelastine may contribute to the treatment of asthma in addition to its antiallergic actions.

Quantitative structure-activity studies of pyrethroids: 16. Physicochemical and structural effects of tetramethrin and related compounds on the decay of sodium tail currents in crayfish giant axons

Effects of tetramethrin and related compounds that have a lactam or an imide structure in the alcohol moiety on sodium currents in internally perfused giant axons of crayfish were examined. The sodium currents were measured with a step depolarizing pulse under voltage clamp conditions using the double sucrose gap method. The compounds decreased the rate of fall of the tail current that was induced upon the step repolarization from the depolarizing phase of the axonal membrane. Variations in the activity to decrease the rate of fall were quantitatively analyzed with reference to the physicochemical parameters of the compounds. The activity of compounds with the lactam structure in the alcohol moiety was lower than that with the imide structure, other things being equivalent. Representing and separating this structural factor with an indicator variable, variations in the activity of the compounds were parabolically related to the steric bulkiness of their alcohol moiety. The whole-body symptomatic activities of the compounds against American cockroaches and their extracellularly measured neurophysiological activities on the cockroach nerve cords were correlated to the activity to decrease the rate of fall of the tail current when transport factors of compounds were considered using the hydrophobicity parameter.

Effect of noradrenaline on current- and voltage-clamped muscle cells of the guinea-pig vas deferens in normal Krebs solution

The ionic mechanism underlying the time-dependent alteration in the action potential configuration by noradrenaline (NA) was studied using vas deferens muscles voltage-clamped according to the double sucrose gap method. Early exposure to NA enhanced the inward Ca 2+ current (I Ca) by increasing conductance whereas late exposure diminished I Ca by reducing the driving force. The degree of increase in the peak outward K + current (I K′) was higher with a greater enhancement of I Ca by NA but the late outward K + current (I k) was insensitive to NA.

Paradoxical reversion of the inhibitory effects of dihydropyridine enantiomers on the calcium current in frog heart by CGP 28861

A dihydropyridine CGP 28861 (5 x 10(-6) M) did not change slow inward Ca current as measured by the double sucrose gap method in frog atrial fibres but decreased the agonist effects of Bay K 8644, CGP 28392, (+)-(S)-202-791 and the antagonist effects of nifedipine and (+)-(PN)-200-110. Paradoxically, the weak antagonists (-)-(R)-202-791, (-)-(PN)-200-110 and (+)-Bay K 8644 increased Ca-current after washout of CGP 28861. These data suggest that CGP 28861 can convert the dihydropyridine Ca-channel receptor from an antagonistic site into an agonistic one.

Evidence for adenosine triphosphate as an excitatory transmitter in guinea-pig, rabbit and pig urinary bladder.

1. The effects of alpha,beta-methylene ATP (alpha,beta-MeATP) on membrane properties and excitatory junction potentials (EJPs) were examined in smooth muscle cells of the guinea-pig, rabbit and pig bladder. 2. Intracellular recording with microelectrodes was used to record membrane electrical activity from the guinea-pig bladder. ATP (10(-3) M) produced a rapid, large depolarization with a marked increase in spike frequency, while carbachol (10(-4) M) or acetylcholine (ACh; 10(-4) M) produced only a small or no depolarization with a smaller increase in spike frequency. alpha,beta-MeATP produced a similar response to that of ATP but at a much lower concentration (5 x 10(-6) M), and the response was transient even in the continuous presence of this agent. 3. Changes in the membrane potential and conductance elicited by alpha,beta-MeATP were also measured with the double sucrose-gap method. alpha,beta-MeATP (5 x 10(-6) M) depolarized the membrane and increased the membrane conductance in all three species, but both parameters returned to control values during continuous exposure to this agent. 4. Intracellular recording with microelectrodes showed that in the guinea-pig bladder treatment with alpha,beta-MeATP abolished the response to ATP, while the response to ACh was unchanged. 5. With the double sucrose-gap method, EJPs were elicited by transmural nerve stimulation of strips of the guinea-pig, rabbit and pig bladder and had spikes superimposed, leading to contractions. Desensitization of P2-purinoceptors by alpha,beta-MeATP (3-5 x 10(-6) M) abolished the EJPs and spikes, and reduced the contraction. Atropine (10(-6) M) alone did not alter the EJPs but reduced the contraction. Combined application of both agents abolished the contraction. 6. It is concluded that in the guinea-pig, rabbit and pig bladder ATP is an excitatory transmitter with ACh and EJPs are mediated by ATP.

Pre- and post-junctional actions of procaterol, a beta 2-adrenoceptor stimulant, on dog tracheal tissue.

1. The effects of procaterol, a beta 2-adrenoceptor agonist, on excitatory neuro-effector transmission in the dog trachea were investigated and the findings were compared to those seen with isoprenaline, with microelectrode, double sucrose gap and tension recording methods. 2. Procaterol (10(-10)-10(-9) M) and isoprenaline (10(-9) M) had no effect on the resting membrane potential or on the input resistance of the smooth muscle cells of dog trachea. However with increased concentrations (greater than 10(-8) M), these agents hyperpolarized the membrane and decreased the input resistance of the membrane. 3. Procaterol (10(-10)-10(-7) M) and isoprenaline (10(-9)-10(-7) M) dose-dependently reduced the amplitude of the twitch contractions evoked by field stimulation in the combined presence of indomethacin (10(-5) M) and guanethidine (10(-6) M). In parallel with actions on twitch contractions, procaterol (10(-10)-10(-7) M) and isoprenaline (10(-9)-10(-7) M) reduced the amplitude of the excitatory junction potentials (e.j.ps), evoked by single pulse field stimulation in the dog trachea. 4. Procaterol (10(-8) M) had no effect on the post-junctional response of smooth muscle cells to exogenous acetylcholine (ACh) (10(-7)-10(-6) M). 5. Pretreatment with ICI-118551, a beta 2-adrenoceptor blocking agent, reduced the inhibitory action of procaterol on the amplitude of twitch contractions evoked by field stimulations in the dog trachea. 6. These results indicate that procaterol in low concentrations has a prejunctional action inhibiting the excitatory neuro-effector transmission in addition to a postsynaptic action, presumably by suppressing transmitter release from the vagus nerve terminals through beta 2-adrenoceptors in the dog tracheal tissue. The pre- and post-junctional actions of procaterol explain its potent bronchodilator effects in clinical use.

Autoregulation of acetylcholine release from vagus nerve terminals through activation of muscarinic receptors in the dog trachea.

1. The effects of pirenzepine and gallamine on the membrane and contractile properties of smooth muscle cells and on excitatory neuro-effector transmission in the dog trachea were investigated by means of microelectrode, double sucrose gap and tension recording methods. 2. Pirenzepine (10(-7) M) and gallamine (10(-5) M) had no effect on the resting membrane potential or the input resistance of the smooth muscle cells. 3. Pirenzepine (10(-10)-10(-9) M) and gallamine (10(-7) M) enhanced the amplitude of twitch contractions evoked by field stimulation in the combined presence of indomethacin (10(-5) M) and propranolol (10(-6) M). At higher concentrations pirenzepine (10(-8) M) inhibited the twitch contractions in a dose-dependent manner. Both pirenzepine and gallamine in doses over 10(-7) and 10(-5) M, respectively, reduced muscle tone. 4. Pirenzepine (10(-10)-10(-9) M) and gallamine (10(-7) M) enhanced the amplitude of excitatory junction potentials (e.j.ps) evoked by field stimulation (single or repetitive stimulation). However, a high concentration of pirenzepine (10(-8) M) reduced the amplitude of e.j.ps. In parallel with its action on e.j.ps, pirenzepine (over 10(-9) M) reduced the response of smooth muscle cells to acetylcholine (ACh), in a dose-dependent manner. Gallamine (5 X 10(-5) M) markedly enhanced the amplitude of e.j.ps but also reduced the response of muscle cells to ACh. 5. ACh (10(-10)-10(-9) M) inhibited twitch contractions evoked by field stimulation, with a slight increase of resting tension. 6. Gallamine enhanced the summation of e.j.ps during repetitive field stimulation at a high frequency (20 Hz), but was without effect on the depression phenomena of e.j.ps observed during double stimulus experiments at different time intervals (5-60 s). 7. These results indicate that both pirenzepine and gallamine have dual actions on pre- and post-junctional muscarinic receptors in dog tracheal tissue. At low concentrations both agents potentiate excitatory neuro-effector transmission, presumably due to enhancement of release of ACh from vagal nerve terminals through blockade of a negative auto-regulatory process activated by endogenous ACh. At higher concentrations, these agents inhibit the response of smooth muscle cells to ACh through post-junctional muscarinic receptors and relaxation of the muscle tissue occurs.

O-211 - Autoregulation of acetylcholine release from vagus nerve terminals through activation of muscarinic receptors in the dog trachea

1. The effects of pirenzepine and gallamine on the membrane and contractile properties of smooth muscle cells and on excitatory neuro-effector transmission in the dog trachea were investigated by means of microelectrode, double sucrose gap and tension recording methods. 2. Pirenzepine (10(-7) M) and gallamine (10(-5) M) had no effect on the resting membrane potential or the input resistance of the smooth muscle cells. 3. Pirenzepine (10(-10)-10(-9) M) and gallamine (10(-7) M) enhanced the amplitude of twitch contractions evoked by field stimulation in the combined presence of indomethacin (10(-5) M) and propranolol (10(-6) M). At higher concentrations pirenzepine (10(-8) M) inhibited the twitch contractions in a dose-dependent manner. Both pirenzepine and gallamine in doses over 10(-7) and 10(-5) M, respectively, reduced muscle tone. 4. Pirenzepine (10(-10)-10(-9) M) and gallamine (10(-7) M) enhanced the amplitude of excitatory junction potentials (e.j.ps) evoked by field stimulation (single or repetitive stimulation). However, a high concentration of pirenzepine (10(-8) M) reduced the amplitude of e.j.ps. In parallel with its action on e.j.ps, pirenzepine (over 10(-9) M) reduced the response of smooth muscle cells to acetylcholine (ACh), in a dose-dependent manner. Gallamine (5 X 10(-5) M) markedly enhanced the amplitude of e.j.ps but also reduced the response of muscle cells to ACh. 5. ACh (10(-10)-10(-9) M) inhibited twitch contractions evoked by field stimulation, with a slight increase of resting tension. 6. Gallamine enhanced the summation of e.j.ps during repetitive field stimulation at a high frequency (20 Hz), but was without effect on the depression phenomena of e.j.ps observed during double stimulus experiments at different time intervals (5-60 s). 7. These results indicate that both pirenzepine and gallamine have dual actions on pre- and post-junctional muscarinic receptors in dog tracheal tissue. At low concentrations both agents potentiate excitatory neuro-effector transmission, presumably due to enhancement of release of ACh from vagal nerve terminals through blockade of a negative auto-regulatory process activated by endogenous ACh. At higher concentrations, these agents inhibit the response of smooth muscle cells to ACh through post-junctional muscarinic receptors and relaxation of the muscle tissue occurs.

The action of procainamide and quinidine on the α1-receptor-operated channels in smooth muscle cells of guinea-pig taenia caeci

The effect of procainamide (2.0–5.0 mM) and quinidine (0.2-1.0 mM) on the α 1 response evoked by adrenaline (3 × 10 −6 M) in smooth muscle cells of guinea-pig taenia caeci (22° C) was studied in the presence of yohimbine (3 × 10 −6 M), propranolol (3 × 10 −6 M) or atropine (10 −6 M). The electrotonic potential elicited by the application of a constant current to the preparation was slightly increased (about 10%) by procainamide (5.0 mM) but not by quinidine (1.0 mM). The double-sucrose gap method was used for measurements. The α 1 response evoked by adrenaline in the absence of extracellular calcium (15 min) was represented by a transient hyperpolarization of the muscle cells, while the hyperpolarization elicited in the presence of calcium was sustained. The hyperpolarization is caused by enhancement of the potassium efflux assumed to be linked with mobilization of calcium form a cellular structure. Superfusion of the preparation with calcium-containing solution to replenish the calcium store in the presence of procainamide (10 min) before the α 1 response evoked in the absence of calcium and procainamide did not affect the transient hyperpolarization. Quinidine, however, suppressed the α 1 response when the same procedure was followed. Both the transient and the sustained hyperpolarization evoked in smooth muscle cells in the presence of procainamide (15 min) or quinidine in calcium-containing or in calcium-free solution, respectively, were inhibited. The α 1 response was reflected by a depolarization of the muscle cells after the potassium channels had been blocked with apamin (3 × 10 −7 M, 20 min). The transient depolarization observed after α 1-receptor stimulation under calcium-free conditions was also suppressed by procainamide and quinidine. These results can be explained by assuming that calcium mobilization, activated after stimulation of the α 1-receptors, is inhibited by procainamide and quinidine. A possible additional effect of quinidine on replenishment of the calcium store from the extracellular space is not excluded.

Lack of involvement of Ca 2+ conductance change in ATP-induced depolarization of the guinea-pig vas deferens

The double sucrose-gap method was used to examine the electrial responses of the guinea-pig vas deferens to ATP and their possible dependence on external Ca 2+. Normally ATP induced a depolarization and an increase in membrane conductance and both effects were concentration-dependent. The reversal potential of the 10 −4 M ATP-induced depolarization was 27.1 mV positive to the resting membrane potential of the tissue. This value was quite similar to that previously obtained for the 3 × 10 −5 M ATP-induced depolarization. The smooth muscle membrane was depolarized by 5.9 mV in a Ca-free medium, in which ATP also caused a depolarization, associated with an increase in membrane conductance. The reversal potential of the depolarization induced by ATP (10 −4 M) in the Ca-free medium was 26.5 mV positive to the resting membrane potential. The results suggest that, in this tissue, ATP induces membrane depolarization with little effect on Ca 2+ conductance.

Electrophysiological and Pharmacological Study on Innervation of the Aganglionic Colon in Hirschsprung's Disease of Human and Murine Model

A true role of extrinsic nerve fibres proliferating abundantly in the aganglionic colon of Hirschsprung's disease has not yet been established. Electrophysiological and pharmacological studies were carried out on rectal muscle strips of piebald lethal mice and their siblings. Muscle strips of both aganglionic and normal colon obtained from fifteen patients with Hirschsprung's disease were also investigated for the purpose of determining whether these extrinsic nerve fibres innervate the smooth muscle cells in the aganglionic colon or not. Junction potentials and contractile tensions evoked by electric stimulations were recorded employing the double-sucrose gap method, and examined the effects of nerve blocking agents on junction potentials. In the normal colon, excitatory junction potentials and inhibitory junction potentials were recorded following stimulation in both man and mouse. Excitatory junction potentials were accompanied by muscle contraction and abolished by atropine or tetrodotoxin. Inhibitory junction potentials were resistant to phentolamine and propranolol, and abolished by tetrodotoxin. It was concluded that the normal colon of both mouse and man is innervated by cholinergic excitatory and non-adrenergic inhibitory nerves. In the aganglionic colon, on the other hand, neither junction potentials nor contractions were evoked by stimulations in both mouse and man. These evidences led to the conclusion that in the aganglionic colon in Hirschsprung's disease of man and murine model, there may be a deficiency in neuronal regulation.

Evidence for suppression of potassium conductance by noradrenaline in smooth muscle of guinea-pig vas deferens.

The ionic dependency was examined in the reversal potential of the NA-induced membrane depolarization of smooth muscle of the guinea-pig vas deferens, using the double sucrose-gap method. In the control (5.9 mM K) solution and solutions containing 20 and 60 mM K ions, replaced with equimolar amount of Na ions, the reversal potential was negative to the membrane potential at the steady state in each solution, by 19.5, 15.1 and 13.2 mV, respectively. Taking account that the higher external K concentration causes the larger membrane depolarization, these results indicate that the reversal potential shifted toward the positive direction as the external K concentration was increased. On the other hand, the calculated equilibrium potential of K ions in those solutions is -88, -52 and -24 mV, respectively. In those solutions, furthermore, the equilibrium potential of Na ions is 42, 38 and 29 mV, respectively. However, the equilibrium potentials of Ca and Cl ions should be unchanged. Therefore, it is suggested that the reversal potential of the NA-induced depolarization varied in the given different solutions compatibly only with the change in the K equilibrium potential. This provides evidence that in this tissue, NA induces a reduction in the K conductance of the cell membrane.

Pre- and post-junctional actions of prostaglandin I2, carbocyclic thromboxane A2 and leukotriene C4 in dog tracheal tissue.

Effects of carbocyclic thromboxane A2 (cTxA2), prostacyclin (PGI2) or leukotriene C4 (LTC4) on the membrane and contractile properties of the smooth muscle cells and on the excitatory neuro-effector transmission in the dog trachea were observed by means of the microelectrode, double sucrose gap and tension recording methods. cTxA2, PGI2 or LTC4 at a concentration of 10(-7)M had no effect on the membrane potential of smooth muscle cells of the dog trachea. At 10(-6)M, cTxA2 and LTC4 slightly depolarized, and PGI2 hyperpolarized the membrane. cTxA2 (greater than 2.7 X 10(-10)M) evoked a sustained contraction, while the amplitude of the twitch contractions evoked by field stimulation in the presence of indomethacin (10(-6)M) and propranolol (10(-6)M) was inhibited, dose-dependently. PGI2 (greater than or equal to 2.7 X 10(-7)M) reduced the muscle tone and the amplitude of twitch contractions evoked by field stimulations. cTxA2 or PGI2 (10(-10)-10(-7)M) reduced the amplitude of the excitatory junction potentials (e.j.ps) evoked by field stimulation with no change in the membrane potential, input membrane resistance or the sensitivity of the muscle cells to acetylcholine (ACh). LTC4 (1.6 X 10(-8)M) evoked a sustained contraction of the dog trachea; however, this agent did not affect either the amplitude of the twitch contractions or the e.j.ps evoked by field stimulation. The amplitude of the e.j.p. was dependent on the external concentration of Ca2+, and the inhibitory actions of cTxA2 on e.j.ps were partly overcome by increasing the concentrations of [Ca]o. When the amplitudes of e.j.ps were plotted against [Ca]o on a double log scale, the above relation yielded a straight line with a slope of 1.7 or 1.0, in the absence or presence of cTxA2, respectively. After treatment with Ca2+-free 2 mM EGTA-containing solution, cTxA2 or LTC4 did not evoke a contraction in the dog trachea, whereas ACh (10(-7)-10(-6)M) did. These results indicate that cTxA2 and PGI2 have dual actions on pre- and post-junctional membranes of the dog tracheal tissue, i.e. both agents inhibit the excitatory neuro-effector transmission in the dog trachea, presumably by inhibiting the release of ACh from the vagal nerve terminal. cTxA2 and LTC4 or PGI2 evoke contraction or relaxation of the muscle tissue, respectively, apparently through direct actions on the smooth muscle cells.