Effects of enkephalin and endorphin on the inhibitory junction potentials in the duodenal smooth muscle cells of the guinea-pig

Inhibitory junction potentials (i.j.p.s) evoked by field stimulation were recorded from the smooth muscle cells of the guinea-pig duodenum intracellularly. The membrane potential was -54.3 mV. The parameters of the i.j.p. were as follows: latency, 71 msec; time to peak, 146 msec; amplitude, 15.5 mV; rate of hyperpolarization, 107 mV/sec; and half decay time of the i.j.p., 193 msec. Met-enkephalin (10(-7)-10(-6) M) had no effect on the membrane potential and the i.j.p. The membrane potential was decreased by beta-endorphin (1.7 X 10(-7)-6.8 X 10(-7) M). Increase in the latency and the time to peak and decrease in the amplitude and the rate of hyperpolarization of the i.j.p. were observed for beta-endorphin. "Spontaneous" excitatory junction potentials (e.j.p.s) were generated by beta-endorphin. Naloxone (3.1 X 10(-6)-3.1 X 10(-4) M) hyperpolarized the membrane of the muscle cells. At high concentrations of naloxone (3.1 X 10(-4) and 3.1 X 10(-3) M), inhibition of the i.j.p. was observed. Levallorphan (2.3 X 10(-4) M) prolonged the latency and the time to peak and reduced the amplitude of the i.j.p. The membrane potential was slightly decreased by levallorphan. "Spontaneous" e.j.p.s were generated by levallorphan in a certain population of the cells. It is concluded that Met-enkephalin does not contribute to the non-adrenergic inhibitory transmission and that beta-endorphin acts as a modulator in the control mechanism of the intestinal motility. The effects of naloxone and levallorphan on the i.j.p. are discussed.     

The effect of cations on the electrical properties of the smooth muscle cells of the guinea-pig vas deferens

1. A study has been made of the effects of Na(+) and Ca(2+) on the responses generated by intracellular current pulses in smooth muscle cells of the guinea-pig vas deferens and upon the propagated action potential.2. Reduction of the extracellular Na(+) activity (a(Na)) to less than 30 mM did not alter the characteristics of the spike-like response to intracellular current pulses (the active response) or of the propagated action potential.3. Reduction of the extracellular Ca(2+) activity (a(Ca)) to 0.1 mM decreased the resting input resistance of the cells but increased the input resistance during the crest of the active response. Reduction of Ca(2+) decreased the overshoot of the action potential by 22 mV per tenfold change in Ca(2+) and the resting potential by 25 mV per tenfold change.4. Reduction of the extracellular Ca(2+), keeping the product a(Ca)/a(2) (Na) constant, did not change the resting potential, but decreased the action potential overshoot by 20 mV per tenfold change.5. It is suggested that part of the current responsible for the rising phase of the action potential is carried by Ca(2+).     

Effects of polyamines on contractility of guinea-pig gastric smooth muscle

This study was designed to investigate the effects of polyamines on mechanical contraction and voltage-dependent calcium current (VDCC) of guinea-pig gastric smooth muscle. Mechanical contraction and calcium channel current I(Ba) were recorded by isometric tension recording and whole-cell patch clamp technique. Spermine, spermidine and putrescine inhibited spontaneous contraction of the gastric smooth muscle in a concentration-dependent manner. Spermine (2 mM) reduced high K+ (50 mM)-induced contraction to 16+/-6.4% of the control (n=9), and significantly inhibited I(Ba) in a reversible manner (p<0.05; IC50=0.8 mM). Pre- and post-treatment of tissue with spermine (2-5 mM, n=10) also inhibited acetylcholine (10 microM)-induced phasic contraction to 5+/-6.4% of the control. Inhibitory effect of spermine on I(Ba) was observed at a wide range of test potentials of current/voltage (I/V) relationship (p<0.05), and steady-state activation of I(Ba) was shifted to the right by spermine (p<0.05). Spermidine and putrescine (1 mM each) also inhibited I(Ba) to 51+/-5.7% and 81+/-5.3% of the control, respectively. And putrescine (1 mM) inhibited I(Ba) at whole tested potentials (p<0.05) without significant change of kinetics (p<0.05). Finally, 5 mM putrescine also inhibited high K+-induced contraction to 53+/-7.1% of the control (n=4). These findings suggest that polyamines inhibit contractions of guinea-pig gastric smooth muscle via inhibition of VDCC.     

Rebound excitation of the smooth muscle cells of the guinea-pig taenia coli after stimulation of intramural inhibitory nerves

1. A study has been made of the increase in the rate of action potential firing in spontaneously active cells and of the initiation of action potential firing in quiescent cells of the taenia coli after stimulation of the intramural inhibitory nerves.2. In the majority of cells which fired action potentials spontaneously at intervals of about 1 sec, stimulation of the intramural inhibitory nerves with single pulses gave an inhibitory junction potential (I.J.P.) which was followed by action potentials which occurred at intervals as small as 0.5 sec. The increased rate of firing lasted up to 30 sec.3. A small number of cells were either not spontaneously active or only fired action potentials at intervals greater than 5 sec. After stimulation of the intramural inhibitory nerves with either single or repetitive pulses, the quiescent cells gave I.J.P.S which were followed by either a single action potential or a burst of action potentials.4. The rate of firing of action potentials after an I.J.P., and the duration of this enhanced rate of firing increased with an increase in the mean amplitude of the hyperpolarization during the I.J.P. As the amplitude of the I.J.P. increases with an increase in frequency of stimulation of the nerves, the rebound excitation increases with an increase in the frequency of stimulation of the inhibitory nerves.     

Effects of ruthenium red on membrane ionic currents in urinary bladder smooth muscle cells of the guinea-pig

 Three major ionic currents, Ca²⁺-dependent K⁺ current (I _K-Ca), delayed rectifier type K⁺ current (I _kd) and Ca²⁺ current (I _Ca), were activated by depolarization under whole-cell clamp in single smooth muscle cells isolated from guinea-pig urinary bladder. Externally applied ruthenium red (RuR) reduced the amplitude of I _K-Ca and I _Ca at 0 mV (IC₅₀ values were 4.2 and 5.6 μM, respectively), but did not affect I _Kd. Spontaneous transient outward currents (STOCs) and caffeine-induced outward currents (I _caf) at –30 mV were reduced by external 10 μM RuR. When 10 μM RuR was added to the pipette solution, I _K-Ca during depolarization, STOCs and I _caf significantly decreased with time. RuR did not change the unitary current amplitude of the large-conductance Ca²⁺-dependent K⁺ (BK) channels, but reduced the open probability of the channel under excised patch-clamp recording mode. RuR reduced the channel activity more effectively from the cytosolic face than from the other. This inhibition decreased when the cytosolic Ca²⁺ concentration was increased. These results indicate that RuR blocks BK and Ca²⁺ channels in urinary bladder smooth muscle cells. The decrease in I _K-Ca, STOCs and I _caf by RuR is attributable to the direct inhibition of BK channel activity, probably in addition to the inhibition of Ca²⁺ release from storage sites. The direct inhibition of BK channel activity by RuR may be related to the interaction of RuR with the Ca²⁺-binding sites of the channel protein. Received: 15 October 1997 / Received after revision and accepted: 25 November 1997     

Ghrelin signalling in guinea-pig femoral artery smooth muscle cells

Aim: Our aim was to study the new signalling pathway of ghrelin in the guinea‐pig femoral artery using the outward I_K as a sensor. Methods: Whole‐cell patch‐clamp experiments were performed on single smooth muscle cells, freshly isolated from the guinea‐pig femoral artery. The contractile force of isometric preparations of the same artery was measured using a wire‐myograph. Results: In a Ca²⁺‐ and nicardipine‐containing external solution, 1 mmol L^?1 tetraethylammonium reduced the net I_K by 49 ± 7%. This effect was similar and not additive to the effect of the specific BK_Ca channel inhibitor iberiotoxin. Ghrelin (10^?7 mol L^?1) quickly and significantly reduced the amplitudes of tetraethylammonium‐ and iberiotoxin‐sensitive currents through BK_Ca channels. The application of 5 × 10^?6 mol L^?1 desacyl ghrelin did not affect the amplitude of the control I_K but it successfully prevented the ghrelin‐induced I_K decrease. The effect of ghrelin on I_K was insensitive to selective inhibitors of cAMP‐dependent protein kinase, soluble guanylyl cyclase, cGMP‐dependent protein kinase or a calmodulin antagonist, but was effectively antagonized by blockers of BK_Ca channels, phosphatidylinositol‐phospholipase C, phosphatidylcholine‐phospholipase C, protein kinase C, SERCA, IP₃‐induced Ca²⁺ release and by pertussis toxin. The ghrelin‐induced increase in the force of contractions was blocked when iberiotoxin (10^?7 mol L^?1) was present in the bath solution. Conclusions: Ghrelin reduces I_K(Ca) in femoral artery myocytes by a mechanism that requires activation of Gα_i/o‐proteins, phosphatidylinositol phospholipase C, phosphatidylcholine phospholipase C, protein kinase C and IP₃‐induced Ca²⁺ release.     

Effects of some autonomic drugs on duodenal smooth muscle.

Longitudinal muscle strips (LMS) and circular muscle strips (CMS), 2 mm wide and 1.5--2 cm long, from opossum duodenum were exposed to some autonomic agonists. The cholinergic agonists, acetylcholine, carbachol, methacholine, and bethanechol stimulated only tonic contractions in LMS and tonic followed by phasic contractions in CMS. These effects were abolished by atropine 10(-6) M. The ED50S of all cholinergic agonists for LMS were significantly lower than for CMS. Norepinephrine caused initial contraction (abolished by phenoxybenzamine, 10(-4) M), followed by relaxation (abolished by propranolol, 10(-5) M), and isopropylnorepinephrine caused relaxation (abolished by propranolol, 10(-5) M) in both layers. There were no differences in relative potencies for adrenergic agonists between the layers. Tetrodotoxin did not affect the response to adrenergic agonists. Thus, the potency of cholinergic agonists is greater in longitudinal than in circular muscle, and the layers respond differently to cholinergic agonists. The alpha-adrenergic receptors mediate contraction and beta-adrenergic receptors mediate relaxation on the duodenal smooth muscle.     

Transmission from intramural inhibitory nerves to the smooth muscle of the guinea-pig taenia coli

1. Membrane potential changes of smooth muscle cells were recorded during stimulation of the intramural inhibitory nerves to the taenia coli.2. Stimulation across the taenia coli with single pulses of 200 musec duration excites the intramural nerves and not the muscle directly.3. The membrane potential changes due to stimulation of the intramural inhibitory nerves were different from those produced by perivascular inhibitory nerve stimulation in the following ways: hyperpolarizations (i.j.p.'s) of up to 25 mV were produced in response to single pulses; the latency, i.e. the time taken for the membrane to hyperpolarize after a stimulus of maximal strength, was as short as 80 msec; when the nerves were stimulated repetitively the membrane was hyperpolarized by up to 35 mV and all spontaneous activity was abolished; the mean hyperpolarization due to repetitive stimulation increased with the frequency of stimulation up to 10 pulses/sec and then remained constant; the hyperpolarization due to stimulation at frequencies greater than 5 pulses/sec was not maintained but decreased after 3-5 sec of stimulation; and finally when stimulation had ceased action potentials commenced firing at frequencies greater than normal.4. The amplitude and rate of hyperpolarization of the i.j.p. increased with increasing strength of stimulation until a maximum amplitude and rate of hyperpolarization was reached. The recovery or depolarizing phase of the i.j.p. was exponential with a time constant which varied from about 250 msec to 500 msec and could not therefore be due to the discharge of the membrane capacitance. In some cases there was an inflexion on this depolarizing phase and in these cases recovery led directly into an action potential.5. Spontaneous hyperpolarizations of the membrane were seen in some cells, and these hyperpolarizations were similar to those recorded on submaximal stimulation of the intramural nerves.6. There were no changes in the characteristics of the i.j.p. in the presence of guanethidine or bretylium.     

Transmission from perivascular inhibitory nerves to the smooth muscle of the guinea-pig taenia coli

1. Membrane potential changes of the smooth muscle cells of the taenia coli were recorded during stimulation of the perivascular inhibitory nerves.2. Some spontaneous action potentials were preceded by a slow pacemaker-like potential. Others began at or near the maximum level of the membrane potential and were not preceded by pacemaker-like potentials.3. There were no changes in the membrane potential of smooth muscle cells when the inhibitory nerves were stimulated with a single pulse. Stimulation at frequencies greater than 5 pulses/sec caused a hyperpolarization of the smooth muscle membrane. This resulted in a decrease in spike frequency and relaxation.4. When the frequency of stimulation of the inhibitory nerves was increased there was an increase in the amplitude and rate of rise of the hyperpolarization and a decrease of the latency. The latency varied from 150 to 300 msec, and the largest hyperpolarization recorded was 16 mV.5. The effect of the hyperpolarization due to nerve stimulation in cells showing pacemaker-like activity was to increase the level of the membrane potential at which the action potentials began and to increase the membrane potential to which the action potentials repolarized. Action potentials which occurred during hyperpolarizations of the membrane had greater rates of rise and fall and larger amplitudes than did the action potentials which occurred before hyperpolarization.6. The effect of the hyperpolarization due to nerve stimulation in cells which did not show pacemaker-like activity depended on the amplitude of the hyperpolarization. Small hyperpolarizations exposed small depolarizations of the membrane which occurred when an action potential would normally have been expected. Large hyperpolarizations blocked the action potentials entirely.7. Action potentials did not begin firing again at the normal rate immediately after stimulation ceased. The time taken for the rate of firing of action potentials to return to normal increased with increasing frequency of stimulation.8. The hyperpolarization in response to perivascular inhibitory nerve stimulation was blocked by guanethidine and bretylium.     

Action potentials and net membrane currents of isolated smooth muscle cells (urinary bladder of the guinea-pig)

1. Cells were isolated by incubating chunks of tissue from the urinary bladder of the guinea-pig in a high potassium, low chloride medium containing 0.2 mM calcium plus the enzymes collagenase and pronase. After isolation, the cells were superfused with a physiological salt solution (PSS) containing 150 mM NaCl, 3.6 mM CaCl₂ and 5.4 mM KCl (35°C). Patch electrodes filled with an isotonic KCl-solution were used for whole cell recordings. With a single electrode voltage clamp we measured a capacitance of 50±5 pF per cell, an input resistance of 200±25 kOhm ·cm² and a series resistance of 44±4 Ohm·cm².
2. The cells had resting potentials of ?52±2 mV. They did not beat spontaneously but responded to stimuli with single action potentials (APs) which rose from the threshold (?38 mV) with a maximal rate of 6.5±1.8 V/s to an overshoot of 22±3 mV. The AP lasted for 36±4 ms (measured between threshold and ?40 mV). Continuous cathodal current produced repetitive activity, a pacemaker depolarization followed the AP and preceded the next upstroke.
3. Net membrane currents evoked by clamp steps to positive potentials were composed of an inward and an outward component. The inward component generating the upstroke of the AP was carried by Ca ions (i _Ca, Klöckner and Isenberg 1985). The repolarization resulted from a potassium outward currenti _K. Ca-channel blockers (5 mM NiCl₂) reducedi _K suggesting that (part of)i _K was Ca-activated.
4. i _K rose within about 100 ms to a peak of 40–200 μA/cm² from which it inactivated slowly and incompletely. The inactivatingi _K followed a bell-shaped voltage-dependence, the noninactivatingi _K an outwardly rectifying one. Both parts had similar steady state inactivation curves with a half maximal inactivation potential at ?36 mV and a slope of 9 mV.
5. Repolarization to ?50 mV induced outward tail currents which reversed polarity at ?85 mV (the calculated potassium equilibrium potential). The amplitude and the time course of the envelope of the tail currents varied in proportion toi _K during the prestep. Thus, the tail current is suggested to reflect the turning off of a potassium conductance which had been activated during the prepulse.
6. i _K was largely reduced but not blocked by 20 or 150 mM tetraethylammonium (TEA). TEA did not significantly change the resting potential, but it prolonged the AP and facilitated upstroke and overshoot.i _K could be blocked by loading the cells with Cs released from Cs-filled patch electrodes.
7. We compare the results with the data from multicellular tissue (Creed 1971). The more negative resting potential and the absence of spontaneous APs are mainly attributed to the absence of transmitter release from nerve terminals. The isolated cell is suggested as a model of the postsynaptic membrane properties.

     

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.2. In Ca-free Locke solution the membrane was depolarized, the membrane resistance became low, the spike amplitude became small and the mechanical response decreased. In most preparations the electrical and mechanical activity was abolished within 10 min, but in some preparations the electrical activity continued for more than 30 min.3. In Ca-free solution containing 0.1 mM-EGTA, the membrane was depolarized and the electrical and mechanical activity was abolished within 5 min in every preparation. When NaCl was replaced with sucrose, the effects of Ca removal on the spike activity and contraction appeared very slowly and the membrane potential and membrane resistance remained unchanged.4. When Ca was replaced with Mg (2 mM) the spike was blocked within 1 min without depolarization or reduction of the membrane resistance. In Na-deficient (sucrose) solution, the presence of Mg accelerated the disappearance of the spike caused by Ca removal.5. In Ca-free solution containing 0.5 mM-Mg, a spike-like activity was observed without accompanying mechanical response. This activity was blocked by increasing the Mg concentration above 2 mM. It was Na-dependent, since it was abolished by removing Na from the external solution, but it was not influenced by tetrodotoxin (2 x 10(-6) g/ml.).6. It was concluded that calcium has at least two functions, one as current carrier for the action potential and another as controller of the Na permeability of the membrane. It was also suggested that the Ca which is bound at the membrane may be utilized as a source of Ca ions to carry the current for the action potential.     

Effects of polyamines on voltage-activated calcium channels in guinea-pig intestinal smooth muscle

Effects of polyamines on the spontaneous mechanical and electrical activity of guinea-pig intestinal smooth muscle were studied. Spermine and spermidine inhibited action potential generation and contractions, while putrescine had no effect. Single smooth muscle cells were isolated from the longitudinal muscle layer of the guinea-pig ileum. Whole-cell voltage-clamp experiments were carried out to investigate the effects of polyamines on current through voltage-activated Ca²⁺ channels. Spermine and spermidine (0.1–1 mM) reduced the inward current in a concentration-dependent manner. Spermine blocked current activated by the dihydropyridine agonist BAY K 8644 (1 M), whereas no additional inhibition by spermine was seen after blockage of dihydropyridine-sensitive channels by nifedipine (0.1 M). Inhibition by spermine or spermidine did not shift the peak of the current voltage relation of the inward current. Steady-state activation and inactivation relationships were not affected and thus the amplitude, but not the voltage dependence, of the window current responsible for Ca²⁺ inflow during sustained depolarization was affected. Putrescine (1 mM) had no significant effect on the inward current. These results suggest that spermine and spermidine inhibit contraction in spontaneously active intestinal smooth muscle by inhibiting Ca²⁺ current responsible for generation of action potentials.     

Effects of tetraethylammonium chloride on the membrane activity of guinea-pig stomach smooth muscle

1. The effects of tetraethylammonium (TEA) on the membrane activity of the antral circular muscle of the guinea-pig stomach were investigated with micro-electrode and double sucrose gap methods.2. In a concentration of 1-1.5 x 10(-3) g/ml. (3-5 mM), the membrane potential was not influenced; the membrane resistance measured by inward current pulses remained the same but the rectifying property of the membrane was suppressed.3. TEA (1-1.5 x 10(-3) g/ml.) enhanced the spike amplitude markedly even from fibres which generated graded responses.4. TEA (1-1.5 x 10(-3) g/ml.) did not increase the maximum rate of rise of the spike but decreased the maximum rate of fall of the spike markedly.5. In Na-free (Tris or sucrose) solution, in K-deficient and excess-K solutions, TEA (1-1.5 x 10(-3) g/ml.) suppressed the rectifying property of the membrane and enhanced the spike amplitude.6. Atropine (10(-6) g/ml.) had no effect on the enhancement of the spike amplitude produced by TEA.7. The minimum concentration of Ca ions required for the effect of TEA on the spike amplitude was one fifth of the normal concentration. TEA also enhanced the spike amplitude in Sr-Krebs.8. The possible role of TEA on the membrane activity is considered to be due to suppression of the K conductance when the membrane is depolarized. Alternative possible roles of TEA on the spike amplitude are also discussed.     

Effects of removing the external potassium on the smooth muscle of guinea-pig taenia coli

1. Removal of the external K ions depolarized the membrane of the guinea-pig taenia coli by about 5 mV, and increased the frequency of the spontaneous spike activity. After about 20 min in K-free solution, the frequency gradually decreased and spontaneous activity stopped after about 60 min.2. Readmission of K produced a transient hyperpolarization by 10-15 mV for 10-20 min, and lowered the excitability. The membrane resistance decreased during the hyperpolarization. When the external K concentration was returned to normal before the spontaneous activity had disappeared in K-free solution, there was a complete suppression of the spontaneous electrical and mechanical activities, accompanied by hyperpolarization of the membrane. The duration of this suppression of electrical and mechanical activities depended on the exposure time to K-free solution, the recovery being delayed by longer exposure.3. The effects of removing external K were essentially the same in the presence of tetrodotoxin (10(-7) g/ml.), of atropine (10(-6) g/ml.), and also in low Cl (2-7 mM).4. The effects of ouabain (10(-6) g/ml.) were similar to those of zero K but appeared more slowly. After treatment with ouabain, the membrane was hyperpolarized by removing the external K and there was no hyperpolarization nor suppression of the mechanical activity on readmission of external K.5. Rb substitution for K produced little effect and addition of 5.9 mM-Rb after K-removal produced a typical suppression of activity as the readmission of K. When K was replaced with an equimolar concentration of Cs, the effects were similar to those produced by K-free solution.6. The pattern of the response to a change in the external K concentration was different below 23 degrees C. The spontaneous activity was suppressed by removal of K, and increased by returning the external K.7. It is concluded that, in the taenia, the membrane potential can be modified by an electrogenic pump, the activity of which is inhibited by removing K, by ouabain, and by lowering the temperature to about 23 degrees C. During the recovery from the exposure to K-free solution or to ouabain, the pump activity is potentiated, causing hyperpolarization of the membrane by 10-20 mV. Rb can, Cs cannot, replace K in activating the pump at the same concentration as K.     

Effects of extracellular calcium and other divalent cations on mechanical response of frog skeletal muscle

In order to investigate the mechanism by which elevated extracellular Ca ions decrease twitch and tetanus tension in frog skeletal muscle we made mechanical and electrophysiological measurements on single fibers or small bundles from twitch muscles. High concentration of Ca caused a hyperpolarization and an increase in the duration of action potential. The mechanical threshold, estimated by using the strength-duration curve, was shifted upward by adding Ca ions. These effects were fully reversible. Steady state twitch tension was slightly increased by replacing Mg and Ni with Ca and decreased by elevating their concentrations, although Ba resulted in a marked twitch augmentation and a positive correlation with the ion concentration. By contrast, the strength-duration curve was shifted upward by Ni while Mg and Ba showed no shift. These evidences point to a failure of the early step of excitation-contraction coupling, including the T-membrane depolarization, as the primary mechanism of action of high concentration of Ca, Mg, and Ni ions, whereas Ba ion has an additional intracellular potentiating effect.