Non-neural electrical responses of smooth muscle cells of the rabbit basilar artery to electrical field stimulation

In smooth muscle cells of the rabbit basilar artery, field stimulation evoked a depolarizing response which consisted of a fast (1-3 s duration) and a following slow (1-4 min duration) component. The amplitude of these responses increased in an intensity-dependent manner and, when exceeding 10-15 mV, a spike potential was generated. During generation of the slow depolarization, ionic conductances of the membrane were increased. When outward current pulses with long duration (2-3 s) were applied to the smooth muscle using the partition stimulating method, electrotonic potentials and spike potentials were generated. The cessation of the current pulse caused repolarization of the membrane with time constant of 250-350 ms. The depolarizing responses were resistant to tetrodotoxin, sympathetic transmission blocking agents (guanethidine, bretylium, or 6-hydroxydopamine treatment), receptor antagonists for 5-hydroxytryptamine (methysergide), dopamine (haloperidol), ACh (atropine), noradrenaline (phentolamine), ATP (alpha,beta-mATP) or histamine (mepyramine), blockade of synthesis of prostaglandins or thromboxane A2 (indomethacin) or high Mg2+, low Ca2+ solution. Smooth muscle cell membrane of the basilar artery was depolarized by 5-hydroxytryptamine (above 0.1 microM) or histamine (above 10 microM) but not by ACh (up to 100 microM) or noradrenaline (up to 10 microM). The depolarization induced by 5-hydroxytryptamine or histamine was antagonized by methysergide or mepyramine, respectively. Denervation of the vessel by storing in a cold condition (4 degrees C) decreased but did not abolish the depolarizing response. The decrease in amplitude of the depolarizing response during cold storage was attributed to associated depolarization of the smooth muscle membrane. Internal perfusion of the vessel with distilled water abolished generation of the depolarizing response, and this procedure also abolished the endothelium-dependent relaxation induced by ACh during the potassium contraction. The results suggest that the depolarizing response evoked by field stimulation is generated by substances released from non-neural components, possibly from the endothelial cells.     

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.     

Inhibitory actions of indomethacin on electrical and mechanical responses produced by nerve stimulation in circular smooth muscle of the guinea-pig gastric fundus.

The effects of indomethacin on electrical and mechanical responses produced by transmural nerve stimulation (TNS) were investigated in isolated circular smooth muscle of the guinea-pig gastric fundus. TNS evoked a cholinergic excitatory junction potential (e.j.p.). The e.j.p.s were inhibited by 1-10 microM indomethacin, in a concentration-dependent manner, with no marked alteration of the resting membrane potential. Exogenously applied acetylcholine caused a depolarization of the membrane that was not altered by indomethacin. TNS evoked a cholinergic twitch contraction at low frequencies (0.1 Hz). A train of TNS's at high frequency (1 Hz) produced a transient contraction with a subsequent sustained relaxation. Indomethacin reduced the resting tension and inhibited these TNS-induced contractions. Application of Nomega-nitro-L-arginine (NOLA), an inhibitor of nitric oxide (NO) synthesis, increased the amplitude of twitch contractions, and altered transient contractions to tetanic contractions during TNS at a frequency of 1 Hz, also with an increased amplitude. In the presence of NOLA, indomethacin (5 microM) again reduced the resting tension and inhibited TNS-induced contractions. This inhibition was greater for twitch contractions than for tetanic contractions. Nifedipine reduced the TNS-induced contractions, while addition of indomethacin further reduced the amplitude of contractions. Contractions produced by low concentrations of acetylcholine (0.1 microM) were inhibited by indomethacin, while those produced by 1 microM were not. These results indicate that the inhibitory actions of indomethacin on TNS-induced contractions do not involve enhanced production of NO or selective inhibition of voltage-gated Ca-channels. Prejunctional autoregulatory mechanisms may also not be altered by indomethacin. As indomethacin inhibits the enzyme cyclooxygenase, it is speculated that endogenously produced prostaglandins exert excitatory actions on gastric smooth muscle, and act mainly postjunctionally to facilitate spontaneous and neurogenic electrical and mechanical activity.     

Mechanical activity of frog esophagus muscle in response to electrical stimulation of intramural nerves.

Microscopic observation of intramural nerves in the frog esophagus, fixed and stained with OsO(4) and ZnI(2), revealed that nerve cell bodies and bundles connecting the nerve cell bodies formed loose and irregular networks. The nerve cell bodies were mostly lying singly in the nerve bundles, with occasional observations of two closely linked nerve cell bodies. Isolated circular and longitudinal segments of esophageal muscle were spontaneously rhythmically contractile, with a frequency of 2.2-3.0 per min. This was not altered by tetrodotoxin (TTX). In longitudinal muscle segments, transmurally applied electrical stimulation produced contractile responses which were not inhibited by atropine or guanethidine, but were reduced in amplitude by TTX, suggesting a nonadrenergic-noncholinergic (NANC) excitatory innervation in the esophagus muscle. In circular muscle segments, transmural application of brief electrical stimulation evoked two types of mechanical response: a biphasic response consisting of an initial relaxation and a following contraction (type I) and a contraction alone (type II). These mechanical responses were not modulated by either atropine or guanethidine. In the type I response, TTX abolished the relaxation component, suggesting that this was produced by non-adrenergic non-cholinergic (NANC) inhibitory nerve excitation. In about half of the type II responses, the amplitude of the contraction was significantly reduced by TTX, suggesting that a part of the contraction was produced by activation of NANC excitatory nerves. Thus, the esophageal smooth muscle of the frog demonstrates myogenic activity, and is innervated by both excitatory and inhibitory NANC nerves.     

Non-adrenergic, non-cholinergic inhibitory responses to nerve stimulation in rat colonic circular muscle.

The nerve-mediated response to electrical field stimulation (EFS) in rat colonic circular muscle was investigated using the single sucrose-gap technique. EFS with a single pulse (0.4 ms, supramaximal voltage) elicited transient TTX-sensitive hyperpolarization (IJP) often followed by an 'off' depolarization associated with muscular contraction. No relaxation associated with the IJP could be seen unless tone was pharmacologically induced by carbachol (10(-6) M). IJPs were due to non-adrenergic, non-cholinergic (NANC) nerve activation since they were not affected by atropine (10(-7) M) or guanethidine (10(-6) M) superfusion. The mechanism underlying the IJP was presumably an increase in K+ conductance, and the NANC neurotransmitter might open largely apamin-sensitive, Ca(2+)-dependent K+ channels. Purines or vasoactive intestinal polypeptide (VIP) did not mimic the effects of NANC nerve stimulation. Therefore, the NANC inhibitory system, producing IJPs, in rat colonic circular muscle is not purinergic or VIPergic in nature.     

The effects of exogenous amino acids on the relaxant responses of pig urethral smooth muscle evoked by stimulation of the inhibitory nitrergic nerves

Inhibitory innervation of urethral smooth muscle is mediated partly through release of NO. We investigated the mechanisms involved in the supply of the substrate l-arginine to NO synthase by examining the relaxant response of the muscle to electrical field stimulation (EFS) and the effects of addition of amino acids to the bathing medium. Relaxant responses persisted during hours of repetitive stimulation but were enhanced rapidly by addition of l-arginine (the arginine paradox). Addition of l-lysine (competes with l-arginine for transport on the y⁺ carrier) and l-glutamine (competing on the y⁺L carrier) attenuated the enhancement. Enhancement persisted after washing but was reversed by application of l-lysine, suggesting that exogenous l-arginine fills an intracellular pool and that l-lysine can trans-stimulate its efflux from the pool. After prolonged depolarization in high-K⁺, Na⁺-free solution the relaxant response became purely nitrergic. Addition of l-arginine during the exposure continued to enhance the subsequent responses but l-glutamine added with l-arginine, could no longer reduce this enhancement. The results show the arginine paradox in inhibitory nerves and suggest the involvement of y⁺ and y⁺L carriers in the transport of l-arginine.     

Effects of chloroquine on smooth muscle contracted with noradrenaline or high-potassium solutions in the rat thoracic aorta.

The effects of chloroquine on the smooth muscle of isolated rat aortic segments were investigated in preparations contracted with either noradrenaline or high-potassium. At rest, chloroquine (up to 10(-4) M) produced no mechanical response, while noradrenaline (10(-6) M) produced a sustained contraction. In the presence of 10(-4) M chloroquine, however, the amplitude of contractions produced by noradrenaline was attenuated by about 70%, with no alteration of the resting tension. In preparations contracted either with noradrenaline or with high-K solutions, chloroquine produced a concentration-dependent relaxation. The tension decreased below resting level as a result of the co-application of these stimulants. The relaxing actions of chloroquine were not altered by methylene blue (an inhibitor of guanylate cyclase), suggesting that the cyclic GMP-related mechanism was not involved. The ratio of the amplitude of chloroquine-induced relaxation was similar in contractions produced by different concentrations of potassium ions, suggesting that chloroquine did not cause relaxation as a result of membrane hyperpolarization. These results suggest that the inhibition of aortic smooth muscle contraction caused by chloroquine is different to that produced by endothelium-derived vasodilating factors. It is possible that the inhibition of aortic smooth muscle contraction by chloroquine involves modulation of the contractile systems and of their regulatory proteins.     

Changes in the lability of smooth muscle during stimulation of the sympathetic and parasympathetic nerves

Summary The effect of sympathetic and parasympathetic nerves of a smooth muscle—the retractor penis—was investigated. Stimulation of the sympathetic nerve extends the limits of the optimal and pessimal frequency of stimulation towards greater frequencies, the action current frequency and amplitude increase, while the value of the resting current decreases.On stimulation of the parasympathetic nerve, the action current frequencies become decreased, their amplitude declines and the resting current rises.A suggestion is made that the antagonism of the sympathetic and parasympathetic innervation of the smooth muscle apparently is due to the action exerted on the functional properties of the muscle, particularly on its lability.(Presented by Active Member AMN SSSR V. V. Parin) Translated from Byulleten' éksperimental'noi biologii i meditsiny Vol. 49, No. 2, pp. 22–26, February, 1960.     

Metabolic and cardiovascular responses during voluntary pedaling exercise with electrical muscle stimulation

Purpose

We aimed to test the effect of additional electrical muscle stimulation (EMS) during moderate-intensity voluntary pedaling exercise on metabolic and cardiovascular responses.

Methods

Eleven healthy male subjects performed moderate-intensity pedaling exercise at a constant workload (80 % of ventilatory threshold) for 20 min while EMS was applied to thigh muscles from 5 to 10 min and from 15 to 20 min during the exercise.

Results

A significantly higher oxygen uptake (VO₂), heart rate, and respiratory gas exchange ratio were observed during the exercise periods with EMS despite the constant workload. These changes were accompanied by an elevated blood lactate concentration, suggesting the existence of additional fast-twitch motor unit (MU) recruitment during the exercise with EMS.

Conclusion

Our data suggest that the use of intermittent EMS during a constant load exercise mimics the high-intensity interval training, possibly due to additional fast-twitch MU recruitment and co-contractions of the quadriceps and hamstrings muscles, leading to higher anaerobic metabolism and a lower mechanical efficiency.     

Effects of 5-hydroxytryptamine on electrical responses of circular smooth muscle isolated from the guinea-pig gastric antrum.

The effects of 5-hydroxytryptamine (5-HT) on electrical responses of the membrane were investigated in circular smooth muscle isolated from the guinea-pig stomach antrum. Small segment of circular muscle tissue produced a periodical generation of slow potentials at frequency of 0.1-2 cycles min(-1), during random generation of unitary potentials. Application of 5-HT (10(-7)-10(-5) M) hyperpolarized the membrane and either increased or decreased the frequency of slow potentials, both with associated increase in amplitude of slow potential. These effects of 5-HT were abolished by methysergide. N(omega)-nitro-L-arginine (L-NA) increased the frequency of spontaneously generated slow potentials and also increased the frequency of slow potentials generated during stimulation with 5-HT, suggesting an involvement of the increased production of nitric oxide (NO) by 5-HT. Atropine did not alter spontaneous and 5-HT-induced electrical responses. The hyperpolarization produced by 5-HT was associated with a decrease in input resistance and time constant of the membrane. The amplitude of the 5-HT-induced hyperpolarization was increased in low [K(+)](o) solution and decreased in high [K(+)](o) solution or in the presence of glybenclamide, suggesting that the hyperpolarization was produced by activation of ATP-sensitive K-channels. The increase in amplitude of slow potentials by 5-HT may be secondary due to hyperpolarization of the membrane. The inhibition by 5-HT of the frequency of slow potentials may be partly due to the increased release of NO, however the mechanism by which dual effects of 5-HT on the frequency of slow potentials remains unsolved.     

Electrical responses of the smooth muscle of the guinea-pig cerebral artery to brief electrical stimulation

Electrical responses to brief electrical stimulation were investigated in the cerebral artery of a guinea-pig using a microelectrode. A single brief stimulus (0.05 ms) induced a spike potential followed by a depolarizing slow-potential, and these events were associated with muscle contraction. An outward current injected into the smooth muscle cell induced spike potential but failed to induce depolarizing slow-potential. These activities persisted in the presence of TTX (10(-6) M), guanethidine (5 X 10(-6) M), or atropin (10(-5) M). TEA (5 mM) enhanced the amplitude of the spike potential, but not that of the depolarizing slow-potential. When the external Na was reduced, the membrane transiently hyperpolarized. During this period, the depolarizing slow-potential could be evoked. In a Cl-deficient solution, the membrane depolarized and the amplitude of the depolarizing slow-potential decreased. From these observations it is believed that the contribution of K, Na, or Cl is minor. In a 20 mM-Ca solution, a brief stimulation induced neither spike potential nor depolarizing slow-potential, but did induce a hyperpolarizing slow-potential. The hyperpolarizing slow-potential was also induced in a Na-deficient solution, but only after completion of Na re-distribution across the membrane. These observations suggest that a substance released by brief stimulation produces a prolonged change in ionic conductances of the smooth muscle membrane, allowing the muscle to contract for a certain period.     

Some electrical properties of the rabbit anococcygeus muscle and a comparison of the effects of inhibitory nerve stimulation in the rat and rabbit

1. Simultaneous recordings of mechanical activity and membrane potential of individual smooth muscle cells have been made in the rabbit anococcygeus muscle and the effect of field stimulation on these examined.2. In the absence of tone the mean resting membrane potential was - 48 mV. In the stretched muscle spontaneous tone and rhythmic activity quite frequently appeared and this was associated with depolarization of the muscle cells.3. The response to field stimulation depended on the frequency of stimulation, the level of membrane potential and the presence of myogenic tone. The usual response to single pulses or low frequency stimulation was a hyperpolarization of up to 30 mV (mean 14+/-6.8 mV) after a latency of 185 msec and accompanied by muscle relaxation. Higher frequencies (over 8 Hz) produced an initial depolarization often with a spike potential and followed by hyperpolarization. The mechanical response in these instances was contraction or contraction followed by relaxation. At all frequencies rebound depolarization and an associated contraction followed the end of stimulation).4. Phentolamine (5x10(-6)M) and guanethidine (10(-6)M) blocked the initial depolarization and contraction but had no effect on hyperpolarization, muscle relaxation or rebound depolarization and contraction.5. The effect of field stimulation in the presence of guanethidine (4x10(-5)M) was re-examined in the rat anococcygeus. Single pulses were ineffective, repetitive stimulation produced muscle relaxation but no hyperpolarization comparable to the rabbit. Any oscillations in membrane potential were damped during field stimulation and sometimes a small hyperpolarization was produced with a maximum amplitude of 13 mV and a mean of 1.9+/-1.2 mV.6. The transmembrane potential at the peak of hyperpolarization in the rabbit was rarely more than -70 mV. Passive displacement of the membrane potential by current pulses altered the amplitude of the hyperpolarization and suggested that there was a reversal potential at between -80 and -90 mV.7. No change in input resistance could be measured during inhibitory nerve stimulation in either the rabbit or the rat but measurements based on electrotonic potentials indicated a reducation in membrane resistance, small in the rat but greater in the rabbit.8. These experiments suggest that in both species muscle relaxation is associated with an increase in ionic permeability and a move, at least in the rabbit muscle, towards an equilibrium potential of -80 to -90 mV. In view of the much smaller effect in the rat it is not clear whether this is the cause or at least the sole cause of the muscle relaxation.     

Coordinated interlimb compensatory responses to electrical stimulation of cutaneous nerves in the hand and foot during walking

It has been shown that stimulation of cutaneous nerves innervating the hand (superficial radial, SR) and foot (superficial peroneal, SP) elicit widespread reflex responses in many muscles across the body. These interlimb reflex responses were suggested to be functionally relevant to assist in motor coordination between the arms and legs during motor tasks such as walking. The experiments described in this paper were conducted to test the hypothesis that interlimb reflexes were phase-dependently modulated and produced functional kinematic changes during locomotion. Subjects walked on a treadmill while electromyographic (EMG) activity was collected continuously from all four limbs, and kinematic recordings were made of angular changes across the ankle, knee, elbow, and shoulder joints. Cutaneous reflexes were evoked by delivering trains of electrical stimulation pseudorandomly to the SP nerve or SR nerves in separate trials. Reflexes were phase-averaged according to the time of occurrence in the step cycle, and phasic amplitudes and latencies were calculated. For both nerves, significant phase-dependent modulation (including reflex reversals) of interlimb cutaneous reflex responses was seen in most muscles studied. Both SR and SP nerve stimulation resulted in significant alteration in ankle joint kinematics. The results suggest coordinated and functionally relevant reflex pathways from the SP and SR nerves onto motoneurons innervating muscles in nonstimulated limbs during walking, thus extending observations from the cat to that of the bipedal human.     

Mechanical response with reversed electrical response to noradrenaline by Ca-deprived arterial smooth muscle

1. When sheep carotid arteries had become electrically active after 30 min in Ca-free saline, noradrenaline 0.1 mM caused almost as much contraction as it did with Ca 1.25 mM present; it also caused slight electrical depolarization, usually with increased spike frequency, followed by electrical quiescence.2. In Ca-free saline with EDTA the arteries became profoundly depolarized, and their mechanical responses greatly reduced, within a few minutes. The mechanical responses to noradrenaline that remained were accompanied by electrical repolarization or, after longer periods in EDTA, by no electrical change.3. This residual mechanical responsiveness to noradrenaline, always tested at 36 degrees C, declined on average 26 times as rapidly during exposure to EDTA at 36 degrees C as at 5 degrees C and was not reduced by increasing the concentration of EDTA from 1.25 to 12.5 mM. This temperature-sensitivity was significantly too high to be explained by a diffusion-limited process.4. The results suggest that most of the tissue Ca responsible for contractility in simple Ca-free saline was either free in the extracellular space or in cellular stores that were discharged within a few minutes when free extracellular Ca was removed. They also indicate a small resistant Ca store which did not communicate with the exterior by diffusion, and part of which noradrenaline could utilize for contraction by means not dependent on depolarization conducted from the cell membrane.     

An analysis of the transmission of excitation from autonomic nerves to smooth muscle

1. An analysis has been made of the transmission of excitation from the hypogastric nerve to the smooth muscle cells of the guinea-pig vas deferens.2. Depolarization of single muscle cells with current pulses from an intracellular electrode gave local depolarizations of the cell membrane which were not propagated. The total membrane resistance after 100 msec of depolarization was 15 MOmega for depolarizations between 10 and 40 mV.3. Depolarization of some cell membranes with a current pulse during the excitatory junction potential (E.J.P.) decreased the amplitude of the E.J.P. from about 10 mV at 20 mV depolarization, to nearly zero at 60 mV depolarization. In some cells the E.J.P. was unchanged during depolarizations of 50 mV.4. The action of transmitter on the smooth muscle cell membrane continued for the duration of the E.J.P. Action potentials which occurred at various times during the E.J.P. failed to remove the remaining phases of the E.J.P.5. It was shown that the slow time course of the E.J.P. could not be due to the instantaneous and simultaneous release of transmitter from a number of relatively distant sources.6. It was shown that each smooth muscle cell was innervated by several axons. The serial sections examined with the electron microscope showed that a smooth muscle had either a single axon terminating within 200 A of the muscle or no axons terminating on it at all. Therefore transmitter must be released along the length of the axons as well as at the terminations of the axons.     

Effects of endogenous and exogenous nitric oxide on electrical responses of circular smooth muscle isolated from the guinea-pig stomach antrum.

The effects of endogenous and exogenous nitric oxide (NO) on electrical activity were investigated in circular smooth muscle preparations isolated from the guinea-pig stomach antrum. The actions of endogenous NO were evaluated from the effects of inhibition of NO synthesis by N(omega)-nitro-L-arginine (nitroarginine), while those of exogenous NO were assessed from the effects of SIN-1, an NO donor. Antral circular smooth muscle generated slow potentials periodically at a frequency of about 1 cycle per min (cpm), and unitary potentials were also generated in a random fashion in the interval between slow potentials. Application of nitroarginine (10(-5) M) increased the frequency of slow potentials, with no significant alteration of the resting membrane potential and amplitude of slow potentials. Frequency analysis of unitary potentials revealed that nitroarginine also increased the spectral density at 0.01-1 Hz frequency. The refractory period for the generation of slow potentials evoked by depolarizing pulses was about 10 s, but was decreased to 6 s by nitroarginine. In the presence of nitroarginine, SIN-1 (10(-9)-10(-7) M) reduced the amplitude and frequency of slow potentials: low concentrations (<10(-8) M) reduced only the frequency of slow potentials, while higher concentrations (10(-8)-10(-7) M) reduced both the amplitude and frequency of slow potentials, in a concentration-dependent manner, before abolishing the slow potentials. The power spectrum of the unitary potentials indicated that SIN-1 (>10(-8) M) reduced the spectral density at 0.01-1 Hz frequency. The refractory period for the generation of slow potentials was increased again to about 10 s by SIN-1. Thus, the excitatory effects of nitroarginine could be antagonized by SIN-1, suggesting that the inhibitory effects of endogenous NO are comparable to those of exogenous NO produced by SIN-1. The results also suggested that the effects of NO on smooth muscle are insignificant and NO selectively inhibits the activity of intramuscular interstitial cells of Cajal (ICC-IM).