Potassium Contracture Research Articles

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.

L-glutamate and potassium-induced contractures in denervated cockroach muscles

1. The effects of denervation on the mechanical responses to various concentrations of l-glutamate in retractor unguis muscle of cockroach ( Perilpaneta americana) was examined, comparing them with contractures induced by high potassium saline. 2. The dose-response curve was shifted to the lower concentrations of l-glutamate after 9 days of denervation. 3. Following a transient increase in the maximum contracture tension, it decreased with days after denervation and reached a constant level by several days. However, from 16 to 20 days after denervation, the tension ratios of the maximum glutamate to potassium contractures were significantly higher than that of the innervated muscles. 4. A sustained contracture was observed on and after treatment of l-glutamate in the denervated muscle. Pretreatment of the muscle by concanavalin A facilitated to induce l-glutamate contracture. 5. It was suggested that the sensitivity of the muscle membrane to l-glutamate was increased in the denervated muscle.

A strain-gauge myograph for isometric measurements of tension in isolated small blood vessels and other muscle preparations

Isometric tension measurements in vitro on small vessels are important in studies of pharmacodynamic vascular effects of drugs and autacoids. The present study describes a precision myograph equipped with a newly constructed highly isometric strain-gauge transducer used in conjunction with a very sensitive, low-noise and virtually drift-free dc-amplifier. The isometric quotient of the transducer, defined as deflection in micrometer per g of force, is about 5.2. The transducer resonance frequency is 341 c sec with a damping constant of 1.05. The highest sensitivity of the transducer in connection with the matching dc-amplifier is about 170 mVg/V force. The practical working range of the myograph is from 2 mg to about 10 g of applied tensile force. Tubular segments of vessels with internal diameters down to 150 μm can be mounted in the myograph on two stainless-steel pins submerged in a 5 ml preparation bath. Precise mounting and setting of passive (resting) tension is assured by the use of micrometer-equipped adjustable miniature mounting tables suspended by roller bearings. The myograph may be provided with electrodes for field-effect stimulation of the vessel preparations. Other muscle preparations may also be mounted in the myograph. Application of the myograph in studies on rabbit coronary arteries is demonstrated by a length-tension diagram obtained in experiments with tubular segments (inner diameter about 500 μm) of the circumflex branch on the left coronary artery, which shows a typical course in development of active and passive tension of the smooth musculature when exposed to alternating potassium depolarization and increasing mechanical stretching. The potassium contracture of the coronary vessels and the log-concentration/response relationship at accumulating potassium concentrations were also investigated.

TMB-8 can block twitches without blocking high K + or caffeine induced contractures in frog's skeletal muscle

TMB-8 [8-(diethylamino)-octyl-3,4, 5trimethoxybenzoate] is known to inhibit calcium ion dependent processes in several tissues by stabilizing some intracellular stores of membrane-bound calcium. TMB-8 was used to study the excitation-contraction (E-C) coupling process in frog's skeletal muscle. TMB-8 (5 × 10 −5 − 10 −4 M) blocked electrically evoked twitches but not high K + (123 mM)- or caffeine (2.36 mM)-induced contractures in isolated, curarized toe muscles. TMB-8 (10 −4 M) produced a small decrease (16%) in the action potential of frog's sartorius muscle fibres. However, reducing extracellular Na + to 44.7 mM produced a similar reduction (17%) in action potential amplitude but did not suppress the twitch; i.e. it produced only a small increase (about 10%) in twitch amplitude. It is known that potassium contractures are produced by extracellular Ca ++ ions which enter through calcium channels in the t-tubules and that caffeine produces contractures by sensitizing the sarcoplasmic reticulum to Ca ++-induced Ca ++ release. The present results suggest that TMB-8 blocks twitches by preventing the release of Ca ++ ions bound to the intracellular surface of the t-tubular membrane which is often called the store of ‘trigger-calcium’ ions.

Effect of oxytocin on spontaneous electrical and mechanical activities in pregnant human myometrium

Spontaneous intracellular electrical activity and contraction of pregnant human myometrium were recorded by the single sucrose-gap method, and the effects of oxytocin on the muscle were studied. In pregnant human myometrium at term, both plateau and spike types of action potentials were observed. All contractions were well synchronized with each action potential. Oxytocin, 10−2 U/ml, potentiated spontaneous contractions by enhancing the plateau part of action potentials; spike-type configuration became plateau. When extracellular ionized calcium was removed, spontaneous activities disappeared, while 10−2 U/ml of oxytocin could evoke action potentials and contractions but these were smaller than those of the controls. Spontaneous activities also disappeared when ionized calcium was increased to 5 mmol/L, but oxytocin evoked plateau potentials and contractions remarkably. Diltiazem (ionized calcium antagonist), 10−6 gm/ml, suppressed the spontaneous activity, but oxytocin evoked action potentials and contractions in high frequency, the duration of the action potential being short and the contraction being small. In the presence of 10−4 gm/ml of diltiazem, 10−2 U/ml of oxytocin could not evoke any action potentials but did evoke small and long contractures, while in a high ionized potassium contracture experiment, oxytocin potentiated the tonic phase. These results suggest that oxytocin can increase spontaneous contractions by enhancing plateau potentials and that this effect requires sufficient extracellular ionized calcium. In this potentiation, the effects on frequency and amplitude of contractions might vary. It is also suggested that oxytocin may evoke a contracture in the absence of an action potential by releasing calcium from intracellular storage sites.

Effects of external calcium on potassium contractures in tonic muscle fibers of the frog (Rana pipiens).

K+ contractures of tonic bundles from cruralis muscle of the frog were studied with different K+ concentrations (10-120 mM). K+ contractures had an initial transient phase followed by a sustained tension. The amplitude of the sustained tension diminished with high K+ concentration (80-120 mM). However, in all cases, tension was maintained for several minutes. External Ca2+ reduction practically abolished the sustained phase of the K+ contractures. The initial phase was also reduced and tension spontaneously relaxed. The curve relating the peak tension with log [K+]o, showed that the threshold was not affected but the peak tension was reduced to about 70% in low-Ca2+ saline (0 Ca2+ + 3 mM-Mg2+) and 50% in Ca2+-free saline (1 mM-EGTA + 3 mM-Mg2+). The dependence of the sustained tension on external Ca2+ was further confirmed by Ca2+ withdrawal and re-establishment and/or by Ni2+ substitution for Ca2+ before or during K+ contractures. These results indicate that external Ca2+ had to be continuously present to maintain the tension during K+ contractures and that Ni2+ was not able to restore the normal temporal course of K+ contracture. The sustained phase was diminished by blocking agents of Ca2+ channels, such as nifedipine (1 microM) and diltiazem (1-10 microM). The present results can be explained by a direct control of the Ca2+ currents on K+ contracture or by specific interactions between external Ca2+ and Ca2+-binding sites in the membrane.

Phasic contractions of canine and human coronary arteries induced by potassium channel blockers.

To gain insight into mechanisms underlying phasic coronary vasospasm in patients with variant angina pectoris, we studied whether phasic contractions could be induced in isolated canine and human coronary arteries by agents which block potassium channels. Phasic contractions of canine coronary arteries were always induced by 3,4-diaminopyridine (10(-2) M) and less frequently by 4-aminopyridine (10(-2) M). These agents also caused phasic contractions in human, swine and monkey coronary arteries and in canine basilar, carotid, renal and femoral arteries. The cycle length of phasic coronary contractions ranged from 30 sec to 1 hour, and the developed tension was 2.5 times greater than for potassium contractions. The contractions continued for more than 11 hours. Morphologically, perinuclear vacuolization, a characteristic change of vasospasm, appeared in the coronary smooth muscles. The phasic contractions were not eliminated by tetrodotoxin, atropine, phentolamine or yohimbine, but they were eliminated by nicorandil which activates potassium channels and nifedipine which blocks slow calcium channels. The results indicate that potassium channel blockers can induce phasic arterial contractions.

Nifedipine inhibits human bronchial smooth muscle contractions induced by leukotrienes C4 and D4, prostaglandin F2 alpha, and potassium.

The effects of nifedipine, a calcium channel blocker, on human bronchial smooth muscle contractions induced by leukotrienes C4 and D4 (LTC4, LTD4), prostaglandin F2 alpha (PGF2 alpha), and potassium were studied in vitro. The LTC4, LTD4, PGF2 alpha, and potassium caused bronchial smooth muscle contractions. After incubation with nifedipine at 2.9 X 10(-6)M, the contractions caused by LTC4, LTD4, PGF2 alpha, and potassium were significantly decreased. Nifedipine also significantly reversed human bronchial smooth muscle contractions previously initiated by these agonists. The effect of nifedipine on potassium contraction was significantly greater than that on LTC4, LTD4, and PGF2 alpha-induced contractions. The pharmacologic feature of nifedipine is that it inhibits the calcium influx associated with membrane depolarization. Therefore, these results suggest that there are two mechanisms of calcium movements in human bronchial smooth muscle contractions induced by LTC4, LTD4, and PGF2 alpha: membrane-depolarization-dependent and -independent mechanisms.

Excitation-contraction coupling and contractile properties in denervated rat EDL and soleus muscles.

The long-term (up to ten weeks) effects of denervation on isometric tension and potassium (K) contractures were studied in isolated bundles of fibres from rat extensor digitorum longus (EDL) and soleus muscles, at 21 degrees C, bathed in solutions with low concentrations of chloride ions (to reduce the effects of high membrane chloride conductance). The usual increases in twitch time course and twitch for tetanus ratio were attributed to changes in excitation-contraction coupling because both developed between one to three weeks after denervation. Transient changes during the first week in the time course of the twitch and twitch to tetanus ratio in EDL, and post-tetanic twitch size and specific tension in both muscles, were attributed to the surface membrane electrical properties which are maximally altered within three days after denervation. In contrast to results obtained in solutions of normal chloride concentration, the resting membrane potentials of chronically denervated fibres were hyperpolarized and spontaneous action potentials and fibrillations were seen. The time to the peak of the K-contracture was faster than normal and very slow inactivation kinetics appeared in the decay phase. Mechanical repriming was normally slower in EDL than in soleus but similar rates were seen in the two muscles after denervation. These changes in the K-contracture were not due to a change in the relationship between membrane potential and potassium ion concentration. It is concluded that denervation caused changes in the kinetics of mechanical activation, inactivation and repriming and that these aspects of excitation-contraction coupling are normally controlled by an influence of the motor nerve.

Calcium influx in contracting and paralyzed frog twitch muscle fibers.

Calcium uptake produced by a potassium contracture in isolated frog twitch fibers was 6.7 +/- 0.8 pmol in 0.7 cm of fiber (mean +/- SEM, 21 observations) in the presence of 30 microM D600. When potassium was applied to fibers paralyzed by the combination of 30 microM D600, cold, and a prior contracture, the calcium uptake fell to 3.0 +/- 0.7 pmol (11): the fibers were soaked in 45Ca in sodium Ringer for 3 min before 45Ca, in a potassium solution, was added for 2 min; each estimate of uptake was corrected for 5 min of resting influx, measured from the same fiber (average = 2.3 +/- 0.3 pmol). The calcium influx into paralyzed fibers is unrelated to contraction. This voltage-sensitive, slowly inactivating influx, which can be blocked by 4 mM nickel, has properties similar to the calcium current described by several laboratories. The paired difference in calcium uptake between contracting and paralyzed fibers, 2.9 +/- 0.8 pmol (16), is a component of influx related to contraction. Its size varies with contracture size and it occurs after tension production: 45Ca applied immediately after contracture is taken up in essentially the same amounts as 45Ca added before contraction. This delayed uptake is probably a "reflux" refilling a binding site on the cytoplasmic side of the T membrane, which had been emptied during the prior contracture, perhaps to initiate it. We detect no component of calcium uptake related to excitation-contraction coupling occurring before or during a contracture.

The cellular distribution of calcium in freeze-dried rabbit vas deferens using EM autoradiography.

The role of calcium in initiating smooth muscle contraction is widely accepted. The sources of this calcium are thought to be located both intracellularly and extracellularly. We have recently developed a method by which the cellular localization of calcium in smooth muscle can be determined. This method involves exposing the tissue to 45Ca, rapidly freezing and vacuum dehydrating the tissue, and preparing the tissue for electron microscopic autoradiography (EM ARG). In the present study the distribution of calcium in control and potassium-contracted tissue of the rabbit vas deferens was compared. No significant differences in distribution were observed in the two treatments. This finding provides morphological support for the hypothesis that the calcium used in potassium-induced contraction is primarily of extracellular origin. In addition, significant sequestration by intracellular organelles does not occur during a potassium contraction. In other experiments, the effect of rinsing tissue in cold calcium prior to freezing was investigated. From these data is appears that calcium is removed from the cytoplasmic matrix, plasma membrane, and organelles in a nonuniform manner. Further investigation into these findings should enable us to characterize more precisely the intracellular redistribution of calcium that occurs as a result of a variety of physiological manipulations.

Effects of conditioning depolarization, repriming, and external calcium reduction on the potassium contractures in frog single twitch muscle fibers.

The inactivation of potassium (K) contractures induced by prolonged conditioning depolarization and repriming after 190 mM K+ contractures were studied at both room and low temperatures using frog single twitch muscle fibers. Effects of conditioning K+ depolarization and external Ca2+ reduction on the biphasic K contractures were also studied. When the conditioning depolarization with 15-30 mM K+ was prolonged, the peak tension of test 190 mM K+ contractures was inhibited in parallel with the shortening of plateau duration at both room and low temperatures. Such parallel changes were also observed during the early stage of repriming after 190 mM K+ contracture at both temperatures. On the other hand, the peak tension of the secondary component of the test 80 mM K+ contractures was rather potentiated by the conditioning depolarization, even though the time course of the component was markedly shortened. The foregoing changes were also illustrated in a diagramatic way. These results suggested that the peak tension and the time course of the test K contractures after conditioning depolarization and during repriming may be determined by a balance between the activation and the inactivation processes of the contractures. This view is supported by the results obtained under conditions in which the concentration of external Ca2+ was reduced. Furthermore, based on additional experimental results, it was suggested that the initial component is not accompanied by an inactivation process and that Ca uptake by sarcoplasmic reticulum may partially contribute to determining the spontaneous relaxation phase of the secondary component, at least at room temperature.

Effects of sodium vanadate on the smooth muscle of the guinea-pig vas deferens.

Effects of sodium vanadate on electrical and mechanical activities of smooth muscle of the guinea-pig vas deferens were investigated. Sodium vanadate of concentrations higher than 5 X 10(-6) M caused an elevation of basal tension and, at concentrations higher than 5 X 10(-4) M, initiated spontaneous contractions. These effects were not blocked by treatments with reserpine, tetrodotoxin or phentolamine. Treatment with ouabain also did not block the tension development by sodium vanadate. Sodium vanadate caused slight depolarization of membrane and potentiated spike activities. The phasic contraction of potassium contracture was potentiated by sodium vanadate in a similar manner to pretreatment with 15 mM K+ or with ouabain, both treatments occasioning slight depolarization. Sodium vanadate also caused tension development in K-depolarized preparetions. Contrastingly, drug-induced contractions were not significantly affected by sodium vanadate. It is suggested that sodium vanadate acts directly on smooth muscles and causes tension development without relation to Na, K-ATPase activity. Changes in the membrane electrical activities may be part of the cause of contraction. However, it can also initiate tension development without membrane potential changes, presumably acting on intracellular Ca binding sites.

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

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

Ruthenium red: differential effects on excitation and excitation-contraction coupling in frog skeletal muscle.

The effects of purified ruthenium red (RR6+) on action potentials, resting potentials, twitches, tetani and potassium contractures in fibre bundles dissected from frog leg muscles were compared to the effects of supranormal concentrations of Ca2+. The effects of RR6+ include a shift of action potential threshold and reduction in rates of depolarization, repolarization and conduction velocity. Resting potentials and action potential overshoots are unaffected by RR6+ in concentrations of 10 microM or less. Higher RR6+ concentrations cause membrane depolarization. Low concentrations of RR6+ cause twitch potentiation but do not affect tetanus tension. Unlike Ca2+, RR6+ has no effect on the mechanical activation curve. However, crude preparations of RR6+ contain an unidentified contaminant which does shift the mechanical activation curve to the right, i.e. it increases mechanical threshold. RR6+ itself appears to be largely excluded from the t-system.

Potassium contractures and mechanical activation in rat skeletal muscle: effects of multivalent cations, temperature and tetracaine.

The effects of cations, temperature and tetracaine on potassium-induced contractures of rat soleus and extensor digitorus longus (e.d.l.) muscles were investigated. In the soleus, the threshold for the potassium contracture was lower (10-20 vs 20-40 mM), the peak amplitude was up to fourteen times larger, and the time course was about one half that in the e.d.l. muscle. The extent of inactivation of a test potassium contracture was directly related to the concentration of potassium in the conditioning solution and the period of exposure. Removal of calcium reduced the amplitude and time course of potassium contractures in both preparations. Addition of cobalt (10 mM) reduced the amplitude but prolonged the time course of contractures. Exposure of muscles to tetracaine (10(-5)-10(-6) M for 30 min) increased, but higher concentrations reduced, the amplitude of potassium contractures. When present for one minute, tetracaine (1 mM) moved the potassium activation curve to higher, and the potassium inactivation curve to lower, potassium concentrations.

Synaptic and non-synaptic effects of molluscan cardioexcitatory neuropeptides on locust skeletal muscle

Neurally evoked contractions of the locust jumping muscle are potentiated by the molluscan cardioexcitatory peptide (FMRFamide) and related peptides. This effect is due to a synaptic and a non-synaptic action of the peptides: (1) excitatory synaptic potentials are increased; (2) excitation-contraction coupling becomes more efficient, as indicated by an increased strength of potassium contractures. Octopus heptapeptide (YGGFMRFamide), which contains the entire enkephalin structure (YGGFM), is 100 times more potent than FMRFamide. Structure-activity studies indicate that the observed actions are not mediated by opioid receptors but are dependent on the C-terminal structure of the peptides.

Mechanism of action of barium ion on rat aortic smooth muscle.

The mechanism of action of barium ion on the aortic smooth muscle of the normal rat was investigated using in vitro calcium-depleted aortic strips. Aortic strips were depleted of calcium by repeated exposure to norepinephrine in a calcium-free bathing solution. Although calcium depletion abrogated the response of strips to catecholamines and depolarizing agents, the response to barium chloride remained quantitatively intact. The calcium influx blocker D 600 prevented the contractile response to barium but not to catecholamines, whereas phentolamine prevented the response to catecholamines but not barium. The strip response to barium was depressed by a twofold increase in extracellular magnesium concentration whether the strip was intact or calcium depleted. Although increased concentrations of calcium in the extracellular medium inhibited the contractile response to potassium ion, increases in barium merely potentiated the potassium contracture. These findings indicate that barium produces its contractile effect on vascular smooth muscle by a direct intracellular interaction with the contractile or regulatory proteins. Barium enters these cells via calcium influx channels and is probably not sequestered in a physiologically releasable pool. Unlike calcium, barium does not stabilize the smooth muscle sarcolemma when present in high concentration.

Effect of sodium gradient on the rate of relaxation of rat mesenteric small arteries from potassium contractures.

We have measured the relaxation of rat mesenteric small arteries (internal diameter ca. 200 micron) from a potassium contracture under conditions where the transplasmalemmal sodium gradient was altered. Adjustment of the sodium gradient was made either by exposing vessels to ouabain (1 mM) for different periods to vary the intracellular sodium concentration (Nai), or by varying the sodium concentrations of the relaxation solutions (Nao). Under normal conditions (where we have previously shown Nai is about 19 mM), vessels relaxed completely within 1 min in low sodium solutions (Nao = 25 mM) using either sucrose, choline-Cl or MgCl2 as substitutes. However, the relaxation (measured after 1 min) was reduced when sucrose substitution was used, and further experiments were made using this substitute. Here it was found that exposing vessels to ouabain, for example 90 min (thus increasing Nai to about 73 mM) and then performing the relaxation in a low sodium solution gave only about 50% relaxation after 1 min. Experiments with intermediate sodium concentrations in the relaxation medium showed that reduction to 100 mM had no significant effect on the relaxation, and that it was only below 50 mM that effects were most marked. The effects of altered sodium gradient were eliminated if the calcium concentration of the relaxation medium was zero. The results indicate that under normal physiological conditions, small changes in sodium gradient have little effect on calcium metabolism of rat mesenteric small arteries, but also that under extreme conditions net calcium extrusion does appear to be reduced by a reduced sodium gradient.

Effects of external calcium reduction on biphasic potassium contractures and action of divalent cations on the calcium reduction in frog single twitch muscle fibers.

The effects of external Ca2+ reduction on the biphasic potassium (K) contractures and the action of divalent cations under conditions of the Ca2+ reduction were examined in detail, using frog single twitch muscle fibers. The peak tension of the initial component of 80 mM K+ contractures was greatly potentiated by exposing the fiber to low Ca2+ solution for 30 sec, and the degree of this potentiation was decreased with increasing the exposing time. In contrast, the peak tension of the secondary component was rapidly inhibited by Ca2+ reduction. The potentiation of the initial component was removed by 3 mM Mg2+ or 0.5 mM Ni2+. The inhibition of the secondary component, especially the shortening of its time course, was reversed partially by 3 mM Mg2+ and almost completely by 0.5 mM Ni2+. The difference between the inhibitory effect of 10 mM Mg2+ or 1-3 mM Ni2+ on the initial component and that on the secondary component was also demonstrated. The tension development of the secondary component was completely inhibited by external Ca2+ reduction for 20 min, but it was observed in the presence of 3 mM Mg2+, although its time course was shorter. These results indicate that the actions of Mg2+ and Ni2+ on the initial component differ from those on the secondary component and suggest the possible mechanisms of the actions of these divalent cations on the biphasic K contractures.