The influence of high potassium depolarization and acetylcholine on calcium exchange in the rat uterus

Net and radioactive calcium movements were studied in the rat uterus during stimulation with acetylcholine and high potassium solutions. High potassium did not affect the efflux of intracellular Ca⁴⁵, but was able to release Ca⁴⁵ from a small parallel Ca fraction which was believed to be located in the cell membranes. High potassium did markedly slow the influx of Ca⁴⁵ and caused a net calcium efflux. Acetylcholine had no effect on calcium movements in polarized myometrium, but it increased the Ca⁴⁵ influx in depolarized uteri. Ca⁴⁵ taken up during contraction exchanged more slowly during subsequent efflux than Ca⁴⁵ taken up at rest. The results were interpreted as supporting the hypothesis that myometrial contraction is induced by a release of calcium from the inside of the cell membrane and the endoplasmic reticulum, and relaxation follows the removal of ionic cytoplasmic calcium by these same structures.     

Induction of Actin-Based Cytoplasmic Contraction in the Siphonous Green Alga Acetabularia (Chlorophyceae) by Locally Restricted Calcium Influx

Perfused cell segments dissected from the stalk or from detached cap ray chambers of Acetabularia were used as an experimental system to study the induction of cytoplasmic contractions and concurrent cytoskeletal changes in plant cells. Immunofluorescence microscopy revealed that the actin cytoskeleton quickly rearranges upon induction of contraction by forming bundles oriented circumferentially around the affected area, whereas microtubules were not detected. Contraction is blocked by cytochalasin D or N-ethylmaleimide but is unaffected by microtubule specific inhibitors. Contraction requires external Ca²⁺ at concentrations of 1 μM or more, but fails to occur below 0.1 μM. Higher concentrations of Ca²⁺ up to 10 mM have no adverse effect. Contraction is prevented in the presence of micromolar Ca²⁺ by either 1 mM of the calcium channel blocker LaCl₃ or 10 μM of the calmodulin inhibitor fluphenazine. Calcium ionophore A 23187 (1 μM) does not perturb wound contraction per se but causes the entire cytoplasm of wounded or unwounded cells to contract slowly. These data suggest that a localized influx of calcium ions at the wound edge causes major rearrangements in the distribution of cytoskeletal actin prior to contraction in Acetabularia. An involvement of calmodulin in calcium signaling is proposed.     

Differential calcium dependence of contractile responses and 86Rb efflux from the rabbit aorta induced by vasoactive stimuli

⁸⁶Rb was used to monitor potassium movements in strips of rabbit aorta simultaneously with measurements of tension. Histamine, noradrenaline, the prostaglandin endoperoxide analogue U46619, angiotensin II, and 144 mM K⁺ each induced an increase in ⁸⁶Rb efflux concomitantly with contraction. For the first four agonists there was a rank-order correlation between the contractile response and ⁸⁶Rb efflux, but 144 mM K⁺ induced a massive increase in ⁸⁶Rb efflux although it was the weakest contractile stimulus. Contraction and increase in ⁸⁶Rb efflux-induced K⁺ were both reduced by verapamil, which blocks voltage-sensitive calcium channels, implying that both effects of K⁺ were mediated mainly by a depolarisation-induced influx of calcium. Noradrenaline increased both tension and ⁸⁶Rb efflux through an action on alpha-adrenoceptors, but its effect on efflux, unlike its effect on tension, was apparently totally dependent on the presence of extracellular calcium. Experiments performed in the presence of lanthanum, which blocks calcium influx, showed that the intracellular store of calcium released by noradrenaline apparently played no role in inducing ⁸⁶Rb efflux, although it could trigger contraction. Lanthanum also blocked contraction induced by K⁺ but less effect on the increase in ⁸⁶Rb efflux induced by K⁺. Thus, agonist-induced vascular contraction and ⁸⁶Rb efflux can be dissociated, but under normal conditions all the contractile stimuli tested induced ⁸⁶Rb efflux.     

Contraction of protoplasm. IV. Cinematographic analysis of the contraction of some peritrichs

The contraction and relaxation of Vorticella difficilis, V. campanula and Carchesium sp. were studied by high speed cinematography. In Vorticella it was shown that coiling of the stalk usually started near the zooid and spread downwards; the point of initiation bore no relation to the position of the stimulating electrodes. Contraction took about 5 msec to complete, and the fully contracted animals were 29 ± 3.9% of their original lengths. The zooids were 66 ± 5.0% and the stalks 14 ± 6.0% of their original lengths (V. difficilis). The shortening of the stalk was mostly in the form of coiling. Measurement of the myoneme length demonstrated that its real shortening was less than 10%. Thus the contraction is virtually isometric, producing a helical deformation of the stalk. As the stalk contracts it takes the form of a steeply pitched helix. This change in shape should produce rotational forces on the zooid (torque). Physical models of similar proportions produced about 1.5 revolutions of torque for similar changes in pitch. However during contraction no turning of the zooid was detected, though rotation did occur after the completion of contraction. In Carchesium the contraction is not so isometric, the myoneme apparently shortening by 20%. While the coiled shape of the contracted Vorticella stalk can be explained by its acentric structures, the stalk of Carchesium is much more symmetrical in cross-section, demonstrating that a high acentricity is not necessary for helical coiling. In all three species there seems to be some separation of the control of zooid and stalk contraction.     

Use-dependent decline in rat aorta sensitivity to contraction by potassium.

Aortic rings excised from rats at 12 weeks of age showed a decrease in responsiveness during repeated contraction by increasing potassium concentration. By comparison, aortic rings obtained from rats at 22 and 26 weeks exhibited less loss or an increase in responsiveness to high potassium concentration during repeated contraction. The decrease in responsiveness to potassium in aortae of young rats was not due to the extended interval of incubation of these tissue in vitro. Aortic rings incubated without stimulation for 4 h following a reference contraction showed no change in contractile response to potassium. However, the magnitude of loss in responsiveness to potassium did appear to be related to the potassium concentration and the length of time the tissues were exposed to the high potassium solutions. Contraction of the tissue at 60 versus 30 mM KCl or extending the interval in depolarizing solution from 15 to 60 min significantly enhanced the decline in tissue responsiveness to potassium. The interruption of a series of potassium-induced contractions by exposure of the tissue to contractile (serotonin, norepinephrine) or relaxant (acetylcholine, isoproterenol) stimuli had no effect on the loss in responsiveness to potassium. However, injection of the calcium channel agonist, Bay K 8644, into the incubation media restored responsiveness to potassium. Concentration-response curves indicated that both sensitivity and the maximal response to potassium were reduced in aortic rings repeatedly contracted with potassium.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of STIM1 and SOCE in smooth muscle contractility

Contraction is a central feature for skeletal, cardiac and smooth muscle; this unique feature is largely dependent on calcium (Ca²⁺) signaling and therefore maintenance of internal Ca²⁺ stores. Stromal interaction molecule 1 (STIM1) is a single-pass transmembrane protein that functions as a Ca²⁺ sensor for the activation store-operated calcium channels (SOCCs) on the plasma membrane in response to depleted internal sarco(endo)plasmic (S/ER) reticulum Ca²⁺ stores. STIM1 was initially characterized in non-excitable cells; however, evidence from both animal models and human mutations suggests a role for STIM1 in modulating Ca²⁺ homeostasis in excitable tissues as well. STIM1-dependent SOCE is particularly important in tissues undergoing sustained contraction, leading us to believe STIM1 may play a role in smooth muscle contraction. To date, the role of STIM1 in smooth muscle is unknown. In this review, we provide a brief overview of the role of STIM1-dependent SOCE in striated muscle and build off that knowledge to investigate whether STIM1 contributes to smooth muscle contractility. We conclude by discussing the translational implications of targeting STIM1 in the treatment of smooth muscle disorders.     

Relationship between cellular electrolytes and the contraction force of isolated guinea-pig atria with special regard to the extracellular space volume

A micromethod is described which allows the quantitative evaluation of the extracellular space volume of isolated guinea-pig atria (> 15 mg tissue wet weight) by means of an enzymatic estimation of inulin. In addition, the acidic tissue extracts are analysed for the potassium, sodium, magnesium, calcium and ⁴⁵calcium content. Taking into account the individual extracellular space volumes, it is possible to calculate the cellular electrolyte content and relative specific ⁴⁵calcium activity of each atrium. Our experiments reveal under steady state conditions a significant correlation between the force of contraction and the cellular electrolyte content. Atria with a higher contraction force were found to contain a significantly elevated cellular potassium content and a lower content of cellular sodium and calcium than atria with a smaller contraction force. The decrease of cellular calcium content is exclusively caused by the lowering of the fraction of unexchanged tissue calcium. The significance of the results is discussed with respect to the events leading to contraction and relaxation of the heart muscle.     

Anodal Contraction of Paramecium Protoplasm*

SYNOPSIS. Paramecia immobilized on an agar surface were stimulated with pulses of 2–8 mamps/cm² with intervals ranging from 1.0 to 1000 msec. High speed cine films (1500-6000 frames per second) of the animals' responses were analyzed to determine changes in body length and the effects on cilia and trichocysts. At current densities in the range of 1.0 mamp/cm², rapid ciliary reversal occurred. Above this value the paramecia shortened. This body contraction can be very rapid, resulting in shortening to 95% of the body length in 0.95 msec. At all levels of stimulation, contraction of the body length occurred 1st anodally, then cathodally. Extensive trichocyst extrusion occurred 1st at the anode and later at the cathode at current densities greater than 5 mamp/cm² in the agar. Results of this study indicate that the protoplasm of paramecia is capable of very fast contraction in response to electric stimulation and that the initial response is always anodal.     

Two calcium currents in the somatic membrane of mollusc neurons

Two new types of calcium channels were discovered during research in ionic currents in the somatic membrane ofHelix pomatia neurons, using an intracellular perfusion technique. Apart from the principal calcium current described in the literature with a holding potential of about –110 mV, an additional calcium current was observed activated at depolarizations of –40 to –80 mV and was not reduced when the cell was perfused with solutions containing fluoride anions. The kinetics of this current were well described in the context of the Hodgkin and Huxley model with a time constant of activation of 6–8 msec and of inactivation of 300–600 msec. It increased in amplitude as the Ca⁺⁺ rose in the cellular environment but was reduced by extracellular addition of the Ca⁺⁺ antagonists Co⁺⁺, Ni⁺⁺, and Cd⁺⁺, and the organic blockers nifedipine and verapamil. The association constants of these substances with corresponding channels determined from the maximum of the current-voltage relationship were 2 (Ca⁺⁺), 3 (Co⁺⁺), 0.06 (nifedipine), and 0.2 mM (verapamil). The properties detected in this component of calcium conductance are compared with those of calcium channels in other excitatory formations and its possible functional role is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 627–633, September–October, 1985.     

Control of contractility in Spirostomum by dissociated calcium ions

The freshwater protozoan, Spirostomum ambiguum, exhibits generalized contraction when electrically stimulated with a DC pulse. Light and electron microscopic studies show a subcortical filamentous network, believed responsible for generating contractile tension, in association with vesicles which were shown to accumulate calcium oxalate precipitates. Organisms microinjected with the calcium-sensitive, bioluminescent protein, aequorin, emit light when stimulated to contract. Analyses of cinefilm records of electrically induced contraction indicate that contraction may occur up to 25 msec after the onset of stimulation at a point when the calcium-aequorin light emission is at a peak. The evidence shows that calcium release from an interval compartment is directly associated with the onset of contraction in Spirostomum, and that the removal of calcium, through some internal sequestering mechanism, signals relaxation.     

Positive and negative inotropic effects of elevated extracellular potassium level on mammalian ventricular muscle

The effect of moderate elevation in extracellular potassium concentration (up to 12 mM) on contraction of cat ventricular muscle was examined. Isometric force development was recorded from eight excised trabeculae and from six coronary-perfused in situ papillary muscle preparations. Contraction in the steady state was variably affected, sometimes decreasing monotonically, sometimes remaining unchanged, with increasing potassium level. In 11 of these 14 preparations, the steady state was preceded by a transient period in which the contraction was augmented. In addition, eight excised trabeculae were used in an experimental arrangement designed to distinguish between inotropic effects caused by potassium-induced alterations in the action potential and other, more direct, effects of this ion on contraction. The negative inotropic effect is attributable to a potassium-induced reduction in the amplitude and/or duration of the action potential plateau. The positive inotropic effect was found in experimental arrangements where effects of the potassium-rich medium on action potential time-course were effectively "buffered." The positive inotropic effect thus depends on the presence of the elevated potassium concentration and can occur independently of effects on the action potential time-course.     

Measurements of contraction latencies to mechanical and electrical stimulation of the protozoan, Spirostomum ambiguum.

Measurements made on contraction latencies in Spirostomun suggest that mechanical stimulation causes contractions to be initiated by the release of small amounts of calcium from a store tightly coupled to the contractile apparatus. Contraction to electrical stimulation appears to result from the gross electrophoretic mobilization of large amounts of calcium from a loosely coupled store. Contraction latencies to mechanical stimulation were three milliseconds and were independent of stimulus strength, previous stimulation, and contraction probability. For 0.5-millisecond biphasic electrical stimulation the contraction latencies varied widely. Latencies to initial contractions were dependent on stimulus strength: from 1.0 milliseconds for a stimulus that caused a 100% probability of contraction to 2.0 milliseconds for a stimulus that caused a 10% probability of contraction. Latencies of contraction to electrical stimulation were also dependent upon previous stimulation, lengthening to over 300 milliseconds after ten minutes of stimulation. Initial contraction latencies were not affected by previous stimulation to the other (electrical or mechanical) stimulus modality. Repeated electrical stimulation also reduced the animal's resting length and slowed the rate of post contraction re-extension, whereas mechanical stimulation did not have these effects.     

Electrical and Mechanical Responses in Deep Abdominal Extensor Muscles of Crayfish and Lobster

Electrical and mechanical studies have been made of the deep abdominal extensor muscles, medial (DEAM) and lateral (DEAL), of crayfish and lobster. The medial muscle responds to direct (intracellular) and indirect stimulation with a transient membrane depolarization which exhibits the properties of a propagated non-decremental action potential but does not overshoot the zero level. The amplitude is about 30 mv in crayfish and 50 mv in lobster. It is followed by a fast all-or-none twitch whose duration at 20°C is 30 to 50 msec. and whose developed tension is 500 gm/cm² or about half the tetanic value. Membrane potential is K⁺-dependent and immersion in high K⁺ induces a brief transient tension rise as in other twitch-type muscles. The action potential and twitch are normal even if all external Na⁺ is replaced with sucrose but vary with external Ca⁺⁺, the action potential increasing 8 to 10 mv for a twofold increase in Ca⁺⁺. The lateral muscle (DEAL) is much slower and responds to intracellular stimulation only with an electrotonic or a local response. Mechanical responses and relaxation speeds are slow with minimal duration of contraction of 0.5 to 2 seconds. Immersion in high K solutions induces large maintained tensions. Sarcomere length in the fast DEAM is uniform and about 2 µ at rest, but in the DEAL speed is less and sarcomere length is greater averaging about 4.5 µ but with a mixed population of fibers.     

The effect of ions upon the response of smooth muscle to cooling

The slow tonic responses of the anterior byssus retractor of Mytilus edulis to rapid cooling were investigated by simultaneously recording tension and resting potential changes after soaking the muscle in banthine, a powerful neuromuscular blocking agent. The quantitative relations between the amount of cooling and the amount of associated depolarization necessary for contraction at various concentrations of potentiating potassium can be expressed in a family of curves. The plateaus of the curves for sea water and for potassium-free sea water were beneath the depolarization value necessary for contraction, so that it is clear that no amount of cooling with sea water alone or with potassium-free sea water would ever be effective. When the muscle is treated with subthreshold amounts of potassium and rapidly cooled in various concentrations of sodium ion and calcium ion, respectively, the sodium and calcium do not affect the amount of depolarization. Acetylcholine, in subthreshold amounts, has a potentiating effect, but, unlike potassium and cooling, acts through the nervous apparatus. Mytilus muscle will respond to cooling with tonic contraction whenever a critical threshold amount of depolarization is achieved. Cooling alone cannot trigger the contraction since it cannot bring about sufficient depolarization. Cooling can result in contraction, however, if used in conjunction with some other subthreshold depolarizing agent. Cooling affects the contractile mechanism by first causing membrane breakdown and depolarization.     

Ca++ controlled contraction-relaxation cycle in glycerinated amoeboid cells

Summary Contraction and relaxation in glycerinated amoeba models are controlled by the same factors which are known to control streaming in demembranated, living cytoplasm of amoeba. Threshold calcium concentration for the transition from relaxed to contracted state of glycerinated model was found to be about 10^–6 M.     

The Time Course of the Loss and Recovery of Contracture Ability in Frog Striated Muscle Following Exposure to Ca-Free Solutions

Using area under the contracture curve to quantitate contractures, the diffusion coefficient of calcium ions within the frog toe muscle during washout in a calcium-free solution and subsequent recovery after reintroduction of calcium to the bathing solution was calculated to be about 2 x 10^-6 cm²/sec. The diffusion coefficient measured during washout was found to be independent of temperature or initial calcium ion concentration. During recovery it was found to decrease if the temperature was lowered. This was likely due to the repolarization occurring after the depolarizing effect of the calcium-free solution. The relation between contracture area and [Ca]_o was found to be useful over a wider range than that between maximum tension and [Ca]_o. The normalized contracture areas were larger at lower calcium concentrations if the contractures were produced with cold potassium solutions or if NO₃ replaced Cl in the bathing solutions. Decreasing the potassium concentration of the contracture solution to 50 mM from 115 mM did not change the relation between [Ca]_o and the normalized area. If the K concentration of the bathing solution was increased, the areas were decreased at lower concentrations of Ca.     

An analysis of physiological states responsible for antero-posterior disintegration in Planaria dorotocephala

Summary Planaria were treated with equi-molal solutions of ammonium, potassium, sodium, magnesium, and calcium chlorides, made up in distilled water and the rates of cytolysis compared with cytolysis in distilled water. Potassium and ammonium accelerate cytolysis; some protection is afforded by sodium; still more by magnesium, and complete protection by calcium in the concentrations employed.In distilled water solutions of calcium chloride no cytolysis occurs in concentrations from M/500 to M/40,000; cytolysis is distinctly delayed in M/100,000. The protective action of M/1,000,000 is detectable.Potassium oxalate accelerates disintegration in hypotonic solutions.One per cent ethyl alcohol in distilled water causes cytolysis more rapidly than does distilled water alone, but in M/500 molal calcium chloride the alcohol solution is much less effective.Ringer's solution minus calcium affords no protection against death due to absence of calcium and death due to potassium oxalate but completely protects against cytolysis. Death in Ringer's solution minus calcium and in Ringer's solution with potassium oxalate occurs first in the anterior region and describes an antero-posterior gradient.Cytolysis in distilled water, in potassium oxalate solutions, in alcohol solutions, and in hypotonic calcium solutions of extreme dilution is initiated in the anterior end and describes an antero-posterior gradient within a zooid.Earlier work of the writer on the disintegrative action of lipoid solvents, heat, KNC, hyper- and hypotonic solutions is discussed. It is concluded that inPlanaria dorotocephala the antero-posterior gradient in cytolytic disintegration represents an antero-posterior differential in sensitivity to disturbance of the calcium-lipoid-water relation in the organism.     

Separation of sodium and calcium channels in the surface membrane of molluscan nerve cells

By intracellular dialysis of isolated neurons of the mollusksHelix pomatia andLimnaea stagnalis and by a voltage clamp technique the characteristics of transmembrane ionic currents were studied during controlled changes in the ionic composition of the extracellular and intracellular medium. By replacing the intracellular potassium ions by Tris ions, functional blocking of the outward potassium currents was achieved and the inward current distinguished in a pure form. Replacement of Ringer's solution in the extracellular medium with sodium-free or calcium-free solution enabled the inward current to be separated into two additive components, one carried by sodium ions, the other by calcium ions. Sodium and calcium inward currents were found to have different kinetics and different potential-dependence: _mNa=1±0.5 msec, _mCa=3±1 msec, _hNa=8±2 msec, _hCa=115±10 msec (V_m=0), G_Na=0.5 (V_m=–21±2 mV), G_Ca=0.5 (V_m=–8±2 mV). Both currents remained unchanged by tetrodotoxin, but the calcium current was specifically blocked by cadmium ions (2·10^–3 M), verapamil, and D=600, and also by fluorine ions if injected intracellularly. All these results are regarded as evidence that the soma membrane of the neurons tested possesses separate systems of sodium and calcium ion-conducting channels. Quantitative differences are observed in the relative importance of the systems of sodium and calcium channels in different species of mollusks.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 2, pp. 183–191, March–April, 1976.     

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca²⁺]_cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.