A study on the preservation of the beating rhythm of single myocardial cells in vitro

Single myocardial cells from fetal mouse heart beat spontaneously in monolayer culture. In standard medium they maintained constant beating rates for at least 5 h at 37 °C. When the beats of single myocardial cells were stopped for a short time by treatment with EGTA, or slowed down by incubating the cells in medium of low pH, the original beating rates could be restored by replacing the cells in the original medium. The same procedure also restored the rates after they had been disturbed by incubating the cells in medium of low sodium and high potassium ion content. Moreover, the original beating rates could be restored after keeping the cells at 10 °C for 22–24 h, but not after keeping them at 37 °C for 22–24 h.     

The relationship of calcium and parathyroid hormone in their effect on heart cells

Parathyroid hormone (PTH), the plasma concentration of which is raised in uremia, has been suggested as one of the agents responsible for the myocardial changes commonly seen in uremia. The effect of intact [1–84] PTH on rat heart cells grown in tissue culture has been studied. Addition of the hormone to the media significantly stimulated beating rate. The stimulation was directly proportional to the amount of PTH in the medium. Excessively high concentration of PTH caused immediate cessation of the beating, which was reversed by the addition of calcium to the medium. The extent of stimulation by PTH was inversely proportional to the calcium concentrations in the medium. Isoproterenol and phenylephrine at excessively high concentrations in the medium did not mimic the PTH effect either alone or together with PTH. When beating ceased due to verapamil the effect was not reversed by the addition of calcium to the medium.Calcium added to the myocytes seen after beating ceased reversed the effect and the cells started to beat again. Cells kept for a longer period in the arrested state were not revived by the addition of calcium.     

Beating of multinucleated giant myocardial cells in culture

Cultured mouse myocardial cells grown as cell sheets in Petri dishes fused together and formed multinucleated giant cells on treatment with HVJ (Sendai virus). The giant cells had well organized myofibrils and beat spontaneously and rhythmically. The spontaneous beating activity of the giant cells changed in response to changes of the external potassium and calcium concentrations and on addition of ouabain in the same way as the beating of cultured myocardial cells not treated with HVJ. When a microelectrode was inserted into giant cells that exhibited spontaneous beating, action potentials were easily recorded.     

EFFECTS OF CORTISOL ON CULTURED RAT HEART CELLS : Lipase Activity, Fatty Acid Oxidation, Glycogen Metabolism, and ATP Levels as Related to the Beating Phenomenon

This paper reports the determination of the ability of rat heart cells in culture to release [¹⁴C]palmitate from its triglyceride and to oxidize this fatty acid and free [¹⁴C]palmitate to ¹⁴CO₂ when the cells are actively beating and when they stop beating after aging in culture. In addition, the levels of glucose, glycogen, and ATP were determined to relate the concentration of these metabolites with beating and with cessation of beating. When young rat heart cells in culture are actively beating, they oxidize free fatty acids at a rate parallel with cellular ATP production. Both fatty acid oxidation and ATP production remain constant while the cells continue to beat. Furthermore, glucose is removed from the growth medium by the cells and stored as glycogen. When cultured cells stop beating, a decrease is seen in their ability to oxidize free fatty acids and to release them from their corresponding triglycerides. Concomitant with decreased fatty acid oxidation is a decrease in cellular levels of ATP until beating ceases. Midway between initiation of cultures and cessation of beating the cells begin to mobilize the stored glycogen. When the growth medium is supplemented with cortisol acetate and given to cultures which have ceased to beat, reinitiation of beating occurs. Furthermore, all decreases previously observed in ATP levels, fatty acid oxidation, and esterase activity are restored.     

Respiratory control in hyperpermeable adult heart muscle cells Effects of calcium

Spontaneously beating myocardial fragments prepared by mechanical disaggregation have hyperpermeable sarcolemmae. Such preparations were used to study mitochondrial function in situ. The myocardial fragments suspended in a phosphate-buffered salt solution containing 1–3 mM MgCl₂ showed a low rate of oxygen uptake. Addition of succinate, pyruvate plus malate or glutamate was followed by an increase in the rate of O₂ uptake. Addition of ADP to fragments engaged in State 4 respiration was followed by initiation of more rapid State 3 respiration, with respiratory control ratios routinely greater than 3 for succinate and glutamate. If the fragments were suspended in the same medium containing 3 mM ATP (a medium in which contractile activity occurs), State 3 was initiated upon addition of substrate. The suspension medium used in these experiments contained about 8 μM calcium as contamination. Addition of calcium chloride to give a final concentration of 0.14 to 0.57 mM stimulated State 4 respiration of the myocardial fragments. In contrast, similar additions made during State 3 inhibited respiration. The maximum degree of inhibition brought respiration close to the State 4 rate. If calcium was added prior to ADP, respiratory stimulation by the nucleotide was diminished. Respiratory function of myocardial fragments and of mitochondria isolated from them was similar in terms of response to substrate, ADP, and calcium addition in State 4. Response to calcium in State 3 was different in that inhibition was long-lived only at low [P_i] in the case of mitochondria, but at low or high [P_i] in the case of the fragments.     

The effect of monovalent cations on calcium efflux in yeasts

The properties of the calcium efflux system in the yeast Saccharomyces cerevisiae were investigated. After growing the cells overnight in medium containing ⁴⁵Ca, the cells were transferred to medium containing glucose, Hepes buffer (pH 5.2) and monovalent cations. The presence of potassium or sodium in the medium induced efflux of calcium from the cells. The magnitude of the efflux was dependent on the concentration of these cations in the medium. The time course of calcium efflux was analyzed, and two types of exchangeable calcium pools, which turned over at different rates, were detected: ‘Fast turnover’ and ‘slow turnover’. Increase in the concentration of monovalent cations in the medium caused an increase in the fraction of cellular calcium which turned over at a fast rate, and activation of calcium efflux from the ‘slow turnover’ calcium pool. The specific changes in the parameters of calcium efflux induced by monovalent cations were different from those reported previously to be induced by divalent cations. Both processes, i.e. activation of calcium efflux by monovalent and by divalent cations, were found to be additive, indicating that they operate via different mechanisms. Experiments using the respiratory inhibitor Antimycin A, showed that stimulation of calcium efflux by monovalent cations is energy dependent. Lanthanum ions which are known to inhibit calcium influx into yeast cells, inhibitted the activation of calcium efflux by both divalent and monovalent cations. Determination of the cationic composition of the cells indicated that the stimulation of calcium efflux was accompanied by influx of potassium or sodium into the cells.     

The coordinate roles of branchial nerve activity and potassium in the stimulation of ciliary activity in Mytilus edulis: observations with phenoxybenzamine, bromolysergic acid and fluorescence histochemistry.

Potassium concentrations in excess of 30 mM increase the rate of beating of lateral cilia on the gill of Mytilus edulis. Cilioexcitation produced by low frequency (5 beats/s) electrical stimulation was potentiated with potassium but blocked with bromolysergic acid (a serotonergic inhibitor). Cilioinhibition produced by high frequency (50 beats/s) stimulation was decreased with potassium and phenoxybenzamine (a dopaminergic inhibitor). Phenoxybenzamine enhanced the cilioexcitation produced by potassium. Potassium doses incapable of maintaining a basal rate of beating (less than 30 mM) could increase ciliary activity if phenoxybenzamine was also added. After transection of the branchial nerve, the yellow-fluorophore (serotonergic storage) and cilioexcitatory effect of potassium gradually decrease. This study shows that the potassium effect on ciliary activity (a) increase with low frequency nerve stimulation, presumably through the release of serotonin and (b) decreases with high frequency nerve stimulation, presumably through the release of dopamine.     

Galvanic Stimulation of Volvox globator II. Mechanism of Galvanic Response, an Effect of Calcium Displacement

Slight increases or decreases in calcium ions in solutions which supported the growth of Volvox globator colonies caused the colonies to fall to the bottoms of their containers. High speed cinematography (600 frames/sec) showed that the flagella beat normally (21/sec) in balanced electrolyte solutions which have calcium concentrations between 0.5 and 1.0 mM. When colonies were placed in 10.0 or 0.0 mM CaCl₂ solutions, flagellar beating disappeared within 1 hr. The cessation of flagellar beating was reversible when colonies were replaced in the balanced solution. The Volvox cell wall has been shown to be a fairly good cation-exchanger with calcium ions acting as the counterion to the fixed negative change. Colonies that were photopositive and gave a cathodal galvanotaxis responded to DC electrical potentials by producing solution patterns that were indicators of colony electronegativity. Colony resistance to electroosmotic flow was compared in potassium and calcium solutions. When colonies were placed in darkness for 24 hr and stimulated by DC electrical potentials, their cation-exchange properties became reduced and the cell walls appeared thinner. Application of a high DC electrical potential to dark-adapted colonies caused the colonies to shrink on their anode sides (anodal contraction). Other workers have found that the flagella on the anodal sides of dark-adapted colonies ceased beating during DC electrical stimulation. It is hypothesized that the electric current caused an increase of calcium ions on the anodal side of the colony that inhibited the flagellar mechanism of beating on that side. It is also hypothesized that the galvanotaxis associated with light-adapted (photopositive) colonies was due to calcium displacements in the colony cell walls that affected the flagellar beating on both sides of the colony.     

Ischemic myocardial injury in cultured heart cells: preliminary observations on morphology and beating activity

An in vitro model of myocardial ischemia has been established with primary monolayer cultures of neonatal rat heart cells. Ischemic conditions were simulated in vitro by subjecting the heart cell cultures to various levels of oxygen and glucose deprivation. After the ischemic treatments, cultures of beating muscle (M) cells were evaluated for functional and morphological changes. The experimental protocol consisted of treatment with 20% or 0% O2 and 1000, 500 or 0 mg glucose per 1 of medium for 4, 12 or 24 hr. Control cultures were treated with 20% O2 and 1000 mg glucose. The morphological alterations induced by the deficiency of O2 and glucose in the medium were the formation of pseudopodia and cytoplasmic vacuoles; increased cytoplasmic granulation; and the formation of abnormal cell shapes, such as long, spindly shaped M cells. There was a time-dependent decrease in beating activity as the M cells were exposed to longer durations of ischemic conditions. However, if the cultures were replenished with complete medium (1000 mg glucose) and 20% O2, the cells regained their ability to beat.     

The effects of CGRP on calcium transients of dedifferentiating cultured adult rat cardiomyocytes compared to non-cultured adult cardiomyocytes: possible protective and deleterious results in cardiac function

CGRP has potent cardiovascular effects but its role in heart failure is unclear. Effects of CGRP on calcium concentrations in fresh adult rat cardiomyocytes, cultured adult cardiomyocytes and neonatal cardiomyocytes were determined by real time fluorescence spectrophotometry. Treatment of cultured adult cardiomyocytes with CGRP resulted in a rapid cessation of beating and a reduction in intracellular calcium. Similar results were obtained in cultured neonatal myocytes. However, rod-shaped adult cardiomyocytes revealed a number of responses; (a) non-beating cells began to beat with increased intracellular calcium; (b) spontaneously beating cells exhibited increased intracellular calcium content and a faster beating rate or (c), myocytes increased their beating rate and became arrhythmic, suggesting that CGRP action on cultured dedifferentiated adult and neonatal myocytes depletes intracellular calcium, whereas in the rod-shaped mature myocytes calcium is retained, pointing to a different mode of action for CGRP on developing and dedifferentiating cardiomyocytes, compared to fully developed cardiomyocytes.     

Cytosolic calcium, calcium fluxes, and regulation of alveolar macrophage superoxide anion production

The recently available compound quin-2, which acts as a high affinity fluorescent indicator for calcium in the cytosol, was used to examine the role of calcium mobilization in the alveolar macrophage during the stimulation of 0-2 production by the tripeptide N-formyl norleucyl leucyl phenylalanine (FNLLP). After preloading with quin-2, the production of 0-2 was measured in conjunction with the transfer of 45Ca+2 and changes in quin-2 fluorescence upon stimulation with FNLLP. When cells were maintained in low (10 microM) extracellular calcium medium the presence of 1.5 mM quin-2 in the cytosolic space partially inhibited the rate of 0-2 production upon stimulation by FNLLP. Addition of 1 mM Ca+2 to the medium prior to stimulation rapidly restored the cell's capability to produce 0-2 upon stimulation at rates equal to control and extended the duration of stimulated 0-2 production as well. Quin-2 fluorescence measurements indicated an increase in cytosolic Ca+2 upon stimulation with FNLLP. This increase was lowest under conditions in which 0-2 production was inhibited. The addition of 1 mM Ca+2 to the medium caused by itself a rapid but transient increase in cytosolic Ca+2 as measured with quin-2 without stimulating 0-2 production. This intracellularly redistributed calcium was determined to be the source of the greater increase in cytosolic calcium during stimulation in the presence of high extracellular calcium. Measurements of 45Ca+2 transfer demonstrated a buffering of cytosolic Ca+2 changes by quin-2, which in low calcium medium could deplete calcium stores. It is suggested that this effect, prior to stimulation, was responsible for the mitigated 0-2 response for those cells maintained in low calcium medium, wherein calcium stores could not be replenished. These results suggested that the cell's mechanism for regulating cytosolic and bound calcium concentrations may also play an integral role in its normal mechanism for stimulated 0-2 production. They further support the postulate that the commonly observed rise in the concentration of calcium in the cytosol upon formyl peptide stimulation is a concomitant but nonregulatory event only.     

Ciliary beat co-ordination by calcium

Motile cilia in the airway epithelium are the engine for mucociliary clearance, the mechanism responsible for cleaning the airways from inhaled particles. Human airway epithelial cilia appear to have a slow constitutive rate of beating, driven by inherent and spontaneous dynein ATPase activity. Additionally, cilia can increase their beating frequency by activation of several different control mechanisms. One of these controllers is calcium. Its intracellular concentration is regulated by purinergic and acetylcholine receptors. Besides the rate regulatory effect of calcium on ciliary beat, calcium is also involved in synchronizing the beat among cilia of one single cell as well as between cilia on different cells. This article gives an overview of the complex effects of calcium on the beating of motile cilia in the airways.     

Rat pancreas adenylate cyclase VII. Effect of extracellular calcium on pancreozymin-induced cyclic AMP formation

1. 1. The effect of stimulation of adenylate cyclase by pancreozymin-C-octapeptide on the cyclic AMP level of rat pancreatic fragments has been investigated.

2. 2. In normal Krebs-Ringer bicarbonate medium pancreozymin-C-octapeptide causes a slight increase in pancreatic cyclic AMP level; this increase can be considerably enhanced by incubation in a calcium-free incubation medium.

3. 3. The dose-responce curve for pancreazymin-C-octapeptide in calcium-free medium is shifted to lower peptide concentrations, compared to the curve in normal Krebs-Ringer bicarbonate medium.

4. 4. The maximal stimulatory effect of pancreozymin-C-octapeptide id obtained at a 1-methyl-3-isobutylxanthine concentration of 10 mM.

5. 5. It suffices to lower the Ca²⁺-concentration of the medium from 2.5 to 1.5 mM to get the maximal increase in cyclic AMP content under influence of pancreozymin-C-octapeptide.

6. 6. It is concluded that extracellular calcium antagonizes the stimulation of adenylate cyclase by pancreozymin-C-octapeptide. This suggest that a low cytoplasmic Ca²⁺-concentration is required for the maximal response of acinar cell adenylate cyclase to pancreozymin.

Mechanisms of calcium transient and action potential alternans in cardiac cells and tissues

Alternation of cardiac action potential duration (APD) from beat to beat and concurrent alternation of the amplitude of the calcium transient are regarded as important arrhythmia mechanisms. These phenomena are causally interrelated and can be reliably evoked by an increase in beat frequency or by ischemia. The first part of this historical review deals with the physiology of APD alternans. Sections recounting the evolution of knowledge about calcium-activated ion currents and calcium transient alternans are interspersed among sections describing the growth of the so-called "restitution hypothesis," which involves time-dependent recovery of potassium channels (including their passage through pre-open states) as a function of diastolic interval. Major developments are generally in chronological order, but it is necessary to move back and forth between the two theories to respect the overall time line, which runs from about l965 to the present. The concluding two sections deal with the pathophysiology of calcium transient and APD alternans during ischemia, which may be the basis for out-of-hospital cardiac arrest during the initial stages of acute myocardial infarction.     

Electrophysiological control of ciliary motor responses in the ctenophorePleurobrachia

Prey capture by a tentacle of the ctenophore Pleurobrachia elicits a reversal of beat direction and increase in beat frequency of comb plates in rows adjacent to the catching tentacle (Tamm and Moss 1985). These ciliary motor responses were elicited in intact animals by repetitive electrical stimulation of a tentacle or the midsubtentacular body surface with a suction electrode. An isolated split-comb row preparation allowed stable intracellular recording from comb plate cells during electrically stimulated motor responses of the comb plates, which were imaged by high-speed video microscopy. During normal beating in the absence of electrical stimulation, comb plate cells showed no changes in the resting membrane potential, which was typically about -60 mV. Trains of electrical impulses (5/s, 5 ms duration, at 5-15 V) delivered by an extracellular suction electrode elicited summing facilitating synaptic potentials which gave rise to graded regenerative responses. High K+ artificial seawater caused progressive depolarization of the polster cells which led to volleys of action potentials. Current injection (depolarizing or release from hyperpolarizing current) also elicited regenerative responses; the rate of rise and the peak amplitude were graded with intensity of stimulus current beyond a threshold value of about -40 mV. Increasing levels of subthreshold depolarization were correlated with increasing rates of beating in the normal direction. Action potentials were accompanied by laydown (upward curvature of nonbeating plates), reversed beating at high frequency, and intermediate beat patterns. TEA increased the summed depolarization elicited by pulse train stimulation, as well as the size and duration of the action potentials. TEA-enhanced single action potentials evoked a sudden arrest, laydown and brief bout of reversed beating. Dual electrode impalements showed that cells in the same comb plate ridge experienced similar but not identical electrical activity, even though all of their cilia beat synchronously. The large number of cells making up a comb plate, their highly asymmetric shape, and their complex innervation and electrical characteristics present interesting features of bioelectric control not found in other cilia.     

Effects of cardioactive drugs on the beating activity of myocardial cell cultures isolated from offspring of trained and untrained pregant rats

Summary Primary cultures of beating myocardial cells were obtained from 5-d-old offspring of trained (T) and untrained (UT), pregnant, Sprague-Dawley rats. The myocardial cells from the T and UT groups were evaluated for their beating responses to three cardioactive drugs: verapamil (V), isoproterenol (ISO), and propranolol (PRO). The myocardial cell cultures from the UT group showed complete loss of beating when the calcium (Ca⁺⁺) antagonist, V, was added to the cultures for 1 h or more; the T group was able to show some beating at comparable concentrations and durations of exposure with V. The beta agonist, ISO, markedly stimulated the beating rate of both the T and UT groups, but the beating rates were higher in the UT group at comparable concentrations and durations of exposure than with the T group. When the cultures were pretreated with the beta blocker, PRO, before treatment with ISO, a concentration inhibitory effect on the beating rate was observed in both groups. However, the T cultures were more sensitive to the inhibitory effects of PRO. These results demonstrate that primary cultures of rat myocardial cells isolated from the offspring of trained and untrained pregnant rats show differential beating responses to three well-known cardioactive drugs. This study was supported by grants from the American Heart Association, Texas Affiliate, and the University of Texas Research Institute.     

Calcium activation of macrocilia in the ctenophoreBeroë

1. Macrocilia on the lips of the ctenophore Bero? are usually quiescent, but can be activated to beat rapidly and continuously by various stimuli. 2. During feeding, macrocilia beat actively and serve to spread the lips of Bero? over its prey. 3. Vigorous, repetitive mechanical stimulation of the lips evokes widespread activation of macrocilia via a pathway that is probably neural. 4. Extracellular electrical stimulation (DC or bipolar pulse-trains) elicits immediate activation of macrocilia on lip pieces, but not on dissociated cells. 5. Macrocilia on lip pieces are activated to beat by high KCl artificial sea water (ASW), but not by high KCl Ca-free ASW. Continuous beating for long periods is also elicited by high Ca ASW or Mg-free ASW, but not by Ca-Mg-free ASW. Addition of La, Cd, Co or Mn (10 mM) to high KCl ASW reversibly blocks activation. Verapamil, D-600, nifedipine, or BAY K 8644 (10 microM) has no effect on KC1-induced activation, but the anticalmodulin drug W-7 (10 microM) reversibly inhibits beating. 6. Mild heat treatment dissociates macrociliary cells from lip tissue. Such isolated macrociliary cells usually beat continuously in normal sea water, and swim in circular paths. Ca-free ASW, or addition of Co or Mn to ASW, inhibits beating of dissociated cells. High KCl ASW activates beating of quiescent, isolated macrociliary cells. 7. Ca-Mg-free ASW inhibits beating of dissociated macrociliary cells, and return to Mg-free ASW activates motility, allowing one to activate macrocilia on isolated cells simply by addition of Ca.(ABSTRACT TRUNCATED AT 250 WORDS)     

On The Mechanism of Spontaneous Impulse Generation in the Pacemaker of the Heart

Rhythmic activity in Purkinje fibers of sheep and in fibers of the rabbit sinus can be produced or enhanced when a constant depolarizing current is applied. When extracellular calcium is reduced successively, the required current strength is less, and eventually spontaneous beating occurs. These effects are believed due to an increase in steady-state sodium conductance. A significant hyperpolarization occurs in fibers of the rabbit sinus bathed in a sodium-free medium, suggesting an appreciable sodium conductance of the "resting" membrane. During diastole, there occurs a voltage-dependent and, to a smaller extent, time-dependent reduction in potassium conductance, and a pacemaker potential occurs as a result of a large resting sodium conductance. It is postulated that the mechanism underlying the spontaneous heart beat is a high resting sodium current in pacemaker tissue which acts as the generator of the heart beat when, after a regenerative repolarization, the decrease in potassium conductance during diastole reestablishes the condition of threshold.     

Effects of Ca2+ and Mg2+ on ATP-dependent 45Ca2+ influx in A-431 human epidermoidal carcinoma cells

Upon stimulation with 10(-6) -10(-3) M ATP, A-431 human epidermoidal carcinoma cells incorporated radioactive calcium from their medium in a temperature-dependent manner. The rate of incorporation of 45Ca2+ was rapid for the initial 5 min, but decreased immediately thereafter. The preincubation of cells for 2 h in medium depleted of both Ca2+ and Mg2+ abolished the ATP-dependent 45Ca2+ incorporation, irrespective of whether or not the subsequent incubation medium contained Mg2+ ions. ATP-dependent 45Ca2+ incorporation could be restored by a second preincubation (1 h) in medium containing 1 mM Mg2+, but no Ca2+. The Mg2+ ions in the second preincubation medium could be replaced by Ca2+, Co2+, or Cu2+ for restoration of such activity. Elevation of inositol trisphosphate (InsP3) was observed in cells depleted of either Ca2+ or Mg2+, but not in cells depleted of both ions. A parallel effect was observed in changes in [Ca2+]i. Since the concentration of cytosolic calcium ions does not change by incubation of cells in medium depleted of and (or) restored with calcium ions, we conclude that either calcium or magnesium ions associated with some cellular component(s) are responsible for production of InsP3, which then supposedly mobilizes Ca2+ and provokes 45Ca2+ influx.