influence of Ca channel blockers on the positive inotropic effect of ionophore A23187 in the myocardium

Calcium ionophore A23187 (10(-5) M) increases the force of contraction of the frog atrium up to 27 + 4.8%. The calcium antagonists d-600 (5 X 10- M), Zn2+ (2 X 10(-5) M), Mn2+ (2 X 10(-4) M) decrease the force of contraction 50, 10 and 20%, respectively, and inhibit the positive inotropic effect of ionophore A23187. Inhibition of the ionophore effect by the blockers is determined by the ability of D-600, Zn2+ and Mn2+ to form complexes with the ionophore. Besides, the affinity of these blockers to the ionophore is higher than that of Ca2+. It is assumed that Zn2+, Mn2+ and D-600 possess higher affinity to ionophore A23187 as compared with myocardial Ca-channels. Fenigidin interacts with Ca-channels to a larger degree than with ionophore A23187.     

The mitogenic effect of A23187 in human peripheral lymphocytes.

The mitogenic action of the divalent ionophore A23187 was confirmed and shown to be very sensitive to changes in extracellular calcium ion concentration. At optimal calcium and ionophore concentrations, an increase in [3H]-thymidine incorporation was seen that was similar to that seen after phytohemagglutinin addition. A calcium-dependent stimulation of alpha-aminoisobutyric acid transport was also seen after A23187 addition. Studies with three inhibitors demonstrate a similarity between proliferation induced by phytohemagglutinin and by A23187. Isoproterenol (10(-4) M) and ouabain (10(-7) M) blocked the effects of phytohemagglutinin and A23187. A drug, D-600 that has been shown to block calcium channels in cardiac muscle, inhibited proliferation induced by either phytohemagglutinin or A23187. This concentration of D600 had no effect on either phytohemagglutinin- or A23187-induced 45Ca2+ uptake. Furthermore, the (+) and (-) isomers separated from racemic D600, which have been shown to block sodium and calcium channels respectively in smooth muscle, had equal potency in blocking lymphocyte proliferation.     

Effect of calmidazolium (R 24571) on the electrical and contractile properties of smooth muscle cells in the guinea pig ureter

Calmidazolium in macromolecular concentrations inhibited the electric and contractile activity of smooth muscle cells (SMC). The concentrations causing a 50% inhibition of oscillations on the action potential (AP) plate were equal to 1 X 10(-6) microM, AP amplitude was 3 X 10(-6) microM and contraction amplitude was 1 X 10(-6) microM. Calcium ionophore A 23187/8 X 10(-7) microM, added to the normal Krebs solution, decreased rapid AP components amplitude and increased the contraction power of the isolated SMC strip by 62 +/- 9%. A 23187, though to a lesser extent, increased smooth muscle contractions during the action of calmidazolium. With combined use of A 23187 and calmidazolium, rapid AP components were depressed to a greater extent than each of them taken separately. The data obtained point to the presence of calmodulin or similar protein in SMC of the calcium channels.     

The mitogenic effect of A23187 in human peripheral lymphocytes

The mitogenic action of the divalent ionophore A23187 was confirmed and shown to be very sensitive to changes in extracellular calcium ion concentration. At optimal calcium and ionophore concentrations, an increase in [³H]-thymidine incorporation was seen that was similar to that seen after phytohemagglutinin addition. A calcium-dependent stimulation of α-aminoisobutyric acid transport was also seen after A23187 addition.Studies with three inhibitors demonstrate a similarity between proliferation induced by phytohemagglutinin and by A23187. Isoproterenol (10^?4 M) and ouabain (10^?7 M) blocked the effects of phytohemagglutinin and A23187. A drug, D-600, that has been shown to block calcium channels in cardiac muscle, inhibited proliferation induced by either phytohemagglutinin or A23187. This concentration of D600 had no effect on either phytohemagglutinin- or A23187-induced ⁴⁵Ca²⁺ uptake. Furthermore, the (+) and (?) isomers separated from racemic D600, which have been shown to block sodium and calcium channels respectively in smooth muscle, had equal potency in blocking lymphocyte proliferation.     

Effects of ruthenium red, A23187 and D-600 on steroidogenesis in Y-1 cells.

The effects of the calcium antagonists ruthenium red and D-600 and the cation ionophore A23187 on steroidogenesis were investigated. Steroidogenesis triggered by corticotrophin and cyclic AMP was inhibited by each of the agents. Incubation of Y-1 cells with an excess of ethyleneglycol-bis-(beta-amino-ethylether)-N,N'-tetraacetic acid (EGTA) abolished the steroidogenic response to corticotrophin while the response to cyclic AMP was unaffected. The ability of ruthenium red and D-600 (1 . 10(-5) M), and A23187 (6 . 10(-6 M) to inhibit a response which does not require the presence of extracellular calcium (cyclic AMP induced steroidogenesis) suggests that they are altering intracellular calcium. Neither of the calcium antagonists nor the cation ionophore inhibited the steroidogenic response to exogenous pregnenolone, thereby suggesting that the cells were still viable. Only when A23187 was used in the presence of a 15-fold increase in extracellular calcium (4.8 mM) was the response to pregnenolone diminished. The data are interpreted as a further indication that, in intact cells, intracellular calcium plays a role in the steroidogenic pathway.     

The role of the sodium-calcium exchanger for calcium extrusion in coronary arteries

Calcium ionophores, such as the A23187, cause endothelium-dependent relaxation of arterial strips with intact endothelium, whereas the effect of the ionophore should result from the combination of a relaxation caused by the endothelium-dependent factors and of a contraction of the smooth muscles. In addition, the application of a calcium ionophore to a strip of pig coronary arteries without endothelium does not change cytosolic free calcium concentration and force developed by the smooth muscle cells. To explain these paradoxes, the hypothesis that active calcium extrusion would match the entry of extracellular calcium caused by the ionophore was tested. We see that the sodium-calcium exchanger extrudes calcium that enters the smooth muscle cells in the absence of the ionophore. This exchanger is efficient enough to expel the increased influx of calcium created by the additional calcium carriers formed by the ionophore. This explains the inefficiency of calcium ionophores to increase cytosolic free calcium of smooth muscle cells and consequently, the fact that the ionophore does not cause a contraction of a strip without endothelium. This makes evident that a calcium ionophore fully relaxes, in an endothelium-dependent manner. an intact strip of porcine coronary artery.     

Mechanism of excitation and contraction uncoupling in frog and guinea pig myocardial fibers during block of slow sodium-calcium channels by compound D-600]

In the experiments performed on the strips of frog's atria and ventricle it was found that rhythmic stimulation facilitated the dissociation of excitation--contraction coupling caused by D-600 compound through the block of calcium channels, i.e. D-600 prevented the increase of tension in the series of contractions or even converted positive staircase into negative one. The first contraction was affected by D-600 to a lesser degree than the subsequent ones. Guinea pig's left auriculus depressing action of D-600 was found to be strongly dependent on the frequency of stimulation. The data obtained were analysed by the model of excitation--contraction coupling. It was concluded that in amphibians as well as in mammalians tension amplitude during rhythmic activity of myocardial cells is determined mainly by Ca inflow through the excitable calcium channels. On the other hand the amplitude of the first contraction depends on the storage content determined by Ca inflow through the Ca channels open at rest. These latter channels have low sensitivity to D-600 and are better presented in atrium than in ventriculum. An increase of contraction tension observed in low sodium medium took place also under the action of D-600; in this case Ca storage was replenished by the Na-Ca exchange diffusion.     

Temperature-dependent inotropic action of A23187 on guinea-pig ventricular muscles

The effects of the Ca2+ ionophore, A23187, on the contraction and membrane action potential of the isolated guinea-pig papillary muscle were examined at various temperatures (30-16 degrees C) and compared to those of isoprenaline and a high calcium medium. A23187 caused a marked positive inotropic effect with a significant prolongation of the action potential duration at an early repolarization phase but not a late repolarization phase at normal temperature (30 degrees C). Such an inotropic effect was completely abolished at low temperature (16 degrees C) where a marked positive inotropic effect of isoprenaline (5 X 10(-8) M) and a high calcium medium (6.2 mM) still remained. These results suggest that the cardiac responsiveness to A23187 was sensitive to a low temperature at which a membrane lipid phase transition may occur.     

K+-induced twitch potentiation is not due to longer action potential

The objective of this study was to determine whether an increased duration of the action potential contributes to the K+-induced twitch potentiation at 37 degrees C. Twitch contractions were elicited by field stimulation, and action potentials were measured with conventional microelectrodes. For mouse extensor digitorum longus (EDL) muscle, twitch force was greater at 7-13 mM K+ than at 4.7 mM (control). For soleus muscle, twitch force potentiation was observed between 7 and 11 mM K+. Time to peak and half-relaxation time were not affected by the increase in extracellular K+ concentration in EDL muscle, whereas both parameters became significantly longer in soleus muscle. Decrease in overshoot and prolongation of the action potential duration observed at 9 and 11 mM K+ were mimicked when muscles were respectively exposed to 25 and 50 nM tetrodotoxin (TTX; used to partially block Na+ channels). Despite similar action potentials, twitch force was not potentiated by TTX. It is therefore suggested that the K+-induced potentiation of the twitch in EDL muscle is not due to a prolongation of the action potential and contraction time, whereas a longer contraction, especially the relaxation phase, may contribute to the potentiation in soleus muscle.     

Electric field-directed cell shape changes, displacement, and cytoskeletal reorganization are calcium dependent

C3H/10T1/2 mouse embryo fibroblasts were stimulated by a steady electric field ranging up to 10 V/cm. Some cells elongated and aligned perpendicular to the field direction. A preferential positional shift toward the cathode was observed which was inhibited by the calcium channel blocker D-600 and the calmodulin antagonist trifluoperazine. Rhodaminephalloidin labeling of actin filaments revealed a field-induced disorganization of the stress fiber pattern, which was reduced when stimulation was conducted in calcium-depleted buffer or in buffer containing calcium antagonist CoCl2, calcium channel blocker D-600, or calmodulin antagonist trifluoperazine. Treatment with calcium ionophore A23187 had similar effects, except that the presence of D-600 did not reduce the stress fiber disruption. The calcium-sensitive photoprotein aequorin was used to monitor changes in intracellular-free calcium. Electric stimulation caused an increase of calcium to the micromolar range. This increase was inhibited by calcium-depleted buffer or by CoCl2, and was reduced by D-600. A calcium-dependent mechanism is proposed to explain the observed field-directed cell shape changes, preferential orientation, and displacement.     

A calcium requirement for electric field-induced cell shape changes and preferential orientation

C3H/10T1/2 mouse embryo fibroblasts stimulated by a steady electric field (10 V/cm) for 30 min exhibited lamellar retraction on the sides facing the electrodes. Some cells elongated and preferentially oriented with their long axis perpendicular to the field direction. Depletion of external calcium or blockage of calcium influx with lanthanum or the calcium channel blocker D-600 resulted in a reduction of the field-induced response. When external calcium was elevated stepwise from 0 to 10 mM, the field-induced response increased correspondingly. Electric stimulation in the presence of the calcium ionophore A23187 resulted in an increase of spindle-shaped cells with no preferential orientation. This response was blocked by calcium depletion and lanthanum, but not by D-600. The anticalmodulin drug W-13 inhibited the field-induced responses observed in normal buffer as well as in the presence of A23187. Some cell death resulted from prolonged electric field exposure, and the mortality was reduced by calcium depletion, lanthanum or D-600, but was not affected by W-13. We postulate that local calcium influx through channels opened by the electric field produces areas of high intracellular calcium which stimulate the cytoskeletal network to induce lamellar retraction. Prolonged field-induced calcium influx may eventually overcome the cell's mitochondrial calcium-buffer system, leading to necrotic calcification.     

Perturbation of the phagocytic response in P388D1 cultured macrophages by agents altering cell calcium

Phagocytosis in adherent P388D₁ (D₁) cells was monitored utilizing formalin treated $\text{Listeria}$$\text{monocytogenes}$ ($\text{Lm}$) previously labeled with ¹²⁵iododeoxyuridine. The dependence of this phagocytic process on calcium was studied by using several agents which alter calcium metabolism. The calcium antagonist ruthenium red (RR) produced a dose and time dependent stimulation (60–70%) of $\text{Lm}$ phagocytosis by D₁ cells. Utilizing another calcium antagonist, D-600, a prolonged inhibition (4 hours) of phagocytosis (40%) was observed. The addition of the cation ionophore A23187 produced a transient stimulatory increase (38% at 2 hours) in the phagocytic response. The concomitant addition of RR and D-600 did not alter the phagocytosis of $\text{Lm}$ by D₁ cells as compared to control cells. However, this complete drug/drug antagonism was not seen with the combinations of A23187 and D-600 or RR and A23187. The addition of A23187 and D-600 resulted in a time dependent inhibition of phagocytosis which did not become maximal until 3 to 4 hours. A23187 and RR produced a time independent stimulation of phagocytosis which was significantly less than that which was observed for RR alone, but was of longer duration than the response produced by A23187 alone. The use of these calcium probes in the P388D₁ macrophage model suggests a role for calcium in the phagocytic process.     

Spike-triggered average torque and muscle fiber conduction velocity of low-threshold motor units following submaximal endurance contractions.

The motor unit twitch torque is modified by sustained contraction, but the association to changes in muscle fiber electrophysiological properties is not fully known. Thus twitch torque, muscle fiber conduction velocity, and action potential properties of single motor units were assessed in 11 subjects following an isometric submaximal contraction of the tibialis anterior muscle until endurance. The volunteers activated a target motor unit at the minimum discharge rate in eight 3-min-long contractions, three before and five after an isometric contraction at 40% of the maximal torque, sustained until endurance. Multichannel surface electromyogram signals and joint torque were averaged with the target motor unit potential as trigger. Discharge rate (mean +/- SE, 6.6 +/- 0.2 pulses/s) and interpulse interval variability (33.3 +/- 7.0%) were not different in the eight contractions. Peak twitch torque and recruitment threshold increased significantly (93 +/- 29 and 12 +/- 5%, P <0.05) in the contraction immediately after the endurance task with respect to the preendurance values (0.94 +/- 0.26 mN.m and 3.7 +/- 0.5% of the maximal torque), whereas time to peak of the twitch torque did not change (74.4 +/- 10.1 ms). Muscle fiber conduction velocity decreased and action potential duration increased in the contraction after the endurance (6.3 +/- 1.8 and 9.8 +/- 1.8%, respectively, P <0.05; preendurance values, 3.9 +/- 0.2 m/s and 11.1 +/- 0.8 ms), whereas the surface potential peak-to-peak amplitude did not change (27.1 +/- 3.1 microV). There was no significant correlation between the relative changes in muscle fiber conduction velocity or surface potential duration and in peak twitch torque (R2= 0.04 and 0.10, respectively). In conclusion, modifications in peak twitch torque of low-threshold motor units with sustained contraction are mainly determined by mechanisms not related to changes in action potential shape and in its propagation velocity.     

In vivo exposure to carbon disulfide increases the contraction frequency of pregnant rat uteri through an indirect pathway

Exposure to CS2, an organic solvent, is associated with an increased rate of abnormal labor or dysmenorrhea. Contraction of quiescent uteri during pregnancy can cause preterm labor. We wish to know the effects of in vivo and in vitro exposures to CS2 on uterine contractions of mid-gestation rats. After 10-d exposure to 300 or 600 mg/kg CS2, uteri of pregnant rats were measured for contractile responses to various stimuli, such as KCl, oxytocin, carbachol or A23187, a calcium ionophore, using standard muscle bath apparatus. CS2 treatment significantly increased the contractile response to KCl, carbachol, and A23187. The increase to A23187 was the greatest. In contrast, in vitro exposure to CS2 immediately suppressed carbachol-induced contraction but did not affect spontaneous and KCl-induced contractions. Results showed the pregnant uterus of the rat is susceptible to CS2. The influence of in vivo exposure to CS2 on uterine contraction was opposite to that in vitro. The increased response of CS2-treated uteri to A23187 suggests that in vivo exposure to CS2 may sensitize contraction machinery to calcium through indirect pathways.     

Impulse propagation and muscle activation in long maximal voluntary contractions

With fatigue, force generation may be limited by several factors, including impaired impulse transmission and/or reduced motor drive. In 5-min isometric maximal voluntary contraction, no decline was seen in the peak amplitude of the tibialis anterior compound muscle mass action potential (M wave) either during or immediately after the voluntary effort, provided maximal nerve stimulation was retained. For first dorsal interosseous (FDI) muscle, M wave amplitudes declined by 19.4 +/- 1.6% during the first 2 min but did not change significantly thereafter, despite the continued force reduction (up to 94% in 5 min for both muscles). The duration of the FDI M waves increased (greater than 30%), suggesting that the small decline in amplitude was the result of increased dispersion between the responses of different motor units. Some subjects kept FDI maximally activated throughout, but when they used tibialis anterior, twitch occlusion and tetanic muscle stimulation showed that most subjects were usually only able to do so for the first 60 s and thereafter only during brief "extra efforts." Thus force loss during isometric voluntary contractions sustained at the highest intensities results mainly from failure of processes within the muscle fibers.     

Calcium dependence of electrical and mechanical activity in rat ileum examined by the sucrose-gap technique

1. Single sucrose gap recordings showed that spontaneous action potentials of rat ileal smooth muscle consisted of slow waves and superimposed spikes which generated rhythmic contractions. As external potassium was raised, the resting potential progressively depolarized.2. Calcium-free salines inhibited spontaneous mechanical activity and inhibited the plateau phase of the action potential, but spontaneous spike depolarizations persisted.3. Verapamil, nifedipine and diltiazem all inhibited spontaneous mechanical activity and the plateau phase of the action potential, while in addition diltiazem augmented spike amplitude.4. Mn ions also inhibited mechanical activity and the action potential plateau, without affecting spike activity while the calcium ionophore A23187 enhanced both mechanical and electrical activity with a pronounced effect on spike amplitude.5. These results are consistent with the view that the plateau phase of the ileal smooth muscle action potential is dependent upon an influx of extracellular calcium possibly through voltage dependent slow calcium channels.     

Twitch interpolation technique in testing of maximal muscle strength: Influence of potentiation, force level, stimulus intensity and preload

The aim was to study the methodological aspects of the muscle twitch interpolation technique in estimating the maximal force of contraction in the quadriceps muscle utilizing commercial muscle testing equipment. Six healthy subjects participated in seven sets of experiments testing the effects on twitch size of potentiation, time lag after potentiation, magnitude of voluntary force, stimulus amplitude, stimulus duration, angle of the knee, and angle of the hip. In addition, the consequences of submaximal potentiation on the estimation of maximal force from twitch sizes were studied in five healthy subjects. We found an increase in twitch size with increasing levels of potentiation and twitch size decreased exponentially following potentiation. We found a curvilinear relationship between twitch size and voluntary force, and these properties were more obvious when the stimulation intensity of the preload was reduced. The relationship between twitch size and force was only linear, for force levels greater than 25% of maximum. It was concluded that to achieve an accurate estimate of true maximal force of muscle contraction, it would be necessary for the subject to be able to perform at least 75% of the true maximal force.     

The Effect of Calcium Antagonists on Auxin-induced Elongation and on the Expression of Two Auxin-Regulated Genes in Pea Epicotyls

Calcium has been implicated in various regulatory roles in plantcells including auxin-induced cell elongation. Treatment ofpea epicotyl segments with the calcium chelators, EGTA and chlorotetracycline(CTC), the calcium ionophore, A23187 [GenBank] , and channel blocker, D-600,inhibits auxin-induced cell elongation. Depletion of tissuecalcium either by EGTA or EGTA and a calcium ionophore doesnot interfere with the induction of the early auxin induciblemRNAs pIAA4/5 and pIAA6. Similarly, an increase in cytosoliccalcium with calcium and calcium ionophore neither induces thehormonally regulated mRNAs nor interferes with their inductionby auxin. The calcium channel blocker, D-600, is without effecton the auxin-regulated mRNA induction. The results indicatethat calcium is not involved in the rapid induction of IAA4/5and IAA6 genes in pea tissue. However, a possible role for calciumin the translation of these mRNAs, or in the expression of otherauxin-regulated genes, is not excluded. ³Present Address: Department of Biology, Tokyo MetropolitanUniversity, Tokyo, Japan. (Received April 8, 1988; Accepted July 30, 1988)     

Intracellular calcium as a modulator of transepithelial permeability to water in frog urinary bladder

The divalent cation ionophore A 23187 was used to evaluate the action of intracellular calcium on net transepithelial water movement across the isolated frog urinary bladder. Incubation with the ionophore increases the net basal water flux in a dose-dependent fashion but independent of the extracellular calcium concentration. Bladders pretreated with A 23187 and exposed thereafter to an increase in calcium concentration exhibit a water permeability that under certain conditions can be comparable to that achieved with antidiuretic hormone (ADH). Lowering the serosal calcium at the peak of the hydrosmotic responses to both ADH and A 23187 inhibited the maintenance of the net water flux. The action of a supramaximal dose of ADH is blunted in bladders pretreated with A 23187, while the hydrosmotic effects of a submaximal dose are enhanced when the ionophore is added together with the hormone. The results show that an increase in transepithelial water movement can be triggered by calcium and that serosal calcium is needed to sustain the response. This hydrosmotic response may be dependent upon the rate at which intracellular calcium concentrations change and on the absolute concentration attained. It is suggested that calcium is involved in the action of ADH on water permeability and may act as a modulator of the hydrosmotic response.     

Posttetanic potentiation and skeletal muscle fatigue: interactions with caffeine

The purpose of this study was to determine the interaction of three factors that modify twitch contraction amplitude in the rat gastrocnemius muscle in situ: posttetanic potentiation, fatigue, and caffeine. Posttetanic (200 Hz for 1 s) twitch responses were observed before and after 15 Hz stimulation for 6 min (group FS), injection of caffeine (75 mg/kg dissolved in saline, group NC), a combination of both repetitive stimulation and caffeine injection (group FC), or no treatment (group NS). Developed tension increased significantly with posttetanic potentiation and caffeine injection and these potentiating factors were additive (group NC). Repetitive stimulation attenuated the twitch response and the fatigued muscle was still responsive to the potentiating factors. Posttetanic potentiation was accomplished primarily by a significant increase in the peak rate of force development whereas caffeine potentiation and fatigue were effected with a proportional change in contraction time. It seems likely that the mechanism of posttetanic potentiation is not the same as the mechanism of caffeine-induced potentiation. Caffeine-induced potentiation is known to be related to increased release of calcium. Because changes in contraction time with fatigue were opposite to those associated with caffeine potentiation, it is proposed that the attenuated twitch response in fatigue results from reduced release of calcium.