Cyclic AMP-mediated inhibition of noradrenaline-induced contraction and Ca2+ influx in guinea-pig vas deferens

The relaxation effects of forskolin and methylxanthines on noradrenaline (NA)-induced contractions were investigated by measuring isotonic contraction and intracellular calcium concentration ([Ca2+]i) in the epididymal side of guinea-pig vas deferens. NA (100 microM) and high K+ (55 mM) induced a biphasic contraction; fast, transient (phasic) and slow, sustained (tonic) phases. Both phases in either NA or high K+ stimulation were abolished in Ca2+-free solution. Pretreatment with 10 microM nifedipine, an L-type Ca2+ channel blocker, reduced both phasic and tonic contractions induced by high K+. In the case of NA-induced contraction, however, nifedipine reduced the phasic contraction but not the tonic contraction. The nifedipine-insensitive tonic contraction was relaxed by the application of polyvalent cations (Mn2+, Co2+, Cd2+ and La3+). These findings indicate that NA-induced biphasic contraction is mainly due to nifedipine-insensitive Ca2+ influx, especially in the tonic phase. Cyclic AMP-increasing agents such as forskolin (0.5-10 microM), IBMX (5-500 microM) and caffeine (1-20 mM) relaxed the NA-induced contraction extensively in a concentration-dependent manner. However, these agents only partially relaxed the high K+-induced contraction. Forskolin (10 microM) and IBMX (100 microM) reduced the [Ca2+]i response to NA, but had no effect on the [Ca2+]i response to high K+. These results suggest that an increase in intracellular cAMP may relax the NA-induced contraction by attenuating a nifedipine-insensitive Ca2+ influx and by a mechanism independent of a reduction in [Ca2+]i.     

Effects of magnesium on contractile activation of skinned cardiac cells.

1. In the presence of a slight buffering of the free [Ca2+] with 0.050 mM total EGTA cyclic contractions were induced by a Ca2+-triggered release of Ca2+ on skinned (sarcolemma-free) segments of single cardiac cells from rat ventricle. The threshold of the free [Ca2+] trigger was elevated when the free [Mg2+] was increased. 2. At a suprathreshold free [Ca2+] increasing the free [Mg2+] resulted in a decrease in frequency and in an increase in amplitude of the phasic contractions. Addition of caffeine at a specified interval after a cyclic contraction produced a larger contraction when free [Mg2+] was higher. It was concluded that an increase of free [Mg2+] increased the capacity and the rate of binding for Ca2+ by the sarcoplasmic reticulum (SR). 3. Small skinned fibres of skeletal muscle which were perfused with 10 mM caffeine yielded results similar to those obtained in skinned cardiac cells. It was concluded that the mechanism of action of free Mg2+ was similar in both preparations, but that the SR of skeletal muscle had a higher capacity and rate of binding for Ca2+ than the cardiac SR. 4. With a strong buffering of the free [Ca2+] with 4-0 mM total EGTA, a smaller tonic tension was developed for a given pCa in the presence of a higher free [Mg2+]. This result was nearly identical in skinned cells from cardiac and skeletal muscle tissue. 5. A decrease of the [MgATP2-] produced a tension in the skinned cardiac cells that were perfused in Ca2+ free media. The maximum tension was observed for [MgATP2-] 10(-5-50)M as in skinned fibres of skeletal muscle. A further decrease of [MgATP2-] resulted in a decrease of tension.     

The effect of substances releasing intracellular calcium ions on sodium-dependent calcium efflux from guinea-pig auricles.

1. 45-Ca efflux and resting tension were measured in isolated guinea-pig auricles under conditions known to change the intracellular free Ca ion concentration. 2. In the presence of [Na]o, caffeine (2mM) increases 45-Ca efflux, but does not produce a contracture, while in the absence of [Na]o and [Ca]o caffeine causes a contracture without increasing 45-Ca efflux. Adrenaline (10-minus5-10-minus 4M) with or without theophylline (0-5-1-0mM) has no effect on either 45-Ca efflux or resting tension. 3. In the presence of caffeine the rate of net efflux of Ca depends on [Na]o-2. Caffeine contractures of muscles in Na-free solution relax upon the addition of [Na]o. Relaxation is correlated with the increase in net efflux of Ca. 4. Cyanide (2mM) produces a variable increase in 45-Ca efflux without a concomitant contracture in Na-containing solutions, but in Na, Ca-free solutions a large contracture occurs without significant increase in 45-Ca efflux. 5. A large increase in 45-Ca efflux and a contracture were observed with the 'Ca-ionophore' X 537 A. 6. Changes in membrane potential (K-depolarization) in hypertonic solutions have no significant effect on Na-dependent 45-Ca efflux, which is an agreement with an electroneutral 2:1 Na-Ca exchange. 7. Cyanide and X 537 A both cause a considerable release of Ca ions from isolated guinea-pig heart mitochondria, while caffeine has no effect. 8. The results suggest a powerful role of the Na-Ca exchange system in reducing the intracellular Ca concentration after Ca release from intracellular stores.     

The effect of sodium and calcium on the action potential of the smooth muscle of the guinea-pig taenia coli

1. Spontaneous spike activity and action potentials evoked by external field stimulation were recorded, intracellularly and with the double sucrose gap method, from the smooth muscle of guinea-pig taenia coli.2. Replacement of external NaCl with sucrose (leaving 10 mM-Na in the buffer) caused hyperpolarization and stopped spontaneous activity within 10 min. Spikes could, however, be evoked for 2-3 hr. The amplitude, the overshoot and rate of rise of the spike were increased.3. In 10 mM-[Na](o) the intracellular Na concentration was reduced from 35 to 24 mM, shifting the Na-equilibrium potential from +34 to -22 mV.4. Excess Ca (12.5 mM) caused hyperpolarization and increased membrane conductance. The amplitude and the rate of rise of the spike were increased, the threshold was raised and the latency of the spike evoked by threshold stimulation became shorter.5. The effect of reducing the external Ca concentration depended on the Na concentration present, being greater with higher external [Na](o). When the membrane was depolarized and spikes deteriorated in low Ca (0.2-0.5 mM) reduction of Na to 10 mM caused repolarization and recovery of the action potential.6. Mn (0.5-1.0 mM) blocked spontaneous spike discharge after 20 min. Higher concentrations (more than 2.0 mM) were required to block the evoked action potential.7. The results indicate that the smooth muscle spike in taenia is due to Ca-entry and that Na influences spike activity indirectly by competing with Ca in controlling the membrane potential.     

Calcium-sodium antagonism on the frog's heart: a voltage-clamp study.

1. In double sucrose-gap voltage-clamped frog atrial fibres the influence of [Ca]o and [Na]o on membrane current and contraction was investigated. 2. The slow (secondary) inward current varied with [Ca]o but was almost insensitive to changes in [Na]o. In contrast, the phasic (transient) contraction initiated by the slow inward current was affected by both [Ca]o and [Na]o. 3. With moderate changes of [Ca]o and [Na]o from normal, the strength of phasic contraction at a given depolarization followed the [Ca]o/[Na]2o ratio approximately. This was best seen at membrane potentials near zero level. 4. Under the same conditions, tonic (sustained) contractions associated with prolonged depolarizations were strictly correlated to the [Ca]o/[Na]2o ratio at any potential. No interrelation between tonic tension and steady-state current was found. 5. With extensive changes in [Ca]o and [Na]o, the sensitivity of both phasic and tonic tension to the [Ca]o/[Na]2o ratio declined, the negative effect of [Na]o becoming smaller than was expected from this ratio. 6. In Na-free choline-Ringer, a strong contracture developed followed by a spontaneous relaxation. Starting from the relaxed state, application of depolarizing clamps gave rise to phasic contractions with a very slow relaxation while tonic contractions were apparently lacking. 7. The results are interpreted in terms of an energy-dependent carrier mechanism exchanging one Ca for two Na ions across the cell membrane. The model implies a strong asymmetry in the rate constants governing the chemical reactions on both sides of the membrane. The system is thought to operate close to equilibrium at any potential, thereby determining the steady level of myoplasmic Ca. The equilibrium itself is considered to shift upon depolarization. Assuming that [Na]i is constant, the steady level of [Ca]i is expected to be proportional to the [Ca]o/[Na]2o ratio, the scale factor being a function of membrane potential. 8. The carrier model suggests the occurrence of a depolarization-induced inward transfer of Ca which might be involved in the generation of tonic contractions. 9. The apparent lack of tonic contractions in the absence of external Na ions may be explained by a suppression of carrier-mediated Ca influx normally occurring upon depolarization. 10. The antagonistic effects of [Ca]o and [Na]o on phasic contraction are understood as being due to alterations of the Ca pumping system rather than changes in slow inward current.     

Caffeine stimulates the reverse mode of Na/Ca2+ exchanger in ferret ventricular muscle.

This study investigated the effect of caffeine on the sarcolemmal mechanisms involved in intracellular calcium control. Ferret cardiac preparations were treated with ryanodine and thapsigargin in order to eliminate the sarcoplasmic reticulum (SR) function. This treatment abolished caffeine contracture irreversibly in normal solution. The perfusion with K-free medium that blocked the Na+--K+ pump resulted in a recovery of slow relaxing caffeine contractures similar to Na-free contractures. The amplitude of caffeine contractures was dependent on the bathing [caffeine]o and [Ca2+]o. Divalent cations Ni2+ and Cd2+, which have an inhibitory effect on the Na+/Ca2+ exchanger, produced dose-dependent inhibition of caffeine responses with apparent Ki of 780 +/- 19 and 132 +/- 5 microM, respectively. Caffeine also caused dose-dependent inhibition of Na-free contractures (Ki=4.62 +/- 1.5 mM), and the reduction or removal of [Na+]o exerted an inhibitory effect on caffeine contractures (Ki=73.5 +/- 17.12 mM). These experiments indicate that the increase in resting tension following exposure to caffeine was mediated by Na+/Ca2+ exchanger, which represents an additional element of complexity in caffeine action on cardiac muscle.     

Effects of sodium depletion on the caffeine-induced contraction of frog's skeletal muscle

Effects of depletion of Na ions from the environmental medium on the caffeine-induced contraction were investigated in both intact and skinned fibers prepared from the frog's fast twitch muscle. In intact muscle fibers, both the twitch and the contracture induced by 5 mM caffeine were enhanced by depleting the external Na at 9-10 degrees C. In contrast, in the mechanically skinned muscle fiber, which was used to examine the capability of the sarcoplasmic reticulum to take up Ca ions from the sarcoplasm and/or the superfusing medium, the contractile tension as well as its fractional area measured by applying 25 mM caffeine were markedly inhibited by Na-depletion at 5 degrees C. The apparent discrepancy between these two conditions is discussed.     

Involvement of a Na−Ca exchange mechanism in contraction induced by low-Na solution in isolated guinea-pig aorta

The possibility of involvement of a Na–Ca exchange mechanism in the contractile responses induced by a reduction of external Na concentration ([Na]₀) has been studied in isolated guinea-pig aorta. Low-Na (11.9 mM) solution (Lisubstituted) produced a contraction in ouabain-treated muscles in the presence of phentolamine (10^–6 M). The magnitude of the contraction was dependent on the duration of the pretreatment with ouabain (2×10^–5 M). Ca-free solution, but not verapamil (10^–6 M), abolished the contraction induced by low-Na solution. The muscles were loaded with various amounts of Na by incubating the tissue with ouabain and varying [Na]₀ (11.9–148.7 mM) in the absence of Ca. The magnitude of the contractions induced in these muscles by low-Na solution containing Ca (2.5 mM) was dependent on the cellular Na content. Loss of cellular Na into low-Na solution followed a single exponential time course and the rate coefficient of Na-loss in the presence of external Ca was about twice as great as in the absence of Ca. Cellular⁴⁵Ca uptake in low-Na solution was significantly greater in Na-loaded tissues (pretreated with ouabain for 3 h) than in normal tissues. The⁴⁵Ca uptake in low-Na solution was not inhibited by verapamil. These results suggest that the contraction induced by low-Na solution is caused by a Ca influx which is dependent on internal Na (a Na–Ca exchange mechanism).     

Effects of Na removal and readmission on the mechanical response in the guinea-pig tracheal smooth muscle

In the guinea-pig tracheal smooth muscle, tonic contraction, maintained in normal medium, was abolished by Ca removal or by indomethacin (10(-6) M). Removal of Na produced a transient contraction followed by a gradual decrease in tension to a level lower than the control. When Na was readmitted, a rapid relaxation was observed. The degree of the relaxation was enhanced with the exposure time to Na-free solution, and with the concentration of Na readmitted. At 5.9 mM K, the contraction produced by Ca readmission with simultaneous Na removal became smaller and slower as the external Na was reduced before Ca readmission, while at 40 mM K, the Ca-induced contraction was less dependent on the Na concentration. The rate of relaxation on Ca removal was slower when the Na concentration was reduced both at 5.9 and 40 mM K. Verapamil (10(-5) M) had a weak suppressing effect in normal solution, but suppressed markedly in 40 mM K medium. In the presence of verapamil, the difference between the effects of Na removal and of Na readmission on Ca-induced contraction was markedly potentiated, particularly at 40 mM K. It was concluded that the Na-Ca exchange mechanism may contribute to the transient response on Na removal and to the relaxation phase following Na readmission. At 5.9 mM K, removal of Na may increase the Ca conductance and also reduce the contribution of prostaglandins. On the other hand, when depolarized by excess K, the effect of Na is less significant; probably due to a high Ca conductance.     

Inhibition of the endothelium-dependent relaxation by 18beta-glycyrrhetinic acid in the guinea-pig aorta.

The effect of 18beta-glycyrrhetinic acid (GA), an agent which interferes with gap junction conductivity, on endothelium-dependent relaxation produced by substance P was investigated in isolated aortic rings of the guinea-pig. In nor-adrenaline (NA)-contracted aortic rings, substance P (10(-7) M) induced an endothelium-dependent, transient relaxation. The relaxation was only slightly reduced by the co-application of nitroarginine and diclofenac. When GA (2x10(-5) M) was applied first, it slightly reduced substance P-induced relaxation, and a subsequent co-application of nitroarginine and diclofenac strongly reduced the relaxation. In aortic rings contracted with high-K solution ([K(+)](o) = 29.4 mM), substance P-induced relaxation was reduced by the simultaneous application of GA, nitroarginine and diclofenac, but not by GA alone. In endothelium-denuded aortic rings, GA reduced the threshold concentration of NA required to produce contractions and increased the amplitude of NA-induced contractions. GA increased the amplitude of contraction produced by small increases of [K(+)](o) (<30 mM) but reduced those produced by higher concentrations of [K(+)](o) (>54 mM). In NA-contracted aortic rings, Y-26763, a K(+)-channel opener, could relax muscles with reduced amplitude in the presence of GA. It is concluded that in guinea-pig aortic rings, GA inhibits mainly the EDHF-induced components of endothelium-dependent relaxation. GA also modulated contractions produced by NA or high-K solutions. The possible effects of inhibition of gap junctions by GA on endothelium-dependent relaxation were discussed.     

Effects of various intracellular Ca ion concentrations on the calcium current of guinea-pig single ventricular cells

The effects of changing the intracellular Ca concentration ([Ca]i) on the calcium current (iCa) were studied in isolated single ventricular cells of the guinea-pig. [Ca]i was varied by an intracellular perfusion technique using a suction pipette. iCa measured from internally perfused cells at a pCa lower than 9.0 was dependent on the extracellular Ca concentration ([Ca]o). Increasing [Ca]o from 1.8 to 5.4 mM increased the amplitude of iCa, and reduction of [Ca]o from 1.8 to 0.01 mM decreased the amplitude. The inactivation time course of iCa became faster as [Ca]o was increased and slower as [Ca]o was reduced. By decreasing the pCa of the internal perfusate from 9.0 to 6.8, the amplitude of iCa was decreased markedly, but no significant change was observed in its time course. Analysis of the I-V curve led to the conclusion that a change in the driving force was not a major factor in the reduction of iCa. The "steady state inactivation" of iCa was measured by a double-pulse method. The amplitude of iCa elicited by the test pulse was smaller at pCa 7.4 than at pCa 9.0 at potentials of between -27 and +33 mV. By normalizing the iCa amplitude, however, the "steady state inactivation" curve in the control and at high [Ca]i overlapped. Similar results were obtained in Sr-Tyrode solution. The degree of "steady state inactivation" of iCa at the potentials positive to +10 mV was larger in Ca-Tyrode than in Sr-Tyrode solution. It is proposed that the reduction in amplitude of iCa at higher [Ca]i is caused by a reduction of the maximum conductance of iCa (gCa) and that Ca ions passing through iCa channels have a remarkable effect on its inactivation.     

Possible involvement of sodium pump in the relaxation of rat aorta induced by alpha-human atrial natriuretic polypeptide

In order to clarify whether the sodium handling of smooth muscle is associated with the relaxing action of alpha-human atrial natriuretic polypeptide (alpha-hANP), we examined the sodium pump-related effects of alpha-hANP on rat aortic smooth muscles. Application of Ca2+ (1.0 to 10.0 mM) to the muscle preincubated in Ca2+-free, and K+-free or 0.5 mM K+ medium for 60 min induced a contraction. Pretreatment with alpha-hANP (1 x 10(-8) M) decreased the contraction evoked in 0.5 mM [K+]o but not that in K+-free medium. After a contraction was elicited by norepinephrine in K+-free solution, an addition of KCl (1.4-5.4 mM) caused a transient relaxation in a concentration-dependent manner, presumably due to the activation of electrogenic Na pump. The alpha-hANP enhanced the relaxation, which was sensitive to ouabain, and the potentiation by alpha-hANP was inversely related to the concentration of K+ added. When alpha-hANP was applied to relax the muscle precontracted by norepinephrine in the varied concentration of external K+, alpha-hANP-induced relaxation was greater in 1.4 or 2.7 mM [K+]o than in 0 or 5.4 mM [K+]o. These results suggest that the vasodilating effect of alpha-hANP is at least partially mediated by the activation of electrogenic Na, K-pump and this effect is prominent when the Na, K-pump is partially suppressed.     

The effect of the internal sodium concentration on calcium fluxes in isolated guinea-pig auricles

1. Calcium efflux from guinea-pig auricles followed saturation kinetics when [Ca](o) and [Na](o) were changed while the ratio [Ca](o)/[Na](o) (2) was kept constant. The Michaelis constant, K(m) (Ca+Na) = 40 mM, suggests that a hypothetical carrier system, responsible for sodium-calcium exchange, is far from saturation with the inside concentrations of these ions.2. [Na](i) was altered in the auricles between 12.5 and 60 mM/kg fibre water while total cellular calcium concentration ([Ca](t)) at the beginning of the influx period was not significantly different in the various groups of preparations.3. (45)Ca influx increased appreciably with increasing [Na](i). (45)Ca influx from sodium-poor solution corresponded to an almost equal increase in [Ca](t), while [Ca](t) did not change much in preparations loaded with (45)Ca in Tyrode solution. When the sodium-activated fraction of calcium influx was plotted against [Na](i) (2) the resulting curve indicated saturation with K(m) (Na) = 3500 (mM [Na](i))(2) and maximal influx rate, J(i, max) (Ca') = 1.35 mM/kg wet weight x 10 min.4. When the preparations were re-equilibrated for various times in normal Tyrode solution after [Na](i) had been increased, both the sodium-activated component of calcium influx and [Na](i) (2) decreased with approximately the same rate constants.5. Calcium efflux from auricles with high [Na](i) was increased when it was measured in Tyrode solution while the efflux in sodium-poor solution was inhibited.6. Auricles with increased [Na](i) showed a positive inotropic contractile response.7. The main conclusion reached by these experiments is that calcium influx is affected by [Na](i) in a way which is compatible with a carrier-mediated sodium-calcium exchange system.     

Comparative effects of Mg, Ca, Sr, and verapamil on the uterine longitudinal muscle of spayed and estrogen-treated rats

A comparison was made of effects of Mg, Ca, Sr, or verapamil on the mechanical and electrical activities in the uterine longitudinal muscles of spayed and estrogen-treated rats. The muscle strips taken from spayed rat exhibited spontaneous rhythmic activity in the Locke solution which did not contain Mg, whereas spontaneous activity was less frequent in the preparation taken from estrogen-treated rat. The resting potentials were -54 and -61 mV in the spayed and the estrogen-treated preparations, respectively. An initial spike potential followed by plateau potential with abortive spikes on the top was generated in both spayed and estrogen-treated preparations. In the spayed preparation, the frequency of rhythmic contractions was reduced, and the base-line tension was lowered when 0.6 mM Mg was added to the solution. The base-line tension was elevated progressively when the external Ca concentration was raised, and reached a maximal value up to 10 mM. The amplitude of phasic contraction was progressively increased by increasing Ca concentrations in the range from 1.25 to 5 mM, and was reduced by Ca higher than 10 mM. In the estrogen-treated preparation, the amplitude of phasic contraction was increased by increasing Ca concentrations in the range from 1.25 up to 17.5 mM. When the amplitude of phasic contraction was increased, the duration of plateau potential became protracted. Substitution of the external Ca with Sr caused an increase in the spike activity generated on the top of plateau potential. However, the amplitude of phasic contraction was diminished in both the spayed and the estrogen-treated preparations. Verapamil (2 microM) caused a stronger depression of electrical and mechanical activity in the spayed preparation. Results were discussed in relation to the genomic effects of estradiol on the membrane properties so as to change the interaction with divalent cations.     

The mechanical response of rat myometrium to adenosine triphosphate in Ca-free solution

The contractile response of the myometrial longitudinal muscle of pregnant and estrogen-treated rats to adenosine triphosphate (ATP) was investigated. ATP (10(-5)-4 X 10(-3) M) added to Krebs solution caused a generation of spontaneous activity consisting of phasic contractions and an elevation of muscle tone in a dose-dependent manner. Effects of the "test solution" consisting of isotonic K, 4(mM) ATP, 4 Mg, 1-20 EGTA, 20 tris(hydroxymethyl)aminomethan (Tris) maleate (pH 6.8) on the contractile response was then investigated. An initial phasic and a following tonic contraction was evoked by the application of the test solution, when applied after the equilibration of the muscle with Krebs solution. ATP was proved to be an agonist to evoke the Ca-free contraction. The phasic contraction was depressed when the muscle was incubated with Ca-free Krebs solution. The amplitude of the tonic contraction became progressively larger when application of the test solution was repeated. The amplitude was 15-70% as large as the tonic component of the K-contracture induced by 40 mM K. Theophylline (10 mM), 0.1 mM papaverine and 1 microM isoprenaline nearly abolished, and 1 mM cAMP partly depressed the tonic contraction of K-contracture, whereas the tonic contraction induced by the test solution was unaffected. A calmodulin antagonist, W-7 (100 microM) strongly suppressed both the K-contracture and the contraction induced by the test solution, whereas trifluoperazine (10-200 microM) preferentially depressed the K-contracture. The tonic contraction induced by the test solution was strongly depressed when Mg was removed and 20 mM EDTA was applied. From these results, it is discussed that some Ca-independent process is involved in the generation of tonic component of Ca-free contraction developed by the application of the test solution.     

Catecholamine release from bovine chromaffin cells: the role of sodium-calcium exchange in ouabain-evoked release

Spontaneous catecholamine (CA) release from bovine chromaffin cells maintained in primary tissue culture has been measured after pre-loading the cells with [3H]noradrenaline. Ouabain inhibited 86Rb+ uptake and increased 3H release in a concentration-dependent manner during a 60 min incubation period. Low external Na+ (5 mM: Li+ substitution) also increased 3H release. Whereas the 3H-releasing action of ouabain was maintained, the Li(+)-evoked release decreased with time. The effects of both ouabain and low Na+ solution on 3H release were completely inhibited by removal of Ca2+ from the external medium even though in Ca2(+)-free solution ouabain further inhibited 86Rb+ uptake into the cells. Readmission of Ca2+ to Na(+)-loaded cells (10-4 M-ouabain in Ca2(+)-free-1 mM-EGTA solution for 60 min) markedly increased the release of 3H. In the additional presence of diphenylhydantoin (DPH, 10-4 M) 3H release was significantly less on Ca2+ readmission. The 3H release from Na(+)-loaded cells was proportional to the concentration of Ca2+ readmitted. The 3H release was further increased from Na(+)-loaded cells in response to Ca2+ readmission when [Na+]o was lowered from 149 to 5 mM (Li+, choline+, Tris+ or sucrose substitution) though Li+ was less effective than the other Na+ substitutes. Potassium removal from the external medium significantly inhibited the 3H release evoked by Ca2+ readmission to Na(+)-loaded cells, even when [Ca2+]o was greater than normal (7.5 mM) or if Ca2+ was readmitted in low [Na+]o solution. Rb+, Cs+ or Li+ could substitute for K+ with the order of potency: Rb+ greater than or equal to K+ greater than Cs+ greater than Li+. A slight increase of external K+ (10.8 mM) potentiated the 3H release from Na(+)-loaded cells on Ca2+ readmission, but a higher concentration of K+ (149.4 mM) had the opposite action. The data is consistent with the hypothesis that ouabain-evoked CA release from bovine chromaffin cells is, in part, a consequence of an internal Na(+)-dependent Ca2+ influx. The evidence also suggests that there is Na(+)-Ca2+ competition at the external arm of the exchanger together with a monovalent cation activation site.     

Sodium-calcium sites in smooth muscle and their accessibility to lanthanum

1. The Na, K, Ca, and Mg contents were determined in longitudinal smooth muscles of the guinea-pig ileum incubated in various physiological solutions with or without ouabain or in a solution containing LaCl(3) 10 mM instead of CaCl(2).2. The Na and K contents were dependent upon the K concentration of the physiological solution. A decrease in [K](o) below 6 m-mole/l. increased tissue Na.3. A rise in [Na](i) was followed by an increase of total cell Ca preceding a secondary decrease of total Ca, as well as by an increased (45)Ca content occurring even when total Ca was decreased.4. Muscles washed in the lanthanum solution contained less Ca and Na than those incubated in the physiological solution. The Mg content was not significantly modified in the presence of La.5. In the La solution, the (45)Ca diffusion space was not different from the [(14)C]inulin diffusion space, indicating that there was no Ca entry within the cell nor Ca binding at superficial sites. (45)Ca content of preloaded muscles decreased; this decrease was blocked after 30 min washout in the La solution: this arrest lasted a further 45 min.6. The cell Ca fraction displaced by increased [Na](i) originated from a Ca compartment which was not blocked by La.     

Effects of Ca removal on the smooth muscle of the guinea-pig taenia coli

1. The effects of removing the external Ca ions on the spontaneous and evoked activity of the smooth muscle of the guinea-pig taenia coli were investigated with the double sucrose-gap method.2. In Ca-free Locke solution the membrane was depolarized, the membrane resistance became low, the spike amplitude became small and the mechanical response decreased. In most preparations the electrical and mechanical activity was abolished within 10 min, but in some preparations the electrical activity continued for more than 30 min.3. In Ca-free solution containing 0.1 mM-EGTA, the membrane was depolarized and the electrical and mechanical activity was abolished within 5 min in every preparation. When NaCl was replaced with sucrose, the effects of Ca removal on the spike activity and contraction appeared very slowly and the membrane potential and membrane resistance remained unchanged.4. When Ca was replaced with Mg (2 mM) the spike was blocked within 1 min without depolarization or reduction of the membrane resistance. In Na-deficient (sucrose) solution, the presence of Mg accelerated the disappearance of the spike caused by Ca removal.5. In Ca-free solution containing 0.5 mM-Mg, a spike-like activity was observed without accompanying mechanical response. This activity was blocked by increasing the Mg concentration above 2 mM. It was Na-dependent, since it was abolished by removing Na from the external solution, but it was not influenced by tetrodotoxin (2 x 10(-6) g/ml.).6. It was concluded that calcium has at least two functions, one as current carrier for the action potential and another as controller of the Na permeability of the membrane. It was also suggested that the Ca which is bound at the membrane may be utilized as a source of Ca ions to carry the current for the action potential.     

The effects of manganese ions on the contraction of the frog's heart

1. The effects of Mn(2+) in particular but also of Ni(2+), Co(2+), Cd(2+), verapamil and D600 on the contraction of isolated frog atrial trabeculae have been investigated. Contraction was initiated either by electrical stimulation, or by raising the [K](o) or by lowering the [Na](o).2. Mn ions like Ca ions cause a hyperpolarization of the cell membrane and a rise in the threshold for the action potential and twitch. Mn ions, particularly at low concentrations, reduce the overshoot of the ventricular action potential.3. Mn ions reduce the strength of the regularly evoked heart beats. Prolonged exposure, in beating hearts, results in a rise in resting tension and often a small recovery of the heart beat.4. In normal Ringer solution and in Ringer free of Na ions, the addition of Mn causes the tension-depolarization curve to be displaced by an amount equivalent to an 18 mV hyperpolarization of the membrane potential for a tenfold increase in the divalent cation concentration.5. Mn, Co, Ni and Cd ions all cause a marked reduction in the tension generated by exclusion of Na ions from the bathing fluid. In the presence of these divalent cations the contracture divides into an initial phasic and a later tonic contraction. This inhibition is reversed by raising the [Ca](o), while the tension developed during the initial phasic contraction varies with the [Ca](o)/[Mn](o) quotient.6. A similar tonic contracture is initiated after exposure to Na-free fluid containing a high [Mn](o) by the addition of a small concentration of Na, Li, hydrazinium or hydroxylammonium ions.7. The organic ;Ca antagonists' verapamil and D600 have little effect on the contracture induced by lowering [Na](o) even after prolonged exposure at relatively high concentrations but they do inhibit the twitch contraction and the K contractures.8. The effects of Mn on the Na-withdrawal contracture of frog heart can be interpreted in terms of an exclusively extracellular effect where Mn ions resemble Na ions in their action, and both antagonize the movement of Ca across the cell membrane.9. The experimental evidence suggests that the K contracture in frog heart is initiated by a mechanism which is, in some ways different to that underlying the Na-withdrawal contracture, and may involve two different sources of activator Ca.10. The several different effects of Mn on the frog heart probably reflects the ability of this cation to interfere with many processes involving Ca, and that there are a number of such processes involved in the results described in this work. The effects of Mn are more complex than might be generally supposed.     

Mechanism of activation of the tonic component of contraction in myocytes of guinea pig heart

AIM: Contractions of myocytes of guinea pig heart consist of a phasic component relaxing independently on the voltage and a tonic component relaxing upon repolarization. We found previously that Ca(2+) activating the tonic component is released from the sarcoplasmic reticulum. In this study, we analysed the mechanism of activation and maintenance of this release. METHODS: Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped myocytes were stimulated by the pulses of the duration of 300 ms to 15-45 s from the holding potential of -40 to +5 mV. [Ca(2+)](i) was monitored as fluorescence of Indo-1 and contractions were recorded with the TV edge-tracking system. RESULTS: Myocytes responded to the short and long pulses with phasic contraction or Ca(2+) transient followed by the sustained contraction or increase in [Ca(2+)](i). Repolarization brought about relaxation. 10 mmol L(-1) Ni(2+) blocking Na(+)/Ca(2+) exchange superfused during the tonic component increased its amplitude. Superfusion of Ca(2+)-free solution during sustained contraction brought about relaxation both in normal cells and in cells superfused with Ni(2+) despite preserved sarcoplasmic reticulum Ca(2+) content assessed with caffeine spritz. Relaxing effect of Ca(2+)-free solution was not affected by carboxyeosin, a blocker of sarcolemmal Ca(2+)-ATPase. Tonic component of contraction and of Ca(2+) transient was inhibited by 200 micromol L(-1) ryanodine, a blocker of Ca(2+) release channels of the sarcoplasmic reticulum and by 20 micromol L(-1) nifedipine, a blocker of L-type I(Ca). CONCLUSION: Tonic component of contraction results from Ca(2+) release via the sarcoplasmic reticulum Ca(2+) channels activated by sustained, nifedipine-sensitive and Ni(2+)-insensitive Ca(2+) influx. Alternatively, the SR Ca(2+) release is activated by voltage, the dihydropyridine receptors acting like voltage sensors.