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.     

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.     

Modulation of intracellular Na+ and Ca2+ induced by the cardiac Na+-Ca2+ exchanger in guinea pig ventricular myocytes

The Na+-Ca2+ exchanger current was measured in single guinea pig ventricular myocytes, using the whole-cell voltage-clamp technique, and intracellular free calcium concentration ([Ca2+](i)) was monitored simultaneously with the fluorescent probe Indo-1 applied intracellularly through a perfused patch pipette. In external solutions, which have levels of Ca2+ (approximately 66 microM Ca2+) thought low enough to inhibit exchanger turnover, the removal of external Na+ (by replacement with Li+) induced both an outward shift of the holding current and an increase in [Ca2+](i), even though the recording pipette contained 30 mM bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), sufficient to completely block phasic contractions. The effects of Na+ removal were blocked either by the extracellular application of 2 mM Ni2+ or by chelating extracellular Ca2+ with 1 mM EGTA. In the presence of 10 microM Ryanodine, the effects of external Na+ substitution with Li(+) on both membrane current and [Ca2+](i) were attenuated markedly in amplitude and at a much slower time course. Reversal potentials were estimated by using ramp pulses and by defining exchange currents as the Ni2+-sensitive components. The experimental values of the reversal potential and [Ca2+](i) were used to calculate cytosolic Na+ ([Na+](i)) by assuming an exchanger stoichiometry of 3Na+ : 1Ca2+. These calculations suggested that in the nominal absence of external Ca2+ ( approximately 66 microM under our experimental conditions), the exchanger operates at -40 mV as though approximately 40 mM Na+ had accumulated in the vicinity of the intracellular binding sites. We conclude that under the conditions of low extracellular Ca2+ and high intracellular Ca2+ buffering, the Na+-Ca2+ exchanger can still generate sufficient Ca2+ influx on the removal of external Na+ to markedly increase cytosolic free Ca2+.     

Reversal of alpha 1-receptor mediated relaxation in intestinal smooth muscle

The alpha 1-receptor agonist phenylephrine relaxed longitudinal rabbit jejunal muscle contracted in vitro by low concentrations of barium ions (1 mM). When the Ba2+ concentration was increased to 10-15 mM the response to phenylephrine was a contraction, and at Ba2+ concentrations in between the high and low range this response was biphasic--a relaxation followed by a contractile phase. The alpha 2-receptor agonist clonidine did not affect the tone of the Ba2+ contracted preparation. When the muscle preparation was contracted by Sr2+ (1-20 mM) in the presence of Ca2+ (2.5 mM), phenylephrine relaxed it, and no contractile response to phenylephrine was observed. In the absence of extracellular Ca2+, 5 mM Ba2+ caused a contraction. Under these conditions phenylephrine had no effect on the tissue tone. When Ca2+ was added in a low concentration (0.2-2 mM), phenylephrine elicited a gradually increasing contractile response. At 5 mM Ca2+ the contractile response was replaced by the normal relaxation. The contractile response to phenylephrine in the presence of 5 mM Ba2+ and 2.5 mM Ca2+ was partially blocked by low concentrations of verapamil. In higher concentrations verapamil abolished the tissue tonus completely. The contractile response to phenylephrine in the presence of 5 mM Ba2+ and 2.5 mM Ca2+ could be reverted to the normal relaxation by the addition of 20 mM Mg2+. Increasing the K+ concentration from the normal 5.9 to 62.9 mM blocked the phenylephrine-induced relaxation. No contractile response to phenylephrine occurred. It is concluded that Ba2+ could reverse the response of alpha 1 receptor stimulation in rabbit jejunum from a relaxation to a contraction and that this contractile response was dependent on the presence of Ca2+.     

Internal free magnesium modulates the voltage dependence of contraction and Ca transient in rabbit ventricular myocytes

 We investigated the effect of altering internal free magnesium concentration (Mg_i) on the contraction and Ca_i transient of patch-clamped rabbit ventricular myocytes. Experiments were performed at 35°C; cells were held at –40 mV to inactivate Na channels and T-type Ca channels, and at this potential (and in the absence of cyclic AMP) ”Ca-induced Ca release” is the primary trigger mechanism. Cells dialysed with a low Mg_i (2.9 μM) had a large and fast phasic contraction and Ca_i transient at positive potentials (+60, +80 mV). Cells dialysed with a high Mg_i (7.1 mM) had a small or absent phasic contraction and Ca_i transient at positive potentials. These effects were due to a change in free Mg_i, and not due to a change in [Mg.ATP]. In cells dialysed with a low Mg_i, application of Ca channel blockers (32 μM nifedipine with 10 μM D600) for a single beat abolished current through L-type Ca channels (I _Ca,L); however, 53% of the Ca_i transient was still elicited. Adding 5 mM Ni to Ca channel blockers abolished the remaining Ca_i transient, indicating that (in the absence of I _Ca,L) the transient might be triggered by reverse Na/Ca exchange. In cells dialysed with a high Mg_i, a single-beat switch to Ca channel blockers was sufficient alone to abolish the Ca_i transient, indicating that under these conditions Ca entry via I _Ca,L is the primary sarcoplasmic reticulum trigger mechanism. These results suggest that raised free Mg_i might partially inhibit the activity of the Na/Ca exchange, or might limit its ability to trigger Ca release. Received: 29 August 1997 / Received after revision: 12 November 1997 / Accepted: 13 November 1997     

Comparative study of the effects of propranolol and tetracaine on cation movements in resealed human red cell ghosts.

1. The effects of two positively charged local anaesthetic amines, tetracaine and propranolol, on cation permeability were studied in resealed human red cell ghosts prepared from metabolically depleted erythrocytes. 2. The K permeability was reduced by tetracaine but increased by propranolol. The effect of tetracaine was independent of extracellular Ca concentration but was raised to 2-5 x 10(-7) M. The effect of propranolol, which was enhanced when the external Ca concentration was raised, could be completely inhibited by lowering the internal free Ca to less than 10(-7) M. 3. Propranolol, but not tetracaine, increased the intracellular Ca ion concentration by releasing up to 20% of the membrane-bound Ca to the cell interior. This increase in intracellular Ca was sufficient to mediate the observed change in K permeability. 4. Tetracaine and propranolol reduced the Ca binding capacity of the ghost membrane by about 20 and 40% respectively. The Ca permeability was increased by propranolol and was slightly reduced by tetracaine. 5. In high concentrations (2-7 mM) propranolol by itself moderately increased K and Na permeability, but supressed completely the Ca-induced increase in K permeability. Tetracaine in concentrations up to 4 mM enhanced the Ca-induced increase in K permeability. Higher concentrations of the drug caused lysis of the cells. 6. Maximally effective concentrations of tetracaine and propranolol inhibited the ATP-dependent Ca outward transport by 30 and 70% respectively. 7. The effects of tetracaine on K permeability were shared by the local anaesthetics prilocaine and lidocaine, those of propranolol were shared by practolol, a beta-adrenergic antagonist and tetraethylammonium, a ganglionic blocking agent. 8. It is suggested that the differences in the effects of tetracaine and propranolol on cation permeability reflect qualitatively different interactions of the two drugs with Ca binding sites on the inner surface of the membrane.     

Activation of the contractile mechanism in the anterior byssal retractor muscle of Mytilus edulis.

1. The electrical and mechanical responses of the anterior byssal retractor muscle (ABRM) of Mytilus edulis to acetylcholine (ACh), high [K]O or the removal of external Ca were examined under a variety of conditions. 2. ACh (10(-6)--10(-3)M) produced contracture tensions larger than those produced by high [K]O (30-300 mM) for a given amount of depolarization. In Ca-free solution, the rate of decline of ACh-contractures was much smaller than that of K contractures, though both ACh- and K-contractures eventually disappeared. 3. 5-HT (10(-4)M) of procaine (1 mM) markedly reduced the height of ACh-contractures, but had little or no effect on K-contractures. The height of K contractures was markedly decreased by Mn ions (20 mM) or low pH (4-5), while ACh-contractures remained unaffected. 4. Partial replacement of [Na]o by choline (30-100 mM) reduced both ACh-induced depolarization and contracture tension, whereas K-contractures remained unchanged even after total replacement of [Na]o by choline. 5. ACh could produce little or no tension when applied during the relaxation phase of K-contractures, while high [K]o produced the maximal contracture tension when applied during the relaxation phase of ACh-contractures. 6. Following the removal of external Ca from solutions containing less than 10 mM-Mg, the ABRM showed a marked tension development associated with repetitive electrical activity superimposed on a gradual decline of membrane potential. 7. These results suggest that ACh-contractures are mainly due to the release of intracellularly stored Ca, while K-contractures are mainly associated with the inward movement of external Ca.     

Contribution of Na+/Ca2+ exchanger to glucose-induced [Ca2+]i increase in rat pancreatic islets

The present study was carried out to elucidate the role of the reverse mode of the Na+/Ca2+ exchanger in an increase in intracellular Ca2+ concentration ([Ca2+]i) induced by a stimulatory concentration of glucose in rat pancreatic islets. The effects of KB-R7943, a selective inhibitor of reverse Na+/Ca2+ exchanger, on Na+o removal-induced [Ca2+]i changes were examined by a microfluorimetric method using fura-2 in perifused preparations of isolated rat pancreatic islets. Na+o removal induced a rapid increase in [Ca2+]i under 100 or 5 mM K+ conditions, respectively. The increases in [Ca2+]i induced by Na+o removal were inhibited by KB-R7943. The net amount of the [Ca2+]i increases during Na+o removal (Delta[Ca2+]i), obtained by subtracting the KB-R7943-independent Delta[Ca2+]i in the presence of KB-R7943 from Delta[Ca2+]i in the absence of KB-R7943, was significantly increased when extracellular K+ was raised. Increasing the external glucose concentration from 3 to 20 mM caused a biphasic increase in [Ca2+]i, which exhibited a transient increase (first phase) followed by a sustained increase (second phase) in [Ca2+]i. KB-R7943 (10 microM) partially inhibited the second phase of the [Ca2+]i increase rather than the first phase. These results suggest that the increase in [Ca2+]i induced by Na+o removal may be enhanced when plasma membrane is depolarized, and consequently, Ca2+ influx through the reverse Na+/Ca2+ exchanger may partially contribute to the glucose-induced [Ca2+]i dynamics in rat pancreatic islet cells.     

Effects of magnesium and temperature during the recovery process from the potassium contracture of the pregnant rat myometrium.

Effects of external ions (Na, K, Mg) on the recovery from K-contracture of the pregnant rat myometrium were investigated in Ca-free media. Relaxation was faster, when the external concentration of Na and K ions was higher. The time course of relaxation within a limited range of 60-20% of the mechanical output followed a single exponential function. The rate of relaxation, given by the reciprocal of the period during which the contraction decayed to 1/e, was 0.3-0.5 min-1 at 34-35 degrees C in isotonic Na solution. Low temperature caused prolongation of the relaxation, and the activation energy estimated on the rate of relaxation was 3-4 kcal-mol-1. Relaxation became faster when Mg (0.5-20 mM) was added to the relaxation media. Relaxation in isotonic K media was slower than in isotonic Na media, and the addition of Mg caused a slight acceleration of the relaxation. Low temperature caused an acceleration of the relaxation, irrespective of the presence or absence of external Mg ions. In low K media, where K ions were replaced by isomolar sucrose, addition of Mg caused a significant acceleration of the relaxation. Activation energy ranged between 13-15 kcal mol-1 in the solutions containing Mg. This favors the existence of a relaxing system in pregnant rat myometrium that is Mg- and metabolism-linked.     

Preferential loss of EC coupling gradation at positive membrane potentials in rat ventricular myocytes

Rat ventricular myocytes switched from a solution containing 1 mM extracellular Ca to one containing 5 mM extracellular Ca exhibited larger Ca currents that inactivated more rapidly. Their Ca transients exhibited a slower decay. In some cells the amplitude of the Ca transients increased markedly, but not in others. Strikingly, in about half the cells examined, the graded nature of the Ca transients over the range of positive membrane potentials was lost, as were Ca transients elicited upon repolarization. These changes were manifest without equivalent loss of gradation over the range of negative membrane potentials (-35 to 0 mV). Such preferential loss of gradation at positive membrane potentials also occurred in solutions devoid of extracellular and/or intracellular Na, and was abolished by nifedipine. The results suggest that Ca-induced Ca release from the SR at positive potentials can saturate or become regenerative if the Ca entry trigger is increased. The lack of a similar effect at negative potentials indicates that large numbers of activated L-type channels are critical for induction of regenerative behavior.     

Calcium spike and calcium-dependent potassium conductance in mechanosensory neurons of the lamprey

Action potentials (APs) of long duration (up to 1 s) followed by prolonged (0.5-5 s) hyperpolarizing afterpotentials (HAP) were recorded in lamprey primary mechanosensory neurons (dorsal cells) in isolated spinal cords exposed to either or both of the potassium channel blockers, tetrathylammonium (TEA) and 3,4-diaminopyridine (DAP). The membrane events underlying the prolonged AP and HAP were investigated in current clamp studies and were shown to be a Ca spike- and a Ca-dependent K conductance, respectively. The prolonged AP was accompanied by an increased membrane conductance and, unlike the normal Na AP in these cells, was not blocked by tetrodotoxin (TTX) or by replacement of external Na with choline or TEA. Reduction of [Ca]o from 10 to 0 mM reduced the amplitude and duration of the prolonged TTX-resistant AP but did not eliminate it within the 15-min washout period, probably because of Ca buffering in the spinal cord. The overshoot of the prolonged AP varied in amplitude as a linear function of the log of the external Ca concentration (2.5-10 mM) with a slope of 31.5 mV for a 10-fold change in Ca concentration, a value close to the 28 mV expected from the Nernst relation. Co (2 mM) and Cd (1 mM) blocked the prolonged APs. Ba and Sr substituted for Ca. The APs in Ba were extremely long lasting (up to 40 s). The HAPs following Ca spikes were 0.5-5 s in duration (peak to half amplitude) and were accompanied by an increased membrane conductance. The HAP varied in amplitude with the extracellular K concentration, reversed in sign at the presumed K equilibrium potential (-90 mV), and was insensitive to injected Cl. We conclude that HAP is a result of increased K conductance. The increase in K conductance during the HAP appeared to be dependent on Ca influx, because the amplitude and duration of the HAP varied with the extracellular Ca concentration and increased in duration during repetitive Ca spike activation, presumably as a result of accumulation of Ca intracellularly. Further, the HAP was absent following even very long lasting spikes in Ba, an ion that in other cells does not activate the Ca-dependent K conductance. Small regenerative depolarizations sometimes followed Ca spikes in dorsal cell somata. These are believed to reflect Ca spikes in discrete axonal regions at various electrotonic distances from the soma.     

Effects of sodium depletion on contractions evoked in intact and skinned muscles of the guinea-pig mesenteric artery

In the guinea-pig mesenteric artery, reduction in [Na]o by 30 mM (substituted by choline or sucrose; 137 mM [Na]o in Krebs solution) generated contraction with no change in membrane potential. In NaCl-free solution (15 mM [Na]o), the amplitude of phasic contraction reached 0.8 times the contraction evoked by 118 mM [K]o with only a slight depolarization. In NaCl-free solution, the amplitude of phasic contraction evoked by noradrenaline (NA) 5 X 10(-5) M or caffeine 5 mM increased to roughly twice the amplitude of the contraction evoked in the control solution. In Ca-free solution, the K-, NaCl-free- or Na-free-induced contractions rapidly ceased, but NA-induced contraction ceased within 5 min and the caffeine-induced contraction persisted for more than 15 min. In a skinned fiber, increase of [Na]o from 10 to 60 mM suppressed the pCa-tension relationship in the ranges of 10(-7) and 10(-5) M free Ca but not with a dose of 30 mM [Na]o. NA (10(-5) M) had no effect on skinned fibers. Increase in Na concentration (60 mM) had no effect on Ca accumulation in the store site or on Ca release by caffeine. Possible Na-related mechanisms on the development of mechanical response are discussed in relation to Ca on the surface and in the internal membrane structure. The NaCl-free-induced contraction in smooth muscles of the guinea-pig mesenteric artery is postulated to be due to influx of Ca through the Na channel, rather than the Ca channel.     

Contribution of intracellular stored calcium to contractile activation in contractures of stomach circular muscle of Bufo vulgaris formosus

Contractile responses of stomach circular muscle of Bufo to high-K, to acetylcholine (ACh) in normal Ringer or in high-K solution, and to calcium in Ca-free high-K solution showed a phasic contraction which relaxed completely in 30-45 sec. K-induced contracture was abolished in Ca-free solution containing 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) within 20 sec, while ACh-induced contracture was not abolished and 10-25% of control tension was kept up to 40 sec. This response increased to 40-50% when all extracellular Na was replaced with tris(hydroxymethyl)-aminomethane (Tris). K-induced contracture was inhibited completely by 1 mM La. ACh-induced contracture in the muscle depolarized by high-K solution was dependent on the depolarization time, 0-10, 60-70, and nearly 100% of control after 1, 3, and 10 min depolarization, respectively. These ACh-induced contractures were not inhibited by 1 mM EGTA or La. All contractures mentioned above were markedly inhibited by 5 mM procaine. These results suggest that activation of both contractures induced by high-K and ACh were, at least partly, dependent on the Ca at the intracellular Ca storage sites. Ca-induced contracture was dependent on depolarization time as was ACh-induced contracture, when the muscle was depolarized by Ca-free high-K solution without pre-treatment with Ca-free Ringer solution. These results suggest that activation of Ca-contracture is also dependent on intracellular stored Ca.     

Contribution of Na+/Ca2+ exchanger to the regulation of myogenic tone in isolated rat small arteries.

The contribution of the Na+/Ca2+ exchanger to the myogenic vascular tone was examined in rat isolated skeletal muscle small arteries (ASK) with pronounced myogenic tone and mesenteric small arteries (AMS) with little myogenic tone. Myogenic tone was assessed by the vascular inner diameter at transmural pressures of 40 and 100 mmHg. To depress the Na+/Ca2+ exchanger, the extracellular Na+ concentration ([Na+]o) was lowered from 143 to 1.2 mM by substituting choline-Cl for NaCl. The ASK developed significant myogenic tone and constricted further in low [Na+]o. Nifedipine (1 microM) reduced both myogenic tone and low [Na+]o-induced contraction. Because the membrane potential of ASK was not changed by low [Na+]o (-35 +/- 2 mV at 143 mM [Na+]o, -37 +/- 3 mV at 1.2 mM [Na+]o), depolarization-induced Ca2+ influx was not a cause of the low [Na+]o-induced contraction. The AMS did not develop significant myogenic tone. Although low [Na+]o also constricted AMS, the magnitude of constriction was significantly weaker than that in ASK (17 +/- 4 vs. 47 +/- 6%, P < 0.01, at 58 mM Na+). With Bay K 8644, AMS developed myogenic tone, and low [Na+]o-induced constriction was significantly increased. In conclusion, Na+/Ca2+ exchanger may play an important role in regulating myogenic tone, likely via mediating Ca2+-extrusion.     

External K+ increases Na+ conductance of the hyperpolarization-activated current in rabbit cardiac pacemaker cells.

The hyperpolarization-activated current (I(f)) was recorded from single myocytes dissociated from rabbit sinoatrial node. Although I(f) is usually carried by both Na+ and K+, removal of the minor K+ component from physiological saline suppresses inward component. This inward Na+ current through I(f) channel increases on raising the extracellular K+ concentration. The Na+ conductance relative to K+ conductance (PNa/PK), as measured from the reversal potential, increases and saturates near 5 mM K+. This effect is different from the current increase caused by raising the concentration of carrier ion K+, which saturates at 70 mM with a half-maximal value (K1/2) of 10 mM. It is suggested that the I(f) channel has multiple, interactive binding sites for cation permeation.     

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.     

The secretory mechanism in adrenal chromaffin cells by nitrophenol compounds: possible involvement of the change in the surface potentials

The rabbit adrenal gland was perfused with a modified Locke's medium and the mechanism of adrenaline secretion induced by nitrophenol compound was investigated. The secretory response to 2,4-dinitrophenol (DNP) or trinitrophenol (TNP) showed the following peculiar features. (1) Prolonged exposure to DNP or TNP caused an immediate, and long-lasting increase of secretion only in the presence of Ca. (2) The response to DNP depended on the concentration of Ca, but that to TNP was largest in the presence of 0.5 mM Ca. (3) Re-addition of 2 mM Ca during prolonged exposure to DNP or TNP produced a larger response than did the simultaneous addition of Ca and either DNP or TNP. (4) The response to DNP in the presence of 2 mM Ca was markedly reduced by removal of external Na, but the Na dependency became less marked in the presence of 0.1 mM Ca. The response to TNP in the presence of 0.5 mM Ca was slightly reduced in the absence of Na. (5) DNP or TNP caused a much larger response during depolarization with a high K, Rb, or Cs medium than that with a 5 mM KCl medium. (6) The response to DNP was largely potentiated in the absence of most ions in the presence of only 0.1 mM Ca and that to TNP was potentiated in the presence of only 0.5 mM Ca under the same condition. (7) Re-addition of Ca 5 min after the removal DNP or TNP still caused substantial secretion. These results suggest that nitrophenol compounds stimulate secretion by two independent mechanisms: one is related to its effect on the surface potentials of the plasma membrane and the other unknown except for the possible dependence of Na and Ca.     

The effect of Na, K and Cl ions on the resting membrane potential of sino-atrial node cell of the rabbit

The resting membrane potential of S-A node cell was investigated by observing the response of the membrane potential to change in [K+]O or [Cl-]O under the presence or absence of Na ion. The slope of the change in membrane potential per decade change in [K+]O increased from 12.3 to 44 mV by removal of Na ion from the external medium, suggesting an extensive contribution of Na ion to the resting membrane potential. To determine the relative conductance between Na and K ions, Cl ion in Tyrode solution was substituted with SCN ion, which is 2.1 times more permeable than Cl ion, in order to eliminate the contribution of Cl concentration cell to the resting membrane potential. The contribution of the Cl ion to the resting membrane potential could be examined only in Na-free medium. Acetate ion has been reported to be 0.5 times less permeable than Cl ion. The replacement of Cl ion by acetate ion in various proportions caused a transient depolarization. The slope of this transient depolarization per decade change of [Cl-]O between 10 to 100 mM was determined to be -6.8mV. Under the assumption that the resting membrane potential could be determined by the parallel concnetration cells for Na, K and Cl, relative conductance ratio between K, Na and Cl was calculated to be 1:0.58:0.15.     

Effects of removal of Na(+) and Cl(-) on spontaneous electrical activity, slow wave, in the circular muscle of the guinea-pig gastric antrum

In the circular muscle of the guinea-pig gastric antrum, a decrease in the external Na(+) to less than 20 mM produced depolarization of the membrane with transient prolongation of the slow wave. This was followed by a high rhythmic activity. The activity was inhibited by reapplication of Na(+) before recovery. The depolarization in Na(+)-deficient solution was prevented and rhythmic activity continued at about 5/min for at least 6 min by simultaneous removal of K(+), Ca(2+), or Cl(-). After exposure to a Na(+)- and Cl(-)-deficient solution for a few minutes, reapplication of the Na(+) in Cl(-)-deficient solution inhibited generation of the slow wave until Cl(-) reapplication. Similar results were obtained when Na(+) and Cl(-) were reapplied in the absence of K(+) after exposure to a Na(+)-, K(+)-free, and Cl(-)-deficient solution, although the inhibition was weaker than Na(+) reapplication in a Cl(-)-deficient solution. In the presence of furosemide or bumetanide, a strong inhibition of activity was produced by the reapplication of Na(+) and Cl(-) after exposure to an Na(+)- and Cl(-)-deficient solution. A hypothesis is presented that intracellular Ca(2+) concentration ([Ca(2+)](i)) is the most important factor determining the generation and frequency of the slow wave and that [Ca(2+)](i) is regulated by the Na(+) concentration gradient across the plasma membrane. The recovery of the Na(+) concentration gradient by Na(+) reapplication after removal of Na(+) and Cl(-) is mainly controlled by a Na(+)-K(+)-Cl(-) co-transport.     

Suppression of cellular injury during the calcium paradox in rat heart by factors which reduce calcium uptake by mitochondria

Isolated Langendorff perfused rat hearts were used to study changes in the Ca, Na and K content, contractile force and the loss of cellular material during the Ca paradox. Five minutes perfusion with Ca-free solution containing 1 mM EGTA, followed by 10 min of reperfusion in 1.8 mM Ca causes irreversible contracture, K loss, increase in Na and Ca and a massive release of myoglobin and other cellular material into the perfusate (the calcium paradox). During the Ca-free perfusion the ventricles gain Na but the K content decreases slightly. The size of the Na gain appears to depend upon the buffer used and is larger in bicarbonate than in Tris. When HCO3- or H2PO4- ions are omitted from the bathing solution (in Tris, HEPES, or TES buffered salines) the adverse effects of Ca readmission are reduced. Tris buffer gives the best protection. Metabolic inhibition with FCCP (5 X 10(-7) M), or with CN-(2 X 10(-3) M) together with iodoacetic acid (2 X 10(-3) M), decreases Ca uptake during the Ca paradox and inhibits the release of cellular material. In both cases a contracture is observed. Ruthenium red (10(-4) M) does not inhibit the Ca readmission contracture but reduces the release of cellular material and the gain of Ca and Na. The results suggest that the loss of cellular constituents during the calcium paradox, is related to an active uptake of Ca by the mitochondria and may lead to massive changes in the cellular ion concentration, during Ca-repletion.