Relative Contribution of Superficially Bound and Extracellular Calcium to Activation of Contraction in Isolated Rat Portal Vein

The spontaneous electrical and mechanical activity of the isolated rat portal vein is abolished after only 2–3 min in nominally Ca-free medium, and after 5–6 min there is no contractile response to depolarizing (122 mM K⁺), Ca-free solution. In the present study we have examined the electrical and mechanical responses of the portal vein to depolarization with simultaneous readministration of Ca²⁺ (2.5 mM) after periods of variable length in Ca-free standard solution. After 30 to 60 min of Ca depletion a slow contracture occurred in response to the high-K⁺ solution with 2.5 mM Ca²⁺. When the period in Ca-free medium was reduced below 30 min an early, faster phase appeared in the contracture response, and this phase was more rapid the shorter the time of Ca depletion. It is suggested that the slow contracture obtained after 30 min or more uses mainly extracellular Ca for activation and that the faster phase seen after shorter periods of Ca depletion is due to release of superficially bound Ca. This latter pool of tissue bound Ca does not alone produce contraction in response to depolarization, suggesting that extracellular Ca is required to trigger the release perhaps through a regenerative process.     

Time- and Na-dependent effects of Ca depletion on potassium contracture in frog twitch muscle fiber.

The effect of the extracellular Ca depletion on potassium contracture was investigated in single fibers isolated from frog semitendinosus muscle mainly in relation to its time and Na dependency. The shortening of plateau duration and the increase in the rate of relaxation of the potassium contracture appeared within 3--5 sec and 15 sec, respectively, after the fiber was immersed in Ca-free Na Ringer solution containing 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) (EGTA-Na Ringer solution) or Ca-free choline Ringer solution containing 1 mM (EGTA (EGTA-choline Ringer solution). These effects were independent of the presence or absence of extracellular Na. In EGTA-Na Ringer solution, the potassium contracture tension was inhibited only by about 20% after 20--90 min and was abolished after 120 min. The inhibition of the peak tension was accelerated by the depletion of extracellular Na; in EGTA-choline Ringer solution, the tension was gradually inhibited by about 20% during the first 7 min and abolished after 10--12 min. When the peak tension of potassium contracture was abolished in EGTA-choline Ringer solution, the depolarization by Ca depletion was about 10 mV and the caffeine contracture was sufficiently produced. The results suggest that the inhibition of the potassium contracture tension in EGTA-choline Ringer solution is due to the dissociation of excitation-contraction coupling. On the basis of these results, an aspect of the inactivation of the potassium contracture was proposed.     

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.     

On the roles of calcium ion during potassium induced contracture in the smooth muscle cells of the rabbit main pulmonary artery

The half decay time of the K-induced contracture of rabbit main pulmonary artery following pretreatment with Ca-free EGTA containing solution was 110 sec. A Ca-free K-solution did not generate contraction while noradrenaline, acetylcholine and prostaglandin F2alpha-containing solution did evoke contracture. The decays of the chemically induced mechanical response in Ca-free solution against the exposure times could be classified into three components (2 min, 28 min and over 100 min, respectively). When the membrane depolarization produced by excess K+ was simulated in Krebs solution by application of current, the generated mechanical response was smaller than that produced by 118 mM K+. When the membrane potential was clamped at the resting level before, during and after application of the excess K+, and excess K+ still evoked contracture. The amplitudes of contracture depended on [K]o. The effects of various [K]o on the length constant of the tissue were also observed in relation to the clamping condition. It is postulated that the mechanical response of the pulmonary artery induced by excess K is mainly due to influx of Ca++ and the depolarization plays only a minor role. This means that release of stored Ca by depolarization is not an essential factor in generation of K-induced contracture in this tissue.     

Mechanical response with reversed electrical response to noradrenaline by Ca-deprived arterial smooth muscle

1. When sheep carotid arteries had become electrically active after 30 min in Ca-free saline, noradrenaline 0.1 mM caused almost as much contraction as it did with Ca 1.25 mM present; it also caused slight electrical depolarization, usually with increased spike frequency, followed by electrical quiescence.2. In Ca-free saline with EDTA the arteries became profoundly depolarized, and their mechanical responses greatly reduced, within a few minutes. The mechanical responses to noradrenaline that remained were accompanied by electrical repolarization or, after longer periods in EDTA, by no electrical change.3. This residual mechanical responsiveness to noradrenaline, always tested at 36 degrees C, declined on average 26 times as rapidly during exposure to EDTA at 36 degrees C as at 5 degrees C and was not reduced by increasing the concentration of EDTA from 1.25 to 12.5 mM. This temperature-sensitivity was significantly too high to be explained by a diffusion-limited process.4. The results suggest that most of the tissue Ca responsible for contractility in simple Ca-free saline was either free in the extracellular space or in cellular stores that were discharged within a few minutes when free extracellular Ca was removed. They also indicate a small resistant Ca store which did not communicate with the exterior by diffusion, and part of which noradrenaline could utilize for contraction by means not dependent on depolarization conducted from the cell membrane.     

Stimulation of45Ca efflux from smooth muscle cells by metabolic inhibition and high K depolarization

Summary The characteristics of the extracellular and cellular calcium exchange in taenia coli have been studied by efflux experiments under different experimental conditions. The exchange of extracellularly bound calcium is accelerated by the presence of calcium in the external solution. If a Ca-free solution is used as washing solution, the slowly exchanging extracellular calcium also contributes appreciably to the later phase of the Ca efflux and obscures the changes of the cellular calcium exchange. There is no evidence for a Ca–Ca exchange diffusion.Most of the⁴⁵Ca bound at extracellular binding sites can be released by a 10 min exposure to 2 mM EGTA or to 10 mM La³⁺. This La concentration moreover largely inhibits the release of⁴⁵Ca from the cellular compartment by metabolic depletion. A release of cellular⁴⁵Ca can be induced by metabolic depletion or by K depolarization. Both procedures probably act at the same sequestering sites. However, while DNP+IAAa cts in the absence of external Ca, it is observed that K depolarization can only cause a Ca release if external Ca can enter the cells.This work was supported by research grant 20.487 from the F.W.G.O. (Belgium).Established investigator of the American Heart Association     

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.     

Mechanisms of endothelin-induced augmentation of the electrical and mechanical activity in rat portal vein

Actions of porcine endothelin (ET) on the electrical and mechanical activity of the rat portal vein were investigated by means of the intracellular microelectrode and isometric tension recording techniques, ET (> 0.1 nM) enhanced the amplitude and frequency of the spontaneous contractions which ceased in the presence of 100 nM dihydropyridine derivatives (nifedipine or nicardipine). ET (0.15 nM) increased the frequency of the spontaneous action potentials, with no change in the basal membrane potential. Higher concentrations of ET (≧ 0.3 nM) further depolarized the membrane potential and increased the spike frequency. After blocking the spontaneous action potentials with nifedipine (100 nM), ET still depolarized the membrane. The depolarization was associated with a reduction in the electrotonic potential and was blocked in a Na-deficient solution (15.5 mM) but not in Ca-free, K-deficient or Cl-deficient solutions. In a Na-deficient solution, ET still evoked action potentials without depolarization. In Ca-free solution, ET depolarized the membrane potential with small oscillations, which were blocked by nifedipine (100 nM). The results indicate that in the rat portal vein, ET enhances electrical and mechanical responses through activation of the dihydropyridine-sensitive and voltage-dependent Ca channels. Acceleration of the Ca entry induced by ET can occur with or without depolarization of the membrane and can enhance the pacemaking mechanism.     

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.     

Ca concentration and flux in Ca-deprived arteries

1. Extracellular Ca and EDTA concentrations in arterial smooth muscle in Ca-free solutions with and without EDTA were estimated from (45)Ca and [(14)C]EDTA washout studies. Ca concentrations were calculated from the measured rate of (45)Ca washout at a given moment, combined with separate determinations of the coefficient of diffusion of Ca in the tissue, while EDTA concentrations were calculated by analysing an exponential phase of [(14)C]EDTA washout and checked by separate determinations of coefficient of diffusion of EDTA in the tissue.2. Extracellular Ca concentrations after 30 min in simple Ca-free saline at 36 degrees C was 0.049 mM at the centre of the tissue and averaged 0.033 mM throughout the tissue; these were well above the value of 0.005 mM-Ca that was found to be the threshold external level for contraction after tissue stores of Ca were removed.3. Extracellular EDTA concentration at the centre of the tissue reached 6.25 mM after 1.9 min in EDTA 12.5 mM at 36 degrees C or after 4.9 min at 5 degrees C. It reached 12.45 mM after 30 min at 5 degrees C, enough to keep free extracellular Ca at this point well below threshold even if all of the Ca in the tissue, determined as 0.42 mumole/g, were suddenly released on warming.4. Ca efflux increased greatly on warming after long periods in cold EDTA, and all measurable Ca left the tissue within 30 min in EDTA 12.5 mM at 36 degrees C although tissue Mg did not fall significantly during this time.5. Contractions elicited by noradrenaline in Ca-free saline, with or without EDTA present, were not associated with any increases in the rate of external loss of Ca greater than 0.001 mumole.g(-1).min(-1).6. Electronmicrographs showed numerous microvesicles that communicated with the extracellular space; also smooth endoplasmic reticulum among other structures that might have contained non-communicating Ca stores.7. The results provide evidence that large responses given by the arteries to noradrenaline in simple Ca-free saline were due to persisting extracellular Ca or to labile Ca stores dependent on this, while small responses obtained after long periods in cold EDTA depended on non-communicating Ca stores whose loss was too temperature-dependent to be limited by diffusion.     

Mechanisms involved in contraction of smooth muscles of the rat portal vein as induced by sodium depletion

Responses to external Na depletion were investigated in the rat portal vein, using a microelectrode and an isometric force transducer. Mechanical response to Na depletion was characterized by a large tonic contraction with phasic contractions in a choline solution, and by phasic contractions without a tonic contraction in a Li solution. An identical depolarization of the membrane occurred in either choline or Li solution. After the tonic contraction was established in the Na-free choline-solution, the vein all but completely relaxed with partial re-admission of Na, while maintaining constant the concentration of choline. The Na-free choline-solution at 13 degrees C did not induce the tonic contraction. In nominal Ca-free solution, no contraction occurred with a depletion of Na. A tonic contraction was also induced by Na depletion in the presence of Mn but not Ca. It is concluded that with depletion of both external and internal Na, Ca and Mn may enter the cell through channels usually occupied by Na.     

Sodium flux and electrical activity of arterial smooth muscle

1. The intracellular concentration and transmembrane flux of Na in smooth muscle cells of sheep carotid arteries were measured by identifying a fraction of tissue Na whose efflux was markedly sensitive to removal of external Ca. This allowed intracellular Na to be distinguished from extracellular bound Na exchanging with a similar time constant.2. When the arteries were in solution containing Na 148 mM and Ca 2.5 mM the mean intracellular Na concentration was 7.3 m-mole/kg cell water and the mean transmembrane flux of Na was 0.18 p-mole cm(-2) sec(-1), both values being much lower than reported values for intestinal smooth muscle.3. During electrical activity induced by Ca deprivation the influx of Na increased to 3.2 p-mole cm(-2) sec(-1); a 50% reduction in Na concentration stopped electrical activity and reduced influx by more than 50%, and the excess Na influx per action potential was calculated to be at least 1.0-1.5 p-mole cm(-2).4. Arteries in physiological saline (Ca 2.5 mM) contained 3.48 mumole Ca/g, much of which was bound extracellularly; in Ca-free saline 1.39 mumole Ca/g left the tissue within 3 min, showing that extracellular dissolved Ca diffused freely out of the tissue.5. The results provide further evidence that Na is the principal ion carrying the depolarizing current of action potentials in this smooth muscle.     

Contractile inactivation in frog single denervated muscle fibers.

After preliminary conditioning depolarizations, single muscle fibers of the frog were tested for ability to contract in response to depolarization by 100 mM K+ Ringer solution. Denervated fibers (6-42 days) lose their ability to produce a 100 mM K+ contracture more rapidly than do control fibers. This decrease in 100 mM K+ contracture size (inactivation) is dependent on length of exposure to and magnitude of the conditioning depolarization and on the calcium concentration in the external medium. At 0.4 mM Ca++, the inactivation is 3 times faster than at 1.5 mM Ca++. The rate of contracture loss is not correlated with fiber diameter or the number of days after failure of neuromuscular transmission, and the preliminary conditioning depolarizations do not affect the rate of terminal relaxation from the 100 mM K+ contractures.     

Effects of sodium on the calcium paradox in rat hearts

Reduction of the Na concentration in the Ca-free perfusion solution reduces the amount of myoglobin released by the cells when Ca is readmitted if sucrose is used to replace NaCl under mild hypothermia. When salts like cholinechloride or LiCl are used instead of sucrose, no protection is seen at any temperature. The temperature threshold above which myoglobin loss sharply increases is lowered by prolonged Ca depletion or by the addition of EGTA to the Ca-free solution. Protection by sucrose does not occur in the presence of EGTA. An increase of cell Na induced by strophanthidin during the Ca depletion phase has no effect on myoglobin release. The exponential decline in twitch tension in the early phase of Ca deprivation has the same half-live (T _1/2) for Ca-free solutions containing 145 mM Na or 35 mM Na (110 mM Li or choline), but itsT _1/2 is prolonged if sucrose is used to replace NaCl. When 5 mM EGTA is added to the Ca-free solutions, theT _1/2 is shortened and is not changed by the replacement of NaCl with sucrose. The rate of washout of Ca within the first 20 s of Ca depletion has a similar time course in a normal Na or in a Li and low Na solution. In a sucrose and low Na solution the rate of the Ca efflux is reduced. The addition of EGTA increases this rate and abolishes the slowing effect of a sucrose and low Na solution. Therefore myoglobin release during the Ca paradox does not depend on the Na gradient across the sarcolemma. Na⁺, like other cations, probably enhances the displacement of Ca²⁺ from critical binding sites during Ca-free perfusion, which predisposes the cells to the paradox.     

Calcium entry in response to maintained depolarization of squid axons

1. Intracellular aequorin was used to monitor changes in Ca entry in response to maintained depolarization either produced electrically or by exposure to K-rich solutions.2. External K concentrations greater than 50 mM produce a phasic light response. The light rises to a peak in a few sec and then falls in 0.5-5 min to a new steady level that is always greater than the level in the absence of K.3. The phasic light response does not result from depletion of available aequorin at the periphery of the axon, but rather seems to reflect a phasic entry of Ca in response to depolarization.4. Similar phasic responses are produced by prolonged electrical depolarization. These results are consistent with depolarization serving both to activate and also to inactivate Ca entry.5. Following inactivation and after return to normal sea-water, there is an appreciable relative refractory period during which the response both to K-rich sea-water and electrical depolarization is reduced in size. Complete recovery takes 10-15 min.6. The response to 410 mM-KCl is dependent on the previous treatment of the preparation. Pre-treatment with 100 or 200 mM-KCl reduced the response to 410 mM-KCl. The potential for half inactivation was about -25 mV in 112 mM-Ca and -40 mV in 20 mM-Ca.7. The rate of onset of inactivation is potential dependent and is faster for depolarizations to zero potential than for smaller ones.8. The phasic Ca entry produced by K-rich solutions is insensitive to external tetrodotoxin and internal tetraethylammonium ions, but is blocked by external Mn(2+), Co(2+) and Ni(2+) ions and by the drugs D-600 and iproveratril. This suggests that the phasic Ca entry involves the late Ca channel.9. Recovery of the outward K current after a long depolarization is much faster than recovery of the late Ca entry system. This provides further support for the view that the late Ca channel and the K channel are distinct.     

Ionic requirements for arterial action potential

1. Strips of smooth muscle from common carotid arteries of sheep were electrically quiescent in solution containing Na 148 mM and Ca 2.5 mM.2. When Ca was removed they became electrically active. Addition of low concentrations of Ca (0.025-0.075 mM) or Mg (0.025-0.750 mM) stopped their activity while ethylenediamine tetra-acetate (EDTA) (1.25 mM) accelerated it.3. Replacement of Na by Tris or choline stopped the activity in Ca-free solution. After partial replacement of Na electrical activity could be restored by lowering the resting potential but after complete replacement of Na it could not.4. In the presence of Ca (2.5 mM) small spikes could sometimes be induced after 20 min in Na-free Tris solution by lowering the resting potential by an increase in the external K concentration.5. The results indicate that the depolarizing current of action potentials in this smooth muscle was largely carried by Na, although a little may have been carried by Ca in Ca-containing solutions.6. The arteries in general resembled striated muscle rather than intestinal smooth muscle in these respects, but unlike striated muscle their action potentials were not stopped by tetrodotoxin.     

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.     

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.     

Prolonged potentials in gastrointestinal muscles induced by calcium chelation.

When stomach muscles of skate, toad, or frog or intestinal muscle of cat are treated with Ca-free physiological solutions containing 2-5 mM EGTA or EDTA, spontaneous spikes and slow waves disappear reversibly. With continued treatment, depolarization of 25-30 mV from resting potentials of -65 mV occurs and rhythmic prolonged potentials of several seconds duration appear. They show rapid depolarization to near zero and rapid repolarization and they may continue for several hours. The prolonged potentials disappear when Na is replaced by Li, Tris, or choline. They are insensitive to TTX. The EGTA-induced waves are abolished by Mn, Co, La, verapamil, and D 600. After 10-15 min in 5 mM EGTA, voltage-current and abolition of anomalous rectification. It is concluded that when bound Ca is removed by a chelator, nonspecific reduction in resistance occurs and Na ions may enter rhythmically through channels normally used by Ca.     

Catecholamine release from bovine adrenal medulla in response to maintained depolarization.

Prolonged exposure of venous-perfused bovine adrenal glands to high K in the presence of external Ca produces a transient increase in catecholamine output that reaches a maximum after about 1 min and then declines with a half-time of about 1-2 min. 2. The time course of the transient secretory response to high K does not depend appreciably on the total catecholamine output which indicates that depletion of releasable catecholamine is unlikely to be responsible for the transient nature of the response. 3. Application of 3-6 mM-Ba stimulates secretion from a gland after many minutes exposure to high K, when catecholamine output has declined close to resting levels. This provides further evidence that depletion does not play a major role in the transient response and shows that maintained depolarization does not inhibit the secretory mechanism. 4. Exposure to high K solutions in which Ca has been replaced isomotically by Mg does not evoke any catecholamine output. Subsequent application of Ca always elicits some secretion although the size of this response to added Ca declines rapidly during exposure to Ca-free, high K solutions. The failure of the secretory response in these experiments is more rapid, and earlier in onset than the declining phase of the normal secretory response evoked in the presence of calcium. 5. Pre-treatment with Ca-free solutions of intermediate K content reduces the response to subsequent simultaneous application of high K and Ca. There is a roughly sigmoidal relation between the reduction in response and the logarithm of the K concentration used for pre-treatment. 6. Thin slices of bovine adrenal medulla show qualitatively similar responses on exposure to high K. Examination of the flourescent signal from slices dyed with the potential-sensitive dye DiS-C(3)-(5) suggests that maintained exposure to high K produces a stable depolarization. 7. The most likely explanation for these results is that K-depolarization first activates and subsequently inactivates a potential-sensitive Ca permeability channel. This inactivation is time and possibly potential dependent. 8. The effect of high K on calcium movements in medullary slices was examined. Exposure to 72 mM-K increases (45)Ca uptake, the increase being greatest during the first 10 min. The efflux of Ca is also increased on exposure to high K in the presence of Ca. The net Ca uptake in 72 mM-K is smaller than the tracer uptake of Ca. These findings indicate that K depolarization stimulates a Ca-Ca exchange process. They are also consistent with, but do not offer strong positive support for, the idea that K-depolarization first activates and subsequently inactivates Ca entry. 9. It is suggested that Ca inactivation might play a role in the modulation of neurosecretion and neurotransmitter release by changes in membrane potential.