M Valdeolmillos

The effects of metabolic inhibition on intracellular calcium and pH in isolated rat ventricular cells.

1. Intracellular calcium concentration [( Ca2+]i) and pH (pHi) were measured in single, isolated rat ventricular myocytes using, respectively, the fluorescent indicators Fura‐2 and BCECF (2,7‐bis(carboxyethyl‐5(6)‐carboxyfluorescein). Contraction was measured simultaneously. The intracellular calibration of BCECF is demonstrated. In a HEPES‐buffered bathing solution of pH 7.4, pHi had a mean value of 7.16 +/‐ 0.05 (mean +/‐ S.E.M.). 2. Addition of NH4Cl (5‐20 mM) produced an intracellular alkalosis that was associated with an increase of contraction amplitude. Removal of NH4Cl produced an acidosis and decrease of contraction. 3. The addition of 2 mM‐cyanide (CN‐) to inhibit oxidative phosphorylation had variable effects on contraction amplitude. Changes of contraction amplitude could largely be accounted for by changes in the systolic Ca2+ transient. 4. CN‐ addition increased lactic acid production. However, in the majority of experiments, this was not accompanied by an intracellular acidosis. 5. Anaerobic glycolysis was inhibited by either removal of glucose, addition of deoxyglucose, or addition of iodoacetate. Under these conditions the application of CN‐ decreased systolic [Ca2+]i and contraction amplitude. This was sometimes preceded by a transient increase of systolic [Ca2+]i and contraction amplitude. 6. When glycolysis was inhibited, the subsequent addition of CN‐ always increased diastolic [Ca2+]i and produced a contracture. The increase of [Ca2+]i occurred before the contracture. However, once the contracture had developed, decreasing [Ca2+]i (by removal of external Ca2+) did not cause relaxation. 7. With glycolysis inhibited, addition of CN‐ resulted in a large (0.51 +/‐ 0.05 pH unit) acidosis that was sometimes preceded by an alkalosis. This acidosis was unaffected by removal of external Ca2+ or external alkalinization. Calculations show that some of this acidosis may result from protons released by ATP hydrolysis. 8. If the acidosis produced by metabolic blockade was partly reversed by adding NH4Cl then a contracture immediately developed. This suggests that the acidosis delays the onset of the contracture. 9. We conclude that metabolic inhibition increases diastolic [Ca2+]i. The accompanying acidosis prevents contraction. Once the contracture has developed it is maintained by factors other than increased [Ca2+]i, possibly by a fall of [ATP].

Effects of metabolic blockade on the regulation of intracellular calcium in dissociated mouse sensory neurones.

1. Impaired intracellular Ca2+ concentration ([Ca2+]i) regulation may underlie alterations in neuronal function during hypoxia or hypoglycaemia and may initiate cell damage. We have used the Ca2(+)‐sensitive fluorophore, Fura‐2, to study the regulation of [Ca2+]i in neurones isolated from mouse dorsal root ganglia. Mean resting [Ca2+]i was 163 +/‐ 11 nM (mean +/‐ S.E.M., n = 38). 2. Depolarization by exposure to 20 or 30 mM‐K+ caused a rapid Co2(+)‐ and Cd2(+)‐sensitive rise in [Ca2+]i, which subsequently declined with a time course usually fitted by the sum of two exponential functions. 3. Interference with mitochondrial function (by CN‐ or FCPP) or with glycolysis (by glucose removal) all raised [Ca2+]i by up to 220%. Addition of FCCP in the presence of CN‐ further increased [Ca2+]i. The response to CN‐ was still seen in the absence of extracellular Ca2+, although it attenuated rapidly, indicating release from an intracellular store. 4. Either CN‐ or glucose removal increased the rise in [Ca2+]i induced by K+ 2‐ to 3‐fold and slowed recovery, suggesting interference with sequestration or extrusion of [Ca2+]i. 5. Resting [Ca2+]i rose when external Na+ was replaced by Li+ or N‐methyl‐D‐glucamine, demonstrating the presence of a Na(+)‐Ca2+ exchange process. However, Na+ replacement had only a slight effect on the handling of a Ca2+ load. 6. We conclude that (i) Ca2+ is released into the cytoplasm from intracellular organelles when energy supplies are reduced: (ii) that the extrusion or sequestration of Ca2+ entering the cell during electrical activity is rapidly impaired by interference with mitochondrial metabolism: and (iii) Na(+)‐Ca2+ exchange makes only a small contribution to intracellular Ca2+ homeostasis. 7. [Ca2+]i would thus be expected to rise in vivo during hypoxia or hypoglycaemia and may initiate alterations in neuronal function. However, if a rise in Ca2+ is an important cause of cell damage in cerebral hypoxaemia, the combination of excitation and hypoxia will lead to the largest increases in [Ca2+]i, while hypoxia alone appears to cause only a small increase in [Ca2+]i in quiescent cells.

A novel method for absolute calibration of intracellular pH indicators.

In this paper we present methods to measure intracellular pH (pHi) with fluorescent indicators. These methods are based on the change in intracellular pH following the addition of weak acids and weak bases to the extracellular medium. The first method requires that the fluorescence of the indicator is proportional to the change in pHi that follows the addition of a weak acid or weak base to the extracellular medium. The second is a null method which uses a mixture of weak acid and weak base that does not change the fluorescent signal. This null method can be used in situations in which the fluorescent signal is a monotonic but non-linear function of pH. The first method depends upon four assumptions. (i) That only the uncharged forms of the weak acids and bases cross the surface membrane. (ii) That the pKa is the same inside and outside the cell. (iii) That the buffering power is constant. (iv) That there is no significant pH regulation on the time scale of the change in pHi. The null method only requires the first two assumptions. We have made estimates of pHi in four different cell types and compared the results obtained with these methods with those obtained from other methods of pHi calibration.

Measurements of intracellular Ca2+ in dissociated type I cells of the rabbit carotid body.

1. The carotid body chemoreceptors are stimulated in situ by cyanide (CN‐), which mimics the effect of hypoxia. We have shown that CN‐ increases a calcium‐dependent potassium conductance (gK(Ca)) in single type I cells dissociated from the carotid body of the rabbit. We have now used the Ca2(+)‐sensitive fluorophore, Fura‐2, to measure intracellular Ca2+ directly in single type I cells. 2. CN‐ reversibly increased [Ca2+]i from approximately 90 nM to a mean of approximately 200 nM. Some of this Ca2+ originated from an intracellular store, which was depleted by exposure to Ca2(+)‐free solutions. Prolonged application of CN‐ caused a sustained increase in [Ca2+]i, suggesting that CN‐ impairs the removal or sequestration of Ca2+. 3. pHi measured with the dye BCECF (2,7‐bis(2‐carboxyethyl)‐5(and‐6)‐carboxyfluorescein) did not change consistently in response to CN‐, although pHi changed predictably in response to both ammonium chloride and to acidification of the superfusate with CO2. 4. Potassium‐induced depolarization (35 mM‐K+) caused a large, cadmium‐sensitive rise in [Ca2+]i. The K(+)‐induced Ca2+ load was used to study the regulation of [Ca2+]i. 5. The clearance of a Ca2+ load was slowed either by removal of [Na+]o or by application of CN‐. This shows that both a Na+‐Ca2+ exchange and an energy‐dependent process or processes contribute to the regulation of [Ca2+]i. 6. Carbachol (CCh, 10‐100 microM), which also hyperpolarizes type I cells, caused a small transient rise in [Ca2+]i, indicating release from an exhaustible intracellular pool. The response to CN‐ was unaffected by prior or continued exposure to CCh, suggesting that the two stimuli operate by distinct mechanisms. 7. The increased gK(Ca) seen in type I cells in response to CN‐ thus reflects a change in cellular Ca2+ homeostasis. The rise in [Ca2+]i presumably underlies the documented increase in transmitter release from the carotid body in response to CN‐. If chemotransduction is a consequence of the release of transmitters from the type I cell, the response of the carotid body to CN‐, and possibly also to hypoxia, is thus a direct consequence of the energy dependence of Ca2+ homeostasis in the type I cell.

Calcium-induced calcium release activates contraction in intact cardiac cells

The “caged” calcium chelator Nitr-5 was Incorporated into isolated rat ventricular myocytes. Brief illumination with ultra-violet light made the cell twitch. The light-induced twitch was inhibited by ryanodine (1–10 μM) suggesting that it resulted from calcium-induced release of calcium from the sarcoplasmic reticulum. Inhibition of the Ca current (Ni, 10 mM) abolished the electrically stimulated twitch but did not inhibit the light-induced twitch. These results provide direct evidence for the importance of Ca-induced Ca release in excitation-contraction coupling in the heart.

A study of intracellular calcium oscillations in sheep cardiac Purkinje fibres measured at the single cell level.

Previous work has shown that an elevation of intracellular calcium concentration [( Ca2+]i) produces spontaneous oscillations of [Ca2+]i. However the fact that the oscillations are unsynchronized between different cells has made it difficult to study them. We have therefore injected only one cell in a Purkinje fibre with aequorin in order to avoid these problems. The addition of strophanthidin (10 microM) produced an increase of mean aequorin light over the course of several minutes. During this period spontaneous oscillations of light developed and, with time, their frequency and magnitude increased. The oscillations could first be seen at levels of [Ca2+]i of less than 1 microM. The amplitude of the oscillations of [Ca2+]i could be up to 10 microM and was modulated at a slow rate (about 0.3‐0.5 Hz). This suggests that, even within one cell, different regions may oscillate at different frequencies. Elevating [Ca+]o, removing extracellular Na+, or depolarization increased the magnitude of the aequorin light oscillations. Converting the records to [Ca2+]i showed that this increase in the magnitude of the aequorin oscillations was accompanied by a real increase of mean [Ca2+]i and of the magnitude of the oscillations [Ca2+]i. The frequency of the oscillations increased up to a point but saturated at a maximum value of 3‐4 Hz. Since previous experiments have used the mean aequorin light to estimate mean [Ca2+]i, we have calculated the error produced in this calculation by the presence of [Ca2+]i oscillations. We estimate that the error is greatest at low levels of Ca2+ loading when the frequency of the oscillations is low. However, at higher Ca2+ loads, when the frequency is above 2 Hz, the error is probably less than 10%. If oscillations were produced by removal of external Na+ after the application of strophanthidin, then either ryanodine or caffeine abolished the oscillations. Furthermore, in both cases, the resulting steady level of [Ca2+]i was similar to the mean level before the addition of the drugs. In another series of experiments we examined the effects of these drugs on oscillations produced by the application of strophanthidin. Caffeine produced a transient increase in both the frequency of the oscillations and mean [Ca2+]i before abolishing the oscillations and decreasing [Ca2+]i to below the level in the absence of caffeine. In contrast ryanodine gradually decreased both the mean [Ca2+]i and the frequency until the oscillations were abolished. During this period of slowing of the oscillations their magnitude was often increased.

The effects of ryanodine on calcium-overloaded sheep cardiac Purkinje fibers.

Prolonged exposure to high concentrations of strophanthidin produces an initial increase followed by a subsequent decrease of twitch tension. The slow decrease is termed calcium overload. The aim of the present work was to investigate the effects of ryanodine (an inhibitor of calcium release from the sarcoplasmic reticulum) on calcium-overloaded sheep cardiac Purkinje fibers. The fibers were voltage-clamped, and tension was measured while monitoring the intracellular calcium concentration with the photoprotein aequorin. When strophanthidin (10 microM) was applied to produce calcium overload, a depolarizing pulse produced twitch, and tonic components of tension and repolarization produced an aftercontraction. These components of tension were accompanied by corresponding increases of aequorin light. Ryanodine (1 microM) gave a transient increase of twitch tension. The twitch then decreased to very low levels. The aftercontraction and its corresponding aequorin light signal decreased monotonically on application of ryanodine. It has been suggested that the fall of force in calcium overload may be due to random diastolic release of calcium from the sarcoplasmic reticulum interfering with subsequent systolic calcium release. We suggest that the positive inotropic effect of ryanodine can be explained if ryanodine decreases the diastolic release of calcium. The transient positive inotropic effect of ryanodine reported here is therefore consistent with the hypothesis that the fall of force in calcium overload is due to diastolic calcium oscillations.

The mechanism of the increase of tonic tension produced by caffeine in sheep cardiac Purkinje fibres.

The effects of caffeine were examined on contraction and membrane current in voltage‐clamped sheep cardiac Purkinje fibres. The photoprotein aequorin was injected into several cells in order to measure the intracellular ionized Ca concentration [( Ca2+]i). When the Na‐K pump was inhibited, depolarization produced a twitch followed by a tonic component of tension. Repolarization produced an after‐contraction. These components of tension were accompanied by corresponding increases of aequorin light. Caffeine (10 mM) decreased both the twitch and the after‐contraction while increasing the tonic component. The application of caffeine also produced a transient increase of aequorin light, both during depolarization and at rest, which was followed by a maintained decrease in all three components of the light signal. In particular, although caffeine decreased the rise of aequorin light during prolonged depolarization it increased the tonic tension. The possibility that the effects of caffeine on tonic tension could be due to suppression of spontaneous Ca oscillations was rejected for the following reasons. (i) Ryanodine (which also abolishes Ca oscillations) decreased the magnitude of the tonic tension. (ii) Caffeine still increased tonic tension when it was added to a fibre exposed to ryanodine (1‐10 microM). In the presence of ryanodine it was possible to measure [Ca2+]i and tonic tension without interference from Ca oscillations. The increase of tonic tension produced by caffeine could not be accounted for by a rise of [Ca2+]i. The results showed that, at a given level of Ca, caffeine increased tension. The results show that a large part of the increase of tonic tension produced by caffeine is due to an increase of the Ca sensitivity of the contractile apparatus rather than to changes of [Ca2+]i. The consequence of this observation for the experimental use of caffeine is discussed.

The effects of membrane potential on active and passive sodium transport in xenopus oocytes

1. The effects of membrane potential on the Na+‐K+ pump were studied by measuring membrane current and 22Na+ efflux in voltage‐clamped Xenopus oocytes. The effects of inhibiting the Na+‐K+ pump with strophanthidin were examined. 2. Strophanthidin produced an inward shift of membrane current which reversed on removal of the drug. In control oocytes the magnitude of this current was not significantly affected by changing membrane potential over the range ‐20 to ‐160 mV. 3. In another series of experiments the intracellular Na+ concentration ([Na+]i) was elevated either by overnight Na+‐K+ pump inhibition (strophanthidin or exposure to K+‐free solutions) or by loading with nystatin. This Na+‐loading increased the magnitude of the strophanthidin‐sensitive current. The ratio of strophanthidin‐sensitive 22Na+ efflux:strophanthidin‐sensitive current was consistent with that expected from a 3Na+‐2K+ exchange. 4. When [Na+]i was elevated the strophanthidin‐sensitive current was sensitive to changes of membrane potential. Hyperpolarization from ‐20 to ‐80 mV decreased the current to 60% of control. It is suggested that the current is not sensitive to membrane potential at normal [Na+]i because the over‐all reaction is rate limited by the availability of intracellular Na+. 5. The application of strophanthidin decreased the rate of 22Na+ efflux. Both the strophanthidin‐insensitive and the strophanthidin‐sensitive components of efflux were sensitive to changes of membrane potential. The strophanthidin‐insensitive component was not greatly affected by hyperpolarization from ‐40 to ‐160 mV but was increased by depolarization to +40 mV. 6. In Na+‐loaded oocytes, the strophanthidin‐sensitive component of 22Na+ efflux was inhibited by hyperpolarization negative from ‐40 mV. Hyperpolarization from ‐40 to ‐160 mV decreased the efflux by 54 +/‐ 5%. Over the limited range of potentials for which a comparison could be made, the effects on 22Na+ efflux were somewhat less than on the electrogenic Na+‐K+ pump current. On average there was no significant effect of depolarizing from 0 to +40 mV. However, in some experiments a clear inhibition of the efflux was observed. If the oocytes were not Na+ loaded there was no significant effect of membrane potential on the strophanthidin‐sensitive Na+ efflux. 7. These results show that the effects of membrane potential on the net reaction of the Na+‐K+ pump (as measured by the electrogenic current) result partly from an inhibition of the forward mode of operation. However, there is also evidence to suggest a contribution from stimulation of the reverse reaction.

Effects of membrane potential on intracellular calcium concentration in sheep Purkinje fibres in sodium-free solutions.

1. The intracellular Ca2+ concentration [( Ca2+]i) was measured in voltage‐clamped sheep cardiac Purkinje fibers while recording tension simultaneously. 2. When [Na+]i was elevated (by Na+‐K+ pump inhibition) depolarization produced an increase of tonic tension. 3. Replacement of external Na+ by Li+ or choline produced a contracture which then relaxed spontaneously. Following this relaxation, depolarization either had no effect on tonic tension or produced a small decrease. 4. When external Na+ was replaced by Ca2+, depolarization (over the range ‐120 to ‐20 mV) produced a decrease of tonic tension and [Ca2+]i. Hyperpolarization increased tonic tension and [Ca2+]i. 5. An after‐contraction and accompanying increase of [Ca2+]i were produced by repolarization in both Na+‐free and Na+‐containing solution. This eliminates the possibility that the stimulus for the after‐contraction is the increase of [Ca2+]i during the depolarization and suggests that the stimulus may be the change of membrane potential. 6. The increase of [Ca2+]i on hyperpolarization seen in Na+‐free solutions persisted in the presence of ryanodine. 7. These results show, in contrast to previous work, that in Na+‐free solutions tonic tension is still sensitive to membrane potential. The results support the hypothesis that, in Na+‐containing solutions, the increase of tonic tension on depolarization results from a voltage‐dependent Na+‐Ca2+ exchange. The reduction of tonic tension on depolarization in Na+‐free solutions may be due to the decrease of the electrochemical gradient for Ca2+ to enter the cell.