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D A Eisner

Publications and source records attributed to D A Eisner.

At least 19 recordsLinked to original sources
PubMed1992

Caffeine inhibits the agonist-evoked cytosolic Ca2+ signal in mouse pancreatic acinar cells by blocking inositol trisphosphate production.

The inhibitory effects of caffeine on receptor-activated cytosolic Ca2+ signal generation in isolated mouse pancreatic acinar cells were investigated. Using the ability of caffeine to quench Indo-1 fluorescence we measured simultaneously the free intracellular Ca2+ concentration ([Ca2+]i) and the intracellular caffeine concentration ([caffeine]i). We also measured inositol 1,4,5-trisphosphate (InsP3) production with a radioreceptor assay. When caffeine was added to the extracellular solution during a sustained receptor-activated increase in [Ca2+]i, [caffeine]i rose to its steady level within a few seconds. This was accompanied by a decrease of [Ca2+]i, which started only after [caffeine]i had reached an apparent threshold concentration (about 2 mM in the case of 0.5 microM acetylcholine (ACh) stimulation). Above this [caffeine]i level there was a linear relationship between [caffeine]i and [Ca2+]i. Throughout the caffeine exposure [Ca2+]i remained at a steady low level. Following removal of caffeine from the bath, [caffeine]i decreased to zero within seconds. There was no significant increase in [Ca2+]i until [caffeine]i had been reduced to the threshold level (about 2 mM at 0.5 microM ACh). Caffeine inhibited Ca2+ signals evoked by ACh, cholecystokinin, and ATP and also inhibited signals generated in the absence of external Ca2+. Caffeine application had the same effect as removal of agonist allowing recovery from apparent desensitization. Caffeine inhibited the agonist-evoked production of InsP3 in a dose-dependent manner. Our results demonstrate the acute and reversible dose-dependent inhibition of agonist-evoked cytosolic Ca2+ signal generation due to rapid intracellular caffeine accumulation and washout. The inhibition can be explained by the reduction of agonist-evoked InsP3 production.

Acetylcholine↗
PubMed1992

The effects of thapsigargin on [Ca2+]i in isolated rat mesenteric artery vascular smooth muscle cells.

The effects of thapsigargin were studied on single cells isolated from side branches of the rat mesenteric artery. Thapsigargin (150 nM) produced a transient increase of [Ca2+]i. This transient rise of [Ca2+]i was unaffected by removing external Ca2+ ions. This suggests that thapsigargin is releasing Ca2+ ions from an intracellular store. In the absence of thapsigargin both noradrenaline and caffeine also produced a transient increase of [Ca2+]i. These increases were abolished by prior exposure to thapsigargin. Correspondingly, the effects of thapsigargin were abolished by prior exposure to caffeine. These results show that thapsigargin releases Ca2+ from the noradrenaline and caffeine-sensitive stores.

Animals↗
PubMed1992

Fluorescence measurements of cytoplasmic and mitochondrial sodium concentration in rat ventricular myocytes.

1. The fluorescent Na+ indicator SBFI was incorporated into isolated ventricular myocytes using the acetoxymethyl (AM) ester. 2. The excitation spectrum was found to be shifted about 20 nm in the cell compared to in vitro. In the cell, an increase of [Na+] decreased fluorescence at 380 nm (F380) and had no effect at 340 nm (F340). The ratio (R = F340/F380) was used as a measure of [Na+]i. 3. In vivo calibration of SBFI for [Na+]i was obtained by equilibrating [Na+] across the plasma membrane with a divalent-free solution in the presence of gramicidin D. 4. Selective removal of the surface membrane with saponin or digitonin released only about 50% of the indicator. Following saponin treatment, cyanide or carbonylcyanide m-chlorphenylhydrazone (CCCP) increased the apparent [Na+] measured by the remaining (presumably mitochondrial) SBFI. It is suggested that mitochondrial [Na+] is normally less than cytoplasmic. 5. Attempts to examine the effects of metabolic inhibition on [Na+]i were hampered by changes of autofluorescence due to changes of [NADH]. It is shown that this effect can be corrected for using the isosbestic signal (excited at 340 nm). 6. Inhibition of both aerobic metabolism (with CN-) and glycolysis (glucose removal or iodoacetate) produced a gradual increase of [Na+]i. This began before the resting contracture developed and may (via Na(+)-Ca2+ exchange) account for some of the rise of diastolic [Ca2+]i seen in previous work. The rise of [Na+]i began at about the same time as the decrease of systolic contraction and therefore at a time when [ATP]i had begun to fall.

Animals↗
PubMed1992

Metabolic changes during ischaemia and their role in contractile failure in isolated ferret hearts.

1. The effects of global ischaemia on phosphorus metabolites, intracellular pH (pHi) and developed pressure were measured in isolated whole ferret hearts using 31P nuclear magnetic resonance (NMR) spectroscopy. 2. Brief (10 min) periods of global ischaemia reduced left ventricular developed pressure (LVDP) to undetectable levels. This fall in LVDP was accompanied by a fall in the intracellular concentration of phosphocreatine (PCr) and increases in the concentrations of inorganic phosphate (Pi) and phosphomonoesters. There was no change in the intracellular ATP concentration ([ATP]i). pHi fell approximately linearly at a rate of 0.04 pH units min-1. 3. When ferret hearts were exposed to cyanide (CN-) in the presence of alpha-cyano-4-hydroxycinnamate (CHC), a blocker of lactate efflux, the changes in pHi and [Pi]i which occurred were similar to those observed during global ischaemia. However, developed pressure only fell to around 15% of the control value. 4. Removing the intracellular acidosis (by reducing the CO2 level of the gas with which the perfusate was equilibrated) during exposure to CN- and CHC caused an increase in developed pressure, consistent with the fall in pHi being responsible for a substantial fraction of the fall in developed pressure. 5. Taken together, these results suggest that most, but not all, of the fall in developed pressure during ischaemia can be explained by the effects of the changes in pHi and [Pi]i on the contractile apparatus. 6. Action potential recordings made with a suction electrode during short periods of global ischaemia showed that there was no decrease in action potential duration over the period when developed pressure was falling, eliminating action potential shortening as a possible cause of the fall in developed pressure. 7. In hearts in which the rate of glycolysis had been reduced by glycogen depletion, global ischaemia led to a marked shortening of the action potential. NMR experiments showed that under these conditions [ATP]i decreased by around 50% over the first 10 jin of ischaemia, while the intracellular acidosis which occurred was smaller than that in a control ischaemic period. 8. The time course of the decline of [ATP]i was examined in several hearts during long (45 min and over) ischaemic periods without prior glycogen depletion. After 45 min of ischaemia [ATP]i fell to around two-thirds of the control value, while pHi declined to approximately 6.1. Resting pressure did not increase. On reperfusion pHi recovered rapidly to control levels. [ATP]i, however, did not recover. 9. If ischaemia was prolonged further, [ATP]i eventually became undetectable after 70-90 min.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1991

Intracellular pH and buffering power measured in isolated single cells from pregnant rat uterus.

Intracellular pH (pHi) affects smooth muscle function yet there have been few direct pHi measurements. Using the fluorescent indicator, 2',7'-bis-2-(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, we report here the first measurements of pHi and buffering power (beta) in single, isolated, uterine smooth muscle cells. Mean pHi in cells from pregnant rat uteri was 7.09 +/- 0.08 (+/- S.E.M., n = 16 cells; 37 degrees C), and beta was found to be 12.8 +/- 2.8 mmol/l per pH unit (n = 15). The cells were able to regulate their pHi in the presence of butyrate when perfused in nominally CO2-free solution.

Animals↗
PubMed1990

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.

Animals↗
PubMed1990

The role of [Ca2+]i and [Ca2+] sensitization in the caffeine contracture of rat myocytes: measurement of [Ca2+]i and [caffeine]i.

1. Fluorescence measurements have been made in single, isolated rat ventricular myocytes using the Ca2(+)-sensitive indicators Fura-2 and Indo-1. In Fura-2-loaded cells, the application of caffeine (2-20 mM) produced a change of fluorescence indicating an increase of [Ca2+]i which then spontaneously decayed to control levels. These changes of [Ca2+]i were accompanied by a contracture. 2. In contrast, in Indo-1-loaded cells, in addition to the changes of fluorescence expected for the transient increase of [Ca2+]i produced by caffeine, there was a maintained decrease of fluorescence. 3. Measurements in vitro showed that caffeine quenches the fluorescence of Indo-1 (but not of Fura-2) in a [Ca2+]-and wavelength-independent manner. Caffeine therefore had no effect on the ratio of Indo-1 fluorescence measured at two wavelengths. This inhibition by caffeine could be described by an apparent Ki of 4 mM. In the cell the Ki was considerably larger (18 mM). 4. We have separated the Indo-1 fluorescence changes into caffeine- and [Ca2+]i-dependent components. The time course of change of intracellular caffeine was calculated. When [caffeine]o was rapidly increased, [caffeine]i changed with a rate constant of 8 s-1 giving an apparent permeability to caffeine of 2 x 10(-3) cm s-1. 5. This method was used to measure [caffeine]i and [Ca2+]i simultaneously during caffeine-induced contractures. The shape of the caffeine contracture was found to depend on both the speed of application of caffeine and the concentration applied. If caffeine was applied quickly then the contracture developed within 1 s to a maximum level and then relaxed to a lower maintained level. With slower application, there was a more complete relaxation of the initial contraction followed by a slower redevelopment of contraction. 6. Despite the difference in contraction time course, irrespective of the flow rate, [Ca2+]i decayed monotonically. The slow secondary development of contraction has the same time course as the increase of [caffeine]i. The caffeine contracture can be reproduced by a model in which both [Ca2+]i and [caffeine]i affect contraction. 7. The increase of [Ca2+]i is not greatly affected by altering the caffeine concentration from 2.5 to 50 mM. In contrast the maintained level of contraction increases over this range showing that the Ca2(+)-independent effects of caffeine on the myofilaments have a low affinity for caffeine.

Animals↗
PubMed1990

A mechanism for the effects of caffeine on Ca2+ release during diastole and systole in isolated rat ventricular myocytes.

1. The fluorescent indicator Indo-1 was used to measure both [Ca2+]i and [caffeine]i in single ventricular myocytes. 2. Caffeine (at concentrations of 1 mM or above) produced a transient increase of resting [Ca2+]i attributed to the release of Ca2+ ions from the sarcoplasmic reticulum (SR). Simultaneous measurement of [caffeine]i showed that the Ca2+ release only began when [caffeine]i had risen to about 1 mM. Subsequently the rate of release was a steep function of [caffeine]i. It is suggested that this results from a positive feedback as the Ca2+ released activates further release. 3. If external Ca2+ was removed the release of Ca2+ produced by caffeine was delayed such that [caffeine]i rose to a greater concentration before release was initiated. This suggests that an increase of [Ca2+]i increases the efficacy of caffeine to release Ca2+ ions from the SR. 4. Lower concentrations of caffeine (50-500 microM) had no effect on diastolic [Ca2+]i. In contrast they increased systolic [Ca2+]i and contraction. This increase was most obvious if the systolic contraction had previously been decreased either by reducing [Ca2+]o from 1 to 0.25 mM or (in voltage-clamped cells) by decreasing the magnitude of the depolarizing pulse. 5. If the exposure to caffeine was prolonged, this increase of systolic [Ca2+]i and contraction was completely transient. On removal of caffeine, systolic [Ca2+]i and contraction decreased to below control before recovering. 6. During these transient changes of systolic [Ca2+]i and contraction there was no change of the sarcolemmal Ca2+ current. 7. It is suggested that the increase of systolic [Ca2+]i is due to caffeine increasing the fraction of the SR Ca2+ content released during the twitch. 8. The above results concerning both diastolic and systolic [Ca2+]i can be explained by a model in which caffeine increases the affinity with which Ca2+ ions activate Ca2(+)-induced Ca2+ release. At high enough [caffeine], the threshold [Ca2+]i for regenerative Ca2(+)-induced Ca2+ release will be reduced to below the resting [Ca2+]i thus producing a diastolic increase of [Ca2+]i. At lower [caffeine] the threshold is higher than resting [Ca2+]i and caffeine only serves to enhance the release produced during systole.

Action Potentials↗
PubMed1990

Local activation of contraction in isolated rat ventricular myocytes.

The aim of this paper was to examine whether contraction (which is thought to result from Ca-induced release of Ca2+ from the sarcoplasmic reticulum) can propagate along a cardiac cell. The experiments were performed on isolated rat ventricular cardiac myocytes. Two techniques were used to initiate contraction in a localized region of the cell. 1) The cells were loaded with the "caged" Ca-containing compound nitr-5. A region of the cell was illuminated with ultraviolet light to increase intracellular Ca2+ concentration ([Ca2+]i) in that area. 2) The cells were superfused with a low (0.1 mM) Ca solution to abolish contraction. Ca was applied to a region of the cell by iontophoresis, and the cell was then electrically stimulated. In both cases a local contraction was produced that did not spread to the rest of the cell. Like the normal twitch, the local contraction was abolished by ryanodine, showing that it is produced by Ca release from the sarcoplasmic reticulum. In the final series of experiments, the cell was stimulated to contract in a control (1 mM Ca2+) solution. Cd2+ was then iontophoresed to a region of the cell. Only the region of the cell exposed to Cd stopped contracting. In conclusion, the above results show that the systolic rise of [Ca2+]i cannot propagate through a cell. This lack of propagation is suggested to be important in stopping a rise of [Ca2+]i in one cell spreading to the rest of the heart.

Animals↗
PubMed1989

The effects of metabolic inhibition on intracellular pH and Ca.

We have investigated the effects of inhibiting aerobic and/or anaerobic metabolism on contraction, intracellular calcium and pH in single rat ventricular myocytes. Inhibition of aerobic metabolism alone (with CN) had little effect on these variables. However, if anaerobic glycolysis was also inhibited, then the application of CN decreased systolic [Ca2+]i and increased diastolic [Ca2+]i. There was also a development of a diastolic contracture which lagged behind the increase of diastolic [Ca2+]i. These events were accompanied by an intracellular acidosis. The acidosis was shown to depress contraction and perhaps to account for the fact that diastolic [Ca2+]i increased before the contracture.

Acidosis↗
PubMed1989

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/microM) 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.

Animals↗
PubMed1989

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 extra-cellular 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.

Animals↗
PubMed1989

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].

Action Potentials↗
PubMed1989

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.

Animals↗
PubMed1988

Effects of rapid application of caffeine on intracellular calcium concentration in ferret papillary muscles.

In this paper we investigate the effects of caffeine (5-20 mM) on ferret papillary muscle. The intracellular Ca2+ concentration ( [Ca2+]i) was measured from the light emitted by the photoprotein aequorin, which had previously been microinjected into superficial cells. Isometric tension was measured simultaneously. The rapid application of caffeine produced a transient increase of [Ca2+]i, which decayed spontaneously within 2-3 s and was accompanied by a transient contracture. The removal of extracellular Na+ or an increase in the concentration of intracellular Na+ (produced by strophanthidin) increased the magnitude of the caffeine response. Cessation of stimulation for several minutes or stimulation at low rates decreased the magnitude of the stimulated twitch and Ca2+ transient. These maneuvers also decreased the size of the caffeine response. These results are consistent with the hypothesis that the caffeine-releasable pool of Ca2+ (sarcoplasmic reticulum) is modulated by maneuvers that affect contraction. Ryanodine (10 microM) decreased the magnitude of the caffeine response as well as that of the stimulated twitch. In contrast, the rapid removal of external Ca2+ abolished the systolic Ca2+ transient within 5 s, but had no effect on the caffeine response. From this we conclude that the abolition of twitch by Ca2+-free solutions is not due to depletion of the sarcoplasmic reticulum of Ca2+, but may be due to a requirement of Ca2+ entry into the cell to trigger Ca2+ release from the sarcoplasmic reticulum.

Aequorin↗
PubMed1988

The effects of nickel on contraction and membrane current in isolated rat myocytes.

Depolarization of isolated myocytes for several seconds produces a maintained, tonic component of tension. We have found that this is abolished by 5 mM-Ni2+. Furthermore if Ni2+ is applied after the tonic contraction has developed then its relaxation is prevented. These results are consistent with the hypothesis that Ni2+ inhibits Na+-Ca2+ exchange. Finally, also consistent with an inhibition of Na+-Ca2+ exchange, Ni2+ abolishes the transient inward current while having no effect on the underlying change of [Ca2+]i.

Animals↗