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A J Levi

Publications and source records attributed to A J Levi.

At least 55 records · Page 3Linked to original sources
PubMed1996

A method for making rapid changes of superfusate whilst maintaining temperature at 37 degrees C.

We have developed a technique for making a rapid solution change, whilst at the same time maintaining the temperature of the preparation at 37 degrees C. It is technically difficult to use rapid solution changes when experiments are performed at normal mammalian body temperature. As a solution is heated from room temperature to 37 degrees C, gas bubbles form in the rapid-flowing solution streams, and these disturb a cell or attached recording pipettes. We describe a system that has been developed to eliminate these problems. We show how to construct the different components of the system, and we have designed an electronic circuit to control solution changes. We have performed tests to characterise the function of this system. Solution flow out of the nozzle of the device (0.88 ml min-1, linear flow velocity 11.6 cm s-1) caused a fall in the steady-state temperature at the experimental preparation of only 0.3 degrees C. The device which takes between 0.5 and 1 s to completely change the superfusate of a single cell, was used routinely with five different experimental solutions. This system may be valuable in studies which require rapid solution changes to be performed at a normal mammalian body temperature.

Animals↗
PubMed1996

Low magnesium enhances the pro-arrhythmic effect of low potassium in the hypertrophied rat heart but not in the normal rat heart.

OBJECTIVES: To investigate the influence of external magnesium on wall stress-induced arrhythmias and to establish whether early cardiac hypertrophy affects the sensitivity of the heart to these arrhythmias. METHODS: We used a modified isolated working heart model to compare hypertrophied hearts from the spontaneously hypertensive rat (SHR) with hearts from normotensive control rats from the Wistar and Wistar-Kyoto strains. Young rats (aged 137.9 +/- 2.04 days mean +/- SEM) were used to ensure left ventricular hypertrophy uncomplicated by fibrosis and cardiac failure. Arrhythmias were induced by 20 s increases in ventricular afterload during which the electrocardiogram was recorded. RESULTS: In control experiments SHR hearts showed a significantly greater arrhythmic response than did normotensive control rat hearts during perfusion with low-potassium (2.4 mmol/l) solutions. Concomitant low magnesium (0.4 mmol/l) perfusion significantly increased the arrhythmic response to 2.4 mmol/l potassium in SHR hearts but had no effect on arrhythmias in normotensive control rat hearts. With low magnesium, the number of ventricular premature beats was increased in SHR hearts compared with that in normotensive control rat hearts during perfusion with 4.2 and 2.4 mmol/l potassium. In addition, perfusion with low magnesium and 2.4 mmol/l potassium led to an increase in the complexity of arrhythmias, ventricular tachycardia occurring in nine of 10 SHR hearts but in only two of six normotensive control rat hearts. CONCLUSIONS: There is a synergistic effect of low magnesium and low potassium on wall-stress-induced arrhythmia in the SHR heart. An analogous effect might be important in patients with hypertension and left ventricular hypertrophy, in whom systolic blood pressure is frequently labile, leading to wide fluctuations in ventricular wall stress; combined potassium and magnesium depletion is a common complication of antihypertensive treatment.

Animals↗
PubMed1996

Actions of the digitalis analogue strophanthidin on action potentials and L-type calcium current in single cells isolated from the rabbit atrioventricular node.

1. The atrioventricular node (AVN) of the heart is vital to normal cardiac function and is a major site of antiarrhythmic drug action. This study describes the effects of the cardiac glycoside analogue strophanthidin on spontaneous action potentials and L-type calcium current recorded from single AVN cells isolated from the rabbit heart. 2. With a standard KCl-based internal dialysis solution, exposure to 50 microM strophanthidin produced a progressive depolarization of the maximum diastolic potential and a reduction in action potential amplitude and upstroke velocity. Sustained application resulted in the loss of action potentials and occurrence of spontaneous 'bell-shaped' depolarizations. 3. Cells were whole-cell voltage clamped at -40 mV and depolarizing voltage clamps applied. With a standard KCl-based internal dialysis solution, exposure to 50 microM strophanthidin caused a large reduction of ICa,L at all potentials between -30 and +40 mV (n = 4). At + 10 mV, the mean ICa,L amplitude was reduced from -232 +/- 65 pA to -48 +/- 26 pA (P < 0.05; 1 test; n = 5 cells). 4. To record ICa,L more selectively, cells were dialysed with a Cs-based pipette solution. A short strophanthidin exposure reduced ICa,L amplitude from -250 +/- 31 pA to -88 +/- 19 pA (P < 0.001; n = 8 cells). For both KCl and CsCl-based solutions it was observed that sustained exposure to strophanthidin for several minutes caused spontaneous inward fluctuations in the membrane current record similar to the 'ITI' (arrhythmogenic oscillatory transient inward) current shown for other cardiac cells. 5. When the calcium chelator BAPTA was added to the pipette solution (10 mM), the reduction in ICa,L by strophanthidin was largely eliminated (P > 0.1), and no spontaneous inward current fluctuations were observed after sustained exposure to strophanthidin (n = 8 cells). 6. When external Ca in the perfusate was replaced with Ba, strophanthidin did not significantly reduce the Ba current through L-type calcium channels (n = 5 cells). 7. We conclude that strophanthidin reduces ICa,L by an indirect action, mediated by the rise in intracellular calcium (Cai) which follows inhibition of the Na/K pump caused by cardiac glycosides. The appearance of spontaneous ITI with strophanthidin would also seem to be mediated by a rise in Cai, and may contribute to the spontaneous oscillations in membrane potential observed after prolonged strophanthidin exposure.

Action Potentials↗
PubMed1996

Substrate and inhibitor specificities of the monocarboxylate transporters of single rat heart cells.

We have used the intracellular pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to characterize the substrate and inhibitor specificity of monocarboxylate transport into isolated rat heart cells. Further evidence was obtained for the presence of two lactate carriers present in heart cells (Wang et al., Biochem. J. 290: 249-258, 1993) both distinct from the recently cloned monocarboxylate transporter isoform 1 (MCT-1) found in many other cell types. Only one isoform was potently inhibited by alpha-cyano-4-hydroxycinnamate [CHC; inhibitor constant (Ki) 190 microM] and the stilbene disulfonates 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (Ki 79 microM) and 4,4'-dinitrostilbene-2,2'-disulfonate (Ki of cis- and trans-isomers 38 and 171 microM, respectively; neither isomer inhibits MCT-1). The second carrier had a Ki of approximately 3 mM for CHC and 0.5-2 mM for the stilbene disulfonates. Thus, unlike in many other tissues, in rat heart cells these inhibitors are not effective at blocking lactate transport totally unless used at very high concentrations. Both carriers were inhibited by 3-isobutyl-1-methylxanthine (Ki 340 microM) and neither by 5-nitro-2-(3-phenylpropylamino)benzoate (a potent inhibitor of MCT-1). The overall Michaelis constant (Km) and maximum reaction rate (Vmax) for transport of a variety of substituted monocarboxylates (C2-C5) were determined, although it was not possible to elucidate the kinetic parameters of the two isoforms. Of physiological interest, the ketone bodies D-beta-hydroxybutyrate and acetoacetate had K(m) values of 10 and 5.4 mM, respectively. Vmax values were similar to those of L-lactate and pyruvate and indicate that transport could limit rates of utilization of ketone bodies. No stereoselectivity for L-over D-isomers of 2-chloro or 2-hydroxy acids was observed.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Effect of internal sodium and cellular calcium load on voltage-dependence of the Indo-1 transient in guinea-pig ventricular myocytes.

OBJECTIVE: To investigate the effect of altering internal Na and cell Ca load on the voltage-dependence of the intracellular Ca transient. METHODS: Ventricular myocytes were isolated enzymatically from the guinea-pig heart. They were patch clamped and dialysed internally with pipette solutions which contained either 0 or 10 or 20 mM Na. Intracellular Ca was monitored with Indo-1 and experiments were carried out at 36 degrees C. A standard level of Ca loading was established before each test pulse by applying a train of conditioning pulses. The voltage-dependence of the Cai (Indo-1) transient provided information about the mechanisms which trigger Ca release from the sarcoplasmic reticulum (SR). RESULTS: The voltage-dependence of L-type Ca current (ICa.L) was assessed in separate experiments by dialysing myocytes with a Cs-based solution. ICa.L had a maximum amplitude at 0 mV, declined at more positive potentials and there was little net inward ICa.L at +100 mV. The rapid initial phasic component of the Indo-1 transient was abolished by ryanodine/thapsigargin; therefore, this component reflected the magnitude of SR Ca release. In cells dialysed with 10 mM Na, the voltage-dependence of the Indo-1 transient was different from ICa.L. The Indo-1 transient became maximal at +20 mV, and the decline of the Indo-1 transient at positive potentials was less steep than the decline of ICa.L. A large proportion of the phasic Indo-1 transient could remain at positive potentials where there was no detectable ICa.L. Increasing dialysing Na from 10 to 20 mM led to a marked change in voltage-dependence of the Indo-1 transient. With 20 mM Na, the amplitude of the phasic Indo-1 transient remained large between +20 and +100 mV. Removing Na from the pipette dialysis solution had the opposite effect on voltage-dependence of the transient. For each dialysing [Na], the level of cellular and SR Ca content was altered by varying the potential of conditioning pulses applied before each test pulse. There was no significant effect on voltage-dependence of the Indo-1 transient of either increasing or reducing the cellular Ca content. CONCLUSION: These data are consistent with the hypothesis that the voltage-dependence of the Cai transient results from the sum of the voltage-dependencies of the two main trigger mechanisms--Ica.L and reverse Na/Ca exchange. When a myocyte was dialysed with Na-free solution, the voltage-dependence of the Cai transient became more similar (but not identical) to that for ICa.L. With 20 mM Na dialysis, the altered voltage-dependence of the Cai transient may reflect an increased trigger influence of reverse Na/Ca exchange.

Animals↗
PubMed1995

Calcium transients which accompany the activation of sodium current in rat ventricular myocytes at 37 degrees C: a trigger role for reverse Na-Ca exchange activated by membrane potential?

We investigated the role of the fast sodium current (INa) in triggering Ca release from the sarcoplasmic reticulum (SR), using adult rat left ventricular myocytes, loaded with Fura-2 to measure intracellular Ca (Cai), which were whole-cell patch-clamped at 35-37 degrees C. Before each test pulse, a series of 400-ms conditioning pulses to +10 mV were applied to establish a constant level of SR Ca load. Pulses were applied every 15 s. A test pulse from -80 mV to -50 mV elicited a rapid INa and a phasic Cai transient. When the solution perfusing a myocyte was rapidly switched for 15 s before a test pulse to one containing the L-type Ca channel blocker nifedipine (20 microM), the test pulse still activated INa and a phasic Cai transient, the amplitude of which was not significantly different from control (P > 0.05; t-test). When a rapid switch to 20 microM nifedipine plus 30 microM tetrodotoxin (TTX) was made 15 s before a test pulse, both INa and the Cai transient were completely abolished (n = 6). When a switch was made to Na-free (Li) solution, which contained 20 microM nifedipine to block L-type Ca current, ICa,L, there was no significant difference in the Cai transient amplitude from that of control (P > 0.05; n = 6). Brief depolarising test pulses (-80 mV to +20 mV, 10 ms duration) to simulate membrane potential escape also elicited a Cai transient which attained 90.0% (+/-2.8%; n = 7) of the Cai transient activated by a conditioning pulse to +10 mV. The Cai transient with a brief pulse was not significantly affected by application of 20 microM nifedipine (P > 0.05), but adding TTX with nifedipine reduced the Cai transient amplitude to 76.9% (+/-6.8%; P < 0.02; n = 8). In four cells, the Cai transient remaining in the presence of nifedipine plus TTX was abolished by adding 5 mM Ni. These data are consistent with voltage escape during activation of INa leading to a trigger Ca entry via a mechanism other than L-type Ca channels or subsarcolemmal Na accumulation with reverse Na-Ca exchange. The block by Ni of the Cai transient suggests that a brief membrane potential escape might directly activate reverse mode Na-Ca exchange to trigger SR release, and this mechanism would seem to account largely for the Cai transient which accompanies INa in rat myocytes, under these experimental recording conditions.

Animals↗
PubMed1995

EMD 57033 enhances arrhythmias associated with increased wall-stress in the working rat heart.

1. EMD 57033 produces a positive inotropic effect by increasing the sensitivity of cardiac muscle myofilaments to calcium. Since the elevation in intracellular calcium produced by conventional inotropic compounds is thought to be arrhythmogenic, it is hoped that compounds such as EMD 57033 may increase cardiac output without exacerbating arrhythmias in patients with cardiac failure. This is the first study to examine whether EMD 57033 influences the susceptibility of the heart to ventricular arrhythmias. 2. We used the isolated working rat heart to investigate the effect of EMD 57033 on wall-stress-induced ventricular arrhythmia. Arrhythmias were induced by increases in ventricular afterload, and the effect of 2 mumol/l EMD 57033 on ventricular arrhythmias was investigated. The effect of 2 mumol/l EMD 57033 on contractility and arrhythmias was also assessed in the presence of different levels of perfusate calcium. 3. EMD 57033 was positively inotropic in the working rat heart, but it also produced a reversible increase in wall-stress-induced ventricular arrhythmia. The incidence of both ventricular ectopics and complex arrhythmias such as ventricular tachycardia were significantly increased by EMD 57033. Arrhythmias increased progressively as the level of perfusate calcium was raised within the physiological range. 4. The mechanism by which EMD 57033 increases wall-stress-induced arrhythmia is unclear, but it seems unlikely to be directly due to elevation of intracellular calcium. Further studies of the arrhythmogenic profile of this novel compound are required in a variety of models to assess its suitability and safety as a potentially therapeutic compound in heart failure.

Animals↗
PubMed1995

Na-Ca exchange tail current indicates voltage dependence of the Cai transient in rabbit ventricular myocytes.

INTRODUCTION: In mammalian cardiac myocytes, a rise of intracellular calcium (Cai) is well known to activate Ca extrusion via forward Na-Ca exchange, which generates an inward membrane current. This can be observed as an inward "tail" current (INa-Ca) when the membrane is repolarized after a depolarization-activated rise of Cai. If, during a voltage step, the membrane is repolarized at the time of the peak of the Cai transient, the size of the INa-Ca tail might be expected to reflect the magnitude of the Cai transient. Therefore, it might be possible to estimate the amplitude and voltage dependence of the Cai transient without, for instance, using fluorescent indicators that can interfere with Cai regulation. The first aim of this study was to use INa-Ca tails to investigate the voltage dependence of the Cai transient in whole cell patch clamped rabbit ventricular myocytes dialyzed with a "normal" level of internal Na. The second aim was to investigate how the voltage dependence of the INa-Ca tails varied with changes to the dialyzing Na concentration. The third aim was to test the correlation of voltage dependence of INa-Ca tails with the voltage dependence of the Cai transient obtained using a fluorescent Ca indicator. METHODS AND RESULTS: Experiments were performed at 35 degrees to 37 degrees C using whole cell patch clamp, and the holding potential was set at -40 mV. Depolarization elicited a Cai transient that peaked in 40 to 50 msec. We reasoned, therefore, that membrane repolarization after 50 msec would cause the raised level of Cai to activate an inward current on forward Na-Ca exchange. The amplitude of INa-Ca measured shortly (10 msec) after repolarization should reflect the peak amplitude of the Cai transient elicited by the depolarization. In cells dialyzed with 10 mM Na-containing solution and depolarized for 50 msec to differing test potentials, the INa-Ca tail on repolarization increased progressively after pulses to between -40 and +20 mV. The INa-Ca tail was maximal after a +20-mV pulse and showed no decline after depolarizations to more positive potentials, up to +100 mV (P > 0.1; n = 8). This implies that the Cai transient has a similar amplitude for depolarizing pulses between +20 and +100 mV. When Na-free solution dialyzed the cell, the voltage dependence of the INa-Ca tail became bell-shaped, with a maximum at +20 mV (n = 4). Voltage dependence of the INa-Ca tail was little affected by raising dialyzing Na from 10 to 20 mM (n = 4); but the amplitude of the INa-Ca tail increased. Inhibition of the Na-K pump with strophanthidin in cells dialyzed with 10 mM Na had qualitatively similar effects to increasing dialyzing Na. In Fura-2 loaded cells dialyzed with 10 mM Na, the Cai transient exhibited a similar voltage dependence to the INa-Ca tail (n = 6). CONCLUSION: The results of this study suggest that in cells dialyzed with 10 mM Na, the voltage dependence of the Cai transient is different from the L-type Ca current, since this current declines at potentials > +20 mV. The results obtained using Fura-2 suggest that the INa-Ca tail current measurement tracked the Cai sufficiently well to reflect the voltage dependence of the Cai transient. The data also confirm that the voltage dependence of the Cai transient in rabbit cells can be modulated by altering dialyzing Na concentration.

Animals↗
PubMed1995

The economic and quality-of-life benefits of Helicobacter pylori eradication in chronic duodenal ulcer disease--a community-based study.

A policy of Helicobacter pylori eradication in patients with duodenal ulceration on long-term acid-suppressing therapy was evaluated in a prospective study amongst a general practice population, with particular reference to economic and quality-of-life benefits. One hundred and sixty-eight patients on long-term acid-suppressing therapy had chronic duodenal ulcer disease of whom 88 were eligible for the study; 45 patients attended for review, with 42 testing positive for H pylori (as assessed by 13C-urea breath test). The median duration of acid-suppressing therapy was six years (maximum 15 years); 47.6% of the patients were using additional antacids and 80.9% still experienced epigastric discomfort. Two-thirds (28/42) of the patients eradicated H pylori. Successful eradication was associated with a highly significant reduction in all symptoms. At 12 months follow-up, heartburn had decreased from 28.7% to 7.1%, epigastric discomfort from 75% to 3.6%, nausea from 32.1% to 0% and wind from 50% to 0%. Of the patients that eradicated H pylori 96.4% reported an improvement in their general health compared to none of those that remained H pylori positive. Successful H pylori eradication therapy scored higher on satisfaction ratings than long-term acid-suppressing therapy. Eradication of H pylori resulted in 27/28 patients being able to discontinue acid-suppressing therapy, representing a 5.8% reduction in the use of such drugs per year in the local general practice population. A policy of H pylori eradication in chronic duodenal ulcer disease reduces the use of long-term acid-suppression therapy in general practice. This has important financial implications as well as offering considerable symptomatic benefits to the patients and improving their quality of life.

Adult↗
PubMed1995

Wall stress induced arrhythmia is enhanced by low potassium and early left ventricular hypertrophy in the working rat heart.

OBJECTIVE: The aim was to investigate the effect of lowering external potassium on the sensitivity of the normal and hypertrophied rat heart to arrhythmias induced by increases in ventricular wall stress. METHODS: The isolated working heart model was used to compare hypertrophied hearts from the spontaneously hypertensive rat (SHR) with hearts from normotensive control rats (NCR) from the Wistar and Wistar-Kyoto strains. Young animals [131.5(SEM 0.64) days] were used to ensure uncomplicated left ventricular hypertrophy. Arrhythmias were induced by 20 s increases in ventricular wall stress. The ECG was recorded and the arrhythmic response of each heart was compared during perfusion with Tyrode solutions containing [K] 6, 4.8, 3.6, and 2.4 mM. RESULTS: Hypertrophied SHR hearts showed a significantly greater arrhythmic response than control hearts at all levels of afterload increase when perfused with [K] 3.6 and 2.4 mM (t test P < 0.05 and P < 0.01). Both the number and complexity of arrhythmias were increased in the SHR hearts; ventricular tachycardia occurred in 10/12 compared with 4/12 control hearts whereas ventricular fibrillation occurred in 5/12 hearts but in none of the control hearts. CONCLUSIONS: At higher levels of [K] the sensitivity of SHR and normal hearts to wall stress induced arrhythmias is similar. However, as [K] is lowered to 3.6 mM or below, hypertrophied hearts show a greatly enhanced response to increases in ventricular wall stress. They develop a larger number of ventricular ectopics and more complex ventricular arrhythmias when compared to normal hearts. This may be of relevance to arrhythmic sudden death in hypertensive patients in whom left ventricular hypertrophy, potassium depletion, and blood pressure lability is common. Excessive fluctuations in systolic pressure and therefore ventricular wall stress could provide a powerful arrhythmic stimulus in hypertensive patients with left ventricular hypertrophy, even before ischaemia, cardiac failure, or extensive extracellular fibrosis have developed.

Animals↗
PubMed1994

Long term acid suppressing treatment in general practice.

OBJECTIVE: To determine the current practice in selected general practices for prescribing long term (> 6 months) treatment to suppress gastric acid secretion. SETTING: Seven general practices in the Harrow area that always or usually refer to Northwick Park Hospital. SUBJECTS: 60,148 patients on lists of the general practices. DESIGN: Identification of patients receiving long term treatment through repeat prescribing data, followed by a manual and computer survey of patients' notes for indications and investigations. Patient compliance and views on treatment were sought by a postal questionnaire. MAIN OUTCOME MEASURES: Indications for treatment, treatment given, investigations undertaken before and during treatment. RESULTS: 492 patients (0.82% of the population) were taking long term acid suppressing treatment. The most common diagnosis was duodenal ulcer disease (183 (37%) of all patients); oesophageal disease (118 (24%)) was also common. 93 patients (19%) were treated for abdominal pain where no diagnosis had been reached or who had only a diagnosis of gastritis on endoscopy. Ranitidine was prescribed in 394 (80%) patients. 298 (74%) patients found treatment helpful, but 108 (27%) had a poor understanding of their diagnosis. 317 patients (78%) took their drug as prescribed. 37 patients were also taking prescribed non-steroidal anti-inflammatory drugs and an additional 43 patients took regular aspirin or ibuprofen without prescription. CONCLUSIONS: Long term acid suppressing treatment is common, and a substantial number of patients are taking these drugs long term without a diagnosis having been reached. It is hoped that protocols for investigation and treatment will improve these figures. Patients need to be better informed about their disease and the possible adverse effects of taking non-steroidal anti-inflammatory drugs in acid related upper gastrointestinal disease.

Abdominal Pain↗
PubMed1994

Intracellular calcium transients recorded with Fura-2 in spontaneously active myocytes isolated from the atrioventricular node of the rabbit heart.

We have used the fluorescent Ca indicator Fura-2 to assess the changes in intracellular calcium (Cai) in single spontaneously active myocytes isolated from the rabbit atrioventricular node (AVN). Simultaneous recordings of membrane potential and the Fura-2 ratio signal (which reflects Cai) showed that a transient rise of Cai occurred with each spontaneous action potential (AP). The AP upstroke preceded the rise in Cai and repolarization of the AP occurred faster than the decline of Cai. The level of Cai remained raised and progressively declined towards a baseline diastolic level during the subsequent pacemaker depolarization. The Fura-2 (Cai) transient in spontaneously active AVN cells had a time-to-peak of 49.2 +/- 5.4 ms (mean +/- s.e.m.; n = 7) and declined with a single exponential time course (time constant = 139.8 +/- 23.9 ms; n = 7). Application of 10 microM ryanodine completely and irreversibly abolished the Cai transient, identifying the sarcoplasmic reticulum (SR) as the major source of releasable Ca. Both removal of external Ca and block of L-type Ca channels (with 2 microM nifedipine) also abolished Cai transients, suggesting that Ca entry via L-type Ca-channels is involved in triggering the SR Ca release underlying the Cai transient. Removal of external Na (in the presence of 20 microM nifedipine to block L-type Ca channels) caused a reversible increase in Cai, showing that Na/Ca exchange is present in AVN cells and that it is involved in Cai regulation. Spontaneous Cai transients were abolished by 1 microM acetylcholine, and this was associated with a hyperpolarization of membrane potential and cessation of action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1994

Properties of the fluorescent sodium indicator "SBFI" in rat and rabbit cardiac myocytes.

INTRODUCTION: Although some properties of the fluorescent sodium indicator "SBFI" are known, there is no accepted method by which the SBFI signal might be calibrated for intracellular Na (Nai) in cardiac cells. The first aim of this study was to characterize the loading and compartmentalization of this indicator in single cardiac myocytes. The second aim was, from experimental observation, to develop a rational calibration method for SBFI. The third aim was to use this Na indicator to study the dependence of tonic contraction on Nai in voltage-clamped ventricular myocytes. METHODS AND RESULTS: SBFI was incorporated into myocytes by incubating with the acetoxymethyl ester (10 microM) for 2 hours. This led to an intracellular concentration of SBFI free acid of 122 +/- 17 microM. We considered a number of issues with respect to compartmentalization of indicator and, under our conditions, we found the majority (71%) of indicator was situated in the cytoplasm. Therefore, SBFI indicates mainly changes of cytoplasmic Na. Calibration of the indicator for Nai was performed by equilibrating internal and external Na. We investigated the conditions required for optimum transmembrane Na equilibration and found it necessary to use a calibration solution free of both Ca and magnesium (Mg). The Na ionophores gramicidin D and monensin were both required, and it was also necessary to inhibit the Na/K pump for optimum Na equilibration. Using these conditions, the Nai concentration in quiescent rat ventricular myocytes was 10.9 +/- 0.74 mM (mean +/- SEM, n = 40; equivalent to an Na activity of 8.3 mM). The concentration of Nai was significantly lower in quiescent rabbit myocytes: 3.8 +/- 0.23 mM (n = 24; equivalent to an Na activity of 2.9 mM). In voltage-clamped rabbit myocytes, inhibition of the Na/K pump caused a rise of Nai; there were also marked effects on the tonic shortening elicited by ramp depolarizations. As Nai rose, the magnitude of tonic shortening increased and its voltage dependence also changed. CONCLUSION: These results confirm the suitability of SBFI for measuring Nai in cardiac cells. Provided that steps are taken to optimize transmembrane Na equilibration, the indicator can be calibrated for Nai. The different Nai of rat and rabbit myocytes has implications for the function of sarcolemmal Na/Ca exchange in each cell type. An Nai of 10.9 mM in rat myocytes gives a calculated reversal potential for the exchange of -35 mV. In comparison, an Nai of 3.8 mM in rabbit myocytes leads to a reversal potential for the exchange +45 mV. Therefore, relatively small changes of Nai can shift the reversal potential of the exchange to values that are substantially more positive or negative than zero. The behavior of voltage-dependent tonic contraction in rabbit myocytes was consistent with the Na/Ca exchange reversal potential being more positive than zero in these cells.

Animals↗
PubMed1994

A faster twitch contraction of soleus in the spontaneously hypertensive rat is partly due to changed fibre type composition.

Contractile properties and fibre type composition of slow-twitch soleus muscles from spontaneously hypertensive rats (SHR) were compared with those from a control normotensive strain (Wistar-Kyoto, WKY). Male rats aged around 100 days were used, and muscle tension was recorded isometrically in vitro. Tetanic tension was reduced by about 15% in SHR; this decrease was highly significant when tension was expressed relative to muscle cross-sectional area. Twitch contraction and relaxation were 12-15% faster in SHR. There were no differences in the rate of rise of tension of either tetani or twitches, but the relaxation of tetanic tension was 17% faster in SHR. Twitch:tetanus ratios were 20% smaller in SHR. Histochemistry showed that SHR muscles had a threefold higher proportion of fast type II fibres compared to WKY. There were no significant changes in the contractile properties of the fast-twitch extensor digitorum longus (EDL) muscles of SHR compared with WKY. Whilst the higher proportion of type II fibres could account for some of the contractile changes in SHR soleus muscles, the lack of any difference in the rate of rise of twitch and tetanic tension is not consistent. The faster rate of relaxation of both twitch and tetanus in SHR suggests that a higher rate of removal of calcium, either from the myofilaments or from the cytoplasm, may be an important factor in causing the changes in SHR soleus. The faster relaxation may be compared with a similar change reported in SHR cardiac myocytes.

Animals↗
PubMed1994

Depolarization-induced Ca entry via Na-Ca exchange triggers SR release in guinea pig cardiac myocytes.

In mammalian heart muscle, Ca entry through L-type Ca channels is thought to be the primary trigger for the sarcoplasmic reticulum (SR) Ca release, which initiates contraction. The results of this study show that, in guinea pig myocytes with a normal internal Na (10 mM Na in pipette), another trigger mechanisms for SR release and contraction exists. A crucial feature of these experiments was the ability to change rapidly the extracellular environment of a single myocyte so that alterations of intracellular Ca and SR Ca load were minimized for each solution change. We found the following results. 1) A switch to Na-free solution 50 ms before depolarization led to an increase of phasic contraction without increasing L-type Ca current (Ica) or Ca loading of the SR. 2) Although rapid application of 20 microM nifedipine 3 s before a + 10-mV pulse blocked ICa completely, 43 +/- 11 (SE) % of the phasic contraction remained. Similar results were obtained by rapid switching to 150 microM Cd to block ICa. 3) Phasic contraction and ICa had different voltage dependence. With steps to positive potentials there was little ICa but still a substantial phasic contraction. 4) Under action potential conditions, 64.6 +/- 7.9% of the control phasic contraction remained after switching to 20 microM nifedipine to block ICa. 5) The contraction remaining with nifedipine was unaffected by adding 100 microM Ni. Because 100 microM Ni blocks T-type Ca channels, this shows that Ca entry via T-type Ca channels is not involved in triggering SR release. 6) The phasic contraction remaining after a rapid switch to nifedipine was blocked completely by adding 5 mM Ni. Because this concentration of Ni is known to block the Na-Ca exchange, this result suggests that the exchange plays a role in triggering SR release. Taken together, the present results indicate that depolarization-induced Ca entry on the Na-Ca exchange is able to trigger SR release and phasic contraction. This explanation can account for increased phasic contraction after a rapid switch to Na-free solution, persistence of a phasic contraction in the complete absence of ICa, substantial phasic contraction at positive test potentials where there is no ICa, and abolition of nifedipine-resistant contraction by 5 mM Ni.

Action Potentials↗
PubMed1994

L-type calcium current in rod- and spindle-shaped myocytes isolated from rabbit atrioventricular node.

The atrioventricular node (AVN) is vital to normal cardiac function. The present report describes the properties of L-type calcium current (ICa) in rod- and spindle-shaped myocytes isolated from the rabbit AVN. With depolarizing voltage clamps from a holding potential of -40 mV, a rapidly activating ICa was observed, which peaked at +10 mV in most cells and exhibited a "bell-shaped" current-voltage relation. ICa was abolished by nifedipine (2-20 microM) and cadmium (100-200 microM) and was greatly reduced by manganese (1 mM). At +10 mV, time to peak ICa was 3.3 +/- 0.15 (SE) ms (n = 12) and ICa current density was 9.3 +/- 1.2 pA/pF (n = 9). Steady-state activation and inactivation curves for ICa showed half-maximal activation at -3.6 mV [slope factor (k) = 6.6 mV] and half-maximal inactivation at -25.8 mV (k = 6.5 mV). The time course of decay of ICa during a depolarizing pulse was voltage dependent and biexponential. The time course of recovery of ICa from inactivation was also biexponential (with two time constants tau 1 = 194.7 and tau 2 = 907.4 ms). Under current clamp, spontaneous action potentials from AVN cells were blocked by nifedipine as well as by cadmium, suggesting that L-type ICa was largely responsible for the action potential upstroke.

Animals↗
PubMed1994

Kinetics of the sarcolemmal lactate carrier in single heart cells using BCECF to measure pHi.

The pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure lactate transport in single cardiac myocytes. Addition of lactate externally caused a rapid fall of intracellular pH (pHi), which was largely inhibited by 5 mM alpha-cyano-4-hydroxycinnamate (CHC), a specific inhibitor of the lactate carrier. Stilbene disulfonates such as 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) only partially inhibited the response, with inhibition being greater in guinea pig than rat myocytes. The data are consistent with two isoforms of the lactate carrier, one sensitive and one insensitive to DBDS, coexisting within a single myocyte and both having a stoichiometry of 1 lactate:1 proton. The initial rate of pHi fall was used to determine carrier kinetics. Rat myocytes had a Michaelis constant (Km) for external L-lactate of 2.74 mM and a Km for external pyruvate of 0.2 mM. Guinea pig cells had a Km for external L-lactate of 2.2 mM. Kinetics of lactate efflux were also evaluated using the rate of pHi recovery on removing external lactate. The Km and maximal rate values for efflux were both threefold higher than for influx and were related to each other and the transmembrane pH gradient as predicted by the Haldane relationship. It is suggested that under hypoxic conditions, the carrier may be the rate-limiting factor for lactate extrusion.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗