David Redon

Calibration of intracellular Ca transients of isolated adult heart cells labelled with fura-2 by acetoxymethyl ester loading

A procedure for calibration of fluorescence signals from adult rat heart cells loaded with the -AM ester of fura-2 is described. Calibration is complicated by dye compartmentation and potentially incomplete dye hydrolysis. These problems were overcome by subtracting from fluorescence transients the non-cytosolic (mitochondrial) component of fura-2 fluorescence plus any Ca-insensitive component of dye fluorescence, after selectively and sequentially quenching cytosolic and non-cytosolic dye with Mn. The Kd of fura-2 in cells loaded by the -AM ester, in cells depleted of ATP and equilibrated with Ca buffers, was found to be 371 +/- 39 nM at 37 degrees C. We found that calibration values for RMAX and RMIN derived from previously measured cells were of general validity, removing the need to measure RMAX and RMIN on every cell. Once these calibration values are determined, the calibration procedure to measure cytosolic Ca on any cell is a five minute procedure to determine compartmentation, using just one non-toxic and inexpensive solution. Finally, we have calculated how the errors intrinsic to the measurements translate into errors of the calculated Ca concentration and transient peak heights. These calculations allow reasonable parameters for data acquisition to be set.

Negative inotropic effect of ketamine in rabbit ventricular muscle.

The effect of ketamine on myocardial contractile force was examined in rabbit papillay muscles in order to determine the underlying mechanism of action of the anesthetic. Ketamine HCl (20 and 40 mg/L) inhibited rested-state contractions that are dependent on the transsarcolemmal influx of Ca2+ for activation and reduced the upstroke velocity of the slow action potential, which reflects Ca2+ influx through the slow Ca2+ channel. On the other hand, ketamine had a relatively small effect on potentiated-state contractions and no effect on rapid cooling induced contractures, both of which are activated by the release of Ca2+ stored in the sarcoplasmic reticulum. These results suggest that ketamine inhibition of transsarcolemmal Ca2+ influx plays a major role in the negative inotropic action of the anesthetic.

The Influence of Serum Potassium on the Cerebral and Cardiac Toxicity of Bupivacaine and Lidocaine

: The influence of hyperkalemia on the central nervous system and cardiac toxicity of bupivacaine and lidocaine was studied in open-chested mechanically ventilated dogs. The seizure and cardiotoxic doses of intravenously administered lidocaine and bupivacaine were determined in two separate groups of normokalemic (2.7 +/- 0.05 SEM mEq/1) dogs. In the case of both anesthetics, the cardiotoxic dose was found to be approximately four times the seizure dose. Under conditions of hyperkalemia (5.4 +/- 0.08 SEM mEq/1), however, the cardiotoxic doses of both anesthetics were decreased significantly to approximately two times the seizure dose. Hyperkalemia did not change the seizure dose for either anesthetic. The cardiac to seizure dose ratio was decreased significantly for bupivacaine but not for lidocaine. The results of this study suggest that hyperkalemia enhances the cardiotoxic effects of both lidocaine and bupivacaine, with this enhancement being more pronounced in the case of bupivacaine.

Ca uptake by heart cells: I. Ca uptake by the sarcoplasmic reticulum of intact heart cells in suspension.

Electric field stimulation of adult rat heart cells suspended in medium with 0.2 mM Ca and isoproterenol caused 45Ca uptake at a rate (5.25 pmol/mg/beat) proportional to stimulation frequency. Uptake was strongly inhibited by verapamil or thapsigargin. 45Ca autoradiography showed that stimulation dependent verapamil sensitive uptake was associated with the rod shaped cells, while the uptake by round cells was unaffected by stimulation and was verapamil-insensitive. 45Ca efflux measurements revealed a caffeine-sensitive component of uptake which was abolished by thapsigargin, and a caffeine-insensitive component. Part of the latter was sensitive to thapsigargin but not to 30 s of stimulation; another part was sensitive to such stimulation but not to thapsigargin. With longer times of stimulation, the caffeine-insensitive pool increased in size, part of which appeared to be mitochondrial Ca uptake via a thapsigargin-sensitive pool. The caffeine-sensitive pool labelled quickly in stimulated cells and its size and rate of labelling was increased by stimulation frequency (3.87 pmol/mg/beat), while the caffeine-insensitive pool labelled more slowly and was relatively insensitive to stimulation (0.77 pmol/mg/beat). We conclude that essentially all of the SR Ca pool, as defined by its involvement in excitation-contraction coupling, is released by caffeine.

Effects of thiopental and halothane on spontaneous contractile activity induced in isolated ventricular muscles of the rabbit

To see if the known properties of thiopental of reducing Ca2+ and K+ fluxes across the myocardial sarcolemma account for its arrhythmogenic action, we have evaluated the effect of the anesthetic on spontaneous contractile activity induced in isolated rabbit papillary muscles. Thiopental (20 mg/1) prolonged the duration of sustained automaticity induced by stimulation at 1–2 Hz in the presence of 1 μmol/I isoproterenol. Thiopental (10, 20 mg/l) shortened the delay before the onset of Ba2+ ‐induced automaticity, which involves a decrease in a K+ current. The minimum concentration of Ba2+ required to induce automaticity was lowered by thiopental. Whether spontaneous activities were induced by high frequency stimulation in the presence of isoproterenol or by Ba2+, thiopental lowered the frequency of spontaneous beats. Thus, thiopental appears to have both arrhythmogenic and antiarrhythmic actions, and the former may be unmasked when catecholamines counteract the latter by increasing Ca2+ influx. Like thiopental, halothane (1.0%) decreased the frequency and force of Ba2+‐induced automatic beats but, unlike thiopental, prolonged the delay before the onset of Ba2+‐induced automaticity, indicating that halothane acts as a purely antiarrhythmic agent in this type of automaticity.

Isoflurane and halothane inhibit tetanic contractions in rabbit myocardium in vitro.

Rabbit right ventricular papillary muscles were tetanized by rapid stimulation in the presence of 1 microM ryanodine, an inhibitor of sarcoplasmic reticular function. Tetanic contractions elicited in this manner increased in strength as extracellular calcium concentration [Ca+2]ext was raised from 0.5 to 5 mM, exhibited saturation behavior above [Ca+2]ext = 5 mM, and were blocked by nifedipine. Accompanying membrane potentials depolarized to +10 mV and repolarized to -60 mV between stimuli. These data suggest that rabbit myocardial tetany is supported in large part by extracellular calcium influx via slow (L-type) calcium channels, consistent with similar recent findings in the ferret. At [Ca+2]ext = 2.5 mM, isoflurane (0.6-2.3%, gas phase) and halothane (0.4-1.5%) inhibited the strength of tetanic contractions in dose-dependent fashion. At [Ca+2]ext = 20 mM neither isoflurane (1.2%) nor halothane (0.8%) inhibited tetanic contraction strength. These data demonstrate that isoflurane and halothane inhibit contractile activity that is dependent on transsarcolemmal calcium influx via pathways independent of the ryanodine-sensitive sarcoplasmic reticulum. The exact sites of inhibition (e.g., slow channel vs. intracellular transit vs. myofibrillar binding) are not identified, but inhibition by either anesthetic may be competitively reversed by high extracellular calcium concentrations.