Kinetics of dissolution of gaseous halothane in Krebs-Ringer's solution.

The responses of biological tissues to volatile anaesthetics are commonly studied by incubating specimens in a bath containing dissolved anaesthetic. One accepted technique is to bubble an anaesthetic gas into several incubation chambers simultaneously. To assess the validity of this technique in producing dissolved anaesthetic (the biologically active form) at equal rates among the several chambers, we determined the kinetics of dissolution of halothane gas in three tissue incubation chambers containing Krebs-Ringer's solution. We found that (1) the dissolution kinetics were first-order in all three chambers; (2) the rate of halothane dissolution depended on the gas bubbling rate; (3) even with the same bubbling rates, chamber shapes and chamber volumes, the dissolution rates for the three chambers were not equal, suggesting that dissolution rate depended on small differences in chamber geometry; (4) the dissolution rates could be made equal by adjusting chamber bubbling rates according to calculations involving the first-order rate equation; and (5) the maximum coefficient of variation of dissolved halothane concentration was 9% at 63% approach to equilibrium and 3% at equilibrium.

A thermal transition of passive calcium efflux in fragmented sarcoplasmic reticulum.

The temperature dependence of passive Ca2+ efflux from skeletal muscle fragmented sarcoplasmic reticulum (FSR) was studied by dilution of a suspension of the vesicles into which 1 mM (CaCl2 + 45Ca) had been passively incorporated by overnight incubation at 3 degrees. It was found that in the presence of 5 mM Mg2+, Ca2+ efflux could be resolved into two simultaneous first-order processes between 5 degrees and 35 degrees, but only a single first-order process appeared between 37 degrees and 55 degrees. Two independent functional transitions were found at 30 degrees, indicating an abrupt membrane molecular reorganization at that temperature: (1) The two components of Ca2+ efflux at 5 degrees--35 degrees contributed equally to the total observed initial efflux at temperatures up to 30 degrees. Between 30 degrees and 35 degrees, the relative contribution of the fast component progressively diminished until, by 37 degrees, only the slow component remained. (2) The slow component, which persisted throughout the entire temperature range 5 degrees--55 degrees, exhibited a break in its Arrhenius plot at 30 degrees--32 degrees. Elevation of internal Ca2+ concentration to 10 mM failed either to produce saturation kinetics of efflux or appreciably change its first-order rate constant. Omitting Mg2+ in the low temperature range accelerated Ca2+ efflux about 20-fold and eliminated the fast component, whereas including Ca2+ in the external medium in the high temperature range retarded Ca2+ efflux by about the same factor and generated a fast component. Omitting Mg2+ in the high-temperature range, however, had little effect on Ca2+ efflux. The failure of external divalent cation to stimulate Ca2+ efflux thus precludes an obligatory carrier-mediated exchange mechanism. Furthermore, participation of the catalytic turnover function of the Ca2+-ATPase molecule in Ca2+ efflux was unlikely because (1) the 30 degrees transition temperature for efflux did not coincide with those previously determined for active Ca2+ uptake, ATPase activity, and reversal of the Ca2+ pump, and (2) above the transition temperature, the activation enthalpy and activation entropy increased for efflux but decreased for both active Ca2+ uptake and ATPase activity. Ca2+ efflux therefore probably involved simple diffusion through a membrane pore (Ca2+ "leak"). By comparison to the results of others using artificial and biological membranes, the effect of external divalent cation to produce a fast component of Ca2+ efflux from FSR is tentatively attributed to the formation of aggregates of SR vesicles.

The role of oxidized nicotinamide adenine dinucleotide in fluoride inhibition of active sodium transport in human erythrocytes.

The rate coefficient for (22)Na release from previously labeled human erythrocytes was determined in the presence of 0.1-10 mM sodium fluoride (F). The oxidized nicotinamide adenine dinucleotide (NAD(+)) level at the end of 2 hr of incubation in tris(hydroxymethyl)aminomethane (Tris)-Ringer medium was also measured. Both parameters decreased proportionately as F concentration was raised. Both F-induced changes were immediate and were reversed by 10 mM pyruvate. The decrease in NAD(+) concentration following enolase inhibition by F is attributed to a diminished rate of formation in the reaction catalyzed by lactic dehydrogenase (LDH) with undiminished continued utilization in the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It is postulated that the NAD(+) lowering limited the GAPDH step, resulting in proportionate decreases in the rates of phosphoglycerate kinase (PGK) and Na,K-dependent adenosine triphosphatase (Na,K-ATPase), a reaction sequence thought to link glycolysis with active Na extrusion. Adding pyruvate with F increased NAD(+) production at the LDH step, thus reactivating GAPDH, PGK, and Na,K-ATPase and leading to the observed restoration of (22)Na release. The results suggest, therefore, that F inhibits active Na transport in intact human erythrocytes indirectly through a lowering of NAD(+), although, direct inhibition of the Na,K-ATPase by F may possibly occur simultaneously.