Inhibition of the Na,K-ATPase of canine renal medulla by several local anesthetics.

The ATPase activity of Na,K-ATPase-enriched membranes from canine renal medulla was determined in the absence of local anesthetic and in the presence of procaine, chloroprocaine, bupivacaine, mepivacaine, lidocaine, and two quaternary derivatives of lidocaine (QX-222 and QX-314) at 37( composite function)C. Chloroprocaine (IC(50)= 13 mM) had slightly greater potency than procaine (IC(50)= 17.7 mM). Bupivacaine (IC(50)= 6.7 mM) was more potent than its congener mepivacaine (IC(50)> 10 mM, the solubility limit). QX-222 (IC(50)> 600 mM) and QX-314 (IC(50)= 132 mM) had less potency than lidocaine (IC(50)= 30.4 mM). This study supports the interpretation that the uncharged forms of local anesthetics are much more potent inhibitors of Na,K-ATPase activity than the cationic forms.

Effects of local anaesthetics on the activity of the Na,K-ATPase of canine renal medulla.

The purpose of this study is to characterize the effects of local anaesthetics on Na,K-ATPase activity. The ATPase activity of Na, K-ATPase-enriched membranes from canine renal medulla was determined in the absence and in the presence of lidocaine, procaine, tetracaine, benzocaine, bupivacaine, prilocaine, and procainamide at 37 and 25 degrees C. All of these local anaesthetics, except benzocaine, inhibit the activity of the Na,K-ATPase of canine renal medulla at both 25 and 37 degrees C. Benzocaine inhibits Na,K-ATPase activity at 37 degrees C, but stimulates activity at 25 degrees C. The influence of lidocaine on stimulation of Na,K-ATPase activity by Na(+) and K(+) was investigated. Lidocaine increases the apparent K(0.5) of the Na,K-ATPase for both Na(+) and K(+) and decreases the V(max) values for both ions. IC(50) values for lidocaine increase with increasing concentrations of both Na(+) and K(+). The data indicate that lidocaine diminishes the affinity of the Na,K-ATPase for Na(+) and K(+) and that binding of Na(+) or K(+) decreases the potency of lidocaine as an inhibitor of the Na,K-ATPase. Lidocaine markedly decreases the affinity of the Na,K-ATPase for ouabain, but only slightly diminishes the maximum amount of ouabain bound. Unprotonated lidocaine is apparently a more potent inhibitor than is the protonated form.

Effects of general anaesthetics on the activity of the Na,K-ATPase of canine renal medulla.

Several previous studies have reported inhibition of Na,K-ATPase activity by chlorpromazine, phenobarbital and pentobarbital, thiopental, and monoketones. The purpose of this study is to investigate the influences of other general anaesthetics on Na,K-ATPase activity. The ATPase activity of Na,K-ATPase-enriched membranes from canine renal medulla was determined at 37 degrees C in the absence and in the presence of hexanol, diethylether, halothane, and propofol. The influence of hexanol on stimulation of Na,K-ATPase activity by Na+ and K+ was investigated. Hexanol, diethylether, halothane, and propofol inhibited the activity at 37 degrees C of the Na,K-ATPase of canine renal medulla. The IC50 values at 37 degrees C were: hexanol, 12.3 mM; diethylether, 170 mM; halothane, 7.35 mM; propofol, 0.127 mM. Hexanol increased the K0.5 of the Na,K-ATPase for K+ at 37 degrees C, but did not affect the K0.5 for Na+. At lower [K+] hexanol was a more potent inhibitor than at higher [K+].

Differential effects of general anesthetics on the quaternary structure of the Ca-ATPases of cardiac and skeletal sarcoplasmic reticulum.

The effects of the general anesthetics hexanol, halothane, and diethyl ether on Ca-ATPase activity and on the oligomeric state of the Ca-ATPase of sarcoplasmic reticulum (SR) from cardiac and skeletal muscle were investigated. The effects of these general anesthetics on Ca-ATPase activity were similar in cardiac and skeletal SR and were characterized by stimulation of Ca-ATPase activity at lower concentrations of anesthetics and inhibition at higher concentrations. The distribution of the Ca-ATPase among its oligomeric states was estimated from the time-resolved phosphorescence anisotropy (TPA) decay of SR in which Ca-ATPase was covalently labeled with erythrosin isothiocyanate (ERITC) or with erythrosin iodoacetamide (ERIA). In contrast to the similar responses of Ca-ATPase activity, there were marked differences in the responses to general anesthetics of the TPA decay between cardiac and skeletal SR. In cardiac SR hexanol, halothane, and diethyl ether caused pronounced increases in the limiting anisotropy at very long times (r infinity), which indicate increases in the fraction of oligomers too large to rotate on the millisecond time scale of the experiments. In skeletal SR, by contrast, there were no significant changes in r infinity in response to the three general anesthetics. This difference between cardiac and skeletal SR in response to general anesthetics is not due to the presence of phospholamban in cardiac SR, since SR from AT-1 cells, which have the SERCA2a isoform of Ca-ATPase, but only trace levels of phospholamban, have increases in r infinity in response to the general anesthetics that resemble those in cardiac SR. Experiments with cardiac SR labeled with ERIA give similar results, showing that the results with ERITC are not an artifact of the labeling procedure. Increasing the ionic strength with LiCl diminished the proportion of large immobile oligomers of cardiac Ca-ATPase under control conditions but enhanced the formation of large oligomers in response to hexanol.

Anesthetics alter the physical and functional properties of the Ca-ATPase in cardiac sarcoplasmic reticulum.

We have studied the effects of the local anesthetic lidocaine, and the general anesthetic halothane, on the function and oligomeric state of the CA-ATPase in cardiac sarcoplasmic reticulum (SR). Oligomeric changes were detected by time-resolved phosphorescence anisotropy (TPA). Lidocaine inhibited and aggregated the Ca-ATPase in cardiac SR. Micromolar calcium or 0.5 M lithium chloride protected against lidocaine-induced inhibition, indicating that electrostatic interactions are essential to lidocaine inhibition of the Ca-ATPase. The phospholamban (PLB) antibody 2D12, which mimics PLB phosphorylation, had no effect on lidocaine inhibition of the Ca-ATPase in cardiac SR. Inhibition and aggregation of the Ca-ATPase in cardiac SR occurred at lower concentrations of lidocaine than necessary to inhibit and aggregate the Ca-ATPase in skeletal SR, suggesting that the cardiac isoform of the enzyme has a higher affinity for lidocaine. Halothane inhibited and aggregated the Ca-ATPase in cardiac SR. Both inhibition and aggregation of the Ca-ATPase by halothane were much greater in the presence of PLB antibody or when PLB was phosphorylated, indicating a protective effect of PLB on halothane-induced inhibition and aggregation. The effects of halothane on cardiac SR are opposite from the effects of halothane observed in skeletal SR, where halothane activates and dissociates the Ca-ATPase. These results underscore the crucial role of protein-protein interactions on Ca-ATPase regulation and anesthetic perturbation of cardiac SR.

Hexanol and lidocaine affect the oligomeric state of the Ca-ATPase of sarcoplasmic reticulum.

Hexanol at 7 degrees C stimulates the activity of the Ca-ATPase of sarcoplasmic reticulum (SR). Time-resolved phosphorescence spectroscopy studies of SR whose Ca-ATPase is covalently labeled with erythrosin isothiocyanate (ERITC) indicate that at 7 degrees C hexanol (1) cause a concentration-dependent increase in the rate of decay of phosphorescence anisotropy, (2) causes larger oligomers of Ca-ATPase to dissociate into smaller oligomers, and (3) increases the rotational mobility of Ca-ATPase in all its oligomeric states. Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled stearic acid (SASL) in SR suggests that at 7 degrees C hexanol diminishes the fraction of SR lipids in the boundary lipid domain and disorders and fluidizes both the boundary lipid and the unrestricted lipid domain. In protein-free liposomes of extracted SR lipids hexanol increases fluidity and decreases order to a greater extent near the center of the lipid bilayer than near the polar head groups. At 25 degrees C hexanol has biphasic effects on Ca-ATPase activity: at 10 and 20 mM hexanol increases activity, but at 30 mM and especially at 40 mM there is inhibition of Ca-ATPase activity. The influence of hexanol at 25 degrees C on the oligomeric state of Ca-ATPase is also biphasic. At 10 and 20 mM, hexanol promotes the dissociation of larger oligomers into smaller ones, whereas at higher concentrations, 30 and 40 mM, hexanol causes larger oligomers to be formed from smaller ones. Lidocaine at 25 degrees C inhibits Ca-ATPase activity and causes dramatic slowing of the decay of phosphorescence anisotropy of ERITC-labeled SR by causing the formation of larger oligomers of Ca-ATPase from smaller ones. In protein-free liposomes of SR lipids at 25 degrees C, lidocaine disorders and fluidizes the acyl chains near the center of the bilayer (as did hexanol), but has opposite effects near the polar head groups. The opposite effects of hexanol and lidocaine on the oligomeric state of the SR Ca-ATPase provide a new molecular explanation for the opposite effects of hexanol and lidocaine on the activity of the Ca-ATPase. We conclude that the biphasic effects of hexanol on the activity of Ca-ATPase can be accounted for by biphasic effects of hexanol on the oligomeric state of the Ca-ATPase. This study supports the view that anesthetics can alter interactions between membrane proteins.

Identification of heterotrimeric and low molecular weight GTP-binding proteins in rabbit skeletal muscle longitudinal sarcoplasmic reticulum.

Direct photoaffinity labeling of proteins of longitudinal sarcoplasmic reticulum (LSR) of rabbit skeletal muscle with [32P]GTP revealed GTP-binding proteins of about 52, 45 and 30 kDa. ADP-ribosylation with [32P]NAD in the presence of cholera toxin (CTX) or pertussis toxin (PTX) indicates the existence of a CTX substrate (about 45 kDa); no PTX substrates were observed. Western blots of LSR probed with RM/1, an antiserum against a decapeptide from the C-terminus of Gs alpha, showed an immunoreactive band at about 45 kDa. [32P]GTP overlays of Western blots of LSR showed a heavily-labeled protein of about 29 kDa and one or more additional slightly smaller proteins that were more weakly labeled. When LSR was subjected to mild trypsin hydrolysis, the Western blot overlay revealed three bands at about 23, 25 and 29 kDa. Western blots of LSR proteins showed no significant immunoreactivity with the anti-(pan)-ras monoclonal antibodies 142-24E05 and Ras 11. ADP-ribosylation of LSR with [32P]NAD in the presence of C3 exoenzyme of Clostridium botulinum yielded a labeled band at about 23 kDa. Our results indicate the presence in rabbit LSR of a Gs alpha, the absence of Gi and G(o), and the presence of several low molecular weight GTP-binding proteins, distinct from p21 ras, one of which belongs to the rho or rac family.

Lactate transport in rat aorta.

Ouabain (10 mM) and gramicidin (5 micrograms/ml) do not inhibit lactate uptake by rat aortic rings. This supports the interpretation that a Na+ gradient is not involved in lactate transport. Dinitrophenol (25 microM) fails to inhibit lactate uptake, suggesting that oxidative metabolism is not required for lactate uptake. DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonate), quercetin, and alpha-cyano-4-hydroxy-cinnamate-agents that have been reported to inhibit lactate transport in other cell types-were ineffective in inhibiting lactate transport in rat aortic rings. Inhibition of lactate uptake by glyceraldehyde is not stereospecific, does not involve inhibition of glucose phosphorylation, and does not appear to involve interaction with membrane sulfhydryls. PCMBS (p-chloromercuribenzenesulfonate) does not markedly inhibit the initial rate of lactate uptake, but diminishes the lactate space at later times. Pyruvate, phenylpyruvate, and thiolactate inhibit lactate uptake, but propionate and glycolate are poor inhibitors.

Regulation of glycolysis in rat aorta.

Certain factors that might contribute to the regulation of the rate of glycolysis by rat aorta were investigated. Rat aortic rings were incubated with [14C]glucose, and the release of [14C]lactate was determined. There was good agreement between the lactate production estimated by enzymatic assay and by [14C]lactate release, suggesting that almost all the lactate produced under our experimental conditions was derived from exogenous glucose. When the glucose concentration in the medium was 10 mM or higher, the rate of glucose transport did not limit the rate of lactate production. In most cases studies were done both aerobically and anaerobically. In Hanks' Balanced Salt Solution the aerobic rate of lactate production was 18% of the anaerobic rate. We tested the effects on glycolysis of agents that alter ATP generation by mitochondria or ATP splitting by Na+-K+-ATPase or the mitochondrial ATPase. Under aerobic conditions, ouabain (5 mM) caused a 54% decrease in lactate production, and gramicidin (5 micrograms/ml) caused a 45% increase. Under anaerobic conditions, neither ouabain nor gramicidin affected lactate production. Aerobically dinitrophenol (25 microM) and carboxyatractyloside (0.5 mM) caused substantial increases in lactate production, 72 and 98% respectively. Under anaerobic conditions the effects of dinitrophenol and carboxyatractyloside were much smaller, with dinitrophenol causing a 15% increase and carboxyatractyloside a 12% decrease in lactate production. Increasing the concentration of phosphate in the incubation medium caused marked increases in lactate production. Both aerobically and anaerobically, shifting from 1.3 to 50 mM phosphate in the incubation medium caused a 3.5-fold increase in lactate production.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of anesthetic alcohols on membrane transport processes in human erythrocytes.

1. Anesthetic alcohols (pentanol, hexanol and heptanol) were found to increase the fluidity of red cell membrane lipids as monitored by the fluorescence depolarization of diphenylhexatriene. The relative potency of the alcohols was found to be parallel to their relative membrane/water partition coefficients. 2. Hexanol had biphasic effect on erythritol uptake by simple diffusion by red cells. At concentrations less than 9 mM, there was an approximately linear increase in erythritol permeability with increasing alcohol concentration. 3. The facilitated transport of uridine was markedly inhibited by hexanol. Hexanol at 6 mM produced a 65% inhibition of uridine (4 mM) uptake. Hexanol decreased both the apparent Km and V values for the equilibrium exchange of uridine. 4. The facilitated transport of galactose was only slightly inhibited by hexanol. 5. Hexanol was without effect on the passive and active fluxes of Na+ and K+ in red cells with altered cation contents. Cells that were slightly depleted of K+ and cells that were highly K+ -depleted were both insensitive to hexanol.

Mediated transport and metabolism of lactate in rat aorta.

1. Under appropriate conditions L- and D-lactate enter the cells of rat aorta and are metabolized. Oxidation of lactate to CO2 occurs under aerobic conditions. 2. L- and D-lactate are taken up into the cells when oxygen, glucose, or both oxygen and glucose are present in the incubation medium. Both L- and D-lactate are excluded from the cells when neither oxygen nor glucose is present. 3. D,L-Glyceraldehyde prevents the uptake of L-lactate. The effect is apparently not due to the inhibition of glucose metabolism by L-glyceraldehyde. 4. L-lactate (20 mM) markedly inhibits the uptake of 5 mM D-lactate, but 20 mM D-lactate fails to inhibit the uptake of 5 mM L-lactate. 5. Raising the pH of the incubation medium markedly depresses the uptake of L-lactate. 6. The results provide evidence that L- and D-lactate enter the cells of rat aorta by a mediated transport system.