The authors recently showed that plasma membrane Ca(2+)-ATPase (PMCA) activity in cerebral synaptic plasma membrane (SPM) is diminished in a dose-related fashion during exposure in vitro to halothane, isoflurane, xenon, and nitrous oxide at clinically relevant partial pressures. They have now extended their work to in vivo studies, examining PMCA pumping in SPM obtained from control rats decapitated without anesthetic exposure, from rats decapitated during halothane anesthesia, and from rats decapitated after recovery from halothane anesthesia.
Three treatment groups were studied: 1) C, control rats that were decapitated without anesthetic exposure, 2) A, anesthetized rats exposed to 1 minimum effective dose (MED) for 20 min and then decapitated, and 3) R, rats exposed to 1 MED for 20 min and then decapitated after recovery from anesthesia, defined as beginning to groom. Plasma membrane Ca(2+)-ATPase pumping and Ca(2+)-dependent ATPase hydrolytic activity, as well as sodium-calcium exchanger activity and Na+-K+-ATPase hydrolytic activity, were assessed in cerebral SPM. In addition, halothane effect on smooth endoplasmic reticulum Ca(2+)-ATPase (SERCA) was examined.
Plasma membrane Ca(2+)-ATPase transport of Ca2+ into SPM vesicles from anesthetized rats was reduced to 71% of control (P < 0.01) compared with 113% of control for the recovered group (NS). No depression by halothane of SERCA activity, sodium-calcium exchanger, or Na+-K+-ATPase activity was noted among the CAR treatment groups.
Plasma membrane Ca(2+)-ATPase is selectively and stably inhibited in cerebral SPM from rats killed while anesthetized with halothane, compared with rats killed without anesthesia or after recovery from anesthesia. The studies described in this report, in conjunction with previously reported inhibition of PMCA activity in vitro by a wide range of anesthetic agents, indicate a relationship between inhibition of PMCA and action of inhalational anesthetics.
Key words: Anesthetics, volatile: halothane. Brain: synaptic plasma membranes. Enzymes: plasma membrane Calcium2+-ATPase. Mechanism of anesthesia.
CENTRAL nervous system lipid or protein moieties undergoing conformational change in response to inhalational anesthetics are generally believed to revert to a preexposure state with elution of anesthetic from the brain. However, we have shown that at least one process, conversion by methyltransferase Iodine of phosphatidylethanolamine to phosphatidyl-N-monomethylethanolamine, remained enhanced twofold in neural membranes of rats killed while anesthetized with halothane or isoflurane.  This stimulation of methylation persisted despite elution of anesthetic from the synaptosomal fraction during the long separation process. In contrast, phospholipid methylation was found to be normal in rats killed after emergence from halothane or isoflurane anesthesia. We now report persistent anesthetic alteration of another enzyme system found in synaptosomes, the ubiquitous plasma membrane Calcium2+-ATPase pump (PMCA). Free calcium is an important intracellular regulator, and its cytosolic activity, [Ca2+]i, is controlled by at least five major classes of pumps, exchangers, and channels. [2–5] Plasma membrane Calcium2+-ATPase plays a critical role in this regulatory process by ejecting Calcium2+ from the cell. We have shown that Calcium2+ pumping in synaptic plasma membrane (SPM) vesicles is diminished in a dose-related fashion during exposure in vitro to halothane, isoflurane, xenon, and nitrous oxide at clinically relevant partial pressures. [6–9] Moreover, fluorinated anesthetics inhibit ATP hydrolysis by erythrocytic PMCA,  a process recently described in a definitive report.  We have extended our work to examine PMCA pumping activity in SPM obtained from rats killed during and after halothane anesthesia. We found persistent inhibition of Calcium2+ pumping by PMCA in SPM obtained from rats killed while anesthetized, compared with control animals killed while awake. We also found return of PMCA function to normal in SPM obtained from animals allowed to recover from anesthesia before being killed.
Materials and Methods
In Vivo Exposure.
Animal use was approved by the Animal Care Committee of Vanderbilt University. Male Sprague-Dawley rats (250–320 g) were allowed access to food and water until the morning of the experiment. Animals were anesthetized with halothane (Halocarbon Laboratories, River Edge, NJ) delivered in warmed, humidified air and oxygen (FIO2= O.3) from a dedicated, calibrated vaporizer. Halothane concentrations were confirmed by gas chromatography. Induction of anesthesia was carried out with each rat breathing 3% halothane in a 21 chamber. When immobilized, the rat was removed from the chamber and placed on a thermal pad with a heating lamp nearby. A Tygon cylinder attached to a T-piece was fitted snugly over the muzzle for continued delivery of anesthetic as the animal breathed spontaneously.  Temperature was monitored with a rectal probe, and warming devices were used as needed. Halothane concentration was adjusted gradually, and anesthetic effect was monitored by clamping the tail every 30–60 s. After 6–8 min, the lowest concentration that suppressed withdrawal with tail clamping was attained, thus defining the minimum effective dose (MED).  Anesthesia was continued, excluding induction time in the chamber, for 20 min. Three treatment groups were studied: 1) C, control rats that were decapitated without anesthetic exposure, 2) A, anesthetized rats exposed to 1 MED for 20 min and then decapitated, and 3) R, “recovered” rats exposed to 1 MED for 20 min and then decapitated after recovery from anesthesia, defined as beginning to groom.
In Vitro Exposure.
Synaptic plasma membranes were prepared and processed, as described below, from rats decapitated without anesthetic treatment. Synaptic plasma membranes were then exposed in incubation vials placed in a Dubnoff shaker under a gassing hood. [1,9] Halothane, in a warmed, humidified air/oxygen mixture (FIO2= 0.3), was delivered in concentrations up to 1.5 vol% from a dedicated, calibrated vaporizer. Anesthetic concentrations were confirmed by gas chromatography.
Effect of Halothane Anesthesia on Arterial Blood Gas Partial Pressures and pH
In parallel experiments, a series of six rats was anesthetized with halothane as described above, and their femoral arteries were surgically exposed. After 20 min of anesthesia, arterial blood samples (300 micro liter) were withdrawn sequentially through a 27.5-G needle in duplicate or triplicate. Syringes were capped after evacuation of air, placed on ice, and directly analyzed on a Ciba Corning 238 pH/Blood Gas Analyzer (Ciba Corning Diag. Ltd, Halstead, England). Partial pressures of Oxygen2and CO2were measured, pH was determined, and base excess and hemoglobin oxygen saturation were calculated. Rats were then killed with an anesthetic overdose; SPM were not prepared from these animals.
Preparation of Synaptic Plasma Membranes
Evaluation of SPM preparative techniques has been detailed elsewhere. [1,9,12] Brains were dissected on ice. Cerebra from three to eight rats were weighed and pooled in ice-cold 0.32 M sucrose (pH 7.4). Synaptosomes were prepared by gradient ultracentrifugation, and SPM were prepared by osmotic shock of synaptosomes followed by differential ultracentrifugation on a discontinuous sucrose gradient. Final pellets were suspended in isosmotic sucrose (0.32 M, pH 7.4) and used immediately for assay of PMCA Calcium2+ pumping, sodium-calcium exchanger activity, or Sodium sup +- Potassium sup +-ATPase hydrolysis of ATP. Assays of Calcium2+-dependent ATP hydrolysis were done either immediately or within a few days. Microsomes were prepared by ultracentrifugation of the supernatant of the S2 fraction at 100,000g for 20 min. Pellets were suspended in isosmotic sucrose (0.32 M, pH 7.4) and used for measurement of Calcium2+ accumulation studies. Protein content in SPM pellets was estimated by the Bradford method.
Measurement of Calcium2+ Pumping Across SPM Membranes
Calcium2+ uptake by everted SPM vesicles, i.e., transport from the intracellular to extracellular surface (and by smooth endoplasmic reticulum, i.e., microsomes), was performed as described by Moore et al. with several modifications.  The incubation mixture (total volume 4 ml) was comprised of 30 mM imidazole-histidine (pH 6.8), 200 mM KCl, 5 mM MgCl2, 5 mM ATP, 5 mM sodium azide, 5 mM ammonium oxalate, and 20 micro Meter CaCl2containing (final concentration) 0.1 micro Ci/ml of45CaCl2(NEN Products, Boston, MA; specific activity 30.7 mCi *symbol* mg1). The reaction was started by adding 70-micro gram aliquots of SPM protein to each tube and maintained at 37 degrees Celsius. Aliquots of 0.5 ml were removed after 20, 30, and 60 min. Synaptic plasma membrane vesicles were collected on 25-mm cellulose nitrate filters (0.45 micro meter pore size, Gelman Sciences, Ann Arbor, MI) that had been prewashed with 2 ml of 0.25 M KCl and 10 ml of deionized water. After vesicle collection, the filters were washed with 2 ml of 0.25 M sucrose and dried. They were placed in vials containing CytoScint (ICN Costa Mesa, CA), and45Calcium2+ activity was assessed in a Beckman LS3801 beta counter. Results were expressed as nmoles of Calcium2+ accumulated per milligram of SPM protein per minute of incubation time. To confirm that Calcium2+ uptake in these experiments reflected PMCA activity, sensitivity to orthovanadate was tested by measuring Calcium2+ uptake in SPM obtained from C, A, and R rats in the presence and absence of 0.1 mM orthovanadate. .
Determination of Calcium sup 2+-Dependent ATPase Hydrolytic Activity
ATPase hydrolytic activity in the SPM preparations was assessed by measurement of inorganic phosphate (Pi) released from ATP during incubation with the enzyme source. [15,16] Synaptic plasma membrane aliquots (2 micro gram per tube) were suspended in 25 mM Tris-HCl buffer (ph 7.4), 50 mM KCl, 2 mM MgCl2, and 1 micro Meter CaCl2. The reaction was started by adding ATP (2 mM final concentration) in a total reaction volume of 1 ml, and samples were incubated for 30 min at 37 degrees Celsius in a Dubnoff shaker. The reaction was stopped by adding 1 ml of a solution (cooled to 4 degrees Celsius) containing perchloric acid (1.1 M), ammonium molybdate (809.1 mM), ammonium hydroxide (285.3 mM), ammonium metavanadate (20.09 mM), and nitric acid (99.1 micro Meter). Tubes were vortexed and optical densities of reaction mixtures and KH2PO4standards were measured in a UV spectrophotometer at 350 nm. Results were expressed in micro moles of liberated Pi per mg protein per hour.
Effect of Halothane on the Sodium-Calcium Exchanger
Sodium-calcium exchanger activity was estimated by measuring Calcium2+ accumulation in SPM vesicles preloaded with sodium ions.  Synaptic plasma membranes were obtained from rats treated according to the anesthetic protocol described above (in vivo group) or from untreated animals (in vitro group). Synaptic plasma membranes (0.7 mg protein) in 0.9 ml of 0.32 M sucrose (pH 7.4) plus 0.1 ml of 1 M NaCl were preincubated for 15 min at 37 degrees Celsius. One-tenth milliliter of the mixture containin 70 micro gram protein was then added to 3.9 ml of 50 mM Tris HCl buffer (pH 7.4) comprised of 200 mM KCl, 5 mM MgCl2, and 20 micro Meter CaCl2including (final concentration) 0.1 micro Ci *symbol* ml1of15CaCl2(NEN Products, Boston, MA; specific activity 30.7 mCi *symbol* mg sup -1). Aliquots of 0.5 ml were removed after 2.5, 5, 10, 20, and 30 min. Membranes prepared from rats in the in vitro group were exposed to desired concentrations of halothane throughout the preincubation and incubation periods, as described above. Synaptic plasma membrane vesicles were collected on 25-mm cellulose nitrate filters, which were processed as described for measurement of PMCA pumping activity. Results were expressed as nmoles of Calcium2+ accumulated per milligram of SPM protein per minute of incubation time.
Effect of Halothane on Sodium-Potassium-ATPase in Synaptic Plasma Membranes
Sodium-potassium ATPase (Sodium sup +-Potassium sup +-ATPase) hydrolytic activity in the SPM preparation was assessed by measurement of Pi release from ATP during incubation in the presence and absence of the specific Sodium sup +-Potassium sup +-ATPase inhibitor, ouabain (2 mM final concentration).  Synaptic plasma membranes were obtained from rats treated according to the anesthetic protocol described above (in vivo group) or from untreated animals (in vitro group). Synaptic plasma membrane aliquots (2 micro gram) were suspended in (final concentrations) 0.1 M Tris-HCl buffer (pH 7.0), 20 mM KCl, 5 mM MgCl2, 0.1 M NaCl, and 1 mM EDTA. The reaction was started by adding ATP (2 mM final concentration) in a total reaction volume of 1 ml, and samples were incubated for 30 min at 37 degrees Celsius in a Dubnoff shaker. Membranes prepared from rats in the in vitro group were exposed to desired concentrations of halothane throughout the incubation period, as described above. The reaction was stopped by adding 1 ml of a solution (cooled to 4 degrees Celsius) containing perchloric acid, ammonium molybdate, ammonium hydroxide, and ammonium vanadate. Tubes were vortexed, and the optical densities of reaction mixtures and KH2PO4standards were measured in a UV spectrophotometer at 350 nm. Results were calculated as the difference in Pi liberation in the absence and presence of ouabain and expressed in micro moles of liberated Pi per mg protein per hour.
Effect of Method of Killing on Calcium sup 2+-ATPase Activity
Four groups of seven rats in three separate experiments were killed by decapitation or by exposure to lethal concentrations of carbon dioxide, nitrogen, or helium. Each rat was placed in a 21 container equipped with inlet and outlet ports. Euthanasia was carried out by rapid replacement of air in the container with CO2, Nitrogen2, or Helium delivered at > 10 l *symbol* min1. Death, as judged by respiratory arrest preceded by loss of muscle tone, occurred within 30–40 s and was followed by decapitation. Control animals were exposed to air delivered at 10 l *symbol* min1for 40 s before decapitation. Synaptic plasma membranes were prepared from rats in each treatment group for measurement of PMCA activity.
Effect of Discontinuation of In Vitro Halothane Exposure on Plasma Membrane Calcium sup 2+-ATPase Activity
Cerebral SPM from untreated rats were incubated and exposed as described above to an air/oxygen mixture or to 1.5 vol% halothane for 30 min. In one group of experiments, ATP was added to the incubation mixture before exposure began, as usual. In a second group, ATP was added at the conclusion of the 30-min exposure period. All incubation vials were exposed to air/oxygen for another 60 min, for a total incubation time of 90 min. Aliquots were removed from all vials at 30, 60, and 90 min for assay of Calcium2+ uptake. Halothane concentration was measured in representative aliquots at each sampling time by high-performance liquid chromatography. Twenty microliters of the incubation solution were mixed with 20 micro liter of 0.05 nM toluene (serving as an internal standard) and injected directly into a Beckman System Gold HPLC equipped with a Waters C18 Novapack HPLC column (Millipore, Marlborough, MA). The eluent was methanol: water (1:1). The chromatogram was monitored for UV absorption at 210 nm.
Data were examined by multifactorial ANOVA and multiple comparison (Student-Newman-Keuls procedure). Statistical significance was inferred if P < 0.05.
Estimation of the Minimum Effective Dose of Halothane
Minimum effective dose (MED) for halothane was determined in 96 male rats used in this study. A mean MED of 1.49 vol% was obtained, with a standard deviation of 0.12 and a standard error of the mean of 0.012.
Arterial Blood Gas Partial Pressures and pH in Anesthetized Rats
(Table 1) shows arterial oxygen and carbon dioxide partial pressure, pH, base excess, and hemoglobin oxygen saturation measured (or calculated) in duplicate or triplicate, in six rats anesthetized for 20 min. Halothane concentrations were adjusted to deliver 1 MED. Partial pressures of Oxygen2reflected an FIO2of 0.3. All other values were within normal limits.
Plasma Membrane Calcium sup 2+-ATPase Pump Activity in Synaptic Plasma Membranes from Control, Anesthetized, and Recovered Rats
(Figure 1) shows Calcium2+ uptake, plotted against incubation time, in SPM obtained from control (C), anesthetized (A), and recovered (R) rats in six separate experiments, with membranes pooled from three to eight rats in each treatment group and incubated in quadruplicate. Plasma membrane Calcium2+-ATPase inhibition is evident at all sampling times with A treatment compared with C, with a return to normal with R treatment. Orthovanadate (0.1 mM), an inhibitor of PMCA, markedly reduced uptake in all treatment groups. Figure 2shows results from these six experiments with PMCA pump activity expressed as nmoles of transported Calcium2+ per mg of protein per minute for all treatment groups. Multifactor ANOVA and multiple comparison testing indicate that Calcium2+ transport by PMCA was significantly inhibited (P < 0.01) in SPM from animals killed while anesthetized. Transport in membranes from animals recovered from anesthesia did not differ significantly from the unanesthetized control group.
Calcium sup 2+-Dependent ATPase Hydrolytic Activity in Synaptic Plasma Membranes from Control, Anesthetized, and Recovered Rats
(Figure 3) shows that hydrolysis of ATP by Calcium2+ dependent ATPase in SPM was not altered by prior anesthesia, i.e., C, A, and R groups do not differ significantly. Results were derived from four experiments in which hydrolysis was assayed, out of the six studies described in the preceding section in which Calcium2+ pumping was measured.
Effect of In Vivo Halothane Exposure on Calcium sup 2+ Uptake by Microsomes
(Figure 4) shows the effect of prior in vivo halothane treatment (1 MED) on Calcium2+ transport by smooth endoplasmic reticulum Calcium2+-ATPase (SERCA) in the microsomal fraction of rat cerebral homogenates in two separate experiments (eight rats in each). No differences in SERCA activity were noted among the C, A, and R treatment groups.
Effect of In Vivo and In Vitro Halothane Exposure on Sodium-Calcium Exchanger Transport of Calcium sup 2+ and Sodium sup +-Potassium sup +-ATPase Activity in Synaptic Plasma Membranes
(Figure 5(a and b)) show the effects of prior in vivo halothane treatment and concurrent in vitro treatment with different halothane concentrations on sodium-calcium exchange in SPM obtained from seven rats. No discernible inhibition with halothane exposure was noted in either circumstance. Figure 5(c and d) show the effects of prior in vivo halothane treatment and concurrent in vitro treatment at different halothane concentrations on Sodium sup +-Potassium sup +-ATPase activity in SPM obtained from seven rats. Again, no discernible depression with halothane was noted.
Plasma Membrane Calcium sup 2+-ATPase Activity in Synaptic Plasma Membranes Obtained from Rats Killed by Decapitation or by Exposure to Carbon Dioxide, Nitrogen, or Helium
(Figure 6) indicates PMCA transport of Calcium2+ in SPM obtained from rats killed by decapitation or by exposure to one of several gases. No significant differences in PMCA activity, as measured by Calcium2+ accumulation by SPM vesicles at 20, 30, and 60 min, were observed between the rats killed while awake or by exposure to lethal concentrations of CO2, Nitrogen2, or Helium.
Effect of Discontinuing In Vitro Halothane on Plasma Membrane Calcium sup 2+-ATPase Pump Activity
(Figure 7) shows the effects of interrupting in vitro halothane exposure during measurement of Calcium2+ uptake in cerebral SPM. Membranes were prepared from previously unexposed rats. Each indicated value is the mean of triplicate measurements with SPM pooled from ten rats in two experiments. When ATP was added initially, before a 30-min exposure to halothane (air/oxygen for controls), Calcium sup 2+ uptake continued linearly after halothane was replaced with air/oxygen. Pumping was depressed to approximately 80% of control (P < 0.01). Similar results were obtained when ATP was added immediately after 30 min of halothane exposure. Despite replacement with air/oxygen during the remainder of the incubation period, Calcium2+ uptake continued linearly and was depressed to 85% of control (P < 0.01). Halothane concentration in the incubation medium was 0.18 mM at 30 min, reduced to 0.050 mM at 60 min and to 0 at 90 min.
We recently showed that PMCA activity in SPM from rat brain is diminished in a dose-related fashion during exposure in vitro to halothane, isoflurane, xenon, or nitrous oxide at clinically relevant partial pressures. [1,6–9] We have now described studies of PMCA activity in SPM from rats treated in three different ways before killing: no anesthetic exposure, exposure to halothane for 20 min (1 MED), or exposure to halothane for 20 min followed by a recovery period. Animals in the last group were considered to be recovered from anesthesia when they began grooming. One MED was defined as the lowest anesthetic concentration delivered to a rat that suppressed withdrawal with tail clamping. Minimum effective dose levels are highly reproducible in individual rats and are clustered closely in groups of rats. We previously reported MED values of 1.43 vol%(about 1.3 MAC) for halothane in eight rats.  Minimum effective dose determined in 96 rats in the course of the current study averaged 1.49 vol%. At this “surgical” level of anesthesia, rats breathed spontaneously. Adequacy of ventilation in this setting was assessed by arterial blood gas analysis in six similarly anesthetized rats. Table 1shows that these animals were able to maintain a normal pH and partial pressure of CO2, as well as a partial pressure of Oxygen2consistent with breathing 30% oxygen.
As indicated in Figure 1, Calcium2+ accumulation at three sampling times during SPM incubation (20, 30, and 60 min) was depressed in animals killed while anesthetized (P < 0.01). Plasma membrane Calcium2+-ATPase returned to levels that did not differ significantly from control values in SPM prepared from brains of animals allowed to recover from anesthesia before decapitation. Inhibition by low concentrations of orthovanadate is characteristic and specific for P-type pumps, such as PMCA. Orthovanadate inhibited Calcium2+ uptake in all treatment groups, with no difference in residual Calcium sup 2+ accumulation among treatment groups, and thus is indicative of a specific effect of halothane on the PMCA pump. Figure 2compares mean PMCA pumping activity, expressed as nmoles of Calcium2+ transported per minute, for all treatment groups in all experiments. Plasma membrane Calcium2+-ATPase transport in SPM from anesthetized rats was reduced to 71% of control (P < 0.01) compared with 113% of control for the recovered group (NS). It is noteworthy that the depression of PMCA pumping activity in this group of rats anesthetized with 1 MED of halothane is very close to that found with in vitro exposure of SPM from untreated animals to halothane or isoflurane at levels equivalent to 1 MED for each anesthetic gas, i.e., 79% of control values for halothane and 68% of control values for isoflurane. This close agreement among experiments carried out under quite different conditions indicates that there is a common molecular effect of anesthetics on PMCA, regardless of the method of exposure. The most notable observation in these in vivo studies, however, is the persistence of PMCA inhibition in the absence of the precipitating anesthetic agent.
In in vitro studies previously reported, we observed depression of both calcium pumping and ATP hydrolysis in SPM with anesthetic exposure. [6–9] We questioned the precision of ATP hydrolysis as a guide to anesthetic effects on SPM PMCA because anesthetic inhibition of ATP hydrolysis occurred, to some degree, in the presence of orthovanadate. This finding indicates either that orthovanadate affected PMCA pumping and hydrolysis differentially in SPM, possibly because of the presence of diverse isoforms and splice variants, or that other Calcium2+-ATPases, most likely ecto-ATPases, were contributing significantly to the observed effect. The function of ecto-ATPases is unknown, but there is no evidence that they have an ion pumping role. [19,20] A related difficulty with studies of ATP hydrolysis in SPM is the magnitude of ATP hydrolytic activity, much greater than that of Calcium2+ pumping. Although it is possible that hydrolytic activity specific to PMCA may be swamped by the presence of ecto-Calcium2+-ATPases, preliminary data from our lab indicate that a substantial fraction of SPM Calcium2+-dependent nucleotide hydrolysis may be PMCA specific, based on studies showing vanadate inhibition and calmodulin stimulation of one-third of the hydrolytic activity. Why, then, the excessive ratio of Pi release to Calcium2+ transport? One contributing factor may be artifact caused by vesicular leakiness, causing a degree, perhaps a large degree, of repumping of “lost” Calcium2+ back into the interior with associated excess ATP utilization. However, our work and the work of others, [21,22] who also find comparatively high levels of Pi release, indicates uncoupling of a large fraction of nucleotide hydrolytic activity from Calcium2+ transport. Transport requires an intact PMCA molecule properly situated in the plasma membrane, whereas hydrolytic action may proceed under less stringent requirements. Sola-Penna et al.  reported that the simple sugar, trehalose, inhibited PMCA pumping in kidney tubules and yet left PMCA hydrolytic activity unaffected. In a similar vein, the Calcium2+ transport/ATP hydrolysis coupling ratio of sarcoplasmic reticulum Calcium2+-ATPase was affected by the lipid composition of the membrane.  A polypeptide antibiotic, duramycin, that modifies phospholipid-protein interactions in sarcoplasmic reticulum vesicles inhibited ATP-dependent Calcium2+ uptake without altering ATP hydrolysis.  It is interesting that we did not note stable inhibition of Calcium2+-ATPase-dependent nucleotide hydrolysis in SPM obtained from anesthetized rats (Figure 3), in contrast to persistent inhibition of Calcium2+ pumping. This observation indicates differential inhalational anesthetic effects on the hydrolytic and pumping domains of PMCA, with stable anesthetic inhibition of the physiologically relevant and pharmacologically specific Calcium2+ transport moiety. Alternatively, failure of persistent inhibition of ATP hydrolysis may reflect the presence and reversible inhibition of other, nonpumping Calcium2+-ATPases.
We also investigated three other subcellular systems involved in ion transport with respect to the CAR model. Figure 4shows studies of smooth endoplasmic reticulum Calcium2+-ATPase (SERCA) in microsomes prepared from rat brain homogenates. This active intracellular Calcium2+ transport system did not appear to be affected by previous anesthetic treatment of the donor rat. We have previously shown that there is very little contamination of the SPM fraction itself with SERCA. . Figure 5indicates the effects of prior in vivo halothane treatment and concurrent in vitro treatment on the sodium-calcium exchanger in SPM (Figure 5(a and b) and on ATP hydrolysis by Sodium sup +-Potassium sup +-ATPase in SPM (Figure 5(c and d). No discernible inhibition with halothane exposure was noted in either circumstance.
An important question to be considered in the experimental design presented herein is the possibility that observed differences in PMCA activity in the treatment groups merely reflect differences in neuronal stimulation associated with decapitation, per se, of awake versus anesthetized rats. This possibility is made less likely by the observation that in vitro exposure of SPM from untreated rats to an equivalent concentration of halothane (1 MED) resulted in the same degree of PMCA inhibition, i.e., about 30%. To examine this question further, we conducted a series of experiments in which rats were killed by exposure to high concentrations (virtually 100%) of Helium, Nitrogen sub 2, or CO2. These results, summarized in Figure 6, demonstrate that death from exposure to Nitrogen2, Helium, or CO2did not significantly modify PMCA activity compared with that in animals decapitated while awake. Therefore, decapitation, with associated massive discharge from the severed cord, is an unlikely cause of nonspecific PMCA alteration.
These experiments do not rule out nonspecific responses to anoxia resulting from either decapitation or lethal gas exposure in the absence of “protective” general anesthesia. It is noteworthy that animals exposed to CO2appeared to pass through an anesthetic stage before death. Death occurred very quickly with no movement or struggle. Perhaps more important is the observation that none of the other enzyme and transport systems examined in the in vivo model followed the pattern expected if we were merely documenting a response, suppressed by anesthesia, to brain death by decapitation. Smooth endoplasmic reticulum Calcium2+-ATPase is a Calcium2+-dependent enzyme involved in neurotransmitter release, the sodium-calcium exchanger also participates in clearance of Calcium2+ from the cytosol, and Sodium sup +-Potassium sup +-ATPase is essential for the axonal transmission of neuronal impulses, yet none of these ion regulators was affected by the anesthetic state of the animal at the time of death (Figure 4and Figure 5). If the phenomenon we have documented reflects only nonspecific neuronal discharge, we would expect enzymes other than plasma membrane Calcium2+ ATPase to be affected. We conclude that observed differences in PMCA pumping activity among rats in the C, A, and R treatment groups were not confounded by alterations of the PMCA response associated with the method of killing, and that stable depression of PMCA pumping reflects a specific response to halothane.
Strong additional support for the biologic validity of stable depression of synaptic PMCA with halothane exposure comes from in vitro studies (Figure 7). Depression of PMCA pumping activity with a 30-min exposure to halothane persisted during another 60 min of measurement, despite discontinuation of halothane and subsequent elution of agent from the medium. It is noteworthy that anesthetic depression of Calcium sup 2+ dependent ATP hydrolysis is reversible, both in vivo (Figure 3) and in vitro (Franks et al., unpublished observations). These observations in an in vitro setting, in conjunction with our in vivo experiments, indicate that reversal of anesthetic depression of PMCA pump activity can occur only in an intact, living cell.
We have now shown that two enzyme systems in brain synaptic membranes, methyltransferase I and PMCA, are stably altered by prior anesthetic exposure of the donor animal. In contrast to PMCA, methyl transferase 1 activity is enhanced in animals killed while anesthetized.  Each of these systems is restored to normal only in intact rats, as evidenced by the state of enzyme activity in animals that have recovered from anesthesia. Any relationship between phospholipid methylation stimulation and PMCA inhibition is purely speculative at this juncture, but it is noteworthy that PMCA activity is very sensitive to the lipid environment in which it is situated.  One could even postulate that anesthetic-induced diversion of phosphatidylethanolamine to synthesis of phosphatidyl-N-monomethylethanolamine and, ultimately, phosphatidylcholine could occur only at the expense of diverting phosphatidylethanolamine from conversion to acid phospholipids, known to enhance PMCA activity. [2–5] Whether phospholipid methylation with associated PMCA inhibition relates to the anesthetic effect of inhalation agents remains an open question at this time, but one worth continued exploration.
Anesthetic inhibition of PMCA may also derive from a quite different series of molecular events. Inhalational anesthetics may affect one or more elements of select and specific intracellular signal transduction pathways, e.g., protein phosphorylation/dephosphorylation, a system that probably modulates one or more Calcium2+ regulators, including PMCA. Other states and factors that could be involved in anesthetic alteration of PMCA include, singly or in combination, enzyme oligomerization, Calcium2+ modulation through calmodulin, and direct Calcium2+ binding. Nearly all of these potential modulators are candidates for participation in “stable” PMCA inhibition after in vivo anesthetic exposure.
We recently obtained additional evidence for significant involvement of PMCA in the anesthetic response from an animal model of chronic PMCA depression, rats with streptozocin (STZ)-induced diabetes. We found post-STZ reduction of PMCA pumping activity in synaptic plasma membranes that was associated with 35% reduction in halothane MED and 12% reduction in xenon MEDmov(defined as loss of movement in response to pressure chamber motion).  Subsequent experiments have shown equivalent reductions in isoflurane and enflurane MED. In addition, the degree of MED reduction in individual rats correlated well with both PMCA inhibition and percent of glycated hemoglobin.
However affected by anesthetics, directly or indirectly, PMCA is a major regulator of intracellular Calcium2+ concentration and dynamics. Interference with this regulatory process in neurons may alter central neurotransmission,  probably a necessary condition for anesthesia. The studies described in this report support this thesis and offer new evidence that halothane causes PMCA inhibition that persists in its absence, that is reversed only in vivo, and that may affect PMCA function at molecular sites distinct from those involved in ATP hydrolysis.
The authors thank Melanie J. Surber, Vicki E. Janson, and Robert Catlin, for technical assistance with all experiments presented in this study.