Perturbation of neuronal calcium homeostasis may alter neurotransmission in the brain, a phenomenon postulated to characterize the anesthetic state. Because of the central role of plasma membrane Ca(2+)-ATPase (PMCA) in maintaining Ca2+ homeostasis, the authors examined the effect of several inhalational anesthetics on PMCA function in synaptic plasma membranes (SPM) prepared from rat brain.
Ca(2+)-ATPase pumping activity was assessed by measurement of ATP-dependent uptake of Ca2+ by SPM vesicles. ATPase hydrolytic activity was assessed by spectrophotometric measurement of inorganic phosphate (Pi) released from ATP. For studies of anesthetic effects on PMCA activity, Ca2+ uptake or Pi release was measured in SPM exposed to halothane, isoflurane, xenon, and nitrous oxide at partial pressures ranging from 0 to 1.6 MAC equivalents. Halothane and isoflurane exposures were carried out under a gassing hood. For xenon and nitrous oxide exposures, samples were incubated in a pressure chamber at total pressures sufficient to provide anesthetizing partial pressures for each agent.
Dose-related inhibition of Ca(2+)-ATPase pumping activity was observed in SPM exposed to increasing concentrations of halothane and isoflurane, confirmed by ANOVA and multiple comparison testing (P < 0.05). Concentrations of halothane and isoflurane equivalent to one minimum effective dose (MED) depressed PMCA pumping approximately 30%. Xenon and nitrous oxide also inhibited Ca2+ uptake by SPM vesicles. At partial pressures of these two gases equivalent to 1.3 MAC, PMCA was inhibited approximately 20%. Hydrolysis of ATP by SPM fractions was also inhibited in a dose-related fashion. An additive effect occurred when 1 vol% of halothane was added to xenon or nitrous oxide at partial pressures equivalent to 0-1.6 MAC for the latter two agents.
Plasma membranes Ca(2+)-ATPase is significantly inhibited, in a dose-related manner, by clinically relevant partial pressures of halothane, isoflurane, xenon, and nitrous oxide. Furthermore, these anesthetics inhibit PMCA activity in accordance with their known potencies, and an additive effect was observed. How inhalational anesthetics inhibit the PMCA pump is not known at this time. It is noteworthy that the only shared characteristic of this group of agents of widely different structure is anesthetic action. The relevance of this dual commonality, anesthetic action and PMCA inhibition, to actual production of the anesthetic state remains to be determined.
Key words: Anesthetics, gases: nitrous oxide; xenon. Anesthetics, volatile: halothane; isoflurane. Mechanism of anesthesia: plasma membrane Calcium2+-ATPase (PMCA); synaptic plasma membranes.
A low cytosolic concentration facilitates the action of free calcium as an intracellular regulator, and eukaryocytes, including neurons, maintain several classes of Calcium2+ transporting systems that control intracellular calcium concentration ([Calcium2+]). [1–3] A P-type pump, plasma membrane Calcium2+-ATPase (PMCA), plays a major role in maintaining low cytosolic [Calcium2+] by ejecting Calcium2+ from the cell. [1–3] Because general anesthesia may result from interference with information transfer at the synaptic level of brain organization and because signaling between nerve cells ensues with Calcium2+-dependent transmitter secretion by the distal axon, inhalational anesthetics may act by perturbing Calcium2+ flux and concentration within the nerve terminal.  These considerations have evoked interest in the PMCA response to anesthetics in neuronal and other cell membranes. Effects on Calcium2+ pumping and ATP hydrolysis by brain synaptic plasma membrane Calcium2+-ATPase and on ATP hydrolysis by purified erythrocytic Calcium2+-ATPase have been noted. [5–8] Inhibition by halothane, isoflurane, and enflurane of Calcium2+-ATPase isolated from erythrocytes or in red cell ghosts has recently been described in a definitive report.  We describe herein studies of the response of PMCA in isolated brain synaptic plasma membranes (SPM) exposed to several anesthetics of widely different structure. We have focused primarily on ion transport rather than ATP hydrolysis as a measure of anesthetic response. We report that halothane, isoflurane, xenon, and nitrous oxide inhibit PMCA pumping in a dose-related fashion at clinically relevant partial pressures.
Materials and Methods
Preparation of Synaptic Plasma Membranes
All experimental protocols were approved by the Animal Care Committee of Vanderbilt University. Male Sprague-Dawley rats (243–338 g) were allowed food and water ad libitum until the morning of the experiment. Animals were killed by decapitation. Whole brains were dissected on ice, and brain areas (cerebrum, cerebellum, midbrain, and medulla) were weighed and pooled in ice-cold 0.32 M sucrose (pH 7.4). Brain fractions were pooled from 3 to 12 rats, as determined by the amount of synaptic plasma membrane (SPM) required for each experiment. Synaptosomes were prepared by gradient ultracentrifugation.  Synaptic plasma membranes were prepared by osmotic shock of synaptosomes followed by differential ultra-centrifugation 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 activity. Assays of Calcium2+-dependent ATP hydrolysis were done either immediately or within a few days. Protein content in SPM pellets was estimated by the Bradford method.
Measurement of Calcium sup 2+ Pumping Across Synaptic Plasma Membranes
Calcium2+ uptake by everted rat SPM vesicles, i.e., transport from the cytosolic to the plasmic surface, was performed as described by Moore et al. with several modifications.  The incubation mixture (total volume 4 ml) consisted 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/mg). The reaction was started by adding 70-micro gram aliquots of SPM protein to each tube. Temperature was maintained at 37 degrees Celsius. Aliquots of 0.5 ml were removed after 5, 10, 20, 30, and 60 min. For assays of PMCA pumping under hyperbaric conditions, incubation tubes were placed in a Parr Cell Disruption Bomb (PCDB; Parr Instrument Corp., Moline, IL) as described below and incubated for 30 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 0.25M KCl and 10 ml of deionized water. After vesicle collection, the filters were washed with 2 ml of 0.25M 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.
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. [12,13] Synaptic plasma membrane aliquots (2 micro gram/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. For assays of ATP hydrolysis under hyper-baric conditions, incubation tubes were placed in a PCDB. 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 30 min.
Evaluation of Synaptic Plasma Membrane Preparation
Samples of the crude synaptosomal fraction (P2), synaptosomes, and synaptic plasma membranes were fixed in 2% glutaraldehyde and evaluated by electron microscopy. .
Purification of SPM was assessed by measuring the specific activity of plasma membrane enzyme markers in the initial brain homogenates and in subsequent fractions obtained during separation. Enzyme markers included acetylcholinesterase, alkaline phosphatase (Sigma Diagnostic Kit 245, St. Louis, MO), gamma-glutamyl-transpeptidase (Sigma Diagnostic Kit 419), and 5′-nucleotidase. [14–17].
The degree of contaminating mitochondria or endoplasmic reticulum in SPM preparations was assessed by adding 5 mM sodium azide or up to 15 micro gram/ml of oligomycin to the incubation medium to inhibit mitochondrial or, alternatively, by preincubating SPM with 50–300 nM thapsigargin to inhibit microsomal uptake of Calcium2+. .
Orientation of Vesicles.
Inside/outside orientation of SPM vesicles was examined by a method  using specific cleavage by trypsin of Sodium sup +-Potassium sup +-ATPase from the cytoplasmic surface of everted vesicles. Remaining oubain-inhibitable ATPase activity was measured in membranes from trypsin-treated vesicles and compared with activity in membranes from untreated vesicles. Inside surfaces were exposed by rupture of vesicles with sodium dodecylsulfate before enzymatic assay.
Specificity of Calcium sup 2+ Transport.
We used several criteria to demonstrate that active Calcium sup 2+ transport in our SPM preparation shared properties consistent with those known for PMCA from other sources. The PMCA pump is substrate (ATP) specific, requires Magnesium2+, and is inhibited by orthovanadate.  Nucleotide specificity was examined by comparing 5 mM ATP, ADP, CTP, UDP, or no nucleotide substrate in parallel assays of Calcium2+ uptake. In another series of experiments, Calcium2+ uptake was measured in the presence of 0.005–1 mM sodium orthovanadate. Requirement for Magnesium2+ was also examined. To evaluate the relationship of Calcium2+ uptake to its concentration, experiments were carried out with medium Calcium2+ concentration varying from 0.1 to 1000 micro Meter.
Synaptic plasma membrane incubation mixtures were exposed to various partial pressures of halothane, isoflurane, xenon, or nitrous oxide as Calcium2+ uptake (transport) or ATP hydrolytic reactions proceeded. For studies of the effects of potent anesthetics, incubation vials were placed in a Dubnoff shaker under a gassing hood and shaken gently at 37 degrees Celsius, as described previously.  Halothane (Halocarbon Labs, River Edge, NJ) or isoflurant (Abbott Laboratories, North Chicago, IL), in a warmed, humidified air/oxygen mixture (FlO2 = 0.3), was delivered under the hood in the desired concentrations from a dedicated, calibrated vaporizer. Delivered concentrations of potent anesthetics were always confirmed by gas chromatography. For experiments with xenon or nitrous oxide, SPM incubation mixtures were exposed to anesthetic gases in a PCDB placed in a water bath at 37 degrees Celsius and used as a pressure chamber. The PCDB was modified by placement of a low-pressure gauge, 0–50 psi, on the lid. The PCDB was flushed for 2 min at 6 l/min either with helium alone (A-L Compressed Gases Inc., Nashville, TN) or, for interaction studies, with helium mixed with 1 vol% halothane before and after placement of the incubation tubes in the PCDB. These tubes remained in a 100-ml beaker filled with water at 37 degrees Celsius throughout the incubation period; constancy of water temperature was confirmed by measurement after incubation was completed. Xenon (Research Grade from Alphagas; Morrisville, PA) or nitrous oxide (A-L Compressed Gases Inc.) was then added to the PCDB to achieve a partial pressure based on the MAC of each anesthetic gas. Finally, supplemental helium was added as needed to insure constant total pressure despite varying xenon or nitrous oxide partial pressures. After 30 min of exposure, pressure in the PCDB was released slowly to avoid disruption of SPM vesicles, and the degree of Calcium2+ uptake or ATP hydrolysis was assayed as described above. Minimum alveolar concentration equivalents of 0, 1, and 1.6 were used for both xenon (0, 0.95, and 1.55 atm) and nitrous oxide (0, 1.55, and 2.46 atm). These delivered partial pressures were based on reported MAC values of 0.95 atmospheres or 14 psi for xenon in the mouse  and 1.54 atm or 23 psi for nitrous oxide in the rat.  The total pressure (above ambient) to which SPM incubations were exposed was 1.54 atm for xenon and 2.46 atm for nitrous oxide.
Data were examined by multifactorial ANOVA, multiple comparison (Student-Newman-Keuls procedure), linear regression, and, when appropriate, t tests. Statistical significance was inferred if P < 0.05.
Synaptic Plasma Membrane Characterization
Evidence for Isolation of a Synaptic Plasma Membrane-Rich Fraction.
Synaptic plasma membrane enrichment of successive subcellular fractions isolated during the separation process was indicated by increases in specific activity in the SPM fraction of four plasma membrane-associated enzymes (Table 1). Electron microscopy confirmed these biochemical findings, with results comparable with Cotman's. .
Calcium2+ uptake can be measured only in everted SPM vesicles. In experiments based on the method of Marin et al.,  we found that the total activity of available Sodium sup +-Pottasium sup +-ATPase in disrupted vesicles was 25.74 plus/minus 0.34 micro moles Pi *symbol* mg protein sup -1 *symbol* 20 min1, n = 3. When SPMs were treated with trypsin and then disrupted, Sodium sup +-Potassium4-ATPase activity was decreased by approximately 44% to 11.36 plus/minus 0.23 micro moles Pi *symbol* mg protein1*symbol* 20 min1, indicating that about one-half of the SPM vesicles were everted. Freezing and thawing of vesicles may reduce the number of intact inside-out vesicles, because PMCA pumping activity in preparations thawed after 1 day of storage was decreased by about 30%. Therefore, only freshly prepared SPM were used for PMCA pumping measurements. No changes in ATP hydrolytic activity were observed after freezing and thawing.
Specificity of Calcium sup 2+-ATPase in the Synaptic Plasma Membrane Fraction
(Figure 1(a)) shows Calcium2+ uptake by SPM vesicles plotted against incubation time in the presence of several Calcium2+-ATPase substrates. It is evident from the regression slopes that pumping is effective only with ATP as the nucleotide substrate. In other experiments (not shown) uptake did not occur in the absence of ATP or Magnesium2+. In addition to a requirement for ATP and Magnesium2+, inhibition by low concentrations of orthovanadate is characteristic and specific for P-type pumps, such as PMCA. Figure 1(b) shows Calcium sup 2+ uptake with varying amounts of orthovanadate added to the medium. The inhibitory constant, Ki, (concentration of orthovanadate producing 50% inhibition of Calcium2+ uptake) was calculated for each of several incubation times, with Ki5min > 50, Ki10min> 50, Ki sub 20min = 50, Ki30min= 41, and Ki60min= 16 micro Meter. Therefore, only data derived from 20-, 30-, and 60-min aliquots of incubation medium were used for determination of Calcium2+ uptake, thereby maximizing the contribution of P-type pump activity (e.g., PMCA) to the assay. Preincubation of SPM with thapsigargin (50300 nM), an inhibitor of smooth endoplasmic reticulum Calcium2+-ATPase (SERCA), did not alter SPM Calcium2+ uptake, indicating minimal contamination by this intracellular calcium pump.  Some contamination of the SPM preparation with mitochondria was indicated by experiments showing a 15–30% reduction of uptake with addition of azide (5 mM) or oligomycin (5 micro gram/ml), but one or the other of these inhibitors of mitochondrial transport of Calcium2+ was always added to the incubation medium in studies of the anesthetic response. Synaptic plasma membrane uptake of Calcium2+ varies with the concentration of this ion in the medium. Studies using concentrations ranging from 0.1 to 1000 micro Meter indicated that anesthetic effects could best be demonstrated between 10 and 50 micro Meter Calcium2+ in the medium.
Anesthetic Effects on PMCA Pump Activity
Halothane and Isoflurane.
Data summarized in Figure 2illustrate the effects of increasing concentrations of halothane (Figure 2(a)) or isoflurane (Figure 2(b)) on PMCA pumping activity in SPM from the cerebrum. Each treatment group was comprised of three independent experiments in which membranes pooled from three to six rats were exposed, in replicates of six, to six different concentrations of halothane or isoflurane. Mcan values of Calcium2+ uptake from each experiment are shown for each anesthetic concentration, with error bars indicating 95% confidence limits from multifactor ANOVA. Multiple comparison testing confirmed dose effect. Significant differences (P < 0.05) in Calcium2+ uptake were noted among the anesthetic concentrations indicated in Figure 2. Orthovanadate markedly suppressed Calcium2+ uptake by SPM vesicles, with no further inhibition by halothane at concentrations ranging from 0.5 to 1.5 vol%. Of particular interest, illustrated in Figure 2, are the effects on PMCA of exposure to concentrations approximately equivalent to 1 minimum effective dose (MED) of either halothane or isoflurane. One MED, determined in intact rats,  is the lowest deliverable anesthetic concentration that suppresses movement in response to tail clamping. Concentrations of halothane (1.5%) and isoflurane (1.9%), approximating 1 MED in intact animals, produced PMCA inhibition in vitro of similar degree, 29.2% and 31.7%, respectively.
Xenon and Nitrous Oxide.
(Figure 3(a)) shows the effect of xenon on PMCA pumping in SPM isolated from four different rat brain areas. Cerebra, midbrains, cerebella, and medullae were obtained from 12 rats and pooled for SPM preparation in five separate experiments. Incubation mixtures were exposed for 30 min to either helium or to helium plus xenon at a partial pressure equivalent to 1.3 MAC. Mean values for Calcium2+ uptake for SPM from each brain fraction are depicted, with error bars indicating 95% confidence limits. Xenon depressed PMCA pumping activity significantly (P < 0.05) in all brain fractions except the medulla, and to approximately the same degree (20–25%). Nitrous oxide also affected Calcium2+ transport (Figure 3(b)). In experiments with SPM prepared from cerebra pooled from three rats, Calcium2+ uptake was depressed 21%(P < 0.05 ) by a partial pressure of nitrous oxide equivalent to 1.3 MAC.
Anesthetic Effects on Calcium sup 2+-Dependent ATPase Hydrolytic Activity
Another measure of Calcium2+-ATPase activity, one that also has been used to assess PMCA function, is release of inorganic phosphate (Pi) by hydrolysis of the nucleotide substrate. All of the anesthetic agents we have examined inhibit ATP hydrolysis in SPM. Figure 4illustrates additive and dose-related anesthetic inhibition of hydrolysis in membranes isolated from pooled, cerebral homogenates. One treatment group was comprised of four experiments in which membranes pooled from three to six rats were exposed for 30 min, in replicates of four, to 0, 1, or 1.6 MAC equivalents of nitrous oxide with and without 1 vol% of halothane. In the second treatment group, xenon was substituted for nitrous oxide. Mean values of ATP hydrolysis from all experiments are plotted against gaseous anesthetic (nitrous oxide or xenon) concentrations, with error bars indicating 95% confidence limits from ANOVA. Multiple comparison testing confirmed a dose-related response. Significant differences (P < 0.05) in ATP hydrolysis were noted, with a single exception, among different MAC equivalent values for nitrous oxide and for xenon. When 1 vol% of halothane was added to the gaseous anesthetics, further inhibition of ATP hydrolysis was noted in both treatment groups at all gaseous anesthetic MAC values, and significant differences were maintained among these MAC equivalents. Figure 5shows that, although halothane significantly depressed Calcium2+-dependent ATP hydrolysis, halothane inhibition persisted in the presence of the specific PMCA inhibitor, orthovanadate.
Studies of anesthetic effects on plasma membrane Calcium2+-ATPase in brain synaptic plasma membranes depend on reproducible isolation of a subcellular fraction rich in pump-containing membranes. We examined our SPM preparation for increases in specific activity of plasma membrane-associated enzymes and by electron microscopy, with results indicating synaptic plasma membrane concentration in the fraction. A test of SPM vesicle orientation indicated a sufficient number of everted vesicles for determination of PMCA pump activity, and confirmed the common assumption that about one-half of prepared vesicles are everted. Presence of a specific P-type calcium pump in the SPM fraction was further supported by a variety of identifying characteristics: requirement for ATP as substrate, magnesium dependence, and orthovanadate sensitivity. Synaptic plasma membrane fraction contaminants did not appear to distort our measures of Calcium2+ uptake. Mitochondrial uptake was minimized by including azide or oligomycin in the incubation mixture, and SERCA contribution to uptake was essentially zero as indicated by no change in uptake when the inhibitor thapsigargin was added.
These studies evaluating the selectivity of plasma membrane preparation and PMCA specificity indicate that it is unlikely that the inhibitory action of anesthetics is produced by interference with Calcium2+ transport independent of PMCA pumping. However, one other possibility must be considered. Plasma membranes contain a second, independent system for ejecting Calcium2+ from cytosol, the sodium-calcium exchanger. Several considerations make exchanger participation unlikely: low intravesicular Sodium sup + concentration, minimal uptake of Calcium2+ by SPM vesicles in the absence of ATP, and inhibition of Calcium2+ uptake by orthovanadate. Continued Calcium2+ accumulation for 60 min also argues against an exchanger effect. Uptake from this high-capacity system occurs rapidly, usually within 10 min. Finally, we recently showed that Calcium2+ uptake into SPM vesicles is unaffected by halothane under conditions designed to maximize sodium-calcium exchange. .
One feature of PMCA pumping in membranes exposed or not exposed to anesthetics is reproducibility. Studies reported herein have been carried out over several years and with different lots of experimental animals, yet results have proved consistent, as indicated in Figure 2. Inhibition of PMCA occurred within a clinically useful range of anesthetic concentrations, and in a clearly dose-related fashion. Both halothane and isoflurane caused about a 30% inhibition of pumping activity at partial pressures very near 1 MED for each anesthetic. We define MED as the lowest deliverable anesthetic dose that suppresses a reaction to a painful stimulus; these levels are highly reproducible in individual rats and are clustered closely in groups of rats. We have reported MED values of 1.43 vol%(approximately 1.3 MAC) for halothane and 1.9 vol%(approximately 1.4 MAC) for isoflurane. [21,24] Calcium2+ pumping with exposure to 1.3 MAC equivalents of xenon and nitrous oxide was decreased about 20%(Figure 3(a and b)). This degree of depression did not differ greatly from that obtained with halothane and isoflurane; indeed, exposure levels were chosen categorically rather than functionally, and may be less than the true MED values for these two gaseous anesthetics. The estimate for xenon is particularly questionable, because the only published MAC value (0.95 atm) for rodents is derived from mouse studies based on suppression of the righting reflex rather than production of the insensate state.  Xenon depression of PMCA activity was observed in SPM prepared from pooled cerebra, mid-brains, and cerebella, but not medullae. Except for the medullary fraction, no significant differences in the degree of depression were found among the different brain areas. Regional differences in PMCA response, if they exist, were not identified by our relatively crude dissection procedure.
We also looked at anesthetic effects on calcium-dependent ATPase hydrolytic activity in SPM preparations. Again, we found dose-related inhibition of Calcium2+-ATPase activity and an additive effect with mixtures of 1% halothane and either xenon or nitrous oxide (Figure 4). It is noteworthy that most reports of anesthetic or related drug effects on PMCA rely on ATP hydrolysis as a measure of enzyme activity, e.g., in erythrocytic [7,9] and brain synaptic membranes. [25,26] Our findings with the hydrolytic method are certainly consistent with these reports. However, experiments illustrated in Figure 5indicate that inhibition of release of inorganic phosphorous is a consequence of anesthetic effects not only on the P-type pump, PMCA, but also on calcium-dependent enzyme(s) that are not inhibited by orthovanadate. Thus, ATP hydrolysis, at least in neural membranes, may be only partially associated with Calcium2+ pumping by PMCA. In recent years, there has been considerable interest in the finding that there are calcium-dependent ATPases on the outer surface of plasma membranes. [27,28] These ecto-ATPases appear to be of several varieties. Their function is unknown, but there is no evidence that they have an ion-pumping role. Ecto-ATPases can use substrates other than ATP, and they are not inhibited by 0.01 mM orthovanadate. Anesthetic depression of calcium-dependent ATPase(s) other than PMCA is of interest. As noted above and in an additional report,  we found that anesthetics do not depress Sodium sup +-Potassium sup +-ATPase, SERCA, or sodium-calcium exchanger activity in SPM. On the other hand, if anesthetics inhibit ecto-ATPases as well as PMCA, it is important to note this in the literature in view of the wide reliance on an assay that may not be specific for PMCA. There is a possibility that orthovanadate primarily inhibits PMCA pumping and that substrate hydrolysis by PMCA remains partially intact in its presence. This phenomemon must be demonstrated before substrate hydrolysis can truly be relied on as a measure of PMCA activity. Such evidence would be welcome in view of the labor intensity of pumping assays, with their requirement for fresh SPM. Although assays of nucleotide hydrolysis probably provide useful approximations, we have placed primary reliance on measurement of Calcium2+ transport across SPM.
One interesting feature of the response of synaptic PMCA to inhalational anesthetics is persistence of pump inhibition after the agent has been eluted from the membrane. Synaptic plasma membrane prepared from rats anesthetized with halothane demonstrated significant Calcium2+ pump depression, compared with SPM from animals allowed to recover from anesthesia before decapitation. Similarly, SPM prepared from untreated rats and then exposed to halothane in vitro showed continued depression of pump activity after removal of halothane. These observations, described in detail elsewhere,  indicate that reversal of anesthetic inhibition of synaptic PMCA pumping can occur only in the intact, living cell.
We have found that a wide variety of inhalational anesthetics depress brain synaptic plasma membrane PMCA pumping, and in a dose-related fashion. Calcium2+ ATPase-dependent nucleotide hydrolysis is also depressed. Agents effecting these changes include the elemental noble gas, xenon; a simple inorganic compound, nitrous oxide; and two widely used fluorinated organic compounds, halothane and isoflurane. How these anesthetics inhibit the pump is unknown at this time, but it is noteworthy that their only shared characteristic is anesthetic action. The relevance of this commonality to actual production of the anesthetic state remains to be determined. As always when looking at physiologic processes altered by anesthetics, the question of nonspecific, even toxic, side effects unrelated to the anesthesia arises. Two considerations make this implausible in this group of experiments. First, PMCA inhibition occurs at agent partial pressures within pharmacologic ranges, it begins at low pressures, and it increases as anesthetizing pressures are attained. Many side effects occur only at high anesthetic concentrations. Second, it appears highly unlikely that PMCA inhibition is a nonspecific toxic effect of fluorinated anesthetics when it is so clearly reproduced with exposure of membranes to xenon, an agent with few pharmacologic effects other than the production of anesthesia. If an unrelated side effect can be excluded, another possibility is that PMCA serves as a reporter molecule, i.e., PMCA inhibition is an epiphenomenon, indicating, but not participating in, the anesthetic state. Alternatively, PMCA inhibition may follow, as a secondary corrective response, perturbation of cytosolic Calcium2+ dynamics that results from effects on otherwise unrelated processes more fundamental to the production of anesthesia. However, the observation of Kosk-Kosicka et al.  on the direct, inhibiting effects of potent, fluorinated anesthetics on purified erythrocytic Calcium2+-ATPase, in concert with our findings in neural membranes, lends credence to the possibility that PMCA itself, as a fine tuner of [Calcium2+], may play a fundamental role in the processes that lead to the anesthetic state.
The link between rapid shifts in intracellular ([Calcium2+]) and intercellular signaling has been recognized for many years.  Homeostatic mechanisms controlling intracellular [Calcium2+] and dynamics are complex, and regulation is linked with cytosolic ion compartmentalization to a degree that is only now becoming evident.  Despite its relatively low transport capacity compared with other cellular Calcium2+ regulators. PMCA may be strategically located within the cell so as to alter [Calcium2+] in critical submembranal areas, perhaps especially in synapses. PMCA effects on Calcium2+ decay curves and on [Calcium2+] oscillatory dynamics may predominate, with profound effects emerging consequent to anesthetic inhibition.  Benham et al. have provided particularly important information on the effect of PMCA on Calcium2+ decay curves with cultured rat dorsal root ganglion sensory neurons.  By a combination of microfluorimetric and patch-clamp techniques, they showed that the PMCA is significantly more important than either sodium-calcium exchange or caffeine-sensitive intracellular storage for the removal of cytosolic Calcium2+ loads generated by actional potentials. It is apparent that the complexity of intracellular Calcium2+ dynamics and regulation confounds the construction of a simple model explaining these global effects of PMCA inhibition. Further complexity may derive from the presence in the brain, unlike the erythrocyte, of several nonhomogeneously distributed PMCA isoforms and, perhaps, splice variants that vary in their regulatory arm structure and, possibly, in their anesthetic response. A relatively crude look at differential PMCA responses in several brain areas (Figure 3) revealed no apparent differences. A precise analysis of the question presents a formidable task, but may offer important new information. It seems likely that other Calcium2+ regulators, such as the intracellular calcium release channels (ICRC), may also be affected, and these too offer the potential of varying anesthetic susceptibility among nonhomogeneously distributed isoforms.  Perhaps most intriguing is the possibility that anesthetics alter neuronal Calcium2+ homeostasis by direct effects on specific protein kinases, phosphatases, or both, which, in turn, serve as common modulators of several controllers of cytosolic [Calcium2+], including PMCA and ICRC.  Defining anesthetic effects on one or more of these Calcium2+ regulatory systems has great potential for furthering our understanding of how anesthetics alter central neurotransmission.
The authors thank Dr. William Whetsell, Jr., for help with the evaluation of their synaptic plasma membrane preparation by electron microscopy; Melanie J. Surber, Vicki E. Janson, and Robert Catlin, for technical assistance with all experiments presented in this study; Dr. B. V. Rama Sastry for assistance in the course of this project; and Eileen Gifford, for secretarial assistance.