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Nitrous oxide and xenon enhance phospholipid-N-methylation in rat brain synaptic plasma membranes
Life Sciences, Vol. 56, No. 25 pp. PL 455-460,1995 Copyright Q 1995 E!&evierScience Ltd Printed in the USA. All rights resewed CKm-3205/95 $950 + .oo
Pergamon
00243205(95)00220-O
PHARMACOLOGY LETTERS Accelerated Communication
NITROUS
OXIDE AND XENON ENHANCE PHOSPHOLIPID-N-METHYLATION RAT BRAIN SYNAPTIC PLASMA MEMBRANES
IN
J-L. Horn, PK. Janicki, J.J. Franks Department
of Anesthesiology,
(Submitted
Vanderbilt
University USA.
Medical Center, Nashville,
Tennessee,
December 19, 1994; accepted February 17, 1995; received in final form March 20, 19%)
ABSTRACT. Halothane and isoflurane increase the rate of phospholipid methylation (PLM) in rat brain synaptosomal membranes, a process linked to the coupling of neuronal excitation to neurotransmitter release. In contrast, synaptic plasma membrane (SPM) Ca*’ ATPase (PMCA) pumping is reduced by exposure to halothane, isoflurane, xenon and nitrous oxide (N,O). To examine further the relationship between PLM, PMCA and anesthetic action, we investigated the effect of clinically relevant concentrations of two less potent anesthetic gases, N,O and xenon, on PLM in SPM. Biochemical assays were performed on SPM exposed to 1.3 MAC of N,O (2 atm), 1.3 MAC of xenon (1.23 atm) or an equivalent pressure of helium for control. N,O or xenon exposure increased PLM to 115% or 113%, respectively, of helium control (~~0.02). Similar exposures to N,O or xenon depressed PMCA activity to 78% and 85% of control (~~0.05). Observations that PLM and PMCA are both altered by a wide variety of inhalation anesthetic agents at clinically relevant partial pressures lend support to a possible involvement and interaction of these orocesses in anesthetic action.
Key Words:
nitrous oxide, xenon, phospholipid methylation, plasma membrane Ca2+-ATPase
Phospholipid methylation (PLM), a ubiquitous process occurring in cell membranes, utilizes two enzymes, phospholipid-N-methyltransferases I and II (PLMT I and II), which successively methylate phosphatidylethanolamine (PE) to phosphatidyl-N-methylethanolamine (PME), phosphatidyl-N-dimethylethanolamine (PDE) and phosphatidyl-choline (PC). PLMT I, located on the cytoplasmic surface, drives the first, rate-limiting methylation step, producing PME. Associated with successive methylation is rapid translocation of the methylated product from the inner to the outer surface of the plasma membrane. Hirata and Axelrod have described the interaction between biological signal transmission and membrane PLM (1,2). They provided evidence for involvement of PLM in the transduction of receptor-mediated signals through cell membranes (1). PLMT I and II activities have been identified in rat brain synaptosomal membranes (3). Franks et al demonstrated that transmethylation of synaptosomal membrane phospholipids in rat brain was increased by either in vitro or prior in vivo exposure to two inhalation anesthetics (IA), halothane and isoflurane (4). Franks et al recently reported that plasma membrane Ca’+-ATPase (PMCA) pumping was reduced in brain synaptic plasma membranes (SPM) of Sprague Dawley rats by Correspondence: Dr. J.J. Franks, Dept. of Anesthesiology, Nashville, TN 37232-2125, USA.
Vanderbilt
University
Medical Center,
PL-456
Anesthetics and Phospholipid Methylation
Vol. 56, No. 25, 1995
exposure to a wide range of inhalation anesthetics (5,6). Though a mechanistic relationship between stimulation of PLMT I and inhibition of PMCA in the anesthetic response is speculative at the present, the possibility of an interaction would be bolstered by demonstrating that IA of widely differing structure affect both processes. We have therefore examined the effect of xenon and N,O on PLM in cerebral synaptic plasma membranes, comparing that effect with the PMCA response to N,O and xenon exposure (5). Materials and methods Animals. All experimental protocols were approved by the Animal Care Committee of Vanderbilt University. Male Sprague-Dawley rats (240-325 grams) were allowed food and water ad libitum. The number of animals was determine by the amount of membrane required for each experiment. One gram of cerebral tissue provided about 1 mg of SPM. For N,O experiments 18 rats were pooled in 6 groups of 3 and assayed in duplicate. The same membrane preparation was used for PLM and PMCA assays, the latter result reported previously (5). For xenon experiments, PLM was assayed in SPM from a group of 12 rats, pooled by 2 in duplicate. Xenon effects on PMCA were examined in 70 rats pooled by 10. This large number of rats reflects the use of Animals were sacrificed by decapitation. Brains were membrane aliquots for other experiments. dissected on ice, and synaptosomes were isolated by ultracentrifugation (7,5). Synaptosomes were osmotically shocked and placed on a sucrose gradient for further purification. Final pellets were suspended in isosmotic sucrose (0.32 M, pH 7.4) and used immediately for assay of PMCA Ca2’ pumping activity. Membrane aliquots were frozen for PLM assays at a later date. Protein content in SPM suspensions was estimated by the Bradford method. Delivery of anesthesia. SPM were expose to N,O and xenon in a Parr Cell Disruption Bomb (PCDB; Parr Instrument Corp., Moline, IL) placed in a water bath at 37 “C and used as a pressure chamber. The PCDB was modified by placement of a low-pressure gauge, O-3 atm, on the lid. The PCDB was flushed for 3 min at 6 l/mitt either with helium for control (A-L Compressed Gases Inc., Nashville, TN) or with N,O (A-L Compressed Gases Inc.) or xenon (Research Grade from Alphagas; Morrisville, PA), after placement of the incubation tubes in a 100 ml beaker filled with water at 37 “C. (Temperature was confirmed by measurement before and after incubation was completed.) N,O or xenon was then added to the PCDB to achieve a partial pressure of 1.3 of the reported minimum alveolar concentration (MAC). Two atm of N,O (8) or 1.23 atm of xenon (9) were selected. Helium was used as control gas at the same pressure used for each agent studied. Biochemical analysis. PLMT I activity was assessed in duplicate by the incorporation of tritiated methyl groups from (methyl-3H) SAM into PME (3,4,10). After incubation, the phospholipids were extracted, Phospholipids were separated by thin layer washed and concentrated in aliquots of chloroform. chromatography (TLC). Fractions were identified by comparison with standards, scraped into scintillation vials and counted. Results were expressed as femtomoles of SAM incorporated into PME per milligram of protein per minute. Ca*’ uptake by everted rat SPM incubation mixture was maintained at protein to each tube. Aliquots of 0.5 Filters were washed, nitrate filters.
vesicles was performed as described previously (11,5). The 37 “C. The reaction was started by adding 70 ug of SPM ml were removed after 30 min and collected on cellulose dried and placed in scintillation vials, and activity was
PL-457
Anesthetics and Phospholipid Methylation
Vol. 56, No. 25, 1995
measured in a beta counter. Results were expressed as nmoles of Ca2’ accumulated of SPM protein per minute of incubation time.
per milligram
Data were examined by multifactorial ANOVA, t-tests when Statistical analysis. appropriate, and multiple comparison (Student-Newman-Keuls procedure). Significance was inferred if ~~0.05. Results Fig. 1 illustrates the effects of 1.3 MAC-E (minimum alveolar concentration equivalent) of N,O and xenon (2 and 1.23 atmospheres respectively) on the first, rate limiting step of phospholipid methylation, the incorporation of methyl-3H groups from SAM into PE to form PME. Femtomoles of methyl-3H groups incorporated into PME per milligram of protein per min was significantly increased to 115% of control by N,O and to 113% of control by xenon. Results are normalized in Fig. 1 by expressing the value obtained for each agent as a fraction of the helium control (taken as unity). Error bars indicate SEM. Differences between control and agent were significant, pcO.02.
1.30
,,................... * .._..,._.....,,,,........................,.,....
-I-
It
* *
0.80 Helium
W
Xenon
Fig. 1 Normalized PLM activity in SPM prepared from cerebra of male Sprague-Dawley rats and exposed to helium, N,O or xenon (1.3 MAC). Aliquots from 6 preparations of SPM were exposed in duplicate in a PCDB to 2 atm of N,O or 1.23 atm of xenon for 30 min at 37 “C. Control membranes were exposed to helium at 2 or 1.23 atm under the same conditions. * denotes values significantly different from control (~~0.02). Fig. 2 shows, for comparison, normalized results of N,O and xenon effects on PMCA in rat cerebral SPM, taken from previously published data (5) for nitrous oxide. As previously noted, N,O decreased PMCA pumping to 78%. Xenon decreased pumping to 85% of helium control values (pcO.05).
PL-458
Vol. 56, No. 25, 1995
Anesthetics and Phospholipid Methylation
1.10
.,..,,,.,,..............................
.
I
..
. . . . ..
. .
* .,,.......................... * 0.80
*
‘I
0.70
0.60
0.50 Helium
1I N,O
-
Xenon
Fig. 2 Normalized PMCA activity in SPM prepared from cerebra of male SpragueDawley rats and exposed to helium, N,O or xenon (1.3 MAC). Aliquots from 6 preparations of cerebral SPM were exposed in duplicate in a PCDB at 37°C to 2 atm of N,O, and aliquots from 7 preparations of cerebral SPM were exposed under the same condition to 1.23 atm of xenon for 30 min. Control membranes were exposed to helium at 2 or 1.23 atm under the same conditions. * denotes values significantly different from control (~~0.05). Absolute values for PMCA pumping activity, as nmoles of 4sCa2’incorporated into everted SPM vesicles per mg per minutes and PLM activity as fmoles of methyl-3H group incorporated by PLMT I into PE to form PME, are shown in table 1. TABLE I PMCA and PLMT I activity expressed per mg of SPM vesicle protein per minute Control for Xenon Xenon Control for N,O N,O 0.66kO.03 0.56+0.01* 1.11~0.02 0.8720.03* PMCA (nmles) 48.05kO.48 54.26k2.01’ 41.43+1.75# PLMT I (fmoles) 35.94Zk1.64 * denote ~~0.05 compared to their control. # denote ~~0.02 compared to their control.
Discussion We have previously shown that phospholipid-N-methylation (PLM) in synaptosomes is significantly enhanced with exposure to halothane and isoflurane with both in vitro and prior in vivo exposure (4). Phospholipid-N-methyltransferase I (PLMT I) is the rate limiting enzyme of PLM, and assay conditions in our original study and in the current study were designed to selectively optimize PLMT I activity. We have also observed that inhalation anesthetics of varying structure, i.e., halothane, isoflurane, xenon, and N,O, inhibit plasma membrane calcium ATPase (PMCA) activity (5,6). The relationship between enhancement of PLMT I and inhibition of PMCA activity by anesthetics is purely speculative at this juncture, as discussed previously
Vol. 56, No. 2.5,1995
Anesthetics and Phospholipid Methylation
PL459
(6). It is noteworthy that PMCA activity is very sensitive to the lipid environment in which it It is possible that anesthetic-induced diversion of phosphatidylethanolamine to is situated. synthesis of phosphatidyl-N-methylethanolamine and ultimately phosphatidylcholine diverts phosphatidylethanolamine from conversion to phosphatidylserine, an acid-phospholipid known to enhance PMCA activity. If a mechanistic relationship does exist between these two processes, it is important to establish that each process is affected by the same wide range of inhalation anesthetics of varying structure. In this study, we examined the effects of N,O and xenon on PLM in cerebral synaptic plasma membranes. We found significant enhancement, 13 and 15%, respectively, of methyl incorporation into PME. This degree of enhancement of PLM by N,O and xenon is considerably less than that found in SPM with exposure to the potent anesthetics, halothane and isoflurane. This discrepancy may reflect that the MAC values for N,O and xenon which we based our studies on are lower than the true values for the rat. Various values for N,O have been reported in the literature (8,12), and xenon MAC has been reported only for the mouse (9). Furthermore, our estimates of the minimum effective dose (MED) of xenon and N,O, which we have defined as the lowest partial pressure of delivered anesthetic that blocks a response to pain (4,6), were perhaps also underestimated by lack of access to rats in our pressure chamber. N,O and xenon MED as measured by the righting reflex and response to pressure chamber motion may be less than the MED derived from ablation of a painful stimulus. It is also possible that the large partial pressures of N,O and xenon required to produce anesthesia cause physical as well as biochemical changes in the synaptic plasma membrane and thus on PMCA activity and the anesthetic response. However, we believe it is unlikely that the large difference in PLM effects between gaseous and volatile anesthetics can be accounted for by these several possible factors. We have already noted striking differences in PMCA activity in some but not all brain areas of diabetic rats, compared with normoglycemic controls (13). Similar anatomic differences may exist with respect to PLMT I activity, and PLM enhancement by gaseous and volatile anesthetics may be more equivalent in as yet undefined brain areas that are critical for anesthetic action. Other evidence for an interaction between PLM and PMCA in the anesthetic response is provided from our studies in diabetic rats (13,14). Rats with streptozocin-induced diabetes mellitus are characterized by reduced PMCA activity in a variety of tissues, including brain, and both require lower concentrations of IA for ablation of movement in response to noxious stimulation (14). PLMT I activity, as measured by the PLM rate, was elevated in these animals. Panagia et al. have also linked PLM to PMCA changes in STZ-induced diabetes rats, proposing possible affects on the lipid environment of the pump (15). In addition, phosphatidyl serine (PS), an acidic phospholipid which stimulates PMCA, was noted to be decreased in the sciatic nerves of patients with diabetes mellitus (16). We propose that our observations of enhancement of PLM and depression of PMCA in brain SPM by a wide variety of inhalation anesthetic agents at clinically relevant partial pressures lend support to a possible involvement and interaction of these processes in anesthetic mechanisms. Acknowledgements Supported by grant GM 46401 from National Institutes
of Health.
PL460
Anesthetics and Phospholipid Methylation
Vol. 56, No. 25, 1995
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
F. HIRATA and J. AXELROD, Science 209 1082-1090 (1980). F. HIRATA , W.J. STRITTMATTER and J. AXELROD, Proc. Natl. Acad. Sci. USA 76 368-372 (1979). F.T. CREWS, F. HIRATA and J. AXELROD, J. Neurochem 34 1491-1498 (1980). J.J. FRANKS, R.B.V. SASTRY, M.J. SURBER and B.S. ENGLAND, Anesthesiology 73 984-989 (1990). J.J. FRANKS, J.L. HORN, P.K. JANICKI and G. SINGH, Anesthesiology g2 108-117 (1995). J.J. FRANKS, J.L. HORN, P.K. JANICKI and G. SINGH, Anesthesiology 82 118-128 (1995). C.W. COTMAN, Methods in Enzymology 2445-452 (1974). G.B. RUSSELL and J.M. GRAYBEAL, Anesth. Analg. 75 995-999 (1992). H.H. LUTTROPP, R. THOMASSON, S. DAHM, J. PERSSON and 0. WERNER, Acta Anaesthesiol. Stand. 28 121-125 (1994) R.B.V. SASTRY, C.N. STATHAM, R.E. MEEKS and J. AXELROD, Pharmacology 23 21 l-222 (1980). L. MOORE, T. CHEN, H.R. KNAPP, JR. and E.J. LANDON, Journal of Biological Chemistry 250 4562-4568 (1975). C.T. GONSOWSKI and E.I. II EGER, Anesthesia & Analgesia 79 710-712 (1994). P.K. JANICKI, J.L. HORN, G. SINGH, W.T. FRANKS and J.J. FRANKS, Life Sci In press (1995). P.K. JANICKI, J.L. HORN, G. SINGH, W.T. FRANKS and J.J. FRANKS, Life Sci 55 PL359-PL364 (1994). V. PANAGIA, Y. TAIRA, P.K. GANGULY, S. TUNG and N.S. DHALLA, Journal of Clinical Investigation @ 777-784 (1990). D. DRISCOLL, W. ENNIS and P. MENESES, Int J Biochem 26 759-767 (1994).
Pergamon
00243205(95)00220-O
PHARMACOLOGY LETTERS Accelerated Communication
NITROUS
OXIDE AND XENON ENHANCE PHOSPHOLIPID-N-METHYLATION RAT BRAIN SYNAPTIC PLASMA MEMBRANES
IN
J-L. Horn, PK. Janicki, J.J. Franks Department
of Anesthesiology,
(Submitted
Vanderbilt
University USA.
Medical Center, Nashville,
Tennessee,
December 19, 1994; accepted February 17, 1995; received in final form March 20, 19%)
ABSTRACT. Halothane and isoflurane increase the rate of phospholipid methylation (PLM) in rat brain synaptosomal membranes, a process linked to the coupling of neuronal excitation to neurotransmitter release. In contrast, synaptic plasma membrane (SPM) Ca*’ ATPase (PMCA) pumping is reduced by exposure to halothane, isoflurane, xenon and nitrous oxide (N,O). To examine further the relationship between PLM, PMCA and anesthetic action, we investigated the effect of clinically relevant concentrations of two less potent anesthetic gases, N,O and xenon, on PLM in SPM. Biochemical assays were performed on SPM exposed to 1.3 MAC of N,O (2 atm), 1.3 MAC of xenon (1.23 atm) or an equivalent pressure of helium for control. N,O or xenon exposure increased PLM to 115% or 113%, respectively, of helium control (~~0.02). Similar exposures to N,O or xenon depressed PMCA activity to 78% and 85% of control (~~0.05). Observations that PLM and PMCA are both altered by a wide variety of inhalation anesthetic agents at clinically relevant partial pressures lend support to a possible involvement and interaction of these orocesses in anesthetic action.
Key Words:
nitrous oxide, xenon, phospholipid methylation, plasma membrane Ca2+-ATPase
Phospholipid methylation (PLM), a ubiquitous process occurring in cell membranes, utilizes two enzymes, phospholipid-N-methyltransferases I and II (PLMT I and II), which successively methylate phosphatidylethanolamine (PE) to phosphatidyl-N-methylethanolamine (PME), phosphatidyl-N-dimethylethanolamine (PDE) and phosphatidyl-choline (PC). PLMT I, located on the cytoplasmic surface, drives the first, rate-limiting methylation step, producing PME. Associated with successive methylation is rapid translocation of the methylated product from the inner to the outer surface of the plasma membrane. Hirata and Axelrod have described the interaction between biological signal transmission and membrane PLM (1,2). They provided evidence for involvement of PLM in the transduction of receptor-mediated signals through cell membranes (1). PLMT I and II activities have been identified in rat brain synaptosomal membranes (3). Franks et al demonstrated that transmethylation of synaptosomal membrane phospholipids in rat brain was increased by either in vitro or prior in vivo exposure to two inhalation anesthetics (IA), halothane and isoflurane (4). Franks et al recently reported that plasma membrane Ca’+-ATPase (PMCA) pumping was reduced in brain synaptic plasma membranes (SPM) of Sprague Dawley rats by Correspondence: Dr. J.J. Franks, Dept. of Anesthesiology, Nashville, TN 37232-2125, USA.
Vanderbilt
University
Medical Center,
PL-456
Anesthetics and Phospholipid Methylation
Vol. 56, No. 25, 1995
exposure to a wide range of inhalation anesthetics (5,6). Though a mechanistic relationship between stimulation of PLMT I and inhibition of PMCA in the anesthetic response is speculative at the present, the possibility of an interaction would be bolstered by demonstrating that IA of widely differing structure affect both processes. We have therefore examined the effect of xenon and N,O on PLM in cerebral synaptic plasma membranes, comparing that effect with the PMCA response to N,O and xenon exposure (5). Materials and methods Animals. All experimental protocols were approved by the Animal Care Committee of Vanderbilt University. Male Sprague-Dawley rats (240-325 grams) were allowed food and water ad libitum. The number of animals was determine by the amount of membrane required for each experiment. One gram of cerebral tissue provided about 1 mg of SPM. For N,O experiments 18 rats were pooled in 6 groups of 3 and assayed in duplicate. The same membrane preparation was used for PLM and PMCA assays, the latter result reported previously (5). For xenon experiments, PLM was assayed in SPM from a group of 12 rats, pooled by 2 in duplicate. Xenon effects on PMCA were examined in 70 rats pooled by 10. This large number of rats reflects the use of Animals were sacrificed by decapitation. Brains were membrane aliquots for other experiments. dissected on ice, and synaptosomes were isolated by ultracentrifugation (7,5). Synaptosomes were osmotically shocked and placed on a sucrose gradient for further purification. Final pellets were suspended in isosmotic sucrose (0.32 M, pH 7.4) and used immediately for assay of PMCA Ca2’ pumping activity. Membrane aliquots were frozen for PLM assays at a later date. Protein content in SPM suspensions was estimated by the Bradford method. Delivery of anesthesia. SPM were expose to N,O and xenon in a Parr Cell Disruption Bomb (PCDB; Parr Instrument Corp., Moline, IL) placed in a water bath at 37 “C and used as a pressure chamber. The PCDB was modified by placement of a low-pressure gauge, O-3 atm, on the lid. The PCDB was flushed for 3 min at 6 l/mitt either with helium for control (A-L Compressed Gases Inc., Nashville, TN) or with N,O (A-L Compressed Gases Inc.) or xenon (Research Grade from Alphagas; Morrisville, PA), after placement of the incubation tubes in a 100 ml beaker filled with water at 37 “C. (Temperature was confirmed by measurement before and after incubation was completed.) N,O or xenon was then added to the PCDB to achieve a partial pressure of 1.3 of the reported minimum alveolar concentration (MAC). Two atm of N,O (8) or 1.23 atm of xenon (9) were selected. Helium was used as control gas at the same pressure used for each agent studied. Biochemical analysis. PLMT I activity was assessed in duplicate by the incorporation of tritiated methyl groups from (methyl-3H) SAM into PME (3,4,10). After incubation, the phospholipids were extracted, Phospholipids were separated by thin layer washed and concentrated in aliquots of chloroform. chromatography (TLC). Fractions were identified by comparison with standards, scraped into scintillation vials and counted. Results were expressed as femtomoles of SAM incorporated into PME per milligram of protein per minute. Ca*’ uptake by everted rat SPM incubation mixture was maintained at protein to each tube. Aliquots of 0.5 Filters were washed, nitrate filters.
vesicles was performed as described previously (11,5). The 37 “C. The reaction was started by adding 70 ug of SPM ml were removed after 30 min and collected on cellulose dried and placed in scintillation vials, and activity was
PL-457
Anesthetics and Phospholipid Methylation
Vol. 56, No. 25, 1995
measured in a beta counter. Results were expressed as nmoles of Ca2’ accumulated of SPM protein per minute of incubation time.
per milligram
Data were examined by multifactorial ANOVA, t-tests when Statistical analysis. appropriate, and multiple comparison (Student-Newman-Keuls procedure). Significance was inferred if ~~0.05. Results Fig. 1 illustrates the effects of 1.3 MAC-E (minimum alveolar concentration equivalent) of N,O and xenon (2 and 1.23 atmospheres respectively) on the first, rate limiting step of phospholipid methylation, the incorporation of methyl-3H groups from SAM into PE to form PME. Femtomoles of methyl-3H groups incorporated into PME per milligram of protein per min was significantly increased to 115% of control by N,O and to 113% of control by xenon. Results are normalized in Fig. 1 by expressing the value obtained for each agent as a fraction of the helium control (taken as unity). Error bars indicate SEM. Differences between control and agent were significant, pcO.02.
1.30
,,................... * .._..,._.....,,,,........................,.,....
-I-
It
* *
0.80 Helium
W
Xenon
Fig. 1 Normalized PLM activity in SPM prepared from cerebra of male Sprague-Dawley rats and exposed to helium, N,O or xenon (1.3 MAC). Aliquots from 6 preparations of SPM were exposed in duplicate in a PCDB to 2 atm of N,O or 1.23 atm of xenon for 30 min at 37 “C. Control membranes were exposed to helium at 2 or 1.23 atm under the same conditions. * denotes values significantly different from control (~~0.02). Fig. 2 shows, for comparison, normalized results of N,O and xenon effects on PMCA in rat cerebral SPM, taken from previously published data (5) for nitrous oxide. As previously noted, N,O decreased PMCA pumping to 78%. Xenon decreased pumping to 85% of helium control values (pcO.05).
PL-458
Vol. 56, No. 25, 1995
Anesthetics and Phospholipid Methylation
1.10
.,..,,,.,,..............................
.
I
..
. . . . ..
. .
* .,,.......................... * 0.80
*
‘I
0.70
0.60
0.50 Helium
1I N,O
-
Xenon
Fig. 2 Normalized PMCA activity in SPM prepared from cerebra of male SpragueDawley rats and exposed to helium, N,O or xenon (1.3 MAC). Aliquots from 6 preparations of cerebral SPM were exposed in duplicate in a PCDB at 37°C to 2 atm of N,O, and aliquots from 7 preparations of cerebral SPM were exposed under the same condition to 1.23 atm of xenon for 30 min. Control membranes were exposed to helium at 2 or 1.23 atm under the same conditions. * denotes values significantly different from control (~~0.05). Absolute values for PMCA pumping activity, as nmoles of 4sCa2’incorporated into everted SPM vesicles per mg per minutes and PLM activity as fmoles of methyl-3H group incorporated by PLMT I into PE to form PME, are shown in table 1. TABLE I PMCA and PLMT I activity expressed per mg of SPM vesicle protein per minute Control for Xenon Xenon Control for N,O N,O 0.66kO.03 0.56+0.01* 1.11~0.02 0.8720.03* PMCA (nmles) 48.05kO.48 54.26k2.01’ 41.43+1.75# PLMT I (fmoles) 35.94Zk1.64 * denote ~~0.05 compared to their control. # denote ~~0.02 compared to their control.
Discussion We have previously shown that phospholipid-N-methylation (PLM) in synaptosomes is significantly enhanced with exposure to halothane and isoflurane with both in vitro and prior in vivo exposure (4). Phospholipid-N-methyltransferase I (PLMT I) is the rate limiting enzyme of PLM, and assay conditions in our original study and in the current study were designed to selectively optimize PLMT I activity. We have also observed that inhalation anesthetics of varying structure, i.e., halothane, isoflurane, xenon, and N,O, inhibit plasma membrane calcium ATPase (PMCA) activity (5,6). The relationship between enhancement of PLMT I and inhibition of PMCA activity by anesthetics is purely speculative at this juncture, as discussed previously
Vol. 56, No. 2.5,1995
Anesthetics and Phospholipid Methylation
PL459
(6). It is noteworthy that PMCA activity is very sensitive to the lipid environment in which it It is possible that anesthetic-induced diversion of phosphatidylethanolamine to is situated. synthesis of phosphatidyl-N-methylethanolamine and ultimately phosphatidylcholine diverts phosphatidylethanolamine from conversion to phosphatidylserine, an acid-phospholipid known to enhance PMCA activity. If a mechanistic relationship does exist between these two processes, it is important to establish that each process is affected by the same wide range of inhalation anesthetics of varying structure. In this study, we examined the effects of N,O and xenon on PLM in cerebral synaptic plasma membranes. We found significant enhancement, 13 and 15%, respectively, of methyl incorporation into PME. This degree of enhancement of PLM by N,O and xenon is considerably less than that found in SPM with exposure to the potent anesthetics, halothane and isoflurane. This discrepancy may reflect that the MAC values for N,O and xenon which we based our studies on are lower than the true values for the rat. Various values for N,O have been reported in the literature (8,12), and xenon MAC has been reported only for the mouse (9). Furthermore, our estimates of the minimum effective dose (MED) of xenon and N,O, which we have defined as the lowest partial pressure of delivered anesthetic that blocks a response to pain (4,6), were perhaps also underestimated by lack of access to rats in our pressure chamber. N,O and xenon MED as measured by the righting reflex and response to pressure chamber motion may be less than the MED derived from ablation of a painful stimulus. It is also possible that the large partial pressures of N,O and xenon required to produce anesthesia cause physical as well as biochemical changes in the synaptic plasma membrane and thus on PMCA activity and the anesthetic response. However, we believe it is unlikely that the large difference in PLM effects between gaseous and volatile anesthetics can be accounted for by these several possible factors. We have already noted striking differences in PMCA activity in some but not all brain areas of diabetic rats, compared with normoglycemic controls (13). Similar anatomic differences may exist with respect to PLMT I activity, and PLM enhancement by gaseous and volatile anesthetics may be more equivalent in as yet undefined brain areas that are critical for anesthetic action. Other evidence for an interaction between PLM and PMCA in the anesthetic response is provided from our studies in diabetic rats (13,14). Rats with streptozocin-induced diabetes mellitus are characterized by reduced PMCA activity in a variety of tissues, including brain, and both require lower concentrations of IA for ablation of movement in response to noxious stimulation (14). PLMT I activity, as measured by the PLM rate, was elevated in these animals. Panagia et al. have also linked PLM to PMCA changes in STZ-induced diabetes rats, proposing possible affects on the lipid environment of the pump (15). In addition, phosphatidyl serine (PS), an acidic phospholipid which stimulates PMCA, was noted to be decreased in the sciatic nerves of patients with diabetes mellitus (16). We propose that our observations of enhancement of PLM and depression of PMCA in brain SPM by a wide variety of inhalation anesthetic agents at clinically relevant partial pressures lend support to a possible involvement and interaction of these processes in anesthetic mechanisms. Acknowledgements Supported by grant GM 46401 from National Institutes
of Health.
PL460
Anesthetics and Phospholipid Methylation
Vol. 56, No. 25, 1995
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
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