CHAPTER 3 3
Effects of Anesthetics, Depressants, a n d Tranquilizers on C e r e b r a l Metabolism
J. H. Quastel
I. Introduction 517 II. Anesthetics and Respiration of the Brain in vitro 519
III. Anesthetics and Specificity of Oxidative Inhibition 520
IV. Reversibility of Narcotic Action in vitro 521
V. Stimulated Brain Metabolism 521 A. Effects of Anesthetics, etc., on Stimulated Brain Metabolism . . 522
B. Anesthetics and Uncoupling of Phosphorylation 524 C. Anesthetics and Incorporation of Phosphate ( P3 2) into Phos-
pholipids 526 D. Anesthetics and Brain Diaphorases 526
E . Effects of Chlorpromazine on Brain Metabolism in vitro 528 F. Effects of Aliphatic Alcohols on Brain Metabolism 529 G. Effects of Salicylates on Brain Metabolism 530 H. Effects of Amytal on the Incorporation of P3 2 into Adenosine
Triphosphate ( A T P ) , Adenosine Diphosphate ( A D P ) , and
Phosphocreatine (CrP) 531 VI. Mode of Action of Anesthetics and Allied Substances 532
VII. Anesthetics and Glycolysis 533 VIII. Effects of Anesthetics on Biological Acetylations 534
IX. Steroids 534 X. Reserpine 535
References 535
I. INTRODUCTION
Narcotics and anesthetics include a large variety of structural types, such as hydrocarbons, alcohols, ethers, urethans, sulfones, amides, ureides, barbiturates, and nitrous oxide. Their common property of inducing narcosis in animals evidently depends on certain physicochemical char-
517
acters that they have in common rather than on the possession of any special chemical constitution.
It has long been known that narcotics, such as the urethans, as a general rule inhibit enzymic and respiratory processes. It was early shown (1) that when yeast cells or a cell-free heart preparation are aerated in the presence of a substrate and a narcotic, cytochrome b is reduced and cytochromes c and a remain in the oxidized form. Ethylurethan (0.36 M) inhibits the oxidation of succinate mediated through the cytochrome system by about 90% and through phenothiazine methosulfate by about 40%. It thus has two effects on a succinic oxidase system in a heart muscle preparation. It breaks a link between cytochrome b and c, and to a lesser degree it inhibits the enzymic activity of succinic dehydro
genase (2). A concentration of 0.02 Μ ethylurethan only inhibits unstimu
lated rat brain cortex respiration in vitro by 6% (3), while 0.03 Μ ethyl
urethan inhibits guinea pig brain respiration by 20% and does not inhibit yeast respiration at all (4). At the latter concentration it inhibits the oxygen consumption of a guinea pig brain preparation in the presence of glucose by 17% and has no effect in presence of succinate (4). The narcotizing concentration of ethylurethan in the rat is about 0.022 Μ (3), and there can be no doubt that, as a general rule, the concentrations of anesthetics required to induce narcosis in an animal are usually of a far smaller order than those required to inhibit most enzymic reactions.
In the narcotic state, induced by a variety of anesthetics, there is diminished cerebral consumption of oxygen. Anoxia, for example (5), exists in the central nervous system during the anesthesia brought about by barbiturates and other narcotics. Under Pentothal, oxygen consump
tion of the cerebral cortex is decreased more than that of the lower centers (6). With thiopental anesthesia, the average oxygen intake is lowered about 35% ( 7 ) . Ether anesthesia is associated with a decrease in the difference between the oxygen contents of arterial and venous bloods (8).
In the human subject under the influence of Amytal there is a small but definite inhibition of oxygen uptake and dextrose utilization by the brain (9). The depression of cerebral function by barbiturates indeed parallels the reduction of oxygen uptake. This fall in oxygen uptake in vivo, how
ever, may be only a reflection of the diminished cerebral activity obtained under narcosis (19).
It should be pointed out in this connection that the physiological facts point to a very high degree of dependence of mental function on the maintenance of oxygen and glucose supply to the central nervous system.
Any interference with the respiratory activity of the nervous system, or with some important aspect of this, by the action of the drug would be
33. ANESTHETICS, DEPRESSANTS, AND TRANQUILIZERS 519 expected to disturb its functional activity. The action of the metabolic inhibitor, i.e., its biochemical effect, may be highly localized even if its distribution in the brain as a whole is uniform. Accessibilty of the sub- stance to the enzyme systems affected must be dependent on the chemical constition of the cell structures in which these enzyme systems are located.
II. ANESTHETICS A N D RESPIRATION OF THE BRAIN IN VITRO
Anesthetics inhibit, at low concentrations, the respiration of brain tissue either in the form of a mince or of intact thin slices (10, 27).
Studies (4) of the effects of seven alkylbarbiturates on the oxygen up- take of minced guinea pig brain respiring in the presence of glucose have shown that there is a rough parallelism in this series of barbiturates between hypnotic power and inhibitive effect on brain respiration. This parallelism takes place among narcotics of different chemical types (11).
Results shown in Table I, obtained by using brain cortex slices (10), make it clear that a variety of anesthetics, at their narcotizing concentra- tions, produces inhibitions of cerebral respiration varying from 6 to 32%.
The data indicate that a small but definite inhibition of respiration is produced by concentrations of the order of those producing deep narcosis.
The inhibitions recorded represent the effects of the narcotics on the respiration of the entire brain cortex of the animal. Local inhibition will be much higher if the narcotic is localized or specifically absorbed at particular centers. It is known that narcotics have differential effects on the neurons in brain (12). The addition of pentobarbital (0.002 M) brings about a large inhibition of oxygen uptake of human cerebral cortex slices in the presence of glucose, human brain cortex being a little more sensitive than rat brain cortex (IS). When very dilute concentrations of an anesthetic are investigated, the effect sometimes, e.g., with pheno- barbital (14), is to bring about a small increase of respiration (about 5-10%) of the brain cortex slice, the effect being dependent on the calcium and magnesium concentrations.
The results of experiments which combined a dog brain biopsy method and the manometric technique of measuring respiration showed that the oxygen consumption in vitro of brain cortex, basal ganglion, or hypothala- mus may be depressed to the extent of 70% in the presence of 0.04%
pentobarbital (15). Moreover, the measurements of the rate of oxygen consumption of rat brain cortex slices in which the suspension medium
TABLE I
ANESTHETIC CONCENTRATION AND EFFECTS ON THE RESPIRATION OF BRAIN CORTEX SLICES IN A GLUCOSE MEDIUM
Per cent inhibition of Estimated Narcotizing brain tissue respiration narcotic dose concentration by narcotizing
Anesthetic Animal (gm/kg) (M) concentration Ethylurethan Rat 2 0.022 6 Magnesium ions Rat — 0.005 13 Chloral hydrate Rat 0.22 0.0013 10 Luminal Rat 0.2 0.0008 15 Chloretone Rat 0.18 0.001 20 Evipan Guinea pig 0.16 0.00062 17 Avertin Rat 0.3 0.0011 31 Chloretone Guinea pig 0.18 0.0010 32
consisted of whole blood drawn from a dog before and at given intervals after the administration of 36 mg/kg Nembutal showed significant inhibi
tions (25-33%) of brain respiration with blood drawn 3, 15, 30, and 60 minutes after administration of the narcotic {16).
III. ANESTHETICS A N D SPECIFICITY OF OXIDATIVE INHIBITION
Anesthetics do not inhibit all oxidative processes to the same extent.
Sen (17), for example, showed that urethan, at the high concentrations that inhibit succinic dehydrogenase, has no effect on xanthine oxidase. The oxidations of glucose, lactate, and pyruvate are most affected by relatively small concentrations of the anesthetics, while those of succinate and p-phenylenediamine are undisturbed (4).
The rate of oxygen consumption of tissues other than brain is also affected by anesthetics, though not to the same extent. Examination of the inhibitive action of narcotics on the respiration of a variety of tissues in the presence of different substances has shown (10) that anesthetics inhibit the oxidation of glucose, lactate, and pyruvate in tissues such as liver, kidney, or diaphragm to about the same extent as in brain. Anes
thetics will also inhibit the oxidation of fatty acids and amino acids by isolated liver (10). Narcotic inhibition of oxidations is not, therefore, restricted to glucose and its breakdown products.
33. A N E S T H E T I C S , D E P R E S S A N T S , A N D TRANQUILIZERS 521
IV. REVERSIBILITY OF NARCOTIC ACTION IN VITRO
The effects of anesthetics such as the barbiturates and chloretone on the respiration of brain slices are reversible. This is shown simply by washing the brain slices in an anesthetic-free medium after their immer- sion for an hour at 37° in the anesthetic solution. The slow constant rate of oxygen uptake found in the presence of the anesthetic is raised im- mediately to a high level, which remains constant (18). High concentra- tions of anesthetics, however, produce irreversible effects.
There are two effects of an anesthetic on brain respiration in vitro:
1. Rapid attainment of an equilibrium between the anesthetic and a constituent of the respiratory system. The inhibition of respiration is that to be expected from a simple mass action equation (3), as observed with small concentrations of anesthetic producing inhibitions not greater than 40%. This applies to anesthetics such as urethan, chloral, chloretone, barbiturates, Avertin (tribromoethyl alcohol), and magnesium ions.
2. Relatively slow development of irreversible changes, leading to in- creased inhibitions of respiration that cannot be restored to normal by removal of the anesthetic. This takes place with most anesthetics, such as barbiturates or chloretone at relatively high concentrations. It occurs, however, at low concentrations with ether (20). Irreversibility of action also occurs with indole, which is a powerful inhibitor of brain respiration (18). Conceivably, this is due to gradual irreversible denaturation of the proteins with which these narcotics become associated.
V. STIMULATED BRAIN METABOLISM
The respiration of isolated brain cortex slices in a normal physiological (glucose-containing) medium is about half that found in vivo. It may be increased by two methods to values approximating those found in vivo:
(a) alteration of the cationic composition, particularly the K + and C a+ + content, of the medium in which the brain slice is incubated; and
(b) application of electrical impulses.
It was shown many years ago (21, 22) that increasing the potassium ion concentration of the medium in which a brain slice is immersed to 100 meq/liter (which is approximately the concentration of potassium ions in the nerve cell) brings about a marked increase in the rate of respiration, approaching double that of the normal in a glucose medium. That the stimulating effect is due to the balance between potassium and calcium ions rather than to the absolute magnitude of the potassium ion concen-
tration is demonstrated by the fact that a considerable stimulation may occur by removal of calcium ions from a medium containing the usual (5 meq/liter) concentration of potassium ions (23). This phenomenon does not occur in a brain homogenate or mince; it is evident that it is linked with the integrity of the brain cell membranes.
It was equally well known that the respiration of isolated muscle and peripheral nerve is increased on electrical stimulation. Winterstein (24) showed that application of electrical impulses to isolated frog spinal cord led to an increase in its respiration. Bronk and Brink (25), in fact, demon
strated that the increment in the rate of oxygen uptake of frog nerve carrying impulses at the rate of 50 impulses/second is highly anesthetic sensitive. Mcllwain (26) showed that application of electrical impulses to the isolated brain tissue (in the form of slices), in a physiological medium, brings about an increased rate of respiration, approximately double the normal value.
There is little doubt that both methods of respiratory stimulation have a common basis, namely, cationic displacements at the brain cell mem
brane. What is important, however, is the fact that the stimulated respira
tion of isolated brain tissue has the magnitude of brain respiration in vivo and possesses some of the characteristic features of brain in vivo, such as response to drug action. It may be considered a working hypothesis that the stimulated brain slice is an approximation, so far as its biochemi
cal characteristics are concerned, to the functioning brain, that is, to brain tissue in vivo, stimulated by sensory impulses. It must be borne in mind, however, that brain slices, under the best experimental conditions obtained so far, do not show all the electrophysiological responses to stimulation, or the spontaneous activity associated with brain in vivo.
Nevertheless, even as an approximation to the in vivo condition, they are able to yield valuable biochemical data that bear upon the properties of the functioning brain (19).
A. Effects of Anesthetics, etc., on Stimulated Brain Metabolism
The steady state of the diminished respiration of brain slices brought about by small concentrations of anesthetics, such as phenobarbital or chloretone, is greatly dependent on the K + concentration of the medium
(10). The presence of 12.8 meq/liter K + secures a steady inhibition of respiration by chloretone, lower concentrations of K + producing a fluc
tuating and unstable state, possibly due to loss of K + from the brain cells. This unstable state depends on the temperature and is less evident at 29° than at 39°.
33. ANESTHETICS, DEPRESSANTS, AND TRANQUILIZERS 523 Anesthetics (as well as depressants and tranquilizers) exercise much larger inhibitory effects on the respiration of nerve tissue, stimulated either electrically or by the presence of a high content of K + or dimin
ished C a + + , than on that of the unstimulated preparation. The rate of oxygen uptake by resting frog nerve is reduced 15% by 2 mM chloretone but the increment in the rate of oxygen uptake by nerves carrying im
pulses at the rate of 50 impulses/second is decreased by 50% by the same quantity of the narcotic [25). Anesthetics such as the barbiturates and chloral inhibit the respiration of electrically stimulated brain cortex slices at concentrations having relatively little effect on the respiration
T A B L E II
EFFECTS OF LUMINAL ON R A T BRAIN CORTEX RESPIRATION IN THE PRESENCE OF POTASSIUM CHLORIDE (27)
Qo2
Qo2 with added Average % without added KC1 increase due
Additions KC1 (0.1 M) to KC1
Glucose 14.2, 12.6, 11.5 24.5, 20.0, 21.0 70 Glucose
+ 3.3 mM Luminal 9.5, 7.4, 6.9 10.5, 8.5, 7.8 12
T A B L E III
EFFECTS OF CHLORETONE ON THE KCL-STIMULATED RESPIRATION OF R A T BRAIN CORTEX SLICES IN THE PRESENCE OF DIFFERENT SUBSTRATES (10 MM) (27)
Qo2
In the presence No chloretone of 3.3 mM Substrate Condition present chloretone
Glucose Without added KC1 12.5 8.3
With 0.1 Μ KC1 20.5 9.2
Sodium pyruvate Without added KC1 13.2 10.1
With 0.1 Μ KC1 18.8 11.2
Sodium L-glutamate Without added KC1 7.5 5.0
With 0.1 Μ KC1 7.0 5.0
Sodium succinate Without added KC1 10.8 10.5
With 0.1 Μ KC1 9.0 8.5
of the unstimulated tissue (26). Moreover, potassium-stimulated respira
tion is more sensitive to the action of barbiturates and chloral than unstimulated respiration. Luminal inhibition, it was already known, is greater in the absence of Ca+ + than in its presence (10). Removal of C a+ + has effects on anesthetic sensitivity similar to that due to added K+ (23). I t is, however, the increased aspect of brain respiration due to cationic stimulation which is highly sensitive to luminal, chloretone, and ethanol (27), and, in fact, the anesthetic suppresses the K+ stimulation
(see Tables II and I I I ) .
The largest effects of cationic stimulation on rates of oxygen consump
tion are with the substrates glucose, fructose, pyruvate, and lactate, and little is seen with L-glutamate or succinate. The presence of increased potassium ion concentrations or decreased calcium ion concentrations does not affect the action of anesthetics on oxygen consumption in the presence of glutamate or succinate. The enhanced inhibitory effect of anesthetics is restricted to the substrates whose oxidation is accelerated by the increased ratio of K + to C a + + . Recent evidence (28, 28a-28c) indicates that the cationic stimulation of respiration is due to the in
creased rate of operation of the citric acid cycle in the brain cell, the rate-limiting step that is (indirectly) affected being the conversion of pyruvate into acetyl CoA.
Investigations using radioactive glucose and estimating the rates of formation of C1 402 instead of oxygen consumption show very clearly the inhibiting effects, at low concentrations, of Amytal (0.5 m M ) , Doriden (a-ethyl-a-phenylglutarimide) (2.5 mM), and ethanol on potassium- stimulated cerebral respiration (29).
B. Anesthetics a n d Uncoupling of Phosphorylation
Anesthetics at low concentrations will dissociate phosphorylations from oxidations, sometimes without any apparent effect on the respiratory rate.
It is well known that certain drugs, such as dinitrophenol and gramicidin, will exhibit this phenomenon. It has been shown (30) that barbiturates at low concentrations will also bring about the same phenomenon. Defi
nite decreases of the P / O ratio may, however, accompany decreases of the rate of oxygen uptake, contrary to what occurs with low concentra
tions of dinitrophenol. The evidence (31) makes it clear that the behavior of narcotics on metabolic processes in the brain differs from that of a typical uncoupling agent, such as 2,4-dinitrophenol (31b). With liver mitochondria the oxybarbiturates do not depress phosphorylation more than they depress respiration (31a; see also 81c). These drugs do not
33. ANESTHETICS, DEPRESSANTS, AND TRANQUILIZERS 525 activate ATPase in contrast to the effect of 2 : 4 dinitrophenol (SI a, b,d).
Experiments with cell preparations gave rise to the conclusion (82) that the site of action of anesthetics (e.g. chloretone) on brain respiration is located with a process playing an intermediate role between cytochrome oxidase and a flavoprotein concerned with the oxidation of diphosphopyri- dine nucleotide ( N A D H2) . This conclusion is confirmed for oxybar- biturates by results of work on mitochondria (81b). In accordance with this conclusion, it has been found (83, 34) that Amytal or chloretone (35) is a highly effective inhibitor of the oxidation of N A D H2. As it is now well known that the biological oxidation of N A D H2 is accompanied by the phosphorylation of adenosine diphosphate (ADP) to adenosine tri
phosphate ( A T P ) , it follows that the anesthetic is also inhibitory to oxidative phosphorylation. This is shown in the following reaction
[Eq. ( 1 ) ] :
Inhibited b y
N A D H2 + ADP + Pi + Ο > N A D + ATP + H20 (1)
A m y t a l or Chloretone
The suppression of N A D H2 oxidation by Amytal thus has the double effect of suppressing the citric acid cycle (as pyruvate oxidation requires N A D for the formation of acetyl CoA) and A T P production. In this connection it may be noted that when the oxidation of pyruvate by liver mitochondria is stimulated by the addition of a pyrase (which releases A D P ) it is the stimulated oxidation which is more inhibited by bar
biturates than the unstimulated (31a, b). Chloretone is an effective in
hibitor of N A D H2 oxidation in a rat brain homogenate (60% inhibition with 2 m M chloretone) and in dog liver mitochondria (35).
It is of importance to note that succinate reduces intramitochondrial pyridine nucleotide ( P N ) and that Amytal (1.6 mM) not only suppresses the oxidation of P N H2 but slows the rate of reduction (35a).
The effects of anesthetics on A T P synthesis in the brain are shown by their suppression of acetylcholine synthesis (36-38) and by their inhibitory effects (e.g., those of 4 m M chloretone or 1 m M Nembutal) on P3 2 incorporation (from phosphate) into phosphoproteins and or
ganic phosphorus compounds in cat brain slices respiring in presence of glucose (38a). A striking demonstration (28) of the action of a low concentration of anesthetic (0.5 m M Amytal) on A T P formation in brain slices is shown by its ability to suppress glutamine synthesis, a reaction that requires the participation of A T P in the condensa
tion of glutamate and ammonia. Other changes also take place, and these can be satisfactorily explained by the conclusion that Amytal has a
twofold effect (see also 39): (a) diminution of N A D formation from N A D H2, and (b) diminution of A T P formation. Reduced triphosphopyri- dine nucleotide oxidation in brain is not affected by Amytal (40). Amytal has little or no effect, it should be noted, on certain other N A D H oxidiz
ing systems (41-43).
C. Anesthetics a n d Incorporation of Phosphate (P3 2) into Phospholipids
It is known that the incorporation of labeled phosphate ( P3 2) into phospholipids of rat brain preparations is dependent on the supply of metabolic energy and that the isotope is incorporated into A T P prior to its entry into phospholipids (43a). The presence of high concentrations of K + results in an increase of incorporation of P3 2 into phospholipids of rat brain cortex slices in an incubation time of 30 minutes (43b, c) (although continued incubation for 4 hours leads to a decreased incor
poration (43d) possibly due to irreversible effects gradually occurring in the high K + medium). Both Amytal (0.5 mM) and chloretone (2mM) almost abolish the potassium-stimulated incorporation of P3 2 into phos
pholipids in rat brain cortex (43b). Moreover, these anesthetics at the concentrations quoted, as well as ethanol (0.2 M ) , suppress the incor
poration of P3 2 into phospholipids in rat brain cortex slices stimulated either by acetylcholine or by potassium ions (43b, 43c). These results are consistent with the conclusion that the anesthetics reduce the ability of brain cortex in vitro, when stimulated by K + , to form A T P and there
fore to bring about, at normal rates, the incorporation of phosphate into phospholipids.
D. Anesthetics a n d Brain Diaphorases
It has been pointed out in experiments with brain homogenates that anesthetics, such as the barbiturates and chloretone, suppress anaerobic reduction of methylene blue by the brain dehydrogenases, particularly those concerned with the breakdown of carbohydrates, lactate, and pyruvate. They have less effect on the dehydrogenases concerned with succinate or glycerophosphate (44). Moreover, some competition with the anesthetic is evident, as the inhibitory effect of the drug diminishes with increasing concentration of the substrate (e.g. lactate). With a sheep brain preparation, 23 m M chloretone produces 50% inhibition of brain lactic acid oxidation with 20 m M lactate as substrate (44).
The activities of purified preparations from ox brain of diaphorases that catalyze the anerobic oxidation of N A D H and N A D P H by a number
33. ANESTHETICS, DEPRESSANTS, AND TRANQUILIZERS 527 of electron acceptors, including menadione and methylene blue, are sup- pressed by Amytal in a competitive manner (45, 46). However, 55%
inhibition of activity by Amytal required the relatively high concentra- tion of 6.6 m M (46). The purified preparation of N A D H diaphorase is inhibited competitively by a number of barbiturates and other depres- sants, including chloretone, which gave a 20% inhibition at 3.3 m M with a concentration of N A D H2 as low as 0.019 m M (47). Possibly these diaphorase inhibitions are responsible for the recorded earlier observations on narcotic-inhibited brain dehydrogenases (44) ·
While these inhibitions are likely to play a role in the mechanisms in- volved in the suppression of brain respiration in vitro by anesthetics, it is to be noted (10) that the respiration of brain cortex slices is not only much more susceptible to the drug than a brain homogenate, but that increase of the concentration of the substrate in the medium in which the cortex slices are incubated does not diminish the percentage inhibition (10). Increase of the substrate concentration may be expected to increase the N A D H (as it presumably does in a brain homogenate) (44), and this should diminish the anesthetic inhibition. It may be concluded, there- fore, that anesthetic inhibition of brain cortex respiration will involve more than a simple suppression of the activity of the N A D H diaphorase system.
Spectrophotometric evidence (47a, b) obtained with intact mitochon- dria have confirmed the conclusion (32) that Amytal acts upon the site of the respiratory chain concerned with the oxidation of N A D H2. Puri- fied N A D H dehydrogenase is not sensitive to Amytal (47c). N A D H diaphorase, using dichlorophenol-indophenol, ferricyanide, and some quinones, is not affected by Amytal (47d; see also 32, 4V)t m contrast with highly sensitive N A D H oxidase which can be extracted from mitochondria (47d). It has been suggested (47c) that chloretone, which inhibits the activity of pyridine nucleotide-dependent oxidases, brings about some destruction of endogenous pyridine nucleotide.
Inhibitive effects of narcotics, anesthetics, or a tranquilizer, such as chlorpromazine, on A T P synthesis in the brain do not necessarily involve an immediate fall in respiration in the brain cells affected. A situation may arise where the increased concentration of A D P , which has a rate- limiting effect on metabolic reactions involved in glucose breakdown, can give rise to an increased rate of oxygen consumption. This is a frequent effect of the addition of respiratory "uncouplers." Nevertheless, this effect may be transient, depending on a variety of conditions, and the ultimate effect of suppression of so important a process as N A D H2 oxidation must be a suppression of respiration.
Narcotics, such as pentobarbital and ethanol, exercise a larger inhibi
tory effect on respiration in the adult than in the young rat brain (48), a result that is to be expected from the fact that the activity of the citric acid cycle increases with age during the early days of postnatal life and that the effect of K + on aerobic nerve metabolism is greatly de
creased in very young animal brain. There is a significant positive corre
lation between rate of brain respiration at different ages and sensitivity to barbiturates (49). The inhibitory effects of barbiturates on respiration diminish in old age, the maximum effects occurring in the young adult.
E. Effects of Chlorpromazine on Brain Metabolism in vitro
Chlorpromazine at low concentrations reduces the enhanced respiration due to electrical excitation (50, 50a) just as it reduces the increased oxygen uptake found with potassium ion stimulation (51). The electrical- stimulated respiration is apparently more sensitive than the potassium- stimulated respiration. It also brings about some inhibition in vitro of incorporation of glycine-1-C1 4 into rat brain cortex proteins, a process that is A T P dependent, at concentrations (0.2 mM) that have little or no effect on the respiration of brain or on the breakdown of glycine-1- C1 402 into C1 402 (51). The inhibitory effect (50% inhibition) is also brought about by 0.5 m M Amytal and 0.4 Μ ethanol. Thus, it is pos
sible that chlorpromazine may affect physiological action by an in- hibitive or uncoupling action on A T P synthesis at the particular site in the brain where it gains access most easily to the relevant enzyme systems. The addition of chlorpromazine (0.1 mM) to guinea pig brain slices brings about changes in labeling of lipid phosphorus in the presence of P3 2, namely, decreases of phosphatidylethanolamine and phosphatidyl
choline and an increase of phosphoinositide; with higher concentrations of the drug there is a considerable decrease in labeling of lipid phosphorus (52, 52a). Chlorpromazine also exerts an inhibitory activity on the res
piration of brain cortex slices in the presence of glucose, pyruvate, or L-glutamate but not succinate. This inhibitory effect is greatly increased with brain cortex slices stimulated by the presence of potassium ions, a definite inhibition occurring with 0.2 m M chlorpromazine.
Recent results (53) indicate that chlorpromazine uncouples phosphory
lation coupled to the oxidation of ferrocytochrome c and inhibits N A D H - cytochrome c reductase, the effect being mainly due to an effect on the coupled phosphorylation reaction.
Thus, chlorpromazine, like Amytal, brings about an inhibition of oxida
tive phosphorylation in brain cortex respiration in vitro. However, the
33. ANESTHETICS, DEPRESSANTS, AND TRANQUILIZERS 529 action of chlorpromazine in vitro differs from that of the barbiturates in bringing about progressive inhibitions and in its high binding power with tissue proteins (probably lipoproteins). The drug, after combining with tissue components, gradually diffuses into the brain cell bringing about its metabolic inhibitions (51). The conclusion that chlorpromazine affects N A D H2 oxidation (in phosphorylating systems, such as those in brain cortex in vitro) has been confirmed with the use of liver mitochon- drial systems (53). Chlorpromazine 0.2 m M brings about in liver mito- chondria a marked inhibition of incorporation of P3 2 into A T P (see also 53a). These facts do not imply that chlorpromazine and Amytal act at the same locations in the nervous system. The differences between the clinical effects of these two drugs may be due more to their different sites of action than to essential differences in biochemical mechanism.
Chlorpromazine appears to affect cell permeability (53b, c). It dimin- ishes the uptake of circulating labeled noradrenaline into heart and adrenal medulla (53d). Possibly, these effects are due either to combina- tion with membrane constituents or to depression of ATP that may con- trol transport at the cell membrane. Combinations of chlorpromazine with manganese ions (53e) or the strong electron-donor properties of chlorpromazine (53f) have been suggested as possible explanations for its activity in the nervous system.
F. Effects of Aliphatic Alcohols on Brain Metabolism
The addition of ethanol at small concentrations diminishes (27, 54) the oxygen consumption of rat brain cortex slices respiring in a glucose- phosphate medium when this has been stimulated by the presence of 100 meq/liter K + . Potassium-stimulated respiration of rat brain slices is much more affected by ethanol at low concentration than normal respira- tion, the concentration being of the same order as that necessary to bring about the narcotic state in the rat. The behavior with ethanol is similar in this respect to that brought about by anesthetics such as the barbitu- rates or chloretone (54-57). Ethanol also causes a decrease in the respira- tion of electrically stimulated brain tissue in the presence of glucose (57).
The inhibitory effects of the alcohols increase markedly as the length of the carbon chain increases and with increase in concentration, and the potassium ion stimulation of brain cortex respiration is diminished or abolished by concentrations of alcohols that have little effect on the un- stimulated respiration (54). n-Pentanol is much more effective than ethanol in effecting an inhibition of the stimulated respiration, and there seems to take place a rapid establishment of equilibria between the
alcohols and the components that influence the brain respiratory system.
It has been found (54, 58) that brain mitochondrial respiration is rela
tively insensitive to concentrations of alcohols that considerably depress stimulated rat-brain slice respiration. In this respect, the alcohols differ from the barbiturates but resemble, perhaps, the action of chlorpromazine (59). The results support the conclusion that the alcohols exercise their inhibitory effects on brain respiration at some site located in the brain cell membranes. If this is true, it must be concluded that a significant propor
tion of the brain cell respiration is controlled by the cell membrane, where presumably the potassium ion stimulation takes place. The alternative explanation, that the effects are due to oxidation of the alcohols to the highly toxic aldehydes or that the alcohols block the entry of substrates into the cell, is not supported by the available evidence.
It may therefore be concluded that anesthetics and cell depressants have a twofold mechanism of action on brain respiration and metabolism:
(1) an action at the cell membrane, affecting cationic equilibria there and thereby diminishing cationic stimulation of respiration and (2) an action within the cell, once penetration has occurred, affecting N A D H oxidation in the manner already visualized. The two mechanisms are apparently independent, and the first may occur without the second.
G. Effects of Salicylates on Brain Metabolism
Salicylates, like 2,4-dinitrophenol, suppress the formation of radioactive glutamine from radioactive glucose in the presence of rat brain cortex slices. Representative results are shown in Table IV (60). There can be little doubt that the inhibition is due to the suppression of oxidative phos
phorylation (61, 62). The effect, however, is more pronounced with the cation-stimulated metabolism than with the unstimulated metabolism.
The effects of acetylsalicylate and 2,4-dinitrophenol are not identical.
The former strongly depresses labeled alanine formation with but little effect on that of labeled γ-aminobutyrate, whereas the latter has the re
verse effect (Table I V ) . A noteworthy effect of acetylsalicylates (and D N P ) is their bringing about a greatly increased leakage of amino acids from the brain tissue, an effect greatly enhanced under conditions where cell A T P is already diminished (e.g., in the presence of high K + ) . They also suppress uptake of amino acids and of creatine into the brain tissue (60). These results indicate that the major effect of the two drugs is that of uncoupling phosphorylation from respiration, the loss of A T P resulting in changed transport velocities at the cell membrane and in retarded ATP-dependent reactions. The different clinical effects are
33. A N E S T H E T I C S , D E P R E S S A N T S , A N D TRANQUILIZERS 531
T A B L E I V
EFFECTS OF ACETYLSALICYLATE AND 2,4-DINITROPHENOL ( D N P ) ON RADIOACTIVE AMINO ACID FORMATION0 FROM G L U C O S E - U - C1 4 (5 M M ) IN R A T BRAIN CORTEX
SLICES0 (60)
0.05 mM 5 mM Acetyl-
D N P salicylate 5 mM 105 mM 5 mM 105 mM 5 mM 105 mM
Amino acid KC1 KC1 KC1 KC1 KC1 KC1 Glutamate 1032 1078 1140 935 890 770
Glutamine 370 732 254 45 255 40
γ-Aminobutyrate 270 467 400 270 234 350
Aspartate 280 280 520 265 227 135
Alanine 215 233 154 125 57 82
o
2 10.2 15.1 18.2 16.4 14.4 11.5° Amounts expressed as ταμ atom C1 4 incorporated/hour/100 mg wet weight tissue.
presumably to be attributed to the different sites of action in the body (62a).
H. Effects of Amytal on the Incorporation of P3 2 into Adenosine Triphosphate (ATP), Adenosine Diphosphate (ADP), a n d Phosphocreatine (CrP)
It has been shown (63) that electrical stimulation of the brain causes a drop in the content of A T P and a rise in inorganic phosphate, while there is evidence that excitation of the brain increases the A D P / A T P ratio (64). It is now well known that concentrations of A D P (or phos
phate ions) may be rate limiting in mitochondrial respiration. Hence, stimulation of the brain in vitro by reactions increasing A D P (or phos
phate) may result in acceleration of oxygen consumption and other dependent metabolic events.
The organic phosphates of brain slices undergo considerable changes in the presence of increased K + . There is a fall of A T P and CrP (65-67).
This effect of cationic stimulation is shown in Table V (60a), where it is seen that the incorporation of P3 2 from radioactive phosphate is dimin
ished in A T P and CrP, and increased in A D P . This would be expected if cationic stimulation causes an increased rate of conversion of A T P to A D P , with resultant fall in CrP according to the reaction [Eq. (2)]
ADP + CrP <=• ATP + Cr (2)
TABLE V
EFFECTS OF AMYTAL ON THE INCORPORATION OF P3 2 INTO ATP, ADP, AND CrP IN R A T BRAIN CORTEX SLICES INCUBATED AEROBICALLY IN GLUCOSE-RINGER
MEDIUM CONTAINING 10 uM N A2H P3 204 (107 COUNTS/MINUTE)
KC1 present
Addition (mM) ATP* ADP* CrPa
Nil 5 3100 100 1900
Nil 105 2150 550 950
Amytal 5 2950 150 1450
(0.5 mM)
Amytal 105 1250 50 400
(0.5 mM)
° Counts/minute/100 mg wet tissue.
In the presence of Amytal (0.5 m M ) , however, there is, with the normal (5 mM) KC1 concentration, a fall in the labeled CrP which is larger than that of labeled ATP. These effects are greatly increased in the presence of 105 m M KC1. It is evident that at normal concentrations of K + the fall in A T P due to Amytal is diminished by the transphosphorylation due to CrP present. With high concentrations of K + , there is less CrP available to react with A D P (whose phosphorylation is suppressed by Amytal), and hence there is a diminished A T P level.
VI. MODE OF ACTION OF ANESTHETICS A N D ALLIED SUBSTANCES
It is possible to understand the effects of anesthetics, depressants, and tranquilizers so far studied by their effects on (a) mitochondrial oxida
tions ( N A D H2 oxidation) and/or (b) cation equilibria at the brain cell membrane. It is known that, so far as the barbiturates are concerned, there is a reasonable correlation between the anesthetic concentration and those required to give 20% inhibition of mitochondrial respiration
(31a, b). The effects on mitochondrial respiration may be summarized in the following reactions [Eqs. (3) and (4) ] :
Pyruvate + N A D + CoA
Stimulated by increased K* or decreased Ca"*""1" (or electrically) by cytoplasmic in
crease of ADP (at expense of ATP and CrP) ^
Acetyl CoA + N A D H2 + C 02
33. ANESTHETICS, DEPRESSANTS, AND TRANQUILIZERS 533 N A D H2 + ADP + Pi
0 Inhibited by Amytal, etc.
N A D + ATP + H20
(4)
The effects at the brain cell membrane may be understood if the transport carriers at the membrane that are responsible for cation move- ments (essential for the changes of A T P and A D P , with increased K + or diminished C a + + in the medium) associate with or are rendered inert by the responsible drugs. Such effects would result in the diminution or abolition of cationic or electrical stimulation and, hence, would ultimately have an effect similar to that of a respiratory inhibitor acting on the mitochondria. Substances affecting the cell membrane in the manner visualized would not necessarily be "uncouplers" of respiration. It is possible that chlorpromazine and the aliphatic alcohols act primarily in this manner, whereas the barbiturates or chloretone act primarily on mitochondrial N A D H2 oxidation.
It is justifiable to conclude that the anesthetics influence both mitochon- drial and nerve cell respiration by their adsorption or combination with lipid groups present in the membrane, these groups being involved in the establishment of ionic gradients and respiratory metabolism of the cells.
VII. ANESTHETICS A N D GLYCOLYSIS
The mechanism of anaerobic breakdown of glucose in brain involves the interplay of dehydrogenases that are also involved in the aerobic break- down of the sugars. The absence of any effect of chloretone (32) indicates that the narcotic-sensitive oxidative system, important in the aerobic breakdown of glucose, is either absent from or is without influence on the reactions involved in the anaerobic breakdown of glucose by brain.
There is now evidence, indeed, to show that anesthetics increase aerobic glycolysis by suppression of the Pasteur effect. Possibly, this is the expla- nation for the observation (69) that anesthetics at low concentrations accelerate glucose consumption by excised rat superior cervical ganglia.
This conclusion would account for earlier observations (70-72) that in the presence of anesthetics there is an increased rate of breakdown of glucose, though there is suppression of respiration.
Anesthetics that suppress respiration also suppress acetylcholine syn
thesis by brain. This was first shown using ether (36). Depression of synthesis by brain slices is obtained by various drugs (37, 38). There is always a concomitant drop in rate of respiration, but this need not neces
sarily be as large.
Anesthetics do not diminish, or only slightly diminish (37, 38), the anaerobic synthesis of acetylcholine by A T P in the presence of brain extracts. The known phosphorylations by A T P (e.g., those involved in hexokinase activity and in glycolysis) are not impeded by anesthetics at low concentrations. N o inhibitory effect by anesthetics is found on the acetylation of sulfanilamide by enzymes present in the liver.
When the acetylating system is linked with a respiratory system such as that present in brain, the A T P necessary for the acetylation by these systems being formed by the energy of respiration, the presence of anes
thetics secures large inhibitory effects on the acetylation (38). Thus, Nembutal (0.5 mM) and chloretone (4 m M ) , at concentrations that do not affect anaerobic synthesis of acetyl sulfanilamide in the presence of ATP, exert marked effects on the aerobic synthesis in the absence of added ATP. Moreover, the addition of A T P to the aerobic system brings about a considerable alleviation of the inhibitory effect of the narcotic.
The most obvious explanation of the inhibitory phenomena that take place aerobically is that the narcotics inhibit those links in the respiratory chain (e.g., N A D H2 oxidation) that are responsible for the oxidative synthesis of ATP.
IX. STEROIDS
Steroids that have anesthetic potency also affect rat brain (homoge
nate) respiration in the presence of glucose but not in the presence of succinate (73, 74). The inhibitions parallel anesthetic activities. There is evidence (75) to indicate that the site of action of corticosterone, and possibly other steroids, lies in the respiratory chain between the flavo- proteins and cytochrome c, a site already suggested as a point of action of a variety of anesthetics (82).
VIII. EFFECTS OF ANESTHETICS O N BIOLOGICAL
ACETYLATIONS
33. ANESTHETICS, DEPRESSANTS, AND TRANQUILIZERS 535
X. RESERPINE
The well-known tranquilizing effect of reserpine seems to be associated with its ability to diminish the content of amines in the cell, e.g., of sero
tonin, from the brain (rabbit, cat) (68) or from other tissues [e.g., rabbit intestine (68a), blood platelets (63b)], or of catechol amines [e.g., from cat hypothalamus (68c), or adrenergic neurons of rabbits, cats, and dogs (68d), or adrenal medulla (68e)]. Reserpine has the power to release serotonin and noradrenaline wherever they are found in the brain (68f).
Reserpine inhibits uptake of noradrenaline, adrenaline, and serotonin by blood platelets (68b). It seems likely that this inhibition is due to an affinity of the drug for the transport carrier concerned with uptake of the amines and is not due to interference with the energetics of the cells con
cerned. Such inhibition could account for the depleting action of reserpine.
The conclusion that the sedative action of the reserpine group of drugs is due to lowered brain levels of certain amines seems to be supported by the fact that drugs that raise these levels (e.g., monamine oxidase inhibitors) may act as antidepressants (68j). Out of a large number of monamine oxidase inhibitors, those that are active in this respect in vitro and in vivo are usually stimulatory to the central nervous system (68g), a conclusion put forward over 20 years ago (68h). However, there still exists conflicting evidence that must be explained before the relation between amine content and sedation (or excitement) is made clear (68g).
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