Pharmacology 14: 39 46 (1976)
Comparisons between the Antianesthetic Action of Dibutyryl Cyclic AMP and Analeptic Drugs on Amobarbital-lnduced Narcosis in the Rat1 Barry J. Kraynack, Major L. Cohn, Marthe Cohn arid Floyd H. Taylor Department of Anesthesiology, Harvard Medical School, Boston, Mass., Department of Anesthesiology, Magce-Womens Hospital, and Department of Community Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa.
Key Words. Analeptic drugs • Antianesthetic properties • Antidotal • CNS ■Dibutyryl cyclic AMP Abstract. The dose-related antianesthetic and antidotal property of dibutyryl cyclic AMP, devoid of toxic effects, imparts uniqueness to the nucleotide as an arousal agent. Of the analeptic drugs studied (d-amphetamine, picrotoxin, pentylenetetrazol, caffeine, the ophylline, strychnine, ethamivan and doxapram), only picrotoxin demonstrated anti anesthetic properties. However, picrotoxin was associated with severe toxicity at all dose levels tested. No analeptic drug is effective in reversing the central nervous system depression produced by sedative, hypnotic or tranquilizer drug overdosage.
We have reported that dibutyryl cyclic AMP, the dibutyryl analog of adenosine 3':5'-monophosphate (cyclic AMP) administered centrally, doserelatedly regulates the duration of amobarbital-induced narcosis in the rat (5). We have also shown that centrally administered dibutyryl cyclic AMP, doserelatedly regulates tire duration of narcosis induced by other sedative, hypnotic, tranquilizer and anesthetic agents (3). This antianesthetic property of dibutyryl cyclic AMP, devoid of subsequent generalized stimulation of the central nervous system (CNS), suggests that the nucleotide is a true CNS antidote to drugs inducing narcosis (4). This finding has a double significance: (1) easy availability of barbiturates has made acute barbiturate poisoning a constantly increasing source of morbidity and mortality; (2) at the present time there is no true 1 Supported by a National Institute of Mental Health grant DA00605.
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Received: July 24, 1975.
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Table 1. Effects of various analeptic drugs on amobarbital-induced (80 mg k g '1) nar cosis (mean ST in minutes t SE) Treatment group
Control (saline 0.9 %) Dibutyryl cyclic AMP1 Strychnine sulfate Caffeine Theophylline1 d-Amphetamine Pentylenetetrazol1 Ethamivan Doxapram1
Concentration (1CV) 1.2 X 1 0 '2 M
2.4 X 1 0 '2 M
104.0 ± 6.8 39.7* ± 4.6 115.1 ± 5.2 123.42 r 3.8 127.42 1 6.9 128.3s ± 5.2 120.42 ± 6.3 116.2 ± 7.1 185.12 t 18.2
26.62 ± 3.4 111.2 ± 8.6 121.5* ± 12.2 116.2 ± 5.6 123.8* ± 6.4 127.6* * 6.9 117.7 ± 16.0 152.62 ± 10.1
1 Statistically significant difference between the mean sleeping times at the different dose levels for this agent based on t-statistic at the 0.05 level of significance. 2 Statistically significant difference between treated and control groups at 0.05 level of significance based on the Student-Newman-Keuls test. There were 10 rats per group.
pharmacologic antagonist to the depressant action that sedative, hypnotic and tranquilizer drug overdosages exert on the CNS. The purpose of the present work is to compare the antianesthetic properties of the heterogeneous group of drugs called analeptics to those of dibutyryl cyclic AMP.
Sprague-Dawley male rats (Zivic Miller, Pittsburgh, Pa.) weighing 85-125 g were housed under constant environmental conditions. Prior to the experiment, they were al lowed free access to drinking water but were fasted overnight. Between 8.30 a.m. and 10.30 a.m., each rat was weighed and given an intraperitoneal (IP) injection of 1 % solution of sodium amobarbital (80 mg k g '1) (Amytal) (Eli Lilly, Indianapolis, Ind.). Immediately after the loss of the righting reflex (LRR), an opening in the skin was made with a scalpel, revealing the bregma. A small hole was drilled through the roof of the skull with a 25 gauge needle, 1.5 mm to the right of the sagittal suture and 1.5 mm below the coronal suture. The needle of a 25 m1 syringe (Unimetrics Universal Company, Anaheim, Calif.) was inserted 3.0 mm in depth and each rat was injected intracerebroventricularly (1CV) with 15 ¿d of varying doses od dibutyryl cyclic AMP. picrotoxine, d-amphetamine, pentylenetetrazol, caffeine, theophylline, strychnine (Sigma, St. Louis, Mo.); doxapram (Dopram®) (Robbins, Richmond. Va.);cthamivan (Emivan®), the latter generously donated by the US Vitamin and Pharmaceutical Corporation, New York (N.Y.) or saline 0.9 %. Awakening was defined as
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Cyclic AMP, Analeptic Drugs and Arousal
Table II. Modulation of amobarbital-induced narcosis (80 mg k g '1): comparison of the effects of dibutyryl cyclic AMP alone and dibutyryl cyclic AMP + doxapram (mean ST in minutes ± SE) Concentrations of dibutyryl cyclic AMP (ICV)
0M 0.4 X 1 0 '5 M 0.8 X 1 0 '! M 1.2 X 1 0 '2 M
Control group
Treated group
doxapram (ICV) OAf
doxapram (ICV) 1.8 X 1 0 '2 M
103.2 ± 8.7 72.81 ± 9.2 64.5' i 6.0 44.3' i 4.1
141.6' 102.5' 86.8' 65.5'
i 7.5 ± 7.6 t 11.9 t 14.0
' Statistically significant difference between control and treated groups at each con centration of dibutyryl cyclic AMP at 0.05 level of significance based on the StudentNewman-Kculs test. There were 10 rats per group.
regaining the righting reflex (RRR). Sleeping time (ST) defined as the period between LRR and RRR, was recorded for all experiment groups. At the time of RRR, the rats were decapitated. The needle tract was examined in each brain. Rats which did not show a definitive needle tract in the ventricle or had evidence of intracerebral hemorrhage were excluded from the series of experiments. Physiologic changes were carefully observed and noted. Rectal temperatures were recorded at 5-min intervals with a thermistor probe (Bailey Instruments, Saddle Brook, N.J.) until righting occurred. Descriptive statistics of the ST among the various treatment groups are given in tables I and II. In addition, certain tests of significance have been carried out using nonparamctric methods to compare the treated groups with the control. The results arc given in tables I and II (20).
Results
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In amobarbital anesthetized rats, d-amphetamine, pentylenetetrazol, ethamivan and theophylline did not affect the respiratory rate. Doxapram produced a deep but brief hyperventilation that lasted from 1 to 8 min; picrotoxin and caffeine produced a tachypnea that lasted at least 30 min. Besides hyperventilation, at all concentrations tested, picrotoxin induced Straub tail phenomenon, shivering, piloerection, moderate to severe convulsions, micturi tion, defecation, phonation and occasionally death. The picrotoxin-induced symptoms were dose-related; higher doses were followed by severe clonic con vulsions and a mortality of 30 %. No toxic symptoms were induced by the other analeptic agents. In contrast to picrotoxin, dibutyryl cyclic AMP produced no ill effects; neither did it produce any mortality.
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Fig. I. Effects of ICV injections of saline (0.9 %) and d-amphetamine (1.2 X 10~J and 2.4 X 1 0 '2 M) on the rectal temperature of amobarbital-anesthetized rats. There were 10 rats per group. The vcrticle lines indicate ± SE. • = Saline; = d-amphetamine.
Table III. Effects of various concentrations of picrotoxin on amobarbital-induced (80 mg k g '1) narcosis Concentrations (1CV) 0 M (control) 0.1 X 1 0 '3 M
1 X 1 0 '3 M 2 X 1 0 '3 M 4 X 1 0 '3 M
Mean ST, minutes ± SE 109.1 i 7.0 76.4' ± 4.6 42.51 * 5.5 34.41,J ♦ 3.0 36.61’2 t 3.8
1 Difference between the mean levels of ST for each concentration of picrotoxin is statistically significant between treated and control groups at the 0.05 level of signifi cance using the t test. 2 The only pair of mean ST which do not have a statistically significant difference, based on the Student-Newman-Keuls test at the 0.05 level of significance is the pair 34.4 and 36.6.
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The administration of d-amphetamine partially antagonized the amo barbital-induced hypothermia but did not alter sleeping times (fig. 1). No other agent tested reversed the hypothermia. The central administration of picrotoxin or dibutyryl cyclic AMP prior to induction of amobarbital did not delay the onset of narcosis. However, the
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administration of picrotoxin or dibutyryl cyclic AMP following LRR shortened narcosis dose-relatedly (5). In the dose ranges tested, tire other analeptic drugs were ineffective antianesthetic agents (tables I, III). Of the analeptic drugs tested, doxapram prolonged ST most significantly. To investigate this potentiation of narcosis, we administered a solution of doxapram and dibutyryl cyclic AMP to amobarbital anesthetized rats (table II). The usual shortening of narcosis that we found with dibutyryl cyclic AMP (5) was reversed, while the tachypnea was unaltered. Caffeine did not alter the duration of narcosis; picrotoxin shortened and doxapram prolonged it; however, all three agents induced tachypnea. No correlation was found between tire respi ratory rate and the antianesthetic action of the analeptic drugs.
Discussion
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The purpose of the present study is twofold: (1) to determine whether or not centrally administered analeptic drugs possess antianesthetic properties similar to those of dibutyryl cyclic AMP; (2) to determine whether or not a relationship exists between the respiratory, cardiovascular and thermal regula tion by these agents and the duration of narcosis. We chose these parameters because in barbiturate overdosage, three major physiologic systems are involved: the CNS, the respiratory system and the cardiovascular system. While death from barbiturate overdosage is believed to occur as a result of irreparable cardiac, renal or hepatic damage, we have shown that the primary cause of death is CNS depression (4). The results obtained showed that following central administration, picro toxin was the only analeptic drug tested to date that shortened the duration of narcosis. However, at all concentrations tested, picrotoxin was associated with severe toxicity. Doxapram significantly potentiated amobarbital-induced narcosis and antagonized the regulatory effects of dibutyryl cyclic AMP. Such findings suggest, therefore, that the antianesthetic property of an agent is not common to all central nervous stimulants but is a unique property possessed by specific agents. This view is supported by our findings that rats and squirrel monkeys administered amobarbital in the LD10o range showed impressive sur vival rates when treated centrally with dibutyryl cyclic AMP (4). This arousal was accomplished without generalized stimulation of tire CNS, suggesting that the nucleotide may be the first true antidote to barbiturate overdosage. Part of the problem, as we see it, is that the term analeptic has been ill-defined. Goodman and Gilman (6) define an analeptic as a CNS stimulant. However, CNS stimulation and arousal are not the same phenomenon; for example, convulsions, one result of CNS stimulation, may occur even under narcosis. It cannot be shown that CNS stimulation leading to increased cardio
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vascular or respiratory activity also leads to arousal. Arousal must involve some increase in conscious control: analeptic stimulation of the CNS leads to increased systemic activity without changing the level of consciousness. Another part of the problem is that the term analeptic covers a wide range of drugs which appear to operate through different systems and have a rather wide range of noncorrelated effects. For example, it has been reported that picrotoxin blocks the GABA receptor, and that strychnine blocks the glycine receptor (19). It has become increasingly difficult to accept the postulate that this widely varied group of drugs causes an identical biological action: arousal. The definition we prefer for analeptic is: any drug that reverses the hypnot ic effects of an agent at any level of tire CNS. We believe that narcosis, which is induced in the CNS, has to be reversed in the same system. The significance of our definition will become more clear later in our discussion. We have found that ICV-administered norepinephrine raises blood pressure in the rat. Share and Melville (18) reported that ICV-administered picrotoxin also raises blood pressure in the cat. However, norepinephrine prolongs amobarbital-induced narcosis (2), while picrotoxin shortens narcosis. Likewise, the typical barbiturate-induced hypothermia (8) was in no way reversed by either picrotoxin or dibutyryl cyclic AMP. In contrast, d-amphetamine partially blocked the fall of rectal temperatures but lacked antianesthetic properties. Because of the failure to reverse amobarbital-induced hypothermia, we con cluded that the dose-related shortening of amobarbital-induced narcosis by di butyryl cyclic AMP and picrotoxin is not attributable to an increased metab olism of the barbiturate (1). While the analeptic drugs have been used to counter potential brain damage and the depressant effects of the barbiturates (7, 11, 17), the evidence shows overwhelmingly that none alters the clinical course of barbi turate overdosage (9, 11,14). One problem we have had to deal with is distinguishing between secondary system stimulation and/or recovery and CNS recovery. Researchers studying the reversal of barbiturate toxicity in one system have assumed that by antagonizing the depression in that system they will lighten the depth of narcosis. However, our evidence continues to support our contentions that CNS overdosage depression must be antagonized in the CNS. Of course, we have not excluded the possibility of shortened ST as an indirect effect of the analeptic drugs administered peripherally. But altered drug absorption, increased excretion, or biotransformation may shorten narcosis without protecting the brain from residual levels of tire barbiturate. Naik et at. (13) reported that treatment with analeptic drugs may increase the levels of barbiturate in the brain manyfold tire control values and therefore lead to more severe brain damage. Maloney et al. (10), Mousel and Essex (12), Mark (11), Robinson et al. (16); and Picchioni (15) have shown the increased dangers following treatment with analeptics.
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Our data suggest that picrotoxin and dibutyryl cyclic AMP do not arouse by stimulating the respiratory, cardiovascular, or temperature centers but by direct ly affecting the arousal center in the brain. Therefore, stimulation of the wake fulness center(s) of the brain is a specific property not shared by either the convulsant agents, or respiratory and cardiovascular stimulant drugs.
A cknowledgements The authors wish to express their gratitude to Dr. Myron Taube, Miss /Inun D. Francis and Miss Beverley Showman for their contributions to Ibis research.
1 Cohn, M.L.: in Fink Cyclic AMP, thyrotropin releasing factor and somatostatin-key factors in the regulation of narcosis (Raven Press, New York 1975). 2 Cohn, M.L.; Cohn, M.. and Taylor, F.H.: Norepinephrine an antagonist of dibutyryl cyclic AMP in the regulation of narcosis in the rat. Res. Commun. chem. Path. Pharmac. 7: 687-699(1974). 3 Cohn, M.L.: Cohn, M.: Taylor, F.H., and Scattaregia, F.: A direct effect of dibutyryl cyclic AMP on the duration of narcosis induced by sedative, hypnotic, tranquilizer and anesthetic drugs in the rat. Neuropharmacology (in press, 1975). 4 Cohn, M.L.; Taylor, F.H.; Cohn, M., and Yamaoka, H.: Dibutyryl cyclic AMP an effective antidote against lethal amounts of amobarbital in the rat. Res. Commun. chem. Path. Pharmac. 6: 435 445 (1973). 5 Cohn, M.L.; Yamaoka, H.; Taylor, F.H., and Kraynack, B : Action of intracerebroventricular dibutyryl cyclic AMP on amobarbital anesthesia in rats. Neuropharma cology 12: 401-405 (1973). 6 Goodman, L.S. and Gilman, A.: The pharmacological basis of therapeutics; 4th ed. (MacMillan. New York 1970). 7 Hahn, F.: Analeptics. Pharmac. Rev. 12: 447 -530 (1960). 8 Kroner, H. und Staib, W.: Barbital, Körpertemperatur und Proteinsynthese. Z. klin. Chem. 8: 41 - 44 (1970). 9 Locket, S. and Angus, J.: Poisoning by barbiturates; success of conservative treatment. L a n c e t 580-582(1952). 10 Maloney, A.H.; Fitch, R.H., and Tatum, A.L.: Picrotoxin as an antidote in acute poisoning by the shorter-acting barbiturates. J. Pharmac. exp. Ther. 41: 465 482 (1931). 11 Mark, L.C.: Analeptics: changing concepts declining status. Am. J. med. Sei. 254: 296-302(1967). 12 Mouse!, L.H. and Essex, H.E.: Experimental study of effects of respiratory stimulants in animals under pcntothal sodium anesthesia. Anesthesiology 2: 272-280 (1941). 13 Naik, S.R.: Gokhale, S. V., and Chittal, S.M.: Effect of analeptics on brain pento barbital levels in sleeping time in mice. Biochem. Pharmac. 18: 2038-2040 (1969). 14 Nilsson, E.: On treatment of barbiturate poisoning: a modified clinical aspect. Acta med. scand. 139: suppl. 253, pp. 1-127 (1951).
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References
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15 16 17 18
19 20
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Picchioni. A.L.: Clinical status and toxicology of analeptic drugs. Am. J. Hosp. Pharm. 28: 201 203 (1971). Robinson, R.R.: Gunnells, J.C., jr., and Clapp, J.R.: Treatment of acute barbiturate intoxication. Mod. Treatment 8: 561 -579 (1971). Setliy, V.H. and Sheth, U.K.: Factors affecting barbiturate sleeping time. Indian J. med.Sci.22: 507 566(1968). Share, N.N. and Melville, K.I.: Intraventricular injections of picrotoxin following central adrenergic blockade with phenoxybenzamine and dichloroisoproterenol. Int. J. Neuropharmac. 4: 149-156. Smythies, J.R.: Relationships between the chemical structure and biological activity of convulsants. A. Rev. Pharmac. 14: 9 21 (1974). Sokal, R.R. and Rohlf, F.J.: Biometry (Freeman, San Francisco 1969).
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Major L. Cohn, MD, PhD, Department of Anesthesiology, Magec-Womens Hospital, Pittsburgh, PA 15213 (USA)
Comparisons between the Antianesthetic Action of Dibutyryl Cyclic AMP and Analeptic Drugs on Amobarbital-lnduced Narcosis in the Rat1 Barry J. Kraynack, Major L. Cohn, Marthe Cohn arid Floyd H. Taylor Department of Anesthesiology, Harvard Medical School, Boston, Mass., Department of Anesthesiology, Magce-Womens Hospital, and Department of Community Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa.
Key Words. Analeptic drugs • Antianesthetic properties • Antidotal • CNS ■Dibutyryl cyclic AMP Abstract. The dose-related antianesthetic and antidotal property of dibutyryl cyclic AMP, devoid of toxic effects, imparts uniqueness to the nucleotide as an arousal agent. Of the analeptic drugs studied (d-amphetamine, picrotoxin, pentylenetetrazol, caffeine, the ophylline, strychnine, ethamivan and doxapram), only picrotoxin demonstrated anti anesthetic properties. However, picrotoxin was associated with severe toxicity at all dose levels tested. No analeptic drug is effective in reversing the central nervous system depression produced by sedative, hypnotic or tranquilizer drug overdosage.
We have reported that dibutyryl cyclic AMP, the dibutyryl analog of adenosine 3':5'-monophosphate (cyclic AMP) administered centrally, doserelatedly regulates the duration of amobarbital-induced narcosis in the rat (5). We have also shown that centrally administered dibutyryl cyclic AMP, doserelatedly regulates tire duration of narcosis induced by other sedative, hypnotic, tranquilizer and anesthetic agents (3). This antianesthetic property of dibutyryl cyclic AMP, devoid of subsequent generalized stimulation of the central nervous system (CNS), suggests that the nucleotide is a true CNS antidote to drugs inducing narcosis (4). This finding has a double significance: (1) easy availability of barbiturates has made acute barbiturate poisoning a constantly increasing source of morbidity and mortality; (2) at the present time there is no true 1 Supported by a National Institute of Mental Health grant DA00605.
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Received: July 24, 1975.
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Table 1. Effects of various analeptic drugs on amobarbital-induced (80 mg k g '1) nar cosis (mean ST in minutes t SE) Treatment group
Control (saline 0.9 %) Dibutyryl cyclic AMP1 Strychnine sulfate Caffeine Theophylline1 d-Amphetamine Pentylenetetrazol1 Ethamivan Doxapram1
Concentration (1CV) 1.2 X 1 0 '2 M
2.4 X 1 0 '2 M
104.0 ± 6.8 39.7* ± 4.6 115.1 ± 5.2 123.42 r 3.8 127.42 1 6.9 128.3s ± 5.2 120.42 ± 6.3 116.2 ± 7.1 185.12 t 18.2
26.62 ± 3.4 111.2 ± 8.6 121.5* ± 12.2 116.2 ± 5.6 123.8* ± 6.4 127.6* * 6.9 117.7 ± 16.0 152.62 ± 10.1
1 Statistically significant difference between the mean sleeping times at the different dose levels for this agent based on t-statistic at the 0.05 level of significance. 2 Statistically significant difference between treated and control groups at 0.05 level of significance based on the Student-Newman-Keuls test. There were 10 rats per group.
pharmacologic antagonist to the depressant action that sedative, hypnotic and tranquilizer drug overdosages exert on the CNS. The purpose of the present work is to compare the antianesthetic properties of the heterogeneous group of drugs called analeptics to those of dibutyryl cyclic AMP.
Sprague-Dawley male rats (Zivic Miller, Pittsburgh, Pa.) weighing 85-125 g were housed under constant environmental conditions. Prior to the experiment, they were al lowed free access to drinking water but were fasted overnight. Between 8.30 a.m. and 10.30 a.m., each rat was weighed and given an intraperitoneal (IP) injection of 1 % solution of sodium amobarbital (80 mg k g '1) (Amytal) (Eli Lilly, Indianapolis, Ind.). Immediately after the loss of the righting reflex (LRR), an opening in the skin was made with a scalpel, revealing the bregma. A small hole was drilled through the roof of the skull with a 25 gauge needle, 1.5 mm to the right of the sagittal suture and 1.5 mm below the coronal suture. The needle of a 25 m1 syringe (Unimetrics Universal Company, Anaheim, Calif.) was inserted 3.0 mm in depth and each rat was injected intracerebroventricularly (1CV) with 15 ¿d of varying doses od dibutyryl cyclic AMP. picrotoxine, d-amphetamine, pentylenetetrazol, caffeine, theophylline, strychnine (Sigma, St. Louis, Mo.); doxapram (Dopram®) (Robbins, Richmond. Va.);cthamivan (Emivan®), the latter generously donated by the US Vitamin and Pharmaceutical Corporation, New York (N.Y.) or saline 0.9 %. Awakening was defined as
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Table II. Modulation of amobarbital-induced narcosis (80 mg k g '1): comparison of the effects of dibutyryl cyclic AMP alone and dibutyryl cyclic AMP + doxapram (mean ST in minutes ± SE) Concentrations of dibutyryl cyclic AMP (ICV)
0M 0.4 X 1 0 '5 M 0.8 X 1 0 '! M 1.2 X 1 0 '2 M
Control group
Treated group
doxapram (ICV) OAf
doxapram (ICV) 1.8 X 1 0 '2 M
103.2 ± 8.7 72.81 ± 9.2 64.5' i 6.0 44.3' i 4.1
141.6' 102.5' 86.8' 65.5'
i 7.5 ± 7.6 t 11.9 t 14.0
' Statistically significant difference between control and treated groups at each con centration of dibutyryl cyclic AMP at 0.05 level of significance based on the StudentNewman-Kculs test. There were 10 rats per group.
regaining the righting reflex (RRR). Sleeping time (ST) defined as the period between LRR and RRR, was recorded for all experiment groups. At the time of RRR, the rats were decapitated. The needle tract was examined in each brain. Rats which did not show a definitive needle tract in the ventricle or had evidence of intracerebral hemorrhage were excluded from the series of experiments. Physiologic changes were carefully observed and noted. Rectal temperatures were recorded at 5-min intervals with a thermistor probe (Bailey Instruments, Saddle Brook, N.J.) until righting occurred. Descriptive statistics of the ST among the various treatment groups are given in tables I and II. In addition, certain tests of significance have been carried out using nonparamctric methods to compare the treated groups with the control. The results arc given in tables I and II (20).
Results
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In amobarbital anesthetized rats, d-amphetamine, pentylenetetrazol, ethamivan and theophylline did not affect the respiratory rate. Doxapram produced a deep but brief hyperventilation that lasted from 1 to 8 min; picrotoxin and caffeine produced a tachypnea that lasted at least 30 min. Besides hyperventilation, at all concentrations tested, picrotoxin induced Straub tail phenomenon, shivering, piloerection, moderate to severe convulsions, micturi tion, defecation, phonation and occasionally death. The picrotoxin-induced symptoms were dose-related; higher doses were followed by severe clonic con vulsions and a mortality of 30 %. No toxic symptoms were induced by the other analeptic agents. In contrast to picrotoxin, dibutyryl cyclic AMP produced no ill effects; neither did it produce any mortality.
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Fig. I. Effects of ICV injections of saline (0.9 %) and d-amphetamine (1.2 X 10~J and 2.4 X 1 0 '2 M) on the rectal temperature of amobarbital-anesthetized rats. There were 10 rats per group. The vcrticle lines indicate ± SE. • = Saline; = d-amphetamine.
Table III. Effects of various concentrations of picrotoxin on amobarbital-induced (80 mg k g '1) narcosis Concentrations (1CV) 0 M (control) 0.1 X 1 0 '3 M
1 X 1 0 '3 M 2 X 1 0 '3 M 4 X 1 0 '3 M
Mean ST, minutes ± SE 109.1 i 7.0 76.4' ± 4.6 42.51 * 5.5 34.41,J ♦ 3.0 36.61’2 t 3.8
1 Difference between the mean levels of ST for each concentration of picrotoxin is statistically significant between treated and control groups at the 0.05 level of signifi cance using the t test. 2 The only pair of mean ST which do not have a statistically significant difference, based on the Student-Newman-Keuls test at the 0.05 level of significance is the pair 34.4 and 36.6.
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The administration of d-amphetamine partially antagonized the amo barbital-induced hypothermia but did not alter sleeping times (fig. 1). No other agent tested reversed the hypothermia. The central administration of picrotoxin or dibutyryl cyclic AMP prior to induction of amobarbital did not delay the onset of narcosis. However, the
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administration of picrotoxin or dibutyryl cyclic AMP following LRR shortened narcosis dose-relatedly (5). In the dose ranges tested, tire other analeptic drugs were ineffective antianesthetic agents (tables I, III). Of the analeptic drugs tested, doxapram prolonged ST most significantly. To investigate this potentiation of narcosis, we administered a solution of doxapram and dibutyryl cyclic AMP to amobarbital anesthetized rats (table II). The usual shortening of narcosis that we found with dibutyryl cyclic AMP (5) was reversed, while the tachypnea was unaltered. Caffeine did not alter the duration of narcosis; picrotoxin shortened and doxapram prolonged it; however, all three agents induced tachypnea. No correlation was found between tire respi ratory rate and the antianesthetic action of the analeptic drugs.
Discussion
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The purpose of the present study is twofold: (1) to determine whether or not centrally administered analeptic drugs possess antianesthetic properties similar to those of dibutyryl cyclic AMP; (2) to determine whether or not a relationship exists between the respiratory, cardiovascular and thermal regula tion by these agents and the duration of narcosis. We chose these parameters because in barbiturate overdosage, three major physiologic systems are involved: the CNS, the respiratory system and the cardiovascular system. While death from barbiturate overdosage is believed to occur as a result of irreparable cardiac, renal or hepatic damage, we have shown that the primary cause of death is CNS depression (4). The results obtained showed that following central administration, picro toxin was the only analeptic drug tested to date that shortened the duration of narcosis. However, at all concentrations tested, picrotoxin was associated with severe toxicity. Doxapram significantly potentiated amobarbital-induced narcosis and antagonized the regulatory effects of dibutyryl cyclic AMP. Such findings suggest, therefore, that the antianesthetic property of an agent is not common to all central nervous stimulants but is a unique property possessed by specific agents. This view is supported by our findings that rats and squirrel monkeys administered amobarbital in the LD10o range showed impressive sur vival rates when treated centrally with dibutyryl cyclic AMP (4). This arousal was accomplished without generalized stimulation of tire CNS, suggesting that the nucleotide may be the first true antidote to barbiturate overdosage. Part of the problem, as we see it, is that the term analeptic has been ill-defined. Goodman and Gilman (6) define an analeptic as a CNS stimulant. However, CNS stimulation and arousal are not the same phenomenon; for example, convulsions, one result of CNS stimulation, may occur even under narcosis. It cannot be shown that CNS stimulation leading to increased cardio
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vascular or respiratory activity also leads to arousal. Arousal must involve some increase in conscious control: analeptic stimulation of the CNS leads to increased systemic activity without changing the level of consciousness. Another part of the problem is that the term analeptic covers a wide range of drugs which appear to operate through different systems and have a rather wide range of noncorrelated effects. For example, it has been reported that picrotoxin blocks the GABA receptor, and that strychnine blocks the glycine receptor (19). It has become increasingly difficult to accept the postulate that this widely varied group of drugs causes an identical biological action: arousal. The definition we prefer for analeptic is: any drug that reverses the hypnot ic effects of an agent at any level of tire CNS. We believe that narcosis, which is induced in the CNS, has to be reversed in the same system. The significance of our definition will become more clear later in our discussion. We have found that ICV-administered norepinephrine raises blood pressure in the rat. Share and Melville (18) reported that ICV-administered picrotoxin also raises blood pressure in the cat. However, norepinephrine prolongs amobarbital-induced narcosis (2), while picrotoxin shortens narcosis. Likewise, the typical barbiturate-induced hypothermia (8) was in no way reversed by either picrotoxin or dibutyryl cyclic AMP. In contrast, d-amphetamine partially blocked the fall of rectal temperatures but lacked antianesthetic properties. Because of the failure to reverse amobarbital-induced hypothermia, we con cluded that the dose-related shortening of amobarbital-induced narcosis by di butyryl cyclic AMP and picrotoxin is not attributable to an increased metab olism of the barbiturate (1). While the analeptic drugs have been used to counter potential brain damage and the depressant effects of the barbiturates (7, 11, 17), the evidence shows overwhelmingly that none alters the clinical course of barbi turate overdosage (9, 11,14). One problem we have had to deal with is distinguishing between secondary system stimulation and/or recovery and CNS recovery. Researchers studying the reversal of barbiturate toxicity in one system have assumed that by antagonizing the depression in that system they will lighten the depth of narcosis. However, our evidence continues to support our contentions that CNS overdosage depression must be antagonized in the CNS. Of course, we have not excluded the possibility of shortened ST as an indirect effect of the analeptic drugs administered peripherally. But altered drug absorption, increased excretion, or biotransformation may shorten narcosis without protecting the brain from residual levels of tire barbiturate. Naik et at. (13) reported that treatment with analeptic drugs may increase the levels of barbiturate in the brain manyfold tire control values and therefore lead to more severe brain damage. Maloney et al. (10), Mousel and Essex (12), Mark (11), Robinson et al. (16); and Picchioni (15) have shown the increased dangers following treatment with analeptics.
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Kraynack /Cohn /Cohn /Taylor
Cyclic AMP. Analeptic Drugs and Arousal
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Our data suggest that picrotoxin and dibutyryl cyclic AMP do not arouse by stimulating the respiratory, cardiovascular, or temperature centers but by direct ly affecting the arousal center in the brain. Therefore, stimulation of the wake fulness center(s) of the brain is a specific property not shared by either the convulsant agents, or respiratory and cardiovascular stimulant drugs.
A cknowledgements The authors wish to express their gratitude to Dr. Myron Taube, Miss /Inun D. Francis and Miss Beverley Showman for their contributions to Ibis research.
1 Cohn, M.L.: in Fink Cyclic AMP, thyrotropin releasing factor and somatostatin-key factors in the regulation of narcosis (Raven Press, New York 1975). 2 Cohn, M.L.; Cohn, M.. and Taylor, F.H.: Norepinephrine an antagonist of dibutyryl cyclic AMP in the regulation of narcosis in the rat. Res. Commun. chem. Path. Pharmac. 7: 687-699(1974). 3 Cohn, M.L.: Cohn, M.: Taylor, F.H., and Scattaregia, F.: A direct effect of dibutyryl cyclic AMP on the duration of narcosis induced by sedative, hypnotic, tranquilizer and anesthetic drugs in the rat. Neuropharmacology (in press, 1975). 4 Cohn, M.L.; Taylor, F.H.; Cohn, M., and Yamaoka, H.: Dibutyryl cyclic AMP an effective antidote against lethal amounts of amobarbital in the rat. Res. Commun. chem. Path. Pharmac. 6: 435 445 (1973). 5 Cohn, M.L.; Yamaoka, H.; Taylor, F.H., and Kraynack, B : Action of intracerebroventricular dibutyryl cyclic AMP on amobarbital anesthesia in rats. Neuropharma cology 12: 401-405 (1973). 6 Goodman, L.S. and Gilman, A.: The pharmacological basis of therapeutics; 4th ed. (MacMillan. New York 1970). 7 Hahn, F.: Analeptics. Pharmac. Rev. 12: 447 -530 (1960). 8 Kroner, H. und Staib, W.: Barbital, Körpertemperatur und Proteinsynthese. Z. klin. Chem. 8: 41 - 44 (1970). 9 Locket, S. and Angus, J.: Poisoning by barbiturates; success of conservative treatment. L a n c e t 580-582(1952). 10 Maloney, A.H.; Fitch, R.H., and Tatum, A.L.: Picrotoxin as an antidote in acute poisoning by the shorter-acting barbiturates. J. Pharmac. exp. Ther. 41: 465 482 (1931). 11 Mark, L.C.: Analeptics: changing concepts declining status. Am. J. med. Sei. 254: 296-302(1967). 12 Mouse!, L.H. and Essex, H.E.: Experimental study of effects of respiratory stimulants in animals under pcntothal sodium anesthesia. Anesthesiology 2: 272-280 (1941). 13 Naik, S.R.: Gokhale, S. V., and Chittal, S.M.: Effect of analeptics on brain pento barbital levels in sleeping time in mice. Biochem. Pharmac. 18: 2038-2040 (1969). 14 Nilsson, E.: On treatment of barbiturate poisoning: a modified clinical aspect. Acta med. scand. 139: suppl. 253, pp. 1-127 (1951).
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References
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Picchioni. A.L.: Clinical status and toxicology of analeptic drugs. Am. J. Hosp. Pharm. 28: 201 203 (1971). Robinson, R.R.: Gunnells, J.C., jr., and Clapp, J.R.: Treatment of acute barbiturate intoxication. Mod. Treatment 8: 561 -579 (1971). Setliy, V.H. and Sheth, U.K.: Factors affecting barbiturate sleeping time. Indian J. med.Sci.22: 507 566(1968). Share, N.N. and Melville, K.I.: Intraventricular injections of picrotoxin following central adrenergic blockade with phenoxybenzamine and dichloroisoproterenol. Int. J. Neuropharmac. 4: 149-156. Smythies, J.R.: Relationships between the chemical structure and biological activity of convulsants. A. Rev. Pharmac. 14: 9 21 (1974). Sokal, R.R. and Rohlf, F.J.: Biometry (Freeman, San Francisco 1969).
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Major L. Cohn, MD, PhD, Department of Anesthesiology, Magec-Womens Hospital, Pittsburgh, PA 15213 (USA)
