Neuropharmacolof]y, 1975, 14, 483 487. PergamonPress. Printed in Gt. Britain.
A DIRECT EFFECT OF DIBUTYRYL CYCLIC AMP ON THE DURATION OF NARCOSIS INDUCED BY S E D A T I V E , H Y P N O T I C , T R A N Q U I L I S E R AND ANAESTHETIC DRUGS IN THE RAT* M. L. COHN, M. COHN, F. H. TAYLOR and F. SCATTAREGIA Department of Anesthesiology, Magee-Womens Hospital and Department of Community Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213 (Accepted 24 November 1974) Summary The ability of exogenously administered N°,O2'-dibutyryl analogue of cyclic AMP to shorten, in a dose-related manner, the duration of narcosis of eight structurally different agents (chloral hydrate, paraldehyde, diazepam, amobarbital, halothane, ketamine, ethanol and methanol) has been demonstrated in the rat. No other nucleotide, neurotransmitter, biogenic amine or hormone tested, shortened, in a dose-related manner the sleeping time induced by the anaesthetic agents.
SUTHERLAND and RALL (1960) showed that the regulatory agent adenosine Y5'monophosphoric acid (cyclic AMP) controls a number of intracellular processes. It has since been reported that in rats, the N6,O2'-dibutyryl analogue of cyclic AMP, dibutyryl cyclic AMP, shortened, in a dose-related manner, the duration of amobarbitalinduced sleeping time (COHN, YAMAOKA,TAYLOR and KRAYNACK, 1973). To investigate the effects of exogenously administered dibutyryl cyclic AMP on narcosis induced by agents other than the barbiturates, we chose seven structurally different hypnotic agents: the aliphatic hypnotics chloral hydrate, ethanol, methanol, and paraldehyde; the minor tranquilizer diazepam; the phencyclidine derivative ketamine; and the inhalation agent halothane. METHODS
Male Sprague-Dawley rats (Zivic-Miller, Pittsburgh, Pennsylvania) weighing 80-125 g were housed under constant environmental conditions. In the morning, the animals were weighed and anaesthetized by the intraperitoneal injection of one of the anaesthetic agents under study in the following dose schedule: chloral hydrate (Noctec), 350 mg/kg (diluted 1:1 in sterile isotonic saline); diazepam, 30 mg/kg; paraldehyde, 1 ml/kg; ketamine, 150 mg/kg; halothane, 1 ml/kg; ethanol 25~o (v/v) 24 ml/kg; methanol 25~o (v/v) 33 ml/kg. For each of the anaesthetic agents the control group was left untreated, but mean sleeping time was recorded. In the experimental groups, following the loss of the righting reflex, an incision was made in the scalp and the bregma of the skull visualized. A 25 gauge needle was used to drill a hole through the roof of the skull 1.5 mm to the right of the sagittal suture and 1-5 mm below the coronal suture. The needle of a 25 /A syringe (Unimetrics Universal Company, Anaheim, California) partially covered with a plastic tubing shield was inserted to a depth of 3 mm into the right lateral ventricle of the brain. Fifteen #1 of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), cyclic AMP, N6,O2'-dibutyryl adenosfne 3':5' monophosphate (dibutyryl cyclic AMP), guanosine 5'-triphosphate (GTP), guanosine 3':5'-cyclic monophosphate (cyclic GMP), N6,O2'dibutyryl guanosine Y:5'-monophosphoric acid (dibutyryl cyclic GMP), sodium succinate, sodium pyruvate (Sigma Chemical Company, St. Louis, Missouri) or saline * This investigation was supported by NIMH Grant DA 00605. A preliminary report was presented at the Symposium on the Medical Aspects of Prostaglandins and Cyclic AMP, Ann Arbor, Michigan, 13-15 November 1972. 483
484
M.L. COUNet al.
0.9~0 diluted in 10-3 M phosphate buffer, pH 7.3, were injected intracerebroventricularly. The time from the loss of the righting reflex until the recovery of the righting reflex was measured in minutes and defined as the sleeping time. After a 24-hr observation period, the needle tract was examined in each brain. If intracerebral haemorrhage had occurred, or a definitive needle tract was not evident in the ventricle, the animal was excluded from the series of experiments. Statistical methods were applied in seeking evidence for significant differences among mean sleeping times for the groups of rats given varying doses of dibutyryl cyclic AMP for each of the various agents. The Student N e w m a n Kreuls test at the 0.05 level of significance was used (SOKAL and ROnLF, 1969).
RESULTS In every experimental group of anaesthetized rats, the doses of dibutyryl cyclic AMP shortened the sleeping time in a dose-related manner. No other adenine or guanosine nucleotide altered the sleeping time from the control values. Halothane (1 ml/kg) administered intraperitoneally induced narcosis in the nontreated group for an average of 100-3 min. Dose-relatedly, dibutyryl cyclic AMP shortened the sleeping time to 89.4, 69.1 and 48.5 min, respectively (Table 1). It was recently reported (Cony, 1975) that to test the hypothesis that centrally administered dibutyryl cyclic AMP does not alter the duration of narcosis by altered drug uptake, excretion or biotransformation but acts directly on the regulation of narcosis in the brain, rats were anaesthetized with halothane by an inhalational method. Without any interruption in the delivery of halothane (1~), rats treated centrally with dibutyryl cyclic AMP regained the righting reflex. Prior administration of dibutyryl cyclic AMP did not block the onset of the induction of narcosis. The descriptive statistics for the sleeping time (minutes) of rats given various agents in combination with varying levels of dibutyryl cyclic AMP are given in Table 1. The results of the statistical analyses were as follows: Based on the StudenWNewma~Kreuls test at the 0.05 level of significance, there was a statistically significant difference between any two mean levels of sleeping time within each of the various agents. Only intra-agent comparisons have been made (Table 1). Among the regression models considered for each of the agents, a particular model may be satisfactory for several agents but not for all agents. The search for a descriptive model that fits all agents has not been successful. We found a satisfactory model for ethanol and methanol but not for amobarbital. Paraldehyde does not appear to conform to any model proposed. A comparison of a regression model has been made for methanol and ethanol. The slopes of the regression equations for these two agents do not have a statistically significant difference. However, there is statistical evidence for rejecting the hypothesis that the two regression lines can be considered as one line, i.e. there are two distinct lines, having common slope, that describe methanol and ethanol. The results of this experiment confirm those obtained previously in rats anaesthetized with amobarbital (Cony e t al., 1973). Increasing the doses of intracerebroventricularly administered dibutyryl cyclic AMP decreased the sleeping time induced by all the agents tested. Our previous observations (COIN e t al,, 1973) were confirmed: (1) no dose of dibutyryl cyclic AMP, even as large as 180 lLg, affected the sleeping time for the first 20 min; (2) none of the behavioural changes described by GESSA, KRISHNA, FORN, TAGLIAMONTEand BRODIE (1970) following the intracerebroventricular injection of dibutyryl cyclic AMP to nonanaesthetized conscious rats occurred in any anaesthetized rats; (3t the prior administration of dibutyryl cyclic AMP did not delay the onset of narcosis induced by any of these agents; (4) sham and saline treatment, as well as adenosine and guanosine nucleotides administered intracerebroventricularly, did not alter the sleeping time induced by any of these agents from control sleeping time values in rats. The stereotyped behaviour of rats under ketamine anaesthesia
Cyclic AMP and narcosis
485
Table 1. Effect of 15 /11 dibutyryl cyclic A M P administered intracerebroventricularly to rats anaesthetized with various agents*
Treatment
0
Concentrations of dibutyryl cyclic A M P (llg/rat)t 30 60
90
Amobarbital 80 mg/kg
106.2 ± 5.5
76.8 :_+5.5
56-8 ± 3.7
37.7 ± 4.1
Paraldehyde I ml/kg
112-1 + 6-8
75.5 :4- 5-I
36.4 ± 3.6
26.5 _+2.9
Chloral hydrate 350 mg/kg
99.5 ± 8.4
88-2 :4- 5-3
72"5 ± 6-1
53.7 ± 3.7
Halothane 1 ml/kg
100.3 +5.1
89.4 ±6-5
69.1 +_ 10.1
48-5 ±4.1
Methanol (25!',,,) 33 ml/kg
102.0 ± 7.3
56.5 :~ 5.8
40-6 ± 3.2
28-0 ± 3-9
Ethanol t25',%) 25 ml/kg
128.0 ± 53
69.4 :~ 4.6
54.7 ± 4.5
40.2 ± 2.0
Ketamine 175 mg/kg
97.9 ± 6.8
75.3 ± 2.9
63.4 ± 5-0
53.6 ± 3-8
Diazepam 30 mg/kg
91.0 ± 7-2
44.5 1 5-6
34.3 + 4.8
22.7 ± 3.4
* Each group contains 10 rats. + Mean sleeping time (min ± S.E.M.).
was characterized by retention of protective reflexes: corneal, eyelid, laryngeal, pharyngeal. Lacrymation and changes in heart rate and blood pressure were unaltered by treatment. There was no evidence that narcosis induced by the dissociative anaesthetic ketamine, differed from the narcosis induced by the other anaesthetic agents as measured by the response to dibutyryl cyclic AMP. Our data indicated that pharmacological doses of dibutyryl cyclic AMP shortened dose-relatedly the duration of narcosis induced by all seven agents. DISCUSSION
The mechanism of narcosis induced by an anaesthetic, hypnotic, or sedative agent is unknown. No valid theory has been established. The accepted belief has been that the duration of narcosis was regulated solely by concentration of drug present in the brain. However, the supporting evidence is inconsistent (SMITH, 1961; MAZEL and BUSH, 1969). The literature does not support any obvious relationship between barbiturate levels in the brain and the duration of narcosis (JORI, B1ANCHETTI and PRESTINE, 1970; NAIK, GOKnALE and CHATTAL, 1969). Preliminary data obtained in a study currently undertaken confirm that, in rats, the barbiturate contents of the brain are not altered from the control values following central administration of dibutyryl cyclic AMP (personal observations). Prior research in rats has shown that dibutyryl cyclic AMP administered intracerebroventricularly shortened, in a dose-related manner, the duration of narcosis induced by amobarbital (COHN et al., 1973). The ability of dibutyryl cyclic AMP to shorten in a dose-dependent manner the duration of narcosis induced by eight structurally different agents, suggests that the antagonism might be universal to all agents that induce narcosis An empirical model fitting all the sleeping time and dose levels of dibutyryl cyclic AMP has not yet been determined. The complexity of the righting reflex that we chose as the behavioural end point of narcosis in our study may account for some of the difficulties we encountered. The same study with electroencephalographic data replacing the righting reflex has been undertaken but not completed. The finding that ethanol and methanol follow similar slopes even though the regression lines are distinct, encourages us to believe that a mathematical model may be found to fit all the data. In our in vivo system, we have tested a large number of catecholamines, biogenic amines, other neurotransmitters and hormones without obtaining any evidence of their
486
M.L. COHN et al.
antianaesthetic activity (COHN, COHN and TAYLOR, 1974). Confirming the data published by GRUNDEN (1969), we have reported that norepinephrine, believed to elevate in vitro brain levels of cyclic AMP in all areas of the brain (PALMER, SULSER and ROBISON, 1973), when administered centrally, antagonized in vivo the antianaesthetic properties of dibutyryl cyclic AMP (COHN et al., 1974). Recently it has been shown by DE BELLEROCHE, DAS and BRADFORD(1974) that in isolated synaptosomes, norepinephrine does not stimulate adenyl cyclase. With the exception of picrotoxin, analeptic drugs injected intracerebroventricularly into rats failed to demonstrate antianaesthetic properties (COHN, 1975). Recently, PRANGE, BREESE,MARTIN, COOPER, WILSON and PLOTNIKOFF(1974) reported that thyrotropin releasing factor served as a first messenger to the arousal mechanism in pentobarbital-induced narcosis in mice. While our data have confirmed that thyrotropin releasing factor shortens the duration of amobarbital-induced narcosis, no dose relationship was obtained (COHN and COHN, unpublished observations). Moreover, dibutyryl cyclic AMP and thyrotropin releasing factor lead to different behavioural syndromes when administered intracerebroventricularly to an unanaesthetized rat. Because dibutyryl cyclic AMP causes a hyperactive syndrome, and thyrotropin releasing factor causes a very brief span of sedation, the postulate that thyrotropin releasing factor is the first messenger seems improbable (COHN, 1975). PAUL, PAUK and DITZION (1970) and SCHMIDT and ROBISON (1971) have reported that barbiturates lowered levels of cyclic AMP in the brain. ISRAEL, KIMURA and KUR1YAMA(1972) and VOHClER and GOLD (1973) reported the same findings following ethanol-induced narcosis. However, other workers have found that halothane increases whole brain concentrations of cyclic AMP (BIEBUYCK, DEDRICK and SCHERER, 1975). Such inconsistent findings may be due to the fact that cyclic AMP levels in the brain have been measured in whole brain following the induction of narcosis with various drugs. Measurements currently in progress in our laboratory will determine whether or not anaesthetic agents stereotaxically implanted in the neuroanatomic pathways regulating narcosis (LEIGHTON and JENKINS, 1970) alter the levels of cyclic AMP in these specific areas of the brain. We know that: (t) the prior administration of dibutyryl cyclic AMP did not delay the onset of narcosis induced by any of the eight anaesthetic agents; (2) that centrally injected dibutyryl cyclic AMP antagonized the uninterrupted flow of halothane administered by an inhalational method; and (3) that levels of barbiturate in the brain were not altered by the administration of dibutyryl cyclic AMP. Thus, dibutyryl cyclic AMP did not act to decrease the absorption or cause the translocation of the anaesthetic drug. The mechanism of antagonism of narcosis was not a blocked response but rather the enhancement of a physiological opposing system. Although the present study suggests that cyclic AMP might be the common pathway and regulator of narcosis through which many, if not all, hypnotic agents may function, the physiologic role of cyclic AMP in the regulation of narcosis has yet to be determined. Acknowledgements---We are indebted to Dr. MYRON TAUBEand Miss ANNA D. FRANCISfor their contributions to this manuscript.
REFERENCES BIEBUYCK, J. F., DEDRICK, D. F. and SCHERER, Y. D. (t975). Brain cyclic AMP and putative transmitter amino acids during anesthesia. Molecular Mechanisms of Anesthesia (FIYK, B. R.. Ed.). Raven Press, New York. COHN, M. L. (1975). Cyclic AMP, thyrotropin releasing factor (TRF) and somatostatin key factors in the regulation of the duration of narcosis. In: Molecular Mechanisms o1' Anesthesia WINK, B. R., Ed.) Raven Press, New York. COHN, M. L., COHN, M. and TAYLOR, F. H. (1974). Norepinephrine an antagonist of dibutyryl cyclic AMP in the regulation of narcosis in the rat. Res. Comm. chem. Pathol. Pharmac. 7: 687- 699. COHN, M. L., YAMAOKA,H., TAYLOR, F. H. and KRAYNACK, B. (1973). Action of intracerebroventricular dibutyryl cyclic AMP on amobarbital anesthesia in rats. Neuropharmacology 12i 401 405. t)E BELLEROCHE,J. S., DAS, I. and BRADFORD, H. F. ~19741. Absence of an effect of histamine, noradrenaline and depolarizing agents on the levels of adenosine 3',5'-monophosphate in nerve endings isolated from cerebral cortex. Biochem. Pharmac. 23: 835-843.
Cyclic AMP and narcosis
487
GESSA, E. L., KRISHNA, G., FORN, J., TAGLIAMONTE, A. and BRODIE, B. B. (1970). Behavioral and vegetative effects produced by dibutyryl cyclic AMP injected into different areas of the brain. Adv. Biochem. Psychopharmacol. 3: 371-381.. GRUNDEN, L. R. (1969). Action of intracerebroventricular epinephrine on gross behavior, locomotor activity and hexobarbital sleeping times in rats. Int. J. Neuropharmac. 8: 573-586. ISRAEL, M. A., KIMURA, H. and KURIYAMA, K. (1972). Changes in activity and hormonal sensitivity of brain adenyl cyclase following chronic ethanol administration. Experientia 28: 132~1323. JORI, A., BIANeHETTI, A. and PRESTINE, P. E. (1970). Relation between barbiturate brain levels and sleeping time in various experimental conditions. Biochem. Pharmac. 19:. 2687-2694. LEIGHTON, K.-M. and JENKINS, L. C. (1970). Experimental studies of the central nervous systcm related to anaesthesia. IV. Effects of pentobarbital placement in caudata nucleus. Can. Anaesth. Soc. J. 17:. 112 118. MAZEL, P. and BUSH, M. T. (1969). Brain barbital levels and anesthesia as influenced by physiostigminc and epinephrine. Biochem. Pharmac. 18:579 586. NAIK, S. R., GOKHALE, S. V. and CHATTAL. S. M. (t969). Effect of analcptics on brain pentobarbital levels and sleeping time in mice. Biochem. Pharmac. 18:2038 2040. PALMER, G. C., SULSER, F. and RoBISON, G. A. (1973). Effects of neurohumoral and adrenergic agents on cyclic AMP level in various areas of rat brain in rivo. Neuropharmacology 12:327 337. PAUL, M. I., PAUK, G. L. and DITZION, B. R. (1970). Effect of centrally acting drugs on the concentration of brain adenosine 3',5'-monophosphate. Pharmacology (Basel) 3:148 154. PRANGE, A. J., BREESE, G. R., MARTIN, B. R. COOPER, B. R., WILSON', [. C. and PLOTN1KOFF, N. P. (1974). Thyrotropin releasing hormone: antagonism of pentobarbital in rodents. L(le Sci. 14:447 455. SCHMIDT, M. J. and ROBISON, G. A. (197l). The effect of norepinephrine on cyclic AMP levels in discrete rations of the developing brain. Life Sci., part 1 10: 459-464. SOKAL, R. R. and ROHLr, F. J. (1969). Biometry, 1st Edition. 767 pp. W. H. Freeman Company, San Francisco. SMITH, D. L. (1961). The effect of experimentally modified sleeping periods on brain levels of barbital. Biochem. Pharmac. 8:317 322. SUTHERLAND, E. W. and RALL, T. W. (1960). The relation of adenosine-Y,5'-phosphate and phosphorylasc to the actions of catecholamines and other hormones. Pharmac. Rer. 12: 26~299. VOLICER, L. and GOLD, B. I. (1973). Effect of ethanol on cyclic AMP levels in the rat brain. Lift' Sci. 13: 26%280.
A DIRECT EFFECT OF DIBUTYRYL CYCLIC AMP ON THE DURATION OF NARCOSIS INDUCED BY S E D A T I V E , H Y P N O T I C , T R A N Q U I L I S E R AND ANAESTHETIC DRUGS IN THE RAT* M. L. COHN, M. COHN, F. H. TAYLOR and F. SCATTAREGIA Department of Anesthesiology, Magee-Womens Hospital and Department of Community Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213 (Accepted 24 November 1974) Summary The ability of exogenously administered N°,O2'-dibutyryl analogue of cyclic AMP to shorten, in a dose-related manner, the duration of narcosis of eight structurally different agents (chloral hydrate, paraldehyde, diazepam, amobarbital, halothane, ketamine, ethanol and methanol) has been demonstrated in the rat. No other nucleotide, neurotransmitter, biogenic amine or hormone tested, shortened, in a dose-related manner the sleeping time induced by the anaesthetic agents.
SUTHERLAND and RALL (1960) showed that the regulatory agent adenosine Y5'monophosphoric acid (cyclic AMP) controls a number of intracellular processes. It has since been reported that in rats, the N6,O2'-dibutyryl analogue of cyclic AMP, dibutyryl cyclic AMP, shortened, in a dose-related manner, the duration of amobarbitalinduced sleeping time (COHN, YAMAOKA,TAYLOR and KRAYNACK, 1973). To investigate the effects of exogenously administered dibutyryl cyclic AMP on narcosis induced by agents other than the barbiturates, we chose seven structurally different hypnotic agents: the aliphatic hypnotics chloral hydrate, ethanol, methanol, and paraldehyde; the minor tranquilizer diazepam; the phencyclidine derivative ketamine; and the inhalation agent halothane. METHODS
Male Sprague-Dawley rats (Zivic-Miller, Pittsburgh, Pennsylvania) weighing 80-125 g were housed under constant environmental conditions. In the morning, the animals were weighed and anaesthetized by the intraperitoneal injection of one of the anaesthetic agents under study in the following dose schedule: chloral hydrate (Noctec), 350 mg/kg (diluted 1:1 in sterile isotonic saline); diazepam, 30 mg/kg; paraldehyde, 1 ml/kg; ketamine, 150 mg/kg; halothane, 1 ml/kg; ethanol 25~o (v/v) 24 ml/kg; methanol 25~o (v/v) 33 ml/kg. For each of the anaesthetic agents the control group was left untreated, but mean sleeping time was recorded. In the experimental groups, following the loss of the righting reflex, an incision was made in the scalp and the bregma of the skull visualized. A 25 gauge needle was used to drill a hole through the roof of the skull 1.5 mm to the right of the sagittal suture and 1-5 mm below the coronal suture. The needle of a 25 /A syringe (Unimetrics Universal Company, Anaheim, California) partially covered with a plastic tubing shield was inserted to a depth of 3 mm into the right lateral ventricle of the brain. Fifteen #1 of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), cyclic AMP, N6,O2'-dibutyryl adenosfne 3':5' monophosphate (dibutyryl cyclic AMP), guanosine 5'-triphosphate (GTP), guanosine 3':5'-cyclic monophosphate (cyclic GMP), N6,O2'dibutyryl guanosine Y:5'-monophosphoric acid (dibutyryl cyclic GMP), sodium succinate, sodium pyruvate (Sigma Chemical Company, St. Louis, Missouri) or saline * This investigation was supported by NIMH Grant DA 00605. A preliminary report was presented at the Symposium on the Medical Aspects of Prostaglandins and Cyclic AMP, Ann Arbor, Michigan, 13-15 November 1972. 483
484
M.L. COUNet al.
0.9~0 diluted in 10-3 M phosphate buffer, pH 7.3, were injected intracerebroventricularly. The time from the loss of the righting reflex until the recovery of the righting reflex was measured in minutes and defined as the sleeping time. After a 24-hr observation period, the needle tract was examined in each brain. If intracerebral haemorrhage had occurred, or a definitive needle tract was not evident in the ventricle, the animal was excluded from the series of experiments. Statistical methods were applied in seeking evidence for significant differences among mean sleeping times for the groups of rats given varying doses of dibutyryl cyclic AMP for each of the various agents. The Student N e w m a n Kreuls test at the 0.05 level of significance was used (SOKAL and ROnLF, 1969).
RESULTS In every experimental group of anaesthetized rats, the doses of dibutyryl cyclic AMP shortened the sleeping time in a dose-related manner. No other adenine or guanosine nucleotide altered the sleeping time from the control values. Halothane (1 ml/kg) administered intraperitoneally induced narcosis in the nontreated group for an average of 100-3 min. Dose-relatedly, dibutyryl cyclic AMP shortened the sleeping time to 89.4, 69.1 and 48.5 min, respectively (Table 1). It was recently reported (Cony, 1975) that to test the hypothesis that centrally administered dibutyryl cyclic AMP does not alter the duration of narcosis by altered drug uptake, excretion or biotransformation but acts directly on the regulation of narcosis in the brain, rats were anaesthetized with halothane by an inhalational method. Without any interruption in the delivery of halothane (1~), rats treated centrally with dibutyryl cyclic AMP regained the righting reflex. Prior administration of dibutyryl cyclic AMP did not block the onset of the induction of narcosis. The descriptive statistics for the sleeping time (minutes) of rats given various agents in combination with varying levels of dibutyryl cyclic AMP are given in Table 1. The results of the statistical analyses were as follows: Based on the StudenWNewma~Kreuls test at the 0.05 level of significance, there was a statistically significant difference between any two mean levels of sleeping time within each of the various agents. Only intra-agent comparisons have been made (Table 1). Among the regression models considered for each of the agents, a particular model may be satisfactory for several agents but not for all agents. The search for a descriptive model that fits all agents has not been successful. We found a satisfactory model for ethanol and methanol but not for amobarbital. Paraldehyde does not appear to conform to any model proposed. A comparison of a regression model has been made for methanol and ethanol. The slopes of the regression equations for these two agents do not have a statistically significant difference. However, there is statistical evidence for rejecting the hypothesis that the two regression lines can be considered as one line, i.e. there are two distinct lines, having common slope, that describe methanol and ethanol. The results of this experiment confirm those obtained previously in rats anaesthetized with amobarbital (Cony e t al., 1973). Increasing the doses of intracerebroventricularly administered dibutyryl cyclic AMP decreased the sleeping time induced by all the agents tested. Our previous observations (COIN e t al,, 1973) were confirmed: (1) no dose of dibutyryl cyclic AMP, even as large as 180 lLg, affected the sleeping time for the first 20 min; (2) none of the behavioural changes described by GESSA, KRISHNA, FORN, TAGLIAMONTEand BRODIE (1970) following the intracerebroventricular injection of dibutyryl cyclic AMP to nonanaesthetized conscious rats occurred in any anaesthetized rats; (3t the prior administration of dibutyryl cyclic AMP did not delay the onset of narcosis induced by any of these agents; (4) sham and saline treatment, as well as adenosine and guanosine nucleotides administered intracerebroventricularly, did not alter the sleeping time induced by any of these agents from control sleeping time values in rats. The stereotyped behaviour of rats under ketamine anaesthesia
Cyclic AMP and narcosis
485
Table 1. Effect of 15 /11 dibutyryl cyclic A M P administered intracerebroventricularly to rats anaesthetized with various agents*
Treatment
0
Concentrations of dibutyryl cyclic A M P (llg/rat)t 30 60
90
Amobarbital 80 mg/kg
106.2 ± 5.5
76.8 :_+5.5
56-8 ± 3.7
37.7 ± 4.1
Paraldehyde I ml/kg
112-1 + 6-8
75.5 :4- 5-I
36.4 ± 3.6
26.5 _+2.9
Chloral hydrate 350 mg/kg
99.5 ± 8.4
88-2 :4- 5-3
72"5 ± 6-1
53.7 ± 3.7
Halothane 1 ml/kg
100.3 +5.1
89.4 ±6-5
69.1 +_ 10.1
48-5 ±4.1
Methanol (25!',,,) 33 ml/kg
102.0 ± 7.3
56.5 :~ 5.8
40-6 ± 3.2
28-0 ± 3-9
Ethanol t25',%) 25 ml/kg
128.0 ± 53
69.4 :~ 4.6
54.7 ± 4.5
40.2 ± 2.0
Ketamine 175 mg/kg
97.9 ± 6.8
75.3 ± 2.9
63.4 ± 5-0
53.6 ± 3-8
Diazepam 30 mg/kg
91.0 ± 7-2
44.5 1 5-6
34.3 + 4.8
22.7 ± 3.4
* Each group contains 10 rats. + Mean sleeping time (min ± S.E.M.).
was characterized by retention of protective reflexes: corneal, eyelid, laryngeal, pharyngeal. Lacrymation and changes in heart rate and blood pressure were unaltered by treatment. There was no evidence that narcosis induced by the dissociative anaesthetic ketamine, differed from the narcosis induced by the other anaesthetic agents as measured by the response to dibutyryl cyclic AMP. Our data indicated that pharmacological doses of dibutyryl cyclic AMP shortened dose-relatedly the duration of narcosis induced by all seven agents. DISCUSSION
The mechanism of narcosis induced by an anaesthetic, hypnotic, or sedative agent is unknown. No valid theory has been established. The accepted belief has been that the duration of narcosis was regulated solely by concentration of drug present in the brain. However, the supporting evidence is inconsistent (SMITH, 1961; MAZEL and BUSH, 1969). The literature does not support any obvious relationship between barbiturate levels in the brain and the duration of narcosis (JORI, B1ANCHETTI and PRESTINE, 1970; NAIK, GOKnALE and CHATTAL, 1969). Preliminary data obtained in a study currently undertaken confirm that, in rats, the barbiturate contents of the brain are not altered from the control values following central administration of dibutyryl cyclic AMP (personal observations). Prior research in rats has shown that dibutyryl cyclic AMP administered intracerebroventricularly shortened, in a dose-related manner, the duration of narcosis induced by amobarbital (COHN et al., 1973). The ability of dibutyryl cyclic AMP to shorten in a dose-dependent manner the duration of narcosis induced by eight structurally different agents, suggests that the antagonism might be universal to all agents that induce narcosis An empirical model fitting all the sleeping time and dose levels of dibutyryl cyclic AMP has not yet been determined. The complexity of the righting reflex that we chose as the behavioural end point of narcosis in our study may account for some of the difficulties we encountered. The same study with electroencephalographic data replacing the righting reflex has been undertaken but not completed. The finding that ethanol and methanol follow similar slopes even though the regression lines are distinct, encourages us to believe that a mathematical model may be found to fit all the data. In our in vivo system, we have tested a large number of catecholamines, biogenic amines, other neurotransmitters and hormones without obtaining any evidence of their
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M.L. COHN et al.
antianaesthetic activity (COHN, COHN and TAYLOR, 1974). Confirming the data published by GRUNDEN (1969), we have reported that norepinephrine, believed to elevate in vitro brain levels of cyclic AMP in all areas of the brain (PALMER, SULSER and ROBISON, 1973), when administered centrally, antagonized in vivo the antianaesthetic properties of dibutyryl cyclic AMP (COHN et al., 1974). Recently it has been shown by DE BELLEROCHE, DAS and BRADFORD(1974) that in isolated synaptosomes, norepinephrine does not stimulate adenyl cyclase. With the exception of picrotoxin, analeptic drugs injected intracerebroventricularly into rats failed to demonstrate antianaesthetic properties (COHN, 1975). Recently, PRANGE, BREESE,MARTIN, COOPER, WILSON and PLOTNIKOFF(1974) reported that thyrotropin releasing factor served as a first messenger to the arousal mechanism in pentobarbital-induced narcosis in mice. While our data have confirmed that thyrotropin releasing factor shortens the duration of amobarbital-induced narcosis, no dose relationship was obtained (COHN and COHN, unpublished observations). Moreover, dibutyryl cyclic AMP and thyrotropin releasing factor lead to different behavioural syndromes when administered intracerebroventricularly to an unanaesthetized rat. Because dibutyryl cyclic AMP causes a hyperactive syndrome, and thyrotropin releasing factor causes a very brief span of sedation, the postulate that thyrotropin releasing factor is the first messenger seems improbable (COHN, 1975). PAUL, PAUK and DITZION (1970) and SCHMIDT and ROBISON (1971) have reported that barbiturates lowered levels of cyclic AMP in the brain. ISRAEL, KIMURA and KUR1YAMA(1972) and VOHClER and GOLD (1973) reported the same findings following ethanol-induced narcosis. However, other workers have found that halothane increases whole brain concentrations of cyclic AMP (BIEBUYCK, DEDRICK and SCHERER, 1975). Such inconsistent findings may be due to the fact that cyclic AMP levels in the brain have been measured in whole brain following the induction of narcosis with various drugs. Measurements currently in progress in our laboratory will determine whether or not anaesthetic agents stereotaxically implanted in the neuroanatomic pathways regulating narcosis (LEIGHTON and JENKINS, 1970) alter the levels of cyclic AMP in these specific areas of the brain. We know that: (t) the prior administration of dibutyryl cyclic AMP did not delay the onset of narcosis induced by any of the eight anaesthetic agents; (2) that centrally injected dibutyryl cyclic AMP antagonized the uninterrupted flow of halothane administered by an inhalational method; and (3) that levels of barbiturate in the brain were not altered by the administration of dibutyryl cyclic AMP. Thus, dibutyryl cyclic AMP did not act to decrease the absorption or cause the translocation of the anaesthetic drug. The mechanism of antagonism of narcosis was not a blocked response but rather the enhancement of a physiological opposing system. Although the present study suggests that cyclic AMP might be the common pathway and regulator of narcosis through which many, if not all, hypnotic agents may function, the physiologic role of cyclic AMP in the regulation of narcosis has yet to be determined. Acknowledgements---We are indebted to Dr. MYRON TAUBEand Miss ANNA D. FRANCISfor their contributions to this manuscript.
REFERENCES BIEBUYCK, J. F., DEDRICK, D. F. and SCHERER, Y. D. (t975). Brain cyclic AMP and putative transmitter amino acids during anesthesia. Molecular Mechanisms of Anesthesia (FIYK, B. R.. Ed.). Raven Press, New York. COHN, M. L. (1975). Cyclic AMP, thyrotropin releasing factor (TRF) and somatostatin key factors in the regulation of the duration of narcosis. In: Molecular Mechanisms o1' Anesthesia WINK, B. R., Ed.) Raven Press, New York. COHN, M. L., COHN, M. and TAYLOR, F. H. (1974). Norepinephrine an antagonist of dibutyryl cyclic AMP in the regulation of narcosis in the rat. Res. Comm. chem. Pathol. Pharmac. 7: 687- 699. COHN, M. L., YAMAOKA,H., TAYLOR, F. H. and KRAYNACK, B. (1973). Action of intracerebroventricular dibutyryl cyclic AMP on amobarbital anesthesia in rats. Neuropharmacology 12i 401 405. t)E BELLEROCHE,J. S., DAS, I. and BRADFORD, H. F. ~19741. Absence of an effect of histamine, noradrenaline and depolarizing agents on the levels of adenosine 3',5'-monophosphate in nerve endings isolated from cerebral cortex. Biochem. Pharmac. 23: 835-843.
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GESSA, E. L., KRISHNA, G., FORN, J., TAGLIAMONTE, A. and BRODIE, B. B. (1970). Behavioral and vegetative effects produced by dibutyryl cyclic AMP injected into different areas of the brain. Adv. Biochem. Psychopharmacol. 3: 371-381.. GRUNDEN, L. R. (1969). Action of intracerebroventricular epinephrine on gross behavior, locomotor activity and hexobarbital sleeping times in rats. Int. J. Neuropharmac. 8: 573-586. ISRAEL, M. A., KIMURA, H. and KURIYAMA, K. (1972). Changes in activity and hormonal sensitivity of brain adenyl cyclase following chronic ethanol administration. Experientia 28: 132~1323. JORI, A., BIANeHETTI, A. and PRESTINE, P. E. (1970). Relation between barbiturate brain levels and sleeping time in various experimental conditions. Biochem. Pharmac. 19:. 2687-2694. LEIGHTON, K.-M. and JENKINS, L. C. (1970). Experimental studies of the central nervous systcm related to anaesthesia. IV. Effects of pentobarbital placement in caudata nucleus. Can. Anaesth. Soc. J. 17:. 112 118. MAZEL, P. and BUSH, M. T. (1969). Brain barbital levels and anesthesia as influenced by physiostigminc and epinephrine. Biochem. Pharmac. 18:579 586. NAIK, S. R., GOKHALE, S. V. and CHATTAL. S. M. (t969). Effect of analcptics on brain pentobarbital levels and sleeping time in mice. Biochem. Pharmac. 18:2038 2040. PALMER, G. C., SULSER, F. and RoBISON, G. A. (1973). Effects of neurohumoral and adrenergic agents on cyclic AMP level in various areas of rat brain in rivo. Neuropharmacology 12:327 337. PAUL, M. I., PAUK, G. L. and DITZION, B. R. (1970). Effect of centrally acting drugs on the concentration of brain adenosine 3',5'-monophosphate. Pharmacology (Basel) 3:148 154. PRANGE, A. J., BREESE, G. R., MARTIN, B. R. COOPER, B. R., WILSON', [. C. and PLOTN1KOFF, N. P. (1974). Thyrotropin releasing hormone: antagonism of pentobarbital in rodents. L(le Sci. 14:447 455. SCHMIDT, M. J. and ROBISON, G. A. (197l). The effect of norepinephrine on cyclic AMP levels in discrete rations of the developing brain. Life Sci., part 1 10: 459-464. SOKAL, R. R. and ROHLr, F. J. (1969). Biometry, 1st Edition. 767 pp. W. H. Freeman Company, San Francisco. SMITH, D. L. (1961). The effect of experimentally modified sleeping periods on brain levels of barbital. Biochem. Pharmac. 8:317 322. SUTHERLAND, E. W. and RALL, T. W. (1960). The relation of adenosine-Y,5'-phosphate and phosphorylasc to the actions of catecholamines and other hormones. Pharmac. Rer. 12: 26~299. VOLICER, L. and GOLD, B. I. (1973). Effect of ethanol on cyclic AMP levels in the rat brain. Lift' Sci. 13: 26%280.
