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Experimental cerebrovascular disorders: Effects of papaverine and theophylline
EXPERIMENTAL
NEUROLffiY
Experimental
56, 469-479 (1977)
Cerebrovascular Disorders: Effects of Papaverine and Theophylline ESA
Department
of Pharmacology, Received
October
R.
HEIKKINEN'
University 20,1975;
of Oulzl, SF-90220
revision received
March
Oulu 22, Finland 7,1977
This study investigated the role of endogenous adenosine in eliciting the increases in cerebrospinal fluid cyclic adenosine 3’,5’-monophosphate concentrations induced by experimental cerebrovascular disorders in rabbits. One group of animals received theophylline, an adenosine antagonist, and another group papaverine, an inhibitor of adenosine uptake, for 7 successive days before the operations. Control animals were similarly pretreated with physiologic saline. Papaverine significantly augmented the acute increase in cyclic CAMP concentration of cerebrospinal fluid whereas theophylline leveled off this elevation response. The presenti findings apparently imply an important role of adenosine in leading to accumulation of cyclic AMP in the cerebrospinal fluid after the experimental cerebrovascular impairment. A further assessment of various adenosine antagonists in the treatment of such disorders seems to be indicated.
INTRODUCTION Cyclic adenosine 3’,5’-monophosphate (CAMP) concentration in the cerebrospinal fluid was described to be elevated after experimentally induced cerebrovascular disorders (11) and after electrical cerebral injury (20) in rabbits. The rise represents a net effect of various metabolic disturbances on functionally important regions of CAMP metabolism in the central nervous system. Experiments using cerebral slice techniques have revealed an important role of adenosine in the accumulation of CAMP Abbreviations : CAMP-cyclic adenosine 3’,5’-monophosphate, CSF-cerebrospinal fluid, ASAT-aspartate aminotransferase. 1 The author thanks Professor Jorma Hirvonen, M.D., Department of Forensic Medicine, University of Oulu, for his expert guidance in the analysis of the histologic preparations, and Mrs. Pirjo Moisio and Mrs. Liisa Hukkanen for their skillful technical assistance. This study was supported by grants from the Medical Council of the Academy of Finland, the Finnish Culture Foundation, and the Medical Factory Orion. 469 Copyri ht 0 1977 by Academic Press, Inc. All rig ‘i ts of reproduction in any form reserved.
ISSN
0014-4886
470
ESA
R.
HEIKKINEN
elicited by biogenic amines (27), electrical pulses, potassium ions (40)) and depolarizing agents (31). Adenosine formation in tivo is known to increase during anoxia, whereas the amount of phosphorylated adenine nucleotides decreases (2, 3). The increased adenosine concentration may lead to augmented stimulation of the adenyl cyclase system, which in turn might cause enhanced anaerobic glycolysis through phosphorylation of phosphorylases (34,35). The effect of adenosine is blocked by theophylline, an adenosine anatagonist (5, 27)) as well as by other methylxanthines (10) and by adenosine deaminase (15), but not by the ,&adrenergic blocking agent propranolol (33). On the other hand, agents which inhibit uptake of adenosine, e.g., dipyridamole, hexobendine, and papaverine, potentiate the adenosineelicited accumulation of CAMP (16). Studies with OI- and ,&adrenergic agonists and antagonists have led to the suggestion that the effect of catecholamines on CAMP concentration is mediated in guinea pigs by the classical a-adrenergic receptor (28). The results from rat experiments using methoxamine, a relatively pure a-agonist, lend further support to the opinion (32). However, the relative importance of (Y- and ,8-adrenergic agents is known to be dependent on species, strains within a species, and region of the central nervous system (32). Aminophylline, a xanthine derivative like theophylline, was used clinically 10 to 20 years ago in the treatment of stroke as a vasodilator agent (8). However, more recent methods for measuring hemispheric and regional cerebral blood flow have revealed aminophylline to be a rather potent data show that vasoconstrictor agent (9, 26), whereas experimental aminophylline effectively reverses vasospasm (6). A beneficial “reverse steal” phenomenon, i.e., increase of cerebral blood flow in poorly perfused regions, in patients with various cerebrovascular diseases has been attributed by other investigators to some xanthine derivatives and to papaverine-like drugs (14). Papaverine was demonstrated to be a vasodilator in cerebral arteries of stroke patients (24). In addition to the opposite effects of theophylline and papaverine in adenosine-elicited accumulation of CAMP and differences in their effects on cerebral blood vessels, these two drugs share one common property : They both inhibit phosphodiesterases, the enzymes hydrolyzing intracellular cyclic nucleotides. The concentrations of theophylline required to inhibit the enzyme by 50% (Iso) at physiologic CAMP concentrations have been reported to be 500 pM in rat erythrocytes (29) ; in the same range in canine cerebral cortex, 600 pM (30) ; and in rat brain, 580 PM (23), and 120 pM (1) in V&U. The 150 concentration of papaverine is much less, about 10 PM in canine and rat cerebral cortex (23, 30). The present study aimed to clarify the participation of endogenous adenosine in experimentally induced cerebrovascular disorders to CAMP accum~~-
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lation in the CSF. The effects of,papaverine and theophylline on CSF constituents [i.e., CAMP, proteins, and aspartate aminotransferase (ASAT) activity] were recorded before and after experimental cerebrovascular dysfunction in unanesthetized rabbits. MATERIALS
AND
METHODS
Adult albino rabbits of either sex weighing 2.5 to 3.5 kg, were used. One group received 13 mg/kg papaverine (Papaverin, Medica, Finland) as one daily subcutaneous dose for 7 successive days before operation. The rabbits in another group were similarly treated by theophylline (Theophyllaminum, Medica, Finland) 67 “g/kg, subcutaneously. Multiple doses were adopted to ascertain an effective drug concentration in plasma. Control animals received physiologic saline subcutaneously once a day for 1 week. The drug therapies were continued until the second postoperative day. No gross behavioral changes or neurological symptoms were seen to be associated with the drug treatments. The left carotid artery was occluded, followed by intracarotid injection of 0.5 ml liquid paraffin in all animals under surgical ether anesthesia as described previously ( 11). The operations were scheduled to be performed at 10 to 12 AM, 2 h after the last injection of each drug. Cisternal CSF samples of 0.5 to 1.0 ml were collected under light ether anesthesia before the beginning of drug treatment, at the second and fifth day in the course of treatment, immediately before occlusion procedures at the seventh day (preoperative sample), and 1, 3, 6, and 24 h, and 2, 5, and 14 days after the operation. All samples with an erythrocyte count more than 2000 x 103/ml were discarded from further analysis. The rate of successful cisternal punctures was about 80% as described earlier (39). From the CSF samples, the CAMP concentration was assayed by the protein-binding method of Gilman (7) and expressed as picomoles/milliliter or nM. The protein concentration was measured by the biuret method (19) as milligrams per milliliter CSF. The activity of aspartate aminowas assayed by a test kit (Baker Diagnostic Retransferase (ASAT) agents, J. T. Baker Chemicals N.V., Deventer, The Netherlands) and expressed as international units per liter (IU/liter) at 37°C. Cerebral samples were prepared for histological investigations as described previously ( 11) . The results were treated statistically by Student’s t test and by the t test for paired observations. RESULTS Three of six died of the saline-treated animals, within the first 24 postoperative hours or developed neurologic deficits making the continuation
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basal
after pretreatment
41
,
R.
HEIKKINEN
343
6h
443
I day
343
443
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Zdoyr
Sdayr
Zweekr
FIG. 1. Cerebrospinal fluid CAMP concentrations in the basal situation, after drug treatment for 7 days, and at various times after carotid occlusion plus intracarotid injection of liquid paraffin. Open bars-animals treated by physiologic saline (controls), shaded bars-by papaverine, and solid bars-by theophylline. All drug therapies ceased at the second postoperative day. The number of animals is shown below the respective bars and the standard error. Large asteriskI’< 0.05, three large asterisks-P < 0.001 when compared to respective values of controls; small asteriskP < 0.05, two small asterisks-P < 0.01, and three small asterisks-P < 0.001, using the t-test for paired observations.
of the experiments unjustifiable, One rabbit of four died during the second day of theophylline treatment probably because of puncture complications. Another rabbit from the group of four animals treated with papaverine died at the 20th postoperative day, whereas all other animals survived without gross neurologic symptoms or changes in other parameters, e.g., weight gain. Figure 1 depicts CSF CAMP concentration before and during drug therapy and at various times after the experimentally induced cerebrovascular disorder. The CAMP concentrations in the preoperative CSF samplesof theophylline-treated animals were significantly higher than those of controls (P < 0.05), whereas the CSF CAMP values of rabbits receiving papaverine were in the range of control concentrations. The difference between control animals and theophylline-treated rabbits was statistically significant also at the first (P < O.OOl), second (P < O.OOl), and fifth postoperative day (P < 0.05). In all three groups of animals, CSF CAMP concentration was significantly elevated at the 1st postoperative hour (P < 0.05 for controls, P < 0.001 for rabbits treated by papaverine, P < 0.01 for ani-
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mals treated by theophylline ; t tests for paired observations). This finding is in accordance with my previous results from experimental cerebrovascular disorders (11). The extent and duration of CSF CAMP rise was augmented by papaverine treatment, the values differing significantly from their preoperative concentrations at the first (P < 0.001 ), third (P < 0.005), sixth (P < 0.01)) and even at the 24th postoperative hour (P < 0.05 ; t tests for paired observations). Contrary to this, theophylline treatment abolished the CSF CAMP rise also at the third postoperative hour, but the CAMP values were significantly above the initial basal values up to the 14th postoperative day (P < 0.02 ; this significant difference is not shown in Fig. 1). The increase of CSF CAMP concentration from the preoperative value was significantly greater in the papaverine-treated group than in rabbits which had received theophylline. This was evident at the first (P < 0.005) and third postoperative hours (P < 0.01). From Fig. 2 it can be seen that CSF protein concentration was significantly higher than the respective preoperative value in control animals at the third postoperative hour (P < 0.05). An increasing trend was seen also at the sixth postoperative hour both in control and papaverine-treated rabbits. The CSF protein values remained relatively constant during the entire time they were measured after the operation in the animals treated
313
basal
423
after pretreatment
313
,h
21
6h
313
1 day
323
213
223
Ldayr
Sdays
2weeks
FIG. 2. Cerebrospinal fluid protein concentration in the basal situation, after drug treatment for 7 days, and at various times after the experimental cerebrovascular disorders. Symbols are the same as in Fig. 1. Small asterisk--P < 0.05 (t test for paired observations).
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FIG.
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was killed infiltrations
Hippocampal at the 14th were found.
R.
HEIKKINEN
neurons of a rabbit treated with theophylline. postoperative day. Neither microinfarctions nor The cells stained normally.
The animal perivascular
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with theophylline, from 0.2 to 2.3 mg/ml, mean 1.13 + 0.14 (SE, n = 18). There were no significant changes in the ASAT activities of the CSF in relation to the experimental procedures nor to drug treatments. The values were from 6 to 25 III/liter, with means of 13.9 -C 1.4 III/liter (H = 10) in the control animals, 13.8 * 1.2 (n = 18) in the papaverine-treated animals, and 14.2 f 1.5 (n = 12) in theophylline-treated rabbits. Histologic Findings. Severe ischemic changes in neurons, especially in the parietal cortex and hippocampus, were evident in cerbral sections prepared at the 14th postoperative day from control animals. Contrary to this, there was no gross neuronal damage either in samples prepared after 2 weeks (n = 1) or 3 weeks (n = 2) from animals treated by theophylline. Histologic findings in rabbits which had received papaverine also seemed to indicate milder damage than those of controls (cerebral sections prepared after 5 weeks follow-up time). Figure 3 represents typical cerebral histology of a rabbit treated by theophylline. DISCUSSION The cerebral CAMP concentrations of the experimental animals are increased within a few seconds or minutes after exposure to anoxia (18, 34) or mechanical trauma (36). The rise of this cyclic nucleotide concentration in the CSF 1 to 3 hours after electroconvulsive shock (21) or experimentally induced cerebrovascular disorder ( 11)) demonstrated in rabbits, apparently reflects leakage of CAMP out of neural or glial cells and its subsequent diffusion into the surrounding CSF. In clinical studies, a rise in CSF CAMP concentration was reported in epileptic patients shortly after an attack, in humans suffering from active damaging processes of the central nervous system (22), and in patients after cerebral infarction (37, 38) or other severe cerebrovascular disorder (12, 13). The extent of the CSF CAMP rise is suggested to be positively correlated to the severity of acute cerebral damage (20). In the present investigation, the mechanism of the rise in CAMP concentration in the CSF in connection with experimentally induced cerebrovascular disorder in rabbits was studied. Special attention was given to the role of adenosine, which elicits at least partially the effects of various factors on the accumulation of cerebral CAMP. Theophylline, an adenosine antagonist (27), blocked the acute elevation of CSF CAMP concentration after the experimental procedures, leveled off the increase in CSF protein concentration during the entire follow-up time of 14 days, and also prevented the severe ischemic cerebral changes seen in saline-treated control animals. No strict conclusions about the protective effect of theophylline in rerebrovascular disorders can be drawn, because no standard cerebral
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lesion was produced despite the use of identical experimental methods as possible for producing the cerebrovascular dysfunction in all animals. The rise in the baseline concentration of CSF CAMP during theophylline treatment is not clearly explained, but it may be caused by secondary changes in adenine derivative metabolism induced by the antagonism of adenosine. The inhibition of phosphodiesterases does not seem a probable mechanism, because theophylline is shown to be a relatively ineffective phosphodiesterase inhibitor in brain tissue (4, 17). The cause of the delayed increase in CSF CAMP concentration (i.e., from the 1st postoperative day onwards) may refer to an ability of theophylline to protect the brain from anoxic damage at the first postoperative day. In addition, it supports the qualitative result of the inverse correlation of severity of the experimentally induced cerebrovascular disorder to the duration of the rise in CSF CAMP concentration (11). The increase of c,AMP concentration may even be an expression in the brain of reflex defense processes against cerebral ischemia. Papaverine augmented the rise in CAMP concentration induced by the experimental cerebrovascular disorder to as much as the 24th postoperative hour. This observation apparently indicates the ability of papaverine to inhibit adenosine uptake (16)) thus increasing extracellular adenosine and its effect on the adenyl cyclase system. The contribution of phosphodiesterase inhibition is not totally excluded but seems improbable, because no rise in CSF CAMP concentration was seen during the treatment by papapaverine. In addition, Huang and Daly (16) have reported that potentiation of the effects of low concentrations of adenosine by agents which inhibit its uptake parallels more closely their efficacy as inhibitors of adenosine uptake rather than their potency as phosphodiesterase inhibitors. In the present study, papaverine did not inhibit a slight rise of CSF protein concentration, apparently indicating loss of its protective effect against cerebral/cerebellar/blood-brain barrier lesions produced by experimental cerebrovascular disorder. In the study of Watanabe and Passonneau (36)) the P-adrenergic receptor blocking agents, dichloroisoproterenol and pronethalol, did not inhibit the rise in cerebral CAMP concentration after mechanical trauma in mice. The suggestion that norepinephrine is not involved in the rise of CAMP level does not seem to be sufficiently supported by the experimental results, if we assume that norepinephrine-shown to be altered in cerebral tissue by anoxia (18, 25)-p rimarily causes the rise in CAMP concentration by acting on the classical a-receptors. The participation of a-receptors and the role of adenosine in CAMP accumulation are discussed in more detail in the work of Skolnick and Daly (32). In summary, the increase of CSF CAMP concentration after experimental cerebrovascular disorder \vas
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augmented by papaverine treatment and leveled off by theophylline. These findings are consistent with the hypothesis that adenosine has a crucial role in eliciting accumulation of CAMP in the CSF. A further experimental and clinical assessment of various adenosine antagonists in the treatment of cerebrovascular disorder seems to be indicated. REFERENCES 1. BEER, B., M. CHASIN, D. E. CLODY, J. R. VOGEL, AND Z. P. HOROVITZ. 1972. Cyclic adenosine monophosphate phosphodiesterase in brain : effect on anxiety. Science 176 : 428430. 2. BERNE, R. M., R. RUBIO, J. G. DOBSON, JR., AND R. R. CURNISH. 1971. Adenosine and adenine nucleotides as possible mediators of cardiac and skeletal muscle blood flow regulation. Circ. Res. 28, Suppl. 1: 11.5-119. 3. COLLINS, R. C., J. V. POSNER, AND F. PLUM. 1970. Cerebral energy metabolism during electroshock seizures in mice. Amer. J. Physiol. 218: 943-950. 4. DALTON, C., H. J. CROWLEY, H. SHEPPARD, AND W. SCHALLEK. 1974. Regional cyclic nucleotide phosphodiesterase activity in cat central nervous system : effects of benzodiazepines. Proc. Sot. Exp. Biol. Med. 145: 407-410. 5. DALY, J. W. 1976. The nature of receptors regulating the formation of cyclic AMP in brain tissue. Life Sci. 18 : 1349-1358. 6. FLAMM, E. S., J. KIM, J. LIN, AND J. RANSOHOFF. 1975. Phosphodiesterase inhibitors and cerebral vasospasm. Arch. Neurol. 32: 569-571. 7. GILMAN, A. G. 1970. A protein binding assay for adenosine 3’,5’-cyclic monophosphate. Proc. Nat. Acad. Sci. USA 67: 305-312. 8. GOTTSTEIN, U. 1962. Der Hirnkreislauf water dem Einfuss Vasoaktiver Substansen. Dr. Hiithig Verlag, Heidelberg. 9. GOTTSTEIN, U., AND 0. B. PAULSON. 1972. The effect of intracarotid aminophylline infusion on the cerebral circulation. Stroke 3: 560-565. 10. GREEN, R. D., AND L. R. STANBERRY. 1977. Elevation of cyclic AMP in C-1300 murine neuroblastoma by adenosine and related compounds and the antagonism of this response by methylxanthines. Biochem. Pharmacol. 2L: 37-44. 11. HEIKKINEN, E. R. 1977. Experimental cerebrovascular disorders: Effects on cardiovascular functions and on cerebrospinal fluid production and constituents with special reference to cyclic AMP. Exp. Nezlrol. 56 : 451-468. 12. HEIKRINEN, E. R., V. V. MYLLYL~~, E. HOKKANEN, AND H. VAPAATALO. 1976. Cerebrospinal fluid concentration of cyclic AMP in cerebrovascular diseases. Eur. Neural. 14: 129-137. 13. HEIKKINEN, E. R., H. VAPAATALO, V. V. MYLLYLX, AND E. HOKKANEN. 1975. Role of cyclic AMP in neurological diseases. Advan. Cyclic Nucleotide Res. 5: 815. 14. HEISS, W.-D. 1973. Drug effects on regional cerebral blood flow in focal cerebrovascular disease. J. Neural. Sci. 19 : 461-482. 15. HUANG, M., E. GRUENSTEIN, AND J. W. DALY. 1973. Depolarization-evoked accumulation of cyclic AMP in brain slices: Inhibition by exogenous adenosine deaminase. Biochim. Biophys. Acta 329 : 147-151. 16. HUANG, Mz, AND J. W. DALY. 1974. Adenosine-elicited accumulation of cyclic AMP in brain slices: Potentiation by agents which inhibit uptake of adenosine. Life Sci. 14 : 489-503. 17. KAKIUCHI, S., R. YAMAZAKI, Y. TESHIMA, K. UENISHI, AND E. MIYAMOTO.
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1975. Multiple cyclic nucleotide phosphodiesterase activities from rat tissues and occurrence of a calcium-plus-magnesium-ion-dependent phosphodiesterase and its protein activator. Biochem. J. 146: 109-120. KOGURE, K., P. SCHEINBERG, A. MATSUMOTO, R. BUSTO, AND 0. M. REINMUTH. 1975. Catecholamines in experimental brain ischemia. Arch. Neurol. 32: 21-24. LAYNE, E. 1955. Spectrophotometric and turbidimetric methods for measuring proteins. Page 447 ilt S. P. CULOWICK AND N. 0. KAPLAN, EDS. Methods in Enzymology, Vol. 1 Academic Press, New York. MYLLYL~~, V. V. 1976. Effect of cerebral injury on cerebrospinal fluid cyclic AMP concentration. Eur. Neural. 14 : 413-425. MYLLYL;~, V. V. 1976. Effect of convulsions and anticonvulsive drugs on cerebrospinal fluid cyclic AMP in rabbits. Eur. Neural. 14: 97-107. MYLLYL~~, V. V., E. R. HEIKKINEN, H. VAPAATALO, AND E. HOKKANEN. 1975. Cyclic AMP concentration and enzyme activities of cerebrospinal fluid in patients with epilepsy or central nervous system damage. Eur. Neural. 13: 123130. NAHORSKI, S. R., K. J. ROGERS, AND J. BINNS. 1973. Cerebral phosphodiesterase and the dopamine receptor. J. Pharm. Pharmacol. 25: 912-913. OLESEN, J., AND 0. B. PAULSON. 1971. The effect of intraarterial papaverine on the regional cerebral blood flow in patients with stroke or intracranial tumor. Stroke 2: 148-159. PAUSESCU, E., R. LUGOJAN, AND M. PAUSESCU. 1970. Cerebral catecholamine and serotonin metabolism in post-hypothermic brain oedema. Brain 93: 31-36. REGLI, F., T. YAMAGUCHI, AND A. G. WALTZ. 1971. Responses of surface arteries and blood flow of ischemic and nonischemic cerebral cortex to aminophylline, ergotamine tartrate, and acetazolamide. Stroke 2 : 461-470. SATTIN, A., AND T. W. RALL. 1970. The effect of adenosine and adenine nucleotides on the cyclic adenosine 3’,5’-phosphate content of guinea pig cerebral cortex slices. Mol. Pharmacol. 6 : 13-23. SCHULTZ, J., AND J. W. DALY. 1973. Adenosine 3’,5’-monophosphate in guinea pig cerebral cortical slices: Effects of a- and @-adrenergic agents, histamine, serotonin and adenosine. I. Neurochem. 21: 573-579. SHEPPARD, H., AND G. N. WICCAN. 1971. Analogues of 4-(3,4-dimethoxybenzyl)2-imidazolidinone as potent inhibitors of rat erythrocyte adenosine cyclic 3’,5’phosphate phosphodiesterase. Mol. Pharmacol. 7 : 111-115. SHEPPARD, H., AND G. N. WIGGAN. 1971. Different sensitivities of the phosphodiesterases (adenosine-3’,5’-cyclic phosphate 3’-phosphohydrolase) of dog cerebral cortex and erythrocytes to inhibition by synthetic agents and cold. Biothem.
Pharmacol.
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31. SHIMIZU, H., AND J. W. DALY. 1972. Effect of depolarizing agents on accumulation of cyclic adenosine 3’,.5’-monophosphate in cerebral cortical slices. Eur. J. Pharmacol. 17 : 240-252. 32. SKOLNICK, P., AND J. W. DALY. 1975. Stimulation of adenosine 3’,5’-monophosphate formation in rat cerebral cortical slices by methoxamine: Interaction with an alpha-adrenergic receptor. .I. Pharmacol. Exp. Ther. 193: 54%558. 33. SOMERVILLE, A. R., AND A. A. SMITH. 1972. The effects of propranolol and electrical stimulation on the cyclic 3’,5’-AMP content of isolated cerebral tissue. J. Neurochem. 19 : 2003-2006. 34. STEFANOVJCH, V., AND J. P. JOHN. 1974. The increase of cyclic AMP in rat’s brain during anoxia. Res. Commun. Chews. Pathol. Pharmacol. 9: 591-593.
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35. WATANABE, H., AND J. V. PASSONNEAU. 1974. The effect of trauma on cerebral glycogen and related metabolites and enzymes. Brain. Res. 66: 147-159. 36. WATANABE, H., AND J. V. PASSONNEAU. 1975. Cyclic adenosine monophosphate in cerebral cortex. Alterations following trauma. Arch. Ne~rol. 32: 181-184. 37. WELCH, K. M. A., J. S. MEYER, AND A. N . CHEE. 1975. Effects of cerebral infarction in man on cyclic AMP levels in CSF and blood. Advan. Cyclic Nucleotide Res. 5 : 814. 38. WELCH, K. M. A., J. S. MEYER, AND A. N. CHEE. 1975. Evidence for disordered cyclic AMP metabolism in patients with cerebral infarction. Eur. Ncur02. 13: 144-153. 39. YLITALO, P., E. R. HEIKKINEN, AND V. V. MYLLYLX. 1976. Evaluation of successive collections of cisternal cerebrospinal fluid in rats, rabbits, and cats. Exp. Neural. 50 : 330-336. 40. ZANELLA, J., JR., AND T. W. RALL. 1973. Evaluation of electrical pulses and elevated levels of potassium ions as stimulants of adenosine 3’,5’-monophosphate (cyclic AMP) accumulation in guinea-pig brain. J. Pharmacol. Exp. Ther. l&6: 241-252.
NEUROLffiY
Experimental
56, 469-479 (1977)
Cerebrovascular Disorders: Effects of Papaverine and Theophylline ESA
Department
of Pharmacology, Received
October
R.
HEIKKINEN'
University 20,1975;
of Oulzl, SF-90220
revision received
March
Oulu 22, Finland 7,1977
This study investigated the role of endogenous adenosine in eliciting the increases in cerebrospinal fluid cyclic adenosine 3’,5’-monophosphate concentrations induced by experimental cerebrovascular disorders in rabbits. One group of animals received theophylline, an adenosine antagonist, and another group papaverine, an inhibitor of adenosine uptake, for 7 successive days before the operations. Control animals were similarly pretreated with physiologic saline. Papaverine significantly augmented the acute increase in cyclic CAMP concentration of cerebrospinal fluid whereas theophylline leveled off this elevation response. The presenti findings apparently imply an important role of adenosine in leading to accumulation of cyclic AMP in the cerebrospinal fluid after the experimental cerebrovascular impairment. A further assessment of various adenosine antagonists in the treatment of such disorders seems to be indicated.
INTRODUCTION Cyclic adenosine 3’,5’-monophosphate (CAMP) concentration in the cerebrospinal fluid was described to be elevated after experimentally induced cerebrovascular disorders (11) and after electrical cerebral injury (20) in rabbits. The rise represents a net effect of various metabolic disturbances on functionally important regions of CAMP metabolism in the central nervous system. Experiments using cerebral slice techniques have revealed an important role of adenosine in the accumulation of CAMP Abbreviations : CAMP-cyclic adenosine 3’,5’-monophosphate, CSF-cerebrospinal fluid, ASAT-aspartate aminotransferase. 1 The author thanks Professor Jorma Hirvonen, M.D., Department of Forensic Medicine, University of Oulu, for his expert guidance in the analysis of the histologic preparations, and Mrs. Pirjo Moisio and Mrs. Liisa Hukkanen for their skillful technical assistance. This study was supported by grants from the Medical Council of the Academy of Finland, the Finnish Culture Foundation, and the Medical Factory Orion. 469 Copyri ht 0 1977 by Academic Press, Inc. All rig ‘i ts of reproduction in any form reserved.
ISSN
0014-4886
470
ESA
R.
HEIKKINEN
elicited by biogenic amines (27), electrical pulses, potassium ions (40)) and depolarizing agents (31). Adenosine formation in tivo is known to increase during anoxia, whereas the amount of phosphorylated adenine nucleotides decreases (2, 3). The increased adenosine concentration may lead to augmented stimulation of the adenyl cyclase system, which in turn might cause enhanced anaerobic glycolysis through phosphorylation of phosphorylases (34,35). The effect of adenosine is blocked by theophylline, an adenosine anatagonist (5, 27)) as well as by other methylxanthines (10) and by adenosine deaminase (15), but not by the ,&adrenergic blocking agent propranolol (33). On the other hand, agents which inhibit uptake of adenosine, e.g., dipyridamole, hexobendine, and papaverine, potentiate the adenosineelicited accumulation of CAMP (16). Studies with OI- and ,&adrenergic agonists and antagonists have led to the suggestion that the effect of catecholamines on CAMP concentration is mediated in guinea pigs by the classical a-adrenergic receptor (28). The results from rat experiments using methoxamine, a relatively pure a-agonist, lend further support to the opinion (32). However, the relative importance of (Y- and ,8-adrenergic agents is known to be dependent on species, strains within a species, and region of the central nervous system (32). Aminophylline, a xanthine derivative like theophylline, was used clinically 10 to 20 years ago in the treatment of stroke as a vasodilator agent (8). However, more recent methods for measuring hemispheric and regional cerebral blood flow have revealed aminophylline to be a rather potent data show that vasoconstrictor agent (9, 26), whereas experimental aminophylline effectively reverses vasospasm (6). A beneficial “reverse steal” phenomenon, i.e., increase of cerebral blood flow in poorly perfused regions, in patients with various cerebrovascular diseases has been attributed by other investigators to some xanthine derivatives and to papaverine-like drugs (14). Papaverine was demonstrated to be a vasodilator in cerebral arteries of stroke patients (24). In addition to the opposite effects of theophylline and papaverine in adenosine-elicited accumulation of CAMP and differences in their effects on cerebral blood vessels, these two drugs share one common property : They both inhibit phosphodiesterases, the enzymes hydrolyzing intracellular cyclic nucleotides. The concentrations of theophylline required to inhibit the enzyme by 50% (Iso) at physiologic CAMP concentrations have been reported to be 500 pM in rat erythrocytes (29) ; in the same range in canine cerebral cortex, 600 pM (30) ; and in rat brain, 580 PM (23), and 120 pM (1) in V&U. The 150 concentration of papaverine is much less, about 10 PM in canine and rat cerebral cortex (23, 30). The present study aimed to clarify the participation of endogenous adenosine in experimentally induced cerebrovascular disorders to CAMP accum~~-
CEREBROVASCULAR
DISORDERS
AND
ADENOSINE
471
lation in the CSF. The effects of,papaverine and theophylline on CSF constituents [i.e., CAMP, proteins, and aspartate aminotransferase (ASAT) activity] were recorded before and after experimental cerebrovascular dysfunction in unanesthetized rabbits. MATERIALS
AND
METHODS
Adult albino rabbits of either sex weighing 2.5 to 3.5 kg, were used. One group received 13 mg/kg papaverine (Papaverin, Medica, Finland) as one daily subcutaneous dose for 7 successive days before operation. The rabbits in another group were similarly treated by theophylline (Theophyllaminum, Medica, Finland) 67 “g/kg, subcutaneously. Multiple doses were adopted to ascertain an effective drug concentration in plasma. Control animals received physiologic saline subcutaneously once a day for 1 week. The drug therapies were continued until the second postoperative day. No gross behavioral changes or neurological symptoms were seen to be associated with the drug treatments. The left carotid artery was occluded, followed by intracarotid injection of 0.5 ml liquid paraffin in all animals under surgical ether anesthesia as described previously ( 11). The operations were scheduled to be performed at 10 to 12 AM, 2 h after the last injection of each drug. Cisternal CSF samples of 0.5 to 1.0 ml were collected under light ether anesthesia before the beginning of drug treatment, at the second and fifth day in the course of treatment, immediately before occlusion procedures at the seventh day (preoperative sample), and 1, 3, 6, and 24 h, and 2, 5, and 14 days after the operation. All samples with an erythrocyte count more than 2000 x 103/ml were discarded from further analysis. The rate of successful cisternal punctures was about 80% as described earlier (39). From the CSF samples, the CAMP concentration was assayed by the protein-binding method of Gilman (7) and expressed as picomoles/milliliter or nM. The protein concentration was measured by the biuret method (19) as milligrams per milliliter CSF. The activity of aspartate aminowas assayed by a test kit (Baker Diagnostic Retransferase (ASAT) agents, J. T. Baker Chemicals N.V., Deventer, The Netherlands) and expressed as international units per liter (IU/liter) at 37°C. Cerebral samples were prepared for histological investigations as described previously ( 11) . The results were treated statistically by Student’s t test and by the t test for paired observations. RESULTS Three of six died of the saline-treated animals, within the first 24 postoperative hours or developed neurologic deficits making the continuation
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after pretreatment
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,
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6h
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I day
343
443
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Zdoyr
Sdayr
Zweekr
FIG. 1. Cerebrospinal fluid CAMP concentrations in the basal situation, after drug treatment for 7 days, and at various times after carotid occlusion plus intracarotid injection of liquid paraffin. Open bars-animals treated by physiologic saline (controls), shaded bars-by papaverine, and solid bars-by theophylline. All drug therapies ceased at the second postoperative day. The number of animals is shown below the respective bars and the standard error. Large asteriskI’< 0.05, three large asterisks-P < 0.001 when compared to respective values of controls; small asteriskP < 0.05, two small asterisks-P < 0.01, and three small asterisks-P < 0.001, using the t-test for paired observations.
of the experiments unjustifiable, One rabbit of four died during the second day of theophylline treatment probably because of puncture complications. Another rabbit from the group of four animals treated with papaverine died at the 20th postoperative day, whereas all other animals survived without gross neurologic symptoms or changes in other parameters, e.g., weight gain. Figure 1 depicts CSF CAMP concentration before and during drug therapy and at various times after the experimentally induced cerebrovascular disorder. The CAMP concentrations in the preoperative CSF samplesof theophylline-treated animals were significantly higher than those of controls (P < 0.05), whereas the CSF CAMP values of rabbits receiving papaverine were in the range of control concentrations. The difference between control animals and theophylline-treated rabbits was statistically significant also at the first (P < O.OOl), second (P < O.OOl), and fifth postoperative day (P < 0.05). In all three groups of animals, CSF CAMP concentration was significantly elevated at the 1st postoperative hour (P < 0.05 for controls, P < 0.001 for rabbits treated by papaverine, P < 0.01 for ani-
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mals treated by theophylline ; t tests for paired observations). This finding is in accordance with my previous results from experimental cerebrovascular disorders (11). The extent and duration of CSF CAMP rise was augmented by papaverine treatment, the values differing significantly from their preoperative concentrations at the first (P < 0.001 ), third (P < 0.005), sixth (P < 0.01)) and even at the 24th postoperative hour (P < 0.05 ; t tests for paired observations). Contrary to this, theophylline treatment abolished the CSF CAMP rise also at the third postoperative hour, but the CAMP values were significantly above the initial basal values up to the 14th postoperative day (P < 0.02 ; this significant difference is not shown in Fig. 1). The increase of CSF CAMP concentration from the preoperative value was significantly greater in the papaverine-treated group than in rabbits which had received theophylline. This was evident at the first (P < 0.005) and third postoperative hours (P < 0.01). From Fig. 2 it can be seen that CSF protein concentration was significantly higher than the respective preoperative value in control animals at the third postoperative hour (P < 0.05). An increasing trend was seen also at the sixth postoperative hour both in control and papaverine-treated rabbits. The CSF protein values remained relatively constant during the entire time they were measured after the operation in the animals treated
313
basal
423
after pretreatment
313
,h
21
6h
313
1 day
323
213
223
Ldayr
Sdays
2weeks
FIG. 2. Cerebrospinal fluid protein concentration in the basal situation, after drug treatment for 7 days, and at various times after the experimental cerebrovascular disorders. Symbols are the same as in Fig. 1. Small asterisk--P < 0.05 (t test for paired observations).
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was killed infiltrations
Hippocampal at the 14th were found.
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neurons of a rabbit treated with theophylline. postoperative day. Neither microinfarctions nor The cells stained normally.
The animal perivascular
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with theophylline, from 0.2 to 2.3 mg/ml, mean 1.13 + 0.14 (SE, n = 18). There were no significant changes in the ASAT activities of the CSF in relation to the experimental procedures nor to drug treatments. The values were from 6 to 25 III/liter, with means of 13.9 -C 1.4 III/liter (H = 10) in the control animals, 13.8 * 1.2 (n = 18) in the papaverine-treated animals, and 14.2 f 1.5 (n = 12) in theophylline-treated rabbits. Histologic Findings. Severe ischemic changes in neurons, especially in the parietal cortex and hippocampus, were evident in cerbral sections prepared at the 14th postoperative day from control animals. Contrary to this, there was no gross neuronal damage either in samples prepared after 2 weeks (n = 1) or 3 weeks (n = 2) from animals treated by theophylline. Histologic findings in rabbits which had received papaverine also seemed to indicate milder damage than those of controls (cerebral sections prepared after 5 weeks follow-up time). Figure 3 represents typical cerebral histology of a rabbit treated by theophylline. DISCUSSION The cerebral CAMP concentrations of the experimental animals are increased within a few seconds or minutes after exposure to anoxia (18, 34) or mechanical trauma (36). The rise of this cyclic nucleotide concentration in the CSF 1 to 3 hours after electroconvulsive shock (21) or experimentally induced cerebrovascular disorder ( 11)) demonstrated in rabbits, apparently reflects leakage of CAMP out of neural or glial cells and its subsequent diffusion into the surrounding CSF. In clinical studies, a rise in CSF CAMP concentration was reported in epileptic patients shortly after an attack, in humans suffering from active damaging processes of the central nervous system (22), and in patients after cerebral infarction (37, 38) or other severe cerebrovascular disorder (12, 13). The extent of the CSF CAMP rise is suggested to be positively correlated to the severity of acute cerebral damage (20). In the present investigation, the mechanism of the rise in CAMP concentration in the CSF in connection with experimentally induced cerebrovascular disorder in rabbits was studied. Special attention was given to the role of adenosine, which elicits at least partially the effects of various factors on the accumulation of cerebral CAMP. Theophylline, an adenosine antagonist (27), blocked the acute elevation of CSF CAMP concentration after the experimental procedures, leveled off the increase in CSF protein concentration during the entire follow-up time of 14 days, and also prevented the severe ischemic cerebral changes seen in saline-treated control animals. No strict conclusions about the protective effect of theophylline in rerebrovascular disorders can be drawn, because no standard cerebral
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lesion was produced despite the use of identical experimental methods as possible for producing the cerebrovascular dysfunction in all animals. The rise in the baseline concentration of CSF CAMP during theophylline treatment is not clearly explained, but it may be caused by secondary changes in adenine derivative metabolism induced by the antagonism of adenosine. The inhibition of phosphodiesterases does not seem a probable mechanism, because theophylline is shown to be a relatively ineffective phosphodiesterase inhibitor in brain tissue (4, 17). The cause of the delayed increase in CSF CAMP concentration (i.e., from the 1st postoperative day onwards) may refer to an ability of theophylline to protect the brain from anoxic damage at the first postoperative day. In addition, it supports the qualitative result of the inverse correlation of severity of the experimentally induced cerebrovascular disorder to the duration of the rise in CSF CAMP concentration (11). The increase of c,AMP concentration may even be an expression in the brain of reflex defense processes against cerebral ischemia. Papaverine augmented the rise in CAMP concentration induced by the experimental cerebrovascular disorder to as much as the 24th postoperative hour. This observation apparently indicates the ability of papaverine to inhibit adenosine uptake (16)) thus increasing extracellular adenosine and its effect on the adenyl cyclase system. The contribution of phosphodiesterase inhibition is not totally excluded but seems improbable, because no rise in CSF CAMP concentration was seen during the treatment by papapaverine. In addition, Huang and Daly (16) have reported that potentiation of the effects of low concentrations of adenosine by agents which inhibit its uptake parallels more closely their efficacy as inhibitors of adenosine uptake rather than their potency as phosphodiesterase inhibitors. In the present study, papaverine did not inhibit a slight rise of CSF protein concentration, apparently indicating loss of its protective effect against cerebral/cerebellar/blood-brain barrier lesions produced by experimental cerebrovascular disorder. In the study of Watanabe and Passonneau (36)) the P-adrenergic receptor blocking agents, dichloroisoproterenol and pronethalol, did not inhibit the rise in cerebral CAMP concentration after mechanical trauma in mice. The suggestion that norepinephrine is not involved in the rise of CAMP level does not seem to be sufficiently supported by the experimental results, if we assume that norepinephrine-shown to be altered in cerebral tissue by anoxia (18, 25)-p rimarily causes the rise in CAMP concentration by acting on the classical a-receptors. The participation of a-receptors and the role of adenosine in CAMP accumulation are discussed in more detail in the work of Skolnick and Daly (32). In summary, the increase of CSF CAMP concentration after experimental cerebrovascular disorder \vas
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augmented by papaverine treatment and leveled off by theophylline. These findings are consistent with the hypothesis that adenosine has a crucial role in eliciting accumulation of CAMP in the CSF. A further experimental and clinical assessment of various adenosine antagonists in the treatment of cerebrovascular disorder seems to be indicated. REFERENCES 1. BEER, B., M. CHASIN, D. E. CLODY, J. R. VOGEL, AND Z. P. HOROVITZ. 1972. Cyclic adenosine monophosphate phosphodiesterase in brain : effect on anxiety. Science 176 : 428430. 2. BERNE, R. M., R. RUBIO, J. G. DOBSON, JR., AND R. R. CURNISH. 1971. Adenosine and adenine nucleotides as possible mediators of cardiac and skeletal muscle blood flow regulation. Circ. Res. 28, Suppl. 1: 11.5-119. 3. COLLINS, R. C., J. V. POSNER, AND F. PLUM. 1970. Cerebral energy metabolism during electroshock seizures in mice. Amer. J. Physiol. 218: 943-950. 4. DALTON, C., H. J. CROWLEY, H. SHEPPARD, AND W. SCHALLEK. 1974. Regional cyclic nucleotide phosphodiesterase activity in cat central nervous system : effects of benzodiazepines. Proc. Sot. Exp. Biol. Med. 145: 407-410. 5. DALY, J. W. 1976. The nature of receptors regulating the formation of cyclic AMP in brain tissue. Life Sci. 18 : 1349-1358. 6. FLAMM, E. S., J. KIM, J. LIN, AND J. RANSOHOFF. 1975. Phosphodiesterase inhibitors and cerebral vasospasm. Arch. Neurol. 32: 569-571. 7. GILMAN, A. G. 1970. A protein binding assay for adenosine 3’,5’-cyclic monophosphate. Proc. Nat. Acad. Sci. USA 67: 305-312. 8. GOTTSTEIN, U. 1962. Der Hirnkreislauf water dem Einfuss Vasoaktiver Substansen. Dr. Hiithig Verlag, Heidelberg. 9. GOTTSTEIN, U., AND 0. B. PAULSON. 1972. The effect of intracarotid aminophylline infusion on the cerebral circulation. Stroke 3: 560-565. 10. GREEN, R. D., AND L. R. STANBERRY. 1977. Elevation of cyclic AMP in C-1300 murine neuroblastoma by adenosine and related compounds and the antagonism of this response by methylxanthines. Biochem. Pharmacol. 2L: 37-44. 11. HEIKKINEN, E. R. 1977. Experimental cerebrovascular disorders: Effects on cardiovascular functions and on cerebrospinal fluid production and constituents with special reference to cyclic AMP. Exp. Nezlrol. 56 : 451-468. 12. HEIKRINEN, E. R., V. V. MYLLYL~~, E. HOKKANEN, AND H. VAPAATALO. 1976. Cerebrospinal fluid concentration of cyclic AMP in cerebrovascular diseases. Eur. Neural. 14: 129-137. 13. HEIKKINEN, E. R., H. VAPAATALO, V. V. MYLLYLX, AND E. HOKKANEN. 1975. Role of cyclic AMP in neurological diseases. Advan. Cyclic Nucleotide Res. 5: 815. 14. HEISS, W.-D. 1973. Drug effects on regional cerebral blood flow in focal cerebrovascular disease. J. Neural. Sci. 19 : 461-482. 15. HUANG, M., E. GRUENSTEIN, AND J. W. DALY. 1973. Depolarization-evoked accumulation of cyclic AMP in brain slices: Inhibition by exogenous adenosine deaminase. Biochim. Biophys. Acta 329 : 147-151. 16. HUANG, Mz, AND J. W. DALY. 1974. Adenosine-elicited accumulation of cyclic AMP in brain slices: Potentiation by agents which inhibit uptake of adenosine. Life Sci. 14 : 489-503. 17. KAKIUCHI, S., R. YAMAZAKI, Y. TESHIMA, K. UENISHI, AND E. MIYAMOTO.
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