The cyclic AMP system of human brain

Brain Research, 50 (1973) 135-146

135

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

THE CYCLIC AMP SYSTEM OF H U M A N BRAIN

TAKAFUMI KODAMA, YASUHIKO MATSUKADO AND HIROTOSHI SHIMIZU* Kumamoto University Medical School, Department of Neurosurgery and First Department of Pharmacology, Honjo, Kumamoto (Japan)

(Accepted August 6th, 1972)

SUMMARY

In the cortical gray matter of human brains, which were obtained by permissible resection at the time of surgery, cyclic 3',5'-AMP was found in a range of concentrations between 42 and 143 pmole/mg protein. The subcortical white matter and various types of brain tumor contained much lower concentrations of the nucleotide. The level of the cyclic nucleotide in incubated slices of gray matter was elevated 20-50-fold by norepinephrine (0.5-1.0 mM) or veratridine (0.05 mM), 4-7-fold by histamine (1.0 mM) or adenosine (0.2 mM) and 1.5-2-fold by serotonin. With respect to the stimulatory effect on the cyclic AMP level in human brain slices, norepinephrine was inferior to epinephrine or isoproterenol, and was completely antagonized with a fl-adrenergic blocking agent, propranolol, but only slightly by a-blocking agents, such as phentolamine and dibenamine. The particulate fraction of human brain homogenate was capable of synthesizing cyclic AMP from ATP. This enzymic activity was 0.19-0.24 nmole/min/mg protein. This activity was stimulated 2-3-fold by the addition of 10 mM NaF but not by any of the biogenic amines tested. The activity of nucleotide phosphodiesterase of human brain was almost equal to that of rat brain: only a single Km for the substrate cyclic AMP was observed around 0.9 × 10-4 M.

INTRODUCTION

During the past few years many publications have reported the biological importance of adenosine 3',5'-cyclic monophosphate (cyclic AMP) in the central nervous system (see refs. 3, 7, 10, 11, 17, 22 and 23). These findings have arisen mostly * Present address: Nippon Roche Research Center, 200, Kajiwara, Kamakura-City, Kanagawa Prefecture, Japan.

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from data obtained with laboratory animals, and little is yet known about the nucleotide in human brain. Williams et al. 25 have observed enzymic activities of both synthesizing and degrading cyclic AMP in various regions of human brain. They used the broken cell preparation obtained from frozen autopsy materials and, inherently, no influence of various neurohormones was demonstrated on either of the enzymic activities. Working with the intact cell preparation of human brain of Japanese individuals, our research group has reported that the formation of cyclic AMP in human brain in vitro is extremely enhanced by catecholamines, and that human brain may be the most sensitive to catecholamine, with respect to the cylic AMP formation, among the brains of various animals 21. This finding was further confirmed by Fumagalli et al. 9 with Italians. Since the two latter communications were preliminary regarding the number of samples and the assay technique used, we have extended the previous work using additional specimens of human brain and a different assay method. This paper describes further details about the cyclic AMP system of human brain: the concentration of the nucleotide in various parts of the brain, its increase by several biogenic amines in vitro, and properties of enzymes that synthesize or degrade the nucleotide. MATERIALS AND METHODS

Materials

Human brain specimens were obtained at surgical operation from patients suffering from brain tumor or aneurysm. Patients from whom the specimens were obtained were the 6 cases listed in the previous report '21 and 1t additional cases. Many of the experiments were carried out with macroscopically intact brain tissue from the patients who were under inhalation anesthesia with nitrous oxide plus halothane at the time of the operation. Removal of the intact tissues was necessary either for the manipulation at the deeper region or for decompression against the postoperative brain edema. A portion of the intact specimens was always examined under the light microscope, and those specimens that revealed infiltration of unusual cells, such as tumor cells, were excluded from the present data. Every catecholamine used in the present study was the levorotatory form purchased from commercial sources. Purification and assay o f cyclic A M P in tissue

The frozen tissue was homogenized in a glass homogenizer (Kontes, A-25) with 1 ml of 7.5 ~ trichloroacetic acid (TCA) which contained 2 nCi of Jail]cyclic AMP (25 Ci//tmole) to monitor recovery. After centrifugation at 3,000 × g the supernatant layer was separated and treated with benzene and ether to remove lipid and excess TCA. The aqueous solution was dried in vacuo at room temperature, and the dried residue was treated by thin-layer chromatographyt9 (n-butanol-ethyl acetatemethanol-NH4OH, 7:4:3:4, v/v/v/v) to purify cyclic AMP. The area corresponding to cyclic AMP was scraped off, and the nucleotide was extracted from the gel with 2 ml of 50% (v/v) aqueous ethanol. The extract was dried and then further purified by

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paper electrophoresisz° (100 V/cm, 30 min on Whatman No. 1 filter paper, in a buffer of pyridine-acetic acid -water, 5:50:945, pH 3.5). The purified nucleotide was extracted from the paper with 2 ml of 50 % aqueous ethanol and dried. The dried sample was dissolved in 200 #1 water, of which 50 #1 were counted for aH to calculate recovery of the [all]cyclic AMP. Another 50/~1 aliquot was incubated in an enzymic mixture to convert the cyclic AMP to ATP, and the ATP formed was determined by the luciferase assay. This assay method for cyclic AMP is based on the report by Ebadi et al. 8. The incubation was carried out for 5 h at 30 °C in 200 #1 of a reaction mixture containing 1.25 M KCI, 90 mM MgSO4, 625 mM glycylglycine buffer (pH 7.4), 1.2 × 10-aM ATP, 1.25 mM phosphoenolpyruvate, myokinase, pyruvatekinase, and phosphodiesterase, the amount of each enzyme being determined empirically as described in ref. 8. The reaction was terminated by the addition of 5 #1 of 6 % (v/v) H~O~ followed by immediate freezing. On the day when the ATP was assayed, the frozen sample was thawed and kept at 0-2 °C until assay. The assay was done by mixing a 50 #1 portion of the reaction mixture with 100 #1 of a solution of crude firefly lantern extract (Sigma) and by counting the emitted luminescence with a liquid scintillation counter (Aloka, model LSC-601) for a 30 sec period between 10 and 40 sec after initiation of the mixing.

Assay of adenyl cyclase activity with homogenate preparation The enzyme preparation for the cyclase activity study was obtained by the method of Krishna et al. 13. The reaction mixture contained 40 mM Tris-HCl (pH 7.4), 5 mM MgClz, 6.7 mM caffeine, 3 mM [8-14C]ATP and the particulate enzyme suspension (0.3-0.5 mg protein) in a total volume of 0.2 ml. After incubation for 10 min at 30 °C, the reaction was stopped by boiling. The [14C]cyclicAMP formed was purified on a Dowex 50 column 13, followed by paper electrophoretic purification20 in the presence of 0.5 mg of authentic non-labeled cyclic AMP, and radioassayed. Correction was made by recovery of the authentic cyclic AMP which had been added to each reaction mixture immediately after the termination of the reaction.

Method for measuring the effect of biogenic amines on the adenyl cyclase of brain slices This was done by the technique reported previously19. Briefly, the method involves pulse-labeling of ATP in sliced brain tissue with [14C]adenine, followed by incubation in a medium containing the test substance, separation by thin-layer chromatography of [14C]cyclicAMP formed in the slices and radioassay of the formed radioactive cyclic AMP. In some experiments incubated slices of brain were directly exposed to the amine without the pre-labeling, and total amounts of cyclic AMP in slices were determined enzymically as described above. In either method the conditions for incubation of brain slices were the same as reported previously19. Unless otherwise specified, slices were prepared in the cold room (4 °C) with a Mcllwain tissue chopper (Mickle Laboratory, England) with a blade adjustment at 250/~m. The amount o f protein was assayed by the Lowry method 14, using bovine serum albumin as a standard reference, throughout this experiment.

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Assay of activity of cyclic 3',5'-nucleotide phosphodiesterase The human brain specimen used for this particular study was obtained from the right temporal lobe of a patient suffering from astrocytoma, an extensive lobectomy being undertaken. Immediately after removal of an intact part of the brain tissue, the gray matter was cut and homogenized in 5 vol. of water and centrifuged for 30 min at 30,000 × g. The supernatant was adjusted to 50 9/0saturation with a saturated solution of (NH4)zSO4 and centrifuged for 10 min at 30,000 × g. The pellet was taken up with 5 vol./g original wet weight in 60 mM Tris-HCt (pH 8.0) and 5 mM 2-mercaptoethanol, and dialyzed overnight against two changes of an excess of this buffer. This procedure is essentially the same as used for rat brain phosphodiesterase by Brooker

et al. 2. Assay of phosphodiesterase activity was usually carried out in a reaction mixture of 200 #1 containing 40 mM Tris-HC1 (pH 7.5), 1.8 mM MgSO4, 2 mM [all]cyclic AMP (/zCi/mole), snake venom (50 g of protein) and the brain phosphodiesterase preparation for 20 min at 30 °C. The reaction was terminated by 3 rain boiling, followed by the addition of 100/,1 of authentic non-labeled adenosine (10 mg/ml). After centrifugation a quarter of the supernatant was applied to thin-layer chromatography (Silica Gel GF) and developed in n-butanol-methanol-ethyl acetate-isopropanol-ammonium hydroxide (10:1:1:1:3, v/v/v/v/v) to separate the adenosine from cyclic 3',5'-AMP. The adenosine was then extracted from the gel with 2 ml of 50 % aqueous ethanol, and a half portion of the extract was used for the radioassay of [all]adenosine in 10 ml of Bray's solution with the liquid scintillation spectrometer. Recovery of the carrier adenosine was checked by the absorption at 258 nm, and each value of the radioassay was corrected by the recovery. Adenosine was the only radioactive product in this reaction system. RESULTS

Concentration of cyclic AMP in human brain Various types of human brain tissue were dissected during surgical operation, immediately frozen in acetone cooled by dry ice, and homogenized in 7 % TCA. The amount of cyclic AMP in the acid extract was determined as described in the text. The concentration of cyclic AMP in macroscopically intact parts of cerebral gray matter varied between 42 and t43 pmoles/mg protein (Table I), Concentrations in the subcortical white matter and in tumor tissues were much lower, although these tissues always contained definite amounts of the nucleotide.

Adenyl cyclase activity of human cerebrum measured in homogenate preparations The particulate fraction obtained from human cerebrum was capable of forming cyclic AMP from ATP, and this enzymic formation was stimulat~l by NaF which is known to stimulate adenyl cyclase of various tissues of other animals, The enzymic activity was about 0.20 nmole/min/mg protein in the presence of 10 mM N a F (Table II). This enzyme preparation did not show any sensitivity towards neurohormones,

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TABLE I CONCENTRATIONS OF CYCLIC

AMP

IN VARIOUS PARTS OF HUMAN BRAIN AND BRAIN TUMORS

Samples were obtained at surgical operation, frozen immediately in acetone cooled in dry ice, and cyclic AMP was determined as described in Methods.

Patients Age

Sex

Diagnosis

Tissue analyzed

49

male

glioma

49

male

49 58

male male

58

male

58 24

male female

24

female

53 55 65 32 5

male male female male male

gray matter (right frontal lobe) glioma white matter (right frontal lobe) glioma tumor glioblastoma gray matter (right temporal lobe) glioblastoma white matter* (right temporal lobe) glioblastoma tumor glioma gray matter (left temporal) glioma white matter* (left temporal) acoustic neurinoma cerebellum acoustic neurinoma cerebellum meningioma tumor raeningioma tumor medullo-blastoma tumor

Cyclic AMP (pmole/mg protein) 42.0 3.3 5.2 143.0 21.0 5.1 45.0 19.9 75.0 38.0 14.0 5.3 2.3

* Slightly contaminated with gray matter.

such as norepinephrine, histamine or a depolarizing agent veratridine, when examined with and without NaF.

Response of human cerebrum adenyl cyclase to catecholamines Cerebral slices were prepared from the gray matter of temporal lobe obtained from an aneurysm patient. The slices were pre-labeled with [14C]adenine for 40 min, further incubated in the presence and absence of norepinephrine (0.5 m M ) f o r various times, and [14C]cyclic A M P in the slices was radioassayed as described in the text. This time course study is presented in Fig. 1 together with results of the norepinephrine effect observed with cerebral slices from other patients. After exposure to norepinephrine for 6 - i 0 min each sample, of slices contained 20-50-fold higher levels of [14C]cyclic A M P than the control. To confirm the norepinephrine effect some samples of slices obtained from various patients were exposed to norepinephrine for 6 min without the pre-labeling, and the total amount of cyclic A M P in each group of slices was assayed. The norepinephrine effect observed with the two different methods is compared in Table III. Equally dramatic stimulation by norepinephrine was observed with the two different methods. Although the pre-labeling method showed a slightly higher stimula-

T. KODAMA e t a / .

140 T A B L E II ADENYL CYCLASE ACTIVITY OF HUMAN CEREBRUM

The reaction mixture contained 40 m M Tris-HCl (pH 7.4), 5 m M MgCI2, 6.7 m M caffeine, 3 m M [8-1aC]-ATP and the particulate enzyme suspension (0.3-0.5 mg protein) prepared as described in the text in a total volume o f 0.2 ml. Incubation for 10 rain at 30 °C. The enzyme was obtained from the cerebral gray matter (the intact tissue) o f the frontal lobe (exp. 1, 48-year-old, male, meningioma), the temporal lobe (exp. 2, 57-year-old, female, glioma), and the temporal lobe (exp. 3, 58-year-old, male, aneurysm).

Addition

NaF( IO mM)

None None NE* NE** NE* + HN*** NE* + HN*** None None NE* NE* + HN*** NE* + HN*** Veratridine (0.05 m M )

------+ + + + + --

CyclicAMP formed (nmole/min/rng protein) Exp. 1

Exp. 2

Exp. 3

0.19 0.19 0.19 0.18 0.19 ------0.18

0.24 0.24 --0.26 0.27 0.58 0.57 0.58 0.55 ---

0.20 0.18 --0.19 0.18 0.60 0.57 -0.61 0.57 --

* Norepinephrine 0.5 m M . Norepinephrine 0.1 m M . *** Histamine 0.5 m M . **

T A B L E III EFFECT OF NOREPINEPHRINEON THE FORMATIONOF CYCLIC 3 ' , 5 ' - A M P IN INCUBATED SLICES OF HUMAN CEREBRAL CORTEX N u m b e r s in parentheses indicate -E S.D. Each experiment was carried out with a specimen from a different individual.

Cyclic AMP level counts/min/mg protein* pmoles/rag protein* * Control With norepinephrine (0.5 m M ) N u m b e r o f experiments Stimulation

310 (65) 7880 (1030) n = 5 X 25.4

19 (6) 285 (53) n = 4 X 15.0

* Newly formed [laC]cyclic A M P in slices preincubated with [14Cladenine for 40 min. ** Total endogenous cyclic A M P determined enzymically (using the firefly system) as described in the text.

tion, this cannot

be considered

significant because of the great variation= between

samples and because different samples were used for different methods. concentration

of norepinephrine

The optimal

for the stimulation has tentatively been ascertained

as between 0,5-1.0 mM using the pre-labeling method. The effect of norepinephrine

on

c y c l i c A M P IN HUMANBRAIN

141

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40

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IO

|.,~t 2 4 6

0

8

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tO 12

Incubation time (min)

Fig. 1. Time course of [14CJcyclicAMP level in slices of human cerebral cortex after exposure to norepinephrine. Approximately 1 g of the gray matter obtained from the frontal lobulus (case IV in ref. 21) was chopped by hand. (Only this experiment was carried out with the hand-chopped mince and the approximate size of each mince was 0.5 mm× 0.5 mm× 1.0 mm.) The mince was labeled for 40 rain with [14C]adenine (1.0/~Ci, 21.8 nmole in 6 ml buffer), divided into several portions, and further incubated with or without norepinephrine for various times. The levels of [x4C]cyclicAMP were determined radiometrically as described in the text. The result is expressed as the percentage of [14C]cyclic AMP in the total x4C present in the slices (% conversion), and the points obtained in this experiment are connected with solid lines to make clear the time course. Other points, not connected with the lines in this figure, were obtained from separate experiments carried out on different human cortical specimens using the mechanical slicer as described in the text.

the total cyclic A M P level in incubated slices of human brain was also maximal at 0.5 and 1.0 m M concentrations. We have preliminarily reported that the catecholamine effect on the human cerebrum adenyl cyclase might be a fl-adrenergic type 21. We have accumulated data concerning this problem and summarized it in Table IV. In every experiment the order of efficacy at an equimolar concentration was: isoproterenol > epinephrine > norepinephrine. The antagonism with adrenergic blocking agents was examined with human brains of 3 different individuals (Table V). The inhibition by the fl-adrenergic blocker, propranolol, was invariably marked, whereas the a-blockers, such as phentolamine and dibenamine, showed a much weaker effect than propranolol. The blocking agents alone (without catecholamine) did not show any effect at the concentrations indicated in Table V.

Effect of other agents on the pre-labeled slices of human brain The stimulatory effect of serotonin, histamine and veratddine on the human brain cyclic A M P has been reported 21. This effect was confirmed in two additional experiments. The order o f efficacy was always serotonin < histamine < veratridine

r. KODAMA et al.

142

TABLE IV EFFECT OF CATECHOLAMINEAGONISTSON THE FORMATIONOF CYCLIC AMP IN SLICESOF HUMAN BRAIN Data for exp. 1 were obtained from the previous report ~t. The formation of cyclic AMP was measured by the pre-labeling method described in the text. Intact portions of the temporal lobe (58-year-old, male, aneurysm) and the frontal lobe (49-year-old, male, glioma) were used for exp. 2 and 3, respectively.

Experiment Concentration of catecholamine

1

2

3

0.5

0.5

O.1

Stimulator

Cyclic AMP formation ( % conversion)

Norepinephrine Epinephrine Isoproterenol None

36 51 57 1.8

32.8 46.6 48.1 0.8

30 43 43 0.6

(Table VI). In 3 experiments, veratridine (0.05 mM) was more efficacious than norepinephrine (0.5 mM), but in two other experiments the reverse order was observed: the veratridine effect varied from 38.5 to 50.2 % conversion whereas the norepinephrine effect was between 28.7 and 61.1% conversion (data not shown). Although the stimulatory effect expressed as the conversion rate varied considerably as shown here, the extent of the stimulation compared with controls (e.g., percentage of control) was relatively constant for an identical stimulus. In an experiment carried out with a single brain material the veratridine effect was maximal at 0.05 mM. When veratridine (0.05 mM) and norepinephrine (0.5 mM) were added toTABLE V EFFECT OF ADRENERGIC BLOCKING DRUGS ON THE NOREPINEPHRINE-ELICITED FORMATION OF CYCLIC AMP

IN HUMAN CEREBRAL SLICES

Cyclic A M P forrfmtion was measured by the pulse-labeling method as described in the text,

Sample used

Concentration o f Adrenergic blocker norepinephrine (mM) (mM)

Cyclic AMP formation ( % conversion)

Inhibition (%)

Frontal lobe 0.5 (31 year male, glioma) 0.5 0.5 0.5

None None Phentolamine 0.5 Propranolol 0.5

27.6 27.0 25.8 5.1

--5.5 82.6

Temporal lobe (65 year female, meningioma)

0.05 0.05 0.05

None Phentolamine 0.01 Propranolol0.01

32.1 32.1 20.6

-0 37.1

Temporal lobe (32 year male, meningioma)

0.05 0.05 0.05 0.05

None Pbentolamine 0.05 Dibenamine 0.05 Propranolol 0.05

25.7 21.2 14.0 2.6

-17.6 46.2 91.6

CYCLICAMP IN HUMANBRAIN

143

T A B L E VI EFFECT OF VARIOUS AMINES ON THE FORMATION OF CYCLIC A M P IN INCUBATED SLICES OF HUMAN BRAIN Cyclic A M P f o r m a t i o n was m e a s u r e d by t h e pulse-labeling m e t h o d . T h e gray m a t t e r o f t h e f r o n t a l lobe (49-year-old, male, glioma) a n d t h e t e m p o r a l lobe (42-year-old, m a l e , glioma) were u s e d for exp. 1 a n d 2, respectively.

Stimulator

Incubation (rain)

Concen- Exp. 1 Exp. 2 Reported exp. ( ref 21) tration (mM) Conversion Relative Conversion Relative Conversion Relative rate (%) effect rate (%) effect rate (%) effect

None Norepinephrine Histamine Serotonin Adenosine Veratridine

8-12 6 10 6 8 10

-0.5 1.0 1.0 0.2 0.05

0.8 28.7 5.4 1.7 5.8 41.3

1.0 36 7.0 2.1 7.2 52

1.1 51 5.9 1.8 5.3 39

1.0 46 5.4 1.6 4.7 35

2.0 39.3 8.8 4.1 -46.4

1.0 19.7 4.4 2.0 -23.2

gether, the cyclic AMP formation varied between 52.4-78.3 % conversion. Adenosine (0.2 mM) was also effective in producing a 5-7-fold elevation of the [14C]cyclicAMP level.

Properties of Y,5'-nucleotide phosphodiesterase The phosphodiesterase was partially purified from human and rat (a Wistar male) brains as described in Materials and Methods, and the activities of the diesterase from the two animals were compared, under the condition described in the text, with these results: human, 158 nmole/mg protein/min and rat, 181 nmole/mg protein/min. The soluble phosphodiesterase obtained from rat brain in this manner has been reported to have two affinity constants (Kin) for cyclic AMP 2. Therefore we examined the Km of human brain phosphodiesterase carefully over a range of substrate concentrations between 0.004 and 0.5 mM (Fig. 2). Yet we could see only one Kra (-- 0.9 × 10-4 M) for human brain phosphodiesterase (Fig. 2). The activity was competitively inhibited by caffeine. DISCUSSION

The reported concentration of cyclic AMP in mammalian brain varies between 5.0 mmoles/g in rat t5 and 1.3 mmoles/g (wet weight) in mice1. We found a much higher average concentration in human cerebral gray matter. It is not clear, however, whether this difference is due to a species difference or to possible metabolic alterations during the surgical procedure, although special attention was paid to avoid deterioration of the quality of tissue specimens. Concentrations of the nucleotide in the subcortical white matter, glioma, meningioma and medulloblastoma were much lower than in the gray matter. This result might be related to the findings that the activity of adenyl cyclase was the highest in the synaptosome-containingfraction of the

144

-r. KODAMAet al.

3

+4

zg

T"C

._c

I/

2o. ~, 0.4

II

/ +,,,,,ou,°o,+,.y.%__.

"6 -I

I

2

3

4

5

5> 0.2

J -I

I X 10-4M (Cyclic AMP)

Fig. 2. Lineweaver-Burk presentation of the activity of human cerebral 3',5'-nucle.otide phosphodiesterase and its inhibition by caffeine. Preparation of the enzyme and assay of the activity as described in Materials and Methods.

rat homogenate. The sharp contrast between the gray and white matters was also noted in the activation study of the adenyl cyclase of intact cell preparations. For example, the concentration of cyclic A M P in the slice of the gray matter rose to more than 50 times the control after stimulation with either norepmephrine or veratridine, whereas the stimulatory effect in the white matter preparation was less than 5 times the control (unpublished data from human cerebrum). It is noteworthy, however, that the concentration of cyclic AMP in cultured human astrocytoma cells is elevated by catecholamines and histamine 6. The most interesting finding in the present study may be that the cyclic A M P level of human brain slices is dramatically elevated by catecholamines. Catecholamine appears to have the most profound effect on the human brain cyclase among various mammalian brains, e.g. squirrel monkey, guinea-pig (Hartley), rabbit (New Zealand White), rat (Wistar) and mouse (see ref. 18). Our preliminary study showed that, among catecholamine agonists, isoproterenol was the most potent followed by epinephrine and norepinephrine, and that the stimulatory effect of norepinephrine is almost completely blocked by the fl-blocker but only slightly by the a-blocker. Further studies presented in this paper confirmed these results, and indicate that the fl-adrenergic type receptor predominates in human cerebrum adenyl cyclase over the a-type. This is in accord with the report on the rabbit cerebellum 1°, but the reverse situation is reported for the cerebrum o f guinea-pig 4. In human cerebrum, norepinephrine was much more efficacious than histamine in elevating the cyclic AMP level. The cultured human astrocytoma 6, as well as the cortical gray matter of the rat and the mouse, has the same tendency as the human

CYCLIC AMP IN HUMANBRAIN

145

cerebrum whereas histamine is more efficacious than norepinephrine in cerebral slices of the rabbit and the guinea-pig. The adenosine effect has now been observed in brains of all mammals so far examined. Synergism between norepinephrine and veratridine was demonstrated with the human cerebral materials, although the type of synergism was additive rather than potentiative as it was in the slice preparation of guinea-pig cerebrum. The synergism resulted in the conversion of up to 78.3 ~o of the total incorporated 14C to [14C]cyclic AMP. This conversion rate is the highest found with the cerebral tissue of various animals and indicates that the ATP pool, labeled by prior incubation with [14C]adenine, is almost exclusively connected with the adenyl cyclase. This experiment also verified previous reports that the effect or" depolarization and its synergism with biogenic amines are common phenomena throughout brains of various mammals. Properties of cyclic 3',5'-nucleotide phosphodiesterase in various mammalian brains have been documented (see refs. 2, 5, 12, 16 and 24). Inhibition of the enzyme by caffeine is now established with the human preparation as well, the type of inhibition being competitive with the substrate. With a rat brain preparation, Brooker et al. ~ have reported two cyclic AMP phosphodiesterase activities with apparent K m ' s of 1.3 × 10-4 and 1.0 × 10-6 M. Because of this and of the report on the presence of two nucleotide diesterases with different properties2,1~, we have carefully examined the Km of the human diesterase for cyclic AMP using a wide range of substrate concentrations. Our results always gave a single apparent K,n, which was 0.9 × 10-4M. We have not repeated the rat experiment of Brooker et aL 2 under our conditions. In conclusion, although the stimulation by catecholamine was exceptionally high in human brain, the general characteristics of the cyclic AMP system found in human brain are similar to that of other animal brains. Therefore, when any of many hypotheses is substantiated for experimental animals, it could presumably be extrapolated to the human brain. REFERENCES 1 AURBACH,G. D., AND HOUSTON,B. A., Determination of 3',5'-adenosine monophosphate with a method based on a radioactive phosphate exchange reaction, J. biol. Chem., 243 (1968) 59355940. 2 BROOKER,G., THOMAS,L. J., ANDAPPLEMAN,M. M., The assay of adenosine Y,5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate in biological materials by enzymic radioisotopic displacement, Biochemistry, 7 (1968) 4177-4181. 3 BUTCHER,R. W., AND SUTHERLAND,E. W., Adenosine 3',5'-phosphate in biological materials, J. biol. Chem., 237 (1962) 1244-1250. 4 CHASIN,M., RIVraN, I., MAMRAK,F., SAMANIEGO,S. G., AND HESS, S. M., a- and fl-adrenergic receptors as mediators of accumulation of cyclic adenosine 3',5'monophosphate in specific areas of guinea pig brain, J. biol. Chem., 246 (1971) 3037-3041. 5 CHEUNG, W. Y., Properties of cyclic 3',5'-nucleotide phosphodiesterase from rat brain, Biochemistry, 6 (1967) 1079-1087. 6 CLARK, R. B., AND PERKINS,J. P., Regulation of adenosine Y,5'-cyclic monophosphate concentration in cultured human astrocytoma cells by catecholamines and histamine, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 2757-2760. 7 DE ROBERTm,E., RODmGUEZDE LORESARNAIZ,G., ALBERXO,M., BUTCHER,R. W., ANDSUTHERLAND,E. W., Subcellular distribution of adenyl cyclase and cyclic phosphodiesterase in rat brain cortex, J. biol. Chem., 242 (1967) 3487-3493.

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1". KODAMAel al.

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