EXPERIMENTAL

NEUROLOGY

Stimulation

of Cyclic

EDWARD

hTcurologg

LEWIN,

ScrrGce, Unizvrsit~r

422-426

50,

AMP TONY

(1976)

Accumulation

GOLDEN,

?‘rteram Admiwistratior, of Colorado Medical

AND

by Pentyienetetrazol

JONATHAN

E.

WALKER

Hospital and Drpurtr,lent Center, Denz~r, Colorado



of

Ketrrolog.v,

80230

The convulsant agent, pentylenetetrazol (PTZ) , increased the content of cyclic AMP (CAMP) in rat cortical slices incubated in vitro in physiologic media. This increase was partially reversed by theophylline. The addition of PTZ to maximally effective concentrations of either adenosine or 2-chloroadenosine resulted in a significantly greater than additive augmentation of CAMP accumulation, suggesting that PTZ may produce its effect by enhancing the action of endogenous adenosine. The PTZ response was not antagonized by either diphenylhydantoin, phenobarbital. or ethosuximide.

INTRODUCTION Kecent investigations have suggested that cyclic AMP (CAMP) may play a role in epileptogenesis. Dibutyryl CAMP produces seizures in animals when injected intraventricularly (1) and epileptiform discharges when applied to the cerebral cortex ( 12). In addition, epileptogenic cortical foci induced by freezing contain elevated levels of CAMP (13). Electrical stimulation of cortical slices maintained in physiologic media markedly increases CAMP content in the slices, an effect which is blocked by theophylline (3). IAre, therefore, elected to study the effect of the convulsant agent, pentylenetetrazol (PTZ j , on CAMP formation in slices of rat tortes prelabeled with l&C-adenine and incubated in z&o. METHODS Adult female Wistar rats were sacrificed by decapitation, and the brains were rapidly removed. Blocks of somatosensory cortex, approximately 3 x 3 mm, were cut from both hemispheres. These blocks were sliced at a thickness of 0.26 mm with a McIlwain tissue chopper, turned 90” and 1 The Virginia

Copyright All rights

advice Bleck

and counsel are gratefully

provided by acknowledged.

1975 by Academic Press Inc. reproduction in any form reserved.

Dr.

John

Perkins,

Marilyn

Moore,

and

CYCLIC

AMP

FORMATION

423

chopped again, resulting in slices 0.26 mm X 0.26 mm X cortical thickness. Slices from both hemispheres of each animal were combined and prelabeled with %-adenine as described by Shimizu et al. (8) with minor modifications. The medium used was that of McIlwain (5) and contained NaCl, 120 mM; KCl, 3.0 mM; KH2POI, 1.2 mM ; MgCl2, 1.2 mM; CaC12, 0.75 mM ; NaHC03,25 mM ; and glucose, 10 mM, in equilibrium with O2 + COa (95 : 5). After prelabeling 40 min, the slices were washed twice and divided into aliquots. Slices were incubated for an additional 20 min with the experimental drugs. After incubation, the slices were extracted with 1.0 ml 5% TCA containing 50 ~1 unlabeled CAMP, 0.014 M, in order to monitor recovery. The resulting homogenate was centrifuged at 10,000 g for 20 min, and TCA removed by extracting 4 times with three volumes water-saturated ether. The excess ether was boiled off. CAMP was isolated initially on Dowex 50 by the method of Perkins and Moore (6) followed by further purification on aluminum oxide. To the 3 ml-fraction containing CAMP from the Dowex 50 column, 0.1 ml of 1.5 M Tris, pH 8, was added, and the entire fraction was applied to a 3 cm aluminum oxide (Woelm neutral, activity grade 1) column prepared in a Pasteur pipette. Then 1.O ml 0.05 M Tris, pH 8, was added to the column, and the resulting 4 ml effluent was divided into 2 aliquots for counting in a scintillation counter and for reading in a spectrophotometer at 260 rnp to measure the recovery of unlabeled CAMP. The CAMP counts were corrected for recovery, and the results were calculated as percentage conversion to CAMP of the total counts in the tissue extract, i.e., percentage conversion to CAMP = lOO(correctec1 CAMP counts/total tissue extract counts). RESULTS The results obtained with varying concentrations of PTZ are presented i!l Fig. 1. An approximately 35% increase in CAMP was found at 5 mM PTZ (P < 0.02) and at 100 m&f PTZ a 3-fold increase occurred (P < 0.001) . As shown in Table 1, theophylline, which antagonizes the stimulation of CAMP formation produced by adenosine (7)) reduced CAMP accumulation in control medium and partially blocked the effect of PTZ. The addition of PTZ to a maximally effective concentration of adenosine resulted in a significantly greater than additive augmentation of CAMP accumulation (P < 0.01)) and, similarly, 2-chloroadenosine produced a much larger than additive increase in CAMP (P < 0.005). I n contrast, the combination of PTZ and norepinephrine appeared to be merely additive in their effect on CAMP accumulation. In Table 2, the results obtained when several commonly used anticonvulsant agents were added to 100 mM PTZ are shown. It can be seen

424

LEWIN,

FIG. 1. Effect of percentage conversion

GOLDEN,

pentylenetetrazol to CAMP.

Bars

AND

WALKER

on CAMP accumulation. are f standard error

Results expressed of the mean.

as

that the stimulation of CAMP accumulation by PTZ was not reduced by either diphenylhydantoin, phenobarbital, or ethosuximide.

It has been shown that PTZ in concentrations of 5 to 100 mar enhances CAMP accumulation in an i)z vitro system. Although the concentrations of PTZ in brain which result from parenteral injection at the dosages commonly used to induce seizures are not known, they are undoubtedly much lower than those used in these investigations. However, in itz vitro studies of neurons from several invertebrates, concentrations ranging from 30 to I.40 1x1~ have been required to produce spikes and paroxysmal depolarizations (2, IO, 1-C)) and the concentrations which we used are in this range. The stimulation of CAMP accumulation by PTZ was inhibited by theophylline. Theophylline has been shown to block the increase in CAMP TABLE Etfect

of Theophylline, Stimulation

_-..

.

---

Adenosine, of CAMP

Drug - .--.-._

2-Chloroadenosine, and Norepinephrine Accumulation by Pentylenetetrazol No PTZ .-.- _~~-___-~_-~_

-.

None (16) Theophylline, 0.5 m11 (4) Adenosine, 0.1 m&l (4) 2-Chloroadenosine, 0.1 mM (4) Norepinephrine, 0.05 rnM (4) Results expressed as percentage Numbrr of esperiments indicated

1

0.99 0.44 2.50 4.75 2.49

zt f f f f

conversion to criM in parenthcscs.

0.06 0.07 0.22 0.76 0.05 P & standard

on the

PTZ, 100 n131 .___. _ . 3.15 1.63 6.15 14.8 4.87 error

f 0.28 f 0.07 f 0.53 f 2.2 f 0.28 of the mean.

CYCLIC

AMP

TABLE Effect

of Antkonvulsants Formation

2

on the Stimulation by Pentylenetetrazol

Drug Kane (12) Diphenylhydantoin, Phenobarbital, Ethosuximide,

0.3 nlM 1.0 mM (4) 1.0 m&g (4)

Results expressed as percentage Number of experiments indicated

425

FORMATION

of CAMP

No PTZ

(4)

0.81 1.02 0.65 0.72 conversion to CAMP in parentheses.

PTZ,

f 0.06 f 0.08 f 0.13 * 0.03 I!= standard

2.99 2.92 3.01 3.70 error

100 m>l f f f f

0.21 0.25 0.38 0.11

of the mean.

formation produced in cortical slices by adenosine (7), and this result, therefore, suggested that the PTZ response might have been mediated by endogenously released adenosine. If this postulation were true, it was anticipated that PTZ would not augment the effect of an optimal concentration of added adenosine or of its analog, 2-chloroadenosine. However, the addition of PTZ to either adenosine or 2-chloroadenosine produced a significantly greater than additive increase in CAMP accumulation, a finding which suggested that PTZ may potentiate the action of endogenous adenosine at its postulated receptor rather than enhance its release. In contrast, the combination of PTZ and norepinephrine produced merely an additive stimulation of CAMP accumulation, and it, therefore, seems unlikely that norepinephrine participated in the PTZ response. The pharmacological mechanism by which PTZ induces seizures has not been established. As discussed above, the application of dibutyryl CAMP to the cortical surface results in epileptiform discharges, and it is tempting to suggest that an increase in cortex CAMP might participate in the convulsant action of PTZ. The function of CAMP in cerebral cortex has not been elucidated. However, in the cerebellum, CAMP applied by microelectrophoresis to Purkinje cells reduced the discharge rate of these cells (9). Of interest, Purkinje cell output appears to have an inhibitory influence on epileptogenic foci induced in cortex with penicillin, and a decrease in Purkinje cell discharge correlates with intensified epileptiform discharge (3). If ci\MP similarly decreases the discharge rate of selected inhibitory cortical neurons, the effect of an increase in CAMP might be to favor epileptogenesis. The failure of either phenobarbital or ethosuximide to block the CAMP response was disappointing, as both drugs, unlike diphenylhydantoin, raise the threshold to PTZ convulsions in animals (11). The CAMP response to PTZ may be entirely unrelated to its epileptogenic properties, or, alternatively, these two agents may act elsewhere in the epileptic process.

426

LEWIN,

GOLDEN,

AiWl

\VALKER

REFERENCES 1. GESSA, G. L., G. KRISHNA, J. FORK, A. TAGLIAEVIONTE, and B. B. BRODIE. 1970. Behavioral and vegetative effects produced by dibutyryl cyclic AMP injected into different areas of the brain, pp. 371-381. In “Role of Cyclic AMP in Cell Function, Advances in Biochemical Psychopharmacology, Vol. 3.” P. Greengard and E. Costa [Eds.]. Raven Press, New York. 2. JOHNSON, W. L., and J. L. O’LEARY. 1965. Assay of convulsants using single unit activity. Arclz. Nrlfvol. 12 : 113-127. 3. JULIEN, R. M., and L. M. HALPEKN. 1972. Effects of diphenylhydantoin and other antiepileptic drugs on epileptiform activity and Purkinje cell discharge rates. Epilepsia 13 : 38740. 4. KAKIUCHI, S., T. W. RALL, and H. MCILWAIX. 1969. The effect of electrical stimulation upon the accumulation of adenosine 3’,5’-phosphate in isolated cerebral tissue. J. Nezlvorkc:rl. 16 : 485-491. 5. MCILWAIN, H. 1972. Electrical stimulation of specified subsystems of the mammalian brain, as isolated tissue preparations, pp. 269-289. Itz “Experimental Models of Epilepsy-A Manual for the Laboratory Worker.” D. P. Purpura, J. K. Penry, D. Tower, D. M. Woodbury, and R. Walter [Eds.]. Raven Press, New York. 6. PERKINS, J. P., and >I. M. MOORE. 1973. Characterization of the adrenergic receptors mediating a rise in cyclic 3’,5’-adenosine monophosphate in rat cerebral cortex. J. Pharwacol. Exp. Tlacr. 185 : 371-378. 7. SATTIN, A., and T. W. RALL. 1970. The effect of adenosine and adenine nu:leotides on the cyclic adenosine 3’,5’-phosphate content of guinea pig cerebral cortex slices. Mol. Pharrnacol. 6: 13-23. 8. SHIMIZU, H., J. W. DALY, and C. R. CI~EVELIXG. 1969. A radioisotopic method for measuring the formation of adenosine 3’,5’-cyclic monophosphate in incubated slices of brain. J. h’c~~rorhc~~z. 16 : 1609-1619. 9. SIGGINS, G. R., B. J. HOFFER, and F. E. BLOOM. 1969. Cyclic adenosine monophosphate : Possible mediator for norepinephrine effects on cerebellar Purkinje cells. Scielzcc 165 : 1018-1020. 10. SPECKMAXN, E.-J., and H. CAsPEI

Stimulation of cyclic AMP accumulation by pentylenetetrazol.

EXPERIMENTAL NEUROLOGY Stimulation of Cyclic EDWARD hTcurologg LEWIN, ScrrGce, Unizvrsit~r 422-426 50, AMP TONY (1976) Accumulation GOLDE...
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