Effects of chronic morphine and N6-cyclopentyl-adenosine administration on kainic acid-induced status epilepticus

Epilepsy Research 44 (2001) 89 – 96 www.elsevier.com/locate/epilepsyres

Effects of chronic morphine and N 6-cyclopentyl-adenosine administration on kainic acid-induced status epilepticus Agustina Cano-Martı´nez a,b,c, Rafael Villalobos-Molina c, Luisa Rocha b,c,* a

Departamento de Fisiologı´a, Instituto Nacional de Cardiologı´a ‘‘Ignacio Cha´6ez’’, Juan Badiano c 1 CP 14080, Mexico D.F. b Instituto Nacional de Psiquiatrı´a ‘‘Ramo´n de la Fuente’’, A6. Me´xico-Xochimilco 101 CP 14370, Mexico D.F. c Departamento de Farmacobiologı´a, Centro de In6estigacio´n y Estudios A6anzados del IPN, Calzada de los Tenorios 235, Col. Granjas Coapa CP 14330, Mexico D.F. Received 30 June 2000; received in revised form 7 December 2000; accepted 8 December 2000

Abstract The aim of the present study was to investigate if the upregulation of m or A1 receptors modifies the expression of the kainic acid (KA)-induced status epilepticus (SE). Male Wistar rats received one of the following treatments: saline solution (SS) (1 ml/kg, i.p. for 7 days); morphine (M) (20 mg/kg, i.p. for 7 days) or N 6-cyclopentyl-adenosine (CPA) (1 mg/kg, i.p. for 9 days). Twenty-four hours after the last administration rats were sacrificed. Membranes were obtained m and and A1 receptor binding experiments were carried out. Furthermore, an injection of SS (1 ml/kg, i.p.) or KA (10 mg/kg, i.p.) was applied in rats pretreated chronically with M, CPA or SS, 48 h after the last administration. Seizure activity, death rate and a postictal explosive motor behavior were evaluated after KA administration. Chronic M administration increased m receptor number in hippocampus (115%) and cortex (265%), whereas chronic CPA treatment enhanced A1 receptor number in hippocampus (55%), amygdala (39%) and cortex (51%). The pretreatment with M facilitated the KA-induced SE and reduced the death rate as well as the postictal explosive motor behavior. The pretreatment with CPA delayed the SE presentation, increased the death rate and decreased the postictal explosive motor behavior. These findings suggest that upregulation of m receptors enhances the KA seizures, whereas upregulation of A1 receptors depresses these seizures. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Kainic acid; m Receptors; A1 receptors; Status epilepticus; Opioid peptides; Adenosine

1. Introduction Adenosine and opioid peptides have been shown to be involved in the mechanisms by which * Corresponding author. Tel.: + 525-483-2859; fax: + 525483-2863. E-mail address: [email protected] (L. Rocha).

seizure activity spontaneously terminates (Albertson et al., 1984; Dragunow et al., 1985). Studies support that A1 receptor activation mediates the antiepileptic effect of adenosine (Von Lubitz et al., 1993) and that seizure activity following pentylenetetrazol or repeated electroconvulsive shocks enhances these receptors (Gleiter et al., 1989; Pagonopoulou et al., 1993). In fact, the

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increased A1 receptor binding detected in human neocortex of patients with temporal lobe epilepsy has been suggested as a protective mechanism against subsequent seizures (Angelatao et al., 1993). On the other hand, the effects of opioid peptides in epilepsy are at present unclear. Several reports support the anticonvulsant effects of these peptides (Albertson et al., 1984; Bohme et al., 1987; Caldecott-Hazard et al., 1987; Ferna´ ndezGuardiola et al., 1989). In contrast, other experimental evidence suggests that activation of m opioid receptors induce proconvulsant effects (Tortella et al., 1987; Lee et al., 1989). In previous studies, we reported that the naloxone-induced upregulation of m receptors facilitates the development of the amygdala kindling process and increases the postictal seizure suppression when kindling electrical stimulation is applied in pyriform cortex, central and medial amygdaloid nuclei (Rocha et al., 1991). Similarly, chronic morphine administration, a treatment known to upregulate m receptor binding (Holaday et al., 1982; Rothman et al., 1987), also facilitates the amygdala kindling process and enhances the postictal seizure suppression (Rocha et al., 1996). Kainic acid (KA), an agonist of glutamate, produces status epilepticus (SE) when applied systemically or intracerebrally into rats (Lothman and Collins, 1981; Nadler, 1981; Sperk, 1994). The administration of adenosine agonists decreases the KA-induced seizures and brain damage (Arvin et al., 1989; Zhang et al., 1990), whereas adenosine antagonists enhances them (Pinard et al., 1990). With regard to adenosine receptors, it was described that A1 receptor immunoreactivity in CA2/CA3 field begins to decline drastically, approximately 4 weeks after KA administration and remains diminished 8 weeks after treatment (Ochiishi et al., 1999). Concerning opioid peptides, it is known that DAMGO (a m opioid agonist) intensifies the neurotoxicity induced by KA (Lason et al., 1988) and that m binding is enhanced in specific rat brain areas both at 4 and 48 h after KA-induced SE (Perry and Grimes, 1989; Ridd et al., 1998). This group of evidence indicates that adenosine and opioid peptides may have opposite effects in the KA-induced seizure model.

The upregulation of receptors could be associated with a higher sensitization of the agonists effects (Schulz et al., 1979; Yoburn et al., 1989). Therefore, for the present study we assumed that increased m or A1 receptors could intensify the pro- or anticonvulsant effects of the endogenous opioid peptides or adenosine in the KA-induced SE and postictal and interictal depression. Experiments were carried out to investigate the seizure expression following KA administration in rats with upregulation of m or A1 adenosine receptors.

2. Methods

2.1. Animals Male Wistar rats weighing 250–300 g were used in all studies. The animals were housed individually in temperature controlled conditions and were allowed free access to food and water.

2.2. Experimental groups 2.2.1. Chronic administration of morphine or N 6-cyclopentyl-adenosine Rats were treated with morphine (M group, 20 mg/kg, i.p. daily for 7 days, n= 12) or N 6-cyclopentyl-adenosine (CPA group, 1 mg/kg, i.p. daily for 9 days, n= 12) or saline solution (SS group, 1 ml/kg, i.p. for 9 days, n= 12). Animals were killed by decapitation 48 h after the last administration. Their brains were removed immediately and cortex, hippocampus and amygdala were dissected on ice according to the procedures described by Glowinski and Iversen (1966) and Engel and Sharpless (1977). These structures were chosen for evaluation because of their involvement in the appearance and propagation of seizure activity (Goddard et al., 1969; Lothman and Collins, 1981). The tissue from four rats was pooled for each assay, gently blotted before being weighed and then homogenized in ice-cold Tris–HCl (50 mM, pH 7.4) by using a Polytron (PT-10, Brinkman Instruments), at a setting of 6 for 30 s and centrifuged (30000g for 20 min, 4°C); the resulting pellets were spun again in the same conditions.

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For A1 receptors, after a further resuspension, the membranes were incubated with Tris– HCl buffer in the presence of 2 U/ml of adenosine deaminase (type IV, Sigma Chemical Co.) during 30 min at 22°C. For m receptors, the suspensions were incubated in a shaking water bath for 25 min at 25°C to remove endogenous opioids. The membrane suspensions were then centrifuged (30000g for 20 min, 4°C), and the resulting pellets were resuspended in the same buffer at a concentration of 10 mg/ml (original wet weight) and kept on ice before binding assays. An aliquot was used to determine protein concentration with the method by Lowry et al. (1951). Saturation binding assays for A1 and m opioid receptors were performed in triplicate samples with 100 or 500 mg of membrane protein, respectively. Membranes were incubated with 8 increasing concentrations (0.1– 10 nM) of the A1 receptor agonist [3H]CCPA ([3H]chloro-cyclopentyl-adenosine, specific activity = 70 Ci/ mmol; Dupont-NEN); non-specific binding was obtained in the presence of theophylline (1 mM) in a final volume of 0.25 ml during 60 min at 25°C. Samples were filtered through a Brandel cell harvester using Whatman GF/B paper, and washed three times with 5 ml of ice-cold buffer; filters were dried and Insta Gel (Amersham, UK) was added and counted in a Beckman LS6000SC liquid scintillation spectrometer. For m opioid receptor binding, membranes were incubated with the agonist [3H]DAMGO ([3H][D-Ala2-N-methylphe-glycol5][tyrosyl-3-4-3H]enkephalin, specific activity=55 Ci/mmol; Amersham, UK) in the concentration range of 0.1– 10 nM; non-specific binding was obtained in the presence of naloxone (100 nM) in a final volume of 0.25 ml during 60 min at 25°C, filtered and counted. Scatchard plots were obtained from saturable binding curves, using the 8 concentration points (in triplicate) for estimating Bmax and Kd values after EBDA program analysis (Munson and Rodbard, 1980). Both ligands gave a Hill coefficient close to 1. Specific binding was 75– 85% at the Kd for [3H]CCPA and 50 – 70% at the Kd for [3H]DAMGO. [3H]DAMGO binding was determined in membranes obtained from SS and M groups, whereas

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[3H]CCPA binding was estimated in membranes obtained from SS and CPA groups. Statistical analysis was carried out by means of two-way analysis of variance, and differences between group means were evaluated by Student’s t test for paired observations, considered significant when PB 0.05.

2.2.2. Status epilepticus in animals chronically pretreated with morphine or N 6-cyclopentyl-adenosine Rats were pretreated with morphine, N 6-cyclopentyl-adenosine, or saline solution as described above (M+ KA, CPA +KA and SS+ KA groups, respectively, and n= 12 per group). Forty-eight hours after the last administration they were injected with KA (10 mg/kg, i.p.). Animals from control group were manipulated as described above, except that they were always treated with saline solution (SS+SS group, n=12). Immediately after KA injection and during the ictal period, the latency of presentation of behavioral changes was evaluated according with the phases previously reported by Lothman and Collins (1981): I – staring; II – wet dog shakes; III – automatisms-mild limbic convulsions; IV – severe limbic convulsions; and SE, considered established when limbic seizures appeared repetitively at intervals of 3 min, during at least 30 min. The duration of SE, the total number of severe limbic convulsions during SE and death rate were also estimated. Twenty-four hours after SE, the number of animals showing explosive motor jumping (EMJ) by handling was evaluated for each experimental group. This response, considered an excitatory behavior, has been used to estimate the excitability during the postictal period (Caldecott-Hazard et al., 1987). Latency of presentation of behavioral changes and duration of SE after KA administration were evaluated utilizing one-way ANOVA and a posthoc Dunnett’s test. Death rate and EMJ were analyzed applying the  2 test. The Animal Use and Care Committee of the Instituto Nacional de Psiquiatrı´a approved all experiments.

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Fig. 1. Representative Scatchard plots of specific [3H]DAMGO (A) and [3H] CCPA (B) binding to m opioid and A1 hippocampal receptors, respectively. Membranes were obtained from rats treated chronically with SS, M and CPA.

3. Results

3.1. Effects of chronic morphine and N 6-cyclopentyl-adenosine administration on 3 H-DAMGO and 3H-CCPA binding, respecti6ely Specific binding of 3H-DAMGO and 3H-CCPA in all regions and in all experiments was saturable and Scatchard plots were linear (Hill \0.9). With respect to m receptors in animals from M group and in comparison with SS group, an increased number of receptors was found in hippocampus (115%), and cortex (265%), and reduced Kd values in hippocampus (54%) and amygdala (52%). Concerning A1 receptors in rats from the CPA group and when compared with the SS group, experiments revealed enhanced receptor number in hippocampus (55%), amygdala (39%) and cortex (51%), and reduced Kd in hippocampus (34%) (Fig. 1, Tables 1 and 2).

was 1429 4 min, the duration of SE was 2109 15 min and they showed 8% of death rate after suffering SE. The EMJ was observed in 100% of the live animals 24 h after SE (Figs. 2 and 3). In contrast with the SS+ KA group, animals from the M+ KA group displayed a faster evolution of SE, with shorter latency to the first severe limbic convulsion (1179 3 min). Although they presented a higher number of generalized seizures (179 3, PB 0.05), SE duration was not significantly different (1959 15 min) and 100% of animals were alive 24 h after KA administration. The EMJ behavior was observed in 2 rats (16%) (Figs. 2 and 3). Table 1 Effects of chronic administration of morphine on Bmax (fmol/ mg protein) and Kd (nM) values of m receptorsa Group

Hippocampus

Amygdala

Cortex

SS Bmax Kd

32 92 1.1 90.3

33 92 1.7 90.2

71 9 3 1.8 90.2

3.2. Effects of chronic pretreatment with morphine or N 6-cyclopentyl-adenosine on kainic acid-induced status epilepticus

M Bmax Kd

69 9 3* 0.5 90.1*

33 9 2 0.8 90.1*

259 95* 2.2 90.2

Rats from the SS+KA group presented an average of 1292 generalized seizures during SE, their latency to the first generalized convulsion

a Bmax and Kd values are expressed as the mean 9 SEM. SS – control group treated chronically with saline solution; M – group treated chronically with morphine. * PB0.05 when compared with SS group.

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Table 2 Effects of chronic administration of CPA on Bmax (fmol/mg protein) and Kd (nM) values of A1 receptorsa Group

Hippocampus

Amygdala

Cortex

SS Bmax Kd

730 9 30 0.85 90.1

5409 20 0.499 0.2

4409 20 0.61 9 0.1

CPA Bmax Kd

1130 940* 0.56 90.1*

7509 20* 0.669 0.1

660 9 20* 0.62 90.1

a Bmax and Kd values are expressed as the mean 9 SEM. SS – control group treated chronically with saline solution; CPA – group treated chronically with N 6-cyclopentyl-adenosine. * PB0.05 when compared with SS group.

When compared with the SS+ KA group, the CPA + KA group exhibited a longer latency of presentation of the SE behavioral phases as well as the first severe limbic convulsion (1679 4 min, P B0.05). This experimental group showed lower number of generalized seizures (792, P B 0.05) and similar SE duration (180910 min). Nevertheless, 3 rats (25%) from the CPA+ KA group died after presenting SE and none of the 9 live animals (75%) displayed the EMJ behavior (Figs.

Fig. 2. Time in minutes of presentation of behavioral changes after i.p. KA administration. I – staring; II – wet dog shakes; III – automatisms-mild limbic convulsions; IV – severe limbic convulsions; SE – establishment of status epilepticus. SS + KA, kainic acid control group; M + KA, kainic acid group pretreated chronically with morphine; CPA + KA, kainic acid group pretreated chronically with N 6-cyclopentyl-adenosine. Values are expressed as averages 9 SEM. (*) PB 0.05 when compared with the SS + KA group.

Fig. 3. Percentage of rats showing death (mortality) and EMJ behavior after KA-induced status epilepticus. SS + KA, kainic acid control group pretreated chronically with saline solution; M +KA, kainic acid group pretreated chronically with morphine; CPA+KA, kainic acid group pretreated chronically with N 6-cyclopentyl-adenosine. (*) PB0.05 when compared with the SS +KA group.

2 and 3). In fact, all of these rats exhibited an important attenuation of motor activity 24 h after KA administration (data not shown).

4. Discussion The present study indicates that chronic pretreatments with morphine and CPA do not modify the SE duration, however they alter the expression of the seizure activity as well as death rate and EMJ behavior 24 h after SE. Our results are in agreement with the morphine-induced upregulation of m receptor binding previously described by other authors (Holaday et al., 1982; Rothman et al., 1987), a situation associated with supersensitivity to morphine effects (Yoburn et al., 1989). An interesting finding from the present study was that the upregulation of m receptors resulting from chronic morphine administration facilitated the acquisition of KA-induced SE. This result, in addition with those previously obtained with the electrical amygdala kindling process (Rocha et al., 1996), indicate that the morphine-induced enhancement of m receptors may lead to proconvulsant effects. However, as previously described, that increased m receptor binding is associated with enhanced postictal

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seizure suppression (Rocha et al., 1991, 1993). In fact, in the present study, the lack of KA-induced death rate and reduced EMJ behavior presented by morphine pretreated rats (M+KA group), insinuate a protective mechanism of m receptors during postictal and interictal periods. It is possible to presume that m receptor binding plays antiand proconvulsant effects, depending on the state of excitability (Hong et al., 1993). Concerning A1 receptors, this is the first evidence indicating that the repetitive administration of CPA results in enhanced Bmax for 3H-CPA binding. Although the mechanism by which that effect is produced is unknown, it may represent a compensatory response that results from removing repetitive exposure to an A1 agonist. A similar effect has been suggested to explain the increased m receptor binding detected during the morphine withdrawal syndrome (Holaday et al., 1982; Rothman et al., 1987). It has been proposed that adenosine increases inhibitory processes that shorten the convulsive component (Herberg et al., 1993). The administration of CPA delays the onset of NMDA-evoked seizures (Von Lubitz et al., 1993) and increases the pentylenetetrazol seizure threshold (Tchekalarova and Georgiev, 1999). Moreover, administration of adenosine agonists increases the latency of motor seizures, reduces seizure severity and enhances the postictal depression induced by the kindling process (Dragunow et al., 1985; Rosen and Berman, 1985; Whitcomb et al., 1990). The augmented seizure latency following pentylenetetrazol-induced seizures in mouse has been associated with upregulation of A1 receptors (Angelatao et al., 1991). Present results led to suggest that the previous upregulation of A1 receptors in hippocampus, amygdala and cortex as a result of chronic pretreatment with CPA, delays KA-induced SE presentation and reduces seizure severity. It is possible that these effects correlate with a supersensitivity of A1 receptors. Furthermore, the higher death rate after SE, as well as the reduced excitatory behavior and motor activity 24 h after SE displayed by rats pretreated with CPA, could be explained if the sedative effects produced by adenosine (Dunwiddie and Worth, 1982) were intensified by the upregulation of A1 receptors.

According with the Scatchard plot analysis, the range of concentrations we used gave Kd values similar to those reported for [3H]CCPA (Lohse et al., 1988) and for [3H]DAMGO (Mansour et al., 1986). The EBDA-ligand analysis gave a best fit for 1 site when the data were used. This analysis plus the intrinsic variability in the reported values for Kd of CPA suggest that no change in high/low affinity is occurring, even though in some brain regions the change in affinity is statistically significant. More detailed studies would show if the changes we observed here could be attributed to high/low affinity alteration due to treatments and if there is a functional correlation with the observed KA-induced SE presentation. Traditional pharmacological studies have used the administration of agonists or antagonists to elucidate the effects of opioid peptides and adenosine in seizure activity. Unfortunately, this approach is unable to avoid secondary effects of such substances, i.e., dependence and tolerance by opiates, or muscle relaxation and sedation by adenosine agonists. The relevance of the present study is that the upregulation of both, m and A1 receptors may be associated with supersensitivity to endogenous neurotransmitters and with subsequent enhancement of their effects. In fact, it is highly unlikely that effective concentrations of either morphine or CPA, can be found in the circulation or in the interstitial space of the brain at the end of the 2-day wash-out period (Berkowitz, 1976; Pavan and Ijzerman, 1998). The present experiments revealed that the upregulation of m or A1 receptors did not modify SE duration, but it altered excitatory behaviors and death rate 24 h after KA administration. These findings strongly support the idea that activation of opioid peptide and adenosine receptors is more involved in postictal and interictal rather than ictal events. On the other hand, it is relevant to notice that KA-induced neurotoxicity has been correlated with the duration of seizure activity (Routbort et al., 1999) and that adenosine has neuroprotective effects (MacGregor et al., 1998; Matsuoka et al., 1999; Young and Dragunow, 1995), whereas m agonists intensify KA-induced injury (Lason et al., 1988). Future experiments should be performed to evaluate if the effects

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produced by the administration of adenosine and opioid peptide agonists match with those induced by the upregulation of A1 and m receptors respectively, on the KA-induced damage. In conclusion, our results demonstrated that the upregulation of m and A1 receptors have opposite effects in the KA-induced SE. It may be that together, opioid peptide and adenosine receptors play a compensatory effect during the KA-induced SE.

Acknowledgements We thank Mrs. Magdalena Briones, Rau´ l Cardoso and Jose´ Luis Caldero´ n for their excellent technical assistance. Isabel Pe´ rez Montfort corrected the English version of the manuscript. The authors are indebted to Dr. Vero´ nica Guarner for her support. This study was partially supported by CONACyT grants 31702-M (L.R.) and 28553M (R.V.M.).

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