Distribution and co-localization of nitric oxide synthase and argininosuccinate synthetase in the cat hypothalamus

Argininosuccinate synthetase (ASS) and nitric oxide synthase (NOS) comprise part of the cyclic metabolic pathway to produce nitric oxide (NO). ASS is one of the arginine synthesis enzymes which synthesizes argininosuccinate from aspartate and citrulline, and NOS forms NO and citrulline from arginine. This study examines the localization of ASS and NOS in the cat hypothalamus using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and immunohistochemistry against ASS and NOS. NADPH-d positive and/or ASS-immunoreactive neurons were localized in the following areas: the anterior hypothalamic area, the anterior hypothalamic nucleus, the supraoptic nucleus, the suprachiasmatic nucleus, the periventricular complex, the paraventricular nucleus, the parvocellular nucleus, the lateral hypothalamic area, the dorsomedial hypothalamic nucleus, the dorsal hypothalamic area, the posterior hypothalamic area, and the supramammillary nucleus. NOS and ASS double-labeled neurons were found in the anterior hypothalamic area, the supraoptic nucleus, the central part of the paraventricular nucleus of the hypothalamus, the lateral hypothalamic area, ventral part of the parvocellular hypothalamic nucleus, the posterior hypothalamic area, and the supramammillary nucleus. Double-labeled neurons in the hypothalamus comprised 20.7-32.0% of ASS-immunoreactive neurons and 10.2-26.3% of NOS-immunoreactive neurons. The results suggest the existence of the 'NO cycle' in situ and the physiological importance of NO and argininosuccinate in several regions of the cat hypothalamus.     

Determination of the flux control coefficient of nitric oxide synthase for nitric oxide synthesis in discrete brain regions in vivo

The flux control coefficient of nitric oxide synthase (NOS), for the in vivo synthesis of the key biological mediator nitric oxide (NO), was determined in four rat brain regions with varying NOS activities (cerebral cortex, hippocampus, amygdala and cerebellum) using metabolic control theory. Flux control coefficients were calculated from the ratio of the initial slopes of the fractional effect of the NOS inhibitor N omega-nitro-L-arginine (L-NA) on NO pathway flux and NOS activity. Under conditions of normal behaviour in the rat, NOS had a flux control coefficient not significantly different from one in all regions examined. These data demonstrate that the large majority of flux control for the synthesis of NO in the brain resides in NOS itself and not in the transport of its amino acid precursor, L-arginine, across the blood-brain or neuronal cell membranes. This paper describes the first example in which the control of metabolic flux has been quantified in a mammalian system in vivo and demonstrates the power of metabolic control theory to elucidate the distribution of control within a metabolic pathway in vivo.     

Effects of veratridine on the action potentials and contractility of right and left ventricles from normo- and hypertensive rats

Induction of nitric oxide synthase and generation of nitric oxide in pancreatic islet beta-cells may mediate cytokine-induced dysfunction leading to insulin-dependent diabetes mellitus. Nitric oxide generation can be regulated by availability of arginine substrate which, in turn, may be affected by substrate utilization in competing pathways such as the arginase-catalysed formation of ornithine and urea. In this study we have investigated the activity of arginase in the rat insulinoma-derived cell line RINm5F and the effect on this of interleukin 1beta, the nitric oxide synthase reaction intermediate NG-hydroxy-l-arginine and the nitric oxide-generating compounds 3-morpholinosydnonimine and S-nitrosoglutathione. Cytosols from RINm5F cells treated with or without interleukin 1beta (0.1nM, 18h) were incubated (45min, 37 degrees C) with [U-14C]arginine. Radiolabelled products ([14C]citrulline from nitric oxide synthase, [14C]ornithine and [14C]urea from arginase) were separated by high-performance liquid chromatography or ion-exchange chromatography. Interleukin 1beta increased citrulline production (from 0.01+/-0.002 to 0.58+/-0.03 pmol/microg cell protein), indicating induction of nitric oxide synthase, and significantly decreased production of both ornithine (from 4.60+/-0.20 to 3.40+/-0.20 pmol/microg) and urea (0.93+/-0.05 to 0.69+/-0.04 pmol/microg) (P<0.001), indicating decreased activity of arginase. Arginase was significantly inhibited by NG-hydroxy-l-arginine (IC50=50 microM), S-nitrosoglutathione (500 microM: 69+/-7% of control) and 3-morpholinosydnonimine (1 mM: 57+/-7% of control) (P<0.05). We conclude that during cytokine-directed beta-cell assault nitric oxide synthase-catalysed production of NG-hydroxy-l-arginine and nitric oxide may inhibit arginase thereby increasing the availability of arginine for nitric oxide production.     

Recycling of L-citrulline to sustain nitric oxide-dependent enteric neurotransmission

Neurons that synthesize nitric oxide from arginine produce stoichiometric amounts of citrulline. We investigated whether nitric oxide-releasing enteric neurons have the capacity to recycle citrulline to arginine and thereby sustain nitrergic neurotransmission. Argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity, enzymes capable of citrulline to arginine conversion, were both localized in discrete populations of myenteric and submucosal neurons in the canine proximal colon. Argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity co-localized with neuronal beta-nicotinamide adenine dinucleotide phosphate diaphorase staining, a marker for nitric oxide synthase. The functional significance of argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity was shown by testing the effects of exogenous citrulline on responses to enteric inhibitory nerve stimulation, which were assessed by measuring contractions, inhibitory junction potentials and electrical slow waves. As shown previously, arginine analogues (L-nitroarginine methyl ester or L-nitroarginine; 100 microM) inhibited nitric oxide-dependent responses, and excess L-arginine restored inhibitory responses. Citrulline alone (0.1-2 mM) had no effect on nitrergic transmission under control conditions, but in the presence of L-nitroarginine methyl ester or L-nitroarginine, citrulline (0.1-2 mM) restored nitrergic transmission in a concentration-dependent manner. Other neutral amino acids (L-serine, L-leucine) did not mimic the effects of citrulline. Taken together, these data suggest that enteric nitrergic neurons have the enzymatic apparatus and functional capability of recycling citrulline to arginine.     

Oxidation of NG-hydroxy-L-arginine by nitric oxide synthase: evidence for the involvement of the heme in catalysis

The involvement of the protoporphyrin IX heme iron of macrophage nitric oxide synthase (NOS) in the oxidation of NG-hydroxy-L-arginine (L-NHA) to nitric oxide (NO) and citrulline was investigated by carbon monoxide (CO) inhibition studies and binding difference spectroscopy. A CO:oxygen mixture (80:20) was found to inhibit the reaction by 33% with L-NHA as a substrate compared to 57% with L-arginine. Spectral perturbations were observed upon the addition of L-NHA to oxidized NOS, producing a type I binding difference spectrum with a maximum at 384 nm and minimum at 420 nm. In addition, L-NHA was incapable of reducing anaerobic oxidized NOS in the absence of NADPH. These studies support the involvement of the heme in the oxidation of L-NHA to NO and citrulline, indicating that the heme functions in both of the currently characterized oxidative steps of the NOS reaction.     

Estrogen upregulates endothelial constitutive nitric oxide synthase expression in human osteoblast-like cells

The protective effects of estrogen on the cardiovascular system are thought to be mediated, in part, by nitric oxide (NO). Estrogen also has protective effects on bone although the mechanisms of action have not been fully established. Since nitric oxide synthase (NOS) inhibitors have been found to abrogate the protective effect of estrogen on bone in ovariectomised rats, we studied the effects of 17beta-estradiol on NOS activity and NOS mRNA levels in cultured human osteoblast-like cells. 17beta-Estradiol stimulated NOS activity by approximately 2.0 fold and this effect was reversed by the calcium chelator, EGTA, and the NOS inhibitor, L-NMMA, implicating activation of a constitutive, calcium-dependent isoform. Further studies using RT/PCR indicated that only the endothelial nitric oxide synthase (ecNOS) isoform was expressed and RNase protection assays showed that 17beta-Estradiol treatment resulted in a 2.2 fold increase in ecNOS mRNA levels. These findings suggest that estrogen stimulates NOS activity in osteoblastic cells by activation of the ecNOS pathway, and taken together with previous data, is consistent with the possibility that NO may act as a mediator of estrogen actions on bone.     

Pediatric antifungal therapy

We have recently reported the synthesis of urea from ammonia, glutamine and arginine in enterocytes of postweaning pigs. The present study was conducted to determine the compartmentation and kinetics of urea cycle enzymes in these cells. Carbamoyl phosphate synthase I (CPS I) and ornithine carbamoyltransferase (OCT) were located exclusively in mitochondria, whereas argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) were found in the cytosol. Arginase isozymes were present in both the cytosol and mitochondria of enterocytes, and differed in their sensitivity to heat inactivation. Except for OCT, Vmax values of urea cycle enzymes were much lower in enterocytes than in the liver of pigs, and vice versa for their Km values. Because of a low rate of ureagenesis in enterocytes compared with the liver, intestinal urea cycle enzymes may function primarily to synthesize citrulline. The co-localization of CPS I and OCT and a high activity of OCT in enterocyte mitochondria favors the intestinal synthesis of citrulline from ammonia, HCO3- and ornithine. Low activities of cytosolic ASS and ASL minimize the conversion of citrulline into arginine and therefore, the recycling of citrulline into ornithine via arginase in postweaning-pig enterocytes. These kinetic properties of intestinal urea cycle enzymes maximize the net synthesis of citrulline from glutamine and explain the release of large amounts of citrulline by the pig small intestine. The two compartmentally separated arginase isozymes in enterocytes may play an important role in regulating the intestinal metabolism of proline, nitric oxide and polyamines.     

Nitric oxide synthase activity in the gravid rat uterus decreases a day before the onset of parturition

OBJECTIVE: Our purpose was to determine the timing, tissue location, and isoform of the uterine nitric oxide synthase activity decrease at term in gravid rat uteri. STUDY DESIGN: Nitric oxide synthase specific activity was assayed in rat uteri 11 through 22 days' gestation by the difference in radiolabeled arginine to citrulline conversion with and without the cofactor reduced nicotinamide adenine dinucleotide phosphate. Nitric oxide synthase isoform was assessed by calcium sensitivity and subcellular location. RESULTS: Rat uterine nitric oxide synthase activity decreased between days 15 and 21 of gestation but did not decrease further at term (day 22), before and after the onset of labor. Decidual nitric oxide synthase activity exceeded the myometrial activity at 15 days' gestation, but then the two were equal at 18 through 22 days' gestation. The nitric oxide synthase activity was calcium insensitive except for half the decidual cytosolic activity on day 15. CONCLUSION: The decrease in pregnant rat uterine nitric oxide synthase activity coincides with the preparation of the uterus for parturition rather than the final activation of labor.     

Argininosuccinate synthetase: localization in astrocytes and role in the production of glial nitric oxide

An antiserum raised against the peptide representing the partial sequence 196-222 of mouse liver argininosuccinate synthetase (ASS) was used to detect and localize the enzyme in cells of neural primary cultures. No ASS immunoreactivity was detected by Western blotting in homogenates of mouse pure astroglial cultures and rat astroglia-rich cultures. However, when the cultures had been treated with bacterial lipopolysaccharide, interferon-gamma, or a combination of both, ASS immunoreactivity was disclosed. Immunocytochemical examination of rat astroglia-rich cultures revealed a colocalization of ASS with the astroglial marker glial fibrillary acidic protein (GFAP) in many cells. However, there were some GFAP-positive cells showing no specific staining for ASS, and vice versa. Colocalization of ASS with the inducible isoform of nitric oxide synthase in the same cell was shown only occasionally; nitric oxide synthase was predominantly expressed in microglial cells. In rat neuron-rich primary cultures astroglial cells as well as neurons expressed ASS. Cells of mouse pure astroglial cultures were able to synthesize arginine and, consequently, nitric oxide from citrulline, but not from ornithine. The findings demonstrate that ASS is expressed in astroglial cells under conditions that stimulate long-lasting production of nitric oxide; a functional role of this enzyme in the latter process is implicated.     

Corneal penetration of diclofenac from a fixed combination of diclofenac-gentamicin eyedrops

Recent studies suggest a dual role for nitric oxide (NO) in tumour biology. High concentrations of NO can mediate tumouricidal activity, whereas lower concentrations have been shown to promote tumour growth. In this study, NO synthase (NOS) activity was investigated in cells that were prepared from tissue from primary and metastatic sites and from malignant effusions in 41 cases of suspected ovarian cancer. NO biosynthesis, determined by nitrite + nitrate (NOx) accumulation in medium from cultured cells prepared from disaggregated tumours or effusions and indicative of the inducible NO synthase isoform, was detected in 37% of the cases investigated (range 10.2-114 microM). There was a significant relationship between NOx and tumour differentiation (P = 0.014), with NOx being significantly higher for the more differentiated tumours. NOS activity, determined by the conversion of radiolabelled L-arginine to citrulline by tissue or cell extracts, was detected in 29% of cases (range 0.9-6.9 pmol/min per mg of protein), with all samples tested being moderately or poorly differentiated. Seventy percent of this activity was calcium dependent, indicative of constitutive NOS isoforms. Morphological and immunohistochemical assessment of tumour samples indicated a significant relationship between high macrophage content and NOS activity (as NOx biosynthesis) (rs = 0.726, N = 16, P < 0.01). The relationship between NOS expression, immune response, and disease progression is complex and not simply dependent on the differentiation status of ovarian cancer.     

Immobilization-induced stress activates neuronal nitric oxide synthase (nNOS) mRNA and protein in hypothalamic-pituitary-adrenal axis in rats

The purpose of this study was to determine whether immobilization stress can cause changes in the enzyme activity and gene expression of neuronal nitric oxide synthase (nNOS) in the hypothalamus, pituitary, and adrenal gland in rats. NOS enzyme activity was measured as the rate of [3H]arginine conversion to citrulline, and the level of nNOS mRNA signal was determined using in situ hybridization and image analysis. NOS-positive cells were also visualized using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) histochemistry and by immunohistochemistry using an anti-nNOS antibody. A significant increase of NOS enzyme activity in the anterior pituitary, adrenal cortex, and adrenal medulla (1.5-, 3.5-, and 2.5-fold) was observed in the stressed animals (immobilization of 6 h) as compared to non-stressed control rats. Up-regulation of nNOS mRNA expression in anterior pituitary and adrenal cortex was already detectable after stress for 2 h with 1.5- and 2-fold increase, respectively. The nNOS mRNA signals in hypothalamic paraventricular nucleus (PVN) significantly increased after the stress for 6 h. This increase in NOS enzyme activity was confirmed using NADPH-diaphorase staining and immunostaining in the PVN and adrenal cortex. An increase of NOS enzyme activity in adrenal medulla after immobilization for 6 h posited by far longer than in the adrenal cortex and anterior pituitary. The present findings suggest that psychological and/or physiological stress causes NO release in hypothalamic-pituitary-adrenal (HPA) axis and in sympatho-adrenal system. It is suggested that NO may modulate a stress-induced activation of the HPA axis and the sympatho-adrenal medullary system. The different duration of stress-induced NOS activity in HPA axis and the adrenal medulla may suggest NO synthesis is controlled by separate mechanism in the two HPA and the sympatho-adrenal systems.     

Carbon monoxide: an endogenous modulator of the nitric oxide-cyclic GMP signaling system

Carbon monoxide (CO) is an activator of soluble guanylyl cyclase and is implicated as a neuronal messenger. CO production, nitric oxide synthase (NOS) activity, and guanosine 3',5'-monophosphate (cGMP) levels were quantitated in cerebellar granule cell cultures. Metabolic labeling experiments enabled the direct measurement of neuronal CO production in vitro. CO production is significant, and peaked during early stages of culture. NOS activity and cGMP levels synchronously increased as cells matured. Whereas inhibition of NOS depleted cGMP in mature cultures, inhibitors of CO production potentiated the nitric oxide (NO)-mediated cGMP increase. Exogenous CO at similar concentrations to endogenous levels blocked the NO-mediated cGMP increase. These results directly demonstrate that endogenous neuronal CO production is high and indicate that while NO is the major regulator of cGMP in these neurons, CO may modulate the NO-cGMP signaling system.     

Emergency medicine in the New Delhi area, India

OBJECTIVES: To examine the role of nitric oxide in the cardiovascular system in spontaneous hypertension. In particular, we wanted to know whether the production of nitric oxide in the cardiovascular system of the spontaneously hypertensive rat is different from that of the normotensive Wistar-Kyoto rat and whether nitric oxide is biologically effective in this system. DESIGN: We studied various aspects of the L-arginine-nitric oxide pathway in the cardiovascular system of spontaneously hypertensive rats and Wistar-Kyoto rats. METHODS: To address the first objective we analysed the expression of endothelial nitric oxide synthase in the heart by Western blotting and the activity of constitutive nitric oxide synthase in resistance microvessels obtained from the mesenterium, both from spontaneously hypertensive rats and Wistar-Kyoto rats aged 14-18 weeks. We also analysed the concentration of the oxidative product of nitric oxide, nitrate, in plasma from these rats. To address the second objective, that is, to assess the bioactivity of nitric oxide, we studied the accumulation in tissue of cyclic guanosine 3',5'-monophosphate (GMP), as well as the acute and chronic effects of withdrawing the nitric oxide vasodilatory tone with the inhibitor of nitric oxide synthesis NG-nitro-L-arginine methyl ester on Wistar-Kyoto rats and spontaneously hypertensive rats. RESULTS: We found that the expression of endothelial nitric oxide synthase in the heart, the activity of constitutive nitric oxide synthase in resistance microvessels and the concentration of nitrate in plasma were all significantly higher in the spontaneously hypertensive rats. In contrast, neither cyclic GMP levels nor the effects of NG-nitro-L-arginine methyl ester were greater in the spontaneously hypertensive rat than they were in the Wistar-Kyoto rat. CONCLUSIONS: The nitric oxide pathway is upregulated in the resistance circulation and the heart of the spontaneously hypertensive rat by a mechanism involving induction of the constitutive nitric oxide synthase and overproduction of nitric oxide. However, nitric oxide is not sufficiently bioactive to stimulate the formation of cyclic GMP and to maintain an adequate nitric oxide-dependent vasodilatory tone.     

Selective anesthetic inhibition of brain nitric oxide synthase

BACKGROUND: It has been postulated that nitric oxide (NO) is a neurotransmitter involved in consciousness, analgesia, and anesthesia. Halothane has been shown to attenuate NO-mediated cyclic guanosine monophosphate accumulation in neurons, and a variety of anesthetic agents attenuate endothelium-mediated vasodilation, suggesting an interaction of anesthetic agents and the NO-cyclic guanosine monophosphate pathway. However, the exact site of anesthetic inhibitory action in this multistep pathway is unclear. The current study examines effects of volatile and intravenous anesthetic agents on the enzyme nitric oxide synthase (NOS) in brain. METHODS: NOS activity was determined by in vitro conversion of [14C]arginine to [14C]citrulline. Wistar rats were decapitated and cerebellum quickly harvested and homogenized. Brain extracts were then examined for NOS activity in the absence and presence of the volatile anesthetics halothane and isoflurane, and the intravenous agents fentanyl, midazolam, ketamine, and pentobarbital. Dose-response curves of NOS activity versus anesthetic concentration were constructed. Effects of anesthetics on NOS activity were evaluated by analysis of variance. RESULTS: Control activities were 57.5 +/- 4.5 pmol.mg protein-1.min-1 in the volatile anesthetic experiments and 51.5 +/- 6.5 pmol.mg protein-1.min-1 in the intravenous anesthetic experiments. NOS activity was not affected by ketamine (< or = 1 x 10(-4) M), pentobarbital (< or = 5 x 10(-5) M), fentanyl (< or = 1 x 10(-5) M), and midazolam (< or = 1 x 10(-5) M). Halothane decreased NOS activity to 36.7 +/- 2.5 (64% of control, P < 0.01 from control), 23.8 +/- 4.3 (41%, P < 0.01 from control and < 0.05 from 0.5% halothane), 25.2 +/- 3.8 (44%, P < 0.01 from control and < 0.05 from 0.5% halothane), and 19.7 +/- 2.8 (34%, P < 0.01 from control and < 0.05 from 0.5% halothane) pmol.mg protein-1.min-1 at 0.5, 1.0, 2.0, and 3.0% vapor. Isoflurane decreased NOS activity to 48.9 +/- 6.1 (85% of control), 46.0 +/- 3.2 (80%, P < 0.05 from control), 40.3 +/- 5.1 (70%, P < 0.05 from control), and 34.2 +/- 4.0 (60%, P < 0.05 from control and 0.5% and 1.0% isoflurane) pmol.mg protein-1.min-1 at 0.5, 1.0, 1.5, 2.0% vapor, respectively. CONCLUSIONS: Volatile anesthetics inhibit brain NOS activity in an in vitro system, but the intravenous agents examined have no effect at clinically relevant concentrations. This inhibition suggests a protein-anesthetic interaction between halothane, isoflurane, and NOS. In contrast, intravenous agents appear to have no direct effect on NOS activity. Whether intravenous agents alter signal transduction or regulatory pathways that activate NOS is unknown.     

Immunocytochemical analysis of the transport of arginine analogues into nitrergic neurons and other cells in the retina and pituitary

Nitric oxide is formed by the action of nitric oxide synthase upon l-arginine. The efficacy of some exogenously applied arginine analogues in inhibiting nitric oxide synthase and thus nitrergic transmission indicates that neurons producing nitric oxide may possess an arginine transport system. To investigate whether arginine analogues are preferentially transported into nitric oxide-utilising cells or into cells making other neurochemicals, we have raised highly specific antisera against a number of arginine analogues including NG-methyl arginine, D-arginine, NGnitro-L-arginine, NG-nitro-L-arginine methyl ester and canavanine. Retinae were incubated in physiological media containing these analogues and rats were given intraperitoneal injections of the analogues to study the pituitary. Immunocytochemistry and NADPH-diaphorase histochemistry revealed that many of these analogues could be transported preferentially, but not exclusively, into nitric oxide-generating cells. However, some nitric oxide-producing cells apparently lacked the ability to take up some arginine analogues. We conclude that nitric oxide-generating cells in the retina and pituitary possess one or more arginine transporters. Other subsets of neurons that use GABA or glutamate as a neurotransmitter may also accumulate arginine analogues, possibly as a substrate for formation of these neurochemicals.     

Maternal dietary protein deficiency decreases nitric oxide synthase and ornithine decarboxylase activities in placenta and endometrium of pigs during early gestation

Little is known about the mechanism responsible for retarded placental and fetal growth induced by maternal dietary protein malnutrition. On the basis of the recent finding that nitric oxide (NO) and polyamines (products of L-arginine) play an important role in embryonic and placental development, the present study was designed to determine whether protein deficiency decreases placental and endometrial activities of NO synthase (NOS) and ornithine decarboxylase (ODC) (the first and key regulatory enzyme in polyamine synthesis). Primiparous gilts selected genetically for low or high plasma total cholesterol concentrations (low line and high line, respectively) were mated and then fed 1.8 kg/d of isocaloric diets containing 13% or 0.5% crude protein. At d 40 or 60 of gestation, they were hysterectomized, and placenta and endometrium were obtained for incubations, NOS and ODC assays, and measurements of free amino acids and polyamines. Maternal dietary protein restriction decreased arginine and ornithine concentrations, constitutive and inducible NOS activities and NO production, as well as ODC activity and polyamine concentrations in placenta and endometrium of both lines of gilts. Placental NO synthase activity and NO generation were lower in high line gilts than in low line gilts. ODC activities and polyamine concentrations in placenta and endometrium were decreased at d 60 compared with d 40 of gestation. These changes in placental and endometrial synthesis of NO and polyamines during early gestation may be a mechanism responsible for reduced placental and fetal growth in protein-deficient gilts and for altered conceptus development in high line gilts.     

Cloning and expression of the human galanin receptor GalR2

We have previously shown that the novel anticonvulsant levetiracetam exerts potent anticonvulsant activity against both focal and secondarily generalized seizures in fully amygdala-kindled rats, i.e. , a model of temporal lobe epilepsy. We examined whether levetiracetam also exhibits antiepileptogenic activity, i.e., prevents or retards acquisition or development of amygdala-kindling in rats. Before the experiments with chronic administration of levetiracetam at different doses, we determined the pharmacokinetics of the drug after i.p. injection. Levetiracetam had a relatively short half-life (about 2-3 hr) in rats, so that any lasting effects of the drug after chronic administration were certainly not due to drug accumulation. When rats were treated with levetiracetam during kindling acquisition at daily i.p. doses of 13, 27 or 54 mg/kg, the drug dose-dependently suppressed the increase in seizure severity and duration induced by repeated amygdala stimulation. After termination of daily treatment with 54 mg/kg, duration of behavioral seizures and of afterdischarges recorded from the amygdala remained to be significantly shorter compared to vehicle controls, although amygdala stimulations were continued in the absence of drug. These data thus indicate that levetiracetam not simply masked the expression of kindled seizures through an anticonvulsant action, but exerted a true antiepileptogenic effect. Adverse effects were not observed at any dose of levetiracetam tested in kindled rats. The powerful antiepileptogenic activity of levetiracetam in the kindling model indicates that levetiracetam is not only an interesting novel drug for symptomatic treatment of epilepsy but might be suited for pharmacological prevention of this disease in patients with a high prospective risk of the development of epilepsy.