Chronic unilateral inhibition of GABA synthesis in the amygdala increases specificity of conditioned fear in a discriminative fear conditioning paradigm in rats.

Neural activity in the amygdala is critical for fear learning. In anxiety disorder patients, bilateral hyperactivity of the amygdala can be observed. This hyperactivation is often associated with the facilitation of fear learning and/or over-generalization of conditioned fear. In contrast, hypoactivity of the amygdala, e.g. by pharmacological interventions, attenuates or blocks fear learning. To date, little is known about how neural excitability of the amygdala affects specificity or generalization of fear. Therefore, the present study utilized chronic inhibition of GABA synthesis in the amygdala to increase excitability and investigated the effect on the specificity of fear learning. In rats, unilateral cannulas aiming at the amygdala were implanted. The cannulas were connected to subcutaneously implanted osmotic mini pumps that delivered either the GABA synthesis inhibitor L-allylglycine or its inactive enantiomer D-allylglycine. Following one week of chronic GABA synthesis manipulation, the rats were submitted to a discriminative fear conditioning protocol. In addition, anxiety-like behavior in the light-dark box was measured. Our data show that chronic unilateral L-AG infusions into the amygdala improve the specificity of learned fear, support safety learning, and reduce fear generalization and anxiety. This data demonstrates that moderately increased amygdala excitability can be beneficial for the specificity of fear learning and highlights the potential application for therapeutic interventions.

Prefrontal/amygdalar system determines stress coping behavior through 5-HT/GABA connection.

Coping is defined as the behavioral and physiological effort made to master stressful situations. The ability to cope with stress leads either to healthy or to pathogenic outcomes. The medial prefrontal cortex (mpFC) and amygdala are acknowledged as having a major role in stress-related behaviors, and mpFC has a critical role in the regulation of amygdala-mediated arousal in response to emotionally salient stimuli. Prefrontal cortical serotonin (5-hydroxytryptamine (5-HT)) is involved in corticolimbic circuitry, and GABA has a major role in amygdala functioning. Here, using mice, it was assessed whether amygdalar GABA regulation by prefrontal 5-HT is involved in processing stressful experiences and in determining coping outcomes. First (experiment 1), bilateral selective 5-HT depletion in mpFC of mice reduced GABA release induced by stress in basolateral amygdala (BLA) and passive coping in the Forced Swimming Test (FST) (experiment 2). Moreover, prefrontal-amygdala disconnection procedure that combined a selective unilateral 5-HT depletion of mpFC and infusion of an inhibitor of GABA synthesis into the contralateral BLA, thereby to disrupt prefrontal-amygdalar serial connectivity bilaterally, showed that disconnection selectively decreases immobility in the FST. These results point to prefrontal/amygdala connectivity mediated by 5-HT and GABA transmission as a critical neural mechanism in stress-induced behavior.

GABAergic control of retinal ganglion cell dendritic development.

Developing GABAergic neurons mature long before excitatory neurons, and early GABA(A) activity exerts important paracrine effects while neurons extend dendrites and axons and they establish neural connections. One of the unique features of early GABA(A) activity is that it induces membrane depolarization and Ca(2+) influx and it shifts to inhibition when networks mature. Although it has been demonstrated in several systems that early GABA(A) signaling plays a fundamental role in guiding neurite outgrowth, it has never been investigated in the retina. Here we show that chronic GABAergic activity is required for the stabilization and maintenance of newly formed dendritic branches in developing turtle retinal ganglion cells (RGCs) in ovo. Blocking GABA(A) receptors with bicuculline or inhibiting GABA synthesis with l-allylglycine have contrasting effects on dendritic growth and branching in biocytin-labeled RGCs. Dendritic arbor reconstruction shows that bicuculline induces dendritic branch loss without global change in the extent of dendritic fields while l-allylglycine causes the entire tree to shrink. At the same time, multielectrode array recordings and Ca(2+) imaging show that l-allylglycine has similar effects to bicuculline (Leitch et al., 2005) on overall network excitability, preventing the disappearance of immature retinal waves of activity and the GABAergic polarity shift. This study demonstrates for the first time that GABA plays an important role in vivo in stabilizing developing dendrites into mature arbors in the retina. However, the way GABA influences dendritic growth appears to be driven by complex mechanisms that cannot be explained solely on the basis of overall network activity levels.

In vivo neurochemical evidence that newly synthesised GABA activates GABA(B), but not GABA(A), receptors on dopaminergic nerve endings in the nucleus accumbens of freely moving rats.

GABA released from accumbal GABAergic interneurons plays an inhibitory role in the regulation of dopamine efflux through GABA(B) and GABA(A) receptors located on accumbal dopaminergic nerve endings. The cytosolic newly synthesised GABA alters vesicular GABA levels and, accordingly, the amount of GABA released from the neuron. Therefore, we hypothesised that glutamic acid decarboxylase (GAD) which generates GABA in accumbal GABAergic neurons, at least partly determines the GABA receptor subtype-mediated GABAergic tonus. To (in)validate this hypothesis, in vivo microdialysis was used to study the effects of an intra-accumbal infusion of the GAD inhibitor l-allylglycine (allylglycine) on the accumbal dopamine efflux of freely moving rats. The intra-accumbal infusion of allylglycine (50.0, 250.0 and 500.0 nmol) dose-dependently increased the accumbal dopamine levels. The co-administration of tetrodotoxin (720 pmol) suppressed the allylglycine (500.0 nmol)-induced dopamine efflux. The intra-accumbal infusion of GABA(B) receptor agonist baclofen (2.5 and 5.0 nmol) inhibited the allylglycine (500.0 nmol)-induced dopamine efflux. The baclofen's effects were counteracted by GABA(B) receptor antagonist saclofen (10.0 nmol). Neither GABA(A) receptor agonist (muscimol: 25.0 and 250.0 pmol) nor antagonist (bicuculline: 50.0 pmol) altered the allylglycine (250.0 and 500.0 nmol)-induced dopamine efflux. The present study provides in vivo neurochemical evidence that newly synthesised GABA that exerts an inhibitory tonus on the accumbal dopaminergic activity, acts at the level of GABA(B) receptors, but not GABA(A) receptors. The present study also shows that there is an allylglycine-insensitive GABA pool that release GABA exerting an inhibitory control of the accumbal dopaminergic activity, at the level of GABA(A) receptors. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

ONO 3403, a synthetic serine protease inhibitor, inhibits lipopolysaccharide-induced tumor necrosis factor-{alpha} and nitric oxide production and protects mice from lethal endotoxic shock.

ONO 3403, a new synthetic serine protease inhibitor, is a derivative of camostat mesilate and has a higher protease-inhibitory activity. The effect of ONO 3403 on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α and nitric oxide (NO) production in RAW 264.7 macrophage-like cells was examined. ONO 3403 significantly inhibited LPS-induced TNF-α production at a lower concentration than camostat mesilate. It also inhibited LPS-induced NO production. Their inhibition was responsible for the reduced mRNA expression of TNF-α and inducible NO synthase. In LPS-stimulated cells, ONO 3403 prevented the augmentation of MyD88 expression and inhibited the phosphorylation of IκB-α, stress-activated protein kinase (SAPK) and IRF-3, and the production of interferon-ß. ONO 3403 abolished the elevation of the extracellular serine protease activity in response to LPS. Further, it reduced the circulating TNF-α level, hepatic injury and mortality in mice receiving an injection of D-galactosamine and LPS. ONO 3403 was suggested to inhibit LPS-induced inflammatory responses via inactivation of MyD88-dependent and independent pathways.

L-allylglycine dissociates the neural substrates of fear in the periaqueductal gray of rats.

The dorsal (dPAG) and ventral (vPAG) regions of the periaqueductal gray are well known to contain the neural substrates of fear and anxiety. Chemical or electrical stimulation of the dPAG induces freezing, followed by a robust behavioral reaction that has been considered an animal model of panic attack. In contrast, the vPAG is part of a neural system, in which immobility is the usual response to its stimulation. The defense reaction induced by the stimulation of either region is accompanied by antinociception. Although GABAergic mechanisms are known to exert tonic inhibitory control on the neural substrates of fear in the dPAG, the role of these mechanisms in the vPAG is still unclear. The present study examined defensive behaviors and antinociception induced by microinjections of an inhibitor of gamma-aminobutyric acid synthesis, L-allylglycine (l-AG; 1, 3, and 5 microg/0.2 microl), into either the dPAG or vPAG of rats subjected to the open field and tail-flick tests. Passive or tense immobility was the predominant behavior after L-AG (1 or 3 microg) microinjection into the vPAG and dPAG, respectively, which was replaced with intense hyperactivity, including jumps or rearings, after injections of a higher dose (5 microg/0.2 microl) into the dPAG or vPAG. Moreover, whereas intra-dPAG injection of 3 microg L-AG produced intense antinociception, only weak antinociception was induced by intra-vPAG injections of 5 microg L-AG. These findings suggest that GABA mechanisms are involved in the mediation of antinociception and behavioral inhibition to aversive stimulation of the vPAG and exert powerful control over the neural substrates of fear in the dPAG to prevent a full-blown defense reaction possibly associated with panic disorder.

Modelling of tumour--host coexistence In vitro in the presence of serine protease inhibitors.

The activities of cell surface serine proteases are markedly enhanced in malignant tumours. Proteolytic degradation of the extracellular matrix and basal membrane of normal cells is an important event for tumour cell growth and invasion. Two well-known broad-spectrum inhibitors of serine protease, Foy-305 and Ono-3403, were evaluated for their ability to affect the growth rate and survival of MCF7 breast cancer cells co-cultured with MRC5 lung fibroblasts as feeder cells in the absence of serum. Flow cytometry and differential staining demonstrated that in the mixed culture, the rate of tumor growth was dependent upon the presence of the feeder MRC5 lung fibroblasts and could be obviated by the additional presence of the inhibitors of serine proteases.

Disruption of GABAergic tone in the dorsomedial hypothalamus attenuates responses in a subset of serotonergic neurons in the dorsal raphe nucleus following lactate-induced panic.

Panic patients are vulnerable to induction of panic attacks by sub-threshold interoceptive stimuli such as intravenous (i.v.) sodium lactate infusions. Facilitation of serotonergic signaling with selective serotonin reuptake inhibitors can suppress anxiety and panic-like responses, but the mechanisms involved are not clearly defined. We investigated the effects of i.v. 0.5 M sodium lactate or saline, in control and panic-prone rats on c-Fos expression in serotonergic neurons within subdivisions of the midbrain/pontine raphe nuclei. Rats were chronically infused with either the GABA synthesis inhibitor l-allylglycine into the dorsomedial hypo thalamus to make them panic-prone, or the enantiomer d-allylglycine (d-AG) in controls. Lactate increased c-Fos expression in serotonergic neurons located in the ventrolateral part of the dorsal raphe nucleus (DRVL) and ventrolateral periaqueductal gray (VLPAG) of control, but not panic-prone, rats. The distribution of lactate-sensitive serotonergic neurons in d-AG-treated rats is virtually identical to previously defined pre-sympathomotor serotonergic neurons with multisynaptic projections to peripheral organs mediating 'fight-or-flight'-related autonomic and motor responses. We hypothesized that serotonergic neurons within the DRVL/VLPAG region represent a 'sympathomotor control system' that normally limits autonomic/behavioral responses to innocuous interoceptive and exteroceptive stimuli, and that dysfunction of this serotonergic system contributes to an anxiety-like state and increases vulnerability to panic in animals and humans.

Neural pathways underlying lactate-induced panic.

Panic disorder is a severe anxiety disorder characterized by susceptibility to induction of panic attacks by subthreshold interoceptive stimuli such as 0.5 M sodium lactate infusions. Although studied for four decades, the mechanism of lactate sensitivity in panic disorder has not been understood. The dorsomedial hypothalamus/perifornical region (DMH/PeF) coordinates rapid mobilization of behavioral, autonomic, respiratory and endocrine responses to stress, and rats with disrupted GABA inhibition in the DMH/PeF exhibit panic-like responses to lactate, similar to panic disorder patients. Utilizing a variety of anatomical and pharmacological methods, we provide evidence that lactate, via osmosensitive periventricular pathways, activates neurons in the compromised DMH/PeF, which relays this signal to forebrain limbic structures such as the bed nucleus of the stria terminalis to mediate anxiety responses, and specific brainstem sympathetic and parasympathetic pathways to mediate the respiratory and cardiovascular components of the panic-like response. Acutely restoring local GABAergic tone in the DMH/PeF blocked lactate-induced panic-like responses. Autonomic panic-like responses appear to be a result of DMH/PeF-mediated mobilization of sympathetic responses (verified with atenolol) and resetting of the parasympathetically mediated baroreflex. Based on our findings, DMH/PeF efferent targets such as the C1 adrenergic neurons, paraventricular hypothalamus, and the central amygdala are implicated in sympathetic mobilization; the nucleus of the solitary tract is implicated in baroreflex resetting; and the parabrachial nucleus is implicated in respiratory responses. These results elucidate neural circuits underlying lactate-induced panic-like responses and the involvement of both sympathetic and parasympathetic systems.

Panic-prone state induced in rats with GABA dysfunction in the dorsomedial hypothalamus is mediated by NMDA receptors.

Rats with chronic inhibition of GABA synthesis and consequently enhanced glutamatergic excitation in the dorsomedial hypothalamus (DMH) develop panic-like responses, defined as tachycardia, tachypnea, hypertension, and increased anxiety as measured by a social interaction (SI) test, after intravenous sodium lactate infusions, a phenomenon similar to patients with panic disorder. Therefore, the present studies tested the role of the postsynaptic NMDA and AMPA type glutamatergic receptors in the lactate-induced panic-like responses in these rats. Rats were fit with femoral arterial and venous catheters and Alzet pumps [filled with the GABA synthesis inhibitor L-allylglycine (L-AG; 3.5 nmol/0.5 microl per hour) or its inactive isomer D-AG] into the DMH. After 4-5 d of recovery only those rats with L-AG pumps exhibited panic-like responses to lactate infusions. Using double immunocytochemistry, we found that rats exhibiting panic-like responses (e.g., L-AG plus lactate) had increased c-Fos immunoreactivity in DMH neurons expressing the NMDA receptor 1 (NR1) subunit, but not those expressing the glutamate receptor 2 and 3 subunits of the AMPA receptors. To confirm this pharmacologically, we tested another group of rats implanted with l-AG pumps with intravenous lactate infusions preceded by injections of either NMDA [aminophosphonopentanoic acid (AP-5) or (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine maleate (MK-801)] or non-NMDA [CNQX or 4-(8-methyl-9H-1,3-dioxolo[4,5-h][2,3]benzodazepin-5-yl)-benzenamine dihydrochloride (GYKI52466)] antagonists into the DMH. Injections of NMDA, but not non-NMDA, antagonists into the DMH resulted in dose-dependent blockade of the tachycardia, tachypnea, hypertension, and SI responses after lactate infusions. These results suggest that NMDA, and not non-NMDA, type glutamate receptors regulate lactate-induced panic-like responses in rats with GABA dysfunction in the DMH.

Conversion into GABA (gamma-aminobutyric acid) may reduce the capacity of L-glutamine as an insulin secretagogue.

We have carried out a detailed examination of L-glutamine metabolism in rat islets in order to elucidate the paradoxical failure of L-glutamine to stimulate insulin secretion. L-Glutamine was converted by isolated islets into GABA (gamma-aminobutyric acid), L-aspartate and L-glutamate. Saturation of the intracellular concentrations of all of these amino acids occurred at approx. 10 mmol/l L-glutamine, and their half-maximal values were attained at progressively increasing concentrations of L-glutamine (0.3 mmol/l for GABA; 0.5 and 1.0 mmol/l for Asp and Glu respectively). GABA accumulation accounted for most of the 14CO2 produced at various L-[U-14C]glutamine concentrations. Potentiation by L-glutamine of L-leucine-induced insulin secretion in perifused islets was suppressed by malonic acid dimethyl ester, was accompanied by a significant decrease in islet GABA accumulation, and was not modified in the presence of GABA receptor antagonists [50 micromol/l saclofen or 10 micromol/l (+)-bicuculline]. L-Leucine activated islet glutamate dehydrogenase activity, but had no effect on either glutamate decarboxylase or GABA transaminase activity, in islet homogenates. We conclude that (i) L-glutamine is metabolized preferentially to GABA and L-aspartate, which accumulate in islets, thus preventing its complete oxidation in the Krebs cycle, which accounts for its failure to stimulate insulin secretion; (ii) potentiation by L-glutamine of L-leucine-induced insulin secretion involves increased metabolism of L-glutamate and GABA via the Krebs cycle (glutamate dehydrogenase activation) and the GABA shunt (2-oxoglutarate availability for GABA transaminase) respectively, and (iii) islet release of GABA does not seem to play an important role in the modulation of the islet secretory response to the combination of L-leucine and L-glutamine.

Growth inhibition of mouse autochthonous skin cancer by oral administration of new serine protease inhibitor ONO-3403.

The orally active serine protease inhibitor ONO-3403 is an analog of FOY-3403 that has more potent protease-inhibitory activity. In the present study, oral administration of ONO-3403 was used to challenge 3-methylcholanthrene-induced carcinoma. This drug was administered 3 times daily for 9 weeks via a stomach tube at a dose of 10 mg/kg in a 1-ml volume in 6 mice harboring solid tumors. This protease inhibitor significantly inhibited tumor growth (p<0.001) and prolonged survival-time (p<0.01). These results indicated that oral administration of the potent serine protease inhibitor ONO-3403 has an antitumor effect on malignant tumors.

Decrease in growth factor receptors after treatment with serine protease inhibitor ONO-3403.

FOY-305 is a synthetic serine protease inhibitor and ONO-3403 is a derivative with a higher protease-inhibitory activity. The growth-suppressive effects of ONO-3403 were more prominent in Ha-ras-transformed NIH3T3 (ras-NIH) cells than in non-transformed NIH3T3 cells. After treatment of ras-NIH cells with ONO-3403 at 100-200 microg/ml, the percentage of cells found in G(1) phase decreased and, concomitantly, that in S phase increased. Molecular events caused by ONO-3403 were investigated by Western blot analysis using anti-phosphotyrosine antibody. The results showed a marked decrease in the tyrosine phosphorylation level of a 180-kDa protein after treatment with ONO-3403. This 180-kDa phosphotyrosine-containing molecule which was tentatively designated pY-p180 might be platelet-derived growth factor (PDGF) receptor since addition of PDGF to serum-starved NIH3T3 cells induced a marked tyrosine phosphorylation of the same size within 5 min. This was further confirmed by immunoprecipitation of cell extract with anti-PDGF-receptor antibody followed by Western blot analysis using anti-phosphotyrosine antibody. Treatment of T.Tn human esophageal carcinoma cells with ONO-3403 caused also decrease in pY-p180, which appeared to be epidermal growth factor receptor. Thus, ONO-3403 may induce growth suppression by down-regulation of cell surface growth factor receptors.

Growth inhibition of mouse skin tumor by serine protease inhibitor ONO-3403.

The new serine protease inhibitor, ONO-3403 is an analog of FOY-305 (Foypan). The IC50s values of ONO-3403 toward serine proteases, such as trypsin, plasmin, kallikrein and thrombin are much lower than that of FOY-305. To investigate the growth-suppressing effect of ONO-3403 on 3-methylcholanthrene-induced autochthonous solid tumors in mice, ONO-3403 was intraperitoneally administered to mice at a dose of 4 mg/kg twice a day for 5 weeks. All seven mice receiving the drug had a solitary tumor and showed potent growth suppression (p<0.001) without any apparent side effects such as hair loss and body weight loss. The results suggest that ONO-3403 may be useful for the treatment of squamous cell carcinoma.

Chronic oral administration of protease inhibitor decreases CCK-A receptor mRNA expression but increases pancreatic growth in rats.

It is well-known that chronic oral administration of trypsin inhibitors induces pancreatic hypertrophy and hyperplasia via stimulation of endogenous cholecystokinin (CCK) release. Because the growth-promoting effect of CCK on the pancreas is specifically mediated by the CCK-A receptor, we examined the plasma CCK concentrations, the expression of CCK mRNA in the intestine and CCK-A receptor mRNA in the pancreas, and pancreatic growth in rats after chronic oral administration of synthetic protease inhibitor (PI). PI at a dose of 100 mg/kg body weight was administered via an orogastric tube once daily for 20 days. Plasma CCK concentrations at 24 hours after the first PI administration were significantly higher than those in randomly fed rats (6.57 +/- 0.67 pmol/L vs 4.31 +/- 0.51 pmol/L; p < 0.001), and further increased to 14.24 +/- 1.63 pmol/L after PI for 10 days and decreased to 10.05 +/- 0.72 pmol/L after 15 days of PI administration. Treatment with PI for 20 days significantly increased the pancreatic weight, and the total pancreatic protein and DNA content by 190%, 290%, and 170%, respectively, when compared to the control rats. Chronic oral administration of PI, however, reduced CCK-A receptor mRNA expression in the pancreas by 60%. These findings suggest that chronic oral administration of PI induces an elevation of endogenous CCK release and stimulates pancreatic growth, but down-regulates the biosynthesis of CCK-A receptor at the transcriptional level in the pancreas.

Evidence for GABA(A) receptor agonistic properties of ketamine: convulsive and anesthetic behavioral models in mice.

UNLABELLED: We examined the potentiation by ketamine of the gamma-aminobutyric acid(A) (GABA(A)) receptor function using convulsive and anesthetic behavioral models in adult male ddY mice. General anesthetic potencies were evaluated by a rating scale, which provided the data for anesthetic scores, loss of righting reflex, duration, and recovery time. All drugs were administered intraperitoneally. Small subanesthetic doses of ketamine did inhibit tonic seizures induced by a large dose of the GABA(A) receptor antagonist bicuculline (8 mg/kg). The 50% effective dose value was 15 (95% confidence limits 10-22) mg/kg. Even large anesthetic doses (100-150 mg/kg) did not suppress clonic seizures in 50% of the animals. The GABA(A) receptor agonist, muscimol (0.32-1.12 mg/kg), potentiated ketamine-induced anesthesia in a dose-dependent fashion (P < 0.05). Similarly, the benzodiazepine receptor agonist, diazepam (1-3 mg/kg), augmented ketamine anesthesia in a dose-dependent manner (P < 0.05). Bicuculline (2-5 mg/kg) dose-dependently antagonized ketamine-induced anesthesia (P < 0.05). Neither the benzodiazepine receptor antagonist, flumazenil (2-20 mg/kg), nor the GABA synthesis inhibitor, L-allylglycine (200 mg/kg), affected the anesthetic action of ketamine. These results suggest that ketamine has GABA(A) receptor agonistic properties and that ketamine-induced anesthesia is mediated, at least in part, by GABA(A) receptors. IMPLICATIONS: We examined the potentiation by ketamine of the gamma-aminobutyric acid(A) receptor function using convulsive and anesthetic behavioral models in mice. Subanesthetic doses of ketamine-inhibited tonic convulsions induced by the gamma-aminobutyric acid(A) receptor antagonist bicuculline. The gamma-aminobutyric acid(A) receptor agonist, muscimol, potentiated ketamine-induced anesthesia. Bicuculline antagonized ketamine anesthesia, but the benzodiazepine receptor antagonist, flumazenil, and the gamma-aminobutyric acid synthesis inhibitor, L-allyglycine, did not. The effects of ketamine on the gamma-aminobutyric acid(A) receptors appear to correlate with its anesthetic actions.

Oral administration of a synthetic trypsin inhibitor increases pancreatic duct function in CCK-A receptor-deficient rats.

The effects of oral administration of a synthetic trypsin inhibitor on bicarbonate secretion were examined in cholecystokinin A (CCK-A) receptor-deficient (OLETF) rats and compared with Wistar rats. Rats were fed chow containing 0.1% trypsin inhibitor for 7 days. Rats were prepared with cannulae draining bile and pancreatic juice separately and with duodenal and extrajugular vein cannulae after 3-day trypsin inhibitor ingestion. Then the animals were maintained in Bollman cages, and the experiments were conducted 4 days after surgery. After 1.5 h of basal secretion with bile-pancreatic juice return, bile-pancreatic juice was diverted for 2 h. The responses of bicarbonate secretion to bile-pancreatic juice diversion were significantly enhanced in rats treated with trypsin inhibitor compared with those given a control diet, whereas responses of fluid and protein secretion were not affected in OLETF rats. The response of protein secretion, but not those of fluid or bicarbonate secretion, was enhanced in Wistar rats by treatment with trypsin inhibitor. Carbonic anhydrase II gene expression was increased by 7-day treatment with trypsin inhibitor only in OLETF rats, and not in Wistar rats.

LY354740, a potent group II metabotropic glutamate receptor agonist prevents lactate-induced panic-like response in panic-prone rats.

LY354740 is a potent and selective agonist at the group II metabotropic glutamate receptors and is shown to be an effective inhibitor of glutamate release with significant anxiolytic and drug withdrawal alleviating properties in certain animal models. Rats with chronic inhibition of GABA synthesis in the dorsomedial hypothalamus (DMH) are highly anxious and exhibit panic-like responses to peripheral lactate infusions similar to patients with panic disorder. Using these panic-prone rats, we tested the efficacy of LY354740 in preventing the lactate-induced panic-like response, comparing it to alprazolam, a clinically effective anti-panic drug. Rats were fitted with femoral arterial and venous catheters and implanted with Alzet pumps infusing the GABA synthesis inhibitor L-allylglycine into the DMH. After four days of recovery, they were confirmed to be panic-prone to lactate infusions as indicated by increases in heart rate, blood pressure, respiratory rate and "anxiety" measured in the social interaction test. Next, they were pretreated with either vehicle, LY354740 (0.3 and 0.6 mg/kg) or alprazolam (0.5 and 1.0 mg/kg) and re-challenged with lactate infusions. LY354740 treatment was equally efficacious as alprazolam in preventing lactate-induced panic attacks in this model. These data suggest that LY354740 could be a novel anti-panic drug, as effective as alprazolam in acute treatment.

Different effects of trypsin inhibitors on intestinal gene expression of secretin and on pancreatic bicarbonate secretion in CCK-A-receptor-deficient rats.

The effects of oral administration of two synthetic trypsin inhibitors (camostate and ONO-3403) and soybean trypsin inhibitor (SBTI) on cholecystokinin (CCK), secretin gene expression and pancreatic secretion were examined in CCK-A-receptor-deficient (OLETF) rats. The rats were fed chow containing 0.1% trypsin inhibitors for 7 days. To examine pancreatic secretion, the rats were prepared with cannulae to drain the bile and pancreatic juice separately, a duodenal cannula and an external jugular vein cannula. The animals were maintained in Bollman cages and the experiments were conducted 4 days after surgery. The levels of CCK mRNA were significantly increased by each treatment. The levels of secretin mRNA were significantly increased by camostate and SBTI, but not by ONO-3403. Bicarbonate secretion was significantly increased in rats treated with camostate and ONO-3403, but not SBTI, while protein secretion was not affected by any treatment. These observations suggest that increased bicarbonate secretion produced by synthetic trypsin inhibitors in CCK-A-receptor-deficient rats may not be due to secretin but due to ONO-3403 in the circulation.

Effects of a new cholecystokinin antagonist, TS-941, on experimental acute pancreatitis in rats.

The effects of a new benzodiazepine-derivative, cholecystokinin receptor antagonist, TS-941, on experimental acute pancreatitis were studied in rats. Hemorrhagic pancreatitis was induced by an infusion of a mixture of trypsin and taurocholate into the pancreatic duct. Edematous pancreatitis was induced by intraperitoneal injection of 40 microg/kg body weight of cerulein at 0 and 1 h after the start of the experiment. TS-941 (3 mg/kg) was injected subcutaneously immediately and 3 h after the induction of pancreatitis. In trypsin-taurocholate-induced pancreatitis, TS-941, with or without the synthetic trypsin inhibitor ONO-3403, had no beneficial effects on the survival rate, pancreatic wet weight, and serum pancreatic enzymes. In cerulein-induced pancreatitis, the treatment with TS-941 significantly reduced the increases of pancreatic wet weight and serum amylase and lipase. Plasma trypsinogen activation peptide (TAP) significantly rose 1 h after the first injection of cerulein. TS-941 inhibited the liberation of TAP in cerulein-induced pancreatitis. These results show that TS-941 is effective for prevention of cerulein-induced edematous pancreatitis. ONO-3403 has beneficial effects on trypsin-taurocholate-induced hemorrhagic pancreatitis, but the combination of TS-941 and ONO-3403 has no additive effect.