Pranidipine, a 1,4-dihydropyridine calcium channel blocker that enhances nitric oxide-induced vascular relaxation.

Pranidipine, a long acting 1,4-dihydropyridine calcium channel blocker, prolongs nitric oxide (NO)-mediated relaxation of rat aorta; it prolongs acetylcholine-induced relaxation in presence of endothelium as well as nitroglycerin-induced relaxation in absence of endothelium. In rat aorta the effect of pranidipine on NO-mediated relaxation is cyclic guanosine monophosphate (cGMP)-independent, but in guinea pig carotid artery the same effect of pranidipine is cGMP-dependent. It has been reported that in co-cultured human endothelial and smooth muscle cells pranidipine, at a higher concentration (10(-6) M), enhances vasorelaxant effect of NO by blocking NO decomposition. The enhancement of NO action by pranidipine differs from the direct NO-releasing action of other 1,4-dihydropyridines. It is expected that enhancement of NO-induced vasodilatation will lead to a venodilator action in vivo and less peripheral edema. The target organ protective effects of pranidipine are also reviewed in this article.

A single nucleotide polymorphism of CYP2b6 found in Japanese enhances catalytic activity by autoactivation.

A single nucleotide polymorphism (SNP) resulting in a substitution from Gln to His was found in exon 4 of the CYP2B6 gene in Japanese. The frequency of the variant allele was found to be 19.9%. The mutant- and the wild-type enzymes were expressed in Escherichia coli, and the effects of the single amino acid substitution on the catalytic activity were examined by investigating the kinetic profiles of 7-ethoxycoumarin O-deethylase activity. The wild-type enzyme showed typical Michaelis-Menten kinetics, while the mutant-type enzyme represented the sigmoidal kinetics with a higher V(max) value compared to that of the wild-type enzyme. Eadie-Hofstee plots further revealed an existence of allosteric effects for the reaction catalyzed by the variant. This is the first evidence demonstrating that only one amino acid substitution, Gln172His, caused by natural SNP enhances the catalytic activity of CYP by obtaining the character of homotropic cooperativity.

Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on spatial memory and on cholinergic and peptidergic neurons in rats with ibotenate-induced lesions of the nucleus basalis magnocellularis.

We conducted behavioral and neurochemical studies of a novel prolyl endopeptidase inhibitor, (S)2-[[(S)-2-(hydroxyacetyl)-1pyrrolidinyl]carbonyl]-N-(phenylmeth yl)-1-pyrrolidine-carboxamide (JTP-4819), in rats with lesions of the nucleus basalis magnocellularis (NBM-lesioned rats) induced by ibotenate. Administration of JTP-4819 (1 and 3 mg/kg, p.o.), on and after the 8th day, significantly shortened the escape latency in the Morris water maze as compared to the vehicle-treated group. JTP-4819 also significantly increased the path length in the quadrant with the platform removed in the spatial probe trial. Neurochemical studies of brains removed after the Morris water maze task showed that choline acetyltransferase activity in the cerebral cortex, but not the hippocampus, was significantly reduced by NBM lesioning, while there were no changes of muscarinic M1 receptor binding activity detected using [3H]pirenzepine. JTP-4819 had almost no effect on these cholinergic parameters in NBM-lesioned rats. Substance P-like immunoreactivity (LI), thyrotropin-releasing hormone (TRH)-LI, and arginine-vasopressin-LI were not significantly changed in the cerebral cortex and hippocampus of NBM-lesioned rats as compared to sham-operated rats. However, these neuropeptide levels were significantly increased in both brain regions by repeated administration of JTP-4819 (1, 3 and/or 10 mg/kg, p.o.). These results suggest that JTP-4819 ameliorated memory impairment due to NBM lesioning by potentiating SP, TRH and AVPergic neurons secondary to PEP inhibition.

Identification of possible quantitative trait loci responsible for hyperglycaemia after 70% pancreatectomy using a spontaneously diabetogenic rat.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type non-insulin-dependent diabetes mellitus (NIDDM) in humans. The OLETF rat exhibits sustained hyperglycaemia after partial pancreatectomy, while the normal control rat does not. This difference is thought to be genetically determined and to be caused by impairment of beta-cell regrowth, a possible event involved in the pathogenesis of NIDDM. Our investigation was designed to identify quantitative trait loci (QTL) responsible for post-pancreatectomy hyperglycaemia by performing a genome-wide scan in an F2 intercross obtained by mating the OLETF and Fischer-344 (F344) rats. We have identified three possible QTL on rat chromosomes (Chrs) 3, 14 and 19 that account for a total of approximately 75% of the genetic variance in the F2. For the QTL on Chr 14, the OLETF allele corresponds with increased glucose levels, as expected. Surprisingly, for the QTL on Chr 19, the F344 allele corresponds with increased glucose levels. The Chr 3 QTL exhibits heterosis, heterozygotes showing significantly higher glucose levels than OLETF or F344 homozygotes. We also found evidence for interaction (epistasis) between the QTL on Chrs 14 and 19.

Differential properties of the optical-isomers of pranidipine, a 1,4-dihydropyridine calcium channel modulator.

Pranidipine is an optically-active 1,4-dihydropyridine (DHP) voltage-dependent L-type calcium channel inhibitor. Certain enantiomeric pairs display opposite effects, i.e., inhibition and activation of the calcium channel while others exhibit the same qualitative actions. We investigated pranidipine, a new DHP, using a paradigm of vascular smooth muscle reactivity. In isolated rat aorta, depolarized with 80 mM KCl, both isomers of pranidipine caused a right-ward shift of the concentration-contraction curves for extracellular Ca2+. The apparent pA2, values of the S-isomer and R-isomer were 10.03 and 8.36, respectively, providing evidence that the calcium channel blocking action of the S-isomer was 50 times more potent than that of the R-isomer. Antihypertensive actions of these two isomers studied in pentobarbital-anaesthetized spontaneously hypertensive rats, revealed that the S-isomer, at doses of 3-30 microg/kg i.v. decreased blood pressure in a dose-dependent manner, while the R-isomer had no effect on blood pressure at those doses. We conclude that the pair of enantiomers of pranidipine qualitatively display the same Ca2+ channel blocking action and that neither isomer exhibits Bay K 8644-like activation. Pranidipine may be useful in studies on the architecture of the DHP receptor 'pocket'.

Identification of a quantitative trait locus influencing plasma insulin levels after 70% pancreatectomy using the OLETF rat.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type non-insulin-dependent diabetes mellitus (NIDDM) in humans. The OLETF rat has poor capacity for proliferation of pancreatic beta-cells after partial pancreatectomy, which may be the critical pathogenetic event in NIDDM development. The poor pancreatic beta-cell proliferation in this model is characterized by reduction in beta-cell mass and decrease in insulin content in the remnant pancreas. Our investigation was designed to identify quantitative trait loci (QTLs) responsible for beta-cell mass and plasma insulin levels after partial pancreatectomy by performing a genome-wide scan in an F2 intercross obtained by mating the OLETF and the Fischer-344 (F344) rats. We have identified a suggestive QTL for the plasma insulin levels, near D20Mgh5 on rat chromosome 20, with a maximum lod score of 3.75 which accounts for 20% of the total variance, while no QTLs were detected for beta-cell mass. This chromosome 20 QTL, whose OLETF allele is associated with low plasma insulin levels through acting in an incompletely recessive manner, may affect insulin secretion itself rather than beta-cell proliferation.

A major quantitative trait locus co-localizing with cholecystokinin type A receptor gene influences poor pancreatic proliferation in a spontaneously diabetogenic rat.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type, non-insulin-dependent diabetes mellitus (NIDDM) in humans. The OLETF rat has poor capacity for pancreatic proliferation, which may be the critical pathogenetic event in NIDDM development. Our investigation was designed to identify quantitative trait loci (QTLs) responsible for poor pancreatic proliferation by examining compensatory proliferation of the pancreatic remnant after partial pancreatectomy and performing a genome-wide scan in an F2 intercross obtained by mating the OLETF and the Fischer-344 (F344) rats. We identified a highly significant QTL on rat Chromosome 14 with a maximum lod score of 16.7, which accounts for 55% of the total variance. The QTL co-localizes with the gene encoding cholecystokinin type A receptor (CCKAR) which is likely to mediate the trophic effect of cholecystokinin on pancreas and is defective in the OLETF rat.

A novel prolyl endopeptidase inhibitor, JTP-4819--its behavioral and neurochemical properties for the treatment of Alzheimer's disease.

Formation of beta-amyloid and neurofibrillary tangles in the brain due to genetic or other factors is the most frequent cause of Alzheimer's disease. In addition, marked reduction of certain brain neuropeptide levels is a consistent finding in patients with Alzheimer's disease, together with the deterioration of cholinergic neurons. Currently, there is great demand for the development of new drugs to improve memory deficits or to delay the neurodegenerative process in conditions such as Alzheimer's disease. In this report, the pharmacological actions of JTP-4819, a novel specific prolyl endopeptidase (PEP) inhibitor devised for the treatment of Alzheimer's disease, are reviewed with respect to its effects on PEP activity, neuropeptidergic and cholinergic neurons, and memory-related behavior in rats. We also discuss the possible beneficial effect of JTP-4819 on beta-amyloid metabolism and its potential neuroprotective properties.

Effect of a prolyl endopeptidase inhibitor, JTP-4819, on radial maze performance in hippocampal-lesioned rats.

The effect of a novel prolyl endopeptidase (PEP) inhibitor, (S)-2-2[[(S)-2-(hydroxyacetyl)-1-pyrrolidinyl]carbonyl]-N-(phenylmethyl) -1-pyrrolidinecarboxamide (JTP-4819), on spatial learning deficits in rats with dorsal hippocampal (DH) lesions was examined using an eight-arm radial maze. The correct performance remained at chance levels even after 18 acquisition trials in rats with DH lesions. JTP-4819 (3.0 mg/kg, p.o.) significantly ameliorated this learning impairment after 34-41 days of treatment. When DH lesions were created in rats after achievement of learning, postoperative performance deteriorated prominently, but gradually recovered with the repetition of trials. JTP-4819 (3.0 mg/kg, p.o.) significantly decreased the number of trials needed to reattain learning criterion. After the behavioral experiment, the choline acetyltransferase (ChAT) activity and [3H]-pirenzepine binding (Kd, Bmax) in the residual hippocampus and cerebral cortex were analyzed. Neither parameter was significantly affected by JTP-4819. In conclusion, JTP-4819 can improve both learning and relearning deficits of spatial memory in DH-lesioned rats, postulating that enhancement of neuropeptide activity via PEP inhibition may be involved in the mechanism of action of JTP-4819.

Enhanced expression of hepatic acyl-coenzyme A synthetase and microsomal triglyceride transfer protein messenger RNAs in the obese and hypertriglyceridemic rat with visceral fat accumulation.

The liver plays a central role in lipoprotein metabolism. In particular, very-low density lipoprotein (VLDL) is assembled in the hepatocytes and secreted into the blood circulation. The VLDL is then catabolized to low-density lipoprotein by lipoprotein lipase and hepatic triglyceride lipase. Obese subjects, especially those with visceral fat accumulation, are frequently associated with hyperlipidemia, non-insulin-dependent diabetes mellitus (NIDDM), and hypertension. The mechanism of hyperlipidemia in visceral fat obesity has not yet been elucidated. Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model of NIDDM, characterized by obesity with visceral fat accumulation, hyperlipidemia, and late-onset insulin resistance. To elucidate the mechanism of hyperlipidemia observed in OLETF rats, we focused on the production of VLDL by the liver and investigated hepatic messenger RNA (mRNA) levels of microsomal triglyceride transfer protein (MTP), acyl-coenzyme A synthetase (ACS), and apolipoprotein B (apo B), which play important roles in VLDL synthesis and secretion. In 6-week-old OLETF rats, in which insulin resistance had not been manifested, visceral fat weight was already higher and portal free fatty acid (FFA) and VLDL-triglyceride levels were elevated compared with the control rats. Hepatic ACS activity and mRNA levels, and MTP mRNA levels were also increased in OLETF rats, whereas apo B mRNA levels were similar; these results suggest that the enhanced expression of both ACS and MTP genes associated with visceral fat accumulation before developing insulin resistance may be involved in the pathogenesis of hyperlipidemia in obese animal models with NIDDM.

Impaired beta-cell function and deposition of fat droplets in the pancreas as a consequence of hypertriglyceridemia in OLETF rat, a model of spontaneous NIDDM.

Hypertriglyceridemia is known to be a feature of obesity-related NIDDM, but the patho-etiological significance of this association is obscure. The effects of triglycerides (TGs) on beta-cell function and morphological changes in pancreas were examined using in vivo and in vitro approaches in male OLETF rats at ages 6, 12, and 30 weeks, with their diabetes-resistant counterpart, LETO rats, as normal controls. The results showed that, in the fasting state, plasma TGs in OLETF rats were increased 2.5-fold at age 6 weeks, 3.3-fold at age 12 weeks, and 6.2-fold at age 30 weeks, compared with age-matched LETO rats. The TG content in islets from 12-week-old OLETF rats was significantly increased when compared with those from their age-matched counterparts, but this was not the case with the 6-week-old OLETF rats. Therefore, the islets from 6-week-old rats were cultured with either free fatty acids (FFAs; 1.0 mmol/l sodium oleate) or TG (5.0 mmol/l Intralipide) for 72 h. Several abnormalities in OLETF rats were evident, in contrast to the results from control LETO rats: 1) glucose-induced insulin secretion was more inhibited by either FFAs or TGs in the presence of 27.7 mmol/l glucose, a result associated, at least in part, with reduced glucokinase activity in the islets; 2) a marked elevation in TG content was found in the islets; and 3) the deposition of fat droplets in the enlarged islets, even in the beta-cells, was found by Oil Red O-insulin double staining at age 30 weeks. In conclusion, hypertriglyceridemia resulted in significant TG stores in the islets, which subsequently inhibited glucose-induced insulin secretion, at least in part, via reduced glucokinase activity in the islets. Fat droplets in islets, therefore, may play an important role in hastening the development of NIDDM in this rat model.

Specific inhibitor for prolyl endopeptidase suppresses the generation of amyloid beta protein in NG108-15 cells.

A potent and specific prolyl endopeptidase inhibitor, JTP-4819, was used to investigate the role of prolyl endopeptidase in the generation of amyloid beta protein (A beta) from APP by NG108-15 cells. Synthetic substrates, 7-(succinyl-Ile-Ala)-4-methylcoumarinamide and Z(Val-Lys-Met)-4-methylcoumarinamide, respectively, corresponding to the C-terminal and N-terminal portions of A beta, were cleaved by NG108-15 cell lysates. Cleavage of the C-terminal portion, but not the N-terminal, was inhibited by JTP-4819 (IC50 = 0.6 nM). Western blot analysis showed that the A beta level in the culture medium of NG108-15 cells was increased by serum deprivation. JTP-4819 caused concentration (>10(-9) M)- and time-dependent inhibition of this serum deprivation-induced increase of A beta without having any effect on the level of the secretory form of APP. Using both specific anti-A beta (1-40) and anti-A beta (1-42) antisera, the A beta that increased with serum deprivation was confirmed to be A beta (1-40), suggesting that it might be produced by conversion of A beta (1-42) to A beta (1-40). These findings indicate that prolyl endopeptidase may be a key enzyme in the production of A beta by NG108-15 cells and that A beta secretion can be modulated by a prolyl endopeptidase inhibitor.

Plasma membrane content of insulin-regulated glucose transporter in skeletal muscle of the male Otsuka Long-Evans Tokushima Fatty rat, a model of non-insulin-dependent diabetes mellitus.

The male Otsuka Long-Evans Tokushima Fatty (OLETF) rat shows insulin resistance in skeletal muscle and visceral obesity. To obtain information on the mechanism of the insulin resistance in the diabetic rats, we examined the content of insulin-regulated glucose transporter (GLUT4) in skeletal muscles. The results indicate that the total content of the transporter is significantly decreased (P < 0.05) in muscles of the diabetic rats. Plasma membrane content of the GLUT4 protein in muscles of the diabetic rats was increased in the basal state as compared to control rats. Hyperinsulinemic clamps increased GLUT4 levels in the plasma membrane of control rats but failed to do so in the diabetic rats. The distribution of GLUT4 in OLETF rat is reminiscent of the characteristics of human non-insulin-dependent diabetes mellitus.

Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on spatial memory and central cholinergic neurons in aged rats.

The effects of a novel prolyl endopeptidase inhibitor (PEP), (S)-2-[[(S)-2-(hydroxyacetyl)-1-pyrrolidinyl]carbonyl]-N-(phenylmethyl)- 1-pyrrolidinecar-boxamide (JTP-4819), on performance of the Morris water maze task and on central cholinergic function were investigated in aged rats. Spatial memory (escape latency, path length, and swimming speed to the platform) was impaired in aged rats performing the Morris water maze task when compared to young rats. Administration of JTP-4819 (1 mg/kg, p.o.) for 14 days improved this memory deficit in aged rats, as shown by the decrease in escape latency and path length. In addition, when JTP-4819 (at doses of 1 and 3 mg/kg, p.o.) was administered for 3 wk, it reversed the age-related increase of ChAT activity in the cerebral cortex and the decrease of 3H-choline uptake in the hippocampus. These data suggest that JTP-4819 ameliorates age-related impairment of spatial memory and partly reverses central cholinergic dysfunction, possibly due to the enhancement of neuropeptide function by inhibition of PEP mediated degradation of substance P, arginine-vasopressin, and thyrotropin-releasing hormone.

Gene structure of mouse Cyp3a11: evidence for an enhancer element within its 5' flanking sequences.

A mouse Cyp3a11 gene was isolated from a mouse sperm DNA library with mouse Cyp3a11 cDNA as a probe. The nucleotide sequences determined for the gene and the 5' flanking region revealed that the mouse Cyp3a11 gene was composed of 13 exons and 12 introns. The exons spun about 23 kb. The nucleotide sequence of the exons was completely identical to Cyp3a11 cDNA. Within the 5' flanking sequence, putative binding sites of several transcriptional factors were found. Transient transfection assays were carried out with HepG2 cells, a human hepatoma cell line, using constructs containing different lengths of 5' flanking sequence fused to a reporter, chloramphenicol acetyltransferase gene. The results showed that a cis-acting element(s) was located from -1609 to -907.

A novel prolyl endopeptidase inhibitor, JTP-4819, with potential for treating Alzheimer's disease.

The pharmacological actions of JTP-4819, a new prolyl endopeptidase (PEP) inhibitor targeted for the treatment of Alzheimer's disease, are reviewed with respect to its effects on PEP activity, brain neurotransmitters, and memory-related behaviour in rats. JTP-4819 was shown to be a very potent and specific inhibitor of PEP. At nanomolar concentration, JTP-4819 inhibited the degradation of substance P, arginine-vasopressin, and thyrotropin-releasing hormone by PEP in supernatants of the rat cerebral cortex and hippocampus. Repeated administration of JTP-4819 reversed the aging-induced decrease in brain substance P-like and thyrotropin-releasing hormone-like immunoreactivity, suggesting that this drug may be able to improve the imbalance of peptidergic neuronal systems that develops with senescense by inhibiting PEP activity. JTP-4819 increased acetylcholine release from the frontal cortex and hippocampus, regions closely associated with memory, in both young and aged rats. In addition, it improved performance in several memory and learning-related tests (e.g., the Morris water maze task in aged or MCA-occluded rats and the passive avoidance test). This memory-enhancing effect of JTP-4819 may result from prevention of the metabolic degradation of brain neuropeptides by PEP as well as from the enhancement of acetylcholine release. Taken together, these unique and potent pharmacological actions of JTP-4819 suggest that it may have the potential to be used for treating Alzheimer's disease.

Glucose transporter levels in a male spontaneous non-insulin-dependent diabetes mellitus rat of the Otsuka Long-Evans Tokushima Fatty strain.

Otsuka Long-Evans Tokushima Fatty (OLETF) rats are a new strain of spontaneous non-insulin-dependent diabetes mellitus (NIDDM) models. To evaluate the role of glucose transporters (GLUT) in the development of diabetes in this model, we examined the action of insulin on the translocation of GLUT4 and GLUT1 in isolated adipocytes, and the GLUT4 protein levels in muscles. Long-Evans Tokushima Otsuka (LETO) rats were used as a control strain. In adipocytes, the GLUT4 protein levels in OLETF rats at 30 weeks of age (diabetic stage) were considerably lower than those in LETO rats at the same age. At a pre-diabetic stage (7 weeks), there were no significant differences in GLUT4 protein levels in adipocytes between LETO and OLETF rats. However, the degree of GLUT4 translocation in OLETF rats was lower than that in LETO rats at 7 weeks of age. There were no differences in GLUT1 levels in adipocytes between the two strains. In muscles, the decrease in GLUT4 protein was observed in OLETF rats at 30 weeks of age. Whether such a difference is under the influence of hyperglycemia was also examined using rats rendered diabetic by 70% pancreatectomy. OLETF rats aged 7 weeks were subjected to partial pancreatectomy (Px) and sham pancreatectomy (sham). At 4 weeks after surgery, GLUT4 protein levels in adipose tissues and skeletal muscles were determined. GLUT4 decrease was observed for both tissues of hyperglycemic Px rats compared with euglycemic sham. Moreover, we examined the direct effect of glucose on GLUT4 protein using primary cultured adipocytes of OLETF rats at 5 weeks of age. After 7-day culture with normal (5.6 mmol/l) or high (25 mmol/l) concentrations of glucose, the GLUT4 protein levels in adipocytes decreased at 25 mmol/l glucose compared with 5.6 mmol/l glucose. These findings suggest an early defect in the insulin resistance of OLETF rats probably reflects impaired GLUT4 translocation. The GLUT4 decrease, which occurs later in the process appears to be a consequence, rather than a cause of diabetes in OLETF rats.

Application of primary hepatocytes from p53-knockout mice for studies of expression of Cyp3a.

CYP3A rapidly disappears in primary hepatocytes, although the primary cells are suitable for studies of the regulation of CYP3A genes. In the present study, we found that Cyp3a mRNA could be expressed in the primary hepatocytes from p53-knockout mice for at least 2 weeks when the cells were cultured in the presence of dexamethasone. Propoxycoumarin O-depropylase activity, which is known to be mainly catalyzed by CYP3A, was maintained at a level of 50% of the initial activity even after 5 days of culture, and the activity correlated with the expression level of Cyp3a mRNA in the primary hepatocytes from p53-knockout mice. The cells remained morphologically intact during 4 weeks. These results suggest that hepatocytes from p53-knockout mice are a useful tool for studies of the expression of Cyp3a.

Pharmacological studies of a novel prolyl endopeptidase inhibitor, JTP-4819, in rats with middle cerebral artery occlusion.

We studied behavioral and pharmacological effects of a novel prolyl endopeptidase inhibitor, (S)-2-[[(S)-2-(hydroxyacetyl)- 1-pyrrolidinyl]carbonyl]-N-(phenylmethyl)-1-pyrrolidine-car boxamide (JTP-4819), in rats with middle cerebral artery occlusion. Administration of JTP-4819 (0.1 and 1 mg/kg p.o for 7 days) significantly prolonged passive avoidance latency, while the latency of rats with middle cerebral artery occlusion receiving the vehicle was significantly shorter than that of sham-operated rats. The prolonged escape latency in the Morris water maze task in rats with middle cerebral artery occlusion was also significantly reduced by administration of JTP-4819 (0.3 and 1 mg/kg p.o.). Interestingly, administration of JTP-4819 (0.3-3 mg/kg p.o. for 15 days) restored the decreased cortical thyrotropin-releasing hormone (TRH)-like immunoreactivity content of rats with middle cerebral artery occlusion but did not affect the cortical and hippocampal substance P- or arginine vasopressin-like immunoreactivity content. These results suggest that JTP-4819 ameliorates memory impairment due to middle cerebral artery occlusion by restoring the cortical TRH content.

Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on neuropeptide metabolism in the rat brain.

The effect of a novel prolyl endopeptidase (PEP) inhibitor, (S)-2-[[(S)-2-(hydroxyacetyl)-1-pyrrolidinyl] carbonyl]-N-(phenylmethyl)-1-pyrrolidine-carboxamide (JTP-4819), on neuropeptide metabolism was investigated in the rat brain. JTP-4819 exhibited a strong in vitro inhibitory effect on cortical and hippocampal PEP activity, with the IC50 values being approximately 0.58 +/- 0.02 and 0.61 +/- 0.06 nM, respectively. JTP-4819 also inhibited the in vitro degradation of substance P (SP), arginine-vasopressin (AVP), and thyrotropin-releasing hormone (TRH) by rat brain supernatants, with the IC50 values being respectively 3.4, 2.1, and 1.4 nM in the cerebral cortex and 3.3, 2.8, and 1.9 nM in the hippocampus. Oral administration of JTP-4819 at doses of 1 and 3 mg/kg increased SP-like immunoreactivity (LI) and AVP-LI in the cerebral cortex. JTP-4819 also increased hippocampal SP-LI and AVP-LI at doses of 1 and 3 mg/kg, as well as hippocampal TRH-LI at a dose of 3 mg/kg. These findings suggest that JTP-4819 inhibited the degradation of SP, AVP, and TRH in the rat brain secondary to the inhibition of PEP, and thus increased cortical and hippocampal SP-LI and AVP-LI as well as hippocampal TRH-LI.