Pentylentetrazole-induced loss of blood-brain barrier integrity involves excess nitric oxide generation by neuronal nitric oxide synthase.

Dysfunction of the blood-brain barrier (BBB) is one of the major pathophysiological consequences of epilepsy. The increase in the permeability caused by BBB failure is thought to contribute to the development of epileptic outcomes. We developed a method by which the BBB permeability can be demonstrated by gadolinium-enhanced T1 weighted imaging (GdET1WI). The present study examined the changes in the BBB permeability in mice with generalized convulsive seizures (GCS) induced by acute pentylentetrazole (PTZ) injection. At 15min after PTZ-induced GCS, the BBB temporarily leaks BBB-impermeable contrast agent into the parenchyma of the diencephalon, hippocampus and cerebral cortex in mice, and the loss of BBB integrity was gradually recovered by 24h. The temporary BBB failure is a critical link to the glutamatergic activities that occur following the injection of PTZ. PTZ activates the glutamatergic pathway via the NMDA receptor, then nitric oxide (NO) is generated by NMDA receptor-coupled neuronal NO synthase (nNOS). To examine the influence of nNOS-derived NO induced by PTZ on the increases of the BBB permeability, GdET1WI was performed using conventional nNOS gene-deficient mice with or without PTZ injection. The failure of the BBB induced by PTZ was completely protected by nNOS deficiency in the brain. These results suggest that nNOS-derived excess NO in the glutamatergic pathway plays a key role in the failure of the BBB during PTZ-induced GCS. The levels of NO synthetized by nNOS in the brain may represent an important target for the future development of drugs to protect the BBB.

The threshold of pentylenetetrazole-induced convulsive seizures, but not that of nonconvulsive seizures, is controlled by the nitric oxide levels in murine brains.

Alterations in the NO pathway play an important role in the development of convulsive seizures via the glutamatergic and GABAergic systems in acute pentylenetetrazole (PTZ) seizure animals. We previously reported that the background NO levels under physiological conditions negatively regulate convulsive seizures, while excess NO levels under pathologic conditions positively regulate PTZ-induced convulsive seizures. In this study, the NO content in various brain regions after a single dose injection of PTZ was quantitatively and directly measured using the ex vivo X-band electron paramagnetic resonance method with an NO-trapping agent. Experimental data demonstrated the effects of NO on the convulsive seizure threshold: a 1.5-fold increase in the NO level in all brain regions compared to that observed in the control state showed proconvulsive properties without any involvement with nonconvulsive seizures. The distribution of the background NO content in the normal animals was higher in the temporal region of the cerebral cortex, including the amygdala, than in the hippocampus, cerebellum and other regions of the cerebral cortex. However, the levels of NO after the occurrence of acute PTZ-induced convulsive seizures significantly increased by more than 50% in all brain regions, thus suggesting that the NO levels in all brain regions contribute to PTZ-induced convulsions as a seizure threshold. In a pharmacological study, the inhibitor of neuronal NO synthase and antagonists of ionotropic glutamate receptors prevented PTZ-induced convulsions and excessive NO generation. In addition, therapeutic drugs, such as valproate and ethosuximide used to treat generalized seizures not only inhibited the increase in NO generation induced by PTZ, but also prevented both convulsive and nonconvulsive seizures caused by PTZ. We herein provide novel insight into the involvement of NO in PTZ-seizure susceptibility at the whole-animal level.

Effects of sulfaphenazole after collagenase-induced experimental intracerebral hemorrhage in rats.

Treatment of intracerebral hemorrhage is often pointless, although considerable effort has been devoted to developing treatments for ischemic stroke. The purpose of this study was to determine the influence of drugs in improving neurological outcomes with pharmaceutical therapy after intracerebral hemorrhage. The free-radical hypothesis for intracerebral hemorrhage is based on the cytotoxicity triggered by blood components and its degradation products, such as heme and iron as a potent pro-oxidant atom. Sulfaphenazole (SPZ) has a different mechanism such as reactive oxygen species scavenging, in addition to the inhibition of superoxide production by cytochrome P450. The present study investigated the properties of SPZ in collagenase-induced intracerebral hemorrhage rat brain damage. The results show that systemic SPZ treatment after intracerebral hemorrhage reduces striatal dysfunction, the elevation of lipid peroxidation, and brain edema in the rat. These results suggest that SPZ is a potentially effective therapeutic approach for intracerebral hemorrhage as the effect of SPZ was initiated for either 1 h or 3 d post-intracerebral hemorrhage.

Human L1CAM carrying the missense mutations of the fibronectin-like type III domains is localized in the endoplasmic reticulum and degraded by polyubiquitylation.

Any mutations in the human neural cell adhesion molecule L1 (hL1CAM) gene might cause various types of serious neurological syndromes in humans, characterized by increased mortality, mental retardation, and various malformations of the nervous system. Such missense mutations often cause severe abnormalities or even fatalities, and the reason for this may be a disruption of the adhesive function of L1CAM resulting from a misdirection of the degradative pathway. Transfection studies using neuroblastoma N2a cells demonstrated that hL1CAM carrying the missense mutations in the fibronectin-like type III (FnIII) domains most likely is located within the endoplasmic reticulum (ER), but it is less well expressed on the cell surface. One mutant, L935P, in the fourth FnIII domain, was chosen from six mutants (K655 and G698 at Fn1, L935P and P941 at Fn4, W1036 and Y1070 at Fn5) in the FnIII domains to study in detail the functions of hL1CAM(200 kDa) , such as the intracellular traffic and degradation, because only a single band at 200 kDa was detected in the hL1CAM(L935P) -transfected cells. hL1CAM(200 kDa) is expressed predominantly in the ER but not on the cell surface. In addition, this missense mutated hL1CAM(200 kDa) is polyubiquitylated at some sites in the extracellular domain and thus becomes degraded by proteasomes via the ER-associated degradation pathway. These observations demonstrate that the missense mutations of hL1CAM in the FnIII domain may cause the resultant pathogenesis because of a loss of expression on the cell surface resulting from misrouting to the degradative pathway.

Characterization of a novel posttranslational modification in neuronal nitric oxide synthase by small ubiquitin-related modifier-1.

The multifaceted functions of nitric oxide (NO) in the CNS are defined by the activity of neuronal NO synathase (nNOS). The activities of nNOS are modulated by posttranslational modifications, such as phosphorylation and ubiquitination, but whether it is modified by small ubiquitin-related modifier (SUMO) remains unknown. The aim of this study was to elucidate whether nNOS is posttranslationally modified by SUMO proteins. Bioinformatic analyses using SUMOplot and SUMOFI predicted that nNOS had potential SUMO modification sites. When HEK293T cells were transiently co-expressed with nNOS and SUMO-1, two bands corresponding to nNOS-SUMO-1 conjugates were detected. In addition, two nNOS-SUMO-1 conjugates were confirmed by an in vitro sumoylation assay using recombinant proteins. Furthermore, nNOS-SUMO-1 conjugates were identified by MALDI-QIT/TOF mass spectrometry. These findings indicate that nNOS is clearly defined as a SUMO-1 target protein both in vitro and at the cellular level. We next characterized specific enzymes in the nNOS-SUMO-1 conjugation cycle at the cellular level. SUMO-1 conjugation of nNOS depended on Ubc9 (E2). The interaction between nNOS and Ubc9 was facilitated by PIASxß (E3). On the other hand, SUMO-1 was deconjugated from nNOS by SENP1 and SENP2. Overall, this study has newly identified that nNOS is posttranslationally modified by SUMO-1.

The scaffold protein JIP3 functions as a downstream effector of the small GTPase ARF6 to regulate neurite morphogenesis of cortical neurons.

The small GTPase ADP-ribosylation factor 6 (ARF6) plays crucial roles in a wide variety of cell functions. To better understand the molecular mechanisms of ARF6-mediated signaling and cellular functions, we sought new ARF6-binding proteins in the mouse brain. We identified the signaling scaffold protein JNK-interacting protein 3 (JIP3), which is exclusively expressed in neurons, as a downstream effector of ARF6. Overexpression of a unique dominant negative mutant of ARF6, which was unable to interact with JIP3, and knockdown of JIP3 in mouse cortical neurons stimulated the elongation and branching of neurites. These results provide evidence that ARF6/JIP3 signaling regulates neurite morphogenesis.

Important amino acid residues that confer CYP2C19 selective activity to CYP2C9.

Although CYP2C9 and CYP2C19 display 91% sequence identity at the amino acid level, the two enzymes have distinct substrate specificities for compounds such as diclofenac, progesterone and (S)-mephenytoin. Amino acid substitutions in CYP2C9 were made based on an alignment of CYP2C9, CYP2C19 and monkey CYP2C43 sequences. Mutants of CYP2C9 were expressed in Escherichia coli. Sixteen amino acids, which are common to both CYP2C19 and CYP2C43 but different between CYP2C9 and CYP2C19, were substituted in CYP2C9 (CYP2C9-16aa). Next, the mutated amino acids in CYP2C9-16aa were individually reverted to those of CYP2C9 to examine the effect of each substitution on the enzymatic activity for CYP2C marker substrates. In addition, the role of the F-G loop in CYP2C9 and CYP2C19 was examined for substrate specificity and enzymatic activity. Our results showed: (i) CYP2C9-16aa displays 11% (S)-mephenytoin 4'-hydroxylase and full omeprazole 5-hydroxylase activity compared with that of CYP2C19; (ii) residue 286 is important for conferring CYP2C9-like enzyme activity on CYP2C9-16aa and residue 442 in CYP2C19 may be involved in the interaction with NADPH-P450 reductase; (iii) substitution of the F-G loop in CYP2C9 to that of CYP2C19 enhances tolbutamide p-methyhydroxylase and diclofenac 4'-hydroxylase activities and confers partial (S)-mephenytoin 4'-hydroxylase and omeprazole 5-hydroxylase activities, which are attributed to CYP2C19.

Amino acids of the human alpha1d-adrenergic receptor involved in antagonist binding.

Computer simulations of the human alpha(1d)-adrenergic receptor (alpha(1d)-AR) based on the crystal structure of rhodopsin have been combined with experimental site-directed mutagenesis to investigate the role of residues in the transmembrane domains in antagonist binding. Our results indicate that the amino acids Asp176 in the third transmembrane domain (TMD), Glu237 in TMD IV, and Ser258 in TMD V of alpha(1d)-AR were directly involved in prazosin and tamsulosin binding. The Asp176Ala mutant did not exhibit any affinity for [(3)H]prazosin and neither did it show agonist-stimulated inositol phosphates (IP) formation. On the other hand, the Glu237Ala and Ser258Ala mutant alpha(1d)-AR showed increased binding affinity for [(3)H]prazosin. Competition binding experiments showed that prazosin affinity had increased to 5-fold and 3-fold in the Glu237Ala and Ser258Ala mutants, respectively, versus wild-type; and tamsulosin affinity only increased in the Ser258Ala mutant (2-fold vs wild-type). It seems that these two residues constrain the receptor by interaction with other residues and this disruption of the interaction increased the receptor's binding affinity towards antagonists. However, the Glu237Ala and Ser258Ala mutant receptors retained the ability to stimulate the formation of myo-[(3)H]inositol but had activities lower than that of the wild-type receptor. The present results provide direct evidence that these amino acid residues are responsible for the interactions between alpha(1d)-AR and the radioligand [(3)H]prazosin as well as tamsulosin.

Increased production of lipocalin-type prostaglandin D synthase in leptomeningeal cells through contact with astrocytes.

Lipocalin-type prostaglandin D synthase (L-PGDS) is dominantly expressed in the leptomeninges surrounding the brain and secreted into the cerebrospinal fluid as beta-trace, a major cerebrospinal fluid protein. To examine the interaction between the leptomeninges and the brain parenchyma, we co-cultured rat leptomeningeal cells with cells dissociated from the neonatal rat cortex and found that the production of L-PGDS was remarkably increased after the co-cultivation. A similar increase in L-PGDS production was observed by the co-culturing of the leptomeningeal cells with cells dissociated from astrocyte-rich cultures or with 1321-N1 astrocytoma cells. When a crude membrane fraction prepared from 1321-N1 cells was added to leptomeningeal cell cultures, L-PGDS gene expression was slowly increased up to 48 h after the addition. These results indicate that leptomeningeal cells enhance their L-PGDS production by a slow activation of L-PGDS gene expression through their contact with astrocytes.

Monoclonal antibody Rip specifically recognizes 2',3'-cyclic nucleotide 3'-phosphodiesterase in oligodendrocytes.

The antigen recognized with monoclonal antibody (mAb) Rip (Rip-antigen) has been long used as a marker of oligodendrocytes and myelin sheaths. However, the identity of Rip-antigen has yet to be elucidated. We herein identified the Rip-antigen. No signal recognized by mAb-Rip was detected by immunoblot analyses in the rat brain, cultured rat oligodendrocytes, or the oligodendrocyte cell line CG-4. As this antibody worked very well on immunocytochemistry and immunohistochemistry, Rip-antigen was immunopurified with mAb-Rip from the differentiated CG-4 cells. Eight strong-intensity bands thus appeared on 5-20% SDS-PAGE with SYPRO ruby fluorescence staining. To identify these molecules, each band extracted from the gel was analyzed by MALDI-QIT/TOF mass spectrometry. We found an interesting molecule in the oligodendrocytes from an approximately 44-kDa band as 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). To test whether CNP was recognized by mAb-Rip, double-immunofluorescence staining was performed by using Alexa Fluor 488-conjugated mAb-Rip and Alexa Fluor 568-conjugated mAb-CNP in the rat cerebellum, mouse cerebellum, cultured rat oligodendrocytes, and CG-4 cells. The Rip-antigen was colocalized with CNP in these cells and tissues. To provide direct evidence that CNP was recognized by mAb-Rip, rat Cnp1-transfected HEK293T cells were used for double-immunofluorescence staining with mAb-Rip and mAb-CNP. The Rip-antigen was colocalized with CNP in rat Cnp1-transfected HEK293T cells, but the antigen was not detected by mAb-Rip and mAb-CNP in mock-transfected HEK293T cells. Overall, we have demonstrated that the antigen labeled with mAb-Rip is CNP in the oligodendrocytes.

Endogenous ghrelin in pancreatic islets restricts insulin release by attenuating Ca2+ signaling in beta-cells: implication in the glycemic control in rodents.

Ghrelin, isolated from the human and rat stomach, is the endogenous ligand for the growth hormone (GH) secretagogue receptor, which is expressed in a variety of tissues, including the pancreatic islets. It has been shown that low plasma ghrelin levels correlates with elevated fasting insulin levels and type 2 diabetes. Here we show a physiological role of endogenous ghrelin in the regulation of insulin release and blood glucose in rodents. Acylated ghrelin, the active form of the peptide, was detected in the pancreatic islets. Counteraction of endogenous ghrelin by intraperitoneal injection of specific GH secretagogue receptor antagonists markedly lowered fasting glucose concentrations, attenuated plasma glucose elevation, and enhanced insulin responses during the glucose tolerance test (GTT). Conversely, intraperitoneal exogenous ghrelin GH-independently elevated fasting glucose concentrations, enhanced plasma glucose elevation, and attenuated insulin responses during GTT. Neither GH secretagogue receptor antagonist nor ghrelin affected the profiles of the insulin tolerance test. In isolated islets, GH secretagogue receptor blockade and antiserum against acylated ghrelin markedly enhanced glucose-induced increases in insulin release and intracellular Ca2+ concentration ([Ca2+]i), whereas ghrelin at a relatively high concentration (10 nmol/l) suppressed insulin release. In single beta-cells, ghrelin attenuated glucose-induced first-phase and oscillatory [Ca2+]i increases via the GH secretagogue receptor and in a pertussis toxin-sensitive manner. Ghrelin also increased tetraethylammonium-sensitive delayed outward K+ currents in single beta-cells. These findings reveal that endogenous ghrelin in islets acts on beta-cells to restrict glucose-induced insulin release at least partly via attenuation of Ca2+ signaling, and that this insulinostatic action may be implicated in the upward control of blood glucose. This function of ghrelin, together with inducing GH release and feeding, suggests that ghrelin underlies the integrative regulation of energy homeostasis.

Identification of principal cytochrome P-450 in triphenyltin metabolism in rats.

The in vivo and in vitro metabolism of triphenyltin using rat hepatic cytochrome P-450 (CYP) systems was investigated to confirm the specific CYP that is closely related to triphenyltin metabolism. No significant sex differences occurred between the in vivo and in vitro metabolic patterns of the chemical, indicating that the principal CYP for triphenyltin metabolism in rats is not a sex-specific form of CYP. In addition, seven types of complementary DNA (cDNA)-expressed rat CYPs, typical phenobarbital (PB)-inducible forms and the CYP2C subfamily were tested to determine the activity of triphenyltin metabolism. Among the CYP isoforms studied, although CYP2B1 had a small metabolic capacity, a marked dearylation of the chemical was induced by CYP2C6. Furthermore, anti-rat CYP2C6 antibodies and cimetidine, a selective CYP2C6 inhibitor, inhibited triphenyltin dearylation activity in the hepatic microsomes of rats. Taken together, these findings suggest that CYP2C6 is the principal CYP for the triphenyltin metabolism in rats.

Mammalian D-aspartyl endopeptidase: a scavenger for noxious racemized proteins in aging.

The accumulation of D-isomers of aspartic acid (D-Asp) in proteins during aging has been implicated in the pathogenesis of Alzheimer's disease, cataracts, and arteriosclerosis. Here, we identified a specific lactacystin-sensitive endopeptidase that cleaves the D-Asp-containing protein and named it D-aspartyl endopeptidase (DAEP). DAEP has a multi-complex structure (MW: 600kDa) and is localized in the inner mitochondrial membrane of mouse and rabbit, but DAEP activity was not detected in Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans. A specific inhibitor for DAEP was newly synthesized, and inhibited DAEP activity (IC(50), 3microM), a factor of 10 greater than lactacystin on DAEP. On the other hand, the inhibitor did not inhibit either the 20S or 26S proteasome.

Assessment of affinities and dissociation potencies of several 5-HT2 antagonists to and from M2 muscarinic receptor in rat heart membranes.

The aim of the present study was to investigate the binding affinities and dissociation potencies of several 5-HT(2) antagonists in M(2) muscarinic receptor of rat heart membranes using [(3)H]QNB as a radioligand. The 5-HT(2) antagonists used in this study were sarpogrelate, ketanserin and cyproheptadine. The results showed that sarpogrelate and ketanserin had very weak binding affinities to M(2) muscarinic receptor, whereas cyproheptadine had higher binding affinity to this receptor than the muscarinic receptor antagonist, atropine. All of these three 5-HT(2) antagonists as well as muscarinic receptor antagonists (atropine and pirenzepine) were readily dissociated from M(2) muscarinic receptor in rat heart membranes after washing. Therefore, the findings of the present investigation suggest that the dissociation potencies of neither 5-HT(2) antagonists nor muscarinic antagonists used correlate with their binding affinities to M(2) muscarinic receptors in rat heart.

Metabolism of tributyltin and triphenyltin by rat, hamster and human hepatic microsomes.

Tributyltin and triphenyltin are metabolized by cytochrome P-450 system enzymes, and their metabolic fate may contribute to the toxicity of the chemicals. In the current study, the in vitro metabolism of tributyltin and triphenyltin by rat, hamster and human hepatic microsomes was investigated to elucidate the metabolic competence for these compounds in humans. The metabolic reaction using microsome-NADPH system that is usually conducted was not applicable to in vitro metabolism of organotins, especially triphenyltin. We therefore examined the effects of dithiothreitol (DTT), one of the antioxidants for sulfhydryl groups, to determine the in vitro metabolism of tributyltin and triphenyltin. As a result, the treatment with 0.1 mM DTT in vitro increased the activity of the microsomal monooxygenase system for metabolism of tributyltin as well as triphenyltin; the total yield of tributyltin and triphenyltin metabolites as tin increased, respectively, by approximately 1.8 and 8.9 times for rat, 2.1 and 1.2 times for hamster, and 1.6 and 1.5 times for human. It is suggested that the organotins directly inactivate cytochrome P-450 because of the interaction with critical sulfhydryl groups of the hemoprotein. We confirmed the utility of this in vitro metabolic system using DTT in the hepatic microsomes of phenobarbital (PB)-pretreated and untreated hamsters. Thus, the in vitro metabolic system described here was applied to a comparative study of the metabolism of organotins in rats, hamsters and humans. Tributyltin was metabolized more readily than triphenyltin in all the species. In humans, the in vitro metabolic pattern resembled that of hamsters, which were susceptible to in vivo triphenyltin toxicity because of incompetent metabolism. It is possible that the hamster is a qualitatively and quantitatively suitable animal model for exploring the influence of tributyltin and triphenyltin in humans.

AT-1015, a newly synthesized 5-HT2 receptor antagonist, dissociates slowly from the 5-HT2 receptor sites in rabbit cerebral cortex membrane.

The purpose of this study was to investigate the association and dissociation kinetics of [3H]AT-1015 from 5-HT2 receptors in rabbit cerebral cortex membranes using a radioligand binding assay method and to make a comparison with those of [3H]ketanserin binding. Scatchard analysis of [3H]AT-1015 binding in rabbit cerebral cortex membranes indicated the existence of a single class of binding sites (dissociation constant, Kd = 2.18 nM). The specific binding of [3H]AT-1015 increased slowly with time and the association rate constant of [3H]AT-1015 binding (k1 = 0.1229 min-1 nM-1) was two times slower than that of [3H]ketanserin binding (k1 = 0.2451 min-1 nM-1). The dissociation rate constant of [3H]AT-1015 binding (t 1/2 = 37.03 min) was six times slower than that of [3H]ketanserin binding (t 1/2 = 6.29 min), when the addition of excess unlabelled ligands were AT-1015 and ketanserin, respectively. The dissociation rate constant of [3H]AT-1015 was slowed to a greater degree (t 1/2 = 163.40 min and t 1/2 = 198.12 min) by the addition of ketanserin and sarpogrelate as excess unlabelled ligands than was that of [3H]ketanserin (t 1/2 = 17.76 min and t 1/2 = 18.45 min) by the addition of AT-1015 and sarpogrelate as an excess unlabelled ligand, respectively. These findings on the dissociation kinetics of [3H]AT-1015 have confirmed and supported previously reported evidence of the slower dissociation of AT-1015 from 5-HT2 receptors.

Hypoglycemic and hypotriglyceridemic effects of tolbutamide in triphenyltin chloride-induced diabetic rabbits.

Triphenyltin (TPT) induces transient hyperglycemia and hypertriglyceridemia in rabbits and hamsters through inhibition of the insulin release stimulated by glucose. The disturbed site in TPT-diabetes is a result of signal transduction occurring before the voltage-dependent Ca2+ channel. The ATP-sensitive K channel (KATP channel) is located immediately at the upstream signal of voltage dependent Ca2+ channels on the signaling pathway of insulin secretion. KATP channel produces depolarization by a signal of ATP through glucose metabolism and by stimulation from sulfonylurea drugs (tolbutamide, glibenclamide). To clarify if the insulin secretion that a KATP channel mediates is inhibited in vivo, we studied the effects of tolbutamide (a sulfonylurea) on changes in plasma glucose, triglyceride and insulin in TPT-diabetic rabbits prepared by po administration of 100 mg TPT-chloride/kg bw. In TPT-diabetic rabbits, plasma glucose decreased to a minimum at about 50% and plasma triglyceride levels also decreased. Insulin release was detected after injecting = 10 mg tolbutamide/kg, and insulin was secreted much higher than in normal rabbits. These findings suggest that the insulin released by tolbutamide stimulus decreased the plasma glucose and triglyceride levels in the TPT-diabetic rabbits. Moreover, a possible mechanism to be considered is as follows: tolbutamide combines with sulfonylurea receptor; membrane depolarization is induced by a KATP channel with the signal of a sulfonylurea receptor; insulin is released. The inhibition of insulin secretion by TPT may be caused by a glucose metabolic disorder in beta cells before the occurrence of membrane depolarization due to closed KATP channels interacting directly with a sulfonylurea receptor.