Female-specific neuroprotection against transient brain ischemia observed in mice devoid of prion protein is abolished by ectopic expression of prion protein-like protein.

This study was designed to examine the function of cellular prion protein and prion protein-like protein/Doppel, in transient ischemia-related neuronal death in the hippocampus. Two different lines of mice devoid of cellular prion protein, Zrch I Prnp(0/0) and Ngsk Prnp(0/0), were used. The former lacks cellular prion protein whereas the latter ectopically expresses prion protein-like protein/Doppel in the brain in the absence of cellular prion protein. Mice were subjected to 10 min-occlusion of the bilateral common carotid arteries with recovery for 14 days. Less than 10% of the pyramidal neurons in the CA1 subfield were degenerated in male and female wild-type mice. In contrast, more than half of the neurons were lost in male Zrch I Prnp(0/0) and Ngsk Prnp(0/0) mice. Such severe neuronal loss was also observed in female Ngsk Prnp(0/0) mice. However, female Zrch I Prnp(0/0) mice showed mild neuronal loss similar to wild-type mice. Flunarizine, a T- and L-type Ca(2+)-channel antagonist, significantly reduced the neuronal loss in female but not in male Ngsk Prnp(0/0) mice. These results indicate that loss of cellular prion protein renders hippocampal neurons susceptible to ischemic insult specifically in male but not female mice and the ectopic expression of prion protein-like protein/Doppel aggravates the ischemic neuronal death in female prion protein-null mice probably via overloading of Ca(2+)-dependent signaling.

Functions of peripheral 5-hydroxytryptamine receptors, especially 5-hydroxytryptamine4 receptor, in gastrointestinal motility.

The multiple 5-hydroxytryptamine (5-HT, serotonin) receptor subtypes are distinguished. In this article, we described mainly the 5-HT4 receptor of four subtypes of functional 5-HT receptors, 5-HT1, 5-HT2, 5-HT3, and 5-HT4, recognized in the gastrointestinal tract. In-vivo microdialysis experiments determined that activation of the 5-HT4 receptor stimulated intestinal motor activity associated with a local increase in acetylcholine (ACh) release from the intestinal cholinergic neurons in the whole body of dogs. The 5-HT4 receptor-mediated response of ACh release in the antral, corporal, and fundic strips isolated from guinea pig stomach corresponds to the presence of 5-HT4 receptor in the myenteric plexus. In-vitro receptor autoradiograms of the stomach and colon indicate that the distribution of 5-HT4 receptors in human tissues is similar to that in the guinea pig, although density of 5-HT4 receptors in the myenteric plexus of human tissues is lower than that in guinea pig tissues. The 5-HT4 receptors located in the myenteric plexus may participate in gastrointestinal motility, and thus the 5-HT4 agonists and antagonists may be available for treatment of dysfunction of gastrointestinal motility.

Involvement of glial endothelin/nitric oxide in delayed neuronal death of rat hippocampus after transient forebrain ischemia.

1. We examined time- and cell-type-dependent changes in endothelin (ET)-1-like immunoreactivity, ET receptors binding and nitric oxide (NO) synthase (NOS) activity in CA1 subfields of the hippocampus of stroke-prone spontaneously hypertensive rats subjected to a 10-min bilateral carotid occlusion and reperfusion. 2. Microglia aggregated in accord with neuronal death and expressed a high density of ET(B) receptors and an intense NOS activity in the damaged CA1 pyramidal cell layer, 7 days after the induced transient forebrain ischemia. The increased NOS activity and ET(B) receptor in microglia disappeared 28 days after this transient ischemia. 3. In contrast to microglia, astrocytes presented a moderate level of ET-1-like immunoreactivity, ET(B) receptors, and NOS activity in all areas of the damaged CA1 subfields, 7 days after the ischemia. These events were further enhanced 28 days after the ischemia. 4. In light of these findings, the possibility that the microglial and the astrocytic ET(B)/NO system largely contributes to development of the neuronal death and to reconstitution of the damaged neuronal tissue, respectively, in the hippocampus subjected to a transient forebrain ischemia would have to be considered.

Facilitation of acetylcholine release by SK-951, a benzofuran derivative, via the 5-hydroxytryptamine4 receptor in guinea pig stomach.

Facilitation of acetylcholine (ACh) release by SK-951 ((-)4-amino-N-[2-(1-azabicyclo[3.3.0] octan-5-yl)ethyl]-5-chloro-2,3-dihydro-2-methylbenzo[b]furan-7-carboxami de hemifumarate), a benzofuran derivative, via the 5-hydroxytryptamine (5-HT)4 receptor in guinea pig stomach was examined by in vitro receptor autoradiography and functional studies. [125I]SB207710 binding was detected in the myenteric plexus of the gastric corpus. High densities of binding sites were observed in the myenteric plexus and a moderate density in the muscle layer. SK-951 inhibited the binding of [125I]SB207710, a specific 5-HT4-receptor ligand, as in the case of SB204070, a specific 5-HT4-receptor antagonist, thus indicating the presence of 5-HT4 receptors in guinea pig stomach. SK-951 as well as 5-HT enhanced the electrically stimulated twitch contractions of gastric corpus strips, which were sensitive to tetrodotoxin and atropine, and enhanced electrically stimulated release of ACh from corporal strips, which was tetrodotoxin-sensitive and Ca2+-dependent. The enhancements of twitch contractions and ACh release by SK-951 were antagonized by GR113808, a selective 5-HT4-receptor antagonist. Thus, SK-951 binds to 5-HT4 receptors of the guinea pig gastric corpus and may accelerate gastric motility due to facilitation of ACh release.

Differential localization of 5-hydroxytryptamine3 and 5-hydroxytryptamine4 receptors in the human rectum.

The functions of the 5-hydroxytryptamine3 (5-HT3) and 5-hydroxytryptamine4 (5-HT4) receptors in gastrointestinal tract are complex depending on the species and anatomical regions, and the localization of these receptors in the human rectum was unclear. We examined the localization of the 5-HT3 and 5-HT4 receptors in human rectum by in vitro receptor autoradiography using [125I](S)iodozacopride and [125I] SB207710 as a ligand, respectively. Specific [125I](S)iodozacopride binding sites were clearly evident in the myenteric plexus, whereas, low levels of [125I]SB207710 binding sites were distributed over the muscle but not to the myenteric plexus. The 5-HT3 receptor located on the myenteric plexus and the 5-HT4 receptor on the smooth muscle may participate in contractility and relaxation of human rectum, respectively.

Localization of the 5-HT(4) receptor in the human and the guinea pig colon.

The functions of the 5-HT(4) receptor in the gastrointestinal tract are complex, depending on the species and anatomical regions, and localization of the receptor was not clear. The present study attempted to examine the localization of the 5-HT(4) receptor in the colon of human for comparison with that in guinea pig colon. Human specimens of sigmoid colon and the distal colon of guinea pig were used for in vitro receptor autoradiography using [125I]SB207710, (1-n-butyl-4-piperidinyl) methyl-8-amino-7-iodo-1, 4-benzodioxane-5-carboxylate, as a ligand. [125I]SB207710 binding sites were distributed over the muscle layer of human colon, in the myenteric plexus and in the muscle. In the guinea pig colon, a much higher density was detected in the myenteric plexus than in the muscle. Therefore, in the human and guinea pig colon, the 5-HT(4) receptor was located both in the myenteric plexus and in the muscle, and in the guinea pig colon, the receptor was located more predominantly in the myenteric plexus of the muscle than it is in the human colon.

Regional difference in correlation of 5-HT4 receptor distribution with cholinergic transmission in the guinea pig stomach.

Localization and function of 5-HT4 receptors in the stomach were examined in mucosa-free preparations of antrum, corpus and fundus from guinea pig stomach by determination of acetylcholine release and in vitro receptor autoradiography. Specific [125I]SB207710, (1-n-butyl-4-piperidinyl) methyl-8-amino-7-iodo-1,4-benzodioxane-5-carboxylate, binding sites were detected in 3 regions of the stomach. High densities of binding were observed in the myenteric plexus of antrum and corpus, but not fundus. In mucosa-free preparations treated with 5-HT1, 5-HT2 and 5-HT3 receptor antagonists, 5-HT (10(-8)-10(-6) M) potentiated the electrically stimulated (0.5 Hz, 1 ms) outflow of [3H]acetylcholine from antrum and corpus strips preloaded with [3H]choline, but not from fundus strips, and the potentiation was antagonized by SB204070, (1-n-butyl-4-piperidinyl) methyl-8-amino-7-chloro-1,4-benzodioxane-5-carboxylate. Thus, 5-HT4 receptors are located on myenteric cholinergic neurons in the antrum and corpus of guinea pig stomach and their activation evokes the release of acetylcholine.

Ability of mosapride to bind to 5-HT4 receptor in the human stomach.

Ability of mosapride, a gastrokinetic agent, to bind to 5-HT4 receptor was examined in the stomach of human and guinea pig by in vitro receptor autoradiography. [125I]SB207710 binding sites were detected in the muscle layer including the myenteric plexus of the stomach from both humans and guinea pigs, although the binding was observed more clearly and densely in the stomach of guinea pigs than humans. Mosapride as well as SB204070 inhibited the binding of [125I]SB207710. Thus, mosapride possesses the ability to bind to 5-HT4 receptors of human stomach and may modulate the motility, as in the case of guinea pig stomach.

Differential distribution of 5-hydroxytryptamine3 receptor in the colon between human and guinea pig.

Localization of 5-hydroxytryptamine3 (5-HT3) receptor in the human colon was examined by in vitro receptor autoradiography using [125I](S)iodozacopride, and compared with that in the guinea pig colon. [125I](S)iodozacopride binding sites were found with high densities around the myenteric plexus, but with low ones in the muscle layer and mucosa of the human colon, and the binding was abolished by granisetron, a specific 5-HT3 receptor antagonist. While in the guinea pig colon, specific [125I](S) iodozacopride binding was not detected in either the myenteric plexus or the muscle layers. Thus, the 5-HT3 receptors are present in the human colon, especially densely located in the myenteric plexus, but not in the guinea pig colon, and those may participate in the colonic motility. The results of functional studies of 5-HT3 receptor obtained from experiments using guinea pig are not always applying to the human.

Participation of nitric oxide in the mucosal injury of rat intestine induced by ischemia-reperfusion.

The dual role of nitric oxide as a cytoprotective or a cytotoxic free radical gas has been noted in various types of pathophysiological conditions, including the digestive system. The aim of this study was to examine the role of nitric oxide in the mucosal injury induced by ischemia-reperfusion in the rat small intestine. A transient intestinal ischemia was produced in the catheterized ileal segments of rats by occluding the anterior mesenteric artery for 60 min. Nitric oxide metabolites (NO2- and NO3-) and lactate dehydrogenase activity in perfusates of the intestinal lumen were measured over 5 hr periods. The time-course of histological changes in small intestine was also observed. After ischemia-reperfusion, nitric oxide release in the intestinal lumen increased significantly and the dynamics of nitric oxide release correlated with that of lactate dehydrogenase leakage. The administration of NG-nitro-L-arginine methyl ester (1.0-2.5 mg/kg) inhibited this increased nitric oxide release and the lactate dehydrogenase leakage and afforded protection against the mucosal injury induced by ischemia-reperfusion. In conclusion, the nitric oxide production that was accelerated by ischemia-reperfusion of small intestine may possibly participate in the breakdown of intestinal mucosa after ischemia-reperfusion insult.

[The glial endothelin-nitric oxide system in ischemia-related neuronal cell death].

Both endothelin and nitric oxide (NO) have been proposed to act as pathophysiological factors in ischemia-related neural damage. This review is concerned with the participation of the glial endothelin-NO system in ischemia-related neuronal cell death. In the rat brain with cerebral apoplexy, endothelin, endothelin receptors and NO synthase (NOS) were rich in the glial cells of damaged brain areas. The brain subjected to transient forebrain ischemia contained astrocytic endothelins and microglial expressions of the ETB-receptor and NOS aggregating in the damaged CA1 subfield of the hippocampus at 7 days after the ischemia. Astrocytic endothelin, ETB-receptor and NOS became more apparent at 28 days after the ischemia, corresponding to a time when neural tissue-repair/remodeling after damage occurs, whereas no activities of the endothelin-NO system are observed in microglia. In the in vitro experiment, endothelin was found to modulate the release of NO from the hippocampal slices subjected to transient forebrain ischemia. There may be a cross-talk between the endothelin system and NO in the astrocytes and microglia during the process of ischemia-related neuronal cell death and neural tissue-remodeling.

Cloning and tissue distribution of novel splice variants of the rat GABAB receptor.

We have identified two novel splice variants of the metabotropic gamma-aminobutyric acid receptor (GABABR1), designated GABABR1c and GABABR1d, when screening a rat cerebellum cDNA library. GABABR1c has an amino acid sequence identical to GABABR1b, a member of GABABR1 isoforms, and an additional 93-bp insertion that generates an additional 31-amino-acid sequence in the fifth transmembrane region of GABABR1b. Thus, GABABR1c may have a structural variation in the second extracellular loop and fifth transmembrane region. GABABR1d also has an amino acid sequence identical to GABABR1b and an additional insertion of 566 bp that generates a divergent amino acid sequence in the carboxylterminal end. Reverse-transcription polymerase chain reaction analysis showed that in various rat tissues GABABR1c mRNA was ubiquitously expressed and GABABR1d mRNA in forebrain, cerebellum, eye, kidney, and urinary bladder. GABABR1 isoforms may function not only in the central nervous system but also in various peripheral tissues.

Neuronal release of endogenous dopamine from corpus of guinea pig stomach.

Neuronal release of endogenous dopamine was identified in mucosa-free preparations (muscle layer including intramural plexus) from guinea pig stomach corpus by measuring tissue dopamine content and dopamine release and by immunohistochemical methods using a dopamine antiserum. Dopamine content in mucosa-free preparations of guinea pig gastric corpus was one-tenth of norepinephrine content. Electrical transmural stimulation of mucosa-free preparations of gastric corpus increased the release of endogenous dopamine in a frequency-dependent (3-20 Hz) manner. The stimulated release of dopamine was prevented by either removal of external Ca2+ or treatment with tetrodotoxin. Dopamine-immunopositive nerve fibers surrounding choline acetyltransferase-immunopositive ganglion cells were seen in the myenteric plexus of whole mount preparations of gastric corpus even after bilateral transection of the splanchnic nerve proximal to the junction with the vagal nerve (section of nerves between the celiac ganglion and stomach). Domperidone and sulpiride potentiated the stimulated release of acetylcholine and reversed the dopamine-induced inhibition of acetylcholine release from mucosa-free preparations. These results indicate that dopamine is physiologically released from neurons and from possible dopaminergic nerve terminals and regulates cholinergic neuronal activity in the corpus of guinea pig stomach.

Endothelin receptors in kainic acid-induced neural lesions of rat brain.

Seven days after an intracerebroventricular injection of 0.8 microgram kainic acid, a time of neural tissue-repair after damage, we applied our receptor autoradiographic method to examine changes in the endothelin receptors in kainic acid-induced neural lesions of the rat brain. There were belt-shaped areas with the de novo expressed [125I]endothelin-1 binding sites in the damaged hippocampus CA1, CA3, and CA4 subfields. We also noted a homogeneous zone with a low binding-density, the area sandwiched by the belt-shaped areas. In a "remote" area corresponding anatomically to the deep soma layer of the piriform cortex plus lateral parts of amygdaloid complex we noted a well-defined area with "punched hole-figure" of low density [125I]endothelin-1 binding sites. The lesion was surrounded by areas rich in binding sites. The de novo expressed [125I]endothelin-1 binding sites were characterized endothelin B receptor. Microglia were present in the area with "punched hole-figure" and in the hippocampus pyramidal cell layer with neuronal death. In contrast to microglia, astrocytes were rich with hypertrophia in kainic acid-induced neural lesions anatomically corresponding to areas with the de novo endothelin B receptor. Taken together with the present observations of microscopic evidence of cellular distribution, we suggest that the de novo expressed endothelin B receptor was carried by astrocytes aggregating in neural lesions. In light of our findings, the possibility that astrocytes can be activated by the endothelin B receptor in response to neural tissue repair after damage to neurons would have to be considered.

Expression of endothelin receptors and nitric oxide synthase in the brain of stroke-prone spontaneously hypertensive rats with cerebral apoplexy.

Endothelin (ET) receptors, ET-1-like immunoreactivity and nitric oxide synthase (NOS) were examined in the brain of stroke-prone spontaneously hypertensive rats (SHRSPs) with cerebral apoplexy. Our receptor autoradiographic method with 125I-ET-1 and unlabeled selective ligands for ET receptors revealed de novo expressions of ET(A) and ET(B) receptors in areas of neural lesions with cerebrovascular damage in SHRSPs. Immunohistochemical staining for ET-1 showed clear ET-1-like immunoreactivity in areas with highly expressed ET receptors. Histochemical studies on astrocytes and microglia suggested that these glial cells, aggregating in lesions, may carry ET receptors, ET-1-like immunoreactivity. Furthermore, NOS detected histochemically using an NADPH-diaphorase staining method was rich on glial cells in damaged areas of the brain in SHRSPs with cerebral apoplexy. Our data suggest the pathophysiological significance of glial ET(A) and ET(B) receptors, ET-1 and NOS in neural lesions of SHRSPs.

Endothelin receptors in rat pituitary gland.

1. We used the quantitative receptor autoradiographic method plus 125I-endothelin-1 (125I-ET-1), BQ-123, a specific antagonist for the endothelin ETA receptor, and sarafotoxin S6c, a selective agonist for the ETB receptor to investigate the ET receptor in the rat pituitary gland. 2. The method revealed that the BQ-123-sensitive ETA receptor was present predominantly in the anterior lobe and Rathke's pouch. 3. The posterior lobe contained BQ-123-sensitive ETA and sarafotoxin S6c-sensitive ETB receptors, in almost the same proportion. There was no significant 125I-ET-1 binding to the intermediate lobe. 4. Knowledge of the heterogeneous distribution of ET receptor subtypes in the pituitary gland supplies information that will be pertinent to physiological investigations of the gland.

Rat peritoneal macrophages express endothelin ET(B) but not endothelin ET(A) receptors.

The properties of endothelin receptors on rat peritoneal macrophages were examined in in vitro receptor autoradiographic binding experiments and in a reverse transcription-polymerase chain reaction (RT-PCR) study. Dense and specific [(125)I]endothelin-1 binding sites were detected on the macrophages. [(125)I]Tyr13-Suc-[Glu9,Ala(11,15)]-endothelin-1(8-21) , IRL1620, a selective endothelin ET(B) receptor ligand, but not [(125)I](N-[(hexahydro-1-azepinyl)carbonyl])L-Leu(1-Me)D-Trp-D-Tyr , PD151242, a selective endothelin ET(A) receptor ligand, specifically bound to rat macrophages (Kd = 0.75 +/- 0.19 nM, Bmax = 7.77 +/- 2.50 fmol/mg). RT-PCR experiments also showed the expression of endothelin ET(B) receptor mRNA, but not endothelin ET(A) receptor mRNA, in these macrophages. These results indicate that rat peritoneal macrophages apparently express the endothelin ET(B) receptor but not the endothelin ET(A) receptor.

Microglia with an endothelin ETB receptor aggregate in rat hippocampus CA1 subfields following transient forebrain ischemia.

We examined endothelin (ET) receptors in the hippocampus CA1 subfields of stroke-prone spontaneously hypertensive rats subjected to a 10-min bilateral carotid occlusion and reperfusion. When delayed neuronal death had occurred in the pyramidal cell layer at 7 days after transient forebrain ischemia, the quantitative receptor autoradiographic method we used revealed a dramatic increase in number of 125I-ET-1 binding sites in the hippocampus CA1 subfields. The highest number of de novo binding sites appeared in the area corresponding anatomically to the pyramidal cell layer with neuronal death. These binding sites were characteristically the ETB receptor. The de novo 125I-ET-1 binding was mainly present on microglia aggregating with a high density in the damaged pyramidal cell layer. As ET-1- and ET-3-like immunoreactivities were highly expressed within astrocytes in damaged neural tissue, the possibility that microglia with the ETB receptor are activated to participate in the pathophysiology of ischemia-related neural tissue damage by astrocytic ET-1 and ET-3 produced in response to transient forebrain ischemia would have to be considered.

Conflict behavior and dynamics of monoamines of various brain nuclei in rats.

The present study was an attempt to clarify the role of noradrenaline (NA) and of the 5-hydroxytryptamine (5-HT) system in various nuclei in brain, as a component of a proposed neural circuit in the mediation of conflict behavior and the anticonflict action of anxiolytics. The authors investigated changes in the concentrations of NA, 3-methoxy-4-hydroxyphenylethyleneglycol, 5-HT and 5-hydroxyindoleacetic acid in discrete regions of the brain in rats, in correlation with conflict behavior and also the effects of diazepam and suriclone. Noradrenergic neural activity diminished with a conflict situation, in the frontal cortex, central amygdala, mammillary body and dorsal hippocampus. 5-Hydroxytryptaminergic neural activity increased with a conflict situation in the frontal cortex, central amygdala, basolateral amygdala and medial septum. These changes in the frontal cortex, central amygdala, mammillary body and dorsal hippocampus were not observed when diazepam 20 mg/kg (p.o.) and suriclone 40 mg/kg (p.o.) produced anticonflict action. Suriclone normalized the increased 5-HT-ergic activity in the medial septum. The suppression of NA-ergic and the activation of 5-HT-ergic (except for the mammillary body) neural activity in the frontal cortex, central amygdala, mammillary body and dorsal hippocampus seemed to be linked to the mediation of conflict behavior. The facilitatory and inhibitory action on NA and 5-HT (except for the mammillary body) neurons, respectively, in these regions of the brain, may be involved in mechanisms underlying the anticonflict action of anxiolytics (diazepam or suriclone).

Delta 9-tetrahydrocannabinol facilitates striatal dopaminergic transmission.

We examined the effects of delta 9-tetrahydrocannabinol (THC) on striatal dopaminergic neurons in rats. THC inhibited the uptake of 3H-dopamine (DA) into striatal synaptosomes. THC facilitated the release of endogenous DA but not dihydroxyphenylacetic acid (DOPAC) from striatal slices. The concentration of DA in the dorsolateral striatum was reduced by THC. We propose that THC may stimulate nigrostriatal dopaminergic neurotransmission mainly by inhibiting uptake of DA and by facilitating release of DA.