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Myotonia congenita is caused by mutation of the CLCN1 gene, which encodes the human skeletal muscle chloride channel (ClC-1). The ClC-1 protein is a dimer comprised of two identical subunits each incorporating its own separate pore. However, the precise pathophysiological mechanism underlying the abnormal ClC-1 channel gating in some mutants is not fully understood. We characterized a ClC-1 mutation, Pro-480-Thr (P480T) identified in dominant myotonia congenita, by using whole-cell recording. P480T ClC-1 revealed significantly slowed activation kinetics and a slight depolarizing shift in the voltage-dependence of the channel gating. Wild-type/mutant heterodimers exhibited similar kinetic properties and voltage-dependency to mutant homodimers. Simulating myotonic discharge with the voltage clamp protocol of a 50 Hz train pulse, the increment of chloride conductance was impaired in both wild-type/mutant heterodimers and mutant homodimers, clearly indicating a dominant-negative effect. Our data showed that slow activation gating of P480T ClC-1 impaired the increment of chloride conductance during repetitive depolarization, thereby accentuating the chloride conductance reduction caused by a slight depolarizing shift in the voltage-dependence of the channel gating. This pathophysiology may explain the clinical features of myotonia congenita.

Selenium is an essential trace metal element, whereas large doses of selenium exert adverse effects to the human body. We examined the effects of selenium compounds, sodium selenite (Na2SeO3) and sodium selenate (Na2SeO4), on catecholamine secretion from cultured bovine adrenal chromaffin cells. Treatment of chromaffin cells with sodium selenite for 72, 48, and 24 h caused decreases in protein and catecholamine contents, in association with cell damage, at concentrations over 30, 300, and 300 µM, respectively. The cells treated with subtoxic conditions (

Coupling of functional GABAB receptors (GABABR) to G proteins was investigated with an expression system of baby hamster kidney (BHK) cells and Xenopus oocytes. Fluorescence resonance energy transfer (FRET) analysis of BHK cells coexpressing GABAB1a receptor (GB1aR) fused to Cerulean, a brighter variant of cyan fluorescent protein, and GABAB2 receptor (GB2R) fused to Venus, a brighter variant of yellow fluorescent protein, revealed that GB1aR-Cerulean and GB2R-Venus form a heterodimer. The GABABR agonists baclofen and 3-aminopropylphosphonic acid (3-APPA) elicited inward-rectifying K+ currents in a concentration-dependent manner in oocytes expressing GB1aR and GB2R, or GB1aR-Cerulean and GB2R-Venus, together with G protein-activated inward-rectifying K+ channels (GIRKs), but not in oocytes expressing GB1aR alone or GB2R alone together with GIRKs. Oocytes coexpressing GB1aR + Gαi2-fused GB2R (GB2R-Gαi2) caused faster K+ currents in response to baclofen. Furthermore, oocytes coexpressing GB1aR + GB2R fused to Gαqi5 (a chimeric Gαq protein that activates PLC pathways) caused PLC-mediated Ca2+-activated Cl− currents in response to baclofen. In contrast, these responses to baclofen were not observed in oocytes coexpressing GB1aR-Gαi2 or GB1aR-Gαqi5 together with GB2R. BHK cells and Xenopus oocytes coexpressing GB1aR-Cerulean + a triplet tandem of GB2R-Venus-Gαqi5 caused FRET and Ca2+-activated Cl− currents, respectively, with a similar potency in BHK cells coexpressing GB1aR-Cerulean + GB2R-Venus and in oocytes coexpressing GB1aR + GB2R-Gαqi5. Our results indicate that functional GABABR forms a heterodimer composed of GB1R and GB2R and that the signal transducing G proteins are directly coupled to GB2R but not to GB1R.

Among various alcoholic beverages, it has reported that beer has a potent activity to stimulate gastric emptying. Our previous studies showed that beer congener stimulated gastrointestinal motility by directly stimulating muscarinic M3 receptor, present in smooth muscles of the gastrointestinal tract. However, active components that account for the action have yet to be identified. We attempted to isolate the stimulant(s) of gastrointestinal motility in beer.Beer congener was prepared from beer and used to separate and purify active components by a series of liquid chromatography using affinity to muscarinic M3 receptor as an index. Gastrointestinal motility-stimulating activity was evaluated using a test for activity that causes contraction of longitudinal muscles in guinea pig ileum and a test for gastric emptying activity in mice.The active components (compounds A and B) were purified and isolated from beer by four liquid chromatography steps. The IC50 values of two active isolates to muscarinic M3 receptor were 0.65 x 10 g/ml and 2.30 x 10 g/ml, respectively. The concentrations of compounds A and B contained in beer were sufficient to explain most of the muscarinic M3 receptor binding activity of beer. The active fraction that contained both compounds A and B (which was 10 times as active as beer congener in muscarinic M3 receptor binding activity) dose-dependently contracted the longitudinal muscles of guinea pig ileum with an activity that was 20 times as potent as that of beer congener. The same active fraction significantly stimulated gastric emptying in mice with an activity 20 times as potent as that of beer congener.Two active components (compounds A and B) were isolated as gastrointestinal motility stimulants (muscarinic M3 agonists) in beer. These results suggest that the two isolated active components are the active entities of the gastrointestinal motility-stimulating effect of beer.

The objective of this study was to evaluate serum nitrite and nitrate (nitrite/nitrate) concentrations that affect adversely pregnancy outcome. Pregnant rats, from day 2 to day 8 of pregnancy, were daily given subcutaneously several doses (5, 10, and 30 mg/rat) of diethylenetriamine-nitric oxide (DETA/NO). Serum nitrite/nitrate concentrations were measured using an HPLC system. Serum nitrite/nitrate concentrations increased dose-dependently with DETA/NO. Effects of DETA/NO on pregnancy outcome were assessed on day 14 of pregnancy. In rats given 5 mg DETA/NO, there was a significant increase in serum nitrite/nitrate concentrations (49.2 vs 24.6 μmol/l, P

Characterization of the γ-aminobutyric acid (GABA)B receptor involved in the motility of dog small intestine was analyzed by application of the microdialysis method to the small intestine of the whole body of the dog. The reverse transcription-polymerase chain reaction (RT-PCR) was used. Intraarterial administration of muscimol induced acceleration of motility associated with acetylcholine (ACh) release, these responses being antagonized by bicuculline. Intraarterial administration of baclofen induced inhibition of motility associated with ACh release, these responses being antagonized by CGP62349. GABA induced inhibition of motility associated with decrease in ACh release. CGP62349 alone induced acceleration of motility associated with increase in ACh release. RT-PCR revealed the presence of mRNAs for both subunits of GABAB receptor, GABAB1 and GABAB2, in the dog small intestine, although GABAB1 subunits were 6 isoforms of GABAB1 (GABAB1(a) – GABAB1(g)), except GABAB1(d). Thus, the GABAB receptor located at cholinergic neurons as a heterodimer with subunits of GABAB1 and GABAB2 in the dog small intestine operates predominantly relative to the GABAA receptor in physiological motility. Keywords:: GABAB1 subunit, GABAB2 subunit, microdialysis, RT-PCR

Metabotropic G protein-coupled receptors have recently been recognized as targets for anesthetics and analgesics. In particular, Gq-coupled receptors such as muscarinic M1 receptors (M1R) and 5-hydroxytryptamine (5-HT) type 2A receptors have been reported to be targets for anesthetics. Much less is known, however, about the effects of anesthetics on Gi-coupled receptors. Here we report a method to analyze functions of Gi-coupled receptors in Xenopus oocytes expressing a chimeric Gα protein. A chimeric Gαq protein Gαqi5, which contains carboxy-terminus five amino acids of Gαi, enables Gi-coupled receptors to couple to Gq-coupled receptor-mediated downstream pathways such as activation of phospholipase C. We determined acetylcholine (ACh)-induced Ca2+-activated Cl– currents in Xenopus oocytes coexpressing Gi-coupled muscarinic M2 receptors (M2R) with the chimeric Gαqi5. Although ACh did not induce any currents in oocytes expressing M2R alone, it caused robust Cl– currents in oocytes coexpressing M2R with Gαqi5. The EC50 of the ACh-induced Cl– current mediated through Gαqi5 was 0.2 µmol/l, which was 2.2 times higher than that of the ACh-induced G protein-activated inwardly rectifying K+ currents activated by Gβγ subunits liberated from endogenously expressed Gαi in Xenopus oocytes. Other Gi-coupled somatostatin type 2, 5-HT1A and δ-opioid receptors, when coexpressed with Gαqi5 in oocytes, also caused robust Ca2+-activated Cl– currents. In oocytes coexpressing M2R and Gαqi5, a volatile anesthetic halothane inhibited M2R-induced Cl– currents in a concentration-dependent manner with the IC50 of 1.1 mmol/l, suggesting that halothane inhibits M2R-induced cellular responses at clinically relevant concentrations. Treatment with the protein kinase C inhibitor GF109203X produced a 3.5-fold enhancement of the initial Cl– currents induced by 1 µmol/l ACh in oocytes expressing M2R and Gqi5. The rate of halothane-induced inhibition of Cl– currents elicited by ACh, however, was not changed in such oocytes pretreated with GF109203X. These findings suggest that halothane inhibits the M2R-induced signaling by acting at sites other than PKC activity. Collectively these findings suggest that the use of oocyte expressing Gαqi5 would be helpful to examine the effects of anesthetics or analgesics on the function of Gi-coupled receptors in the Xenopus oocyte expression system.

Although a potential target site of general anesthetics is primarily the GABA A receptor, a chloride ion channel, a previous study suggested that the intravenous general anesthetic propofol attenuates the M1 muscarinic acetylcholine receptor (M1 receptor)-mediated signal transduction. In the present study, we examined the target site of propofol in M1 receptor-mediated signal transduction. Two-electrode voltage-clamp method was used in Xenopus oocytes expressing both M1 receptors and associated G protein alpha subunits (Gqalpha). Propofol inhibited M1 receptor-mediated signal transduction in a dose-dependent manner (IC50 = 50 nM). Injection of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) into oocytes overexpressing Gqalpha was used to investigate direct effects of propofol on G protein coupled with the M1 receptor. Propofol did not affect activation of Gqalpha-mediated signal transduction with the intracellular injection of GTPgammaS. We also studied effects of propofol on l-[N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding and M1 receptor-mediated signal transduction in mammalian cells expressing M1 receptor. Propofol inhibited the M1 receptor-mediated signal transduction but did not inhibit binding of [3H]NMS. Effects of propofol on Gs- and Gi/o-coupled signal transduction were investigated, using oocytes expressing the beta2 adrenoceptor (beta2 receptor)/cystic fibrosis transmembrane conductance regulator or oocytes expressing the M2 muscarinic acetylcholine receptor (M2 receptor)/Kir3.1 (a member of G protein-gated inwardly rectifying K(+) channels). Neither beta2 receptor-mediated nor M2 receptor-mediated signal transduction was inhibited by a relatively high concentration of propofol (50 microM). These results indicate that propofol inhibits M1 receptor-mediated signal transduction by selectively disrupting interaction between the receptor and associated G protein.

Involvement of protein kinase C (PKC) in the release of gamma-aminobutyric acid (GABA) was examined in Xenopus laevis oocytes injected with mRNA from rat cerebellum, as compared with findings in slices of rat cerebellum. The mRNA-injected oocytes preloaded with [3H]GABA showed spontaneous release of [3H]GABA, approximately 0.5% of GABA content per 1 min. Stimulation with either Ca2+ ionophore (A23187) or a high K+ concentration increased the release of [3H]GABA from slices of rat deep cerebellar nucleus and mRNA-injected oocytes but not from noninjected and water-injected oocytes. 12-O-Tetradecanoylphorbol 13-acetate (10-300 nM) but not 4alpha-phorbol 12,13-didecanoate (300 nM) potentiated the A23187-stimulated release of [3H]GABA from slices and from mRNA-injected oocytes, in a concentration-dependent manner. Thus, machinery associated with release processes of GABA can be expressed in oocytes by injecting rat cerebellar mRNA, and PKC participates in GABA release from the functionally expressed GABAergic nerve terminals.

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.

Arginine residue at position 285 (R285) in the intracellular C-terminal domain of inward rectifier potassium channel Kir2.2 is conserved in many species, but missing in previously reported human Kir2.2 sequences. We here identified the human Kir2.2 gene in normal individuals, which contained R285 in the deduced amino-acid sequence (hKir2.2/R285). All 30 individuals we examined were homozygous for Kir2.2/R285 gene. The hKir2.2/R285 was electrophysiologically functional in both mammalian cells and Xenopus oocytes. However, the hKir2.2 missing R285 was functional only in Xenopus oocytes, but not in mammalian cells. Thus, R285 in Kir2.2 is important for its functional expression in mammalian cells.

The mechanism underlying orexin-induced contraction was examined in isolated preparations of guinea pig ileum, in relation to cholinergic transmission. Orexin-A caused contraction of ileal strips in a concentration-dependent manner. 1-(2-Methylbenzoxazol-6-yl)-3-[1,5]napthyridin-4-yl-urea hydrochloride (SB-334867-A) antagonized the orexin-A-induced contraction, with no effects on the acetylcholine-induced contraction and twitch contractions. The orexin-A-induced contraction was inhibited by tetrodotoxin and atropine, but not by hexamethonium, an antagonist of vasoactive intestinal peptide and a mixture of 5-hydroxytryptamine receptor antagonists. Orexin-A evoked an outflow of [3H]acetylcholine from the ileal strips preincubated with [3H]choline, in a concentration-dependent manner, and the orexin-A-evoked outflow was inhibited by tetrodotoxin, indicating that the outflow of [3H]acetylcholine originates from the nerve terminals. The orexin-A-evoked outflow of [3H]acetylcholine was antagonized by SB-334867-A. Thus, orexin-A evokes the release of acetylcholine from the enteric cholinergic neurons due to stimulation of the orexin-1 receptors and then causes contractions of guinea pig ileum.

It has recently been proposed that disturbed gastric adaptive relaxation may play a role in functional dyspepsia (FD). However, the effect of Helicobacter pylori infection, which may be one of multiple factors associated with FD, on gastric relaxation is not clear. The aim of this study was to clarify the influence of H. pylori infection on the responsiveness of smooth muscles of the gastric fundus to agonists or to stimulation of enteric nerves, with particular emphasis on nonadrenergic noncholinergic (NANC) relaxation. We investigated myogenic responses to carbachol (CCH) and sodium nitroprusside (SNP), and neural responses to electrical field stimulation (EFS), in the absence or presence of atropine and guanethidine, in the tissues of the gastric fundus of H. pylori-infected Mongolian gerbils (MGs). H. pylori-infected MGs showed typical gastritis, with H. pylori colonization in the antrum and body. The gastric fundus adjacent to the body was composed of thin gastric mucosa with mild inflammation, which was covered with stratified squamous epithelium, and the muscle layer communicated with that of the gastric body. In the gastric fundus, CCH- and SNP-induced responses were not different in controls and H. pylori-infected MGs. In the absence of any antagonists, EFS-evoked contraction tended to be reduced in H. pylori-infected MGs compared with that in control MGs, albeit that the difference was statistically nonsignificant. Nω-nitro-l-arginine methyl ester inhibited NANC relaxation in the tissues in both groups. EFS-evoked NANC relaxation remained intact in H. pylori-infected MGs. Mild inflammation in gastric fundus associated with H. pylori infection does not cause enteric neuromuscular dysfunction of the site in vitro.

Electrophysiological properties of Xenopus oocytes exposed to alkaline extracellular pH (pHo) were investigated by measuring whole-cell currents using the two-electrode voltage-clamp method. Alkaline pHo (8.5 - 10.5) elicited an outward current in a pHo-dependent manner with a concomitant increase in the membrane conductance. This outward-current response was dependent on K+ because it was suppressed by a K+ channel blocker tetraethy lammonium+ (20 mM), and the reversal potential of the response was in good agreement with the Nernst equation for K+. The response was not affected by pretreatment of oocytes with the acetoxymethyl ester of bis-(o-aminophenoxy)-ethane-N,N,N’N’-tetraacetic acid (10 μM), a membrane-permeant intracellular Ca2+ chelator, but it was augmented by forskolin (0.4 μM), a stimulant of adenylate cyclase. The outward-current response originates in the oocyte but not in the surrounding follicle cells because the current could still be evoked when follicle cells were removed by collagenase or when gap junctions connecting the oocyte membrane and follicle cells were blocked by 1-octanol (1 mM). It is concluded that the outward current elicited by alkaline pHo in Xenopus oocytes is dependent on the activation of K+ channels via the cAMP pathway and that the outward current originates in the oocyte rather than the surrounding follicle cells.

Intestinal motor activity associated with acetylcholine (ACh) release was assessed in the small intestine of anesthetized dogs by simultaneous measurement of motor activity and local ACh concentrations within the intestinal wall with in vivo microdialysis. Basal concentration of ACh measured in the dialysate was 1.12 +/- 0.08 pmol/15 min (n = 10), a value that remained constant until 3 h after perfusion. Intra-arterial infusion of tetrodotoxin reduced dialysate ACh concentration, while the motor activity accelerated at the early phase after infusion of tetrodotoxin and then decreased, thereby suggesting that the motor activity is regulated by not only excitatory cholinergic neurons, but also inhibitory neurons. Intraarterial infusion of atropine increased dialysate ACh concentration but reduced motor activity, thereby indicating that the cholinergic neurons are tonically active and the muscarinic autoreceptors operate to inhibit the ACh release. Intraarterial infusion of norepinephrine reduced, but yohimbine increased both motor activity and dialysate ACh concentration, thereby indicating that the adrenergic neurons regulate the motor activity due to control of cholinergic neuronal activity. This in vivo microdialysis method demonstrated in the whole body of animals that the activity of cholinergic neurons was physiologically regulated by itself and adrenergic neurons.

GABA and glutamate/aspartate amino acids are important neurotransmitters in the neuronal circuits involved in the vestibulo-ocular reflex. Vestibular nuclei are controled by Purkinje cells and opposite vestibular nuclei and affect oculomotor nuclei through GABAergic neurons. There are three types of GABA receptors that are targets of GABAergic nerves: GABAA, GABAB and GABAC receptors. GABAA and GABAB receptors are densely distributed in the cerebellum: GABAA receptor is distributed in cerebellar Purkinje cells, deep cerebellar nucleui and vestibular nucleui, and GABAB receptor is distributed in cerebellar Purkinje cells. GABAC receptor is specifically distributed in the retina. GABAA and GABAC receptors are members of the transmittergate channel superfamily and their compositions are determined by the formation of pentameric structures. GABAA receptor is composed of five kinds of subunit, α(α1-α6), β(β1-β4), γ(γ1-γ3), δand ρ, and possesses binding sites for benzodiazepines, barbiturates and neurosteroids. GABAB receptor is a G-protein coupled receptor, although the receptor is the first discovered receptor forming heterodimers in the Gprotein coupled receptors. The functions of GABAA and GABAB receptors in the neuronal circuits that are involved in the vestiburo-ocular reflex have been investigated by histological and electrophysiological studies, but the role of the receptors in the vestibular functions have not been studied.

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 ETB 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 ETB 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, ETB 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 ETB/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.

1. The role of synaptophysin in the exocytotic release of dopamine (DA) was examined in Xenopus laevis oocytes injected with rat brain mRNA.

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.

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.

IN Xenopus oocytes coinjected with poly(A) + RNA derived from the rat cerebellum and cRNAs for the cloned G protein-gated inwardly rectifying K + channel (GIRK), GIRK1 and GIRK2, the GABA-B agonist baclofen elicited inwardly rectifying K + currents. The inward K + currents elicited by baclofen were inhibited by the selective GABA-B antagonists 2-OH saclofen and CGP 35348, and by the GIRK inhibitor Ba 2+ . In contrast, baclofen caused no currents in oocytes injected with the cerebellar poly(A) + RNA alone, the poly(A) + RNA and cRNA for GIRK1 or GIRK2, or only cRNAs for GIRKI and GIRK2. These findings indicate that GABA-B receptors in the rat cerebellum were functionally expressed in Xenopus oocytes and activated the cloned GIRKs composed of GIRK1 and GIRK2 as heteromultimers.

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.

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.

1. The receptor autoradiographic method done on the rat lower brain stem and cerebellum plus 125I-endothelin-1, BQ-123, an antagonist for the endothelin ETA receptor, and sarafotoxin S6c, an agonist for the ETB receptor, revealed minute amounts of the ETA receptor coexisting with the ETB receptor in the caudal solitary tract nucleus of the rat lower brain stem. 2. The ETB receptor is present predominantly in other parts of the lower brain stem. 3. Knowledge of the heterogeneous distribution of the central endothelin receptor subtypes aids in understanding the neurophysiology of endothelins.

1. Our method of real-time monitoring of dopamine release from rat striatal slices revealed that endothelin (ET)-3-induced dopamine release was inhibited by NG-methyl-L-arginine (L-NMMA; 1 mM), an inhibitor of nitric oxide (NO) synthase, while NG-methyl-D-arginine (D-NMMA; 1 mM), an inactive isomer of L-NMMA, had no effect. 2. The inhibition of L-NMMA (0.1 mM) became apparent when tissues were pretreated with tetrodotoxin (1 microM) for 30 min and subsequently exposed to ET-3 (4 microM). 3. L-NMMA (0.1 and 1 mM) dose dependently protected against ET-3-triggered hypoxic/hypoglycemic impairment of striatal responses to high K+. 4. Thus, NO may work as a promoter in mediation of the stimulatory and neurotoxic action of ET-3 on the striatal dopaminergic system, presumably by interacting with interneurons in the striatum.

1. The type of endothelin (ET) receptor located on the myenteric neurones of guinea-pig ileum was determined by receptor autoradiography and function of the receptor was examined by release experiments of acetylcholine (ACh) from the longitudinal muscle myenteric plexus (LM-MP) preparations. 2. Specific [125I]-ET-1 binding sites were distributed in muscle layers, myenteric and submucous plexuses, and mucosa layers. High-grain densities were detected in both myenteric and submucous plexuses. 3. Binding in the myenteric plexus was abolished by incubation with either IRL 1620 (endothelin ETB receptor agonist) or BQ 788 (endothelin ETB receptor antagonist), but not with BQ 123 (endothelin ETA receptor antagonist). The [125I]-IRL 1620 binding sites were evident in the myenteric plexus. Thus, the endothelin receptor located on the myenteric neurones is of the ETB type. 4. ET-1 (10(-10)-3 x 10(-8) M) and ET-3 (10(-10)-3 x 10(-8) M) evoked 3H outflow from LM-MP preparations of ileum preloaded with [3H]-choline, in a concentration-dependent manner. There was no significant difference between maximum amounts of ET-1-evoked and ET-3-evoked 3H outflow. 5. ET-1 and ET-3 evoked outflow of 3H was BQ 788-sensitive, but BQ 123-insensitive. Both evoked outflows of 3H were Ca(2+)-dependent and tetrodotoxin-sensitive. 6. These results indicate that the endothelin ETB receptor is located on the enteric cholinergic neurones and that stimulation evokes the release of ACh.

Property of receptor for vasoactive intestinal contractor (VIC), a peptide related to the endothelin family, expressed in Xenopus oocytes by injecting mRNA obtained from the intestine of rat, were studied using the voltage-clamp method. Inward-current responses to VIC (1 nM–100 nM) were evoked in a concentration-dependent manner in mRNA-injected oocytes. Non-injected and water-injected oocytes failed to respond to VIC. The reversal potential for the VIC response was around −20 mV and the depolarizing shift was approximately 18 mV, when the external concentration of Cl − was halved, in agreement with the Nernst equation. The response to VTC was suppressed either by the external application of BAPTA/AM (10 μM) or by pertussis toxin (0.5 μg/ml). These results indicate that the receptor for VIC, functionally expressed in Xenopus oocytes injected with rat intestinal mRNA, is coupled to pertussis toxin-sensitive G-protein and its activation leads to mobilization of intracellular Ca 2+ .

TTC-909 is a newly developed isocarbacyclin methyl ester (TEI-9090) incorporated in lipid microspheres. The neuroprotective effect of TTC-909 was histologically examined in the pyramidal cell layer of the hippocampus CA1 subfield 7 days after transient forebrain ischemia using stroke-prone spontaneously hypertensive rats. TTC-909, given intravenously 10 min after the transient forebrain ischemia, dose-dependently protected against ischemia-related delayed neuronal death. The blood pressure remained unchanged following TTC-909 administration. This finding suggests that TTC-909 has a neuroprotective action on ischemic delayed neuronal death in the hippocampus.

Involvement of cyclic AMP-dependent protein kinase in the desensitization of GABAB receptor expressed in Xenopus oocytes was studied. GABA produced an outward current in the presence of bicuculline, under voltage-clamp conditions. Baclofen mimicked the effect of GABA, thereby indicating that the response to GABA in the presence of bicuculline is mediated by stimulation of GABAB receptor. The GABAB receptor-mediated response was suppressed by dibutyric cyclic AMP and forskolin, but not by 1,9-dideoxy-forskolin, and the effect of dibutyric cyclic AMP was inhibited by H-8. Application of GABA for l min at 20-min intervals induced a reliable response, while that at 10-min intervals produced a desensitization. The desensitization was also partially prevented by H-8. Dibutyric cyclic GMP, 8-bromo-cyclic GMP and Na-nitroprusside did not affect the GABAB receptor-mediated response. Thus, cyclic AMP-dependent protein kinase, but not cyclic GMP-dependent protein kinase participates in desensitization of the GABAB receptor expressed in Xenopus oocytes.

The effect of peptide YY on contractility, acetylcholine release and noradrenaline release was examined in the isolated guinea-pig colon, and findings were compared with those for neuropeptide Y. Peptide YY and neuropeptide Y inhibited the twitch contractions mediated by the stimulation of cholinergic neurons. Peptide YY, neuropeptide Y, [Leu31,Pro34]neuropeptide Y and neuropeptide Y-(13–36) inhibited the electrically stimulated release of acetylcholine. Neuropeptide Y, but not peptide YY, inhibited the high K+-stimulated tetrodotoxin-resistant release of acetylcholine, while the inhibitory effect of neuropeptide Y disappeared after treatment with yohimbine. Neuropeptide Y, but not peptide YY or neuropeptide Y analogues, evoked the release of noradrenaline. After desensitization to the effects of neuropeptide Y, peptide YY inhibited electrically stimulated acetylcholine release. Thus, peptide YY inhibits acetylcholine release through stimulation of a receptor, distinct from the site of action of neuropeptide Y, located on cholinergic neurons as well as the neuropeptide Y Y1 and Y2 receptors in the guinea-pig colon. Neuropeptide Y inhibits acetylcholine release due to the noradrenaline release mediated by stimulation of a receptor distinct from neuropeptide Y Y1 and Y2 receptors, located on adrenergic neurons.

Pre-treatment with BAY K 8644, an L-type voltage-gated Ca2+ channels agonist, produced long-term enhancement (LTE) of over 2 h of high K(+)-evoked dopamine release from rat striatal slices. Exposure to BAY K 8644 under Ca(2+)-free conditions did not enhance this release. Thus, a transient activation of L-type voltage-gated Ca2+ channels followed by Ca2+ entry can trigger LTE of the evoked DA release from striatal tissues. This type of LTE in the striatum underlines the importance of presynaptic mechanisms, including L-type voltage-gated Ca2+ channels of 'long-term potentiation' expression.

We investigated mechanisms mediating endothelin-3-evoked dopamine release from rat striatal slices. Endothelin-3 stimulated dopamine release from the slices in a concentration-dependent manner over a range from 1 to 10 microM. Tetrodotoxin suppressed dopamine release, but left 40% of the release unaffected. Nifedipine, a voltage-gated Ca2+ channel (VGCC) antagonist, significantly inhibited dopamine release in the presence and absence of tetrodotoxin. Endothelin-3-evoked dopamine release was attenuated by D-2-amino-5-phosphnovaleric acid or Mg2+, N-methyl-D-aspartate receptor inhibitors, and this attenuation was not observed in the presence of tetrodotoxin, thereby indicating that the tetrodotoxin-sensitive component of dopamine release was partially mediated by glutamatergic pathways. This view was also supported by findings that endothelin-3 evoked glutamate release and the exogenously applied glutamate stimulated dopamine release. Based on these results, we hypothesize that endothelin-3 produces dopamine release through two distinct mechanisms; one is a direct stimulation of dopaminergic nerve terminals and the other was activation of interneurons which promoted the release of glutamate, resulting in dopamine release.

1. Real-time monitoring of dopamine (DA) release from rat striatal slices demonstrated that endothelin (ET)-3 (0.1-10 microM) produced a biphasic DA release consisting of transient and sustained components. When extracellular Ca2+ was removed, the sustained but not transient response remarkably decreased. 2. ET-3 (1-10 microM) stimulated an increase in the intracellular Ca2+ concentration ([Ca(2+)]i), which also consisted of two components. The external Ca2+ depletion inhibited primarily the sustained component of the Ca2+ response to ET-3. 3. ET-3 increased inositol 1,4,5-trisphosphate (IP3) concentrations in striatal slices. This response peaked at 10 to 20 sec and returned to the basal level 2 min after stimulation, an event which was in good accord with a prompt and transient phase of both cytosolic Ca2+ activity and DA release evoked by ET-3. 4. Thus, ET-3 produces a transient and a sustained release of DA from striatal slices by stimulating intracellular Ca2+ mobilization via IP3 formation and extracellular Ca2+ influx, respectively.

We reported here the first evidence that endothelin (ET)-1 and ET-3 produced an increase in intracellular Ca2+ concentrations ([Ca2+]1), consisting of a transient and a sustained component, in cultured neurones and in slices of the rat hippocampus. In the neurones, the removal of Ca2+ or the addition of Cd2+ remarkably inhibited the ET-3-evoked sustained but not the transient response. The [Ca2+]i increased by ETs was observed in the dentate gyrus and in the vicinity of CA1 and CA3 pyramidal cell layer in the hippocampus, findings in good accord with regions where the glutamate receptor agonists have an elevated [Ca2+]i. The possible causal link between ETs and the increased [Ca2+]i in the development of hippocampal neuronal death is discussed.

✓ Endothelin (ET) receptor subtypes (ETA and ETB) in human meningiomas were characterized using quantitative receptor autoradiography. A single class of high-affinity 125I-ET-1 binding sites was localized in all meningioma tissue studied (dissociation constant: 2.4 ± 0.3 nM, maximum binding capacity: 319 ± 66 fmol/mg (mean ± standard error of the mean for 13 tumors)). Unlabeled ET-1 showed a strong affinity for 125I-ET-1 binding to tissue sections of the tumors with a 50% inhibiting concentration (IC50) of 2.9 ± 0.7 × 10−9 M, whereas ET-3 showed a much lower affinity (IC50: 8.4 ± 2.5 × 10−6 M). Sarafotoxin S6c, a selective agonist for the ETB receptor, could not compete for 125I-ET-1 binding to meningiomas. Endothelin-1 significantly stimulated deoxyribonucleic acid (DNA) synthesis in a dose-dependent manner in cultured human meningioma cells. In contrast, no significant stimulation of DNA synthesis occurred with an S6c concentration up to 10−7 M. Pretreatment of the meningioma cells with pertussis toxin, a bacterial toxin that adds adenosine 5'-diphosphateribose to the α subunit of guanine nucleotide binding (G) proteins such as Gi or Go, induced a concentrationdependent reduction in ET-stimulated DNA synthesis in meningioma cells, but did not affect the epidermal growth factor-induced DNA synthesis. These observations suggest that the ETA receptor is predominantly expressed in human meningioma tissue and that ET may act as a growth factor on the meningioma cells by interacting with the ETA receptor and by pertussis toxin-sensitive mechanisms.

✓ Suramin, a polyanionic compound, has been shown to inhibit the binding of various growth factors to cell surface receptors. The effects of suramin on 125I-insulin-like growth factor (IGF)-I binding to human meningioma tissues and IGF-I-induced deoxyribonucleic acid (DNA) synthesis in cultured meningioma cells were examined using the quantitative receptor autoradiographic method and 3H-thymidine incorporation, respectively. Suramin inhibited specific 125I-IGF-I binding to meningioma tissue sections in a concentration-dependent manner, with a 50% inhibiting concentration (IC50) of 8.7 ± 0.5 × 10−5 M. The addition of 10−3 M suramin to the incubation buffer potently dissociated 125I-IGF-I previously bound to meningioma tissue as a function of time (dissociation half-life (T½) 6.8 minutes). After preincubation of tissue sections with 10−3 M suramin for 120 minutes, there was no inhibition of the subsequent 125I-IGF-I binding to meningiomas. Suramin inhibited the IGF-I-induced incorporation of 3H-thymidine into meningioma cells in a dose-dependent manner, with an IC50 of 4.6 ± 1.4 × 10−5 M. The growth rate of meningioma cells (determined 4 days after seeding) was reduced by 10%, 20%, and 50% of the control culture in the presence of 10−6, 10−5, and 10−4 M suramin, respectively. These results suggest that suramin interferes with IGF-I binding to meningioma tissue and inhibits proliferation of cells, at least partially by preventing IGF-I-induced DNA synthesis and probably by interacting with IGF-I directly rather than with its binding sites.

1. We studied the effects of suramin, a nonspecific growth factor antagonist, on epidermal growth factor (EGF) binding to cell surface receptors in surgically excised human meningiomas, using quantitative receptor autoradiographic methods with radioluminography. 2. High concentrations (10−4–10−2M) of suramin inhibited125I-EGF binding to meningioma sections with IC50's of 3.2 ± 0.4 × 10−4M, whereas lower concentrations (10−5–10−4M) of the drug significantly enhanced EGF binding to the tumor. Scatchard analysis of EGF binding profile revealed significant increases in binding affinity following incubation in the presence of 5 × 10−5M suramin, without significant alterations in maximal binding capacity. 3. The addition of 10−3M suramin to the incubation buffer rapidly dissociated125I-EGF previously bound to meningioma tissues as a function of time (dissociation half-life,T1/2 = 12.4 min). 4. Preincubation in the presence of 5 × 10−5M suramin resulted in significant increases in the subsequent binding of125I-EGF to meningiomas, compared to findings in the control. 5. Our data indicate that (a) suramin exerts biphasic effects on EGF binding to the tissue sections of meningiomasin vitro, depending on the concentration of the drug; and (b) low concentrations of suramin enhance the affinity of the EGF receptor in the tumor sections, probably by interacting with the EGF receptor molecule rather than with the EGF peptide. 6. The functional role of increased EGF receptor affinity in meningioma sections in the presence of lower concentrations of suramin remains to be determined.