Kayoko Moroi

Novel Mutations of the Endothelin B Receptor Gene in Patients with Hirschsprung’s Disease and Their Characterization

Hirschsprung’s disease (HSCR) is a congenital intestinal disease, characterized by the absence of ganglion cells in the distal portion of the intestinal tract. Recently, three susceptibility genes have been identified in HSCR, namely theRET protooncogene, the endothelin B (ETB) receptor gene (EDNRB), and the endothelin-3 (ET-3) gene (EDN3). To investigate whether mutations inEDNRB could be related with HSCR in non-inbred populations in Japan, we examined alterations of the gene in 31 isolated patients. Three novel mutations were detected as follows: two transversions, A to T and C to A at nucleotides 311 (N104I) and 1170 (S390R), respectively, and a transition, T to C at nucleotide 325 (C109R). To analyze functions of these mutant receptors, they were expressed in Chinese hamster ovary cells. S390R mutation did not change the binding affinities but caused the decreases in the ligand-induced increment of intracellular calcium and in the inhibition of adenylyl cyclase activity, showing the impairment of the intracellular signaling. C109R receptors were proved to be localized near the nuclei as an unusual 44-kDa protein with the extremely low affinity to endothelin-1 (ET-1) and not to be translocated into the plasma membrane. On the other hand, N104I receptors showed almost the same binding affinities and functional properties as those of the wild type. Therefore, we conclude that S390R and C109R mutations could cause HSCR but that N104I mutation might be polymorphous.

Characterization of RGS5 in regulation of G protein-coupled receptor signaling.

RGS proteins (regulators of G protein signaling) serve as GTPase-activating proteins (GAPs) for G alpha subunits and negatively regulate G protein-coupled receptor signaling. In this study, we characterized biochemical properties of RGS5 and its N terminal (1-33)-deleted mutant (deltaN-RGS5). RGS5 bound to G alpha(i1), G alpha(i2), G alpha(i3), G alpha(o) and G alpha(q) but not to G alpha(s) and G alpha13 in the presence of GDP/AIF4-, and accelerated the catalytic rate of GTP hydrolysis of G alpha(i3) subunit. When expressed in 293T cells stably expressing angiotensin (Ang) AT1a receptors (AT1a-293T cells), RGS5 suppressed Ang II- and endothelin (ET)-1-induced intracellular Ca2+ transients. The effect of RGS5 was concentration-dependent, and the slope of the concentration-response relationship showed that a 10-fold increase in amounts of RGS5 induced about 20-25% reduction of the Ca2+ signaling. Furthermore, a comparison study of three sets of 293T cells with different expression levels of AT1a receptors showed that RGS5 inhibited Ang II-induced responses more effectively in 293T cells with the lower density of AT1a receptors, suggesting that the degree of inhibition by RGS proteins reflects the ratio of amounts of RGS proteins to those of activated G alpha subunits after receptor stimulation by agonists. When expressed in AT1a-293T cells, deltaN-RGS5 was localized almost exclusively in the cytosolic fraction, and exerted the inhibitory effects as potently as RGS5 which was present in both membrane and cytosolic fractions. Studies on relationship between subcellular localization and inhibitory effects of RGS5 and deltaN-RGS5 revealed that the N terminal (1-33) of RGS5 plays a role in targeting this protein to membranes, and that the N terminal region of RGS5 is not essential for exerting activities.

Analysis of two pharmacologically predicted endothelin B receptor subtypes by using the endothelin B receptor gene knockout mouse

This study was performed to clarify whether the endothelin (ET) receptor subtypes mediating two pharmacologically heterogeneous response to ETB receptor agonists in normal mice are the product(s) of a single ETB receptor gene. Vasodilator responses to sarafotoxin S6c (S6c) in the thoracic aorta and contractile responses to ET‐1 and IRL1620 in the stomach were examined in tissues from normal and ETB receptor gene knockout mice, in the absence and presence of an ETA receptor antagonist, BQ‐123, or an ETA/ETB receptor antagonist, PD142893. In the normal mouse aorta precontracted with phenylephrine, S6c (0.1100 nm) caused concentration‐dependent relaxations (pD2=8.4). BQ‐123 had no effect on these responses. However, PD142893 almost abolished the relaxations induced by 0.1300 nm S6c. In aortae taken from ETB receptor gene knockout mice, S6c up to 1 μm failed to cause relaxations, confirming that ETB receptors are involved in mediating this response. In normal mouse gastric fundus, 0.1 nm1 μm ET‐1, S6c or IRL1620 caused dose‐dependent, BQ‐123‐insensitive contractions, which were much more resistant to PD142893 than S6c‐induced relaxations of the aorta. The pD2 values for S6c in the absence and presence of PD142893 (10 μm) were 8.12±0.11 and 7.70±0.11, respectively. In the gastric fundus of the ETB receptor gene knockout mouse, S6c and IRL1620 caused no contractions. ET‐1 (0.1 nm1 μm) caused contractions sensitive to both BQ‐123 and PD142893, indicating that only ETA receptors mediate ET‐1‐induced contractions of the knockout mouse gastric fundus. Since both the PD142893‐sensitive vasodilator response of the aorta and the PD142893‐resistant contractile response of the gastric fundus to S6c were completely absent in the ETB receptor gene knockout mouse, we conclude that the two pharmacologically heterogeneous responses to S6c are mediated by receptors derived from the same ETB receptor gene.

Enzyme induction by repeated administration of tetrachlorvinphos in rats

The capability of enzyme induction in male rats was studied following the administration of tetrachlorvinphos (TCVP). A single oral dose of either 250 or 1000 mg/kg caused a more marked inhibition of true (erythrocyte) ChE than of pseudo (serum) and brain ChE activities. Dose-related increases in the activities of two hepatic microsomal enzyme activities, aminopyrine demethylase and EPN (o-ethyl O-p-nitro-phenyl phenylphosphonothioate) detoxification enzyme, were observed after 10 days of successive administration of TCVP at doses of 60 and 250 mg/kg, po. The liver to body weight ratio of rats increased on the first 7 days and then decreased on Day 10. The repeated administration of TCVP induced the enzymes responsible for the metabolism of TCVP up to 7-fold in microsomes and 1.2-fold in the supernatant fraction. The results demonstrate that the repeated application of TCVP as an insecticide may decrease its toxicity by its capacity to induce metabolizing enzymes.

A simple and sensitive colorimetric assay of benzylhydrazine

Abstract A new and simple procedure for the colorimetric determination of benzylhydrazine is described. It is based on the color development by p-dimethylaminobenzaldehyde in the presences of metaphosphoric acid and glacial acetic acid. The assay of microgram quantities of benzylhydrazine in plasma and tissues of rat treated with isocarboxazid is reported.

Comparison between procaine and isocarboxazid metabolism in vitro by a liver microsomal amidase-esterase

Abstract Characteristics of a liver microsomal amidase with isocarboxazid (ISOC) as substrate were compared to characteristics of a liver microsomal esterase with procaine (PROC) as substrate. Both amidase and esterase activities were mainly localized in the microsomal fraction with low or null activities in other fractions. Higher specific activities were found in smooth endoplasmic reticulum (s-ER) compared with rough endoplasmic reticulum (r-ER). The microsomal amidase-esterase has a pH optimum of 8.5 to 9.0 and a K m for ISOC of 0.19 mM and for PROC of 0.53 mM. On the basis of sensitivity to certain esterase inhibitors, this amidase-esterase was considered to be a carboxylesterase type; however, no divalent cations were found to activate the amidase-esterase. Since there were no significant differences between ISOC and PROC in their enzymic properties, i.e. subcellular localization, stability, pH optimum and susceptibility to inhibition, it is possible to speculate that this amidase-esterase is responsible for the metabolism of various drugs possessing amido or ester linkage.

Solubilization, purification and properties of isocarboxazid hydrolase from guinea pig liver.

Abstract An enzyme preferentially hydrolyzing isocarboxazid was solubilized from guinea pig liver with trypsin. The enzyme was purified by fractionation with ammonium sulfate, followed by chromatography on Sephadex G-200 and DEAE-cellulose. An overall purification of 92-fold was achieved, and molecular weight was estimated to be about 210,000 ± 10,000. Polyacrylamide gel electrophoresis of the final enzyme preparation showed a single band. The purified enzyme is very stable for at least 2 months in the cold (−20°). The K m and V max , of the purified enzyme for isocarboxazid are 2.1 × 10 −4 M and 3.6 nmoles product/mg protein/min respectively. No cofactors are required. The enzyme is significantly inhibited by p -chloromercuribenzoate, dibucaine, SKF 525-A and divalent metal ions, i.e. Hg 2+ , Zn 2+ , Cu 2+ and Co 2+ , but not by EDTA. The pH optimum is 8.0–8.5. The purified preparation catalyzes the enzymatic hydrolysis of β-naphthylamides of three amino acids (leucine, alanine and phenylalanine) and two synthetic penicillins, phenethicillin and propicillin, other than isocarboxazid.

Potent and selective inhibition of angiotensin AT1 receptor signaling by RGS2: Roles of its N-terminal domain

Emerging evidence indicates that R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in functional regulation in the cardiovascular system. In this study, we compared effects of three R4/B subfamily proteins, RGS2, RGS4 and RGS5 on angiotensin AT1 receptor signaling, and investigated roles of the N-terminus of RGS2. In HEK293T cells expressing AT1 receptor stably, intracellular Ca(2+) responses induced by angiotensin II were much more strongly attenuated by RGS2 than by RGS4 and RGS5. N-terminally deleted RGS2 proteins lost this potent inhibitory effect. Replacement of the N-terminal residues 1-71 of RGS2 with the corresponding residues (1-51) of RGS5 decreased significantly the inhibitory effect. On the other hand, replacement of the residues 1-51 of RGS5 with the residues 1-71 of RGS2 increased the inhibitory effect dramatically. Furthermore, we investigated functional contribution of N-terminal subdomains of RGS2, namely, an N-terminal region (residues 16-55) with an amphipathic α helix domain (the subdomain N1), a probable non-specific membrane-targeting subdomain, and another region (residues 56-71) between the α helix and the RGS box (the subdomain N2), a probable GPCR-recognizing subdomain. RGS2 chimera proteins with the residues 1-33 or 34-52 of RGS5 showed weak inhibitory activity, and either of RGS5 chimera proteins with residues 1-55 or 56-71 of RGS2 showed strong inhibitory effects on AT1 receptor signaling. The present study indicates the essential roles of both N-terminal subdomains for the potent inhibitory activity of RGS2 on AT1 receptor signaling.

Species differences in induction and inhibition of liver isocarboxazid hydrolase

Abstract The administration of phenobarbital caused approximately 1.7-fold increase in liver isocarboxazid hydrolase of rats and mice, but not guinea pigs and rabbits. These species differences in enzyme activity were also noted in the inhibition of the enzyme by SKF 525-A in in vitro experiments. In addition, this hydrolase activity was induced to the same extent in both weanling and adult rats by phenobarbital.

Comparative studies on the inhibition of liver amidases, aminopeptidase and serum cholinesterases by EPN

Abstract The hydrolysis of isocarboxazid, a monoamine oxidase inhibitor, by rat liver homogenate was inhibited after treatment of rats with EPN at doses below those which inhibited serum cholinesterase. After single doses of EPN (5–15 mg/kg), almost complete inhibition of isocarboxazid amidase occurred sooner, and persisted longer, than cholinesterase inhibition. Tissue monoamine oxidase inhibition by isocarboxazid was reduced by pretreatment with EPN in rats. Administration of EPN for 3 consecutive days at lower doses (0.5–2.5 mg/kg) caused no inhibition of cholinesterase but produced a small, consistent, inhibition of liver isocarboxazid amidase. In this study the liver isocarboxazid amidase was shown to be a more sensitive indicator of exposure to EPN intoxication than serum cholinesterase inhibition.