Misato Takimoto

Motor discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice

To study the function of GLAST, a glutamate transporter highly expressed in the cerebellar Bergmann astrocytes, the mouse GLAST gene was inactivated. GLAST‐deficient mice developed normally and could manage simple coordinated tasks, such as staying on a stationary or a slowly rotating rod, but failed more challenging task such as staying on a quickly rotating rod. Electrophysiological examination revealed that Purkinje cells in the mutant mice remained to be multiply innervated by climbing fibres even at the adult stage. We also found that oedema volumes in the mutant mice increased significantly after cerebellar injury. These results indicate that GLAST plays active roles both in the cerebellar climbing fibre synapse formation and in preventing excitotoxic cerebellar damage after acute brain injury.

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.

Autocrine receptors for endothelins in the primary culture of endothelial cells of human umbilical vein

Human umbilical vein endothelial cells (HUVECs) in primary culture produced and secreted endothelin I (ET‐1) actively. Specific binding of [125I]ET‐1 to these cells was not detectable because of the saturation of ET receptors with endogenously produced ET‐1. However, addition of phosphoramidon, an inhibitor of ET‐converting enzyme, to the medium reduced the production of ET‐1 and thus the receptors on HUVECs were made available for exogenously added [125I]ET‐1. Binding studies using phosphoramidon‐treated HUVECs indicated the existence of a non‐isopeptide‐selective type (ETB) or ET receptor with a K d of 17 pM. This receptor is thought to be involved in ET‐induced vasodilation in autocrine manner in vivo.

Both ETA and ETB receptors are involved in mitogen‐activated protein kinase activation and DNA synthesis of astrocytes: study using ETB receptor‐deficient rats (aganglionosis rats)

Endothelin (ET) is known to be a potent mitogen in astrocytes. However, the contribution and signalling pathway of ETA and/or ETB receptor to the proliferation of astrocytes remain unclear. We investigated ET‐induced DNA synthesis in astrocytes using ETB receptor‐deficient mutant rats (aganglionosis rats: sl/sl). Western blotting with anti‐ET receptor subtype‐specific antibodies and Scatchard analysis of binding revealed that ETB receptor expression in astrocytes depended on gene dosage (+/+: sl/+: sl/sl = 2: 1: 0), whereas ETA receptor expression was unchanged among the three genotypes. ET‐1 (10 nm) stimulated [3H]thymidine incorporation and mitogen‐activated protein kinase (MAP kinase) activity not only in +/+ via both ETA and ETB receptors, but also in sl/sl astrocytes via ETA receptor with about half the extent of those observed in +/+ astrocytes. Treatment with pertussis toxin (PTX) suppressed the ET‐1‐induced increases in the incorporation and MAP kinase activity in +/+, but not sl/sl astrocytes, indicating that the ETB receptor‐, but not the ETA receptor‐, mediated pathway to DNA synthesis involves PTX‐sensitive G proteins, e.g. Gi and/or Go (Gi/o). In +/+ astrocytes, ET‐1 (1 nm) stimulated cAMP accumulation, and the ETB receptor‐selective agonist IRL 1620 (1 nm) suppressed 10 μm forskolin‐induced cAMP accumulation, suggesting Gs coupling to the ETA receptor and Gi/o coupling to the ETB receptor. On the other hand, ET‐1 did not increase cAMP accumulation in sl/sl astrocytes, although ET‐1 (1 nm) suppressed the forskolin‐induced response, suggesting Gi/o coupling to the ETA receptor. Our results suggest the possibility that the selectivity of G protein for ETA receptor is changed from Gs to Gi/o in ETB receptor‐deficient astrocytes.

Contraction of smooth muscle by activation of endothelin receptors on autonomic neurons

Endothelin receptors, predominantly of the ETB type, were localized to cell bodies, processes, and varicosities of cholinergic and adrenergic intramural autonomic neurons that were present in primary cultures of guinea pig tracheal smooth muscle. Stimulation of the neuronal ETB receptor produced a tetrodotoxin‐sensitive increase in the intracellular calcium concentration in neurons which was followed by contraction of the neighboring smooth muscle cells. These observations suggest that endothelins can induce smooth muscle contraction by means of a neuronally mediated mechanism, in addition to their direct actions on the smooth muscle.

INCREASES OF VASCULAR ENDOTHELIN-CONVERTING ENZYME ACTIVITY AND ENDOTHELIN-1 LEVEL ON ATHEROSCLEROTIC LESIONS IN HYPERLIPIDEMIC RABBITS

The aim of this study was to investigate vascular endothelin-converting enzyme activity and the tissue level of endothelin-1 in the aorta related to atherosclerotic lesions in high cholesterol diet-fed rabbits. Rabbits were fed two atherogenic diets, 0.5% and 1.5% cholesterol, and a normal diet for 16 weeks. Vascular endothelin-converting enzyme activity in the aortic arch and thoracic aorta was significantly increased (2.0-4.4 times) by the atherogenic diet as compared with the normal diet group as well as the levels of lipids and lipid peroxide in plasma were significantly increased. Tissue endothelin-1 levels in both aortas were also elevated (2.3-6.8 times), corresponding well to the increased tissue enzyme activity. In contrast, plasma endothelin-1 levels increased only in the 1.5% cholesterol diet group (2.7 times). These results indicate that the endothelin-converting enzyme activity and the corresponding endothelin-1 level in the vascular walls increase in association with the development of atherosclerotic lesions.

Effect of a novel bifunctional endothelin receptor antagonist, IRL 3630A, on guinea pig respiratory mechanics.

This study characterized the in vitro pharmacological properties of a newly developed endothelin receptor antagonist, N-butanesulfonyl-[N-(3, 5-dimethylbenzoyl)-N-methyl-3-[4-(5-isoxazolyl)-phenyl]-(D)- alanyl]-( L)-valineamide sodium salt (IRL 3630A), and its in vivo effects on respiratory mechanics were determined. IRL 3630A showed highly balanced affinities to human endothelin ET(A) and ET(B) receptors, giving apparent K(i) values of 1.5 and 1.2 nM, respectively. This compound also potently antagonized the endothelin-1-induced intracellular Ca(2+) increases in both embryonic bovine tracheal (EBTr) cells expressing endothelin ET(A) receptors and human Girardi heart (hGH) cells expressing endothelin ET(B) receptors. In guinea pig isolated tracheas having both endothelin ET(A) and ET(B) receptors, IRL 3630A greatly inhibited endothelin-1-induced contraction (pA(2)=7.1), which was partially or scarcely suppressed by the endothelin ET(A) receptor antagonist cyclo[-(D)-Trp-(D)-Asp-(L)-Pro-(D)-Val-(L)-Leu-] (BQ-123) or the endothelin ET(B) receptor antagonist N-(3, 5-dimethylbenzoyl)-N-methyl-3-(4-phenyl)-(D)-phenylalanyl-(L)-t ryptop han (IRL 2500), respectively. Bolus i.v. injections of IRL 3630A administered into anaesthetized guinea pigs at 10 and 30 microg/kg inhibited endothelin-1 (1.3 microg/kg)-induced changes in respiratory resistance and compliance in a dose dependent manner, whereas both sodium 2-benzo[1, 3]dioxol-5-yl-4-(4-methoxy-phenyl)-4-oxo-3-(3,4, 5-trimethoxy-benzyl)-but-2-enoate (an endothelin ET(A) receptor antagonist: PD 156707) and IRL 2500 at doses of up to 30 microg/kg did not affect endothelin-1-induced changes in respiratory mechanics, reflecting the in vitro results. IRL 3630A is thus an effective bifunctional endothelin receptor antagonist, and will be useful in clarifying the role of endothelin in pulmonary diseases such as bronchial asthma.

Expression, secretion, and inhibition of angiotensin-converting enzyme in cultured human bronchial epithelial cells

The purpose of this study was to determine whether angiotensin-converting enzyme is present in cultured human bronchial epithelial cells and which types of epithelial cells possess this enzyme. It is well known that serum promotes squamous differentiation of airway epithelial cell culture in vitro. We found that whole-cell homogenates of both basal (serum-untreated) and squamous-differentiated bronchial epithelial cells degraded hippuryl-L-histidyl-L-leucine, a synthetic substrate for angiotensin-converting enzyme. Analysis of RNA expression by reverse transcription-polymerase chain reaction (RT-PCR) showed the presence of mRNA for angiotensin-converting enzyme in both types of cells. In addition, we found that squamous cells secreted the enzyme into the culture medium more than basal cells did. Angiotensin-converting enzyme inhibitors (imidaprilat, enalaprilat) inhibited the enzyme activity in bronchial epithelial cells with an IC50 of 0.9-3.6 nM. Exogenously added bradykinin was degraded to bradykinin-(1-5), an inactive fragment, in the squamous cell cultures. Our data indicate the presence of angiotensin-converting enzyme in cultured human bronchial epithelial cells and also that the enzyme is secreted by squamous differentiated cells.