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In the inhibition of specific binding of [~:Sl]endothelins (ETs) to membranes from various tissues of rats, guinea pigs, pigs and humans, [Cys ~'-Cyst~]-ET-l( I 1-21 ), IRL 1038, has a much higher affinity for ETa receptors (Ki -6-11 nM) than for ET,., receptors (Ka = 0.4-0.7,a M). In contraction a.~ays, with ET-3 as a stimulant, 3 ,aM IRL 1038 antagonized the ET~ receptor-mediated contraction of guinea pig ileal and tracheal smooth muscle without any significant agonistie activity, but did not effect the ETA receptor-mediated contraction of rat aortic smooth muscle. IRL 1038 is, therefore, considered to be the first antagonist selective to the ETn receptor.

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

Excessive release of glutamate, from glial cells as well as neurons, is thought to be a major cause of neuronal death in ischemia. To investigate glutamate release from glial cells, we measured glutamate efflux from cultures of rat astrocytes preloaded with L-[ 3H]glutamate. Glutamate efflux was induced by either 60 mM KCI or Na-free medium, suggesting that the efflux is due to the reversed operation of a Nat-and K~-coupled glutamate uptake machinery. While investigating various neuropeptides and neurotransmitters, we found that endothelin (ET) specifically induced efflux of glutamate. Northern blot analysis and binding study showed that the FT type B receptor (ETB-A) subtype was expressed two to three times more densely than the ET type A receptor (ETA-A) in astrocytes. The ETB-R antagonist AL 2500 partially inhibited efflux of glutamate induced by 1 nM ET-1 in a concentration-dependent manner, causing a maximal inhibition of 60% at 1 1jM. However, the ETA-A antagonist BQ-123 did not cause significant inhibition even at 10 1iM. Combination of both antagonists completely inhibited the ET-1 -induced efflux. These results indicate that both receptor subtypes are involved in efflux of glutamate with a major contribution from the ETB-R. Our findings suggest that ET, which is known to be released in ischemia, may exacerbate neurodegeneration by stimulating efflux of glutamate. KeyWords: Brain ischemia-Neuronal cell death-Glutamate transporter-IRL 2500-IAL 1620. Abbreviations used: ET, endothelin; ET-R, endothelin receptor; ETA-R, endothelin type A receptor; ETB-R, endothelin type B receptor; HBSS, 20 mM HEPES (pH 7.4)-buffered salt solution; MSO, L-methionine sulfoximine; SSC, saline-sodium citrate.