Wc studied the role of non.selecttv© tyi~ (ET.) of ¢ndothelin (ET} receptor in the vaseulature, using a ligand specific to the fiT. receptor) [Olu~] • sarafotoxin S6b ([Olu'*lgR'rh}. Endothelium.containln8 rat thoracic aorta pos~s~d specific binding sites For ~)l.[OhP]SRTb. which were almost diminatcd by removal ctf the endothelium, while ET.3-sl~cific binding sites were not detected in tl' c endothdium.intaet rat aorl=. Only ET, receptor was deIected in the membranes From the endothdium of porcine thoracic aorta. [GIu'~ISR.Tb exerted only vasodilalion in rat aortic rinB. These findings indicate that El?. receptors are located on vascular endothellum and linked to vasodilation.

The hypoxia-inducible factor (HIF)-1␣ and the HIFlike factor (HLF) transcription factors are regulated at multiple levels including protein stabilization, nuclear import, and activation of transactivation, resulting in recruitment of coactivators such as the cAMP-response element-binding protein (CREB)-binding protein (CBP)/ p300 and SRC-1. During low oxygen tension these proteins modulate a network of genes that are necessary for angiogenesis, erythropopoiesis, and glycolysis. We report here that the C-terminal transactivation domain of HLF is phosphorylated on multiple sites and that phosphorylation on threonine 844 of HLF is necessary for the transcriptional activation function of the protein independently of the hypoxia condition. Importantly, using the mammalian two-hybrid system we demonstrate that a substitution of threonine 844 to an alanine decreased the enhanced transcriptional activation function mediated by CBP/p300.Low oxygen tension (hypoxia) plays an important role in diseases such as cancer, cerebral and myocardial ischemia, and chronic lung and heart diseases (reviewed in Ref. 1). Oxygen tension is also an important factor for regulation of mammalian genes that are involved in angiogenesis, vasculogenesis, glucose metabolism, and apoptosis (for a review see Ref. 1). These oxygen-sensitive genes are regulated by the hypoxia-inducible factor (HIF)-1␣, 1 (2, and reviewed in Ref.3) or the homologue protein HIF-like factor (HLF) (4), which also is termed EPAS1, HIF-2␣, HRF, or MOP2 (reviewed in Ref. 5). The mechanism of activation of oxygen-regulated genes has been determined mainly by studies of HIF-1␣; however, the important functional domains in HIF-1␣ are highly conserved in HLF (4). Moreover, HIF-1␣ appears to have a general role by conferring oxygendependent regulation to the transcription machinery in all cells, whereas HLF is cell type-restricted and plays a more specialized role, according to analysis of knock-out mice of . The N-terminal half of these proteins contains a basic helix-loop-helix domain (bHLH) and a PER-ARNT-SIM (PAS) homology domain. The basic domain is necessary for DNA-binding, and both the helix-loop-helix and the PER-ARNT-SIM homology domain are necessary for dimerization of HIF-1␣ and HLF to their partner factor, the aryl hydrocarbon nuclear translocator (ARNT) protein. Under hypoxic conditions the heterodimer binds to specific consensus DNA sequences and up-regulates genes such as erythropoietin, vascular endothelial growth factor, and glycolytic enzymes (for a review see Ref. 1).The stimulatory effect of hypoxia is caused by stabilization of the oxygen-sensitive HIF-1␣ and HLF. At normoxia these two proteins are rapidly degraded through the ubiquitin-proteasome machinery that involves the von Hippel-Lindau tumor suppressor (pVHL) E3 ubiquitin ligase complex (9 -11). pVHL interacts with the HIF proteins through a short peptide motif containing a hydroxylated proline (12-15). The prolyl hydroxylases that are necessary for post-translational modification are oxygen-depend...

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The positive inotropic effect (PIE) of endothelin (ET) isoforms, ET-1 and ET-3, was similar in that 1) the PIE was associated with prolongation of isometric contractions, 2) the maximal response was approximately 60% of that to isoproterenol (Isomax), 3) the PIE was associated with acceleration of PI hydrolysis, and 4) it was selectively antagonized by phorbol 12,13-dibutyrate. Because the concentration-response curve for ET-1 was biphasic (whereas that for ET-3 was monophasic), ET-1 had a PIE greater than ET-3 up to 10(-8) M. ET-1 induced a PIE at 3 x 10(-14) M and higher, which reached a plateau of 10-20% of Isomax at 10(-12) M (first phase); the curve became steeper at 10(-9) M and higher (second phase), achieving the maximal response at 10(-7) M to 3 x 10(-7) M. An ETA-selective antagonist, BQ-123, did not affect the PIE of ET-1 up to 10(-7) M; it abolished the first phase at 10(-6) M but did not affect the second phase. BQ-123 at 10(-8) to 10(-6) M antagonized the PIE of ET-3, [Thr2]sarafotoxin S6b, and [Glu9]sarafotoxin S6b in a concentration-dependent manner. The PIE of ET-3 was abolished by 10(-6) M BQ-123. An ETB-selective partial agonist IRL-1620 neither elicited a PIE nor affected the PIE of ET-3. These findings indicate that the PIE of ET receptor agonists on rabbit ventricular myocardium cannot be totally explained by occupancy of the ETA or ETB receptor.