Chikahisa Takasaki

Presence of non-selective type of endothelin receptor on vascular endothelium and its linkage to vasodilation

We studied the role of non‐selective type (ET14) of endothelin (ET) receptor in the vasculature, using a ligand specific to the ET14 receptor, [Glu9]‐sarafotoxin S6b ([Glu9]SRTb). Endothelium‐containing rat thoracic aorta possessed specific binding sites for 125I‐[Glu9]SRTb, which were almost eliminated by removal of the endothelium, while ET‐3‐specific binding sites were not detected in the endothelium‐intact rat aorta. Only ET14 receptor was detected in the membranes from the endothelium of porcine thoracic aorta. [Glu9]SRTb exerted only vasodilation in rat aortic ring. These findings indicate that ET14 receptors are located on vascular endothelium and linked to vasodilation.

Sarafotoxins S6: several isotoxins from Atractaspis engaddensis (burrowing asp) venom that affect the heart.

Three isotoxins, named sarafotoxins S6a1, S6b and S6c, with strong cardiotoxic activity were isolated from the venom of a snake, Atractaspis engaddensis. All three sarafotoxins are homologous peptides (four or less than four residue replacements) consisting of 21 amino acid residues. Their structure and activity are novel among snake venom components.

The Transcriptional Activation Function of the HIF-like Factor Requires Phosphorylation at a Conserved Threonine*

The hypoxia-inducible factor (HIF)-1α and the HIF-like 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.

Multiple subtypes of endothelin receptors in porcine tissues: characterization by ligand binding, affinity labeling and regional distribution

To clarify the existence and the distribution of endothelin (ET) receptor subtypes, we have examined the pharmacological properties and the molecular weight (Mr) of 125I-ET-1 and 125I-ET-3 binding sites in various tissues of pigs. ET-1 and ET-2 showed almost identical potencies in displacing the bound 125I-ET-1 in all the tissues examined. ET-3, sarafotoxin S6b (SRT-b) and sarafotoxin S6c (SRT-c) displaced the 125I-ET-1 with the same sensitivity as ET-1 (IC50 = 0.1-1.4 nM) in brain, kidney, liver and adrenal, whereas the three peptides showed very weak competition (IC50 = 40-500 nM) against 125I-ET-1 binding in cardiac atria, aorta, lung, stomach and uterus. The computer analyses of the binding data suggested the presence of high (Kd1 = 0.04-0.29 nM) and low (Kd2 = 60-190 nM) affinity binding sites for ET-3 and SRT-b in lung and stomach. 125I-ET-3 bound to the high affinity sites in lung and stomach was displaced by ET/SRT isopeptides almost equipotently. Two proteins with Mr of 47,000 and 35,000 were affinity-labeled with 125I-ET-1 in cerebellum, while a protein with Mr of 123,000, in addition to the two proteins, was predominantly labeled in lung. The above findings indicated that two distinct subclasses of ET receptors, namely, ET-1-specific and ET/SRT family-common receptors were distributed in various proportions in mammalian tissues, and suggested that their molecular forms are also different.

HIF-1α-prolyl hydroxylase: molecular target of nitric oxide in the hypoxic signal transduction pathway

Abstract We have investigated inhibitory mechanisms of hypoxic activation of HIF-1α by nitric oxide (NO). Using a Hep3B cell-derived cell line, HRE7 cells, we found that the inhibition of HIF-1α activity by NO requires a substantial amount of oxygen, albeit at a lower level. We further investigated the effect of NO on the binding activity of the von Hippel–Lindau tumor suppressor protein (pVHL) to the N-terminal activation domain (NAD) overlapping the oxygen-dependent degradation domain (ODD) of HIF-1α, because this reaction involves prolyl hydroxylation in NAD that requires oxygen. Although we could not detect any binding activity when NAD was incubated with whole cell extracts from cells treated with CoCl 2 or desferrioxamine, the binding capacity was manifested when Hep3B cells were treated together with NO. This activation was also observed when whole cell extracts from CoCl 2 -treated cells were incubated with NO. The prolyl hydroxylase from Hep3B cells treated with CoCl 2 was partially purified about 80-fold, and several enzymatic properties were examined. The enzyme required ferrous ion and 2-oxoglutaric acid. Strong activation of the prolyl hydroxylase by NO was observed without further addition of ferrous ion.

Amino acid sequences of eight phospholipases A2 from the venom of Australian king brown snake, Pseudechis australis

The amino acid sequences of eight phospholipases A2 (Pa-1G, Pa-3, Pa-5, Pa-9C, Pa-10A, Pa-12A, Pa-12C and Pa-15) which had been isolated from the venom of Australian king brown snake (Pseudechis australis) were elucidated. Pa-1G, Pa-3 and Pa-15 showed micro-heterogeneity at the 103rd position and Pa-5 was separated into two components, Pa-5a ([Pro-18 and Tyr-61]Pa-5) and Pa-5b ([ Ser-18 and Phe-61]Pa-5). All the phospholipase A2 molecules except Pa-1Ga and Pa-1Gb which lack the 118th residue, consisted of a single chain of 118 amino acid residues including 14 half-cystine residues and all the common residues among phospholipases A2 from other sources. From comparison studies, Asp-50, Lys-58 and Asp-90 seem to be important for the toxicity, and we propose that the domain for the presynaptic toxicity consists of seven hydrophilic residues, i.e. Arg-43, Lys-46, Asp-50, Glu-54, Lys-58, Asp-90 and Glu-94.

Purification and properties of several phospholipases A2 from the venom of Australian king brown snake (Pseudechis australis)

Thirteen isoenzymes of phospholipases A2 were purified from the venom of Australian king brown snake, Pseudechis australis. They (except phospholipase A2 Pa-9C) showed normal properties of snake venom phospholipases A2; the apparent mol. wts were about 13,000, the optimum pH values were around 8, calcium ion was indispensable for the enzymatic activity and the optimum calcium ion concentrations were more than 5 mM. Phospholipase A2 Pa-9C had a lag period at the initial stage of the enzymatic reaction. The enzymatic activities determined by the titration method using 1,2-dipalmitoylglycerophosphocholine as a substrate at 37 degrees C were 10,500 units/mg for Pa-1G and 75 units/mg for Pa-13. The lethal activities measured by i.v. injections in mice were 0.09 micrograms/g body wt for Pa-5 and 6.8 micrograms/g body wt for Pa-13. The lethal activity correlates with the enzymatic activity (correlation coefficient of 0.92), and both activities showed no relationship to the basicity of the enzyme. Pa-1G is the first acidic phospholipase A2 (pI = 6.4) with high neurotoxicity (0.13 micrograms/g body wt).

Neuromuscular effects of three phospholipases A2 from the venom of the Australian king brown snake Pseudechis australis

Three single chain phospholipases A2 (Pa-10A, Pa-11 and Pa-13) isolated from Australian king brown snake (Pseudechis australis) venom were tested for effects on neuromuscular transmission and muscle contractility on chick biventer cervicis and mouse diaphragm preparations. At 1 microgram/ml (about 85 nM) and higher, Pa-10A and Pa-11 reduced responses of both preparations to indirect stimulation in a concentration-dependent manner. Responses to direct muscle stimulation were generally reduced more slowly. Pa-11 also decreased membrane potentials of chick biventer muscle fibres and caused damage visible by light microscopy. Pa-13, which is about 50 times less active as a phospholipase A2, was also less potent in its pharmacological effects: 20 micrograms Pa-13 per ml were required to reduce responses of either preparation. The phospholipases A2 also caused a slow contracture. After block of responses to nerve stimulation, responses of the chick preparation to acetylcholine, carbachol and KCl could be obtained, although they were smaller than control and highly variable in different preparations. It is concluded that Pa-10A and Pa-11 produce muscle paralysis by reducing acetylcholine release and by a direct blockade of muscle fibre contractility. Pa-13 has similar, though less pronounced, activities.

Isolation and properties of two phospholipases A2 from the venom of an Australian elapid snake (Pseudechis australis).

Two phospholipases A2 (Pa-11 and Pa-13) were purified from the venom of an Australian elapid snake (subfamily Acanthophiinae) Pseudechis australis (king brown snake) by chromatography on CM-cellulose CM-52 followed by gel filtration on a Sephadex G-75 column. The apparent molecular weights of the two phospholipases A2 (Pa-11 and Pa-13) were 14,000 and 13,500, respectively, by gel filtration analysis on a Sephadex G-75 column. Each enzyme molecule consists of a single polypeptide chain of 118 amino acid residues. The isoelectric points of Pa-11 and Pa-13 were 10.5 and 10.0, respectively. The optimum pH values of Pa-11 and Pa-13 for hydrolysis of egg-yolk phosphatidylcholine were 7.8 and 7.5, respectively. Pa-11 was lethal to mice (LD50 0.23 micrograms/g body weight), whereas Pa-13 showed no lethal activity at a dose level of 7.4 micrograms/g mouse. Each enzyme was inactivated by reaction with p-bromophenacylbromide on the sole histidine residue (Pa-11) or on one of the two histidine residues (Pa-13). Oxidation of the tryptophan residues in Pa-11 and Pa-13 with N-bromosuccinimide led to a decrease in the phospholipase A activity. A complete loss of both enzymic and lethal activities of Pa-11 was observed upon oxidation of one of the two tryptophan residues of the molecule.

Structure-activity relationship in vasoconstrictor effects of sarafotoxins andendothelin-1

Sarafotoxins (SRTa, SRTb and SRTc) as well as endothelin‐1 (ET‐1) produced vasoconstrictions in rat thoracic aorta, rat isolated perfused mesentery and pithed rat in various of extents. The potency was ET‐1 > SRTb > SRTa > SRTc at lower doses, but SRTb > ET‐1 > SRTa > SRTc at higher doses. [Nitrophenylsulfenylated Trp21]SRTb and SRTb(1‐19) caused no vasoconstriction. Either the reduction and carboxymethylation of Cys residues, the destruction of the intramolecular loop or the production of the non‐natural disulfide bond, eliminated the constrictor activity. These results indicate that Trp21 and the intramolecular loop structure are essential, and Lys9 and Tyr13 may play some important roles for the vasoconstrictor activity of these peptides.