Kazuhiro Kitazumi

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.

Antiallergic Effect of Epinastine (WAL 801 CL) on Immediate Hypersensitivity Reactions: (I) Elucidation of the Mechanism for Histamine Release Inhibition

Epinastine caused an inhibition of histamine release from rat peritoneal mast cells induced by both antigen-antibody reaction and compound 48/80. Epinastine was similarly effective in inhibiting compound 48/80-induced histamine release not only from isolated rat peritoneal mast cells but also from rat mesenterial pieces. Also, histamine release from lung pieces obtained from actively sensitized guinea pigs after exposure to antigen challenge was markedly inhibited by epinastine. The drug was effective in inhibiting not only Ca2+ uptake into lung mast cells in actively sensitized guinea pigs but also Ca2+ release from the intracellular Ca store of rat peritoneal mast cells exposed to both compound 48/80 and substance P. No significant changes were observed in phosphodiesterase activity in rat peritoneal mast cells treated with epinastine, while adenylate cyclase activity was augmented by epinastine. Epinastine has no inhibitory effect on histamine release induced by Ca2+ or IP3 from permeabilized mast cells. However, the drug significantly and dose-dependently suppressed calmodulin activity suggesting that histamine release inhibition due to epinastine may be partly attributable to Ca(2+)-calmodulin dependent process(es). The drug caused no visible changes in thermodynamic behavior of lipids, either in order parameter or in differential scanning calorimetry, indicating that the drug has no influence on membrane fluidity.

The effects of histamine and some related compounds on conditioned avoidance response in rats

When histamine (Hi) and other agonists were applied intraventricularly, Hi caused a dose-dependent inhibition of the avoidance response in rats; its ED50 was 3.60 micrograms. 1-methylHi, 1-methylimidazole acetic acid and imidazole acetic acid which are major metabolites of Hi produced no inhibitory effect even at 50 micrograms. H1-agonists (2-methylHi and 2-thiazolylethylamine) also depressed the avoidance response; their dose-response lines run parallel to that of Hi. The depressant effects of H2-agonists (4-methylHi and dimaprit) were relatively weak; their dose-response lines were not parallel to that of Hi. When antagonists were pretreated intravenously, Hi action was clearly antagonized by diphehydramine and pyrilamine, but not by cimetidine or ranitidine. Intraventricular injection of Hi mixed with cimetidine or ranitidine did not change the effect induced by Hi alone. The avoidance response was not affected by noradrenaline, dopamine or 5-hydroxytryptamine. Although acetylcholine (ACh) suppressed the avoidance response dose-dependently, its effect was much weaker than that of Hi. Pretreatment with cholinergic blocking drugs (atropine and scopolamine) antagonized ACh action but not Hi action. From these results, it is assumed that the inhibitory effect of Hi on the avoidance response is preferentially linked to the H1-receptor. After intraventricular application of 3H-Hi, the highest radioactivity was determined in the hypothalamus.

Binding and neuropharmacological profile of zaleplon, a novel nonbenzodiazepine sedative/hypnotic

The binding properties of CL284,846 (zaleplon), a novel nonbenzodiazepine sedative/hypnotic, at benzodiazepine receptor subtypes were evaluated. Zaleplon was 14.3 times more potent at inhibiting [3H]flunitrazepam binding to membrane preparations of the cerebellum than to membrane preparations of the spinal cord. The gamma-aminobutyric acid (GABA) ratio of zaleplon was 2.07. Zaleplon produced significant increases in muscimol binding similar to those of diazepam, and it was antagonized by flumazenil. Furthermore, zaleplon showed little affinity for other receptors. Spectral analysis of the electroencephalogram (EEG) of rabbits showed that zaleplon and 3-methyl-6-[3-(trifluoromethyl) phenyl]-1,2,4,-triazolo [4,3-beta] pyridazine (CL218,872), an omega(1) receptor-selective compound (1 mg/kg, i.v., respectively), produced large increases in energy of the delta frequency band without affecting the energy of the alpha and beta frequency bands. In contrast, intravenous administration of triazolam and zopiclone increased the energy of the beta frequency band at doses of 0.1 and 2 mg/kg, respectively. In addition, the zaleplon-induced increase in the energy of the delta frequency band was antagonized by pretreatment with flumazenil (1 mg/kg, i.v.), which did not affect the spontaneous EEG alone. The present results clearly demonstrate that zaleplon is a selective full agonist of the omega(1) receptor subtype, and thus, zaleplon may induce responses closely resembling the physiological pattern of slow wave sleep.

The role of c-Jun protein in thrombin-stimulated expression of preproendothelin-1 mRNA in porcine aortic endothelial cells

Treatment of porcine aortic endothelial cells with thrombin induced a time- and dose-dependent expression of preproendothelin-1 (PPET-1) mRNA. The thrombin-induced expression of PPET-1 mRNA was markedly inhibited by calphostin C, a specific inhibitor of protein kinase C, and phorbol 12-myristate 13-acetate (TPA) induced the expression of PPET-1 mRNA dose-dependently, but 4 alpha-phorbol 12, 13-didecanoate, an inactive enantiomer of phorbol ester, had no effect on the expression of PPET-1 mRNA. On the other hand, challenge of the endothelial cells with thrombin induced a marked and time-dependent increase in the binding activity of nuclear extract to the TPA-responsive element. Furthermore, thrombin elicits synthesis of c-Jun protein as well as triggering its dephosphorylation. From these results, it is concluded that thrombin-stimulated expression of PPET-1 mRNA in porcine aortic endothelial cells can be induced not only by c-Jun protein synthesis but also by initial dephosphorylation in response to activation of protein kinase C.

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.

Antiallergic effect of epinastine (WAL 801 CL) on immediate hypersensitivity reactions: (II). Antagonistic effect of epinastine on chemical mediators, mainly antihistaminic and anti-PAF effects.

Anti-histamine and anti-PAF effects of epinastine were tested in rats, guinea pigs and rabbits. Epinastine showed a potent histamine H1-blocking effect, but the potency was slightly less than that of ketotifen in histamine-induced contraction of guinea pig ileum and histamine-induced cutaneous reactions in rats. In histamine-induced dye leakage into the nasal cavity tested in rats, the drug was slightly more potent than ketotifen and azelastine. Epinastine as well as ketotifen suppressed rabbit platelet aggregation induced by PAF at higher concentrations compared with WEB 2086, a specific PAF-antagonist. In the bronchospasm induced by PAF in guinea pigs, epinastine was more effective than ketotifen in inhibiting the bronchoconstriction, while it showed no remarkable effect on the hypotension induced by PAF. Epinastine caused a potent antagonistic effect on LTC4-induced contraction of isolated guinea pig trachea. In conclusion, the potent anti-histamine, anti-PAF and anti-LT effects of epinastine may significantly contribute to its antiallergic activity.

Involvement of the microtubular system in the endothelin-1 secretion from porcine aortic endothelial cells

The effects of certain microtubule-disrupting agents on endothelin-1 (ET-1) secretion from porcine aortic endothelial cells were studied. When endothelial cells were treated with thrombin (1 unit/mL), a significant increase in ET-1 secretion was detected in the incubation medium, while ET-1 secretion in the medium was diminished when the cells were treated simultaneously with either colchicine or vinblastine (10(-8)-10(-6) M). In such cases, however, the ET-1 content detected in the cells increased dose-dependently in accordance with the concentrations of the microtubule-disrupting agents. The intracellular accumulation of ET-1 was observed both in mitochondrial and microsomal fractions. On the other hand, thrombin produced a significant increase in polymerized tubulin content without affecting the total tubulin content. A thrombin-induced increase in the intracellular Ca2+ concentration of endothelial cells was inhibited by treatment with either colchicine or vinblastine. These results seem to indicate that the microtubular system may play an important role in ET-1 secretion from endothelial cells.

Thrombin-stimulated phosphorylation of myosin light chain and its possible involvement in endothelin-1 secretion from porcine aortic endothelial cells.

Thrombin-stimulated secretion of endothelin-1 (ET-1) from porcine aortic endothelial cells was inhibited in the presence of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). 1-(5-Chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) also prevented the thrombin-stimulated secretion of ET-1 but it enhanced the accumulation of ET-1 in the endothelial cells. When the endothelial cells were treated with thrombin, the phosphorylation of a 20-kDa protein which was identified as myosin light chain (MLC) was detected. Phosphorylation was augmented in a time-dependent manner. As in the case of ET-1 secretion, MLC phosphorylation was prevented by TMB-8, trifluoperazine, W-7 and ML-9 at the same concentrations which were effective in inhibiting the ET-1 secretion. The site of phosphorylation of MLC was identified as a serine residue. Parallel to the phosphorylation of MLC, thrombin increased the amounts of the 43- and 200-kDa proteins in the Triton-insoluble fraction; these proteins were identified as actin and myosin heavy chain, respectively. These results suggest that the MLC phosphorylation elicited by MLC kinase may facilitate the formation of filamentous myosin and actin which are probably involved in ET-1 secretion, possibly in the transport of ET-1-containing vesicles in thrombin-stimulated endothelial cells.

Structure-activity relationships of sarafotoxins: chemical syntheses of chimera peptides of sarafotoxins S6b and S6c

The three chimera peptides of sarafotoxins S6b (SRTb) and S6c (SRTc), [Thr2]SRTb, [Asn4]SRTb and [Glu9]SRTb, were synthesized chemically. From the comparisons of lethality, vasoconstrictor activity and receptor binding activity of SRTb, SRTa [( Asn13]SRTb), SRTc [( Thr2,Asn4,Glu9,Asn13]SRTb), [Thr2]SRTb, [Asn4]SRTb and [Glu9]SRTb, it appears that the Lys9 to Glu9 substitution greatly diminishes these activities while the Lys4 to Asn4 substitution does not affect them, and the Ser2 to Thr2 substitution or the Tyr13 to Asn13 substitution slightly diminishes these activities. These results suggest that the very low activities of SRTc are caused mainly by the Lys9 to Glu9 substitution, but not by the Ser2 to Thr2 substitution, which was suggested to be responsible for the weak bioactivities of SRTd [( Thr2,Ile19]SRTb).