Mitsunobu Mio

Effects of short-acting hypnotics on sleep latency in rats placed on grid suspended over water.

The present study was performed to develop a new sleep disturbance model for evaluating hypnotic potencies by placing rats on a grid suspended over water up to 1 cm under the grid surface. When rats were placed on the grid, significant increases in sleep latency and amount of wakefulness were observed compared with those of rats placed on sawdust. However, the amounts of non-rapid eye movement (non-REM) sleep and rapid eye movement (REM) sleep of rats placed on the grid were significantly decreased compared with those of rats placed on sawdust. Four short-acting hypnotics (triazolam, zopiclone, brotizolam, lormetazepam) caused significant decreases in sleep latency, and the effects of hypnotics in rats placed on the grid were more potent than those in rats placed on sawdust. In conclusion, the present model can serve as a new sleep disturbance model and may also be useful for evaluating the sleep-inducing effects of short-acting hypnotics.

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.

Role of microtubules on Ca2+ release from the endoplasmic reticulum and associated histamine release from rat peritoneal mast cells

In order to study the role of cytoskeletons on histamine release from mast cells, the effects of cytoskeleton-inhibiting agents were investigated. Since neither colchicine, vinblastine nor cytochalasin D was effective in inhibiting the IP3 formation, it is possible that neither microtubules nor microfilaments of rat peritoneal mast cells participate in the initial membrane events of the histamine release. However, both colchicine and vinblastine, but not cytochalasin D, were effective in inhibiting Ca2+ release from the intracellular Ca store. It was accordingly suggested that the microtubules, rather than microfilaments, are intimately related to the Ca2+ releasing process from the endoplasmic reticulum. The fluorescence intensity of the mast cells stained with FITC-labeled anti-tubulin antibody reflects the amount of tubulin polymers inside the cell, and colchicine treatment decreased the fluorescence intensity, indicating that colchicine is effective in depolymerizing the microtubules of rat mast cells. By contrast, the amount of tubulin polymer in the mast cells increased by compound 48/80, indicating that the rearrangement of microtubules took place in the mast cells, leading to histamine release. When permeabilized mast cells were exposed to potassium antimonate solution, microtubules attached themselves to the endoplasmic reticulum and many Ca antimonate dots were observed. From the present results, it was concluded that microtubules play an important role in the processes leading to Ca2+ release from the intracellular Ca store and subsequent histamine release.

Histamine-induced differentiation of HL-60 cells. The role of cAMP and protein kinase A.

When HL-60 cells were stimulated with histamine, a significant differentiation of the cells toward neutrophils was elicited. Histamine increased phagocytic activity, but it reduced myeloperoxidase activity of HL-60 cells. Histamine-induced differentiation in HL-60 cells was inhibited not only by H2 antagonists, such as cimetidine, ranitidine and famotidine, but also by an inhibitor of protein kinase A (A kinase), KT-5720. Histamine increased the cAMP level and A kinase activity in HL-60 cells; both increases preceded the cell differentiation. Histamine also enhanced phosphorylation of a 160 kD protein in HL-60 cells, while H2 antagonists and KT-5720 inhibited this phosphorylation. The results of the present study indicate that an activation of A kinase via H2 receptor stimulation may cause the phosphorylation of a 160 kD protein and that this phosphorylation is probably involved in the process leading to differentiation of HL-60 cells.

Development of Amygdaloid Kindling in Histidine Decarboxylase–deficient and Histamine H1 Receptor–deficient Mice

Summary:  Purpose: This study attempted to clarify the role of histamine or histamine H1 receptors in the development of amygdaloid kindling by using histidine decarboxylase (HDC)‐deficient and histamine H1 receptor (H1R)‐deficient mice.

Cortisol secretion induced by substance P from bovine adrenocortical cells and its inhibition by calmodulin inhibitors

When primary cultured bovine adrenocortical cells were treated with substance P (SP) at concentrations higher than 10 pM, cortisol output increased in a dose-dependent fashion. Although other neurokinins, such as neurokinin A (NKA) and neurokinin B (NKB), were also effective in secreting cortisol, SP was the most potent among the tested neurokinins, the potency order being SP greater than NKA much greater than NKB. This suggests that the NK-1 type receptor on adrenocortical cells may be the site of action of SP on cortisol secretion. The maximal response in SP-induced cortisol secretion was comparable to that elicited by adrenocorticotropic hormone (ACTH). SP-induced cortisol secretion was dependent upon extracellular Ca2+ concentrations, and 45Ca2+ uptake into adrenocortical cells treated with SP was long-lasting. While, in the case of ACTH, 45Ca2+ uptake proceeded transiently, the increase in intracellular cAMP content was much greater compared with that of SP. Although KT-5720, an inhibitor of protein kinase A, inhibited potently ACTH-induced cortisol secretion, SP-induced secretin was not affected by this inhibitor at all. On the other hand, calmodulin inhibitors, such as calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, were not more effective in inhibiting SP-induced cortisol secretion than secretion induced by ACTH. The present study indicates that SP may be one of the physiological stimulants of cortisol secretion and that an increase in intracellular Ca2+ concentration and the subsequent activation of calmodulin may precede SP-induced cortisol secretion.

Ultraviolet B (UVB) light-induced histamine release from rat peritoneal mast cells and its augmentation by certain phenothiazine compounds

When rat peritoneal mast cells were exposed to ultraviolet (UV) light (UVA, UVB and UVC), histamine release was evoked in a dose (intensity X time) dependent manner. The potency order of UV light in inducing the histamine release was UVC > UVB >> UVA. In this study, we focused on the effect of ultraviolet B (UVB) on histamine release from rat mast cells. The UVB-induced histamine release occurred at doses higher than 7.8 kJ m(-2), even at 4 degrees C. At a UVB dose of 18.8 kJ m(-2), where a 51.9+/-4.8% histamine release and a 58.8+/-6.8% degranulation took place, Trypan blue-stained cells accounted for 14.4+/-1.3% of the cells, and the lactate dehydrogenase (LDH) release was about 4.9+/-2.8%. This suggests that the membrane permeability to low molecular weight substances was increased by UVB exposure. The UVB-induced histamine release was inhibited by ascorbic acid at concentrations higher than 500 microM, suggesting the involvement of a radical reaction in the process. The UVB-induced histamine release was enhanced by some phenothiazine compounds, i.e., promethazine, trimeprazine, mequitazine, chlorpromazine, trifluoperazine, ethopropazine and thioridazine. We conclude that the phototoxicity of phenothiazine compounds may be due in part to an enhancement of UVB-induced histamine release from mast cells.

Sequencing and cloning of the cDNA of guinea pig eosinophil major basic protein.

Major basic protein (MBP) purified from guinea pig eosinophils elicited histamine release from rat peritoneal mast cells at concentrations higher than 3 μg/ml both in the presence and in the absence of extracellular Ca2+. After reverse‐phase high‐performance liquid chromatography, it was revealed that MBP was composed of two different proteins with quite similar molecular weights and pl values, although the amino acid compositions were slightly different. The partial amino acid sequence of one of these MBPx was determined and the primers for the polymerase chain reaction (PCR) were synthesized according to the partial amino acid sequence. Using these primers and the cDNAs obtained from guinea pig eosinophils, the PCR was carried out in order to synthesize the hybridization probe of MBP for screening the cDNA library. After screening with 8 × 103 clones, a positive clone, which encoded a full length of pre‐proMBP, was obtained. According to the sequencing data of this clone, it was revealed that pre‐proMBP was composed of 3 domains; signal peptide, acidic domain and mature MBP. The predicted pI value of mature MBP was 11.7, though that of proMBP was 7.8. The homology in the amino acid sequence between guinea pig proMBP and human proMBP was 49.4%, while guinea pig mature MBP was more homologous (58%) to human mature MBP.

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.

Substance P-induced histamine release from rat peritoneal mast cells and its inhibition by antiallergic agents and calmodulin inhibitors

Substance P-induced histamine release and Ca2+ release from the intracellular Ca store of rat peritoneal mast cells were inhibited by both antiallergic drugs and microtubule inhibiting agents. It was found that in the case of antiallergic compounds, histamine release inhibition may be intimately related to the inhibition of Ca2+ release from the intracellular store in which the microtubules play an important role. When mast cells were pretreated with either theophylline or dibutyryl cAMP, the inhibition of histamine release was closely related to the inhibition of Ca2+ release from the intracellular Ca store. Calmodulin inhibitors were also effective in inhibiting histamine release from mast cells induced by substance P. The inhibitory potencies of calmodulin inhibitors on histamine release from mast cells were closely correlated with those exerted on calmodulin activity.