Angiogenesis in rat sponge implants, as determined from the concentration of hemoglobin in the sponge granuloma tissues, was gradually increased over a 14-day experimental period. The inducible cyclooxygenase COX-2 was detected in the sponge granuloma tissues at day 4 by Western blot analysis using specific mouse COX-2 antibody. Angiogenesis in the sponge implants was enhanced by daily topical injections of human recombinant basic fibroblast growth factor (bFGF) or human recombinant epidermal growth factor (EGF) (100 or 1000 ng/sponge/day) for 4 days. These treatments clearly enhanced the expression of COX-2 in the sponge granuloma tissues. In immunohistochemical studies, COX-2-positive staining was mainly observed in the endothelial cells of the neovasculature and in the fibroblasts of the granuloma capsule. Administration of the selective COX-2 inhibitor NS-398 (p.o., 3 mg/kg, 3 times a day) for 14 days significantly inhibited the angiogenesis. The angiogenesis enhanced with bFGF or EGF (day 4) was inhibited by administration of indomethacin or NS-398, both in the above regimen, and fell to the level obtained without growth factor treatment. These results suggest that COX-2 induced in the sponge granuloma tissues may participate in neovascularization through prostaglandin formation.

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Histamine at concentrations higher than 10^-9 M significantly elicited cortisol secretion from bovine adrenocortical (BAC) cells co-incubated with bovine adrenal medullary (BAM) cells, suggesting that BAM cells are responsible for histamine-induced cortisol secretion. Cortisol secretion from BAC cells coincubated with BAM cells was also elicited by both an H₁ agonist, 2-methylhistamine, and an H₂ agonist, 4-methylhistamine. However, 4-methylhistamine was much less effective than 2-methylhistamine. Histamine-induced cortisol secretion was inhibited not only by H₁ antagonists (pyrilamine and diphenhydramine) but also by H₂ antagonists (cimetidine and ranitidine). Histamine effectively increased ⁴⁵Ca uptake and IP₃ production in BAM cells. These responses were antagonized by the H₁ antagonist but not by the H₂ antagonist. Histamine-induced cortisol secretion from BAC cells co-incubated with BAM cells was inhibited by β-adrenoceptor antagonists, propranolol and timolol, as well as an NK₁-receptor antagonist, DArg¹-D-Trp^{7, 9}-Leu¹¹-substance P. These results indicate that histamine can induce cortisol secretion from BAC cells at physiological concentrations through H₁ receptors on BAM cells, and catecholamine and substance P may participate in histamine-induced cortisol secretion.

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[³H]Serotonin (5-HT) uptake by synaptosomes of rat brain was dose-dependently inhibited by nitric oxide (NO) donors such as sodium nitroprusside (SNP), 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine, 3-morpholinosydnonimine and S-nitroso-L-cysteine (NO-CYS). The inhibitory effect was blocked by reduced hemoglobin. The effect was not mimicked by ferrocyanide and ferricyanide. 8-Bromoguanosine 3'', 5''-cyclic monophosphate (8-bromo cGMP) did not affect [³H]5-HT uptake into rat cortical synaptosomes. The reduced activity of [^3H]5-HT uptake into the cortical synaptosomes pretreated with NO-CYS was partially reversed by washing the preparation after the treatment. Kinetic analysis showed that NO-CYS (100 μM) decreased the V_max value without any change in the K_m value. NO-CYS did not affect the specific binding of [³H]paroxetine, a ligand that binds to the 5-HT transporter, in membranes. NO-CYS and SNP, like iodoacetic acid and sodium cyanide, decreased the ATP content in cortical synaptosomes, but the effect on ATP content was not related to that on [³H]5-HT uptake. These findings suggest that NO inhibits reversibly [³H]5-HT uptake into rat brain synaptosomes without affecting the recognition site of the 5-HT transporter in a cGMP-independent manner, and the observed effect is not due to its metabolic effect.

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We studied the effects of (±)-4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6, 9-dimethyl6H-thieno[3, 2 f][1, 2, 4]triazolo[4, 3-a][1, 4]diazepine (Y-24180), a long-acting antagonist for platelet-activating factor (PAF), on antigen-induced eosinophil infiltration and interleukin-5 (IL-5) release in the bronchoalveolar lavage fluid (BALF) of mice. Mice actively sensitized with ovalbumin (OA) were challenged by injecting intratracheally OA 3 times every fourth day. Both the number of eosinophils and level of IL-5 were significantly increased in the BALF 24 hr after the last OA challenge. Either Y-24180 or prednisolone was orally administered once a day for 10 days beginning one day before the first OA challenge. WEB2086, another PAF antagonist, was orally administered once or twice a day for 10 days. Y24180 (0.3 3 mg/kg) suppressed the eosinophil infiltration in a dose-dependent manner and suppressed the IL-5 release at the highest dosage. Prednisolone (10 mg/kg) significantly suppressed both the eosinophil infiltration and IL-5 release. In contrast, WEB2086 affected neither the eosinophil infiltration nor IL-5 release when administered once a day (10 100 mg/kg/day). This drug never affected the IL-5 release but significantly suppressed eosinophil infiltration even when administered twice a day (30-200 mg/kg/day). These results indicate that the suppressive effect of Y-24180 on allergic pulmonary eosinophilia is due to not only to its long-lasting PAF-antagonism but also due to its suppressive effect on IL-5 release.

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γ-Carboxyglutamic acid (Gla)-containing protein, synthesized in the presence of vitamin K, has been found in atherogenic plaques, but the pharmacological effect of vitamin K on atherosclerosis is unclear. We examined whether vitamin K₂ (menatetrenone) could affect the progression of both atherosclerosis and hypercoagulability in hypercholesterolemic rabbits. Vitamin K₂ in daily doses of 1, 10 and 100 mg/kg was given with a 0.5% cholesterol diet for 10 weeks to 8 rabbits each. The plasma levels of totalcholesterol in the vitamin K₂-treated groups were clearly lower than that of the hypercholesterolemic control group. The excessive dose of vitamin K₂, even at the high dose of 100 mg/kg/day for 10 weeks, did not accelerate the progression of atherosclerosis and did not promote the coagulative tendency in the rabbits. In contrast, the vitamin K₂ treatment (1 to 10 mg/kg/day) suppressed the progression of atherosclerotic plaques, intima-thickening and pulmonary atherosclerosis, the increase of ester-cholesterol deposition in the aorta, and both the elevation in plasma factor X level and increase in Hepaplastin ® test value in the rabbits. These results indicate that the pharmacological dose of vitamin K₂ prevents both the progression of atherosclerosis and the coagulative tendency by reducing the total-cholesterol, lipid peroxidation and factor X activity in plasma, and the ester-cholesterol deposition in the aorta in hypercholesterolemic rabbits.

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In the previous study, we have demonstrated that nicotine hardly induces catecholamine release from adrenal medulla of 21-day-old rats. The present study examined the responsiveness of the adrenal chromaffin cells to acetylcholine in vitro and neostigmine and oxotremorine in vivo in 21-day-old and 8-week-old rats. As assessed by electron microscopy, the number of the chromaffin granules was markedly decreased and the content of adrenaline in adrenals was diminished significantly by oxotremorine treatment in 8-week-old rats, whereas these changes did not occur in 21-day-old rats. Morphological changes of the adrenal chromaffin cells, with respect to exocytosis, were not observed in neostigmine-treated 21-day-old rats and acetylcholine-treated adrenal slices prepared from 21-day-old rats. Catecholamine release was hardly evoked by acetylcholine in these slices as judged by measuring the catecholamine content in the medium. These results indicate that the sensitivity of the chromaffin cells to these secretagogues in 21-day-old rats is very low when compared to that in young adult rats.

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We evaluated the effects of rolipram, a selective inhibitor of phosphodiesterase (PDE) 4, on the survival of dopaminergic neurons in 13-day culture. Rolipram did not affect the survival of dopaminergic neurons in the absence of forskolin, but significantly enhanced the survival of dopaminergic neurons in the presence of 10^-5 M forskolin in a concentration-dependent manner (10^-8-10^-5 M). Rolipram also enhanced the neurotrophic effect of forskolin on total neurons including dopaminergic and nondopaminergic neurons at a high concentration (10^-5 M), but did not affect the survival of cells containing glutamate or γ-aminobutylic acid. A non-selective PDE inhibitor, 1-isobutyl-3-methylxanthine, caused a marked increase of dopaminergic neurons, whereas selective inhibitors of PDE2 and PDE3 showed far weaker effects. A PDE1 inhibitor, on the other hand, caused non-specific cell death in the presence or absence of forskolin. These findings suggest that rolipram has a potential to enhance the survival of dopaminergic neurons selectively by way of PDE4 inhibition.

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Investigation of the identity and modes of action of neurotransmitters in the mammalian central nervous system can be facilitated by simultaneous intracellular recording of membrane potential and extracellular iontophoresis of agonists and antagonists. We describe here techniques for conveniently constructing a compound microelectrode, originally described by Sonnhof (Pflugers Arch 341, 351-358, 1973), suitable for such studies. The Sonnhof electrode consists of two components, a centraxial micropipette for recording membrane potential surrounded by a cylindrical array of 6 pipettes for iontophoresis. The cylindrical array tapers coaxially and terminates in 6 contiguous, crescent-shaped orifices surrounding the terminal portion of the central pipette, 25-50 μm from the tip. Pipettes were constructed from borosilicate glass tubing of 1-mm wall thickness having a 10-mm or 16-mm outer diameter. The resistances, flux and transport numbers for iontophoresis of glycine were measured for pipettes constructed from both sizes of glass. Flux increased with increasing levels of current, and transport number decreased with increasing micropipette resistance. A spherical diffusion model points out the steep dependence of steady state concentration on diffusional distance, stressing the importance of diminishing the distance between the iontophoresis source and the recording site. This is particularly true when brief pulses of current are used.

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The aim of this study was to determine the involvement of the different α₁-adrenoceptor subtypes in the α₁-adrenoceptor mediated increase in ⁸⁶Rb⁺ efflux from rat hearts. Isolated hearts were perfused in the presence of a, β-adrenoceptor antagonist (1 μM timolol). After loading with ⁸⁶Rb⁺, the efflux was measured during α1-adrenoceptor stimulation by phenylephrine (30μM). Phenylephrine increased the ⁸⁶Rb⁺ efflux by about 30%. Pretreatment with the preferentially α_1B-adrenoceptor inhibitor chloroethylclonidine (CEC), reduced the response to phenylephrine by about 50%. The preferential α_1D-adrenoceptor inhibitor BMY 7378 inhibited the response to phenylephrine by 35%, with a pK_I=8.4 (95% C.I. 8.2-8.6). The response was sensitive to the preferential α_1A-adrenoceptor inhibitors (+)niguldipine, 5-methylurapidil (5-MU) and WB-4101 at relatively high concentrations, and 5-MU inhibited the response with a pKI = 7.7 (95% C.I. 7.2-8.0) in CEC pretreated hearts. In conclusion, the phenylephrine stimulated increase in ⁸⁶Rb⁺ efflux in the rat heart is not specifically linked to only one of the α₁-adrenoceptor subtypes, but involves the α_1B and the α_1D-adrenoceptor subtypes, and probably the α_1A-adrenoceptor subtype as well.

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An electrophysiological study was performed to investigate the effects of cis-N-[4-[4-(1, 2-benz-isozole-3-yl)-1-piperazinyl]butyl]cyclohexane-1, 2-dicarboximide hydrochloride (perospirone), a novel antipsychotic agent with high affinities for D₂/5-HT₂-receptors, on the dopaminergic (DA) neurons in the ventral tegmental area (VTA) using chloral hydrate-anesthetized rats. DA neurons and non-DA neurons in VTA were identified according to the configurations of their action potentials and firing rates. Spontaneous firing of DA neurons was dose-dependently decreased by i.v. injection of methamphetamine (MAP). Most non-DA neurons were unaffected by MAP up to 2 mg/kg, but the firing was increased with MAP in 2 of 7 neurons. Perospirone injected intravenously reversed the MAP-induced decrease in spontaneous firing of DA neurons in a dose-dependent manner. In addition, i.v. injection of perospirone also inhibited the MAP-induced increase in firing of the 2 non-DA neurons. Similarly, inhibition of spontaneous firing in DA neurons by microiontophoretically applied DA was antagonized during iontophoretic application of perospirone. However, the firing of non-DA neurons, which were insensitive to DA, was not affected by iontophoretically applied perospirone. Since the DA neurons are inhibited by DA via D₂-receptors, these findings suggest that perospirone acts on the D₂-receptors to antagonize the dopaminergic inhibition of DA neurons in VTA.

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To clarify the mode of action of aminosalicylates, which are generally used as therapeutic agents for ulcerative colitis, we investigated the effect of some of the aminosalicylates on lipid peroxidation in the large intestinal mucosa after mesenteric ischemia/reperfusion in the rat. Lipid peroxidation was assessed by measuring the level of thiobarbituric-acid-reactive substances. It was found that aminosalicylates dose-dependently inhibited the elevation of the level of thiobarbituric-acid-reactive substances in the large intestinal mucosa after ischemia/reperfusion. This effect may partly contribute to the therapeutic actions of aminosalicylates in ulcerative colitis.

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To clarify the mechanisms of duodenal ulcerogenic activity of non-steroidal anti-inflammatory drugs (NSAIDs), the effects of indomethacin (IND) on acid-stimulated duodenal bicarbonate secretion and histamine-induced duodenal ulcerogenic responses were studied in comparison with NS-398, a selective cyclooxygenase (COX)-2 inhibitor, in rats. IND (1 and 5 mg/kg, s.c.) significantly decreased duodenal bicarbonate secretion and potentiated duodenal lesion in a dose-dependent manner. On the other hand, NS-398 had no effect on these parameters. These findings suggest that duodenal ulcerogenicity of IND in the presence of histamine is mainly due to the inhibitory action on acid-stimulated bicarbonate secretion mediated by COX-1, but not by COX-2.

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In human neuroblastoma SH-SY5Y cells, treatment with immunosuppressants such as FK506, cyclosporin A or rapamycin for 4 days induced the enhancement of the 27-kDa Bcl-2α protein level. Among immunosuppressants, rapamycin has most potency. Treatment with herbimycin A or wortmannin also enhanced Bcl-2 expression, but the BB type of platelet-derived growth factor decreased the level. These results suggest that Bcl-2 expression is probably regulated by the cascade of tyrosine kinase, phosphatidylinositol 3-kinase and rapamycin-sensitive p70 S6-kinase in human neuroblastoma SH-SY5Y cells.

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The effects of concanavalin A (Con A) on liver cytokine gene expression was studied in mice. The CD4 mRNA expression in normal liver suggests the presence of CD4⁺ T cells. The administration of Con A induced interleukin (IL) 1β, IL-2 and IL-2 receptor mRNAs, which implies lymphocyte activation in the liver. Interferon-γ and tumor necrosis factor-α mRNA expressions were increased gradually. The present results showed that Con A induced liver cytokine genes. This cytokine gene induction might have been the result of lymphocyte activation in the liver.

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The distribution of mRNA for pituitary adenylate cyclase-activating polypeptide receptor (PACAP-R) was examined by in situ hybridization in rat adrenal gland. In the adrenal medulla, PACAP-R mRNA was expressed in almost all chromaffin cells without any significant expression in the cortical region. Using the reverse transcription-polymerase chain reaction, we analyzed the mRNA expression of PACAP-R splice variant forms in the adrenal gland. The predominant forms observed were the variant having a 28amino acid insert in the third intracellular loop (termed PACAP-R-hopl). As PACAP is localized in the noradrenaline secreting cells of the adrenal chromaffin cells, and stimulates catecholamine release, the present results suggest that PACAP may serve as a paracrine or autocrine regulatory factor for the chromaffin cells through PACAP-R.

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We investigated the effect of lomerizine, an anti-migraine drug, on the Ba²⁺ current through voltage-gated Ca²⁺ channels in rat pheochromocytoma (PC12) cells using a whole-cell voltage-clamp technique. Lomerizine inhibited the Ba²⁺ current with an IC₅₀ value of 1.9 μM. Lomerizine and nicardipine were > 4 times more potent than flunarizine, diltiazem, verapamil and dimetotiazine. The time course of inactivation induced by lomerizine was similar to that induced by nicardipine and flunarizine. These data indicate that lomerizine may inhibit the Ca²⁺ channel in a similar manner to nicardipine and flunarizine, and its potency is almost equal to that of nicardipine.

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