Immunization with outer membrane protein A from Salmonella enterica serovar Enteritidis induces humoral immune response but no protection against homologous challenge in chickens.

Vaccination of poultry is one promising strategy to mitigate Salmonella infection in poultry and, in turn, humans as well. We evaluated the efficacy of outer membrane protein A (OmpA) as a novel vaccine candidate against Salmonella in poultry. Native OmpA purified from Salmonella enterica serovar Enteritidis was mixed with adjuvant and administered intramuscularly to 41-d-old chicks. The vaccinated birds showed no decrease in cecal excretion and tissue colonization compared with the unvaccinated birds after oral challenge with 10(9) cfu of the homologous strain at 28 d postimmunization. However, this vaccination induced an increased level of serum anti-OmpA IgG. Similar results were obtained in the replication experiments using a recombinant OmpA with single and double doses. For the development of more effective component vaccines for avian salmonellosis, the vaccine efficacy of outer membrane proteins other than OmpA and route of immunization other than parenteral administration should be evaluated with regard to protection and immune responses, including mucosal IgA.

Morphology of the cockerel's comb after androgen administration.

1. Androgen receptor (AR) expression and morphological changes in blood capillaries were investigated in the comb of cockerels, both untreated controls and after the administration of testosterone (T) or the androgen antagonist flutamide (F) for 7 weeks. 2. Twenty-six male Single Comb White Leghorn roosters were divided into T-treated, F-treated and untreated groups. Tissue sections were examined histologically, immunohistochemically, and comb blood vessel castings were analysed by scanning electron microscopy. 3. Histologically, the capillaries of the peripheral dermis layer in the T-treated group were dilated compared with controls. Many red blood cells were seen in the lumen. Although the capillary diameter in the F-treated group did not show a significant difference as compared with control, blood capillaries with small diameters were often observed, and there were few red blood cells in the capillaries. Some capillary castings were extended markedly in the T treated group, and small blood vessels were observed arborising from the extended blood capillaries. In contrast, all capillaries were slender in the F-treated group, and the casting surface was rough. 4. Immunoreactivity for AR was found in capillary endothelial cells in the peripheral dermis layer of the comb. The intensity of staining in these cells was increased in the T-treated group but was reduced in the F-treated group. 5. It is concluded that immunoreactivity for AR was found in capillary endothelial cells in the peripheral dermis layer of the rooster's comb. The intensity of staining in these cells was increased in the T-treated group but reduced in the F-treated group. Thus, the capillary endothelial cells in the peripheral dermis layer of the comb are androgen targets.

Expression of the T cell receptor Vbeta repertoire in a human T cell resistant to asbestos-induced apoptosis and peripheral blood T cells from patients with silica and asbestos-related diseases.

To explore the effects of asbestos and silica on the human immune system, an experimental model of low-dose and long-term exposure was established using a human HTLV-1-immortalized polyclonal T cell line, MT-2 (MT-2Org). MT-2 cells were continuously exposed to asbestos at a concentration (10 microg/ml) which does not induce complete cell death during short-term exposure. After acquiring resistance to CB-induced apoptosis (designated MT-2Rst), an immunological comparison was made between the MT-2Org and MT-2Rst lines in terms of T cell receptor-Vbeta (TcR-Vbeta) expression. MT-2Rst cells showed excess expression of various TcR-Vbeta, although TcR-Vbeta-overpresenting cells were characterized as undergoing apoptosis due to first contact with CB. Patients with asbestos-related diseases (ARD), such as asbestosis and malignant mesothelioma, were compared with silicosis (SIL) patients as a disease control and with healthy donors (HD). SIL and ARD not only differed in their causative materials, silica and asbestos as mineral silicates, but also in terms of complications; autoimmune disorders in SIL and tumors in ARD. ARD patients showed a restricted overpresentation of TcR-Vbeta without clonal expansion, whereas SIL patients revealed significant overpresentation of TcR-Vbeta 7.2. These experimental and clinical analyses indicate the superantigenic and dysregulation of autoimmunity-inducing effects of asbestos and silica, respectively.

Asymmetrical membrane fluidity of bovine adrenal chromaffin cells and granules and effect of trichosporin-B-VIa.

We examined membrane fluidity of bovine adrenal chromaffin cells and chromaffin granules using cationic trimethylammonium derivative of diphenylhexatriene (TMA-DPH) as a fluorescence probe. After adding TMA-DPH to the suspension of chromaffin cells and that of granules, it first bound to the outer layer of the plasma membrane of the cells and that of the granule membrane, then gradually penetrated the inner layer of each membrane and distributed to both leaflets of the respective membranes. Accompanying increases in the ratio of incorporated probe on the cytoplasmic side of the chromaffin cell membrane, its fluorescence anisotropy gradually decreased. However, in chromaffin granules, the fluorescence anisotropy gradually increased with increases in the ratio of incorporated probe. These findings suggest that the inner layer of the plasma membrane and outer layer of the granular membrane are more fluid than the corresponding side of each membrane, which is suitable for the fusion between both membranes. We also examined the effect of trichosporin-B-VIa, a fungal ion channel forming alpha-aminoisobutyric acid-containing peptide, on the fluidity of chromaffin cells using TMA-DPH. The peptide decreased the fluorescence anisotropy and increased the fluorescence intensity in the concentration range that induced Ca2+ dependent catecholamine secretion, suggesting that a change in lipid dynamics of the lipid bilayer of the plasma membrane was induced by this peptide.

Pathway for Ca2+ influx into cells by trichosporin-B-VIa, an alpha-aminoisobutyric acid-containing peptide, from the fungus Trichoderma polysporum.

Trichosporin (TS) -B-VIa, a fungal alpha-aminoisobutyric acid (Aib) -containing peptide consisting of 19 amino acid residues and a phenylalaninol, produced both 45Ca2+ influx into bovine adrenal chromaffin cells and catecholamine secretion from the cells. The secretion induced by TS-B-VIa at lower concentrations (2-5 microM) was completely dependent on the external Ca2+, while that induced by TS-B-VIa at higher concentrations (10-30 microM) was partly independent of the Ca2+. The concentration-response curves (2-5 microM) for the TS-B-VIa-induced Ca2+ influx and secretion correlated well. The TS-B-VIa (at 5 microM) -induced secretion was not antagonized by diltiazem, a blocker of L-type voltage-sensitive Ca2+ channels. The treatment of fura-2-loaded C6 glioma cells with TS-B-VIa (2-5 microM) led to an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner but the stimulatory effects of TS-B-VIa on [Ca2+]i were only slightly observed in Ca(2+)-free medium, indicating that TS-B-VIa causes Ca2+ influx from the external medium into the C6 cells. The TS-B-VIa-induced increase in [Ca2+]i in the C6 cells was not antagonized by diltiazem and by SK&F 96365, a novel blocker of receptor-mediated Ca2+ entry. High K+ increased neither [Ca2+]1 in the C6 cells nor Mn2+ influx into the cells, while TS-B-VIa increased Mn2+ influx. Also in other non-excitable cells, bovine platelets, similar results were obtained. These results strongly suggest that the mechanism of Ca2+ influx by TS-B-VIa at the lower concentrations is distinct from the event of Ca2+ influx through receptor-operated or L-type voltage-sensitive Ca2+ channels in both excitable cells (the chrornaffin cells) and non-excitable cells (the C6 cells and the platelets) and that TS-B-VIa per se may form Ca(2+)-permeable ion channels in biological membranes. On the other hand, the peptide at the higher concentrations seems to damage cell membranes.

Effects of extract and ingredients isolated from Magnolia obovata thunberg on catecholamine secretion from bovine adrenal chromaffin cells.

The crude extract of magnolia bark, an herbal drug, inhibited the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh) in a concentration-dependent manner (200-900 microg/mL). The extract also diminished the secretion induced by high K(+), which is a stimulus directly depolarizing the plasma membranes, but its inhibition was weaker than that of ACh-evoked secretion. beta-Eudesmol, honokiol, magnolol, and bornyl acetate, but not alpha- and beta-pinenes, all of which are ingredients of magnolia bark, greatly reduced ACh-evoked secretion. beta-Eudesmol and magnolol also inhibited high K(+)-induced secretion to an extent similar to that of ACh-evoked secretion. However, honokiol and bornyl acetate inhibited the secretion induced by high K(+) much less than the secretion evoked by ACh. ACh-induced Na(+) influx and ACh- or high K(+)-induced Ca(2+) influx into the cells were diminished by beta-eudesmol or honokiol. These results indicate that magnolia bark contains some effective components inhibiting the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by ACh due to the antagonism of Na(+) and Ca(2+) influxes into the cells. However, inhibition by the extract of magnolia bark seems to be attributable to honokiol and bornyl acetate. Furthermore, the results indicate that the inhibitory effect of magnolia bark may be associated with its pharmacological effect on activities of the nervous system.

Role of Ca2+/phospholipid-dependent protein kinase in catecholamine secretion from bovine adrenal medullary chromaffin cells.

The role of Ca2+/phospholipid-dependent protein kinase (protein kinase C) in catecholamine secretion from bovine adrenal medullary chromaffin cells was examined using four protein kinase C inhibitors: polymyxin B, sphingosine, staurosporine, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). For this purpose, digitonin-permeabilized chromaffin cells were used. Secretion of catecholamines from these cells was stimulated by the addition of micromolar amounts of exogenous free Ca2+. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and arachidonic acid, activators of protein kinase C, enhanced the catecholamine secretion evoked by Ca2+. But phorbol-12, 13-diacetate, a phorbol ester analog that does not activate protein kinase C, had no effect on Ca2(+)-evoked secretion. Polymyxin B at a low concentration (1 microM) abolished the enhancement of secretion by TPA or arachidonic acid without affecting the secretion evoked by Ca2+. However, polymyxin B at higher concentrations (10-100 microM) greatly reduced Ca2+-evoked catecholamine secretion. Sphingosine 10 microM-1 mM), Staurosporine (100 nM-1 microM, and H-7 (100-500 microM) inhibited TPA- or arachidonic acid-enhanced secretion but not Ca2(+)-evoked secretion. In cells in which protein kinase C was down-regulated by TPA, specific binding of [3H]phorbol-12,13-dibutyrate to the cells almost disappeared and the enhancement of secretion by TPA was no longer observed, whereas Ca2(+)-evoked secretion was maintained. These results strongly suggest that protein kinase C is not essential for the Ca2(+)-dependent catecholamine secretion from bovine adrenal chromaffin cells, but acts instead as a modulator.

Characterization of ginseng saponin ginsenoside-Rg(3) inhibition of catecholamine secretion in bovine adrenal chromaffin cells.

Since ginsenoside-Rg(3), one of the panaxadiol saponins isolated from the ginseng root, significantly inhibited the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh), the properties of ginsenoside-Rg(3) inhibition were investigated. Although ginsenoside-Rg(3) inhibited the secretion evoked by ACh in a concentration-dependent manner, it affected the secretion stimulated by high K(+) or veratridine, an activator of the voltage-sensitive Ca(2+) or Na(+) channels, only slightly. The ACh-induced Na(+) and Ca(2+) influxes into the cells were also reduced by ginsenoside-Rg(3). The inhibitory effect of this saponin on the secretion of catecholamines was not altered by increasing the external concentration of ACh or Ca(2+). The ACh-evoked secretion of catecholamines was completely restored in cells that were preincubated with 10 microM ginsenoside-Rg(3) and then incubated without the saponin, whereas secretion was not completely restored in cells that were preincubated with 30 microM of this compound. Above 30 microM ginsenoside-Rg(3) increased the fluorescence anisotropy of diphenylhexatriene in the cells. Furthermore, the inhibitory effect of ginsenoside-Rg(3) at 30 microM on the ACh-evoked secretion of catecholamines was dependent upon the preincubation time, but this was not the case at 10 microM. These results strongly suggest that ginsenoside-Rg(3) blocks the nicotinic ACh receptor-operated cation channels, inhibits Na(+) influx through the channels, and consequently reduces both Ca(2+) influx and catecholamine secretion in bovine adrenal chromaffin cells. In addition to this action, the ginsenoside at higher concentrations modulates the fluidity of the plasma membrane, which probably contributes to the observed reduction in the secretion of catecholamines.

PCBs, PCQs and PCDFs in tissues of yusho and yu-cheng patients.

All five samples of oil involved in the recent yu-cheng outbreak were heavily contaminated with PCBs, PCQs and PCDFs at levels, on the average, of 62, 20 and 0.14 ppm, respectively. The samples not only had roughly one-tenth of the contamination by PCBs, PCQs and PCDFs but also three to four times lower ratios of PCQs and PCDFs to PCBs than samples of oil involved in yusho in Japan. PCBs, PCQs and PCDFs present were all composed of similar congeners to the ones found in the yusho specimens, though some variation of the component ratios of PCBs and PCDFs were observed. On the other hand, five patients with yusho who died 1 to 10 years following poisoning had markedly higher tissue levels of PCDFs and PCQs than did a worker occupationally exposed to PCBs. Taking great differences in the process of the healing and the tissue levels of PCBs, PCQs and PCDFs between the two poisoning cases into consideration, PCDFs and PCQs--especially the former--and not PCBs are deduced to be strongly associated with the development of yusho.

PCBs, PCQs and PCDFs in blood of yusho and yu-cheng patients.

Individual blood samples obtained from yusho and yu-cheng patients who had been poisoned by ingesting contaminated cooking oils, from workers occupationally exposed to polychlorinated biphenyls (PCBs) and from unexposed individuals were analyzed for PCBs, polychlorinated quaterphenyls (PCQs) and polychlorinated dibenzofurans (PCDFs) by gas chromatography and mass spectrometry. PCBs were found in the blood of all samples. PCQs were detected in the blood of 54 of 56 living yusho patients 11 years after the outbreak, and in all yu-cheng patients 6 months following poisoning. These facts indicate that the presence of PCQs in the blood was a good mark of past ingestion of the toxic oil. In the yu-cheng cases, PCDFs as well as PCBs and PCQs were detected in all blood samples. These identified isomers have been reported to be remarkably highly toxic compounds, i.e., both the 2,3,7,8-tetrachlorinated and 2,3,4,7,8-pentachlorinated compounds are toxicologically hundreds to thousands of times more toxic than PCB. In view of the high toxicity of PCDFs found in the yu-cheng patients' blood, we must deduce that they are the primary causal agents of yusho as well as of the yu-cheng incident.

Biological effect of PCBs, PCQs and PCDFs present in the oil causing yusho and yu-cheng.

Male Sprague-Dawley rats were daily given orally for 22 days a regimen consisting of polychlorinated biphenyls (PCBs), 1 mg/day; polychlorinated quaterphenyls (PCQs), 1 mg/day; polychlorinated dibenzofurans (PCDFs), 10 micrograms/day; or a mixture of PCBs, PCQs and PCDFs (Mix-1, 1 mg + 1 mg + 10 micrograms/day). Female Cynomolgus monkeys were daily administered PCBs (5 mg), PCQs (5 mg) or a mixture (Mix-2) containing 5 mg PCBs + 20 micrograms PCDFs for 20 weeks. The PCBs, and PCDFs had the components of PCBs, PCQs and PCDFs similar to those contained in Japanese yusho oils, respectively. The PCB-treated rats and monkeys showed hepatic hypertrophy, immunosuppression and increased drug-metabolizing enzyme activities in hepatic microsomes, but were devoid of the dermal symptoms characteristic of yusho. PCQs caused an increase in drug-metabolizing enzyme activities in hepatic microsomes and immunosuppression in monkeys, but these effects were much smaller than those found with PCBs treatment. On the other hand, treatment with PCDF or Mix-1 or Mix-2 caused hypertrophy of the liver, immunosuppression, increase in drug-metabolizing enzyme activities of hepatic microsome to much greater extent than observed with PCBs, being more than 100 times as effective as PCBs. In addition PCDFs and the mixtures containing PCDFs caused weight loss and thymic atrophy. PCDFs and Mix-2-treated monkeys showed the dermal symptoms that are characteristic of yusho patients but were not observed in monkeys treated with PCBs and PCQs alone. These results suggest that PCDFs are the primary causative agent of yusho.

Effects of gentamicin on catecholamine levels in the striatum, hypothalamus, adrenal gland and vas deferens.

The effects of gentamicin, an aminoglycoside antibiotic, on changes in the catecholamine levels in the rat striatum, hypothalamus, adrenal medulla and vas deferens were studied. When rats were i.v. injected with gentamicin (1.0 mg/kg), the catecholamine content of all tissues increased 2-4 h after injection. The increases in catecholamine levels induced by gentamicin were about 1.4- to 2.3-fold those induced by saline. The effect of gentamicin was observed at 0.1 or 0.5 mg/kg, and was maximal at 2.0 mg/kg. The activity of tyrosine hydroxylase, a rate-limiting enzyme in catecholamine biosynthesis, was markedly increased in all four tissues of rats treated with gentamicin (1.0 mg/kg, 4 h). However, direct addition of gentamicin to the tyrosine hydroxylase assay medium did not affect tyrosine hydroxylase activity. These data indicate that gentamicin administration to rats increases the catecholamine content of both central and peripheral catecholamine-containing tissues. The results also suggest that the effect of gentamicin is due to an indirect activation of tyrosine hydroxylase.

Properties of ginseng saponin inhibition of catecholamine secretion in bovine adrenal chromaffin cells.

To investigate the relationship between the inhibitory effects of ginseng saponins (ginsenosides) on acetylcholine-evoked secretion of catecholamines and the structures of ginsenosides, we examined the effects of ginsenoside-Rg3 and -Rh2, which are panaxadiol saponins, 20(R)- and 20(S)-ginsenoside-Rg2, which are epimers involving the hydroxyl group at C-20 of sapogenin, and other plant saponins on the acetylcholine-evoked secretion of catecholamines from cultured bovine adrenal chromaffin cells. The ginsenoside-Rg3 (1-100 microM) and -Rh2 (10-100 microM) greatly reduced the acetylcholine-evoked secretion in a concentration-dependent manner comparable to that of ginsenoside-Rg2, a panaxatriol saponin, which was the most potent inhibitor in our previous study. 20(R)- and 20(S)-ginsenoside-Rg2 (1-100 microM) similarly reduced the acetylcholine-evoked secretion. In contrast, saikosaponin-a, glycyrrhizin and the cardiac glycosides (100 nM-100 microM), digitoxin and digoxin, had no significant inhibitory effect on catecholamine secretion. Saikosaponin-c (10-100 microM), however, had an inhibitory effect, which was less than that of ginsenoside-Rg2 and -Rg3. These results strongly suggest that the inhibitory effects of ginsenosides on the acetylcholine-evoked secretion of catecholamines from bovine adrenal chromaffin cells are a unique property of ginseng. Further, the relationship between the inhibitory effects and the structures of ginsenosides is discussed.

Cisplatin, an antineoplastic drug, inhibits catecholamine secretion from bovine adrenal chromaffin cells.

Long-term pretreatment (12-120 h) of cultured bovine adrenal chromaffin cells with cis-diamminedichloroplatinum (cisplatin, Pt(NH3)2Cl2) (33 microM), an antineoplastic drug, resulted in a decrease in the secretion of catecholamines from the cells stimulated by acetylcholine. Acetylcholine-induced 45Ca2+ influx into the cells was also reduced in the cells pretreated with cisplatin for 48 h. The concentration-response curves (3-66 microM) for cisplatin inhibition of the secretion and 45Ca2+ influx were quite similar. Pretreatment of cells with 33 microM Pt4+ or carboplatin, an analog of cisplatin, for 48 h also led to a decrease in acetylcholine-evoked secretion, but not with 33 microM Pt2+ or other metals (Au+, Au3+, Ni2+, Os3+, Pd2+, Ir3+, and Ir4+) that have properties similar to Pt4+. These results strongly suggest that in bovine adrenal chromaffin cells, cisplatin (3-66 microM) inhibits catecholamine secretion by the suppression of the Ca2+ influx into the cells evoked by acetylcholine and that the inhibitory effect of cisplatin is attributable to the tetravalent platinum ion in its molecule.

Effects of ginseng saponins on responses induced by various receptor stimuli.

We investigated the effects of four ginseng saponins, ginsenoside-Rb1, -Rg2, -Rg3 and -Ro, on the responses induced by receptor stimulation of various stimuli. Ginsenoside-Rg2 (1-100 microM) reduced the secretions of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine and gamma-aminobutyric acid but not by angiotensin II, bradykinin, histamine and neurotensin. In guinea-pig, the ginsenoside also diminished the nicotine-induced secretion of catecholamines from the adrenal chromaffin cells, but it did not affect the muscarine- and the histamine-induced ileum contractions. On the other hand, ginsenoside-Rg3 (1-100 microM) reduced not only the acetylcholine-, the gamma-aminobutyric acid- and the neurotensin-induced secretions but also, at a higher concentration (100 microM), the angiotensin II-, the bradykinin- and the histamine-induced secretions from the bovine chromaffin cells. Furthermore, the saponin (3-100 microM) significantly inhibited the muscarine- and the histamine-induced ileum contractions of the guinea-pig. Ginsenoside-Rb1 and -Ro had no marked effect on their responses. These results strongly suggest that ginsenoside-Rg2 is a potent selective blocker of nicotinic acetylcholine and gamma-aminobutyric acid receptors (ionotropic receptors) and ginsenoside-Rg3 is not only a blocker of ionotropic receptors but also an antagonist of muscarinic or histamine receptors.

Effects of SK&F 96365, an inhibitor of receptor-mediated Ca2+ entry, on Ca2+ influx and catecholamine secretion in bovine adrenal chromaffin cells.

SK&F 96365 (1-[3-(4-methoxyphenyl)propoxyl]-1-(4-methoxyphenyl)ethyl-1H- imidazole HCl) (1-50 microM), an inhibitor of receptor-mediated Ca2+ entry, reduced both acetylcholine (ACh)- and 56 mM K+ (high K+)-induced 45Ca2+ influxes and catecholamine secretion in bovine adrenal chromaffin cells. However, SK&F 96365 at lower concentrations (1-3 microM) inhibited only ACh-induced 45Ca2+ influx and secretion but not their high K(+)-induction. In Na(+)-free medium, ACh-induced 45Ca2+ influx and secretion were also considerably decreased by SK&F 96365 at low concentrations (1-3 microM). These results suggest that in the chromaffin cells, not only voltage-sensitive calcium channels but also ACh receptor-operated calcium channels at least in part contribute to the influx of Ca2+ which triggers the catecholamine secretion from the cells when the cells are exposed by ACh.

Inhibitory effect of polymyxin B on catecholamine secretion from cultured bovine adrenal medullary cells.

Incubation of cultured bovine adrenal medullary cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), was associated with increased secretion of catecholamine (CA) from the cells. Polymyxin B (PMB, 30-300 microM), a preferential inhibitor of protein kinase C, inhibited the TPA-induced secretion of CA. PMB also inhibited CA secretion induced by other secretagogues, the Ca2+ ionophore ionomycin (10 microM), 56 mM K+ or acetylcholine (ACh). Ionomycin, 56 mM K+ or ACh increased the concentration of intracellular free Ca2+ ([Ca2+]i) (measured using the fluorescent calcium indicator quin2), whereas TPA did not increase [Ca2+]i. PMB blocked the increase in [Ca2+]i induced by 56 mM K+ or ACh at concentrations similar to those inhibiting the secretion of CA. In contrast, PMB did not affect ionomycin-induced increase in [Ca2+]i. These results strongly suggest that CA secretion induced by TPA or ionomycin is mediated via activation of protein kinase C. The results further indicate that in 56 mM K+- or ACh-evoked CA secretion, PMB inhibits the secretion by blocking Ca2+ influx into the cells.

Characterization of the functional subunit combination of nicotinic acetylcholine receptors in bovine adrenal chromaffin cells.

The combination of nicotinic acetylcholine receptors (nAChRs) subunits connecting with the secretion of catecholamines in bovine adrenal chromaffin cells was pharmacologically investigated using selective agonists and antagonists for their nAChRs. The EC(50) values (microM) for the agonists that stimulate the catecholamine secretion and the rank order were as follows: nicotine (3.3)> or =1,1-dimethyl-4-phenylpiperazinium (3.5) > (E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine (14) > cytisine (23) > or =acetylcholine (25). However, because both the rank order and the EC(50) values differed considerably from those in the various subunits' combinations expressed in Xenopus oocytes or mammalian cells (e.g. alpha2beta2, alpha3beta4, alpha4beta4, etc.), we could not compare them. On the other hand, the IC(50) values (microM) for the antagonists that inhibit the secretion and the rank order were mecamylamine (0.08) > alpha-conotoxin-MII (alpha-CTX-MII) (0.71) > dihydro-beta-erythroidine (DHbetaE) (48) > alpha-bungarotoxin (alpha-BTX) (no effect). Mecamylamine is a highly selective antagonist for alpha3beta4 nAChRs, and alpha-CTX-MII and alpha-BTX are specific antagonists for alpha3beta2 and alpha7 nAChRs, respectively. DHbetaE is a selective antagonist for the alpha4beta2. It has already been shown that the mRNAs for alpha3, alpha5, alpha7 and beta4 subunits are expressed in the chromaffin cells. Therefore, the subunit combination of nAChRs associated with the catecholamine secretion from bovine adrenal chromaffin cells is suggested to be at least alpha3beta4 or alpha3beta4alpha5. Further, the results indicate that the utilization of the nicotinic agonists as selective ligands for the subunit combination of nAChRs may be not suitable for the characterization of nAChRs.

Interferon-alpha reduces catecholamine secretion from bovine adrenal chromaffin cells stimulated by acetylcholine.

A long-term pretreatment (72 h) of bovine adrenal chromaffin cells with recombinant human interferon (IFN) -alpha-2b (1500 units/ml) produced a decrease in the secretion of catecholamines from the cells stimulated by acetylcholine (ACh) (25 micromol/l) but not that with human fibloblast IFN-beta (3000 units/ml) or recombinant human IFN-gamma (3000 units/ml). IFN-alpha-2b inhibited the ACh-induced secretion in a concentration- (30-1500 units/ml) and time-dependent manner (18-72 h). The content of catecholamines in the cells treated with IFN-alpha-2b for 72 h did not change. The inhibitory effect of IFN-alpha-2b on the secretion was abolished when the cells were simultaneously treated with anti-IFN-alpha antibody, and it was overcome by the increase in the external ACh concentration. IFN-alpha-2b also inhibited ACh-induced Ca2+ influx into the cells in a concentration-dependent manner similar to that of the IFN-alpha-2b inhibiting ACh-induced secretion. On the other hand, IFN-alpha-2b failed to reduce the secretion from the cells induced by high K+. These results strongly suggest that IFN-alpha-2b reduces the ACh-induced secretion of catecholamines from bovine adrenal chromaffin cells due to modulating the gene expression of the nicotinic ACh receptor-operated cation channels rather than due to directly affecting the channels. The results further indicate that the IFN-alpha-2b inhibition may be associated with the psychiatric side effects of IFN-alpha (depression, neurasthenica and somnolence, etc.), and that immune systems may regulate the function of (autonomic) nervous systems or adrenal medulla via IFN-alpha in vivo.

Effect of polychlorinated biphenyls and polychlorinated quaterphenyls in Cynomolgus monkey (Macaca fascicularis).

Female Cynomolgus monkeys (Macaca fascicularis) with P-KC-400, Y-PCB, PY-PCB or polychlorinated quaterphenyls (PCQ) received a daily dose of 5 mg for 20 weeks, and some monkeys received a daily dose of 10 mg of Y-PCB or 0.5 mg of PCQ. The chemical compositions of the polychlorobiphenyls (PCB) used for the oral administration were as follows: P-KC-400, PCB from which polychlorodibenzofurans (PCDF) have been removed from Kanecklor 400, largely contains tri- and tetrachlorobiphenyls and no PCDF. Whereas, Y-PCB and PY-PCB, PCB with constituents similar to PCB ingested by yusho patients, largely contain penta- and hexachlorobiphenyls, in addition, PCDF of 400 ppm was present only in Y-PCB, but not in PY-PCB. There were immunosuppression, enlargement and histopathological changes of the liver (such as interstitial inflammation, and proliferation of epithelial cells of biliary duct, etc.) in the groups fed P-KC-400 and PY-PCB (free of PCDF). In the group fed Y-PCB (with PCDF), there were more apparent decreases in body weight, immunosuppression, fatty liver and histopathological changes than in the groups P-KC-400 and PY-PCB. In addition, there were hair loss, acneform eruptions, edema of the eyelid, congestion and abscess of the Meibomian gland, and cornifications of the skin, characteristic dermatological findings of yusho disease.