Regulation of decidualization in human endometrial stromal cells through exchange protein directly activated by cyclic AMP (Epac).

INTRODUCTION: Human endometrial stromal cells (ESCs) undergo differentiation during the decidualization process. Decidualization is characterized by their enhanced production of IGF binding protein-1 (IGFBP-1), prolactin (PRL), and the forkhead transcriptional factor FOXO1, and transformation into more rounded cells, during the secretory phase of the menstrual cycle and subsequent pregnancy. Protein kinase A (PKA)-mediated cAMP signaling is crucial for this process. The present study was undertaken to examine the involvement of a mediator of cAMP signaling, exchange protein directly activated by cAMP (Epac), in decidualization of cultured ESCs. RESULTS: Treatment of ESCs with the Epac-selective cAMP analog 8-CPT-2-OMe-cAMP (CPT) had no effect on IGFBP-1, PRL, and FOXO1 mRNA expression. However, CPT potentiated IGFBP-1 and PRL expression stimulated by the PKA-selective cAMP analog N(6)-Phe-cAMP (Phe) and activated Rap1, a downstream regulator of Epac signaling. Knock-down of Epac1, Epac2, or Rap1 significantly inhibited the Phe- or Phe/CPT-induced increase in IGFBP-1 and PRL expression, as well as Rap1 activation. Furthermore, CPT enhanced IGFBP-1 and PRL expression and the morphological differentiation induced by ovarian steroids, whereas Epac1, Epac2, or Rap1 knock-down suppressed these events. CONCLUSION: These data provide evidence for the involvement of the Epac/Rap1 signaling pathway in cAMP-mediated decidualization of human ESCs.

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.

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.

A comparative study of characteristics of current-type and conventional-type cationic bactericides.

We have synthesized new polycationic bactericides, polyloxyethylene(dimethyliminio)trimethylene(dimethyliminio)ethylene dichloridel (OXD) and poly(hexamethyleneguanidine phosphate) (HEP), in order to develop more active but less skin-irritative bactericides. The effects of these bactericides on Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus (MRSA) and the degree of their irritations on skin were compared with those of a widely used low molecular-weight cationic bactericide, benzalkonium chloride (BAC), and a polycationic bactericide, poly[2-hydroxyethylene(dimethyliminio)methylene chloride] (2HYC). The minimum bactericidal concentration (MBC) of OXD for 10 min contact incubation was 16 microg/ml against P. aeruginosa, E. coli, S. marcescens and K. pneumoniae, and >1000 microg/ml against MRSA. The MBC of HEP for 10 min contact incubation was 16 microg/ml against P. aeruginosa, 32 microg/ml against E. coli and K. pneumoniae, and 64 microg/ml against S. marcescens and MRSA. Itch, edema, erythema, heat, injury, desquamation and keratinization caused by skin irritation were examined in 21 subjects by patch tests. Only one subject treated with OXD experienced edema, and one subject with HEP experienced keratinization. However, BAC caused itch in 3 subjects, edema in 1, erythema in 10 and desquamation in 2, indicating that the incidence of skin irritation of BAC was higher than that of OXD or HEP. OXD and HEP had sterilization ability similar to BAC, however, they were less skin-irritative than BAC. This indicates that OXD and HEP can be used as safe bactericides.

Acquired epileptiform opercular syndrome: a case report and results of single photon emission computed tomography and computer-assisted electroencephalographic analysis.

We report here a girl aged 5 years 3 months with cryptogenic localization-related epilepsy who showed a prolonged episode characterized by dysarthria, dysphagia, drooling and paresis of the right arm associated with almost continuous diffuse sharp-slow wave complexes during sleep. These symptoms were not directly related to seizures or to each sharp-slow wave complex revealed by examination during the video electroencephalographic (EEG) recording. The interictal single photon emission compute tomography showed a localized high perfusion area in the left posterior frontal region. The introduction of clonazepam completely controlled the clinical symptoms as well as the EEG abnormality within 2 weeks. After 4 months of remission, a similar episode recurred which was associated with aggravation of EEG. The clinical and EEG characteristics of this patient were identical to those of acquired epileptiform opercular syndrome (AEOS), a newly proposed epileptic syndrome, in which a transient operculum syndrome develops in association with continuous spike-and-wave activity during slow sleep (CSWS). Computer-assisted EEG analysis demonstrated that the epileptic EEG focus was located in the left sylvian fissure, and produced secondary bilateral synchronous sharp-slow complexes. The present study further supports the hypothesis that the electrical interference by CSWS creates bilateral opercular dysfunction through the mechanism of secondary bilateral synchrony, thus producing AEOS.

Stimulation of catecholamine synthesis in cultured bovine adrenal medullary cells by leptin.

Recently, we characterized leptin receptors in bovine adrenal medullary cells (Yanagihara et al. 2000). Here we report the stimulatory effect of leptin on catecholamine synthesis in the cells. Incubating cells with leptin (10 nM) for 20 min increased the synthesis of 14C-catecholamines from [14C]tyrosine, but not from L-3,4-dihydroxyphenyl [3-14C]alanine. The stimulation of catecholamine synthesis in the cells by leptin was associated with the phosphorylation and activation of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis. The incubation of cells with leptin resulted in a rapid activation of the mitogen-activated protein kinases (MAPKs). An inhibitor of MAPK kinase, U0126, nullified the stimulatory effect of leptin on the synthesis of 14C-catecholamines. Leptin potentiated the stimulatory effect of acetylcholine on 14C-catecholamine synthesis, whereas leptin failed to enhance the phosphorylation and activation of tyrosine hydroxylase induced by acetylcholine. These findings suggest that leptin stimulates catecholamine synthesis via the activation of tyrosine hydroxylase by two different mechanisms, i.e., one is dependent on tyrosine hydroxylase phosphorylation mediated through the MAPK pathway and the second is independent of enzyme phosphorylation.

Evidence for the degradation of maleate moiety in chlorpheniramine maleate solution using a stability-indicating HPLC method.

The degradation phenomenon of maleate moiety of chlorpheniramine maleate in solution has been demonstrated by means of a peculiar ion-pair HPLC method developed by the authors, which permits the simultaneous determination of chlorpheniramine and maleate. A commercial cough drug containing chlorpheniramine maleate was dissolved in water with m-hydroxybenzoic acid as an internal standard, and then kept for several days at room temperature. It was recognized that the maleate content in the drug solution had gradually decreased, whereas chlorpheniramine content had not decreased. A simple solution of maleic acid was also kept for several days at room temperature, and it was also recognized that the maleate content in the solution preserved at the same concentration as the solution of the commercial cough drug had gradually decreased, and the percent of remaining maleate reached zero. The degraded peaks on HPLC chromatogram were not detected at all by UV detector, and the disappearance of maleate was ascertained by GC-MS. No detectable example of maleate of chlorpheniramine maleate in a commercial cough syrup has suggested that maleate moiety of chlorpheniramine maleate decomposed to carbon dioxide.

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.

Effects of interferons on cortisol production in bovine adrenal fasciculata cells stimulated by adrenocorticotropin.

The effects of interferons (IFNs) IFN-alpha, IFN-beta and IFN-gamma on the production of cortisol in bovine adrenal fasciculata cells have been investigated. Pretreatment of the fasciculata cells with recombinant interferon-alpha-2b from man (over 300 units mL(-1)), but not with fibroblast IFN-beta or recombinant IFN-gamma from man, reduced the production of cortisol in cells stimulated with adrenocorticotropin (ACTH) (1 nM). IFN-alpha-2b inhibited ACTH-induced cortisol production in a concentration- (300-15000 units mL(-1)) and time- (2-24h) dependent manner. The inhibitory effect of IFN-alpha-2b on the production was abolished when the cells were simultaneously treated with anti-IFN-alpha antibody, and it was reversible. IFN-alpha-2b also inhibited dibutyryl cyclic AMP-induced production of cortisol but not pregnenolone-induced production. The effect of IFN-alpha-2b was not influenced by increases in external ACTH and Ca2+ concentrations and IFN-alpha-2b did not affect the ACTH-induced increase in cyclic AMP level in the cells. These results strongly suggest that IFN-alpha-2b reduces ACTH-induced production of cortisol in bovine adrenal fasciculata cells by affecting the early process of cortisol synthesis. The results also indicate that IFNs might not directly affect steroidogenesis in the adrenal cortex in-vivo, because of the requirement of high concentrations of IFN-alpha-2b for inhibition, and because of the ineffectiveness of IFN-beta and IFN-gamma.

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.

Preparation and characteristics of antibacterial sepiolite containing povidone-iodine as a useful pharmaceutical product for patient care.

Povidone-iodine (PVP-I), an antibacterial medicine, was infiltrated in sepiolite (SPL). The available iodine content in this new pharmaceutical product, a sepiolite preparation containing povidone-iodine (PVP-I-SPL), was retained at 98.9 and 98.3% during storage at 40 degrees C for 3 and 6 months, respectively. The effective removal of various gasses, including ammonia, hydrogen sulfide, ethylmercaptan and acetaldehyde, was achieved by use of PVP-I-SPL. Especially, the concentration of ammonia gas was reduced more than half after 30 min of exposure, suggesting that PVP-I-SPL has excellent ability to adsorb ammonia gas. The satisfactory antibacterial effect of PVP-I-SPL was also obtained by testing its minimum bactericidal concentration (MBC). No irritation reactions to the rabbit auricle or ophthalmic mucosa or to human skin were observed by the skin irritation test. The PVP-I-SPL preparation has bactericidal activity and gas-adsorbing ability; therefore, this pharmaceutical product should be useful for the prevention of infections and deodorization in hospital rooms and houses, as well as in nursing homes for elderly people.

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.

A possible role of nitric oxide formation in the vasodilatation of rabbit ear artery induced by a topically applied Capsaicin analogue.

Effects of topical application of a capsaicin analogue, nonylic acid vanillylamide (NVA, 0.032-10.0 mM) on the arterial diameter in the ear skin were examined in conscious rabbits using a precise dial caliper. In addition, the possibility of nitric oxide (NO) participating in a vasodilatation induced by low concentrations of NVA was tested by an NO synthase inhibitor. At the lowest concentration of NVA (0.032 mM), no significant change in the diameter was observed after external application of the NVA ointment. At concentrations of 0.32 mM or more, NVA produced a significant vasodilator response. However, at higher concentrations of 3.2 and 10.0 mM, -NVA induced substantial shrinkage in the arterial diameter and oedema formation, which was not affected by L-NAME (NG-nitro-L-arginine methyl ester, 3 mg/kg, i.v.), suggesting fluid leakage induced by oedema from the vessels might suppress the vasodilatation. Thus, the concentration-response curve for NVA was bell-shaped. NVA (0.32 mM)-induced vasodilatation was not significantly affected by atropine (1 mg/kg, i.v.) or propranolol (80 micrograms/kg, i.v.). However, the NVA-induced vasodilatation was completely suppressed by an NO synthase inhibitor, L-NAME (3 mg/kg, i.v.) which had no influence on the resting diameter, but not by an inactive stereoisomer, D-NAME (3 mg/kg, i.v.). These findings suggest that vanilloid receptor activation results in the release of sensory neuropeptides, which in turn stimulate the synthesis of endothelial NO which is responsible for the vasodilatation.

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.

Proline at position 14 of alamethicin is essential for hemolytic activity, catecholamine secretion from chromaffin cells and enhanced metabolic activity in endothelial cells.

Alamethicin is known to lyse different biological cells and to induce voltage dependent ion channels in lipid bilayers. A set of analogs with proline shifted from position 14 in the native peptide towards the N- and C-terminus was used to investigate the role of proline in: (i) alamethicin induced hemolysis of human red blood cells, (ii) stimulation of catecholamine secretion from bovine adrenal chromaffin cells and (iii) induction of metabolic activity in bovine aortic endothelial cells. Half maximal hemolytic activity was found at 30 microM alamethicin concentration, complete lysis occurred at 100 microM. The stimulation of catecholamine secretion in the presence of extracellular Ca2+ was concentration dependent up to 50 microM alamethicin. At this high concentration mild secretion was also found in the absence of Ca2+ indicating cell membrane damage. Alamethicin transiently stimulated the metabolic rate of endothelial cells in a concentration dependent mode up to 20 microM while the inhibition of metabolism at higher concentrations pointed to a toxic effect. The alamethicin analogs were completely inactive in all the biological assays. The effects correlated with a loss of dye release inducing activities on phosphatidylcholine vesicles and reduction of channel forming properties in lipid bilayers and were associated with modifications of membrane affinity rather than conformational changes of the peptides. The results indicate that proline at position 14 of the native peptide is essential for the interaction with different membrane systems.

Down-regulation of the noradrenaline transporter by interferon-alpha in cultured bovine adrenal medullary cells.

The aim of this study was to investigate the effect of long-term treatment with interferon (IFN)-alpha on the noradrenaline transporter of bovine adrenal medullary cells. Treatment of cultured adrenal medullary cells with IFN-alpha caused a decrease in uptake of [3H]noradrenaline by the cells in time (4-48 h)- and concentration (300-1,000 U/ml)-dependent manners. IFN-beta also inhibited [3H]noradrenaline uptake to a lesser extent than did IFN-alpha, whereas IFN-gamma had little effect. An anti-IFN-alpha antibody reduced the effect of IFN-alpha on [3H]noradrenaline uptake. Saturation analysis of [3H]noradrenaline uptake showed that the inhibitory effect of IFN-alpha was due to a reduction in the maximal uptake velocity (Vmax) values without altering apparent Michaelis constant (Km) values. Incubation of cells with IFN-alpha caused a translocation of protein kinase C from the soluble to the particulate fraction in the cells. The effect of IFN-alpha on [3H]noradrenaline uptake was diminished in protein kinase C-down-regulated cells. Incubation of cells with IFN-alpha for 48 h significantly reduced the specific binding of [3H]desipramine to crude plasma membranes isolated from cells. Scatchard analysis of [3H]desipramine binding revealed that IFN-alpha decreased the maximal binding (Bmax) values without any change in the dissociation constant (K(D)) values. These findings suggest that IFN-alpha suppresses the function of noradrenaline transporter by reducing the density of the transporter in cell membranes through, at least in part, a protein kinase C pathway.

Role of the Gln/Glu residues of trichocellins A-II/B-II in ion-channel formation in lipid membranes and catecholamine secretion from chromaffin cells.

Trichocellins (TC) A-II and B-II, 20-residue peptaibols isolated from conidia of the fungus Trichoderma viride, have the same sequence except for the residue at position 18. Both TCs were found to form voltage-dependent ion-channels in bilayer lipid membranes (BLM) and to induce catecholamine secretion from bovine adrenal chromaffin cells through Ca2+ influx. TC-A-II (Gln18, neutral) was more effective than TC-B-II (Glu18, charged) for macroscopic current induction in BLMs and for catecholamine secretion from chromaffin cells, suggesting that Glu18 is unfavorable for the ion-channel formation in BLMs and chromaffin cell membranes. Nevertheless, single-channel recordings indicated that TC-B-II forms larger pores with longer open lifetimes than those of TC-A-II. This indicates that the negatively charged carboxyl group of Glu at position 18 stabilizes larger pores. The effects of the negative charge of Glu18 on the activities were confirmed by the use of a TC-B-II analog containing the methyl ester of Glu18.

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.

[Effects of ginseng saponins on receptor stimulation-responses].

We investigated the effects of root of Panax ginseng C. A. Meyer on the secretion of catecholamines (CAs) from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh). In two major parts, nonsaponin and crude saponin fractions from the root, the crude saponin but not the non-saponin greatly reduced the ACh-evoked secretion. Furthermore, various purified ginseng saponins (ginsenosides) had a tendency to reduce the secretion. Most effective saponin was ginsenoside (G) RG2. GRg2 also inhibited the ACh-evoked Na+ and Ca2+ influxes into the cells. The GRg2 inhibition of the secretion was overcome by increasing the external Na+ but not Ca2+ concentrations. However, GRg2 did not affect the secretion from the cells induced by high K+, which is regarded as directly depolarizing the cell membranes and causing Ca2+ influx through voltage-sensitive Ca2+ channels. Therefore, the root of Panax ginseng contains ingredients, that is saponins, which inhibit the secretion of catecholamines from the cells stimulated by ACh. The inhibition is probably due to the antagonism of nicotinic acetylcholine receptor-operated cation channels. GRg2 had no effects on other receptor stimulation-responses but GRg3 inhibited them. These may be why the root of Panax gingseng has a variety of pharmacological effects.

Role of proline residue in the channel-forming and catecholamine-releasing activities of the peptaibol, trichosporin-B-VIa.

Trichosporin-B-VIa (TS-B-VIa) has a Pro14-kinked helical structure which is considered to be important for the formation of peptaibol-type ion-channels in lipid bilayer membranes. TS-B-VIa and its analog [Aib14]TS-B-VIa with Pro-->Aib substitution at position 14, resulting in a straight helical structure, were tested for ion-channel-forming activity in planar lipid bilayer membranes and for ability to induce catecholamine secretion from cultured bovine adrenal chromaffin cells. Voltage-dependent multi-channel conductance, which is characteristic of TS-B-VIa, was also observed for [Aib14]TS-B-VIa. In single-channel measurements, current fluctuations induced by [Aib14]TS-B-VIa had a shorter life-time and showed fewer substates than those induced by TS-B-VIa. Catecholamine secretion induced by these peptides at low concentrations is completely Ca(2+)-dependent. At high concentrations, TS-B-VIa-induced secretion was partly independent of external Ca2+, but this was not the case for the analog. The differences of behavior can be explained in terms of the differences of hydrophobicity, and magnitude of dipole moment due to the conformational changes around position 14 and the C-terminal domain caused by the Pro-->Aib substitution.