Microbiological degradation of bile acids, further degradation of a cholic acid metabolite containing the hexahydroindane nucleus by Corynebacterium equi.

1. The further degradation of a cholic acid (I) metabolite, (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid (IIa), by Corynebacterium equi was investigated. This organism effected ring-opening and gave (4R)-4-[2alpha-(2-carboxyethyl)-3beta-(3-carboxypropionyl)-2beta-methylcyclopent-1beta-yl]valeric acid (VI). The new metabolite was isolated as its trimethyl ester and identified by partical synthesis. It was not utilized by C. equi. 2. (4R)-4[4alpha-(2-Carboxyethyl)-3aalpha-decahydro-8abeta-methyl5-oxa-6-oxoazulen-1beta-yl]valeric acid (IVa), which is a hypothetical initial oxidation product in the above degradation, was not converted by C. equi into the expected metabolite (VI), but into 3 - [2beta - [(2S) - tetrahydro - 5 - oxofur - 2 - yl] - 1beta - methyl - 5 - oxocyclopent - 1alpha - yl]-propionic acid (VIII), the structure of which was established by partial synthesis. 3. Both the possible precursors of the metabolite (VI), an isomer of the epsilon-lactone (IVa), the gamma-lactone (XIa), and the open form of these lactones, the hydroxytricarboxylic acid (V), were also not utilized by C. equi. 4. Under some incubation conditions, C. equi also converted compound (IIa) and 3-(3aalpha-hexahydro-7abeta-methyl-1,5-dioxoindan-4alpha-yl)propionic acid (IIb) into 5-methyl-4-oxo-octane-1,8-dioic acid (III), (4R)-4-(2,3,4,6,6abeta,7,8,9,9aalpha,9bbeta-decahydro-6abeta-methyl-3-oxo-1H-cyclopenta[f]quinolin-7beta-yl)valeric acid (VII) and probably a monohydroxy derivative of compound (IIa) and compound (III), respectively. 5. The possibility that an initial step in the degradation of compound (IIa) by C. equi is oxygenation of the Baeyer-Villiger type, yielding compound (IVa), is discussed. Metabolic pathways of compound (IIa) to compounds (III), (VI), (VII) and (VIII) are also considered.     

Endothelium-dependent contraction induced by substance P in canine cerebral arteries: involvement of NK1 receptors and thromboxane A2

We characterized the endothelial responses to substance P (SP) in the isolated canine cerebral artery. SP caused concentration-dependent contraction at 10(-10) - 10(-7) M and relaxation at 10(-10) and 10(-9) M, which were abolished by removal of the endothelium. The SP-induced endothelium-dependent relaxation (EDR) was suppressed, while the endothelium-dependent contraction (EDC) was increased by repeated application. The EDC induced by SP (10(-7) M) was attenuated by SR-140333 (10(-9) - 10(-7) M) and CP-99994 (10(-7) M), both NK1 antagonists, but not by SR-48968 (10(-7) M), an NK2 antagonist, or four antagonistic SP analogues (10(-6) M). The EDC induced by SP (10(-7) M) was attenuated by aspirin (10(-5) M), a cyclooxygenase inhibitor, OKY-046 (10(-5) M), a TXA2 synthetase inhibitor and ONO-3708 (10(-8) M), a TXA2 antagonist. Neurokinin A (10(-7) M) but not neurokinin B (10(-7) M) caused EDC similar to that induced by SP. In conclusion, SP induces EDC via endothelial NK1 receptors and TXA2 production in canine cerebral arteries.     

Role of intracellular Ca2+ in endothelium-dependent contraction and relaxation of rabbit intrapulmonary arteries

We examined whether Ca(2+) mobilizers induce endothelium-dependent contraction and relaxation (EDC and EDR) in isolated rabbit intrapulmonary arteries. Ionomycin (10(-7) M) and A-23187 (10(-7) M), both Ca(2+) ionophores, and thapsigargin (10(-6) M), an endoplasmic reticulum Ca(2+)-ATPase inhibitor, caused a contraction in the non-contracted preparations, and a transient relaxation followed by a transient contraction and sustained relaxation in the precontracted preparations. Endothelium-removal abolished the contraction and transient relaxation (EDC and EDR) but not sustained relaxation (endothelium-independent relaxation, EIR). In the noncontracted preparations, ionomycin-induced EDC was significantly attenuated by quinacrine (10(-5) M), manoalide (10(-6) M), both phospholipase A(2) inhibitors, indomethacin (10(-5) M) and aspirin (10(-4) M), both COX inhibitors, and ozagrel (10(-5) M), a TXA(2) synthetase inhibitor. In the precontracted arteries, EDR was markedly reduced by L-NAME (10(-4) M), a NOS inhibitor, and methylene blue (10(-6) M), a guanylate cyclase inhibitor, and was enhanced by indomethacin, aspirin and ozagrel, probably due to inhibition of EDC. ZM230487, a 5-lipoxygenase inhibitor, had no effect on EDR. EIR was not affected by L-NAME, indomethacin or ZM230487. Arachidonic acid (10(-6) M) evoked EDC sensitive to indomethacin and ozagrel. L-Arginine (10(-3) M) caused EDR sensitive to L-NAME in the ionomycin-stimulated preparations. In conclusion, Ca(2+) mobilizers cause EDC and EDR via production of TXA(2) and NO, respectively.     

Investigation of the mechanism of nicotine-induced relaxation on the sheep sphincter of Oddi

Possible mechanisms for nicotine-induced relaxation were investigated in the isolated sheep's sphincter of Oddi. Sheep's sphincter of Oddi rings were mounted in tissue bath with modified Krebs-Henseleit solution and aerated with 95% oxygen and 5% carbon dioxide. Tension was measured with isometric force transducers, and muscle relaxation was expressed as percent decrease of precontraction induced by carbachol. Nicotine (1 x 10(-5) to 3 x 10(-3) mol/L) produced concentration-dependent relaxation on sphincter of Oddi precontracted by carbachol (10(-6) mol/L). Nicotine-induced relaxation was 72.8 +/- 4.2% of precontraction with carbachol (10(-6) mol/L) (mean pD2 value, 3.76 +/- 0.05 mol/L). Nicotine-induced relaxation was not affected by N(w)-nitro L-arginine methyl ester (L-NAME) (3 x 10(-5) mol/L), methylene blue (10(-5) mol/L), indomethacin (10(-5) mol/L), hexamethonium (10(-5) mol/L), glibenclamide (10(-5) mol/L), 4-aminopyridine (10(-3) mol/L), tetraethylammonium (3 x 10(-4) mol/L), clotrimazole (10(-6) mol/L), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) (10(-6) mol/L), and anthracene-9-carboxylate (9-AC) (10(-6) mol/L), but potentiated by bupivacain (10(-5) mol/L). A calcium-antagonizing effect of nicotine was not observed. The results suggest that nicotine-induced relaxation of the sheep's sphincter of Oddi is not mediated by the release of prostaglandins, nitric oxide (NO), or a related substance; by the activation of potassium channels or chloride channels; or by the stimulation of nicotinic cholinoceptors. Potentiation of the nicotine-induced relaxation by bupivacain indicates that blockade of sodium channels may play a role in this relaxation.     

(+)-12alpha-Hydroxysophocarpine, a new quinolizidine alkaloid and related anti-HBV alkaloids from Sophora flavescens

(+)-12alpha-Hydroxysophocarpine (8), a new quinolizidine alkaloid was isolated from the roots of Sophora flavescens, together with 10 known quinolizidine alkaloids, (+)-oxymatrine (1), (+)-matrine (2), (+)-9alpha-hydroxymatrine (3), (+)-allomatrine (4), (+)-oxysophocarpine (5), (-)-sophocarpine (6), (-)-9alpha-hydroxysophocarpine (7), (+)-lehmannine (9), (-)-13,14-dehydrosophoridine (10), and (-)-anagyrine (11). Their structures were elucidated by spectroscopic methods, and the stereochemistry of 8 was confirmed by X-ray analysis. These alkaloids were tested for anti-hepatitis B virus (HBV) activity in vitro, compounds 5, 6, 9, and 10 showed significant anti-HBV activity with inhibitory potency against HBsAg secretion at 48.3-79.3% and that against HBeAg secretion at 24.6-34.6%.     

Determination of amino acid sequence responsible for suppression of bone resorption by serum calcium-decreasing factor (caldecrin).

We previously reported on the serum calcium-decreasing activity of recombinant protein factor referred to as caldecrin [Tomomura et al. (1995) J. Biol. Chem. 270, 30315-30321]. To address the mechanism of this serum calcium-decreasing activity, we investigated the effect of rat caldecrin on osteoclastic bone-resorbing activity. Wild-type caldecrin suppressed resorption pit formation by osteoclast on a dentine slice in a dose-dependent manner. The suppressive effect on the bone resorption was not affected by treatment of caldecrin with phenylmethyl sulfonyl fluoride or by use of protease-deficient mutant caldecrins. Recombinant procaldecrin (-13-239), and its fragments (-13-125), (1-111), (1-46), (47-111), and (126-239) were expressed as His-tagged thioredoxin fusion proteins and investigated for their ability to suppress bone resorption. The proform (-13-239) and fragment (-13-125) did not affect the suppressive activity, whereas fragments (1-111) and (126-239) did suppress the bone resorption. The bone-resorbing activity was also suppressed by fragment (47-111), not by fragment (1-46). Overlapping fragments (47-62), (47-79), (47-98), (56-111), (71-111), and (85-111) were compared for their suppressive activity. The fragments (47-62) and (85-111) did not affect the activity, but the other fragments suppressed the bone resorption. A synthetic peptide having the (71-79) sequence suppressed the bone resorption. These results suggest that amino acid sequence corresponding to rat caldecrin (aa 71-79) is responsible for the suppression of bone resorption by caldecrin.     

Synthesis of vasoactive intestinal peptide (VIP) via the mixed anhydride method

Porcine VIP was synthesized from three segments. The segments, VIP(1-6), VIP(7-13), and VIP(14-28), were synthesized via the Repetitive Excess Mixed Anhydride (REMA) method. The low solubility of the C-terminal segment was greatly improved by a temporary substitution of Asn28 by a beta-t-butyl aspartic acid ester. The segments VIP(1-6) and VIP(7-13) were purified by HPLC and coupled via the mixed anhydride method. The product was purified by gel filtration. VIP was synthesized from VIP(1-13) and VIP(14-28) by the same procedure. After deprotection, Met17-sulfoxide reduction, and purification by ion-exchange chromatography, the product was found to have the expected amino acid composition and biological potency. A HPLC purified sample was compared with several commercial preparations of varying purity.     

Metabolic pathways from 1 alpha,25-dihydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3-26,23-lactone. Stereo-retained and stereo-selective lactonization

The present study was carried out in order to elucidate the metabolic pathway from 1 alpha,25-(OH)2D3 to 1 alpha,25-(OH)2D3-26,23-lactone. For that purpose, we stereospecifically synthesized the vitamin D3 derivatives 1 alpha,23(S),25-(OH)3D3, 1 alpha,23(S),25(R),26-tetrahydroxyvitamin D3, and 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-lactol. The in vitro metabolism of these compounds was examined in kidney homogenates and intestinal mucosa homogenates from 1 alpha,25-(OH)2D3-supplemented chicks. The naturally occurring 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone was produced (in increasing amounts) from 1 alpha,25-(OH)2D3, 1 alpha,25(R),26-(OH)3D3, 1 alpha,23(S),25-(OH),D3, 1 alpha,23(S),25(R),26-(OH)4D3, and 23(S),25(R)-1 alpha,25-(OH)2D3-26,23-lactol. These results indicated that there are two possible metabolic pathways from 1 alpha,25-(OH)2D3 to 1 alpha,23(S),25(R),26-(OH)4D3: the major one is by way of 1 alpha,23(S),25-(OH)3D3 and the minor one is by way of 1 alpha,25(R),26-(OH)3D3. 1 alpha,23(S),25(R),26-Tetrahydroxyvitamin D3 is further metabolized to 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone via 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactol. In the course of our studies, a new biosynthetic vitamin D3 metabolite was isolated in pure form. This metabolite was identified as 23(S),25(R)-1 alpha,25-(OH)2D3-26,23-lactol by UV spectrophotometry and mass spectrometry. Furthermore, we establish in this report that the lactonization of 1 alpha,23,25,26-(OH)4D3 and 1 alpha,25-(OH)2D3-26,23-lactol occurs in a stereo-retained and stereo-selective fashion.     

Stimulation of Na+/Mg2+ antiport in rat erythrocytes by intracellular Cl-

Mg(2+) efflux from rat erythrocytes was measured in NaCl, NaNO(3), NaSCN and Na gluconate medium. Substitution of extracellular and intracellular Cl(-) with the permeant anions NO(3)(-) and SCN(-) reduced Mg(2+) efflux via Na(+)/Mg(2+) antiport. After substitution of extracellular Cl(-) with the non-permeant anion gluconate, Mg(2+) efflux was not significantly reduced. In Na gluconate medium, an influence of the changed membrane potential and intracellular pH on Mg(2+) efflux could be excluded. The results indicate the existence of Cl(-)-independent Na(+)/Mg(2+) antiport and of Na(+)/Mg(2+) antiport stimulated by intracellular Cl(-). Intracellular Cl(-), as determined by means of (36)Cl(-), was found to stimulate Na(+)/Mg(2+) antiport through a cooperative effect according to a sigmoidal kinetics. The Hill coefficient for intracellular Cl(-) amounted to 1.4-1.8, indicating that two intracellular Cl(-) may be simultaneously active. With respect to specificity, Cl(-) was most effective, followed by Br(-), J(-), and F(-). Stimulation of Na(+)/Mg(2+) antiport by intracellular Cl(-) together with intracellular Mg(2+) may play a role during deoxygenation of erythrocytes and in essential hypertension.     

Adrenomedullin and proadrenomedullin N-terminal 20 peptide induce histamine release from rat peritoneal mast cell

Adrenomedullin (ADM)-induced histamine release from rat peritoneal mast cells was investigated. We compared the ability of full-length ADM to induce histamine release to the fragments ADM-(1-25) and ADM-(22-52), as well as proadrenomedullin N-terminal 20 peptide (PAMP). ADM (10(-8) to 10(-5) M) and PAMP (10(-8) to 10(-5) M) dose-dependently increased histamine release from peritoneal mast cell preparations. The effect of ADM-(1-25) was similar to ADM, whereas ADM-(22-52) did not show any effects. These data suggest the relative importance of the ADM C-terminal fragment, which contains a six-membered ring structure. Histamine release, induced by ADM, was significantly and dose-dependently inhibited by the addition of ADM-(22-52) (10(-5) M), Ca(2+) (0.5 to 2.0 mM), and benzalkonium chloride (3 to 7 microM), a selective inhibitor of Gi type G proteins. In contrast, PAMP (10(-5) M)-induced histamine release was not inhibited by Ca(2+). These results suggest that ADM induce histamine release via a putative ADM receptor in a manner sensitive to Gi-protein function and extracellular Ca(2+) concentration, and that PAMP might produce its effect by a different mechanism than ADM.     

Overexpression of regucalcin suppresses apoptotic cell death in cloned normal rat kidney proximal tubular epithelial NRK52E cells: change in apoptosis-related gene expression

The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death and apoptosis was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 24-72 h in a medium without BS containing either vehicle, tumor necrosis factor-alpha (TNF-alpha; 0.1 or 1.0 ng/ml of medium), lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml), Bay K 8644 (10(-9)-10(-7) M), or thapsigargin (10(-9)-10(-7) M). The number of wild-type cells was significantly decreased by culture for 42-72 h in the presence of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-5) M), or thapsigargin (10(-8) or 10(-7) M). The effect of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-6) M), or thapsigargin (10(-7) M) in decreasing the number of wild-type cells cultured for 24-72 h was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with LPS (1.0 microg/ml), Bay K 8644 (10(-7) M), or thapsigargin (10(-8) M) for 24 h, and this DNA fragmentation was significantly suppressed in transfectants. DNA fragmentation in adherent cells was not seen by culture with TNF-alpha (1.0 ng/ml). TNF-alpha-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M), while LPS- or Bay K 8644-induced decrease in cell number was significantly prevented by caspase-3 inhibitor or N omega-nitro-L-arginine methylester (NAME) (10(-5) M), an inhibitor of nitric oxide (NO) synthase. Thapsigargin-induced decrease in cell number was not prevented in the presence of two inhibitors. Bcl-2 and Akt-1 mRNA levels were significantly increased in transfectants cultured for 24 h as compared with those of wild-type cells, while Apaf-1, caspase-3, or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expressions were not significantly changed in transfectants. Culture with TNF-alpha (1.0 ng/ml), LPS (1.0 microg/ml), Bay K 8644 (l0(-7) M), or thapsigargin (10(-8) M) caused a significant increase in caspase-3 mRNA levels in wild-type cells. LPS (1.0 microg/ml) significantly decreased Bcl-2 mRNA expression in the cells. Their effects on the gene expression of apoptosis-related proteins were not significantly changed in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death and apoptosis induced by various factors which their action are mediated through many intracellular signaling pathways, and that it modulates the gene expression of apoptosis-related proteins.     

Pancreastatin: characterization of biological activity

Pancreastatin (PST) (1-49) was first isolated from the porcine pancreas and can inhibit glucose-induced insulin release. PST (33-49), a PST C-terminal fragment, can also inhibit insulin release. The purpose of this study was to determine the shortest C-terminal biologically active fragment of PST, in terms of inhibition of insulin release from the isolated perfused rat pancreas. Porcine PST (1-49) and C-terminal fragments, PST (33-49), PST (35-49), PST (37-49) and PST (39-49) were synthesized by solid-phase methodology. PST (1-49), PST (33-49) and PST (35-49), at 10 nM, significantly (p less than 0.05) inhibited insulin release from isolated perfused rat pancreas: the first phase was inhibited by 15.6 +/- 2.4, 24.4 +/- 6.5 and 12.5 +/- 1.9% and the second phase, 18.9 +/- 2.7, 25.7 +/- 4.8 and 20.1 +/- 1.9% by PST (1-49), PST (33-49) and PST (35-49), respectively. PST (35-49) shows a dose-dependent inhibition of insulin release. PST (37-49) and PST (39-49) were, however, inactive. Our results indicate that the shortest C-terminal biologically active fragment is PST (35-49). These data further indicate that the C-terminal portion of PST is primarily responsible for the biological activity of PST.     

Synthesis of human pancreatic growth hormone-releasing factor and two omission analogs by segment-coupling method in aqueous solution

The human growth hormone-releasing factor (GRF) peptides [GlyS15]-GRF-(1-15) (IV), trifluoroacetyl-GRF-(20-44) (VI), trifluoroacetyl-GRF-(18-44) (VIII), and trifluoroacetyl-GRF-(16-44) (X) were synthesized by the solid-phase method. Each of the peptides was reacted with citraconic anhydride and the trifluoroacetyl group was removed by reaction with 10% hydrazine in water. The citraconylated GRF-(1-15) peptide was coupled to the (20-44), (18-44) or (16-44) peptides by reaction with silver nitrate/N-hydroxysuccinimide to give GRF-(1-15)-(20-44) (XII), GRF-(1-15)-(18-44) (XIII), or GRF-(1-44), respectively. GRF-(1-44) was shown to stimulate the release of rat growth hormone from rat pituitary cells with an ED50 = 8.8 X 10(-11)M. Peptides XII and XIII were inactive, either as agonists or as antagonists of the action of GRF-(1-44).     

Studies on synthesis and anion recognition properties of (3'-nitrobenzo)[2,3-d]-(3'-nitrobenzo)[9,10-d]-1,4,8,11-tetraazacyclotetradecane-5,7,12,14-tetraone

A novel artificial receptor, (3'-nitrobenzo)[2,3-d]-(3'-nitrobenzo)[9,10-d]-1,4,8,11-tetraazacyclotetradecane-5,7,12,14-tetraone, was designed and synthesized. The interactions of this receptor with different anions were determined by UV-vis and (1)H NMR titration experiments, and their affinity constants to different anions were compared with those of other similar/different systems. The results indicated that this receptor showed a high selective and recognitive ability for F(-) among F(-), Cl(-), Br(-), I(-), AcO(-), OH(-), and H(2)PO(4)(-). Moreover, the low energy configuration of this receptor was further determined by means of theoretical investigations.     

Nitrate absorption by barley: I. Kinetics and energetics

The absorption of NO(3) (-) by barley (Hordeum vulgare L.) was investigated by following the disappearance of NO(3) (-). The absorption was related to several parameters: NO(3) (-) and Ca(2+) concentrations, pH, and the presence of various anions. Absorption rate increased with increasing Ca(2+) concentration, reaching a maximum at approximately 5 mm Ca(2+), and was considerably inhibited by NH(4) (+). Absorption was influenced markedly by pH, and little or not at all by anions (Cl(-), Br(-), SO(4) (2-)), and was decreased by respiratory and oxidative phosphorylation inhibitors.     

An in vivo OctreoScan-negative adrenal pheochromocytoma expresses somatostatin receptors and responds to somatostatin analogs treatment in vitro.

A 52-yr-old woman presented with hypertension, elevated urinary vanillylmandelic acid, metanephrines, normetanephrines, and plasma chromogranin A (CgA), but normal urinary catecholamine levels. Abdominal ultrasonography and subsequent MRI imaging showed a 3 cm nodular lesion of the right adrenal gland also visualized by 123I-meta-iodobenzylguanidine scintigraphy consistent with a pheochromocytoma (PC). Her OctreoScan was negative. The patient underwent right adrenalectomy and histological examination showed a PC. The adrenal medulla tissue was examined for somatostatin (SRIH) receptor subtypes 1 to 5 (SSTR1 to 5) expression by RT-PCR. Cultured tumor cells were treated with either SRIH, Lanreotide (Lan), or an SSTR2 (BIM-23 120) or SSTR5 (BIM-23 206) selective agonist. CgA secretion was measured in the medium by ELISA and catecholamine levels by HPLC after 6h. Cell viability was assessed after 48h. RT-PCR analysis showed that SSTR1, 2, 3 and 4 were expressed. CgA secretion was significantly reduced by SRIH (- 80 %), Lan (- 35 %), and the SSTR2 selective agonist (- 65 %). Norepinephrine secretion was reduced by SRIH (- 66 %), Lan (- 40 %), and BIM-23 120 (- 70 %). Epinephrine and dopamine secretion was also inhibited by treatment with SRIH (- 90 % and - 93 %, respectively) and BIM-23 120 (- 33 % and - 75 %, respectively) but not by Lan. Cell viability was also significantly reduced by SRIH (- 30 %), Lan (- 10 %), and the SSTR2 selective agonist (- 20 %). The SSTR5 selective agonist did not modify either CgA and catecholamine secretion or cell viability. Our data show that SSTRs may be present in a PC although OctreoScan is negative in vivo, and that SRIH and its analogs may reduce both differentiated and proliferative functions in chromaffin cells in vitro. These findings suggest that SRIH analogs with enhanced SSTR2 affinity might be useful in the medical therapy of PC, even when an OctreoScan is negative.     

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of d-glucose at carbon 1 into d-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ((2)H(2)O). The intensity of the d-glucono-1,5-lactone band maximum at 1212 cm(-1) due to CO stretching vibration was measured as a function of time to study the kinetics of d-glucose oxidation. The extinction coefficient epsilon of d-glucono-1,5-lactone was determined to be 1.28 mM(-1)cm(-1). The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V(max), K(m), k(cat), and k(cat)/K(m)) determined by Lineweaver-Burk plot were 433.78+/-59.87U mg(-1) protein, 10.07+/-1.75 mM, 1095.07+/-151.19s(-1), and 108.74 s(-1)mM(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36+/-42.83U mg(-1) protein, 6.47+/-0.85 mM, 1187.77+/-108.16s(-1), and 183.58 s(-1)mM(-1) for V(max), K(m), k(cat), and k(cat)/K(m), respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.     

Cooling-induced contraction of guinea pig tracheal smooth muscle

Cooling of isolated guinea pig tracheal smooth muscle from 38 to 28 degrees C over 2.25 min produced a transient contraction followed by sustained relaxation. The cooling-induced contraction was blocked either by pretreatment with ouabain at concentrations of 10(-5) M or greater or by substitution of normal physiological salt solution with K-free solution. In contrast, the contractile response to cooling was not inhibited by pretreatment with phentolamine (10(-5) M), atropine (10(-5) M), tetrodotoxin (3 X 10(-7) M), diphenhydramine (10(-5) M), cromolyn sodium (10(-3) M), indomethacin (3 X 10(-7) M), nifedipine (10(-7) M), or verapamil (3 X 10(-6) M). Addition of NaHCO3 to the bath during cooling, preventing a change in pH of the physiological salt solution, did not affect the cooling-induced contraction. It is concluded that cooling of isolated guinea pig trachea produces a transient ouabain-sensitive contraction, and that the data suggest the contraction is mediated by inhibition of Na-K-ATPase in the smooth muscle rather than through neuronal stimulation or chemical mediator release.     

Muscarinic activation of Na+-dependent ion transporters and modulation by bicarbonate in rat submandibular gland acinus

To investigate the interaction between the ion channels and transporters in the salivary fluid secretion, we measured the membrane voltage (V(m)) and intracellular concentrations of Ca(2+), Na(+) ([Na(+)](c)), Cl(-), and H(+) (pH(i)) in rat submandibular gland acini (RSMGA). After a transient depolarization induced by a short application of acetylcholine (ACh; 5 muM, 20 s), RSMGA showed strong delayed hyperpolarization (V(h,ACh); -95 +/- 1.8 mV) that was abolished by ouabain. In the HCO(3)(-)-free condition, the V(h,ACh) was also blocked by bumetanide, a blocker of Na(+)-K(+)-2Cl(-) cotransporter (NKCC). In the presence of HCO(3)(-) (24 meq, bubbled with 5% CO(2)), however, the V(h,ACh) was not blocked by bumetanide, but it was suppressed by ethylisopropylamiloride (EIPA), a Na(+)/H(+) exchanger (NHE) inhibitor. Similarly, the ACh-induced increase in [Na(+)](c) was totally blocked by bumetanide in the absence of HCO(3)(-), but only by one-half in the presence of HCO(3)(-). ACh induced a prominent acidification of pH(i) in the presence of HCO(3)(-), and the acidification was further increased by EIPA treatment. Without HCO(3)(-), an application of ACh strongly accelerated the NKCC activity that was measured from the decay of pH(i) during the application of NH(4)(+) (20 mM). Notably, the ACh-induced activation of NKCC was largely suppressed in the presence of HCO(3)(-). In summary, the ACh-induced anion secretion in RSMGA is followed by the activation of NKCC and NHE, resulting an increase in [Na(+)](c). The intracellular Na(+)-induced activation of electrogenic Na(+)/K(+)-ATPase causes V(h,ACh). The regulation of NKCC and NHE by ACh is strongly affected by the physiological level of HCO(3)(-).     

Reactive oxygen species mediate arachidonic acid-induced dilation in porcine coronary microvessels

Reactive oxygen species (ROS) have been proposed to mediate vasodilation in the microcirculation. We investigated the role of ROS in arachidonic acid (AA)-induced coronary microvascular dilation. Porcine epicardial coronary arterioles (110 +/- 4 microm diameter) were mounted onto pipettes in oxygenated Krebs buffer. Vessels were incubated with vehicle or 1 mM Tiron (a nonselective ROS scavenger), 250 U/ml polyethylene-glycolated (PEG)-superoxide dismutase (SOD; an O2- scavenger), 250 U/ml PEG-catalase (a H2O2 scavenger), or the cyclooxygenase (COX) inhibitors indomethacin (10 microM) or diclofenac (10 microM) for 30 min. After endothelin constriction (30-60% of resting diameter), cumulative concentrations of AA (10(-10)-10(-5)M) were added and internal diameters measured by video microscopy. AA (10-7 M) produced 37 +/- 6% dilation, which was eliminated by the administration of indomethacin (4 +/- 7%, P < 0.05) or diclofenac (-8 +/- 8%, P < 0.05), as well as by Tiron (-4 +/- 5%, P < 0.05), PEG-SOD (-10 +/- 6%, P < 0.05), or PEG-catalase (1 +/- 4%, P < 0.05). Incubation of small coronary arteries with [3H]AA resulted in the formation of prostaglandins, which was blocked by indomethacin. In separate studies in microvessels, AA induced concentration-dependent increases in fluorescence of the oxidant-sensitive probe dichlorodihydrofluorescein diacetate, which was inhibited by pretreatment with indomethacin or by SOD + catalase. We conclude that in porcine coronary microvessels, COX-derived ROS contribute to AA-induced vasodilation.