Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line

Addition of either vasoactive intestinal peptide (VIP) or the Ca2+ ionophore, A23187, to confluent monolayers of the T84 epithelial cell line derived from a human colon carcinoma increased the rate of 86Rb+ or 42K+ efflux from preloaded cells. Stimulation of the rate of efflux by VIP and A23187 still occurred in the presence of ouabain and bumetanide, inhibitors of the Na+,K+-ATPase and Na+,K+,Cl- cotransport, respectively. The effect of A23187 required extracellular Ca2+, while that of VIP correlated with its known effect on cyclic AMP production. Other agents which increased cyclic AMP production or mimicked its effect also increased 86Rb+ efflux. VIP- or A23187-stimulated efflux was inhibited by 5 mM Ba2+ or 1 mM quinidine, but not by 20 mM tetraethylammonium, 4 mM 4-aminopyridine, or 1 microM apamin. Under appropriate conditions, VIP and A23187 also increased the rate of 86Rb+ or 42K+ uptake. Stimulation of the initial rate of uptake by either agent required high intracellular K+ and was not markedly affected by the imposition of transcellular pH gradients. The effect of A23187, but not VIP or dibutyryl cyclic AMP, was refractory to depletion of cellular energy stores. A23187-stimulated uptake was not significantly affected by anion substitution, however, stimulation of uptake by VIP required the presence of a permeant anion. This result may be due to the simultaneous activation of a cyclic AMP-dependent Cl- transport system. The kinetics of both VIP- and A23187-stimulated uptake and efflux were consistent with a channel-rather than a carrier-mediated K+ transport mechanism. The results also suggest that cyclic AMP and Ca2+ may activate two different kinds of K+ transport systems. Finally, both transport systems have been localized to the basolateral membrane of T84 monolayers, a result compatible with their possible regulatory role in hormone-activated electrogenic Cl- secretion.     

Purification and Properties of an S-Adenosylmethionine: 2,4-Disubstituted Phenol O-Methyltransferase from Phanerochaete chrysosporium

An enzyme catalyzing the O-methylation of acetovanillone (3-methoxy-4-hydroxyacetophenone) by S-adeno-sylmethionine was isolated from Phanerochaete chrysosporium and purified 270-fold by ultrafiltration, anion-exchange chromatography, and gel filtration. The enzyme exhibited a pH optimum between 7 and 9 and was rapidly denatured at temperatures above 55°C. The K_m values for acetovanillone and S-adenosylmethionine were 34 and 99 μM, respectively. S-Adenosylhomocysteine acted as a powerful competitive inhibitor of S-adenosylmethionine, with a K_i of 41 μM. The enzyme was also susceptible to inhibition by thiol reagents and low concentrations of heavy metal ions. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was monomeric and had a molecular weight of approximately 53,000. Substrate specificity studies showed that 3-methoxy- and 3,5-dimethoxy-substituted 4-hydroxy-benzaldehydes, -benzoic acids, and -acetophenones were the preferred substrates for the enzyme. The corresponding 3,4-dihydroxy compounds were methylated relatively slowly, while the 3-hydroxy-4-methoxy compounds were almost inactive as substrates. Substituents in both the 2 and 4 positions relative to the hydroxyl group appeared to be essential for significant enzyme attack of a substrate. Provided that certain steric criteria were satisfied, the nature of the substituent was not critical. Hence, xenobiotic compounds such as 2,4-dichlorophenol and 2,4-dibromophenol were methylated almost as readily as acetovanillone. However, an extended side chain in the 4 position was not compatible with activity as a substrate, and neither homovanillic, caffeic, nor ferulic acid was methylated. The substrate range of the O-methyltransferase tends to imply a role in the catabolism or detoxification of lignin degradation products such as vanillic and syringic acids.     

(Na+,K+)-cotransport in the Madin-Darby canine kidney cell line. Kinetic characterization of the interaction between Na+ and K+

Confluent monolayer cultures of the differentiated kidney epithelial cell line, Madin-Darby canine kidney cells (MDCK), have been used to study ion transport mechanisms involved in transepithelial transport. We have investigated the previously reported K+-stimulation of 22Na+ uptake by confluent monolayers of Na+ depleted cells (Rindler, M. J., Taub, M., and Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 11431-11439). This component of Na+ uptake was insensitive to ouabain and amiloride, but was strongly inhibited by furosemide or bumetanide. Ouabain-insensitive 86Rb+ uptake was also inhibitable by furosemide or bumetanide and stimulated by extracellular Na+. The synergistic effect of Na+ and 86Rb+ uptake and K+ on 22Na+ uptake was reflected by an increase in the apparent Vmax and a decrease in the apparent Km as the concentration of the other cation was increased. The extrapolated Km for either 86Rb+ or 22Na+ uptake in the absence of the other cation was 30 mM while the Km in the presence of a saturating concentration of the other cation was 9 mM. The absolute Vmax values for 22Na+ and 86Rb+ uptake suggest a cotransport system with a stoichiometry of 2Na+:3K+. However, because of the experimental design, the actual ratio may be closer to 1:1. Competition with, and stimulation by, a variety of unlabeled cations indicated that Na+ could be partially replaced by Li+, while K+ could be fully replaced by Rb+ and partially replaced by NH4+ and CS+. Uptake by this system was dependent upon cellular ATP. Reduction of intracellular ATP to 3% of normal abolished both K+-stimulated 22Na+ uptake and Na+-stimulated 86Rb+ uptake.     

Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene.

Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.     

Brown seaweed species from Strangford Lough: compositional analyses of seaweed species and biostimulant formulations by rapid instrumental methods

The aims of this study were to characterise the composition of five seaweed species (Ascophyllum nodosum, Fucus serratus, Fucus vesiculosus, Laminaria hyperborea and Sargassum muticum), their extracts and commercial formulations, using thermogravimetry (TGA), energy dispersive X-ray microanalysis (EDX), Fourier-transform infrared spectroscopy (FTIR) and pyrolysis gas chromatography/mass spectrometry (Py-GC/MS). Analyses of the samples by TGA and EDX provided information on the proportions of algal cell wall, inorganic fractions and minerals. The main carbohydrate constituents of the five species and extracts were identified by their pyrolysis products, e.g. 1-(2-furanyl) ethanone, 5-methyl-2-furcarboxaldehyde, 2-hydroxy-3-methyl-2-cyclopenten-1-one, diannhydromannitol, 1,6-anhydromannopyranose and 1,6-anhydromannofuranose, using Py-GC/MS. The differences in relative intensities of the infrared bands of the five species were enhanced, especially after acid extraction compared with alkaline or neutral treatments, resulting in improved understanding of the compositional changes. In addition four commercial formulations and two acidic extracts of A. nodosum were evaluated for composition using the techniques. The dry matter, pH, electrical conductivity, ash, carbon and nitrogen content of the six preparations showed significant differences in composition. Variations in fatty acid, alginic acid, mannitol, laminarin and fucoidan content of the six formulations were reported. The results have shown that TGA, EDX, Py-GC/MS and FTIR are complementary techniques for rapid evaluation of seaweed materials and products.     

Halomethane:Bisulfide/Halide Ion Methyltransferase, an Unusual Corrinoid Enzyme of Environmental Significance Isolated from an Aerobic Methylotroph Using Chloromethane as the Sole Carbon Source

A novel dehalogenating/transhalogenating enzyme, halomethane:bisulfide/halide ion methyltransferase, has been isolated from the facultatively methylotrophic bacterium strain CC495, which uses chloromethane (CH₃Cl) as the sole carbon source. Purification of the enzyme to homogeneity was achieved in high yield by anion-exchange chromatography and gel filtration. The methyltransferase was composed of a 67-kDa protein with a corrinoid-bound cobalt atom. The purified enzyme was inactive but was activated by preincubation with 5 mM dithiothreitol and 0.5 mM CH₃Cl; then it catalyzed methyl transfer from CH₃Cl, CH₃Br, or CH₃I to the following acceptor ions (in order of decreasing efficacy): I⁻, HS⁻, Cl⁻, Br⁻, NO₂⁻, CN⁻, and SCN⁻. Spectral analysis indicated that cobalt in the native enzyme existed as cob(II)alamin, which upon activation was reduced to the cob(I)alamin state and then was oxidized to methyl cob(III)alamin. During catalysis, the enzyme shuttles between the methyl cob(III)alamin and cob(I)alamin states, being alternately demethylated by the acceptor ion and remethylated by halomethane. Mechanistically the methyltransferase shows features in common with cobalamin-dependent methionine synthase from Escherichia coli. However, the failure of specific inhibitors of methionine synthase such as propyl iodide, N₂O, and Hg²⁺ to affect the methyltransferase suggests significant differences. During CH₃Cl degradation by strain CC495, the physiological acceptor ion for the enzyme is probably HS⁻, a hypothesis supported by the detection in cell extracts of methanethiol oxidase and formaldehyde dehydrogenase activities which provide a metabolic route to formate. 16S rRNA sequence analysis indicated that strain CC495 clusters with Rhizobium spp. in the alpha subdivision of the Proteobacteria and is closely related to strain IMB-1, a recently isolated CH₃Br-degrading bacterium (T. L. Connell Hancock, A. M. Costello, M. E. Lidstrom, and R. S. Oremland, Appl. Environ. Microbiol. 64:2899–2905, 1998). The presence of this methyltransferase in bacterial populations in soil and sediments, if widespread, has important environmental implications.     

Role of astrocytes and altered regulation of spinal glutamatergic neurotransmission in stress-induced visceral hyperalgesia in rats

Glutamate (Glu) is the primary excitatory neurotransmitter in the central nervous system and plays a critical role in the neuroplasticity of nociceptive networks. We aimed to examine the role of spinal astroglia in the modulation of glutamatergic neurotransmission in a model of chronic psychological stress-induced visceral hyperalgesia in male Wistar rats. We assessed the effect of chronic stress on different glial Glu control mechanisms in the spinal cord including N-methyl-d-aspartate receptors (NMDARs), glial Glu transporters (GLT1 and GLAST), the Glu conversion enzyme glutamine synthetase (GS), and glial fibrillary acidic protein (GFAP). We also tested the effect of pharmacological inhibition of NMDAR activation, of extracellular Glu reuptake, and of astrocyte function on visceral nociceptive response in naive and stressed rats. We observed stress-induced decreased expression of spinal GLT1, GFAP, and GS, whereas GLAST expression was upregulated. Although visceral hyperalgesia was blocked by pharmacological inhibition of spinal NMDARs, we observed no stress effects on NMDAR subunit expression or phosphorylation. The glial modulating agent propentofylline blocked stress-induced visceral hyperalgesia, and blockade of GLT1 function in control rats resulted in enhanced visceral nociceptive response. These findings provide evidence for stress-induced modulation of glia-controlled spinal Glu-ergic neurotransmission and its involvement in chronic stress-induced visceral hyperalgesia. The findings reported in this study demonstrate a unique pattern of stress-induced changes in spinal Glu signaling and metabolism associated with enhanced responses to visceral distension.