Substrate and inhibitor specificity of anion exchangers on the brush border membrane of rabbit ileum

Summary In previous studies we have found that several anions can be transported by an exchange process in rabbit ileal brush border membranes. We demonstrated exchanges of Cl for OH or HCO₃, SO₄ for OH, oxalate for OH, and oxalate for Cl. The purpose of these studies was to determine the number of distinct carriers mediating these exchanges. We utilized substrate and inhibitor specificity studies to distinguish between different anion exchange transporters. We conclude that SO₄OH and oxalate: OH exchange occur on the same carrier because: (i) pH-gradient stimulated transport of both¹⁴C-oxalate and³⁵SO₄ were equally sensitive tocis-inhibition by unlabeled SO₄ or oxalate; and (ii) both were inhibited equally by K. We conclude that oxalate: OH and oxalate: Cl exchanges occur on different carriers because: (i) Cl or SO₄ caused unequalcis-inhibition of these two exchanges; and (ii) as compared to oxalate: Cl exchange, oxalate: OH exchange was more sensitive to inhibition by probenecid and K and less sensitive to inhibition by bumetanide. Finally, we conclude that oxalate: Cl exchange and ClHCO₃ exchange occur on different carriers because: (i) ClHCO₃ exchange was almost completely insensitive tocis-inhibition by oxalate; and (ii) oxalate: Cl exchange was more sensitive to inhibition by DIDS and bumetanide than ClHCO₃ exchange. Thus we have found that there are at least three separate anion exchangers on rabbit ileal brush border: (i) a ClHCO₃ exchanger; (ii) a SO₄OH exchanger, which also transports oxalate; and (iii) an oxalate: Cl exchanger.     

Evidence that a system similar to the recA system of Escherichia coli exists in Vibrio cholerae

Summary Two lines of evidence suggest that a gene analogous to the recA gene of Escherichia coli exists in Vibrio cholerae and that its product serves a proteolytic function in the SOS response. Firstly, Southern blot hybridization using the recA gene of E. coli as a probe revealed a genomic sequence in V. cholerae which hybridized with the probe. Secondly, the SOS-like response in V. cholerae (as measured by beta phage induction) triggered by DNA damaging agents like Furazolidone could be blocked by Antipain, a protease inhibitor known to inhibit RecA protease action in E. coli. Maximal blocking effect of Antipain on beta phage induction occurred at 1 mM. At this concentration neither the viability of the host bacterium nor the lytic growth of a clear plaque mutant of the phage was affected by Antipain.     

Hydrogen peroxide metabolism as an index of water stress tolerance in jute

Two species of jute plants Corchorus capsularis L. (cv. JRC 212) and C. olitorius L. (cv. JRO 632) were subjected to water stress for 2 and 4 days by withholding water. The relative water content (RWC) decreased in both plants under water stress but to a greater extent in C. olitorius . The C. olitorius seedlings also showed greater membrane injury than C. capsularis seedlings under water stress as was evident from injury index data. Water stress increased glycolate oxidase (EC 1.1.3.1.) activity more in C. olitorius than in C. capsularis . The activity of superoxide dismutase (SOD, EC 1.15.1.1.) and catalase (EC 1.11.1.6.) decreased under water stress and their decrease was higher in C. olitorius than in C capsularis . The level of hydrogen peroxide and lipid peroxidation also increased in both plants under water stress and the increase was higher in C. olitorius than in C. capsularis seedlings. Under comparable external water stress, C. capsularis seedlings showed lower membrane damage, lower H₂O₂ accumulation and lower lipid peroxidation than C. olilorius which may be taken as indicative of higher water stress tolerance capacity of the former.     

Phosphate sorption isotherms for evaluating phosphorus requirement of wetland rice soils

Summary Phosphate sorption isotherms were developed for five Philippine wetland rice soils using the conventional technique and a modified one. In the conventional method, P requirements of soils varied between 280 and 810 g P/g soil. In the modified method, they varied from 160 to 540 g P/g soil at 0.2 ppm P in solution. Soils with high P-sorption capacities had vermiculite and halloysite as the dominant clay minerals. Soil reduction by flooding decreased P-sorption by 28–70 percent at 0.2 ppm P in solution. The decrease in P-sorption due to soil reduction was greatest in a crystalline soil with vermiculite and halloysite as the dominant clay minerals and least in a soil with dominant X-ray amorphous silicates in the clay fraction.Desorption of freshly adsorbed P under reduction was greater in HCO ₃ ^– solution than in CaCl₂ and it increased with level of applied P. Desorption patterns of freshly adsorbed P were similar to adsorption patterns but values of P in solution were lower at desorption. Soils varied with respect to desorption of freshly sorbed P. Desorption studies indicate that soils vary in intensity factor with respect to P and thus influence P availability to plants. Use of P-sorption and P-desorption data obtained under reduced soil condition was proposed for detecting P needs of submerged rice soils.Results of a pot study with IR36 at different levels of solution P (reduced) in one soil indicated a high degree of correlation between adjusted P levels and the measured growth parameters. About 0.12 ppm P in the soil solution or 0.46 ppm P desorbed in HCO ₃ ^– solution (equivalent to 100 mg P/kg soil) was adequate for near-maximum plant height, tiller production, total dry matter yield, plant P content, and total P uptake.     

Arachidonic Acid Release and Catecholamine Secretion from Digitonin-Treated Chromaffin Cells: Effects of Micromolar Calcium, Phorbol Ester, and Protein Alkylating Agents

The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin-treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N-ethylmaleimide (NEM) or p-bromophenacyl bromide (BPB) resulted in dose-dependent inhibition of 10 microM Ca2+-stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 microM. The IC50 of BPB for both events was 25 microM. High concentrations (5-20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.     

Effect of Centchroman (67/20) and 78/224 on nucleic acid and protein biosynthesis during implantation in rats

The biosynthesis of nucleic acids and proteins was studied in rat uterus by following the incorporation of [³H]-thymidine, [³H]-uridineand[¹⁴C]-leucinein control and pregnant rats in the presence and absence of two anti-implantation drugs. One of the drugs, 78/224 caused a significant increase in incorporation whereas the other drug, Centchroman, caused an inhibition in incorporation of all the three precursors. The implications of these changes in the light of estrogenicity, agonist and antagonist actions of anti-estrogens have been analysed. The importance of homeostatic mechanisms involved in nucleic acids and proteins for the maintenance of constant internal milieu for blastocyst attachment has been discussed.     

Energy conservation in whole cells of the methylotrophic bacterium Methylophilus methylotrophus

Respiratory chain phosphorylation has been investigated in the methylotrophic bacterium Methylophilus methylotrophus following the addition of oxidisable substrates to aerobic, whole cell suspensions. Initial-rate experiments showed that ATP synthesis occurred at the overall expense of AMP and inorganic phosphate via the sequential action of the ATP phosphohydrolase and adenylate kinase; some of the nascent ATP was rapidly used to synthesis nonadenine nucleoside triphosphates. After being corrected for ATP turnover, Pi/O quotients of 0.46 to 0.54, 0.77 and 1.37 nmol/ng-atom O were obtained for the oxidation of methanol dehydrogenase-linked substrates (methanol, ethanol and acetaldehyde), duroquinol and formate (NAD⁺-linked) respectively. These values were proportional to the H⁺/O and/or K⁺/O quotients exhibited by these substrates, and yielded an average H⁺/ATP (H⁺/Pi) quotient of 4.2 ng-ion H⁺/nmol. Steady-state experiments showed that the extent of cellular energisation varied with the respiration rate but was always in the order methanol > duroquinol > acetaldehyde, thus indicating that under these longer-term conditions methanol was completely oxidised to yield PQQH₂ and 2NAD(P)H. These results are discussed in terms of the various reactions which lead to the generation or utilisation of the protonmotive force in this organism.Abbreviations FCCP carbonylcyanide p-trifluoromethyxyphenyl-hydrazone - bulk phase, transmembrane electrochemical potential difference of protons ( ) - pH bulk phase, transmembrane pH difference (pH_in–pH_out) - bulk phase, transmembrane electrical potential difference (_in - _out) - [P] concentration of anhydride phosphate bonds in adenine nucleotides (2[ATP]+[ADP]) - FPLC fast protein liquid chromatography - PQQ pyrroloquinoline quinone - Gp phosphorylation potential     

Prevention of stress-induced gastric ulcers by dopamine agonists in the rat

Dopamine (DA) and DA agonists have been shown to exert a protective role against the formation of duodenal ulcers. The effect of stimulation of DA receptors on the development of stress-induced gastric ulcers is currently unknown. Accordingly, we evaluated the effect of several DA agonists on the development of gastric ulcers induced by 3 h of cold + restraint stress (CRS) in rats. Apomorphine, d-amphetamine, methylphenidate, and threo-dl-p-hydroxymethylphenidate (an hydroxylated analog of methylphenidate), significantly reduced both the incidence and severity of CRS-induced gastric ulcers. The gastric cytoprotection afforded by these agents was dose-related, and completely antagonized by pretreatment with the peripheally acting DA antagonist domperidone. Because domperidone blocks peripheral, but not central, DA receptors, and since the entry of threo-dl-p-hydroxymethylphenidate across the blood-brain barrier into the brain is restricted to a great extent, we conclude that stimulation of peripheral DA receptors is primarily involved in the gastric cytoprotection induced by dopamimetics.The pathogenesis of stress-induced gastric ulcers remains largely unknown, and significant efforts have been made over the last decade to functionally characterize some of the factors involved in the etiology of this disease. Considerable attention has been focused on gastric acid secretion, but its primary role in stress-induced gastric ulcer disease remains uncertain. In fact, agents which effectively inhibit or neutralize gastric acid secretion such as cimetidine or antacids do not necessarily exert protection against stress-induced gastric ulcers (1,2). Moreover, in our original studies with neurotensin, a brain and gastrointestinal peptide, we have found that central administration of this neuropeptide, which completely prevents the development of cold + restraint stress (CRS)-induced gastric ulcers, does not appreciably alter gastric acid secretion (2). These findings support the contention that gastric acid secretion may not be an important factor in the development of this type of gastric ulcer.There is, however, considerable evidence that the automatic nervous system plays an intermediary role in the development of these ulcers (3,4). In this regard, surgical or pharmacological blockade of the vagal (cholinergic) division of the autonomic nervous system prevents the appearance of stress-associated gastric ulcers (5,6). Direct stimulation of catecholamine receptors, or indirect activation via increased sympathetic outflow to the periphery (7,4,8–11) appears to produce a salutary effect of stress-induced gastric ulcers.Szabo and his associates (12, 13, 14) have extensively studied the anti ulcer effects of dopamine (DA) in duodenal ulcer formation. Whether DA also modifies the development of stress-induced gastric ulcers is currently unknown.We have therefore evaluated the effect of selected DA receptor agonists and antagonists on CRS-induced gastric ulcer formation in rats.