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PubMed1997

Inositol 1,4,5-trisphosphate and basal Ca2+ release is affected by the cytoplasmic concentration of Cl- in endothelial cells.

The effects of varying Cl- concentration in the intracellular bathing medium, on IP3-induced 45Ca2+ release from internal stores, were examined in saponin-permeabilised bovine aortic endothelial (BAE) cells. Results from this study show that the release of Ca2+ from the internal stores is affected by the cytoplasmic concentration of Cl- ions. Complete replacement of Cl- with gluconate augmented IP3 (3 microM)-induced 45Ca2+ release by 33 +/- 8%. Replacement of both Cl- and K+ with gluconate and NMG, respectively, had no significant effect on 45Ca2+ release. However, resting levels of internal 45Ca2+ were found to be affected by Cl- removal. These data suggest that in BAE cells, IP3 and also basal 45Ca2+ release may be regulated by the physiological intracellular Cl- concentration.

Animals↗

PubMed1998

Crystal structure of beta-glucosidase A from Bacillus polymyxa: insights into the catalytic activity in family 1 glycosyl hydrolases.

Family 1 glycosyl hydrolases are a very relevant group of enzymes because of the diversity of biological roles in which they are involved, and their generalized occurrence in all sorts of living organisms. The biological plasticity of these enzymes is a consequence of the variety of beta-glycosidic substrates that they can hydrolyze: disaccharides such as cellobiose and lactose, phosphorylated disaccharides, cyanogenic glycosides, etc. The crystal structure of BglA, a member of the family, has been determined in the native state and complexed with gluconate ligand, at 2.4 A and 2.3 A resolution, respectively. The subunits of the octameric enzyme display the (alpha/beta)8 barrel structural fold previously reported for other family 1 enzymes. However, significant structural differences have been encountered in the loops surrounding the active-center cavity. These differences make a wide and extended cavity in BglA, which seems to be able to accommodate substrates longer than cellobiose, its natural substrate. Furthermore, a third sub-site is encountered, which might have some connection with the transglycosylating activity associated to this enzyme and its certain activity against beta-1,4 oligosaccharides composed of more than two units of glucose. The particular geometry of the cavity which contains the active center of BglA must therefore account for both, hydrolytic and transglycosylating activities. A potent and well known inhibitor of different glycosidases, D-glucono-1,5-lactone, was used in an attempt to define interactions of the substrate with specific protein residues. Although the lactone has transformed into gluconate under crystallizing conditions, the open species still binds the enzyme, the conformation of its chain mimicking the true inhibitor. From the analysis of the enzyme-ligand hydrogen bonding interactions, a detailed picture of the active center can be drawn, for a family 1 enzyme. In this way, Gln20, His121, Tyr296, Glu405 and Trp406 are identified as determinant residues in the recognition of the substrate. In particular, two bidentate hydrogen bonds made by Gln20 and Glu405, could conform the structural explanation for the ability of most members of the family for displaying both, glucosidase and galactosidase activity.

Amino Acid Sequence↗

PubMed2001

Structural assembly of the active site in an aldo-keto reductase by NADPH cofactor.

A 1.9 A resolution X-ray structure of the apo-form of Corynebacterium 2,5-diketo-d-gluconic acid reductase A (2,5-DKGR A), a member of the aldo-keto reductase superfamily, has been determined by molecular replacement using the NADPH-bound form of the same enzyme as the search model. 2,5-DKGR A catalyzes the NADPH-dependent stereo-specific reduction of 2,5-diketo-d-gluconate (2,5-DKG) to 2-keto-l-gulonate, a precursor in the industrial production of vitamin C. An atomic-resolution structure for the apo-form of the enzyme, in conjunction with our previously reported high-resolution X-ray structure for the holo-enzyme and holo/substrate model, allows a comparative analysis of structural changes that accompany cofactor binding. The results show that regions of the active site undergo coordinated conformational changes of up to 8 A. These conformational changes result in the organization and structural rearrangement of residues associated with substrate binding and catalysis. Thus, NADPH functions not only to provide a hydride ion for catalytic reduction, but is also a critical structural component for formation of a catalytically competent form of DKGR A.

Alcohol Oxidoreductases↗

PubMed1995

Influence of hydroxypyridones and desferrioxamine on the mobilization of aluminium from tissues of aluminium-loaded rats.

Significantly increased levels of aluminium were assayed in the liver and various brain regions of male Wistar rats after intra-peritoneal injection of aluminium gluconate for a period of 1-2 months. Cessation of aluminium gluconate administration for one month did not alter the tissue content of aluminium, apart from the spleen and hippocampus, indicating temporal stability of the aluminium loading. Administration of desferrioxamine, or one of the hydroxypyridone chelators, decreased tissue aluminium content in the liver and in all regions of the brain investigated. Desferrioxamine was more effective in mobilizing liver aluminium than either of the two hydroxypyridones, CP20 and CP94, whereas the more hydrophobic of the hydroxypyridones, diethyl hydroxypyrid-4-ones, (CP94), was most effective in mobilizing brain aluminium. From the present study it appears that orally active chelators of appropriate hydrophobicity may be extremely effective in mobilizing brain aluminium.

Aluminum↗

PubMed1996

Structural, metabolic and ionic requirements for the uptake of L-carnitine by primary rat cortical cells.

L-Carnitine (L-C) is involved in the transport of acyl groups into mitochondria for beta-oxidation, although its role in the adult brain is still uncertain. We have shown before that the uptake of L-carnitine into cultured rat cortical neurones was dependent on temperature as well as the Na gradient and is inhibited by compounds resembling its structure, like gamma-aminobutyric acid (GABA), but most potently by specific GABA uptake blockers. In this study we have characterised this uptake process further. We have shown that the uptake of L-carnitine may be dependent on Cl ions, in addition to Na ions, but non on Ca ions. The L-C uptake was inhibited by substituent anions in the order gluconate (83%) > isethionate (32%), with propionate being ineffective, whereas GABA uptake was inhibited most potently by propionate substitution (79%) and equally by isethionate and gluconate (67%). This L-C uptake process was not affected by the amino acids, glutamine or lysine, up to 1 mM concentration, although beta-alanine at 500 microM caused a 38% inhibition. The uptake of L-C was also significantly inhibited by structurally-related compounds, with a carbon chain length of three to six atoms, possessing an amine group and/or a carboxyl group. At a concentration of 500 microM, 3-aminopropane sulphonic acid (53%), gamma-butyrobetaine (31%), gamma-hydroxybutyric acid (34%) and 4 methylaminobutyric acid (33%). Other compounds were effective only at the lower concentration of 10 microM, such as butyric acid (25%), nicotinic acid (26%), isonicotinic acid (26%), hexanoic acid (23%) and at 100 microM, like 6-aminocapric acid (22%). Drugs suggested to affect membrane properties, such as chlorpromazine, was without effect at 1 or 10 microM, whereas flunarizine (FLU) at 1 microM inhibited both L-C (24%) and GABA uptake (17%). Other drugs like the cholinesterase inhibitors, tacrine and eserine, also had a small inhibitory effect on L-C uptake, reducing it at 1 microM by 22 and 21% respectively, although higher concentrations were toxic (> 100 microM). Pretreatment of the cells with neuraminidase (50 U ml-1, 10 min) reduced the subsequent uptake of both L-C (18%) and GABA (42%). Hypoxia (3 h) also significantly attenuated L-C uptake (42%), however part of these effects were related to the loss of cell viability. In summary, L-C uptake occurs by a complex mechanism which at least in part may occur by a Na/Cl cotransport mechanism, which could be similar, to that of GABA or may even in part occur via the GABA transporter.

Amino Acids↗

PubMed1994

A general method for identification of polyhydroxyalkanoic acid synthase genes from pseudomonads belonging to the rRNA homology group I.

Using a 30-mer oligonucleotide probe highly specific for polyhydroxyalkanoic acid (PHA) synthase genes, the respective genes of Pseudomonas citronellolis, P. mendocina, Pseudomonas sp. DSM 1650 and Pseudomonas sp. GP4BH1 were cloned from genomic libraries in the cosmid pHC79. A 19.5-kbp and a 22.0-kbp EcoRI restriction fragment of P. citronellolis or Pseudomonas sp. DSM 1650, respectively, conferred the ability to accumulate PHA of medium-chain-length 3-hydoxyalkanoic acids (HAMCL) from octanoate as well as from gluconate to the PHA-negative mutant P. putida GPp104. An 11.0-kbp EcoRI fragment was cloned from P. mendocina, which restored in GPp104 the ability to synthesize PHA from octanoate but not from gluconate. From Pseudomonas sp. GP4BH1 three different genomic fragments encoding PHA synthases were cloned. This indicated that strain GP4BH1 possesses three different functionally active PHA synthases. Two of these fragments (6.4 kbp and 3.8 kbp) encoded for a PHA synthase, preferentially incorporating hydroxyalkanoic acids of short chain length (HASCL), and the synthases were expressed in either GPp104 and Alcaligenes eutrophus H16-PHB-4, respectively. The PHA synthase encoded by the third fragment (6.5 kbp) led to the incorporation of HAMCL and was expressed in GPp104 but not in PHB-4.

Acyltransferases↗

PubMed1993

Effect of protamine sulfate on the permeability properties of the mammalian urinary bladder.

Protamine sulfate (PS, an arginine-rich protein of molecular weight 5,000) has been reported to affect the ionic permeability of gallbladder epithelium, the permeability of cultured epithelial cells to mannitol, and the permeability of endothelial cell layers to albumin. Although the effect of PS has been widely investigated, the mechanism of its action on membrane permeability is presently unknown. The effect of PS on the rabbit urinary bladder epithelium was studied using both transepithelial and intracellular microelectrode techniques in conjunction with equivalent circuit analysis. The addition of 100 micrograms/ml of PS to a NaCl-containing mucosal solution caused (over a 40-min period) a large increase in the transepithelial conductance (Gt) and a transient hyperpolarization of the transepithelial voltage (Vt) followed by a depolarization of Vt. This secondary depolarization of Vt was not present if the mucosal solution was a KCl or a K-gluconate Ringer. The PS-induced increase in Gt was due to an increase in the apical membrane permeability to both cations (Na+ or K+) and anions (Cl- or gluconate). Further studies revealed the following features of the PS-induced conductance. (i) Trypsin inhibits the PS effect; however, this was due to PS hydrolysis by trypsin and not a membrane effect. (ii) Mucosal PS partially inhibited the PS-induced apical membrane conductance. (iii) The ability of PS to increase the membrane conductance was enhanced when the apical membrane potential was cell interior negative. (iv) The rate of conductance change (at any given membrane potential) was a saturating function of the PS concentration. This finding suggests that PS must interact with a membrane binding site before it can induce a change in the membrane conductance. (v) Lanthanum inhibited the PS-dependent conductance by two different mechanisms. One was as a reversible blocker of the PS-induced conductance. The other was by inhibiting the interaction between PS and a membrane binding site. A kinetic model is developed to describe the steps involved in the increase in membrane conductance.

Animals↗

PubMed1992

Ionic effects on bumetanide binding to the activated Na/K/2Cl cotransporter: selectivity and kinetic properties of ion binding sites.

The loop diuretic bumetanide binds specifically to the Na/K/2Cl cotransporter of many cell types including duck erythrocytes. Membranes isolated from these erythrocytes retain the ability to bind bumetanide when cells are exposed to cotransport-activity stimuli prior to membrane isolation. An extensive study of the effects of ions on specific [3H]bumetanide binding to such membranes is presented here and compared to the activity of these ions in supporting transport function in intact cells. Both Na+ and K+ enhanced bumetanide binding in a saturable manner consistent with a single-site interaction. The Km for each ion was dependent on the concentration of the other cation suggesting heterotropic cooperative interactions between the Na+ and K+ binding sites. Na+ and K+ were partially replaceable, with the selectivity of the Na+ site being Na+ greater than Li+ greater than NH4+; N-methyl-D-glucamine+, choline+ and tetramethylammonium+ also supported a small amount of specific binding when substituted for Na+. The selectivity of the K+ site was K+ approximately Rb+ greater than NH4+ greater than Cs+; N-methyl-D-glucamine+, choline+ and tetramethylammonium+ were inactive at this site. The results of transport experiments revealed a slightly different pattern. Li+ could partially substitute for Na+ in supporting cotransport, but other monovalent cations were completely inactive. The order of potency at the K+ site was NH4+ greater than K+ approximately Rb+ greater than Cs+ much greater than other monovalent cations. The effect of Cl- on bumetanide binding was biphasic, being stimulatory at low [Cl-] but inhibitory at high [Cl-]. As this implies the existence of two Cl- binding sites (termed ClH and ClL for the "high-" and "low-" affinity sites, respectively) each phase was examined individually. Cl- binding to ClH could be described by a rectangular hyperbola with a Km of 2.5 mM, while kinetic analysis of the inhibition of bumetanide binding at high [Cl-] revealed that it was of a noncompetitive type (Ki = 112.9 mM). The selectivity of anion binding to the two sites was distinct. ClH was highly selective with Cl- greater than SCN- greater than Br-; F-, NO3-, ClO4-, MeSO4-, gluconate- and SO4(2-) were inactive. The efficacy of anion inhibition of binding to ClL was ClO4- greater than I- greater than SCN- greater than NO3- greater than Cl-; F-, MeSO4-, gluconate-, and SO4(2-) were inactive. Thus, ClH is much more selective than ClL and largely accounts for the specificity of the system with respect to anion transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗

PubMed1993

Effects of anions and/or cell volume on the permeance of an apical water pathway induced by Hg in toad skin epithelium.

Hg compounds block membrane transport units behaving as water channels. Here we show that Hg induces an apical water pathway in toad skins pretreated with 10(-3) M CH3ClHg or HgCl2, added to the outer bathing medium. Washing with SO4-Ringer caused a several-fold increase in net water flow (Jw) and osmotic permeability coefficient (Pf) that was reversed by re-exposure to Cl- or NO3-Ringer and mimicked by gluconate-Ringer. These Pf changes could be elicited repeatedly and were present if, and only if, anion replacements took place in the inner bathing solution. Such inner polarity was related to the anion permeability of the epidermal basolateral membrane: impermeant anions (SO4, gluconate) increased Pf; permeant anions (Cl, NO3) did not change basal Pf but reversed the high Pf induced by impermeant anions. Hg induced the appearance of aggregates that persisted despite repeated washings of the skins during 4-5 h, and whether Pf was high (SO4-Ringer) or low (Cl-Ringer) before skin fixation. The Hg-induced apical water pathway in toad skin appears to be a unique model for studying the interplay between cell volume, cell ionic composition and water permeability.

Animals↗

PubMed1992

cAMP activation of CF-affected Cl- conductance in both cell membranes of an absorptive epithelium.

Cystic fibrosis (CF) is characterized by abnormal epithelial Cl- conductance (GCl). In vitro studies that have shown that cAMP regulation is an intrinsic property of the CF-affected GCl(CF-GCl) have been carried out previously on cultured secretory cells and on nonepithelial cells. Even though GCl in absorption is defective in CF, a clear demonstration of cAMP regulation of CF-GCl in a purely absorptive tissue is lacking. We studied the cAMP regulation of CF-GCl in the microperfused intact human reabsorptive sweat duct. About 40% of the ducts responded to cAMP (responsive) while the remainder of the ducts did not. In responsive ducts, cAMP-elevating agents: beta-adrenergic agonist isoproterenol (IPR), CPT-cAMP, forskolin, theophylline or IBMX increased Gt by about 2.3-fold (n = no. of ducts = 8). Removal of media Cl-, but not amiloride pretreatment (in the lumen), abolished the cAMP response, indicating exclusive activation of GCl. cAMP activated both apical and basolateral GCl. cAMP hyperpolarized gluconate: Cl- (lumen:bath) transepithelial bionic potentials (delta Vt = -20.3 +/- 5.2 mV, mean +/- SE, n = 9) and transepithelial 3: 1 luminal NaCl dilution diffusion potentials (delta Vt = -8.8 +/- 2.9 mV, n = 5). cAMP activated basolateral GCl as indicated by increased bi-ionic (gluconate:Cl-, bath:lumen) diffusion potentials (by about 12 mV). The voltage divider ratio in symmetric NaCl solutions increased by 60%. Compared to responsive ducts, nonresponsive ducts were characterized by smaller spontaneous transepithelial potentials in symmetrical Ringer's solution (Vt = -6.9 +/- 0.8 mV, n = 24, nonresponsive vs. -19.4 +/- 1.8 mV, n = 22, responsive ducts) but larger bi-ionic potentials (-94 +/- 6 mV, n = 35, nonresponsive vs. -65 +/- 5 mV, n = 17, responsive ducts) and dilution diffusion potentials (-40 +/- 5 mV, n = 11, nonresponsive vs. -29 +/- 3 mV, n = 7, responsive ducts). These results are consistent with an inherently (prestimulus) maximal activation of GCl in nonresponsive ducts and submaximal activation of GCl in responsive ducts. We conclude that cAMP activates CF-GCl which is expressed and abnormal in both apical and basal membranes of this absorptive epithelium in CF.

Adrenergic beta-Agonists↗

PubMed1993

Volume-activated chloride channels in HeLa cells are blocked by verapamil and dideoxyforskolin.

The possible role of Cl- currents in regulatory volume decrease processes has been explored in HeLa cells using the whole-cell recording mode of the patch-clamp technique. Cells showed very small currents in voltage-clamp experiments performed with Cl(-)-rich, permeant-cation-free (N-methyl-D-glucamine replacement) intracellular and bathing solutions. Exposure of the cells to hypotonic solutions visibly swelled the cells and activated, reversibly, an outward rectifying Cl- current, which decayed at the most depolarised voltages used. Replacement of extracellular Cl- by a series of halide anions, SCN- and gluconate was consistent with an anion selectivity sequence: SCN- > I- > Br- > Cl- > F- > gluconate. The volume-regulated Cl- current was effectively inhibited by 100 microM 5-nitro-2-(3-phenyl-propylamino)-benzoic acid and by 100 microM 4,4'-diisothiocyanotostilbene-2,2-disulphonic acid, substances known to block Cl- channels in a variety of cells. Chloride current activation by hypotonicity was dependent on the presence of ATP in the intracellular solution and this requirement could be replaced by the non-hydrolysable analogue ATP[gamma S] and Mg(2+)-free ATP. The data suggest that the channels responsible for the current described are involved in the regulatory volume decrease in HeLa cells. The characteristics of this Cl- current are similar to those of the current associated with expression of multidrug resistance P-glycoprotein. Furthermore, the currents in HeLa cells were inhibited rapidly and reversibly by verapamil and 1,9-dideoxyforskolin, which are known to inhibit P-glycoprotein function.

Adenosine Triphosphate↗

PubMed1977

Pathways of D-fructose and D-glucose catabolism in marine species of Alcaligenes, Pseudomonas marina, and Alteromonas communis.

Cell-free extracts of D-fructose grown cells of marine species of Alcaligenes as well as Pseudomonas marina contained an activity which catalyzed a P-enolpyruvate-dependent phosphorylation of D-fructose in the 1-position as well as activities of the following enzymes: 1-P-fructokinase, fructose-1,6-P2 aldolase, PPi-dependent 6-P-fructokinase, fructokinase, glucokinase, P-hexose isomerase, glucose-6-P dehydrogenase, 6-P-gluconate dehydrase, and 2-keto-3-deoxy-6-P-gluconate aldolase. The presence of these enzyme activites would allow D-fructose to be degraded by the Embden-Meyerhof pathway and/or the Entner-Doudoroff pathway. In cell-free extracts of D-glucose grown cells, the activity catalyzing a P-enolpyruvate-dependent phosphorylation of D-fructose as well as 1-P-fructokinase activity were reduced or absent while the remaining enzymes were present at levels similar to those found in D-fructose grown cells. Radiolabeling experiments suggested that both D-fructose and D-glucose were utilized primarily via the Entner-Doudoroff pathway. Alteromonas communis, a marine species lacking 1-P-fructokinase and the PPi-dependent 6-P-fructokinase, contained all the enzyme activites necessary for the catabolism of D-fructose and D-glucose by the Entner-Doudoroff pathway; the involvement of this pathway was also consitent with the results of the radiolabeling experiments.

Alcaligenes↗

PubMed1987

D(-)3-hydroxybutyrate cotransport with Na in rat renal brush border membrane vesicles.

Which are the driving forces for D(-)3-hydroxybutyrate (HB) transport in rat renal brush border membranes (RBB)? Sodium, even in the absence of gradients, accelerates the unidirectional (1-5 s) flux of HB into rat RBB vesicles. Valinomycin (and Ki = Ko) does not significantly alter the NaCl gradient driven HB influx. Thus, the Na-dependent HB influx is driven by the chemical Na+ gradient but it is not driven by changes in the transmembrane electrical potential. Indeed, in valinomycin-treated membranes, vesicle-inside more negative potentials (K-gluconate in-Na-gluconate out) sufficient to accelerate Na-glucose cotransport, did not stimulate HB influx, in the presence of inwardly directed Na+ gradients, and did not significantly inhibit when in the absence of Na+. Thus, cotransport of HB with Na in rat RBB membranes does not involve the net transfer of positive charge and the passive conductance of this membrane for HB- is not large. However, vesicle inside more negative potentials (induced by inwardly directed NaNO3 gradients or by outwardly directed K+ gradients and valinomycin in the presence of inwardly directed Na+ gradients) inhibited HB influx, suggesting that another potential sensitive mechanism, perhaps redistribution of intramembrane charges, may influence HB influx. Acidification (pHi = pHo = 6.4 vs. 7.4) or inwardly directed H+ gradients (pHo/pHi = 6.4/7.4) did not alter HB influx, in the absence of Na+. Thus there is no evidence for a H+ driven HB influx. HB influx is significantly inhibited by high (100 mEq/l) trans concentration of Na+. Also, influx of 2.25 mM 14C-HB was significantly increased by 5-10 mM intravesicular HB under Na-equilibrated conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxybutyric Acid↗

PubMed1984

Na/H- and Cl/OH-exchange in rat jejunal and rat proximal tubular brush border membrane vesicles. Studies with acridine orange.

The quenching of the acridine orange fluorescence was used to monitor the formation and/or dissipation of a delta pH in brush border vesicles isolated from rat kidney cortex or rat jejunum. Similar findings were obtained with both brush border membrane vesicle preparations. Acridine orange fluorescence was quenched by a preset delta pH (intravesicular acid) or by the ionophore (valinomycin/CCCP) dependent development of a delta pH (intravesicular acid) under conditions of potassium efflux. Under sodium efflux conditions, an acidification of the intravesicular space occurred: a) due to indirect (electrical) coupling of sodium and proton fluxes; b) due to directly coupled sodium/proton exchange. The initial rate of the dissipation of a preset delta pH was accelerated by pulse injections of sodium in a saturable manner; lithium partially replaced sodium. The sodium dependent acceleration in the rate of dissipation of a preset delta pH was not altered by replacing gluconate with chloride. Amiloride was an inhibitor of directly coupled sodium/proton exchange. An inwardly directed chloride gradient did not induce intravesicular acidification. The initial rate of the dissipative proton fluxes (preset delta pH) was slightly accelerated by an outwardly directed chloride gradient. Sodium/proton exchange dependent acidification of the intravesicular space was not altered by replacing gluconate with chloride. These results clearly document the existence of sodium/proton exchange in both renal and intestinal brush border membrane vesicles. In contrast, Cl/OH exchange--under our experimental conditions--must have a much smaller rate than Na/H exchange.

Acridine Orange↗

PubMed1985

The role of anions in cellular volume regulation.

Cellular volume can be varied substantially by replacing medium Cl- isosmotically by other univalent anions. Since K+ content changes in parallel, cellular K+ concentration is well maintained. Gluconate behaves as an impermeant anion so cells shrink. Acetate enters cells apparently by non-ionic diffusion causing marked cellular swelling. These changes in volume are fully reversed when Cl- is again restored to the medium. However, ouabain (10(-3) M) largely prevents this reversal when Cl- replaces acetate, arguing against a ouabain-insensitive volume regulating mechanism. In toad urinary bladder, serosal gluconate inhibits transepithelial Na+ transport and cells shrink. Analysis suggests that cell shrinkage results in a loss of Ba2+-sensitive highly selective basolateral membrane K+ conductance channels.

Animals↗

PubMed1983

Reinvestigation of the transepithelial P.D. in the proximal tubule of Necturus kidney.

Published values of transepithelial potential differences (VTE) in the proximal tubule of Necturus vary from approximately 0 to approximately -15 mV. In view of this disparity, we reinvestigated VTE. Our measured VTE was on the average -1.0 mV in early convolutions and +0.6 mV in terminal proximal segments. In the course of this study, we considered five distinct causes of artifacts. 1) Tip and pre-tip potentials: their occurrence was minimized by using Ringer's filled microelectrodes. 2) Interstitial tip localizations: the position of the tip was ascertained by the shift in potential, resulting in response to peritubular perfusion with gluconate solutions. 3) Leaky impalements: VTE responses to gluconate, input resistance determinations and the presence of positive VTE's rule out the leak hypothesis. 4) Zeroline shifts between pre- and postimpalement stages, and 5) Spontaneous VTE drifts in the positive direction, due to gradual passage of the tip from cell to lumen, or in the negative direction, resulting from tip contact with the lower cellular layer. All five causes of artifacts may be involved in the controversy regarding past VTE estimates.

Animals↗

PubMed1989

Physiological significance and bioenergetic aspects of glucose dehydrogenase.

The regulation of the PQQ-linked glucose dehydrogenase in different organisms is reviewed. It is concluded that this enzyme functions as an auxiliary energy-generating mechanism, because it is maximally synthesized under conditions of energy stress. It is now definitively established that the oxidation of glucose to gluconate generates metabolically useful energy. The magnitude of the contribution of the oxidation of glucose to gluconate via this enzyme to the growth yield of organisms such as Acinetobacter calcoaceticus is not yet clear.

Bacteria↗

PubMed1990

Hemodynamic and permeability characteristics of acute experimental necrotizing enterocolitis.

We examined the local hemodynamic response of intestinal loops during acute necrotizing enterocolitis (NEC) in anesthetized rabbits. NEC was induced in ileal loops by transmural injection of a solution containing casein (10 mg/ml) and calcium gluconate (50 mg/ml) acidified to pH 4.0 with propionic or acetic acid. Control loops received casein only (pH 5.0). Mucosal damage was quantified by the blood-to-lumen movement of [51Cr]EDTA, fluid shifts into the lumen, and histology. Mean arterial pressure and loop blood flow were steady over the 3-hr period, loop fluid volume decreased, and there was no evidence of necrosis or epithelial damage. In loops receiving acidified casein and calcium gluconate, there was an immediate dramatic increase in loop blood flow that returned to baseline by 50 min. In addition, loop fluid volume was dramatically increased, necrosis was noted in the form of blunting and loss of villi, and sevenfold increase in [51Cr]EDTA permeability was evident. Administration of CV 1808 (30 mg/kg/hr), a selective adenosine2 agonist, which maintained and elevated loop blood flow throughout the 3 hr protocol, failed to alter the changes in loop fluid volume or prevent necrosis. Histamine levels in loop fluid levels were significantly elevated 20-30 min after NEC induction when compared to saline controls, indicating an early activation of mucosal defenses with this luminal insult. Thus, this model of NEC is characterized by a transient, acute hyperemia, increased intestinal permeability, and histamine release. As mucosal damage was independent of ischemia and could not be prevented by vasodilatory therapy, this model supports the clinical findings that NEC is correlated with luminal factors related to feeding and independent of cardiovascular stress.

Acid-Base Imbalance↗