Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl-/HCO3- and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl- conductance. We investigated whether Cl- has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current (Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl- stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 +/- 0.03 in a low-Cl- solution. It was increased by 0.21 pH units when luminal Cl- was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl- depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl- solutions. The results show that luminal Cl- can increase the microclimate pH via apical Cl-/HCO3- or Cl-/OH- exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl- effects on Na+ absorption. The data further show that the Cl- conductance of rumen epithelium must be located at the basolateral membrane.     

Solubility and flow of calcium,magnesium and phosphorus in the digestive tract of sheep given maize or alfalfa silages

Four sheep, each fitted with a rumen cannula and a re-entrant cannula in the proximal duodenum and terminal ileum, were used for collection of rumen, duodenal and ileal digesta. Four normal sheep were used for collection of faeces. They were given maize silage, maize silage ensiled with urea, wilted alfalfa silage or formic acid-treated alfalfa silage. Although the solubility of the calcium (Ca) in the duodenal digesta was higher with sheep on maize silages than with those on alfalfa silages, sheep fed on maize silage showed negative apparent absorption (?32.7 and ?16.5%) owing to the low concentration of Ca in whole-plant maize. The apparent absorption of magnesium (Mg) was 40.6–42.3% for maize silages and 24.2–26.8% for alfalfa silages. The differences between silages with respect to apparent absorption of phosphorus (P) were not significant and ranged between 1.5 and 10.9%. There were no appreciable differences in soluble proportions, flow and apparent absorption of Ca, Mg and P between the two maize silages or the two alfalfa silages. There was a net absorption of Ca and Mg and a net secretion of P in the stomach of animals on all silages.     

Divalent cations reduce the electrogenic transport of monovalent cations across rumen epithelium

The rumen epithelium of sheep and goats showed an increase in short circuit current ( Isc) and transepithelial conductance (gt) upon mucosal removal of divalent cations. A divalent-sensitive Isc and gt were present in Na+, K+ or Rb+ buffer, but nearly abolished in mucosal NMDG+ (N-methyl-D-glucamine) buffer. High K buffer, addition of BaCl2 or of ouabain on the serosal side also reduced or abolished the divalent-sensitive Isc. Mucosal Ca2+ was more potent in blocking Isc, but had the same potency as Mg2+ in blocking gt. A prolonged mucosal deprivation of Mg2+ ions increased gt, potential difference and basal as well as the Ca2+-sensitive Isc. Mucosal addition of Mg2+ had a smaller effect on gt after serosal preincubation with Ba. The data suggest that rumen epithelial cells exhibit an apical non-selective cation conductance, which permits the passage of monovalents in the mucosal absence of divalents. The development of a divalent-sensitive Isc in Na buffer requires Na+/K+ pumps and K+ recycling through Ba2+-sensitive K+ conductances on the basolateral side. This Isc is blocked by extracellular Ca2+ and both extracellular and intracellular Mg2+ ions. A prolonged deprivation of mucosal Mg2+ alone seems to affect intracellular Mg2+ in this Mg2+-absorbing tissue.     

Ca2+-binding of S-100 protein in the presence of K+ and Mg2+

Ca2+-binding of S-100 protein was studied using a Ca2+ electrode at pH 6.80. In the presence of 0.1 M KCl and 10 mM MgCl2 (ionic strength 0.13), Ca2+-binding to S-100 protein occurred in three steps with positive cooperativity. The numbers of bound Ca2+ ions in the three steps were 2, 2, and 4. The Ca2+-binding constants were 6.9 x 10(3) M-1, 2.9 x 10(3) M-1, and 3.7 x 10(2) M-1, respectively. The Ca2+-binding constants of the first and second steps obtained in the presence of 33.3 mM MgCl2 or 0.1 M KCl (ionic strength 0.10) were 1.4 times larger than those described above. This suggests that Mg2+ does not inhibit Ca2+-binding of S-100 protein. The increase of KCl concentration from 0.1 to 0.2 M caused a decrease of the Ca2+-binding constants to ca. 50%.     

Disparate efficacy of tobramycin on Ca(2+)-, Mg(2+)-, and HEPES-treated Pseudomonas aeruginosa biofilms.

Mucoid exopolysaccharide (MEP) obtained from Pseudomonas aeruginosa 579 was suspended in 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) pH 7.2 containing 0.1-10.0 mM of CaCl2.2H2O or MgCl2.4H2O. MEP treated with HEPES or < 5.0 mM of the Ca2+ or Mg2+ salts remained soluble and bound tobramycin in an equilibrium dialysis bioassay. MEP treated with 5.0 or 10.0 mM of the Ca2+ or Mg2+ salts did not bind tobramycin. Five and 10 mM Ca(2+)-treated MEP precipitated but Mg(2+)-treated MEP did not. Pseudomonas aeruginosa 579 biofilms formed using a defined growth medium having < 1 mM Ca2+ or Mg2+ were treated for 1 h with 10 mM HEPES +/- 5.0 mM CaCl2.2H2O or MgCl2.4H2O, prior to an 8-h exposure to HEPES, or the defined growth medium, +/- 125 micrograms/mL of tobramycin. The tobramycin kill kinetics for the HEPES-, Mg(2+)-, and Ca(2+)-treated biofilms were similar and gradual from T = 0-6 h. The viability of the HEPES- and Mg(2+)-treated populations declined sharply (from 6 to 8 h). Bacteria dispersed from the MEP in control biofilms at 0 and 8 h did not grow in the presence of 7.81 micrograms/mL of tobramycin. Thus, binding of tobramycin of P. aeruginosa 579 MEP may not be as influential to the impediment of tobramycin diffusion as is the steric hindrance imposed by the Ca2+ condensation of the polymer.     

Ectonucleotide diphosphohydrolase activities in hemocytes of larval Manduca sexta

In this work, we describe the ability of living hemocytes from an insect (Manduca sexta, Lepidoptera) to hydrolyze extracellular ATP. In these intact cells, there was a low level of ATP hydrolysis in the absence of any divalent metal (8.24 +/- 0.94 nmol of Pi/h x 10(6) cells). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 15.93 +/- 1.74 nmol of Pi/h x 10(6) cells. Both activities were linear with cell density and with time for at least 90 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.33 mM MgCl2. This stimulatory activity was not observed when Ca2+ replaced Mg2+. The apparent Km values for ATP-4 and Mg-ATP2- were 0.059 and 0.097 mM, respectively. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.6 and 7.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced by an increase of pH. These ecto-ATPase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, sodium fluoride, tartrate, and levamizole. To confirm the observed hydrolytic activities as those of an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), as well as suramin, an antagonist of P2-purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents. Interestingly, lipopolysaccharide, a component of cell walls of gram-negative bacteria that increase hemocyte aggregation and phagocytosis, increased the Mg2+-dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg2+-independent ecto-ATPase activity.     

Effects of magnesium chloride on smooth muscle actomyosin adenosine-5'-triphosphatase activity, myosin conformation, and tension development in glycerinated smooth muscle fibers

The contractile system of smooth muscle exhibits distinctive responses to varying Mg2+ concentrations in that maximum adenosine-5'-triphosphatase (ATPase) activity of actomyosin requires relatively high concentrations of Mg2+ and also that tension in skinned smooth muscle fibers can be induced in the absence of Ca2+ by high Mg2+ concentrations. We have examined the effects of MgCl2 on actomyosin ATPase activity and on tension development in skinned gizzard fibers and suggest that the MgCl2-induced changes may be correlated to shifts in myosin conformation. At low concentrations of free Mg2+ (less than or equal to 1 mM) the actin-activated ATPase activity of phosphorylated turkey gizzard myosin is reduced and is increased as the Mg2+ concentration is raised. The increase in Mg2+ (over a range of 1-10 mM added MgCl2) induces the conversion of 10S phosphorylated myosin to the 6S form, and it was found that the proportion of myosin as 10S is inversely related to the level of actin-activated ATPase activity. Activation of the actin-activated ATPase activity also occurs with dephosphorylated myosin but at higher MgCl2 concentrations, between 10 and 40 mM added MgCl2. Viscosity and fluorescence measurements indicate that increasing Mg2+ levels over this concentration range favor the formation of the 6S conformation of dephosphorylated myosin, and it is proposed that the 10S to 6S transition is a prerequisite for the observed activation of ATPase activity. With glycerinated chicken gizzard fibers high MgCl2 concentrations (6-20 mM) promote tension in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnesium absorption as influenced by the rumen passage kinetics in lactating dairy cows fed modified levels of fibre and protein

The potassium sensitive magnesium absorption through the rumen wall may be influenced by additional dietary properties, such as diet type, forage type or forage to concentrate ratio. These properties are likely associated to rumen passage kinetics modified by dietary fibre content. The study aimed to assess the effects of rumen passage kinetics on apparent Mg absorption and retention in lactating dairy cows fed modified levels of fibre. Six lactating Red-Holstein and Holstein cows, including four fitted with ruminal cannulas were randomly assigned to a 3 × 3 cross-over design. The experimental diets consisted of early harvested low NDF (341 g NDF/kg DM) and late harvested high NDF (572 g NDF/kg DM) grass silage (80% DM) and of concentrates (20% of DM). As the low-fibre diet was excessive in protein, a third high-fibre diet was formulated to be balanced in digestible protein with the low-fibre diet to avoid any eventual confounding effects of NDF and protein excess. All diets were formulated to contain iso-Ca, -P, -Mg, -K and -Na. Passage kinetics of solid and liquid phase of rumen digesta were evaluated using ruminal marker disappearance profiles. Cows fed the low-fibre diet had compared to the other diets, an up to 40% lower solid and 26% lower liquid phase volume of rumen digesta and a 10% numerically higher fractional rumen liquid passage rate. Rumen pH lost 0.6 units and Mg concentration in the rumen liquid phase tripled when cows were fed the low-fibre diet. Faecal Mg excretion was up to 14% higher in cows fed the low-fibre diet and Mg absorbability was 12% compared to up to 19% in other diets. Urinary Mg excretion in cows fed the low-fibre diet was half of the ones in the other treatments, but Mg retention was not affected. Dietary protein excess neither affected rumen passage kinetics nor Mg absorption and retention. Absorption of Mg was correlated with rumen liquid volume which both decreased with decreasing daily NDF intake (NDFi, 11.8 ± 2.4 l/kg NDFi). Consequently, daily Mg absorption decreased by 1.32 ± 0.28 g/kg decreasing NDFi. To conclude, in addition to the known antagonistic effect of dietary K, the present data indicate that Mg absorption was dependent from NDFi which modified rumen liquid volume, but was independent of dietary protein excess likely associated to low NDF herbages.     

Inhibitory effect of Ca2+ on formation of Mg2(+)-mediated two-dimensional hexagonal lattice structure by an R-form lipopolysaccharide from Klebsiella pneumoniae

When the R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-:K1-), from which cationic material had been removed by electrodialysis, was suspended in 50 mM Tris buffer at pH 8.5 containing 0.1 mM or higher concentrations of MgCl2, it formed an ordered two-dimensional hexagonal lattice structure and its center-to-center distance (lattice constant) depended upon the concentration of MgCl2 and reached the shortest value (14 nm) at 10 mM. In contrast, in the presence of 0.1 to 10 mM CaCl2 in place of MgCl2, the electrodialyzed LPS did not form such an ordered hexagonal lattice structure but formed an irregular network structure with a center-to-center distance of 19 to 20 nm. We investigated interaction of Mg2+ and Ca2+ in formation of the hexagonal lattice structure by the electrodialyzed LPS suspended in 50 mM Tris buffer at pH 8.5. When 0.1 mM or higher concentrations of CaCl2 were mixed with 1 mM MgCl2 or when 1 mM or higher concentrations of CaCl2 was mixed with 10 mM MgCl2, the electrodialyzed LPS did not form the hexagonal lattice structure of the magnesium salt type but formed the irregular network structure of the calcium salt type. In the coexistence of equimolar or higher concentrations of CaCl2 together with 1 or 10 mM MgCl2, the binding of Mg to the electrodialyzed LPS was significantly inhibited and, conversely, the binding of Ca was enhanced as compared with when MgCl2 or CaCl2 was present alone. However, the coexistence of 10 times less molar concentrations of CaCl2 did not significantly inhibit the binding of Mg to the electrodialyzed LPS. Therefore, the inhibition of formation of the Mg2(+)-mediated hexagonal lattice structure of the electrodialyzed LPS by equimolar or higher concentrations of CaCl2 accompanied the inhibition of binding of Mg but that by 10 times less molar concentrations of CaCl2 did not accompany it.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.     

Effect of crude protein and phosphorus level on growth performance, bone mineralisation and phosphorus, calcium and nitrogen utilisation in grower-finisher pigs

Two experiments in a 2 x 2 factorial arrangement were conducted to evaluate the effect of crude protein (CP) (130 vs. 200 g/kg) and phosphorus (P) (4.0 vs. 6.0 g total P/kg) level in a phytase supplemented diet (500 FTU [phytase units]/kg) in grower-finisher pigs. Owing to the design of the experiment, as dietary P level increased, there was also an increase in dietary calcium (Ca) level in order to maintain a dietary Ca to P ratio of 1.6:1. In Experiment 1, four diets were fed to 56 pigs (n = 14, initial body weight [BW] 36.7 +/- 4.2 kg) to investigate the interaction between CP and P on growth performance, bone mineralisation and digesta pH. Experiment 2 consisted of 16 entire male pigs (n = 4; offered identical diets to that offered in Experiment 1) for the determination of total tract apparent digestibility and nitrogen (N), P and Ca utilisation. There was an interaction between CP and P level on bone ash, bone P and bone Ca concentrations (p < 0.05). Pigs offered low CP-low P diets had a higher bone ash, P and Ca concentrations than pigs offered high CP-low P diets. However, there was no effect of CP level at high P levels on bone ash, P and Ca concentrations. Pigs offered low P diets had a lower ileal pH compared with pigs offered high P diets (p < 0.05). In conclusion, offering pigs a high CP-low P, phytase-supplemented diet resulted in a decrease in bone mineralisation.     

Cationic specificity of a Ca2+-accumulating system in smooth muscle cell mitochondria

In the experiments conducted with application of an isotopic technique (45Ca2+) on the myometrium cells suspension treated by digitonin solution (0.1 mg/ml) some properties of Ca ions accumulation system in the mitochondria--cationic and substrate specificity as well as effects of Mg2+ and some other bivalent metals ions on the Ca2+ accumulation velocity have been estimated. Ca ions accumulation from the incubation medium containing 3 mM sodium succinate Na, 2 mM Pi (as potassium K(+)-phosphate buffer, pH 7.4 at 37 degrees C), 0.01 mM (40CaCl2 + 45CaCl2) and 100 nM thapsigargin--selective inhibiting agent of endoplasmatic reticulum calcium pump were demonstrated as detected just only in presence of Mg, while not Ni, Co or Cu ions. The increase of Mg2+ concentration from 1 x 10(-6) to 10(-3) M induced the ATP dependent transport activation in the myometrium mitochondria. Under [Mg2+] increase till 40 mM this cation essentially decreased Ca2+ accumulation (by 65% from the maximal value). The optimum for Ca2+ transport in the myometrium cells suspension is Mg2+ 10 mM concentration. Ka activation apparent constant along Mg2+ value (in presence 3 mM ATP and 3 mM sodium succinate) is 4.27 mM. The above listed bivalent metals decreased Mg2+, ATP-dependent accumulation of calcium, values of inhibition apparent constants for ions Co2+, Ni2+ and Cu2+ were--2.9 x 10(-4) M, 5.1 x 10(-5) M and 4.2 x 10(-6) M respectively. For Mg2+, ATP-dependent Ca2+ transport in the uterus myocytes mitocondria a high substrate specificity is a characteristic phenomenon in elation to ATP: GTP, CTP and UTP practically fail to provide for Ca accumulation process.     

A Mg-dependent ecto-ATPase in Leishmania amazonensis and its possible role in adenosine acquisition and virulence.

The plasma membrane of cells contains enzymes whose active sites face the external medium rather than the cytoplasm. The activities of these enzymes, referred to as ectoenzymes, can be measured using living cells. In this work we describe the ability of living promastigotes of Leishmania amazonensis to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (5.39 +/- 0.71 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 30.75 +/- 2.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. The addition of MgCl(2) to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 1.21 mM MgCl(2). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2) or SrCl(2). The apparent K(m) for Mg-ATP(2-) was 0.98 mM and free Mg(2+) did not increase the ecto-ATPase activity. In the pH range from 6.8 to 8.4, in which the cells were viable, the acid phosphatase activity decreased, while the Mg(2+)-dependent ATPase activity increased. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. A comparison between the Mg(2+)-dependent ATPase activity of virulent and avirulent promastigotes showed that avirulent promastigotes were less efficient than the virulent promastigotes in hydrolyzing ATP.     

Comparative enzymatic properties of avian liver and skeletal muscle D-fructose-1,6-bisphosphate 1-phosphohydrolase.

The enzymatic properties of purified preparations of chicken liver and chicken skeletal muscle fructose bisphosphatases (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) were compared. Both enzymes have an absolute requirement for Mg2+ or Mn2+. The apparent Km for MgCl2 at pH 7.5 was 0.5 mM for the muscle enzyme and 5 mM for the liver enzyme. Fructose bisphosphate inhibited both enzymes. At pH 7.5, the inhibitor constants (Ki) were 0.18 and 1.3 mM for muscle and liver fructose bisphosphatases, respectively. The muscle enzyme was considerably more sensitive to AMP inhibition than the liver enzyme. At pH 7.5 and in the presence of 1 mM MgCl2, 50% inhibition of muscle and liver fructose bisphosphatases occurred at AMP concentrations of 7 X 10(-9) and 1 X 10(-6) M, respectively. EDTA activated both enzymes. The degree of activation was time and concentration dependent. The degree of EDTA activation of both enzymes decreased with increasing MgCl2 concentration. Ca2+ was a potent inhibitor of both liver (Ki, 1 X 10(-4) M) and muscle (Ki, 1 X 10(-5) M) fructose bisphosphatase. This inhibition was reversed by the presence of EDTA. Ca2+ appears to be a competitive inhibitor with regard to Mg2+. There is, however, a positive homeotropic interaction among Mg2+ sites of both enzymes in the presence of Ca2+.     

Resolution and calcium-binding properties of the two major isoforms of troponin C from crayfish

Crayfish tail muscle troponin C (TnC) has been fractionated into its five components and the Ca2+-binding properties of the two major isoforms (alpha and gamma) determined by equilibrium dialysis. alpha-TnC contains one Ca2+-binding site with a binding constant of 1 x 10(6) M-1 and one Ca2+ site with a binding constant of 1 x 10(4) M-1. In the complex of alpha-TnC with troponin I (TnI) or with TnI and troponin T (TnT), both sites bind Ca2+ with a single affinity constant of 2-4 x 10(6) M-1. gamma-TnC contains two Ca2+-binding sites with a binding constant of 2 x 10(4) M-1. In the gamma-TnC.TnI and gamma-TnC.TnI.TnT complexes, the binding constant of one of the sites is increased to 4-5 x 10(6) M-1, while Ca2+ binding to the second site is hardly affected (KCa = 4-7 x 10(4) M-1). In the presence of 10 mM MgCl2, the two Ca2+-binding sites of both TnC isoforms exhibit a 2-3-fold lower affinity. Assuming competition between Ca2+ and Mg2+ for these sites, their binding constants for Mg2+ were 120-230 M-1. In the absence of Ca2+, however, alpha-TnC and gamma-TnC bind 4-5 mol of Mg2+/mol with a binding constant of 1 x 10(3) M-1. These results suggest that the effect of Mg2+ on Ca2+ binding at the two Ca2+ sites is noncompetitive, i.e. Mg2+ does not bind directly to these sites (Ca2+-specific sites). Since the formation of the complex of crayfish TnI with alpha-TnC or gamma-TnC increases significantly the affinity of one of their two Ca2+-specific sites, I conclude that the binding of Ca2+ to only one site (regulatory Ca2+-specific site) controls the Ca2+-dependent interaction between crayfish TnCs and TnI.     

Aminoglycosides inhibit hormone-stimulated Mg2+ uptake in mouse distal convoluted tubule cells

The clinical use of aminoglycosides often leads to renal magnesium wasting and hypomagnesemia. Of the nephron segments, both the thick ascending limb of Henle's loop and the distal tubule play significant roles in renal magnesium conservation but the distal convoluted tubule exerts the final control of urinary excretion. An immortalized mouse distal convoluted tubule (MDCT) cell line has been extensively used to study the cellular mechanisms of magnesium transport in this nephron segment. Peptide hormones, such as parathyroid hormone (PTH), glucagon, calcitonin, and arginine vasopressin (AVP) stimulate Mg2+ uptake in MDCT cells that is modulated by extracellular polyvalent cations, Ca2+ and Mg2+. The present studies determined the effect of aminoglycosides on parathyroid hormone (PTH)-mediated cAMP formation and Mg2+ uptake in MDCT cells. Gentamicin, a prototypic aminoglycoside, elicited transient increases in intracellular Ca2+ from basal levels of 102 +/- 13 nM to 713 +/- 125 nM, suggesting a receptor-mediated response. In order to determine Mg2+ transport, MDCT cells were Mg(2+)-depleted by culturing in Mg(2+)-free media for 16 h and Mg2+ uptake was measured by microfluorescence after placing the depleted cells in 1.0 mM MgCl2. The mean rate of Mg2+ uptake, d([Mg2+]i)/dt, was 138 +/- 24 nM/s in control MDCT cells. Gentamicin (50 microM) did not affect basal Mg2+ uptake (105 +/- 29 nM/s), but inhibited PTH stimulated Mg2+ entry, decreasing it from 257 +/- 36 nM/s to 108 +/- 42 nM/s. This was associated with diminished PTH-stimulated cAMP formation, from 80 +/- 2.5 to 23 +/- 1 pmol/mg protein x 5 min. Other aminoglycosides such as tobramycin, streptomycin, and neomycin also inhibited PTH-stimulated Mg2+ entry and cAMP formation. As these antibiotics are positively charged, the data suggest that aminoglycosides act through an extracellular polyvalent cation-sensing receptor present in distal convoluted tubule cells. We infer from these studies that aminoglycosides inhibit hormone-stimulated Mg2+ absorption in the distal convoluted tubule that may contribute to the renal magnesium wasting frequently observed with the clinical use of these antibiotics.     

Subacute ruminal acidosis suppressed the expression of MCT1 in rumen of cows

Subacute ruminal acidosis (SARA) is characterized by the depression of ruminal pH and an increase in the concentrations of short-chain fatty acids (SCFAs) and lipopolysaccharide (LPS) in the rumen of cows. The onset of SARA was linked to the accumulation of SCFAs. However, the mechanism of SCFAs transport is unknown. The proton-linked monocarboxylate transporter (MCT1) plays a vital role in the transportation of SCFAs. The goal of this study was to elucidate the distribution of MCT1 along the gastrointestinal tract of calves and adult cows; the expression change of MCT1 in SARA cows and the effect of ruminal pH, SCFAs, and LPS on MCT1 expression in rumen epithelial cells in vitro. The results indicated the presence of MCT1 along the gastrointestinal tract of calves and adult cows, most abundantly expressed in the rumen. Importantly, the expression of MCT1 was decreased in the rumen epithelium of SARA cows, and the expression of MCT1 was restored in the SARA treatment group. In vitro, LPS, low rumen fluid pH, high concentrations of SCFAs (90 mM acetate, 40 mM propionate, and 30 mM butyrate), and high concentrations of acetate, propionate, and butyrate, respectively, inhibited the expression of MCT1 in rumen epithelial cells. Taken together, these results indicated that LPS, low ruminal pH, and high concentrations of SCFAs decreased the expression of MCT1, further aggravating the accumulation of SCFAs in the rumen by decreasing the absorption of SCFAs.     

The suitability of a faecal suspension of sheep as inocula for the estimation of utilizable crude protein of feeds by in vitro incubation

The objective of the experiments was to study the suitability of using a faecal suspension of sheep for the estimation of the utilizable crude protein (uCP) of feeds for sheep by an in vitro incubation. Twenty-four single feeds and eight feed mixtures were used as incubation substrates. In Experiment 1, the gas production after the in vitro incubation with rumen fluid or with a faecal suspension of a sheep were compared using the Hohenheim gas test. It was found that there were significant linear regression between the 24, 48 and 72 h gas production with rumen fluid and those with faecal suspensions of 35, 50, 100 and 150 g wet faeces of sheep (which were 18.6, 23.5, 52.0 and 70.5 g faeces DM, respectively) per litre McDougall's buffer (P < 0.0001). The highest regression coefficient (r2) was calculated between the gas production after inoculation with a suspension of 100 g wet faeces per litre McDougall's buffer (x, ml x 200 mg (-1) feed DM) for 48 h and the gas production after inoculation with rumen fluid (y, ml x 200 mg (-1) feed DM) for 24 h: y = 0.82 (+/- 0.07)x + 9.87 (+/-3.83), r2 = 0.82, n = 32, P < 0.0001. Based on these results, in Experiment 2 the estimation of utilizable crude protein (uCP) of feeds was compared by using the in vitro incubation technique of Zhao and Lebzien (2000), where feeds were inoculated either with rumen fluid or with a faecal suspension (100 g wet faeces of sheep, i.e. 52 g faeces DM per litre McDougall's buffer). The results indicated that there were no significant differences of the estimated uCP after inoculation with rumen fluid or the faecal suspension (P > 0.05). A significant regression was found between the uCP after incubation for 48 h with 100 g wet faeces (x, g x kg (-1) DM) and the uCP after incubation for 24 h with rumen fluid (y, g x kg(-1) DM): y = 0.95 (+/-0.10)x - 4.90 (+/-26.70), r2 = 0.75, n = 32, Although this regression was significant, the coefficient r2 was not high. Therefore, further research is needed before sheep faeces could replace rumen fluid as an inocula for the estimation of uCP by the in vitro incubation technique.     

Effect of level and origin of rumen degradable nitrogen on rumen microbial growth and nitrogen utilisation efficiency of animals fed maize silage at maintenance.

Isoenergetic maize silage diets, fed at maintenance to 24 suckling cows (Exp. 1) and to 3 (Exp. 2) or 4 (Exp. 3) rumen fistulated sheep, were compared with a urea and controlled release NPN diet (Exps. 1 and 2) and with a protein-N supplemented diet (Exp. 3). Supplementation increased blood urea concentrations (44.7 +/- 22.3 vs. 97.6 +/- 23.7 mg urea-N x L(-1)) (Exp. 1) and renal urea excretion (2.5 +/- 1.1 vs. 7.6 +/- 1.8 g urea-N x d(-1)) (Exps. 2 and 3), whereas blood allantoin concentrations (286.7 +/- 77.0 micromol x L(-1)) (Exp. 1) and renal excretion of purine derivatives (357.6 +/- 90.7 mg purine-N x d(-1)) (Exps. 2 and 3) were not affected, indicating additional N supplementation did not improve rumen microbial growth. However, some deficiency of rumen degradable N might occur in non supplemented diets as suggested by the reduced rumen NH3-N and RNA concentrations (868 +/- 270 vs. 1466 +/- 466 mg RNA x kg(-1) rumen contents).     

Effects of isobutyrate on rumen fermentation, urinary excretion of purine derivatives and digestibility in steers

The objective of this study was to evaluate the effects of isobutyrate supplementations on rumen fermentation, urinary excretion of purine derivatives and feed digestibility in steers. Eight ruminally cannulated Simmental steers were used in a replicated 4 x 4 Latin square experiment. On DM basis, diet consisted of 60% corn stover and 40% concentrate. Dry matter intake (averaged 9 kg/d) was restricted to 90% of ad libitum intake. The four treatment groups received a daily dose of 0 (control), 8.4, 16.8 or 25.2 g isobutyrate per steer. With increasing isobutyrate supplementation total VFA concentration (range 64.2-74.0 mM) was significantly enhanced. The ratio of acetate to propionate (range 2.72-4.25) was also significantly increased due to the increase in actate production and decrease in propionate production. With increasing isobutyrate supplementation the ruminal degradation of NDF from corn stover was improved but the CP degradability of soybean meal was decreased. Furthermore, the isobutyrate supplementation caused a significantly increased urinary excretion of purine derivatives. Similarly, digestibilities of OM, NDF and CP in the total tract were significantly increased. The present results indicate that dietary supplementation with isobutyrate improved rumen fermentation and feed digestion in beef cattle in a dose-dependent manner. According to the conditions of this experiment, the optimum daily dose of isobutyrate was about 16.8 g/animal.