Chronic metabolic acidosis increases NaDC-1 mRNA and protein abundance in rat kidney.
(41/1622)
BACKGROUND: Chronic metabolic acidosis increases, while alkali feeding inhibits, proximal tubule citrate absorption. The activity of the apical membrane Na+/citrate cotransporter is increased in metabolic acidosis, but is not altered by alkali feeding. METHODS: Renal cortical mRNA and brush border membrane protein abundances of sodium/dicarboxylate-1 (NaDC-1), the apical membrane Na+/citrate transporter, were measured. RESULTS: By immunohistochemistry, NaDC-1 was localized to the apical membrane of the proximal tubule. Chronic metabolic acidosis caused an increase in NaDC-1 protein abundance that was maximal in the S2 segment and that increased with time. Metabolic acidosis also increased NaDC-1 mRNA abundance, but this was first seen at three hours and correlated with the severity of the metabolic acidosis. Alkali feeding had no effect on NaDC-1 protein or mRNA abundance. CONCLUSIONS: Chronic metabolic acidosis increases renal cortical NaDC-1 mRNA abundance and apical membrane NaDC-1 protein abundance, while alkali feeding is without effect on NaDC-1. (+info)
The effects of diphenyleneiodonium and of 2,4-dichlorodiphenyleneiodonium on mitochondrial reactions. Mechanism of the inhibition of oxygen uptake as a consequence of the catalysis of the chloride/hydroxyl-ion exchange.
(42/1622)
1. Increasing the substrate concentration only decreased the inhibition of mitochondrial oxidations by diphenyleneiodonium or by 2,4-dichlorophenyleneiodonium by a small amount. 2. Diphenyleneiodonium and 2,4-dichlorodiphenyleneiodonium lowered the amounts of succinate, citrate and glutamate accumulated in the matrix of mitochondria in the presence of Cl-, but not in its absences. 2,4-Dichlorodiphenyleneiodonium decreased the accumulation of substrates by mitochondria oxidizing glycerol 3-phosphate. 3. Diphenyleneiodonium caused an alkalinization of the medium with an anaerobic suspension of mitochondria, which was only partly reversed by Triton X-100. 4. The rate of proton extrusion by mitochondria oxidizing succinate was not altered by diphenyleneiodonium or by 2,4-dichlorodiphenyleneiodium, although the rate of decay of proton pulses was increased. 5. 2,4-Dichlorodiphenyleneiodonium shifted the pH optimum for succinate oxidation by intact mitochondria from pH 7.2 to 8.0, whereas there was no effect on that of freeze-thawed mitochondria, which was pH 8.0. 6. The concentration of 2,4-dichlorophenyleneiodonium required to inhibit respiration by 50% is less the higher the absolute rate of oxygen uptake. 7. EDTA, but not EGTA [ethanedioxybis(ethylamine)-tetra-acetic acid] increased the inhibition of respiration by diphenyleneiodonium, 2,4-dichlorodiphenyleneiodonium and by tri-n-propyltin. 8. It is concluded that diphenyleneiodonium and 2,4-dichlorodiphenyleneiodonium limit respiration in Cl--containing medium by causing an acidification of the matrix, and that there are pH-sensitive sites in the respiratory chain between NADH and succinate, and between succinate and cytochrome c. (+info)
Inhibition of Escherichia coli by bovine colostrum and post-colostral milk. II. The bacteriostatic effect of lactoferrin on a serum susceptible and serum resistant strain of E. coli.
(43/1622)
Two strains of Escherichia coli were inhibited by complement-inactivated cow serum and to a lesser extent by precolostral calf serum devoid of specific antibodies. They were not inhibited by undiluted colostral whey or milk but colostral whey became bacteriostatic after dialysis or dilution in Kolmer saline and addition of precolostral calf serum or lactoferrin. The inhibition in all these fluids was due to iron-binding proteins (transferrin or lactoferrin). Undiluted dialysed milk was not inhibitory because of its low content of lactoferrin but became inhibitory after addition of 1 mg/ml of lactoferrin. The lack of inhibition in undiluted whey is due to the high concentration of citrate in colostral whey (and milk) and it is suggested that citrate competes with the iron-binding proteins for iron and makes it availabe to the bacteria. Addition of bicarbonate, which is required for the binding of iron by transferrin and lactoferrin, can overcome the effect of citrate; hence, the bacteriostatic effect of cow serum and precolostral calf serum is due to the presence of both transferrin and bicarbonate as well as the low lefel of citrate. (+info)
Glucose-regulated anaplerosis and cataplerosis in pancreatic beta-cells: possible implication of a pyruvate/citrate shuttle in insulin secretion.
(44/1622)
The hypothesis proposing that anaplerosis and cataplerosis play an important role in fuel signaling by providing mitochondrially derived coupling factors for stimulation of insulin secretion was tested. A rise in citrate coincided with the initiation of insulin secretion in response to glucose in INS-1 beta-cells. The dose dependence of glucose-stimulated insulin release correlated closely with those of the cellular contents of citrate, malate, and citrate-derived malonyl-CoA. The glucose-induced elevations in citrate, alpha-ketoglutarate, malonyl-CoA, and the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium reduction state, an index of beta-cell metabolic activity, were unaffected by the Ca2+ chelator EGTA. Glucose induced a rise in both mitochondrial and cytosolic citrate and promoted efflux of citrate from the cells. The latter amounted to approximately 20% of glucose carbons entering the glycolytic pathway. Phenylacetic acid, a pyruvate carboxylase inhibitor, reduced the glucose-induced rise in citrate in INS-1 cells and insulin secretion in both INS-1 cells and rat islets. The results indicate the feasibility of a pyruvate/citrate shuttle in INS-1 beta-cells, allowing the regeneration of NAD+ in the cytosol and the formation of cytosolic acetyl-CoA, malonyl-CoA, and NADPH. The data suggest that anaplerosis and cataplerosis are early signaling events in beta-cell activation that do not require a rise in Ca2+. It is proposed that citrate is a signal of fuel abundance that contributes to beta-cell activation in both the mitochondrial and cytosolic compartments and that a major fate of anaplerotic glucose carbons is external citrate. (+info)
Fatty acid oxidation and the regulation of malonyl-CoA in human muscle.
(45/1622)
Questions concerning whether malonyl-CoA is regulated in human muscle and whether malonyl-CoA modulates fatty acid oxidation are still unanswered. To address these questions, whole-body fatty acid oxidation and the concentration of malonyl-CoA, citrate, and malate were determined in the vastus lateralis muscle of 16 healthy nonobese Swedish men during a sequential euglycemic-hyperinsulinemic clamp. Insulin was infused at rates of 0.25 and 1.0 mU x kg(-1) x min(-1), and glucose was infused at rates of 2.0 +/- 0.2 and 8.1 +/- 0.7 mg x kg(-1) x min(-1), respectively. During the low-dose insulin infusion, whole-body fatty acid oxidation, as determined by indirect calorimetry, decreased by 22% from a basal rate of 0.94 +/- 0.06 to 0.74 +/- 0.07 mg x kg(-1) x min(-1) (P = 0.005), but no increase in malonyl-CoA was observed. In contrast, during the high-dose insulin infusion, malonyl-CoA increased from 0.20 +/- 0.01 to 0.24 +/- 0.01 nmol/g (P < 0.001), and whole-body fatty acid oxidation decreased by an additional 41% to 0.44 +/- 0.06 mg x kg(-1) x min(-1) (P < 0.001). The increase in malonyl-CoA was associated with 30-50% increases in the concentrations of citrate (102 +/- 6 vs. 137 +/- 7 nmol/g, P < 0.001), an allosteric activator of the rate-limiting enzyme in the malonyl-CoA formation, acetyl-CoA carboxylase, and malate (80 +/- 6 vs. 126 +/- 9 nmol/g, P = 0.002), an antiporter for citrate efflux from the mitochondria. Significant correlations were observed between the concentration of malonyl-CoA and both glucose utilization (r = 0.53, P = 0.002) and the sum of the concentrations of citrate and malate (r = 0.52, P < 0.001), a proposed index of the cytosolic concentration of citrate. In addition, an inverse correlation between malonyl-CoA concentration and fatty acid oxidation was observed (r = -0.32, P = 0.03). The results indicate that an infusion of insulin and glucose at a high rate leads to increases in the concentration of malonyl-CoA in skeletal muscle and to decreases in whole-body and, presumably, muscle fatty acid oxidation. Furthermore, they suggest that the increase in malonyl-CoA in this situation is due, at least in part, to an increase in the cytosolic concentration of citrate. Because cytosolic citrate is also an inhibitor of phosphofructokinase, an attractive hypothesis is that changes in its concentration are part of an autoregulatory mechanism by which glucose modulates its own use and the use of fatty acids as fuels for skeletal muscle. (+info)
Exercise diminishes the activity of acetyl-CoA carboxylase in human muscle.
(46/1622)
Studies in rats suggest that increases in fatty acid oxidation in skeletal muscle during exercise are related to the phosphorylation and inhibition of acetyl-CoA carboxylase (ACC), and secondary to this, a decrease in the concentration of malonyl-CoA. Studies in human muscle have not revealed a consistent decrease in the concentration of malonyl-CoA during exercise; however, measurements of ACC activity have not been reported. Thus, whether the same mechanism operates in human muscle in response to physical activity remains uncertain. To investigate this question, ACC was immunoprecipitated from muscle of human volunteers and its activity assayed in the same individual at rest and after one-legged knee-extensor exercise at 60, 85, and 100% of knee extensor VO2max. ACC activity was diminished by 50-75% during exercise with the magnitude of the decrease generally paralleling exercise intensity. Treatment of the immunoprecipitated enzyme with protein phosphatase 2A restored activity to resting values, suggesting the decrease in activity was due to phosphorylation. The measurement of malonyl-CoA in the muscles revealed that its concentration is 1/10 of that in rats, and that it is diminished (12-17%) during the higher-intensity exercises. The respiratory exchange ratio increased with increasing exercise intensity from 0.84 +/- 0.02 at 60% to 0.99 0.04 at 100% VO2max. Calculated rates of whole-body fatty acid oxidation were 121 mg/min at rest and 258 +/- 35, 264 +/- 63, and 174 +/- 76 mg/min at 60, 85, and 100% VO2max, respectively. The results show that ACC activity, and to a lesser extent malonyl-CoA concentration, in human skeletal muscle decrease during exercise. Although these changes may contribute to the increases in fat oxidation from rest to exercise, they do not appear to explain the shift from mixed fuel to predominantly carbohydrate utilization when exercise intensity is increased. (+info)
The citrate ion increases the conformational stability of alpha(1)-antitrypsin.
(47/1622)
Sodium citrate has previously been shown to convert native alpha(1)-antitrypsin into the inactive latent state and cause alpha(1)-antitrypsin to polymerize via the C-sheet pathway instead of the more common A-sheet pathway. In order to begin to understand these dramatic effects, we have examined the influence of low concentrations of sodium citrate upon the structure, stability and function of alpha(1)-antitrypsin. In 0.5 M citrate, the midpoint of guanidine hydrochloride-induced unfolding was increased by 1.8 M and the rate of heat inactivation was decreased approximately 30-fold compared with Tris or phosphate buffer. alpha(1)-Antitrypsin was fully active in the presence of a range of citrate concentrations (0. 1-0.5 M), forming a stable 1:1 complex with chymotrypsin. The association rate constant between alpha(1)-antitrypsin and chymotrypsin was decreased with increasing citrate concentration. Fluorescence and circular dichroism spectroscopy demonstrated no significant changes in the tertiary structure due to the presence of citrate. However, the insertion rate of exogenous reactive-center loop peptide increased with increasing citrate concentration, indicating some structural changes in the A beta-sheet region. Taken together, these data suggest that in the presence of 0.5 M citrate alpha(1)-antitrypsin adopts a highly stable but active conformation. (+info)
Molecular cloning, chromosomal organization, and functional characterization of a sodium-dicarboxylate cotransporter from mouse kidney.
(48/1622)
The sodium-dicarboxylate cotransporter of the renal proximal tubule, NaDC-1, reabsorbs filtered Krebs cycle intermediates and plays an important role in the regulation of urinary citrate concentrations. (1) Low urinary citrate is a risk factor for the development of kidney stones. As an initial step in the characterization of NaDC-1 regulation, the genomic structure and functional properties of the mouse Na(+)-dicarboxylate cotransporter (mNaDC-1) were determined. The gene coding for mNaDC-1, Slc13a2, is found on chromosome 11. The gene is approximately 24.9 kb in length and contains 12 exons. The mRNA coding for mNaDC-1 is found in kidney and small intestine. Expression of mNaDC-1 in Xenopus laevis oocytes results in increased transport of di- and tricarboxylates. The Michaelis-Menten constant (K(m)) for succinate was 0.35 mM, and the K(m) for citrate was 0.6 mM. The transport of citrate was stimulated by acidic pH, whereas the transport of succinate was insensitive to pH changes. Transport by mNaDC-1 is electrogenic, and substrates produced inward currents in the presence of sodium. The sodium affinity was relatively high in mNaDC-1, with half-saturation constants for sodium of 10 mM (radiotracer experiments) and 28 mM at -50 mV (2-electrode voltage clamp experiments). Lithium acts as a potent inhibitor of transport, but it can also partially substitute for sodium. In conclusion, the mNaDC-1 is related in sequence and function to the other NaDC-1 orthologs. However, its function more closely resembles the rabbit and human orthologs rather than the rat NaDC-1, with which it shares higher sequence similarity. (+info)