Ouabain-sensitive O2 Consumption Research Articles

Selection for Body Composition Does Not Affect Energetic Efficiency of Jejunal Glucose Uptake in Mice

Five-wk-old male mice from three lines were used to examine whether the apparent energetic efficiency of active jejunal glucose uptake in mouse jejunum is altered by genetic selection for different body composition. The mice lines were selected as follows: HE, high percentage of body fat with no change in body weight as a constraint; LF, low percentage of body fat; and RS, randomly bred control. Body weight was similar in all lines. Total jejunal O2 consumption and ouabain-sensitive O2 consumption were used to estimate the energy expenditure associated with glucose absorption and Na+/K+-ATPase activity. Tritiated 3-O-methyl-D-glucose was used to determine glucose uptake by mouse jejunum. Line LF, when compared with line HE, had lower body fat as indicated by epididymal fat pad weight (143 vs. 362 mg/mouse, P < 0.001). There were no significant differences in small intestinal weight, length and density (mg/cm) between LF and HE lines. Jejunal villus width was greater in line LF compared with line HE (115 vs. 92 µm, P < 0.002). Jejunal glucose transport and O2 consumption were not different between LF and HE lines. Ouabain-sensitive O2 consumption was not significantly different among the three lines. No differences were noted in the apparent energetic efficiency of active glucose uptake among lines.

Interaction between NO synthase and NADPH oxidase in control of sodium transport by the renal thick ascending limb during diabetes

During type 1 diabetes (T1D), the medullary thick ascending limb (mTAL) displays an NADPH oxidase-dependent increase in sodium transport, in concert with increased NO production by NO synthase 1 (NOS1) and NOS2. We hypothesized that NOS1- and/or NOS2-derived NO blunts T1D-induced activation of sodium transport in the mTAL. T1D was induced by streptozotocin injection (STZ rats); sham rats received vehicle. Three-to-four weeks later, mTAL were isolated from both groups for assay of nitrite and superoxide production, and O2 consumption in the absence or presence of various inhibitors. Apocynin (NADPH oxidase inhibitor) normalized superoxide production and ouabain-sensitive O2 consumption and furosemide-sensitive O2 consumption by mTALs from STZ rats, without altering O2 consumption by mTALs from sham rats. Apocynin also unmasked a T1D-induced increase in nitrite production. NOS inhibition did not alter superoxide production in either group. In sham mTAL, total NOS inhibition, but not isoform-specific inhibition of NOS1 or NOS2, increased ouabain- and furosemide-sensitive O2 consumption, confirming a tonic inhibitory impact of NOS3 on sodium transport. In contrast, neither total nor isoform-specific NOS inhibition altered O2 consumption by STZ mTAL. Apocynin treatment of STZ mTAL unveiled the ability of isoform-specific NOS inhibition to significantly increase O2 consumption, without further increase in O2 consumption with total NOS inhibition. Under normal conditions, NOS3-derived NO inhibits sodium transport in the mTAL. T1D dismantles the impact of NOS-mediated inhibition of sodium transport as a result of NADPH oxidase-dependent NO scavenging. Inhibition of NADPH oxidase to preserve NO bioavailability reveals an inhibitory impact of NOS1- and NOS2-derived NO on sodium transport in the mTAL.

NADPH Oxidase and PKC Contribute to Increased Na Transport by the Thick Ascending Limb During Type 1 Diabetes

Type 1 diabetes triggers protein kinase C (PKC)-dependent NADPH oxidase activation in the renal medullary thick ascending limb (mTAL), resulting in accelerated superoxide production. As acute exposure to superoxide stimulates NaCl transport by the mTAL, we hypothesized that diabetes increases mTAL Na(+) transport through PKC-dependent and NADPH oxidase-dependent mechanisms. An O(2)-sensitive fluoroprobe was used to measure O(2) consumption by mTALs from rats with streptozotocin-induced diabetes and sham rats. In sham mTALs, total O(2) consumption was evident as a 0.34±0.03 U change in normalized relative fluorescence (ΔNRF)/min per mg protein. Ouabain (2 mmol/L) reduced O(2) consumption by 69±4% and 500 μmol/L furosemide reduced O(2) consumption by 58±8%. Total O(2) consumption was accelerated in mTAL from diabetic rats (0.74±0.07 ΔNRF/min/mg protein; P<0.05 versus sham), reflecting increases in ouabain- and furosemide-sensitive O(2) consumption. NADPH oxidase inhibition (100 μmol/L apocynin) reduced furosemide-sensitive O(2) consumption by mTAL from diabetic rats to values not different from sham. The PKC inhibitor calphostin C (1 μmol/L) or the PKCα/β inhibitor Gö6976 (1 μmol/L) decreased furosemide-sensitive O(2) consumption in both groups, achieving values that did not differ between sham and diabetic. PKCβ inhibition had no effect in either group. Similar inhibitory patterns were evident with regard to ouabain-sensitive O(2) consumption. We conclude that NADPH oxidase and PKC (primarily PKCα) contribute to an increase in O(2) consumption by the mTAL during type 1 diabetes through effects on the ouabain-sensitive Na(+)-K(+)-ATPase and furosemide-sensitive Na(+)-K(+)-2Cl(-) cotransporter that are primarily responsible for active transport Na(+) reabsorption by this nephron segment.

Calcium-sensing receptor signaling pathways in medullary thick ascending limb cells mediate COX-2-derived PGE2production: functional significance

We determined the functional implications of calcium-sensing receptor (CaR)-dependent, Gq- and Gi-coupled signaling cascades, which work in a coordinated manner to regulate activity of nuclear factor of activated T cells and tumor necrosis factor (TNF)-alpha gene transcription that cause expression of cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) synthesis by rat medullary thick ascending limb cells (mTAL). Interruption of Gq, Gi, protein kinase C (PKC), or calcineurin (CaN) activities abolished CaR-mediated COX-2 expression and PGE2 synthesis. We tested the hypothesis that these pathways contribute to the effects of CaR activation on ion transport in mTAL cells. Ouabain-sensitive O2 consumption, an in vitro correlate of ion transport in the mTAL, was inhibited by approximately 70% in cells treated for 6 h with extracellular Ca2+ (1.2 mM), an effect prevented in mTAL cells transiently transfected with a dominant negative CaR overexpression construct (R796W), indicating that the effect was initiated by stimulation of the CaR. Pretreatment with the COX-2-selective inhibitor, NS-398 (1 microM), reversed CaR-activated decreases in ouabain-sensitive O2 consumption by approximately 60%, but did not alter basal levels of ouabain-sensitive O2 consumption. Similarly, inhibition of either Gq, Gi, PKC, or CaN, which are components of the mechanism associated with CaR-stimulated COX-2-derived PGE2 synthesis, reversed the inhibitory effects of CaR on O2 consumption without affecting basal O2 consumption. Our findings identified signaling elements required for CaR-mediated TNF production that are integral components regulating mTAL function via a mechanism involving COX-2 expression and PGE2 production.

Calcium sensing receptor (CaR)‐mediated TNF production in the medullary thick ascending limb (mTAL) is G i ‐coupled

We tested the contribution of CaR-mediated Gi-coupled signaling to TNF production in mTAL cells. CaR activation induced a 3-fold increase in TNF production at 6 hr that was abolished by a selective Gi inhibitor, pertussis toxin (PTX; 100 ng/ml; n=3; p<0.001). CaR increased calcineurin (CN) activity by approximately 3-fold (n=4; p<0.01). PTX prevented CaR-mediated increases in CN activity (n=4; p<0.01), an NFAT cis-reporter construct (n=5; p<0.001), and a TNF promoter construct (n=3; p<0.01). The interaction between Gi and PKC was determined, as we previously showed that CaR-mediated TNF production was CN- and NFAT-mediated and Gq-dependent. CaR activation increased PKC activity by 2-fold, an effect abolished by transient transfection with a dominant negative CaR construct, R796W (n=4; p<0.001) or pretreatment with PTX (n=4; p<0.001). Inhibition with the pan specific PKC inhibitor, GF 109203X (20 nM) abolished CaR-mediated increases in activity of CN, an NFAT reporter (n=3; p<0.001), and a TNF promoter construct (n=3; p<0.05). PTX reversed CaR-mediated decreases in ouabain-sensitive O2 consumption, suggesting that a Gi-coupled mechanism is important in regulating mTAL transport function. Collectively, the data suggest that Gi-coupled signaling contributes to NFAT-mediated TNF production, in a CN- and PKC-dependent manner, and is part of a CaR mechanism to regulate mTAL function.

Expression and function of NFAT5 in medullary thick ascending limb (mTAL) cells

The distribution of distinct NFAT isoforms and their potential contribution to the regulation of tumor necrosis factor-alpha (TNF) gene expression in mTAL cells is unclear. RT-PCR analysis was performed on primary cultures of mouse mTAL cells and freshly isolated mTAL tubules to determine which NFAT isoforms are present in this nephron segment. Primer pairs were designed, based on published sequences for mouse NFAT1-5, to produce fragments of approximately 200 bp. Analysis of PCR products by gel electrophoresis and subsequent DNA sequencing indicated that cells and tubules contained mRNA for all five NFAT isoforms. The relative expression of NFAT isoforms was then determined using quantitative real-time RT-PCR. The data indicate that NFAT isoforms 1 and 5 are the predominant isoforms present in mTAL cells and tubules. Overexpression of NFAT5 in mTAL cells increased calcium sensing receptor (CaR)-mediated TNF production (n=3; p< 0.05). The functional implications of inhibiting NFAT activity in mTAL cells also were evaluated. Preincubation for 30 min with either cyclosporine A or the peptide inhibitor, VIVIT, attenuated the CaR- and TNF-dependent inhibitory effects on ouabain-sensitive O2 consumption in mTAL cells. Collectively, these data suggest that activation of NFAT5 is part of a TNF-dependent pathway that inhibits ion transport mechanisms in the mTAL after activation of CaR.

L-ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture.

The addition of L-ascorbic acid 2-phosphate (AscP) to primary cultures of rabbit renal proximal tubular cells (RPTC) grown under improved culture conditions resulted in an extended growth phase and increased cellular density (1.3-fold increase in monolayer DNA and protein contents). AscP reduced glycolysis, increased net lactate consumption by 38%, and stimulated net glucose production by 47%. Basal O2 consumption increased by 39% in RPTC grown in the presence of AscP and was equivalent to that in freshly isolated proximal tubules. AscP increased ouabain-sensitive O2 consumption (81%) and Na(+)-K(+)-ATPase activity (2.5-fold), which suggested increased active Na+ transport. Addition of AscP increased Na(+)-dependent glucose uptake by 43% and brush-border enzyme marker activities by 46%. It is concluded that supplementation of media with AscP further improves RPTC culture conditions by promotion of cellular growth and stimulation of in vivo-like respiration, lactate utilization, and net glucose synthesis. These changes are accompanied by an increase in brush-border enzyme activities and stimulation of active Na+ transport and Na(+)-dependent glucose transport, which demonstrate an improved expression of brush-border membrane functions in RPTC.

Amino Acid Metabolism and Water Transport Across the Seawater Eel Intestine

ABSTRACT To elucidate how intracellular L-alanine enhances water transport across the seawater eel intestine, effects of various metabolic inhibitors were examined. The L-alanine-induced water flux was inhibited by amino-oxyacetate, an inhibitor of aminotransferase. After blocking the synthesis of pyruvate from L-alanine with this drug, water transport was stimulated with pyruvate, whose effects were inhibited by oxythiamine, an inhibitor of pyruvate dehydrogenase. 2,4-Dinitro-phenol (DNP) also inhibited the effects of L-alanine. Furthermore, L-alanine enhanced ouabain-sensitive O2 consumption in this tissue, and the enhancement in O2 consumption preceded that in the transepithelial potential difference (PD) and the net water flux. These results indicate that L-alanine is metabolized through the citric acid cycle to produce ATP, and that a metabolic product stimulates ion and water transport. L-Glutamine also seems to be metabolized just like L-alanine because: L-glutamine acted from inside the enterocyte; DNP inhibited the effects of L-glutamine; neither of the effects of L-glutamine and L-alanine were additive but they were mutually complementary; L-glutamine also enhanced ouabain-sensitive O2 consumption; and the increment in O2 consumption preceded that in the PD and the net water flux. The effects of L-glutamine on the PD and the net water flux depended on glutamine concentration and the concentration-response curve was of the Michaelis-Menten type, indicating that the rate of L-glutamine uptake into the enterocyte limits the overall rate of L-glutamine metabolism. A regulatory role of amino acids for ion and water transport is discussed.

The relationship between renal metabolism and proximal tubule transport during ontogeny.

The proximal tubules of newborn and adult animals reabsorb a similar fraction of the filtered load of Na+ and H2O (65%-70%). In tubules from adult animals, transcellular, active Na+ reabsorption accounts for one-third of the total, while two-thirds occur passively through the paracellular pathway, driven by hydrostatic and oncotic forces (one-third) and by cell-generated effective osmotic and ionic gradients (one-third). Since two-thirds of the Na+ is reabsorbed passively and does not require energy, the mature proximal tubule has a high Na+/O2 molar ratio (48 Eq of Na+/mol of O2). Measurements of ouabain-sensitive oxygen consumption in suspensions of proximal tubules indicate that in newborn, aerobic metabolism can support about 50% of the net Na+ transport rate compared with the 33% in tubules from adult animals. Independent confirmation of the direct and proportional relationship between active Na+ transport and ouabain-sensitive O2 consumption exists for the adult but not for the newborn. However, measurements of epithelial conductances and of transepithelial hydrostatic and oncotic pressure differences indicate that passive paracellular fluxes can account for the remaining 50% of the proximal Na+ reabsorption in newborn. The high permeability of the proximal tubules of newborn animals to small molecular weight solutes suggests that cell-generated osmotic and ionic transepithelial gradients are minimal in the tubules of newborn animals. Yet in the newborn, the osmolality of the end proximal tubule fluid was found to exceed that in plasma. This indicates that osmotic gradients due to differences in reflection coefficients for preferentially reabsorbed solutes and Cl- do exist across the proximal tubules of the newborn and suggests that these gradients may contribute to Na+ and H2O reabsorption. If this is indeed the case, then the contribution of active and of hydrostatic and oncotic pressure-driven flows to the overall reabsorption of Na+ and fluid has been overestimated. Resolution of this discrepancy requires measurements of the reflection coefficients for HCO3- and Cl- in the proximal tubule of the newborn. The metabolic processes by which energy is supplied to renal proximal cells during development are also incompletely characterized. There is evidence that maturation of aerobic metabolism, Krebs cycle enzymes activity, and of the mitochondrial membrane surface area precede the development of net reabsorptive transport (Na+, H2O, HCO3, glucose). By contrast, maturation of Na(+)-K(+)-ATPase activity at the basolateral cell membrane follows that in reabsorptive transport and does not limit its development.(ABSTRACT TRUNCATED AT 400 WORDS)

Level of alimentation and line of breeding on oxygen uptake by ovine jejunal mucosa.

Targhee rams from a selected and control line of breeding were provided with the same diet at either 100 or 40% of ad libitum feed intake levels for 28 days. The rams were killed in a serial fashion over a 35-day period. A sample of the jejunum mucosa was taken, and O2 consumption rates with and without ouabain were measured. Total O2 consumption and ouabain-sensitive respiration rates of the jejunal mucosa taken from rams of the select line were greater than for samples taken from the control rams. Line of breeding did not influence the percent of the total O2 consumption that was ouabain sensitive and the proportion of the jejunum that was mucosa. Differences in rates of total and ouabain-sensitive O2 consumption attributed to level of feed intake were not detected as significant. Both the percentage of the total respiration rates that was ouabain sensitive and the proportion of the jejunum that was mucosa were greater for rams fed at high intake levels compared with rams fed at low intake levels. Thus both breeding sheep on the basis of improved growth potential and level of alimentation influenced aspects of jejunal mucosa respiration that was Na+-K+-adenosinetriphosphatase dependent.

Amiloride analogues inhibit proximal tubule metabolism

The effects of two amiloride analogues, ethylisopropylamiloride and methylisopropylamiloride, were tested on active Na+ transport and oxidative metabolism of a rabbit proximal renal tubule suspension. These two analogues have been recently reported to inhibit Na+-H+ antiport activity of various tissues with greater potency than amiloride. In proximal tubules, no effects were detected at concentrations less than 10(-4) M. At concentrations greater than 10(-4) M, both of these compounds inhibited the ouabain-sensitive O2 consumption (a direct measure of Na+ pump activity) with a greater potency than amiloride. Investigations into possible metabolic effects revealed that both amiloride analogues inhibited mitochondrial production of ATP at these concentrations, whereas amiloride did not directly affect metabolism. The amiloride analogues inhibited the nystatin-stimulated O2 consumption (which measures Na+-K+-ATPase activity in the intact cells) as well as the ATP content. These results suggest that the primary effects of these analogues on rabbit proximal tubules are to inhibit both the Na+-K+-ATPase and oxidative metabolism.

Relationship among parathyroid hormone, cAMP, and calcium on proximal tubule sodium transport.

Parathyroid hormone (PTH), through the generation of cAMP, inhibits the transport of sodium, bicarbonate, and water in the proximal convoluted tubule. The present studies were designed to determine whether the response to PTH or dibutyryl cAMP by proximal renal tubules requires an influx of calcium into the cells or an alteration in the cytosolic concentration of calcium. O2 consumption was determined in an enriched suspension of rabbit proximal convoluted tubules, and the ouabain-sensitive component of O2 consumption was taken as a measure of sodium transport. PTH (1 IU/ml) inhibited the ouabain-sensitive component of O2 consumption from 14.2 +/- 1.6 to 8.9 +/- 1.1 nmol O2 X min-1 X mg protein-1 (P less than 0.005). Dibutyryl cAMP (10(-4) M) inhibited ouabain-sensitive O2 consumption from 12.0 +/- 1.1 to 8.4 +/- 0.8 nmol O2 X min-1 X mg protein-1 (P less than 0.005). In the presence of lanthanum (5-50 microM) or verapamil (20-200 microM), PTH inhibited ouabain-sensitive O2 consumption by 21.3 +/- 3.4% (P less than 0.025) and 33.9 +/- 5.5% (P less than 0.025), respectively. Dibutyryl cAMP inhibited the ouabain-sensitive O2 consumption by 22.6 +/- 7.1% (P less than 0.025) in the presence of lanthanum and 35.4 +/- 3.1% (P less than 0.01) in the presence of verapamil. To more directly assess the cytosolic concentration of calcium, the fluorescent intensity of quin 2-loaded tubules was determined. As compared with timed controls, exposure to PTH resulted in a lower cytosolic concentration of calcium over the 10 min of incubation. Dibutyryl cAMP had a similar effect.(ABSTRACT TRUNCATED AT 250 WORDS)