Renal brush border membrane lipid composition in Basenji dogs with spontaneous idiopathic Fanconi syndrome.

To comprehend the renal defect underlying idiopathic Fanconi syndrome in the Basenji dog, we have focused on delineating the lipid profiles of renal brush border membranes isolated from affected and normal Basenji dogs to establish any physical or compositional changes underlying previously observed transport and membrane-fluidity changes. The lipid composition was studied with respect to total lipid, cholesterol, and phospholipid content, cholesterol to phospholipid ratio, distribution of the major phospholipid classes, and fatty acid composition. Total phospholipid of the isolated renal brush border membranes from Fanconi syndrome dogs analyzed by 31P nuclear magnetic resonance showed no difference compared with that of normal dogs. Examination of total fatty acids in both membranes using gas-liquid chromatography analysis of fatty acid methyl esters showed no difference in the mole percents of the major fatty acids. Our data suggest that changes in bulk membrane fluidity of the Fanconi syndrome dog renal brush border as measured by 1,6-diphenyl-1,3,5-hexatriene cannot be attributed to phospholipid and fatty acid compositional change. In the membranes isolated from affected dog kidney, the cholesterol content determined by gas-liquid chromatography analysis was 66 mol% higher than in membranes isolated from normal dog kidney. This correlates with the higher cholesterol to phospholipid molar ratio of 0.82 +/- 0.08 in the affected animal as compared with 0.58 +/- 0.04 in the normal. Cholesterol content and its microdomain in the membrane bilayer may be important in modulating transport functions. Increased membrane cholesterol content may affect the conformational motility of membrane transport proteins and thus affect their function.

The effect of glucose and galactose toxicity on myo-inositol transport and metabolism in human skin fibroblasts in culture.

Myo-inositol transport and metabolism were studied in cultured human skin fibroblasts exposed to potentially toxic levels of glucose or galactose. Although variable among 11 different cell lines, the myo-inositol level in confluent cells, ranging from 10-50 nmol/mg protein, was constant with passage. A high-affinity transport system for myo-inositol had an apparent Kt of 55 microM and Vmax of 16 pmol/min/mg protein. No obvious relationship existed between cellular levels and transport capacity. Dependency on sodium was complex. When medium sodium was lowered to 23 mM, myo-inositol uptake ceased after about 1 h. However, the initial rate of myo-inositol uptake only showed a sodium dependence at low myo-inositol concentrations. Both phloretin and phloridzin inhibited myo-inositol uptake. Phloridzin had a Ki of 60 microM, and phloretin was either a noncompetitive or uncompetitive inhibitor. Glucose and galactose were only weak competitive inhibitors, with a Ki of 30 mM and 65 mM, respectively. After 24 h of incubation with myo-[2-3H]inositol, only 10% of the total cell label was incorporated into phospholipid. Compared with control media with 5 mM glucose, the incubation of confluent cells in media with 20 mM glucose had little effect on intracellular glucose and sorbitol, whereas cells incubated in control media supplemented with 5 mM galactose showed a large increase in galactose and polyol levels. In media with more than 200 microM of myo-inositol, neither treatment had an effect on myo-inositol levels after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

myo-inositol transport and metabolism in fetal-bovine aortic endothelial cells.

The myo-inositol transport system in confluent fetal-bovine aortic endothelial cells was characterized after 7-10 days in subculture, at which time the myo-inositol levels and rates of myo-[2-3H]-inositol uptake and incorporation into phospholipid had reached steady state. Kinetic analysis indicated that the uptake occurred by both a high-affinity transport system with an apparent Kt of 31 microM and Vmax. of 45 pmol/min per mg of protein, and a non-saturable low-affinity system. Uptake was competitively inhibited by phlorhizin, with a Ki of 50 microM; phloretin was a non-competitive inhibitor, with half-maximal inhibition between 0.2 and 0.5 mM. Glucose was a weak competitive inhibitor, with a Ki of 37 mM; galactose failed to inhibit uptake. A weak dependence on Na+ for the initial rate of uptake was observed at 11 microM myo-inositol. When fetal-bovine-serum (FBS)-supplemented medium, which contained 225 microM myo-inositol, was used, the cells contained about 200 nmol of myo-inositol/mg of DNA. With adult-bovine-serum (ABS)-supplemented medium, which contained 13 microM myo-inositol, the cells contained about 110 nmol/mg of DNA. Transport of 11 microM myo-[2-3]inositol was 18 and 125 pmol/min per mg of DNA for cells grown in FBS and ABS respectively. Kinetic analysis showed that for the cells grown in FBS the Vmax. of the high-affinity system was decreased by 64%, whereas the Kt remained essentially unchanged. Increased cell myo-inositol levels were not associated with an increased rate of phosphatidylinositol synthesis. After prolonged exposure of fetal endothelial cells to a myo-inositol concentration which approximated to a high fetal as opposed to a low adult blood level, cell myo-inositol levels doubled and high-affinity transport underwent down-regulation.

Kinetic evidence for compartmentalization of myo-inositol in hepatocytes.

myo-Inositol uptake and conversion to phosphatidylinositol (PI) was studied in isolated rat hepatocytes. Uptake of myo-[2-3H]-inositol into the trichloroacetic acid (TCA)-soluble fraction showed no evidence of saturation, while incorporation into lipid had an apparent Km of 0.28 mmol/L for external myo-inositol. With 50 mumol/L myo-[2-3H]-inositol, approximately half of the radiolabel was found in lipid at 30 minutes. Glucose and galactose were weak inhibitors, while phlorizin at 1 mmol/L reduced uptake by 50%. Metabolic inhibitors reduced incorporation of myo-[2-3H]-inositol into lipid, but had no effect on uptake. Hepatocytes maintained myo-inositol levels of 0.4 mmol/L for 60 minutes when incubated with 50 mumol/L myo-inositol, but levels increased when incubated with 1 mmol/L myo-inositol. Efflux of label was studied in hepatocytes prelabeled for 20 minutes with myo-[2-3H]-inositol. Loss of label was initially rapid, but had slowed by 20 minutes, with much of the label remaining in the cells. Phlorizin inhibited the loss of myo-[2-3H]-inositol, while increasing myo-inositol concentration in the medium enhanced efflux. The effects of these agents on the rate of efflux was found in lipid rather than in the TCA-soluble myo-inositol fraction. These findings suggest that myo-inositol is compartmentalized within hepatocytes, with a bulk metabolically inert pool and a smaller active pool that equilibrates with extracellular myo-inositol via an energy-independent carrier-mediated mechanism, and is preferentially available for efflux or for synthesis of phosphoinositides.

Maturational regulation of inositol 1,4,5-trisphosphate metabolism in rabbit airway smooth muscle.

Airway reactivity has been shown to vary with age; however, the mechanism(s) underlying this process remain unidentified. To elucidate the role of ontogenetic changes in phosphoinositide-linked signal transduction, we examined whether age-related differences in tracheal smooth muscle (TSM) contractility to carbachol (CCh) are associated with developmental changes in the production and metabolism of the second messenger, inositol 1,4,5-trisphosphate (Ins (1,4,5)P3). In TSM segments isolated from 2-wk-old and adult rabbits, both the maximal isometric contractile force and sensitivity (i.e., -logED50) to CCh (10(-10)-10(-4) M) were significantly greater in the immature vs. adult tissues (P less than 0.001). Similarly, Ins(1,4,5)P3 accumulation elicited by either receptor-coupled stimulation with CCh (10(-10)-10(-4) M) or post-receptor-mediated guanine nucleotide binding protein activation of permeabilized TSM with GTP gamma S (100 microM) was also significantly enhanced in 2-wk-old vs. adult TSM. Measurement of the activities of the degradative enzymes for Ins(1,4,5)P3 demonstrated that: (a) mean +/- SE maximal Ins(1,4,5)P3 3'-kinase activity was significantly reduced in the immature vs. adult TSM (i.e., approximately 71.7 +/- 6.0 vs. 137.8 +/- 10.0 pmol/min per mg protein, respectively; P less than 0.005); (b) by contrast, maximal Ins(1,4,5)P3 5'-phosphatase activity was significantly increased in the immature vs. adult TSM (i.e., 27.9 +/- 1.2 vs. 15.6 +/- 1.5 nmol/min per mg protein, respectively; P less than 0.001); and (c) the Km values for Ins(1,4,5)P3 5'-phosphatase were 14- and 19-fold greater than those for Ins(1,4,5)P3 3'-kinase in the 2-wk-old and adult TSM, respectively. Collectively, the findings suggest that the age-related decrease in agonist-induced rabbit TSM contractility is associated with a diminution in Ins(1,4,5)P3 accumulation which is attributed, at least in part, to ontogenetic changes in the relative activities of the degradative enzymes for Ins(1,4,5)P3.

Phosphatidylinositol:myo-inositol exchange activity in intact nerve endings: substrate and cofactor dependence, nucleotide specificity, and effect on synaptosomal handling of myo-inositol.

Micromolar concentrations of CMP produced a large increase in Mn2+-dependent phosphatidylinositol:myo-inositol exchange activity in isolated nerve endings or synaptosomes. The apparent Km for CMP was 2 microM, and that for myo-inositol was 38 microM. Only cytidine nucleotides were capable of enhancing activity, and this effect is probably specific for CMP, because the synaptosomal preparation rapidly converted CTP or CDP to CMP. Manganese did not affect the uptake of myo-inositol into the synaptosomal cytosolic fraction or myo-inositol levels. Determinations of myo-inositol specific activity showed that the Mn2+-enhanced labeling of phosphatidylinositol was not accompanied by a decrease of label content in free myo-inositol. This lack of an effect on intrasynaptosomal myo-inositol and the dependence of exchange on cytidine nucleotides whereas cytidine itself was previously found to be without effect show that for the bulk of Mn2+-dependent exchange activity, it is the myo-inositol in the incubation medium that is being directly incorporated into membrane-bound phosphatidyl-inositol. Because CMP dependence is the hallmark of exchange catalyzed by CDP-diacylglycerol:inositol phosphatidyl transferase, this enzyme is likely to be responsible for most of the exchange activity in synaptosomes. The strong affinity of this exchange system for CMP suggests that endogenous levels of this nucleotide might support Mn2+-dependent exchange in the absence of added nucleotide.

Effect of elevated potassium on phospholipid and inositol metabolism of isolated nerve endings.

Synaptosomes were isolated from rat cerebra, and incubated in the presence of labelled phosphate and inositol. When the potassium concentration of the medium was increased by replacing NaCl with KCl, there was a marked increase in phosphate labeling of phosphatidic acid (PA) and phosphatidylinositol (PI). This was evident with [K(+)] above 12 mM and peaked at about 40 mM KCl. In normal calcium buffers, phosphate labeling of PI but not PA declined sharply with [KCl] above 40 mM. In low calcium buffers, the phosphate labeling response was greatly attenuated for both lipids, but PI labeling did not decline at higher [K(+)]. The phosphate labeling response was confined to PA and PI, and was specific for the increase in [K(+)](0). The same response was seen in constant (105 mM) sodium buffers, and atropine had no effect. The specific radioactivity of ATP was increased by elevated potassium, but not enough to account for the increased labeling of PA. Further, this appeared to be a result of the loss of stored ATP rather than an increase in turnover. Increasing [K(+)](0) produced a decline in [(3)H]inositol incorporation into PI in parallel with the increase in its labeling by (33)PO(4). This was the same in constant sodium and in low calcium buffers. It could be attributed to an inhibition of synaptosomal uptake of labelled inositol from the medium. Synaptosomal inositol content was unaffected. Elevated potassium had a greater effect on PA labeling than on PI, and it was more effective in increasing phosphate labeling of PA than was acetylcholine (ACh). When ACh and elevated potassium were combined at their maximally effective concentration, they acted synergistically to stimulate phosphate incorporation into PA but elevated potassium blocked the increase in [(3)H]inositol incorporation into PI normally produced by ACh. These results indicate that elevated potassium and ACh act upon the same population of synaptosomes, but affect different biochemical steps. Elevated potassium probably effects phospholipid labeling by a calcium dependent increase in diglyceride production from lipids other than PA or PI.

High-performance liquid chromatography of phospholipids: quantitation by phosphate analysis.

Quantitation of individual phospholipids separated by HPLC from tissue extracts by colorimetric analysis of phosphate was investigated. Elution of inorganic phosphate and breakthrough of lecithin were determined using radioisotopes. A substance which interfered with sample phosphate determinations was found in the column eluant, and a method to minimize its effect was developed. This method allows accurate quantitation of individual phospholipids present at a minimum of 20 nmol phosphate.

CMP-dependent phosphatidylinositol:myo-inositol exchange activity in isolated nerve-endings.

Greatly enhanced manganese-dependent phosphatidylinositol:myo-inositol exchange activity was observed when isolated, intact nerve-endings were incubated with the nucleotide, CMP, suggesting that the enzyme, CDP-diglyceride:inositol phosphatidyl transferase, catalyzes this exchange. CMP, at 10 microM, produced as much myo-[2-3H] inositol incorporation into phosphatidylinositol as did 1 mM. This CMP-stimulated exchange activity may reside on the plasma membrane.

The effect of phentolamine on synaptosomal phosphatidylinositol in experimental galactose toxicity.

Abnormal myo-[2-3H]inositol incorporation into phosphatidylinositol has been found in phentolamine-treated synaptosomes that were isolated from the cerebral hemispheres of galactose toxic rats and incubated with [33P]Pi and myo-[2-3H] inositol. In galactose toxic rats phentolamine-stimulated myo-[2-3H]inositol labeling of phosphatidylinositol was 70% greater than in normal animals. This enhanced labeling of synaptosomal phosphatidylinositol in galactose toxic rats during stimulation with phentolamine is in marked contrast to the depressed myo-inositol labeling of phosphatidylinositol reported with acetylcholine stimulation.

High-performance liquid chromatography of phospholipids with UV detection: optimization of separations on silica.

Chromatography of phospholipids was performed on silica columns with detection by absorbance at 205 nm using mixtures of hexane--isopropanol--water in which the role of water and isopropanol in elution was investigated. One system was developed which provided adequate separation of most major phospholipid species. However, lipids with several ionizable groups were not well separated and gave multiple broad peaks. A second system was developed utilizing sulfuric acid for ion suppression. The behavior of phospholipids in this system was found to be dependent on the presence of quaternary ammonium, amino, or hydroxyl groups. Except for plasmalogen, phospholipids were recovered intact. This system was optimized to provide baseline resolution of essentially all phospholipid species commonly found in mammalian tissues.

Experimental galactose toxicity: effects on synaptosomal phosphatidylinositol metabolism.

Experimental galactose toxicity was induced by weaning rats onto an isocaloric 40% galactose diet. Synaptosomes were prepared from cerebra of rats at 2-9 weeks post-weaning and incubated with [33P][i and myo-[2-3H]inositol in the presence or absence of 0.2 mM-acetylcholine. The acetylcholine-stimulated [33P]Pi labeling of phosphatidylinositol and the changes in amounts of phosphatidylinositol were similar in the normal and galactose-toxic rats; however, acetylcholine-stimulated myo-[2-3H]inositol labeling of phosphatidylinositol was markedly decreased in the galactose-toxic rats. The impairment of acetylcholine-stimulated myo-[2-3H]inositol incorporation into phosphatidylinositol observed after 2 weeks on the diet did not vary after more prolonged exposure to galactose.

Evaluation of the binding of serotonin by isolated CNS acidic lipids.

Binding of serotonin by rat lipids was examined in an organic solvent-aqueous partition system. Only phospholipids and sulfatide were found to have appreciable activity; this technique was unsuitable for gangliosides due to their poor extractibility. Binding by phospholipid was abolished and that by sulfatide was greatly inhibited by increasing ionic strength in the aqueous phase. At an ionic strength of 0.3 M the apparent affinity of sulfatide for serotonin was about 3 X 10(3) M. Both tryptamine and 5-methoxytryptamine were much more effective than serotonin in inhibiting the binding of radioactive serotonin, suggesting that the observed binding is simply a charge neutralization with little specificity. Binding of serotonin by mixed brain gangliosides was examined in an equilibrium dialysis system. Without adequate precautions, the chemical lability of serotonin was found to produce spurious data when binding was assessed by the distribution of radiolabel. Binding of serotonin by ganglioside was also greatly inhibited by increasing ionic strength: at 0.3 M an apparent affinity of about 10(3) M was found. While dopamine did not inhibit the binding of radioactive serotonin, tryptamine, 5-methoxytryptamine, and serotonin were equally effective inhibitors.

Evaluation of continuous solvent extraction of organic acids from biological fluids.

We evaluated the efficiency of continuous solvent extraction with ether for the analysis of organic acids by gas chromatography using a representative group of organic acids and urine from several normal children. Variables examined were the time of extraction, volume of sample, and the quantity and chemical class of acid present. In terms of the decreased time required and invariant extraction of acids of pathologic significnace continuous solvent extraction compares favorably with the more time consuming albeit less discriminatory ion-exchange procedure. Continuous solvent extraction appears especially well suited for analysis of short chain aliphatic acids by gas chromatography.