Presence of the NHE3 isoform of the Na+/H+ exchanger in human gallbladder.

1. In man and in various animal species, absorption of NaCl from bile by the gallbladder mucosa is associated with luminal proton secretion. A similar absorption of NaCl in small intestine, colon and renal tubule is related, at least in part, to the presence of the NHE3 isoform of the Na+/H+ exchanger. This work was designed to find out whether NHE3 is also present in human gallbladder. 2. At surgery, 100-200 mg of the gallbladder wall was obtained from patients treated by cholecystectomy for gallstones. After isolation of the mucosa, total RNA was extracted and submitted to reverse transcription-polymerase chain reaction with two primers: 5'-AAGCCICTGGTGCAGTGGCTGAAGG-3' and 5'-GGAGTCCTTIAAGTCGGCIAAGCTGGGC-3', designed to amplify a sequence of 645 bp of rabbit NHE3 mRNA (642 bp in man). RNA from human liver and from rabbit heart, neither of which contain NHE3, and human ileal RNA, which does contain NHE3, were used as controls. 3. RNA extracted from the mucosal moiety of the gallbladder wall gave an amplification product of about 645 nucleotides. Controls gave the expected negative or positive results. Sequencing of the amplified RNA showed it was almost identical to previously determined sequences of NHE3 in other human tissues. 4. It is concluded that the mucosa of human gallbladder contains the mRNA of NHE3 isoform. This isoform could therefore play a role in sodium absorption from bile.

Quantitative multistandard RT-PCR assay using interspecies polymorphism.

The use of RT-PCR to quantify mRNA is often compromised by the variability of reverse-transcription and amplification reactions as well as by the difficulty of assessing the amount and/or the integrity of input RNA. Use of a competitor RNA or the coamplification of an endogenous standard are widespread methods of monitoring these steps. Taking advantage of both sequence conservation between homologous genes in related animal species and interspecific polymorphism, a protocol that may be regarded as a compromise between these two methods is described here. Total RNA samples, extracted from even minute amounts of tissue belonging to a first animal species, were supplemented with a constant amount of total RNA prepared from a second animal species, which thus acts as a multistandard source. The mixture was reverse-transcribed using hexa-random primers. Separate PCRs were then undertaken so that, for each mRNA of interest, products from both origins could be distinguished. Since the ratio between amplified mRNAs is constant in the standard preparation, an accurate normalization in the assay samples of most variations inherent to PCR is obtained. This protocol allows quantification of several mRNAs species, whose amounts may be very different, in a single cDNA preparation.

High-affinity interaction of long-chain fatty acids with serum albumin in nephrotic syndrome.

1. We have examined the effect of hypoalbuminaemia, a hallmark of nephrotic syndrome, on the albumin-fatty acid equilibrium in the plasma of 11 adult patients with nephrosis compared with 12 healthy subjects and six subjects with normoalbuminaemic hyperlipoproteinaemia. 2. We used a dialysis exchange rate method which allows the evaluation in relative terms of the binding affinity of albumin for plasma fatty acid and the fatty acid availability, tentatively equated with the unbound fatty acid fraction. 3. In nephrotic patients, an increase (P < 0.001) in albumin affinity for fatty acid was seen compared with healthy subjects, which was negatively correlated with albuminaemia (r = -0.69, P < 0.02). No change in fatty acid availability was seen for the group as a whole, but individual values showed a wide scatter, with the highest values in four patients with the highest fatty acid-albumin molar ratios. The increase in albumin affinity for fatty acid was specific to nephrotic syndrome since no such effect was seen in subjects with hyperlipoproteinaemia, who only showed a moderate increase (P < 0.01) in fatty acid availability. 4. The increased albumin affinity for fatty acid in nephrotic syndrome supports the hypothesis that an albumin component with lower affinity for fatty acid might filter out through the diseased glomerular membrane and leave the high-affinity albumin in plasma.

Triacylglycerol in biomembranes.

A better knowledge of the biochemical and biophysical properties of cell membranes has revealed fundamental concepts concerning the regulation of cell functions by intrinsic components of the lipid matrix. Membrane lipids exhibit high chemical heterogeneity, with hundreds of distinct chemical species; studies of structure-function relationships have unraveled new roles for an increasing number of these lipids as determinants of membrane structure, anchors for membrane-associated proteins or signalling agents. Recent observations have confirmed triacylglycerol (TG) as a quantitatively minor intrinsic membrane component which seems to play a specific role in important metabolic events such as cell stimulation or transformation and metastatic processes. The rapid turnover of the acyl chains into TG of cell membranes suggests an active metabolism. In the plasma membrane, TG appears to be implicated in the generation of transient non-bilayer domains suspected to be associated with specific cellular events. This paper summarizes the current information on TG metabolism and focuses on the potential role of this neutral lipid species on the structure and function of cell membranes.

Effects of ethanol on phospholipid acylation in rat erythrocytes: a model for a biochemical approach to membrane adaptation.

Challenging intact erythrocytes from naive rats with ethanol resulted in dose-dependent decreases in rates of acylation of phosphatidylcholine and phosphatidylethanolamine. In erythrocytes from ethanol-treated animals, the responses were of lesser magnitude, indicating a lower sensitivity to ethanol. This relative resistance, typical of the state of tolerance, was not associated with increased baseline rates of acylation of PC and PE, nor with changes in fatty acid specificity of acylation reactions. Taken together, the data suggest that (1) intact rat erythrocytes represent a reliable and easily reproducible model for studying biochemical correlates of the adaptive response to ethanol; (2) phospholipid acylation reactions are implicated in the initial sensitivity and subsequent acquisition of tolerance to ethanol in membrane erythrocytes; (3) on the basis of the measured acylation reactions, rat erythrocytes appear to develop tolerance, but not dependence, to ethanol.

Estrogen modulates phospholipid acylation in red blood cells: relationship to cell aging.

Ethinyl estradiol administered in vivo to female rats resulted in a mild anemia with a 120% increase in reticulocytosis. Consistent with a previous study, the red blood cell cholesterol-to-phospholipid molar ratio was decreased by 25%, whereas fatty acyl incorporation was significantly increased into phosphatidylethanolamine (PE) and not into phosphatidylcholine (PC), the major acyl acceptor in red blood cells. Analysis of this estrogen-dependent acylation increase as a function of cell age indicated that it was not expressed in reticulocytes but in erythrocytes and was associated with cell aging. Estrogen was further shown to increase the red blood cell susceptibility to peroxidation generated by incubation with H2O2. Altogether, the results suggest that estrogen indirectly increases phospholipid acylation in red blood cells by decreasing protection against oxidative damage, thereby favoring the action of endogenous phospholipases against oxidized substrates. This occurs predominantly in PE of oldest cells because 1) PE, being more unsaturated than PC, is more sensitive to oxidation, and 2) susceptibility to oxidation increases with cell age.

Triacylglycerol in the human erythrocyte: quantification and fatty acyl turnover in normal subjects and chronic alcoholics.

1. Triacylglycerol in erythrocytes from normal human subjects was estimated to average 2.7 +/- 0.7 nmol/10(10) cells, equivalent to 0.07% of total lipids or 0.3% of neutral lipids. 2. The specific activity of triacylglycerol labelling attained by incubating intact erythrocytes with [3H]oleic acid was 10 nmol/mumol, a value 20-fold higher than that of the highest labelled phospholipid, sphingomyelin; as isolated by ultracentrifugation over a density gradient, the youngest erythrocytes exhibited a labelling rate 10-fold higher than that of older cells. 3. The triacylglycerol content was not modified in erythrocytes from chronic alcoholics, whereas the mean rate of triacylglycerol labelling was 31% (P less than 0.05) higher than that of control subjects, and did not normalize 4 weeks after alcohol withdrawal. 4. These results indicate that triacylglycerol, although a quantitatively minor component, is one of the most active metabolites in the lipid matrix of the human erythrocyte membrane and appears to be implicated in the membrane response to antagonistic agents.

Pattern of the adaptive response of erythrocyte glycerolipid acylation processes during chronic alcoholism.

Challenging intact erythrocytes from normal human subjects with ethanol resulted in dose-dependent decreases in rates of acylation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) while triacylglycerol (TAG) acylation was stimulated. In erythrocytes from alcoholic subjects, the responses were of lesser magnitude, indicating a lower sensitivity to ethanol. This in vitro resistance, typical of the state of tolerance, was associated in erythrocytes from alcoholic subjects with increased baseline rates of acylation of PC, PE and TAG, suggesting high levels of glycerolipid fatty acid turnover. These results suggest that (1) intact human erythrocytes are qualitatively and quantitatively valid for studying the adaptive response to ethanol; and (2) chronic alcoholism is associated with increases in turnover rates of the acyl moieties of lipid membrane components regardless of the pattern of initial sensitivity to ethanol. Increased acylation rates during chronic alcoholism might modify the remodeling of the lipid matrix and thereby the membrane function.

Altered acylation of erythrocyte phospholipids in alcoholism.

The composition and metabolism of erythrocyte lipids were studied in 10 chronic alcoholic patients within 48 hr after discontinuation of alcohol intake and in 10 nonalcoholic control subjects. Chronic alcoholism produced no change in contents of cholesterol, total phospholipids, and proportions of phosphatidylcholine and phosphatidylethanolamine in erythrocyte phospholipids. The mean values of the rates of acylation of phosphatidylcholine and phosphatidylethanolamine with oleic acid were increased respectively by 59% (p less than 0.001) and 38% (p less than 0.05) as compared with the controls. There was no correlation between acylation rates and mean cellular volumes. Increases in acylation rates normalized over several weeks after alcohol withdrawal and were not related to a direct effect of alcohol on the intact erythrocyte, suggesting that these alterations result from ethanol-induced changes in the membrane during erythrocyte formation. The increased rates of acylation might modify the remodeling of the lipid matrix and thereby the membrane function.