Formation of phosphatidylethanol in rat brain by phospholipase D

The mechanism of phosphatidyl [14C]ethanol formation was studied in rat brain microsomal fraction. Phospholipase D and base-exchange enzymes were assayed with [14C]ethanol as substrate. Phospholipase D was found to catalyse the formation of phosphatidylethanol. The reaction was dependent on sodium-oleate as activating factor. Phosphatidylethanol formation by phospholipase D has previously only been reported to occur in plant tissues. Stimulation of base-exchange enzymes with calcium in the presence of [14 C]ethanol did not induce any formation of phosphatidylethanol. These findings indicate that phosphatidylethanol formation in ethanol intoxicated rats is catalysed by phospholipase D.     

Norepinephrine-induced diuresis in chronically ethanol-treated rats

Previous research from this laboratory indicated that noradrenergic mechanisms might mediate ethanol diuresis. Experiments described here examined changes in sensitivity of noradrenergic mechanisms in animals chronically treated with ethanol. Norepinephrine hydrochloride (0-12 micrograms intracerebroventricularly) produced dose-dependent diuresis in control (dextrin maltose) and ethanol (8-11 g/kg/day) treated rats on the first day of treatment. Tolerance to ethanol diuresis was present after 10 days of ethanol treatment. Lack of responsiveness to norepinephrine-induced diuresis was evident only on the 20th day of treatment in both the ethanol and dextrin-maltose groups of rats. These results indicate a temporal dissociation between the tolerance to ethanol-induced and norepinephrine-induced diuresis and suggest that norepinephrine may not play a primary role in the development of tolerance to the diuretic action of ethanol.     

Glucosidase II from control and ethanol-treated rats. Purification and properties

Liver glucosidase II from control and ethanol-treated rats was purified and its physical and catalytic properties studied. No significant variation was found in the purification and properties of the enzyme from either source (ethanol-treated and control rats), except for activity after storage. Glucosidase II was purified to electrophoretic homogeneity from liver microsomes by solubilization, protamine sulphate precipitation, and anion exchange (DEAE-Sephacel) and affinity (Con A-Sepharose-4B) chromatographies. The native enzyme molecule is a tetramer (Mr 425 +/- 10 kDa) with identical subunits (Mr 106 +/- 3 kDa). Km values, determined at pH 6.8 for the p-nitrophenyl-glucosidase activity of glucosidase II from control and ethanol-treated rats, were 1.20 +/- 0.12 and 1.14 +/- 0.13 mM, respectively. The Arrhenius plot was linear, and the value for the apparent activation energy, calculated from this plot, was 56.64 kJ/mol. The p-nitrophenyl-glucosidase activity of glucosidase II from control and ethanol-treated rats was inhibited to the same extent by NH4+, by the divalent cations Ca2+ and Mg2+, and by methanol, ethanol, 2-propanol, n-propanol, isobutanol and n-butanol.     

Formation of biogenic amines by isolated kidneys of spontaneously hypertensive rats

Kidneys form dopamine (DA) from L-dopa and serotonin from L-5-hydroxytryptophan (L-5-HTP) via aromatic L-amino acid decarboxylase. We compared the ability of isolated perfused kidneys from adult (20-week-old) spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) to form these biogenic amines. Renal vascular resistance (RVR) was greater in perfused kidneys from SHR (n = 10) than WKY (n = 8) (p less than 0.01). Slight decreases in RVR were observed during L-dopa infusion but these were unrelated to DA formation. L-Dopa infusion was associated with greater DA output in SHR than WKY in both the renal venous and urinary effluents although the latter did not achieve statistical significance. L-5-HTP increased RVR to a greater degree in SHR than WKY kidneys. This was associated with larger quantities of serotonin in the urinary and venous effluents and greater pressor responses to exogenous serotonin in SHR than WKY kidneys; however, either parameter alone was not significantly increased. Our findings do not support a deficiency of intrarenal DA formation as a pathogenic factor for hypertension in SHR. Biogenic amine formation is as great if not greater in SHR than WKY kidneys and appears to contribute largely to the greater increases in renal resistance seen in SHR kidneys on infusion of L-5-HTP. Enhanced renal serotonin formation may elevate blood pressure, whereas enhanced renal DA formation would favor blood pressure lowering, perhaps as a compensatory mechanism.     

Hydroxyl-radical production and ethanol oxidation by liver microsomes isolated from ethanol-treated rats.

In order to distinguish between the mechanism of microsomal ethanol oxidation and hydroxyl-radical formation, the rate of cytochrome P-450 (P-450)-dependent oxidation of dimethyl sulphoxide (Me2SO) was determined in the presence and in the absence of iron-chelating compounds, in liver microsomes from control, ethanol- and phenobarbital-treated rats. Ethanol treatment resulted in a specific increase (3-fold) of the microsomal ethanol oxidation and NADPH consumption per nmol of P-450. A form of P-450 was purified to apparent homogeneity from the ethanol-treated rats and characterized with respect of amino acid composition and N-terminal amino acid sequence. Specific ethanol induction of a cytochrome P-450 species having a catalytic-centre activity of 20/min for ethanol and consuming 30 nmol of NADPH/min could account for the results observed with microsomes. Phenobarbital treatment caused 50% decrease in the rate of ethanol oxidation and NADPH oxidation per nmol of P-450. The rate of oxidation of the hydroxyl-radical scavenger Me2SO was increased 3-fold by ethanol or phenobarbital treatment when expressed on a per-mg-of-microsomal-protein basis, but the rate of Me2SO oxidation expressed on a per-nmol-of-P-450 basis was unchanged. Addition of iron-chelating agents to the three different types of microsomal preparations caused an 'uncoupling' of the electron-transport chain accompanied by a 4-fold increase of the rate of Me2SO oxidation. It is concluded that ethanol treatment results in the induction of P-450 forms specifically effective in ethanol oxidation and NADPH oxidation, but not in hydroxyl-radical production, as detected by the oxidation of Me2SO.     

Quantitative analysis of metabolism of hepatic triglyceride in ethanol-treated rats.

An acute intraperitoneal injection of ethanol (0.7 or 2.1g/kg body wt.) causes the reversible, dose-dependent accumulation of hepatic triglyceride in rats. By using a pulse of [14C]palmitate injected into a tail vein, it was found that ethanol (2.1g/kg)had no effect on the flux of unesterified fatty acid of serum (4.3mumol/min per 100g body wt.). However, either dose increased the fraction of the total flux going to liver from 0.16 to0.27 as rapidly as could be measured (30s), and it remained elevated until all ethanol had been cleared from the blood. The fraction of the total radioactivity in lipids of liver that was in triglyceride increased linearly for 1 h from 30 to 50% and there was a simultaneous decrease in phospholipid from 60 to 40%. The rate of synthesis of hepatic triglyceride derived directly from unesterified fatty acid of serum was calculated by using the flux rate of unesterified fatty acid in serum, the fractional hepatic uptake of this flux, and the percentage of liver fatty acid esterified to triglyceride. This contribution is related to the total synthetic rate of hepatic triglyceride (rate of accumulation+rate of release) to determine quantitatively how much of the developing fatty liver is attributable to increased uptake of unesterfied fatty acid of serum. At the higher dose of ethanol, about half of the accumulating triglyceride is derived from this source, whereas with the lower dose of ethanol it can account for all of the build-up.     

Mechanism of increased hepatic uptake of unesterified fatty acid from serum of ethanol-treated rats.

Studies were made on the mechanism by which livers of ethanol-treated rats take up an increased fraction of the total flux of unesterified fatty acid in serum. It was found that ethanol (0.7g/kg) causes a twofold rise in the serum content of liver, and that this serum is in rapid equilibrium with the general circulation. The fractional hepatic uptake from serum of group of compounds with varying uptake mechanisms and metabolic fates was studied in control and ethanol-treated animals. All the compounds tested, including unesterified fatty acid, showed an enhanced uptake when ethanol was given. For one of the compounds, carbon tetrachloride, a dose/response relationship was established between the amount administered, the amount taken up by liver, and the amount metabolized. These findings were interpreted to mean that this dose of ethanol causes the liver to receive an increased flow of blood, and as a result all compounds present and capable of being taken by liver are taken up at an increased rate. Hepatic blood flow was measured by a technique that monitors the rate of clearance of a colloidal lipid emulsion. It was found that ethanol increased hepatic blood flow by about 60%. This effect of ethanol on hepatic blood flow provides an explanation for the fatty liver and the synergistic effect between an acute dose of ethanol and carbon tetrachloride. A hypothesis to explain why a moderate dose of ethanol causes triglyceride to accumulate in liver is presented.     

Increased chemiluminescence and superoxide production in the liver of chronically ethanol-treated rats

Rats fed ethanol (1.74 +/- 0.12 g/day/100 g body wt for 12 weeks) showed a 45% increased microsomal production of O-2 (2.23 +/- 0.14 nmol/min/mg protein) and a 28% increased content of endoplasmic reticulum protein (26.8 +/- 1.4 mg/g liver). This could lead, at substrate saturation, to a 86% increased cytosolic production of O-2 which is not compensated by cytosolic superoxide dismutase levels that remain normal. It is claimed that this unbalance between O-2 production and superoxide dismutase leads to a peroxidative stress in agreement with the 54% increased spontaneous liver chemiluminescence (37 +/- 2 cps/cm2) measured in the ethanol-treated rats. Hydroperoxide-induced chemiluminescence was 57, 43, and 28% higher, respectively, in homogenates, mitochondria, and microsomes isolated from ethanol-treated rats as compared with controls. Vitamins E and A were more effective inhibitors of the hydroperoxide-stimulated chemiluminescence in the liver homogenates from ethanol-treated rats as compared with the effect on the homogenates from control animals. The results are consistent with a peroxidative stress in chronic alcoholism leading to increased lipoperoxidation and decreased levels of antioxidants.     

Formation of phosphatidic acid and subclasses of phosphatidylethanol in human neutrophils upon interleukin-8 stimulation

Previous studies showed that interleukin-8 (IL-8) stimulates phospholipase D hydrolysis of phosphatidylcholine to generate phosphatidic acid in human neutrophils. Phosphatidylcholine in these cells contains diacyl, alkylacyl and alkenylacyl subclasses. No studies have examined phospholipase D hydrolysis of the three subclasses of phosphatidylcholine in interleukin-8-stimulated neutrophils. We used a non-radioactive but very sensitive method to assess the relative distribution of the subclasses in phosphatidylethanol, which is derived from phospholipase D activity in ethanol-exposed neutrophils. We present evidence that the relative abundance of diacyl and alkylacyl subclasses in phosphatidylethanol is similar to that in phosphatidylcholine. Alkenylacyl subclass was also detectable in the phosphatidylethanol fraction, albeit as a minor subclass. Our findings suggest that phospholipase D catalyses the hydrolysis of diacyl, alkylacyl and alkenylacyl subclasses of phosphatidylcholine in neutrophils upon IL-8 stimulation.     

Formation of serotonin by rat kidneys in vivo

Renal formation of serotonin by decarboxylation of its amino acid precursor L-5-hydroxytryptophan (L-5-HTP) has been demonstrated with renal tissue homogenates and isolated perfused rat kidneys. Our objective in the present study was to determine whether the conversion of L-5-HTP to serotonin was associated with functional changes by kidneys in vivo. Renal clearance studies were conducted in anesthetized, volume-expanded male Sprague-Dawley rats receiving either saline (n = 9) or L-5-HTP (15 and 75 micrograms/min iv, n = 9). No change in mean arterial pressure was measured during infusions of L-5-HTP at either dose, whereas glomerular filtration rate (GFR), as measured by the clearance of inulin, and effective renal plasma flow (CPAH) decreased by 34 +/- 5% (mean +/- SE, P less than 0.001) and 26 +/- 7% (P greater than 0.07), respectively. Urine flow and sodium excretion decreased by 41 +/- 9% (P less than 0.01). Serotonin and 5-HTP were determined in urine and plasma using HPLC. High levels of 5-HTP were present in plasma, but not urine. Urinary serotonin increased in the rats receiving L-5-HTP without concomitant increases in plasma serotonin. More than 20% of the infused L-5-HTP was recovered in the urine as serotonin. The decarboxylase inhibitor carbidopa (20 micrograms/min) markedly reduced urinary serotonin excretion in the rats which received L-5-HTP and reversed the changes in GFR, CPAH, urine flow, and sodium excretion. Infusions of the amino acid precursor of L-5-HTP, L-tryptophan (n = 7), did not alter kidney function or increase plasma or urinary 5-HTP or serotonin levels. These results are consistent with the intrarenal formation of serotonin by renal decarboxylase with attendant alterations in renal hemodynamics and salt and water excretion.     

G-proteins in kidneys of spontaneously hypertensive rats

G(s alpha)-, total G(i alpha)- and G(q/11alpha)-protein concentrations were investigated by quantitative immunoblotting in membranes of total kidney, renal cortex and medulla as well as in cortical tubules and glomeruli of Spontaneously Hypertensive Rats (SHR) and normotensive Wistar Kyoto rats (WKY), aged 5 weeks, 3 or 8 months. We found that total kidney of 5 week old SHR possess less G(s alpha)-, G(i alpha)- and G(q/11alpha)-proteins than controls. For G(s alpha)-proteins, differences found in total kidney were mirrored both in cortex (tubules and glomeruli) and in medulla. Decreased G(i alpha)-concentrations were accompanied by lower tubular but higher glomerular levels, while medullar levels were also increased. Decreased G(q/11alpha)-concentrations were reflected in decreased glomerular and medullary concentrations. Kidneys of 3 month old SHR and WKY possessed similar concentrations of all G(alpha)-species. In 8 month old SHR similar G(i alpha)-, but decreased G(s alpha)-and G(q/11alpha)-concentrations were observed. The G(s alpha)-decrease was reflected in cortex and medulla, the G(q/11alpha)-decrease in the medulla. We conclude that the main strain-related differences in G(alpha)-concentrations are seen in prehypertensive SHR.     

The fluidity of plasma membranes from ethanol-treated rat liver

Summary Male Wistar rats were maintained for 35–40 days on a liquid diet containing 36% of calories as ethanol. Ethanol was replaced by carbohydrates in the isocaloric diet fed to control animals. The effect of ethanol consumption has been studied on the fluorescence polarization of rat liver plasma membranes and artificial lipid vesicles and on the lipid composition of the membranes. Fluorescence polarization in both membranes and vesicles was determined using DPH and TMA-DPH as fluorescence markers; from these data, the polarization term (r_o/r–1)^–1 and flow activation energy (E) were calculated. The ethanol consumption induces a more fluid environment within the membrane core of liver plasma membranes; the ethanol-fed rat membranes are more resistant to the in vitro effect of ethanol disordering the membrane structure. Vesicles obtained with lipids from either control membranes or ethanol-fed rat membranes were treated with ethanol and the changes in polarization paralleled to those exhibited by the membranes. The absence of phase transitions and of E changes was also shown in temperature-dependence studies. The lower cholesterol content found in ethanol-fed rat plasma membranes might be responsible for observed variations in the microviscosity.Abbreviation OG octyl -D-glucopyranoside     

Microwave thawing of frozen kidneys: A theoretically based experimentally-effective design

Equipment was designed and fabricated for uniformly thawing frozen canine kidneys using single-frequency electromagnetic radiation. Complete and uniform warming of frozen kidneys from ?70 to +14 °C over periods ranging from 1.5 to 4.5 min was achieved without “cooking” or experiencing thermal runaway. Dielectric measurements of renal slices (medulla and cortex) were performed as a function of temperature at a frequency of 918 MHz for a Me₂SO cryoprotectant concentration of 5% (0.7 M). Results of these measurements were then employed as an input to analytical computer models which were used to predict the internal field intensities and power distribution results for both frozen and thawed kidneys. From these predictions, a 918-MHz EM illuminator for thawing canine kidneys was designed and fabricated. Twenty-seven kidneys were thawed using this illumination system. Of these, excellent uniformity of thawing was achieved for 17 kidneys, good uniformity for 8 kidneys, and for only 2 kidneys was thawing uniformity fair to poor.     

Impaired prostaglandin release from the kidneys of salt-loaded and hypertensive rats

Prostaglandin (PG) release by the isolated perfused kidney of the rat has been stimulated by nor-adrenaline infusion and measured by bioassay. There was no basal output of PGE₂-like activity, but stimulated release reached mean concentrations of 9.1 ng/g kidney/ml perfusate in kidneys from female albino rats drinking water and 2.9 ng/g/ml in those from animals given 1.5% NaCl to drink. Kidneys from uninephrectomised animals with mock-clipped renal arteries released 7.3 ng PG/g/ml and those from rats with uninephrectomy and constricted renal arteries 3.3 ng/g/ml.     

Loss of oxidative phosphorylation in mitochondria isolated from kidneys of mercury poisoned rats

Mercurials at low concentrations suppress oxidative phosphorylation in isolated rat kidney mitochondria. The kidneys of rats administered toxic doses of HgCl₂ have been found to accumulate mercury selectively compared e.g. to liver. Mitochondria isolated from such kidneys are defective in coupled ATP synthesis. Providing the time subsequent to the injection of a toxic dose of HgCl₂ does not exceed a critical time, the defect in the mitochondria isolated from the kidneys of such poisoned rats is correctable by addition of Mg++ to the assay medium. Acute mercury toxicity in rats appears to be referable to the impairment of kidney function in turn caused by impairment of mitochondrial function.