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P Churchill

Publications and source records attributed to P Churchill.

At least 37 records · Page 2Linked to original sources
PubMed1990

The role of adenosine in HgCl2-induced acute renal failure in rats.

It has been proposed that adenosine mediates the renal hemodynamic changes in acute renal failure (ARF) and that these changes are pathogenic in reducing glomerular filtration rate. Consistently, adenosine-receptor antagonists such as theophylline have been shown to have protective effects in several experimental models of ARF. The present experiments were designed to explore the potential role of adenosine in HgCl2-induced ARF in rats. In isolated perfused rat kidneys, HgCl2 increased adenosine production and induced a concentration-dependent vasoconstriction. However, the vasoconstriction was unrelated to adenosine production and was not antagonized by theophylline. During the initiation phase of HgCl2-induced ARF in intact rats (first 4 h after injection), theophylline failed to reverse the reduction in inulin clearance, and this failure could not be attributed to a loss of vascular responsiveness to adenosine, since N6-cyclohexyladenosine, a receptor agonist, produced a further reduction in inulin clearance. Furthermore, theophylline actually had deleterious effects during the maintenance phase of HgCl2-induced ARF in intact unanesthetized rats, as evidenced by higher mean serum creatinine values in theophylline-injected rather than in saline-injected rats, on both the second and third days after HgCl2 injection. Therefore HgCl2 acutely increases renal adenosine production, but increased adenosine does not mediate acute HgCl2-induced renal vasoconstriction, and adenosine-receptor antagonism does not have protective effects during the initiation or the maintenance phases of HgCl2-induced ARF in rats. These results provide no support for the hypothesis that increased adenosine mediates the hemodynamic changes in HgCl2-induced ARF.

Acute Kidney Injury↗
PubMed1989

Developmental regulation of D-beta-hydroxybutyrate dehydrogenase in rat liver and brain.

The activities of ketone-metabolizing enzymes in rat brain increase 3- to 5-fold during the suckling period before decreasing to the adult level after weaning. We have observed that a similar developmental pattern also exists for D-beta-hydroxybutyrate dehydrogenase (BDH) in rat liver. Utilizing antibodies prepared against the purified protein we determined that the changes in BDH activities in both brain and liver are due to changes in the amount of BDH in the mitochondria. In vitro translations of isolated RNA followed by immunoprecipitation revealed that the increase in BDH activity and content was correlated with an increase in the level of functional BDH-mRNA in both liver and brain.

Age Factors↗
PubMed1989

Regulation of D-beta-hydroxybutyrate dehydrogenase in rat hepatoma cell lines.

Quantitation of D-beta-hydroxybutyrate dehydrogenase (BDH) in normal rat hepatocytes was compared with that in two rat hepatoma cell lines, H4-II-EC3 and RLT-3C. BDH activity in normal rat hepatocyte mitochondria was 321 nmol/min/mg, which was greatly reduced to 10.7 nmol/min/mg and 1.7 nmol/min/mg in H4-II-EC3 and RLT-3C cell mitochondria, respectively. The cell growth rate and L-[35S]methionine incorporation rate showed that RLT-3C cells had the highest growth rate (32.4-h doubling time) and the fastest protein biosynthesis rate (2.65 x 10(5) cpm/min/10(6) cells). The H4-II-EC3 cell line grew more slowly (48.5-h doubling time) and had lower protein biosynthesis rate (1.46 x 10(5) cpm/min/10(6) cells). The protein synthesis rate in hepatocytes was 1.25 x 10(5) cpm/min/10(6) cells. These results suggest that there is a reciprocal correlation between BDH activity and cell growth and protein synthesis rates. Immunochemical quantitation of BDH showed the amount of BDH in H4-II-EC3 and RLT-3C cells was about 4.8 and 0.5% of that in normal rat hepatocytes, respectively. Quantitation of BDH by biosynthesis indicated that BDH content in H4-II-EC3 cells and RLT-3C cells was 9.3 and 4.0% of that of normal hepatocytes, respectively. Precursor BDH synthesized by in vitro translation primed with RNA of H4-II-EC3 cells or RLT-3C cells was 3.0 and 1.1% of that translated from normal rat hepatocyte RNA. These results suggest that the decrease in BDH content in hepatoma cells results from a decrease in functional BDH-mRNA. The coupling of a decrease in BDH activity with an increase in activity of succinyl-CoA: acetoacetyl-CoA transferase in hepatoma cells may play a role in generating additional energy required for the rapid growth of tumor cells.

Animals↗
PubMed1989

Isolation of a cDNA encoding functional Drosophila alcohol dehydrogenase in Escherichia coli and purification of the bacterially produced enzyme.

Because of the severe limitations on growing large quantities of Drosophila affinidisjuncta in the laboratory, direct purification of alcohol dehydrogenase (ADH) from this species has proven impossible. As an alternative source of this enzyme, a cDNA encoding functional ADH was isolated from a newly constructed cDNA library made from larval poly(A)-containing RNA. The cDNA was recovered by virtue of its hybridization to a previously isolated genomic ADH gene. Nucleotide sequence analysis confirmed the identity of the newly isolated cDNA. When the cDNA was inserted in the proper orientation downstream of the lac promoter on the vector pUC8, the cDNA directed the synthesis of functional ADH by the bacterial host. The bacterially produced enzyme was purified to homogeneity and used to elicit polyclonal antibodies in rabbits. The purified ADH has identical apparent subunit molecular weight to that of authentic ADH in larval fly extracts as determined by immunoblotting. Further, comparisons of the kinetic parameters of the bacterially produced enzyme and ADH activity in larval fly extracts indicate similar substrate preferences, pH dependencies, and Km values for 2-propanol and NAD. These results show that expression of a cDNA in Escherichia coli is a valid strategy for isolation of an ADH that would otherwise be difficult or impossible to purify.

Alcohol Dehydrogenase↗
PubMed1988

Mechanism of adenosine receptor-induced renal vasoconstriction in rats.

Adenosine analogues selective for the A1 subclass of adenosine receptors, such as N6-cyclohexyladenosine (CHA), induce vasoconstriction in the isolated rat kidney perfused at constant flow. Presumably, the vasoconstriction is mediated by increased Ca2+ concentration in renal vascular smooth muscle cells, but the mechanism by which A1 adenosine receptor occupation leads to increased Ca2+ is unknown. In the present experiments, the isolated, perfused rat kidney vasoconstricted in response to CHA, to K depolarization, and to BAY K 8644 (a Ca2+ channel agonist). All of these responses were completely blocked by methoxyverapamil, which suggests that CHA, like K depolarization and BAY K 8644, induces renal vasoconstriction by increasing Ca2+ influx through potential operated Ca2+ channels. The mechanism of action of CHA was different, however, in that pertussis toxin treatment blocked the response to CHA without affecting the responses to K depolarization or to BAY K 8644. Therefore, a pertussis toxin-sensitive step must intervene between occupation of A1 adenosine receptors on renal vascular smooth muscle cells and increased Ca2+ influx through potential-operated Ca2+ channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Renal effects of SK&F 82526 in anesthetized rats.

Dopamine affects renal hemodynamics, renal tubular functions, and the secretion of renin. We have studied the renal effects of SK&F 82526 (an agonist which is selective for the DA1 subclass of dopamine receptors) in anesthetized rats. Infused intravenously at 0.005 mumol/min/kg, this drug increased renal plasma flow and the clearances of PAH and insulin, effects which are consistent with decreased renovascular resistance. Concomitantly, urine flow and K excretion increased, and Na excretion tended to increase. All these effects of SK&F 82526 were antagonized by intravenous metoclopramide (1 mumol/min/kg). Despite its diuretic effect and despite its lack of effect on arterial blood pressure, SK&F 82526 increased arterial plasma renin concentration, suggesting a stimulatory effect on renin secretory rate. Taken together, our results demonstrate that the renal effects of SK&F 82526 mimic those of dopamine.

Animals↗
PubMed1985

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is a reliable internal standard for radiation-inactivation studies of membranes in the frozen state.

The target size of four soluble enzymes (beta-galactosidase, pyruvate kinase, alcohol dehydrogenase, and glucose-6-phosphate dehydrogenase) in the presence or absence of subcellular membrane fractions has been determined by the radiation-inactivation method using samples in the frozen state. For each of the four enzymes, full activity was recovered after freezing and thawing in the absence of radiation. We found minimal (less than 20%) binding of the enzymes to either submitochondrial vesicles or sarcoplasmic reticulum vesicles. Under the conditions tested, beta-galactosidase, pyruvate kinase, and alcohol dehydrogenase exhibited target sizes which varied according to the experimental conditions, i.e., the buffer selected and also the presence or absence of membrane preparations. For these tetrameric enzymes, the target sizes were generally comparable to either a monomer or a dimer. By contrast, the target size of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was found to be essentially invariant when frozen in a variety of buffers and in the presence or absence of either cryoprotectant (sucrose or glycerol) or different membrane preparations. The target size from 19 separate determinations gave an average value of 104 +/- 16 kDa, which is comparable to the molecular weight of the enzyme (104 kDa). We conclude that glucose-6-phosphate dehydrogenase from L. mesenteroides is a reliable internal standard for radiation-inactivation studies of membrane preparations in the frozen state.

Animals↗
PubMed1984

Interaction of liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in the presence and absence of lipid.

Phospholipid has been reported to be necessary for optimal catalytic activity of a number of mammalian cytochrome P-450 (P-450) systems. We also confirm that a number of individual phospholipids and mixtures, used as soluble monomers or phospholipid vesicles, show activation of 7-ethoxycoumarin O-deethylase activity by an enzyme system composed of rat liver microsomal P-450PB-B and NADPH-P-450 reductase. However, by preincubating a mixture of P-450 and NADPH-P-450 reductase at high concentrations, optimal activity can be obtained in the absence of phospholipid. The catalytic activity of the complex formed is concentration dependent in the absence of lipid or in the presence of soluble lipid. The activity in phospholipid vesicles is optimal and concentration independent. The apparent Km for NADPH-P-450 reductase in P-450-dependent oxidation systems is lowered severalfold in the presence of phospholipid. The apparent Km for the P-450 substrate, 7-ethoxycoumarin, and the temperature dependence of 7-ethoxycoumarin O-deethylase activity were unaffected by the addition of phospholipid to a preformed complex of P-450PB-B and NADPH-P-450 reductase. The effect of lipid on a number of other P-450 isozymes was also examined and in no case did lipid enhance the catalytic activity of the preformed complex. These results lead to the conclusion that the major effect of phospholipids in P-450-based enzyme systems is the facilitation of an active P-450:NADPH-P-450 reductase complex. This is the first report that maximum P-450 supported monooxygenase activity can be obtained in the absence of phospholipid.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1983

Influence of diabetes on rat liver mitochondria: decreased unsaturation of phospholipid and D-beta-hydroxybutyrate dehydrogenase activity.

Liver mitochondria and submitochondrial vesicles have been prepared from rats made diabetic by treatment with streptozotocin (diabetic membranes). The membranes were characterized in terms of phospholipid and fatty acid composition, electron transport functions, and D-beta-hydroxybutyrate dehydrogenase activity and compared with mitochondria and submitochondrial vesicles prepared from control animals (control membranes). No change in the phospholipid composition (44% lecithin, 35% phosphatidylethanolamine, and 21% diphosphatidylglycerol) was found, but a marked alteration in fatty acid composition of both the total phospholipid and lecithin occurred within 3 weeks after streptozotozin treatment and persisted thereafter. In lecithin, the 18:1/18:0 ratio decreases approximately 33% and the 20:4/18:2 ratio decreases approximately 55%. D-beta-hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which has a specific requirement of lecithin for function. In diabetic membranes, there is a progressive decrease in D-beta-hydroxybutyrate dehydrogenase activity with time after streptozotocin treatment to about 40% of control value at 15 weeks. In contrast, succinate oxidase and succinate- or NADH-cytochrome c reductase activities remain essentially unaltered. Further, the Arrhenius plot characteristics differ for D-beta-hydroxybutyrate dehydrogenase in diabetic membranes as compared with control membranes, in that the break point of the biphasic plot increases from 20 +/- 1 degree C in controls to 29 +/- 1 degree C in samples from diabetic animals. The change occurs about 3 weeks after streptozotocin treatment and is correlatable with the increased saturation of the fatty acid moiety of the phospholipids. The observed changes in D-beta-hydroxybutyrate dehydrogenase function and phospholipid composition were prevented by administration of insulin to the diabetic animals and are therefore referable to insulin insufficiency.

Animals↗
PubMed1983

Basis for decreased D-beta-hydroxybutyrate dehydrogenase activity in liver mitochondria from diabetic rats.

Liver mitochondria from rats made diabetic with streptozotocin have a reduced level of D-beta-hydroxybutyrate dehydrogenase (BDH) activity and decreased ratios of oleic/stearic and arachidonic/linoleic acids in the phospholipids of the mitochondrial membrane. This altered activity and lipid environment result from insulin deprivation since maintenance of the diabetic rats on insulin leads to normal characteristics (J.C. Vidal, J.O. McIntyre, P.F. Churchill, and S. Fleischer (1983) Arch. Biochem, Biophys. 224, 643-658). In the present study, the basis for the reduced enzymatic activity of this lipid-requiring enzyme was analyzed using three approaches: (i) Purified D-beta-hydroxybutyrate, dehydrogenase was inserted into membranes from mitochondria, submitochondrial vesicles, and mitochondrial lipids extracted therefrom. The activation was the same and optimal irrespective of whether the preparations were derived from normal or diabetic rat liver. Therefore, the decreased activity does not appear to be referable to an altered lipid composition. (ii) BDH activity can be released from the mitochondria by phospholipase A2 digestion. The released activity was proportional to the endogenous activity in the submitochondrial vesicles from normal and diabetic membranes. (iii) The BDH activity in submitochondrial vesicles was titrated by inhibition with specific antiserum. Less enzyme was found in mitochondria from diabetic rats as compared with those from normal animals. Hence, the lowered enzymatic activity is due to decreased enzyme in the mitochondrial inner membrane and not to the modified lipid environment.

Animals↗
PubMed1983

Target size of D-beta-hydroxybutyrate dehydrogenase. Functional and structural molecular weight based on radiation inactivation.

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which is localized on the inner face of the mitochondrial inner membrane. The apoenzyme has been purified to homogeneity from beef heart; it is devoid of lipid and inactive. It can be functionally reconstituted with lecithin or phospholipid mixtures containing lecithin. The active form of the enzyme is the enzyme-phospholipid complex. Classical target analysis of radiation-inactivation data has now been used to determine the molecular size of the enzyme both in the native membrane (submitochondrial vesicles) and in the reconstituted enzyme inserted into phospholipid vesicles containing lecithin. For both forms of the enzyme, we find the same molecular size, approximately 110,00 daltons. This size is consistent with a tetramer. Radiation results in fragmentation of the polypeptide and the destruction of the polypeptide correlates with loss of enzymic function. A similar size is obtained when purified D-beta-hydroxybutyrate dehydrogenase is inserted into a nonactivating mixture of phospholipid (i.e. in the absence of lecithin). We conclude that: 1) the native enzyme in submitochondrial vesicles and the purified active enzyme in phospholipid vesicles are the same size, approximating a tetramer; 2) radiation of D-beta-hydroxybutyrate dehydrogenase results in loss of activity and fragmentation of the polypeptide; and 3) the role of lecithin in activation of D-beta-hydroxybutyrate dehydrogenase is unrelated to determining oligomeric size of the enzymes since both active and nonactive forms exhibit the same structural size.

Animals↗
PubMed1983

Activation of D-beta-hydroxybutyrate apodehydrogenase using molecular species of mixed fatty acyl phospholipids.

D-beta-Hydroxybutyrate apodehydrogenase is a lipid-requiring enzyme with a specific requirement of lecithin for enzymatic function. The purified enzyme which is devoid of lipid can be reactivated with lecithin or mixtures of natural phospholipid-containing lecithin. However, it is mitochondrial phospholipid which activates the enzyme optimally and with kinetic parameters similar to that of the native membrane-bound enzyme. Mitochondrial phospholipid consists of three classes of phospholipid (lecithin:phosphatidylethanolamine:diphosphatidylglycerol in a ratio of approximately 2:2:1 by phosphorus); each class consists of a multiplicity of different molecular species due to diversity in the fatty acyl substituents. In this study, we have synthesized defined molecular species of mixed fatty acyl phospholipids to evaluate whether multiplicity of phospholipid molecular species are essential for optimal reactivation. We find that: 1) ternary mixtures of single molecular species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylpropan-1,3-diol in the liquid crystalline state mimic the optimal reactivation of the enzyme obtained with mitochondrial phospholipids; 2) although some negatively charged phospholipid appears necessary for optimizing the efficiency of activation, diphosphatidylglycerol can be replaced by phosphatidylpropan-1,3-diol, another negatively charged phospholipid; and 3) biphasic Arrhenius plots can be correlated with the liquid crystalline and gel states of the phospholipid.

Animals↗
PubMed1983

Renal effects of methoxyverapamil in anesthetized rats.

The purpose of these experiments was to determine the renal effects of methoxyverapamil (D-600). Three groups of rats were anesthetized with sodium pentobarbital and given 0, 0.85 or 1.69 nmol/min of methoxyverapamil i.v. Increases in urine flow and Na, K and Ca excretory rates occurred, in an apparently dose-dependent manner. Plasma Na and arterial renin concentration decreased at both doses and, at the higher dose, mean arterial blood pressure and effective renal plasma flow decreased while plasma K increased. Plasma Ca, glomerular filtration rate, filtration fraction and total renal plasma flow were not affected. The findings that methoxyverapamil increased urine flow and electrolyte excretion without changing glomerular filtration rate are consistent with the hypothesis that methoxyverapamil acts directly on tubular reabsorptive mechanisms. These effects, and the effect on plasma renin concentration, could contribute to the beneficial effects of this and other Ca entry antagonists in the treatment of hypertension.

Anesthesia, Intravenous↗
PubMed1983

The effect of denervation diuresis on the severity of glycerol-induced acute renal failure in rats.

The results of several previous studies suggest that high urine flow rate has a protective effect in several models of experimentally induced acute renal failure. However, the methods employed to alter urine flow precluded a paired experimental design, left kidney vs. right kidney in the same experimental animal. The purpose of the present studies was to determine whether the unilateral diuresis that results from renal denervation has a protective effect on that kidney in glycerol-induced acute renal failure. Eight groups of rats were studied. The left kidneys of all were either denervated or sham-denervated; 3 to 5 days later, rats were injected with either 150 mM NaCl (controls) or glycerol; then either 3 or 24 hr after injection, renal function was measured in acute experiments. In saline-injected rats, the inulin clearances of right and left kidneys were virtually identical, whether the left kidney was denervated or not; however, in rats with denervated left kidneys, left kidney urine flow exceeded that of the right kidney whereas the opposite was true in rats with sham-denervated left kidneys. In contrast with saline-injected rats, there were highly significant differences in inulin clearances of right and left kidneys in glycerol-injected rats at both 3 and 24 hr after injection; left kidney inulin clearance exceeded that of the right in rats with denervated left kidneys, whereas right kidney inulin clearance exceeded that of the left in rats with sham-denervated left kidneys. Since the kidney with the higher urine flow had the higher inulin clearance at 3 and 24 hr after glycerol, whether or not the kidney was denervated, these results suggest that urine flow per se has a protective effect in glycerol-induced acute renal failure in rats.

Acute Kidney Injury↗
PubMed1982

Reactivation of D-beta-hydroxybutyrate dehydrogenase with short-chain lecithins: stoichiometry and kinetic mechanism.

D-beta-Hydroxybutyrate dehydrogenase (BDH), purified as soluble, lipid-free apoenzyme (inactive) from either beef heart or rat liver mitochondria, can be reactivated by short-chain lecithins in the monomeric state. The enzyme was reactivated with dihexanoyl- [PC(6:0)], diheptanoyl- [PC(7:0)], and dioctanoyllecithins [PC(8:0)]. The titration curves of enzyme activity as a function of the phospholipid concentration are consistent with a model in which the enzyme contains two identical, noninteracting lecithin binding sites. The simultaneous occupation of these sites (via an equilibrium random mechanism) is required to activate the apoenzyme. Similar results were obtained with both rat liver and beef heart apoenzymes. The maximal velocities obtained with the different lecithins were similar [110-140 mumol of NAD+ reduced min-1 (mg of protein)-1]. The KL values (the apparent dissociation constants of the lecithin-site complexes) were 1.2 X 10(-4) M [PC(8:0)], 1.5 X 10(-3) M [PC(7:0)], and 4.5 X 10(-3) M [PC(6:0)] at 37 degrees C. This was confirmed by using phospholipase A2 to compete with the dehydrogenase for the lecithin monomers. Comparison of the delta G degrees values for complex formation with the different lecithins shows an average contribution of approximately 2.4 kJ/mol (0.9RT) per CH2 group. The interaction of the apolar moiety of lecithin with the protein seems to be essential for effective binding of phosphatidylcholine to apoBDH. The delta G degrees values, when combined with the estimated delta H degrees values, suggest that the binding of lecithin to the apoenzyme is approximately 60% enthalpy and approximately 40% entropy driven.

Animals↗
PubMed1981

Renal vascular and tubular effects of vanadate in the anesthetized rat.

The purpose of these experiments was to establish the renal vascular and tubular effects of vanadate. Three groups of adult rats were anesthetized with Na pentobarbital and given an i.v. infusion of a physiological saline solution at 0.055 ml/min. After a 60-min equilibration period, mean arterial blood pressure was measured, as were clearances of para-amino-hippuric acid (CPAH) and inulin (Cin), urine flow (V) and osmolality (Uosm) and the excretion rates of Na (UNaV), of K (UKV) and of Ca (UCaV). After the initial measurements, the i.v. infusion of physiological saline was continued in group 1 (controls), while vanadate was added to the saline and infused at 0.07 and 0.14 microM/min in groups 2 and 3. Then, all measurements were repeated. Compared with the control group, the following dose-dependent changes were observed in the vanadate-infused rats: increased mean arterial blood pressure, decreased CPAH (effective renal plasma flow), decreased Cin (glomerular filtration rate), increased V, UNaV and UCaV and decreased Uosm and UKV. Both the renal vascular and renal tubular effects of vanadate are similar to the renal effects of ouabain, previously reported by others. These effects can be explained by inhibitory effects of vanadate on Na,K-adenosine triphosphatase (ATPase) and/or Ca/ATPase activities of renal vascular and tubular cells.

Animals↗