Formation Of 3H2O Research Articles

Radiometric assay for cytochrome P-450-catalyzed progesterone 16α-hydroxylation and determination of an apparent isotope effect

In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16α-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, [1,2,6,7,16,17- 3H]progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16α position. The resulting [1,7,16- 3H]progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16α-hydroxylation was measured by the formation of 3H 2O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16α-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of [1,7,16- 3H]progesterone and [4- 14C]progesterone was employed.

Effect of glipizide on hepatic fructose 2,6-bisphosphate concentration and glucose metabolism

Glipizide raised, in a dose-dependent manner, the concentration of fructose 2,6-bisphosphate in hepatocytes isolated from 24-hour fasted rats and incubated in the presence of 10 mmol/L glucose. Simultaneously, the rate of l-lactate production, as well as the rate of 3H 2O formation from (3- 3H)glucose, increased markedly. The concentration of glipizide calculated as corresponding to the half-maximal effect in these metabolic parameters was 12 to 15 μmol/L. In hepatocytes isolated from fed rats, either normal or made diabetic by treatment with alloxan, glipizide inhibited the conversion of both (U- 14C)pyruvate and (U- 14C)lactate to ( 14C)glucose; an inverse correlation was established between hepatocyte fructose 2,6-bisphosphate levels and the rate of gluconeogenesis. The increase of fructose 2,6-bisphosphate concentration elicited by glipizide, which occurs without a significant modification of either 6-phospho-fructo 2-kinase activity or hepatocyte cyclic AMP levels, seems to be related to a significant accumulation of hexose 6-phosphates (glucose 6-phosphate and fructose 6-phosphate) in the hepatic cells.

Prostaglandin H synthase catalyzes regiospecific release of tritium from labeled estradiol

Prostaglandin H synthase (PHS) from ram seminal vesicle microsomes was found to catalyze the release of tritium ( 3H) from estradiol (E 2) regiospecifically labeled in position C-2 or C-4 of ring A but not from positions C-17α, C-16α, or C-6, 7. Formation of 3H 2O from ring A of E 2 is dependent upon native enzyme supplemented with either arachidonic acid, eicosapentaenoic acid, or hydrogen peroxide and proceeds very rapidly as do other cooxidation reactions catalyzed by PHS-peroxidase. The 3H-loss from ring A of E 2 reflecting oxidative displacement of this isotope by PHS increases linearly up to 100 μM under our conditions (8–45 nmol/mg × 5 min). Loss of tritium in various blanks is negligible by comparison. Indomethacin (0.07 and 0.2 mM) inhibited the PHS-dependent release of 3H 2O from estradiol but less efficiently than it inhibited DES-cooxidation measured in parallel incubations under similar conditions. Addition of EDTA (0.5 mM) had no effect on the regiospecific transfer of 3H from E 2 or on DES-oxidation; ascorbic acid (0.5 mM) or NADH (0.33 mM) clearly inhibited both reactions and to a similar extent. These data suggest that estradiol- 2 4 -hydroxylation can be catalyzed by PHS in vitro probably via its peroxidase activity and point to PHS as an enzyme that could contribute to catechol estrogen formation in vitro by tissue preparations in the presence of unsaturated fatty acids or peroxides.

Effects of Guanosine 3′,5′-Monophosphate on Glucose Utilization in Isolated Islets of Langerhans*

Dynamic changes in total glucose utilization in isolated islets of Langerhans of the rat were determined by quantitation of the formation of 3H2O from D-[5-3H]glucose. The addition of 8-bromo-cGMP (8-Br-cGMP) or monobutyryl cGMP to the islets during a linear phase of glucose utilization resulted in concentration- and time-dependent increases in glucose utilization. Effects of the analogs of cGMP on glucose utilization were noted as early as 5 min after the onset of stimulation in the presence of 10 mM glucose. 8-Br-cGMP also increased the utilization of 1 mM glucose within 20 min. Stimulatory effects of 8-Br-cGMP were observed in the presence of cycloheximide or N-acetylglucosamine. Neither 8-bromo-cAMP (8-Br-cAMP) nor monobutyryl cAMP induced significant changes in glucose utilization at 1 or 10 mM glucose. In the presence of 3-isobutyl-1-methylxanthine (IBMX), 8-Br-cGMP, but not 8-Br-cAMP, induced a rapid change in glucose utilization. N-Methyl-N'-nitro-N-nitrosoguanidine, which activates guanylate cyclase, also stimulated glucose utilization in the presence of IBMX by 3-fold. IBMX alone did not change glucose utilization. In contrast, 8-Br-5'-GMP reduced glucose utilization, whereas 8-bromoinosine 3',5'-monophosphate and 8-bromoguanosine did not change glucose utilization. Sodium bromide did not affect glucose utilization. Glucose-stimulated insulin release was potentiated by 8-Br-cGMP, whereas insulin release from islets incubated in the absence of glucose or the presence of glyceraldehyde or 2-ketoisocaproic acid was not altered by 8-Br-cGMP. Thus, glucose utilization in pancreatic islets is modulated by cGMP, and the secretory response to 8-Br-cGMP is glucose dependent.

Catalytic defect of medium-chain acyl-coenzyme A dehydrogenase deficiency. Lack of both cofactor responsiveness and biochemical heterogeneity in eight patients.

Medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCADH; EC 1.3.99.3) deficiency (MCD) is an inborn error of beta-oxidation. We measured 3H2O formed by the dehydrogenation of [2,3-3H]acyl-CoAs in a 3H-release assay. Short-chain acyl-CoA dehydrogenase (SCADH; EC 1.3.99.2), MCADH, and isovaleryl-CoA dehydrogenase (IVDH; EC 1.3.99.10) activities were assayed with 100 microM [2,3-3H]butyryl-, -octanoyl-, and -isovaleryl-CoAs, respectively, in fibroblasts cultured from normal controls and MCD patients. Without the artificial electron acceptor phenazine methosulfate (PMS), MCADH activity in fibroblast mitochondrial sonic supernatants (MS) was 54% of control in two MCD cell lines (P less than 0.05). Addition of 10 mM PMS raised control acyl-CoA dehydrogenase activities 16-fold and revealed MCADH and SCADH activities to be 5 (P less than 0.01) and 73% (P greater than 0.1) of control, respectively. Thus, the catalytic defect in MCD involves substrate binding and/or dehydrogenation by MCADH and not the subsequent reoxidation of reduced MCADH by electron acceptors. 20 microM flavin adenine dinucleotide (FAD) did not stimulate MCD MCADH activity in either the 3H-release or electron-transfer(ring) flavoprotein-linked dye-reduction assays. Mixing experiments revealed no MCADH inhibitor in MCD MS; IVDH activities were identical in both control and MCD MS. In postmortem liver MS from another MCD patient, 3H2O formation from [2,3-3H]octanoyl-CoA was 15% of control. When 3H2O formation was assayed with 200 microM [2,3-3H]acyl-CoAs, 15 mM PMS, and 20 microM FAD in fibroblast sonic supernatants from seven MCD cell lines, SCADH, MCADH, and IVDH activities were 72-112% (P greater than 0.1), 4-9% (P less than 0.01), and 86-135% (P greater than 0.1) of control, respectively, revealing no significant biochemical heterogeneity among these patients.

Inhibition by 2,5-anhydromannitol of glycolysis in isolated rat hepatocytes and in Ehrlich ascites cells.

2,5-Anhydromannitol decreases lactate formation and 3H2O formation from [5-3H]glucose in isolated rat hepatocytes metabolizing high concentrations of glucose. The inhibition of glycolysis is accompanied by a slight decrease in the cellular content of fructose-6-P and a more substantial decrease in the cellular content of fructose-1,6-P2, with no change in the content of glucose-6-P. The 3H2O release data and changes in hexosephosphate distribution indicate possible inhibitions at phosphofructokinase-1 and phosphoglucose isomerase. 2,5-Anhydromannitol also inhibits glycolysis in Ehrlich ascites cells, but the tumor cells, unlike hepatocytes, must be treated with 2,5-anhydromannitol prior to exposure to glucose to obtain the inhibition. The decrease in 3H2O formation from [5-3H]glucose and the metabolite pattern that results from the addition of low concentrations (less than or equal to 0.25 mM) of 2,5-anhydromannitol indicate an inhibition at phosphofructokinase-1 that cannot be attributed to a decrease in the cellular content of fructose-2,6-P2. Higher concentrations (greater than or equal to 0.5 mM) of 2,5-anhydromannitol cause a substantial decrease in the cellular content of ATP that is accompanied by decreases in the content of glucose-6-P and fructose-6-P and transient increases in fructose-1,6-P2. In Ehrlich ascites cells, 2,5-anhydromannitol is metabolized to 2,5-anhydromannitol mono- and bisphosphate. The inhibition of glycolysis caused by 2,5-anhydromanitol decreases with time, because the phosphorylated metabolites formed during the preliminary incubation in the absence of glucose are rapidly dephosphorylated during the incubation in the presence of glucose.

The role of phosphoenolpyruvate carboxykinase in muscle alanine synthesis.

3-Mercaptopicolinic acid (3-MPA) is reportedly a specific inhibitor of phosphoenolpyruvate (PEP) carboxykinase and has hitherto been used accordingly to elucidate the metabolic role of PEP carboxykinase in vitro and in vivo. We show that 3-MPA has multiple effects on intermediary metabolism in hemidiaphragms from 40 h-starved rats. It decreases the release of lactate + pyruvate and alanine in hemidiaphragms provided with no added substrate or with valine, leucine or isoleucine. Moreover, irrespective of the substrate provided (none, valine, leucine, isoleucine, glucose, acetate, oleate), 3-MPA decreases the [lactate]/[pyruvate] ratio. 3-MPA is without effect on 14CO2 production from [U-14C]valine, [1-14C]valine, [1-14C]leucine, [U-14C]isoleucine or [1-14C]oleate, but stimulates 14CO2 production from [U-14C]glucose and [1-14C]pyruvate and inhibits 14CO2 production from [1-14C]acetate. Glycolytic flux (measured as 3H2O formation from [5-3H]glucose) is stimulated by 3-MPA. It is concluded that 3-MPA has site(s) of actions other than PEP carboxykinase and that the putative role of PEP carboxykinase in alanine synthesis de novo in skeletal muscle from tricarboxylic acid-cycle intermediates and related amino acids requires reappraisal.

Adrenergic regulation of glucose metabolism in rat heart. A calcium-dependent mechanism mediated by both alpha- and beta-adrenergic receptors.

Epinephrine treatment of the perfused rat heart led to an increase in glucose uptake, detritiation of [5-3H] glucose, glycogenolysis, and the formation of lactate. The change in the rate of formation of 3H2O from [5-3H]glucose was slower to develop (commencing at approximately 30 s) than changes in cyclic AMP concentration, hexose-6-P concentration, and the phosphorylase a/(a + b) ratio which were maximal at 24 s. Epinephrine plus propranolol (alpha-adrenergic combination) treatment of the perfused heart also led to increases in glucose uptake, detritiation of [5-3H]glucose, and the formation of lactate, but these occurred without significant changes in cyclic AMP concentration, hexose-6-P concentration, or the phosphorylase a/(a + b) ratio. Half-maximal stimulation of glucose uptake occurred at 0.2 microM epinephrine, 1.5 microM methoxamine, and 1 microM isoproterenol. The increase in glucose uptake mediated by 1 microM epinephrine was blocked by 10 microM prazosin but unaffected by 10 microM propranolol. The increase in glucose uptake mediated by 10 microM epinephrine plus 10 microM propranolol was partly blocked by yohimbine and completely blocked by prazosin. A role for Ca2+ in the adrenergic regulation of glucose uptake was indicated by the sensitivity of the epinephrine dose curve to Ca2+ and the dependence of epinephrine on Ca2+. In addition the increases in glucose uptake mediated by 1 microM epinephrine, 1 microM epinephrine plus 10 microM propranolol, 1 microM isoproterenol, and by 10 mM CaCl2 were each blocked by the Ca2+ channel blocker nifedipine (1 microM). It is concluded that Ca2+-dependent alpha- and beta-adrenergic receptor mechanisms are present in rat heart for controlling glucose uptake. At submicromolar levels of epinephrine the predominant receptors utilized appear to be alpha 1.

Catechol estrogen formation and metabolism in brain tissue: comparison of tritium release from different positions in ring A of the steroid.

Catechol estrogens labeled with 3H at different positions in rings A and B of the steroid were synthesized by chemical or enzymatic methods, and their oxidative transformation by male rat brain microsomes was followed by the transfer of 3H into 3H2O. This reaction was shown to occur more readily with the catechol estrogens than with the parent steroid and was also influenced by the position of the radiolabel. Tritium was displaced less readily from C-1 than from C-2 or C-4 of the aromatic ring. Spermine, which is known to increase cytochrome P-450-mediated hydroxylation reactions, had no effect on the release of 3H from ring A of either estradiol or 2-hydroxyestradiol with rat brain microsomes in contrast to liver. Glutathione and other thiols were able to cause a rapid loss of 3H from labeled catechol estrogens, even in the absence of tissue, but in double label experiments with [4-3H]- and [4-14C]2-hydroxyestradiol, the isotope ratio in the recovered catechol estrogen was unchanged. The results illustrate some of the problems in determining accurately the metabolism of estrogens by measuring 3H2O formation when aromatic hydroxylation is involved and also highlight the possible interaction of the catechol estrogens with cellular nucleophiles such as glutathione.

Charcoal-catalyzed transfer of tritium into 3H 2O from regiospecifically-labeled 2-hydroxyestradiol in the presence of thiols

Charcoal was found to catalyze the release of 3H 2O from [1- 3H]2-hydroxyestradiol-17β ([1- 3H]2-OHE 2) or [4- 3H]2-hydroxy-estradiol-17β ([4- 3H]2-OHE 2) and this effect was shown to occur in the presence of glutathione or other thiols and to depend on the concentration of free steroid. The radiometric assay for measuring the formation of 3H 2O was not affected significantly by subsequent treatment of the incubation mixture with charcoal if the ratio of steroid to tissue (rat brain or liver microsomes) was low and only initial rates of 3H release were measured. 2-Hydroxyestradiol did not show the charcoal effect in the presence of tyrosinase, either when it was generated from its parent estrogen or added to the enzyme. The formation of 3H 2O from [4- 3H]2-OHE 2 in the presence of glutathione was inhibited by ascorbic acid but the addition of dextran or albumin did not protect the catechol estrogen from the charcoal-catalyzed loss of tritium. The reaction with glutathione and charcoal occurred even at 4°C but other adsorbants such as alumina, silica or hydroxylapatite were without effect.

Hepatic macromolecular covalent binding of the hepatocarcinogen 2,6-dinitrotoluene and its 2,4-isomer in vivo: modulation by the sulfotransferase inhibitors pentachlorophenol and 2,6-dichloro-4-nitrophenol.

The sulfotransferase inhibitors 2,6-dichloro-4-nitrophenol and pentachlorophenol were used to investigate the role of sulfate ester formation during the in vivo bioactivation of 2,4- and 2,6-dinitrotoluene (DNT). Male F-344 rats were administered one of the sulfotransferase inhibitors (40 mu mol/kg i.p.) 45 min prior to oral administration of 28 mg/kg [ring-14C]-2,4-DNT or [3-3H]-2,6-DNT and killed 12 h later. Pentachlorophenol had no significant effect on the urinary excretion of the benzyl glucuronide or benzoic acid metabolites of 2,6-DNT. The sulfotransferase inhibitors decreased the total hepatic macromolecular covalent binding of 2,4-DNT by 33%, and of 2,6-DNT by 69%. Purification of hepatic DNA by hydroxylapatite chromatography indicated covalent binding of 2,4- and 2,6-DNT at levels of 45 and 94 pmol equivalents/mg DNA, respectively. The sulfotransferase inhibitors decreased the binding of the hepatocarcinogen 2,6-DNT to hepatic DNA by greater than 95%. 2,6-Dichloro-4-nitrophenol decreased the binding of 2,4-DNT to DNA by greater than 84% while the decrease due to pentachlorophenol was 33%. These results suggest that sulfation is important in the biotransformation of 2,4- and 2,6-DNT to reactive metabolites which covalently bind to DNA. 3H2O was detected in the urine of rats administered [3-3H]-2,6-DNT. Pentachlorophenol decreased 3H2O formation to the same extent as it decreased the total hepatic macromolecular covalent binding of 2,6-DNT, suggesting that 3H exchange at the 3 position of 2,6-DNT occurs following sulfate ester formation. These results are consistent with a nitrenium-carbonium ion resonance of the sulfate ester-derived reactive intermediate of 2,6-DNT.

Novel method of evaluating biological 19-hydroxylation and aromatization of androgens

[19C 3H]Androstenedione of high specific activity has been prepared. In liver incubation the isotope was shown to be stable to biological processes other than 19-hydroxylation. Incubation of the new substrate with human placental microsomes yielded 3H 2O, 3HCOOH and estrogens devoid of radioactivity. The formation of 3H 2O and 3HCOOH was close to the expected 2:1 ratio indicating that the material can be used to discriminate between 19-hydroxylation which yields 3H 2O and aromatization which results in 3HCOOH. Comparison of the formation of 3H 2O from [1β,2β 3H]androstenedione and of 3HCOOH from [19C 3H 3]androstenedione in placental microsomal incubation showed that the aromatization of the former was 3.2 times faster indicating an isotope effect of that magnitude for the aromatization of [19C 3H] vs [19CH 3]androstenediones. The new substrate will be an effective probe and discriminant of both 19-hydroxylation and aromatization of androgens in vivo and in vitro , reactions which have been reported to be dissociated in specific tissues.

Ciglitazone, a new hypoglycemic agent. II. Effect on glucose and lipid metabolisms and insulin binding in the adipose tissue of C57BL/6J-ob/ob and -+/? mice.

The fat pads isolated from control and ciglitazone-treated C57BL/6J-ob/ob mice and their lean littermates (-+/?) were incubated in vitro with glucose-1-14C/-5-3H in the presence of insulin. The formation of 14CO2 and 3H2O and the levels of free fatty acids and glycerol in the medium and the incorporation of 14C and 3H into esterified lipids and free fatty acids in the fat pads were measured. The basal rates of glucose-1-14C/-5-3H metabolism per unit weight were increased in the fat pads of ciglitazone-treated mice, more pronouncedly in the treated lean than in the treated obese. The insulin-dependent effects were enhanced in the treated ob/ob mice. To see the dose-response of insulin, a second experiment was carried out in which portions of the fat pads were incubated in vitro with glucose-1-14C in the presence of 0-40 ng/ml insulin and isolated adipocytes were used to estimate for 125I-insulin binding. The basal rates of 14CO2 and 14C-lipids formation per unit weight of fat pads were increased in both treated obese and treated lean groups but insulin-dependent elevation was seen only in the treated obese group. Ciglitazone significantly increased insulin binding capacity at the low-affinity sites in the adipocytes of obese mice but not in those of lean mice. The data showed that ciglitazone increased the basal rate of glucose metabolism, lipogenesis, insulin receptor number, and post-receptor responses in the C57BL/6J-ob/ob mice; it increased the basal rate of glucose metabolism and lipogenesis but not insulin sensitivity in the lean mice.

Estrogen synthesis and metabolism in the hamster blastocyst, uterus and liver near the time of implantation

The steroidogenic potential of hamster tissues, just prior to implantation of the blastocyst in the uterus, was characterized by incubating blastocysts (14) and pieces of endometrium with [1,2- 3H]-androstenedione for 24 h. [ 3H]-2-Methoxyestradiol was synthesized, but intermediate estrogens were not found. To obtain a more quantitative assessment and comparison of steroidogenic activity, especially aromatase activity, in these tissues as well as in the uterine myometrium and liver and to increase the possibility of recovering estradiol, microsomes were isolated from 244 blastocysts and portions of the other tissues. Microsomes were incubated with [1α,2α- 3H]-testosterone plus [1β- 3H]-testosterone for 6 h. During this time [ 3H]-metabolites were synthesized by all tissues as indicated by HPLC. [ 3H]-Androstenedione was noted and values were higher than control levels (medium alone or microsomes from uterine flush fluid) in all samples but liver. [ 3H]-Estradiol was detected at an elevated level only in the blastocyst sample; however, addition of unlabeled estradiol during the subsequent incubation of endometrial, myometrial and liver microsomes increased the recovery of [ 3H]-estradiol. Identities of [ 3H]-2-methoxyestradiol from the first experiment and [ 3H]-androstenedione and [ 3H]-estradiol from the second experiment were confirmed by recrystallization. The formation of 3H 2O from [β- 3H]-testosterone was used as an index of aromatase activity. After subtracting control medium values, blastocysts were 24-fold more active (dpm/μg protein) than the endometrium and myometrium in synthesizing 3H 2O. While there was no difference in synthetic potential between endometrium and myometrium, aromatase activity in these tissues was greater than that of the liver. Microsomes from uterine flush fluid displayed no capacity for synthesizing 3H 2O indicating that the elevated blastocyst levels were not caused by contaminating endometrial cells. These results indicate that all of the tissues examined have the capacity to metabolize C 19-steroids to a variety of hormones, including estrogens, and further, that estrogen metabolism occurs rapidly in these tissues. This capacity may be important for providing a suitable hormonal milieu at the time of implantation.

S-Adenosylhomocysteinase: mechanism of inactivation by 2'-deoxyadenosine and interaction with other nucleosides.

S-Adenosylhomocysteinase (SAHase), a tetrameric enzyme, is inactivated by 2'-deoxyadenosine (2'dAdo) in a time-dependent process [Hirshfield, M. S. (1979) J. Biol. Chem. 254, 22-25]. It has been proposed that inactivation involves oxidation of 2'dAdo at C-3' by enzyme-bound nicotinamide adenine dinucleotide (NAD), subsequent proton abstraction at C-2', and elimination of adenine. This results in irreversible formation of enzyme-bound NADH and of adenine (Ade) and inactivation [Abeles, R. H., TAshjian, A. H., Jr., & Fish, S (1980) Biochem. Biophys. Res. Commun. 95, 612-617]. It has now been established that upon inactivation of SAHase with deoxy[2'(R)-3H]adenosine, 3H2O is formed. This is consistent with the proposed mechanism and of 3H2O release shows that maximally two of the four subunits participate in the reaction that results in 3H2O release. Reaction of SAHase with 2'dAdo results in reduction of two of the enzyme-bound NAD molecules. However, all four NAD molecules can be reduced by NaBH4, but only two are reduced to C-4 NADH. When the enzyme is inactivated with adenine-labeled 2'dAdo, radioactivity corresponding to 0.5-1.0 mumol of 2'dAdo binds tightly per micromole of subunit. This radioactive material is not removed from the enzyme by extensive dialysis but can be displaced by unlabeled 2'dAdo or Ade. After denaturation of the complex, radioactive material is released. Of this material 80-90% is adenine and less than 1% 2'dAdo. 2'dAdo also binds tightly to the enzyme reduced with NaBH4. Upon denaturation mostly adenine (80-90%) is released. Reaction of [2'-3H]2'dAdo with enzyme reduced with NaBH4 does not result in 3H2O formation. We conclude that the enzyme catalyzes the release of adenine from 2'dAdo by two mechanisms: One involves formation of 3'keto-2'dAdo and subsequent elimination of adenine. The other does not involve oxidation of 2'dAdo and probably is a hydrolytic process. It is proposed that the ability of the enzyme to carry out the hydrolytic process is a direct consequence of the manner in which 2'dAdo as well as the normal substrate binds to the enzyme, i.e., hydrogen-bond interaction of the protein with the adenine moiety and distortion of the ribose ring. When adenine-labeled adenosine is added to the enzyme, radioactivity corresponding to 0.5 mumol/mumol of subunit is associated with the protein after gel filtration. Of the radioactive material bound to the protein, 20% is adenine, 15% is adenosine, and the remaining radioactivity is present in unidentified compounds. The adenine bound to the enzyme does not participate in the catalytic process, and we conclude that it is bound to two of the subunits that do not participate in catalysis. Possible, these two subunits have a regulatory function. SAHase probably consists of two nonequivalent pairs of subunits. Only one pair participates in catalysis, but all four subunits probably bind Ado and 2'dAdo. We have confirmed the fact that the carbocyclic analogue of adenosine inactivates SAHase [Guranowski, A., Montgomery, J. A., 110-115]...

Selective inhibition of glucose-stimulated insulin release by dantrolene.

Dantrolene sodium, which interferes with excitation-contraction coupling by inhibiting the Ca2+ release from sarcoplasmic reticulum in muscle, was used to investigate the role of stored calcium in the stimulation of insulin release by various secretagogues. Insulin release was measured simultaneously with 45Ca2+ uptake or 45Ca2+ efflux from isolated rat pancreatic islets. Glucose-stimulated insulin release was inhibited by dantrolene (10-100 microM) as was glyceraldehyde- or mannose-stimulated release. In contrast, dantrolene failed to inhibit insulin release stimulated by leucine, arginine, ouabain, potassium, or 3-isobutyl-1-methylxanthine. Although dantrolene lowered glucose-stimulated 45Ca2+ uptake, nonspecific blockade of voltage-dependent Ca2+ channels may not be a primary action of dantrolene because K+-stimulated 45Ca2+ uptake was not inhibited. Glucose utilization (3H2O formation) was unaffected by dantrolene, whereas glucose oxidation (14CO2 production) was decreased. In the absence of Ca2+, the glucose-inhibited 45Ca2+ efflux was unchanged. At normal Ca2+, dantrolene inhibited glucose-stimulated 45Ca2+ efflux and veratridine induced insulin release. This suggests an interference with mobilization of beta-cell calcium stores. The selective action of dantrolene on insulin release makes it an interesting tool for further studies on stimulus-secretion coupling.

Decreased insulin binding, glucose transport, and glucose metabolism in soleus muscle of rats fed a high fat diet.

The relationship between diet and insulin sensitivity was examined in isolated soleus muscle from 10-wk-old lean Zucker rats. Rats were fed either a high fat or high carbohydrate diet that had 67% of calories as fat or carbohydrate, respectively, for 10 days. Plasma insulin but not plasma glucose concentrations were significantly elevated in high-fat-fed rats, indicating that a state of insulin resistance existed. The mechanisms responsible for the insulin resistance were studied by measuring insulin binding, 2-deoxyglucose uptake, and glucose metabolism in soleus muscle. The soleus muscle from the high-fat-fed rats bound significantly less insulin than high carbohydrate control rats under equilibrium binding conditions. The 35% decrease in insulin binding at maximal insulin concentrations resulted from a decrease in insulin receptor number but no change in receptor affinity. Maximal insulin-stimulated 2-deoxyglucose uptake was reduced in soleus muscle from high-fat-fed rats when compared with high carbohydrate controls. A decrease in postmembrane basal and insulin-stimulated glucose utilization was produced by high rat feeding and varied depending upon the pathway involved. An estimate of glycolysis (3H2O) formation from [5-3H]glucose) and glucose oxidation (14CO2 production from 14C-glucose) demonstrated a greater decrease in basal and insulin-stimulated utilization than in [5-3H]glucose conversion to [3H]glycogen. These results suggest that multiple sites are responsible for the observed insulin resistance in soleus muscle after high fat feeding.

Estrogen synthetase and C 17–20lyase activity in the fetal monkey gonad

In the rhesus monkey ( Macaca mulatta) the fetal testis appears to be more active than the fetal ovary in synthesizing steroids. This occurs despite the relative abundance of potential hormone precursors in the circulation. The possibility was investigated that this discrepancy in activity may be related to the activities of two rate-limiting enzymes, estrogen synthetase (ES) and C 17–20lyase (LA). ES and LA activities were characterized in monkey fetal gonads during late gestation from the rate of formation of 3H 2O from [1,2- 3H]-androstenedione (ES) and metabolism of [4- 14C]-17-hydroxyprogesterone to [ 14C]-androstenedione (LA). Ovaries and testes exhibited LA activity with apparent K m values of 3.4 × 10 −6M and 7.1 × 10 −7 M, respectively. Reaction rate was 1.8 ± 0.3 × 10 −3 nmol/min × mg protein ( n = 3) in the ovary which was significantly less than LA rate in the testis (8.3 ± 1.6 × 10 −3 nmol/min × mg −1 protein, n = 5). ES activity was detected only in the fetal ovary; it averaged 1.5 ± 0.4 × 10 −4 nmol/min × mg −1 protein ( n = 5). The apparent K m was 5.3 × 10 −8 M. Isolation of the [ 3H]-estrogens formed revealed the synthesis of estrone and estradiol-17β. During the 30 min incubation the amount of 3H 2O synthesized diverged from the combined quantity of tritiated estrone and estradiol-17β that was produced suggesting that other estrogens were synthesized. The reason fetal ovarian synthetic capacity is lower than that of the testis may be due to a low level of LA and ES activity in the ovary and additional metabolism of estrone and estradiol-17β.

Pathways of carbohydrate metabolism in peripheral nervous tissue. I. The contribution of alternative routes of glucose utilization in peripheral nerve and brain.

The activities of enzymes of the glycolytic route, the pentose phosphate pathway, the tricarboxylic acid cycle and lipogenesis have been measured in rat sciatic nerve and brain. Parallel studies have been made of the utilization of 14 C-labelled glucose and pyruvate in these two tissues. Comparison of the enzyme profiles and flux through alternative routes was based on activity relative to the rate of glucose phosphorylation as measured by the rate of formation of 3H2O from [2-3H]glucose. The contributions of the pentose phosphate pathway and lipogenesis to glucose utilization were substantially higher in sciatic nerve than brain. The relatively high activity of transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2) suggested a special role for these enzymes in sciatic nerve.

Measurement of tyrosine hydroxylase activity in serve terminals of rat hippocampus, hypothalamus, and striatum using an improved assay for tyrosine hydroxylase

The formation of 3H 2O from L-4- 3H-phenylalanine is used as an index of tyrosine hydroxylase activity in synaptosomes from rat hippocampus, hypothalamus, and striatum. The reactions are linear with respect to time (up to 20 min) and with respect to protein concentration (up to 0.2 mg/ml). Formation of 3H 2O from L-4- 3H-phenylalanine is inhibited by standard tyrosine hydroxylase inhibitors (α-methyl-p-tyrosine, L-3-iodotyrosine, dopamine, L-norepinephrine, and L-apomorphine) and by the tyrosine hydroxylase substrate L-tyrosine as well as by synaptosomal lysis. The blank 3H 2O produced from L-4- 3H-phenylalanine (0.02% of total DPM) is 10-fold less than the blank 3H 2O produced from L-3,5- 3H-tyrosine. The K m values of tyrosine hydroxylase for phenylalanine determined by the production of 3H 2O from L-4- 3H-phenylalanine are 3.1, 1.3, and 1.2 μm in hippocampal, hypothalamic and striatal synaptosomes respectively. The results indicate that analysis of 3H 2O formed from L-4- 3H-phenylalanine is a sensitive and reliable method for quantitating synaptosomal tyrosine hydroxylase activity from tissues with low levels of tyrosine hydroxylase such as synaptosomes from hippocampus and hypothalamus.