Transamination and oxidative decarboxylation of L-isoleucine, L-alloisoleucine and related 2-oxo acids in perfused rat hind limb muscle.

Metabolism of L-isoleucine, L-alloisoleucine and corresponding 2-oxo acids in rat hind limb muscle was comparatively studied under steady-state perfusion conditions. At 0.5 mM L-[1-14C]isoleucine, apparent transamination and 2-oxo acid decarboxylation rates amounted to about 17 and 4 nmol/min per g of muscle, respectively. With L-allo[1-14C]isoleucine, the corresponding rates were about 5- and 10-fold lower, respectively. After addition of dichloroacetate (1-5 mM), the portion of (S)- and (R)-methyl-2-oxopentanoate undergoing further oxidative decarboxylation within the tissue was similarly increased by over 40%. In perfusions with 0.5 mM (R,S)-3-methyl-2-oxopentanoate and tracer doses of 1-14C-labeled (S)- or (R)-enantiomer, the 14CO2 production was comparable (about 0.5 nmol/min per g of muscle). Dichloroacetate caused a several-fold increase in 14CO2 release from either enantiomer, apparent 2-oxo acid transamination rates remaining unaffected. Indications for a racemization of 2-oxo acid were not obtained in the experiments. The results are discussed with respect to the appearance/disappearance of L-alloisoleucine in vivo and to the fact that (R)-3-methyl-2-oxopentanoate, but not L-alloisoleucine, can support growth of rats on a diet deficient in L-isoleucine.

Differential effect of dichloroacetate on branched-chain amino acid catabolism in perfused rat hindlimbs.

The effect of 1 mM and 5 mM dichloroacetate on the catabolism of branched-chain amino acids in isolated rat hindlimbs was investigated in perfusions with 0.5 mM 1-14C-labeled L-leucine or L-valine. The results demonstrate an increasing effect of dichloroacetate on the flux through skeletal muscle branched-chain 2-oxo acid dehydrogenase. A minor effect was observed with the high dichloroacetate concentration. Evidence is presented that this was essentially due to diminished pyruvate supply.

Distribution of progesterone-binding cytochrome P450 and steroid-17 alpha-hydroxylase/C-17,20-lyase within different compartments of the rat testis.

Progesterone-binding capacity of cytochrome P450 and rate of progesterone consumption by cytochrome P450-dependent hydroxylases are highest in the Leydig cell fraction but are also detectable in the non-Leydig cell fraction of interstitial cells as well as in seminiferous tubules from rat testis. The Leydig cell compartment, however, contributes only to a minor extent to the total progesterone binding and hydroxylation within the whole testis.

Effect of adrenergic agonists on phosphoinositide breakdown in rat skeletal muscle preparations.

Adrenergic regulation of phosphoinositide breakdown in rat skeletal muscle was investigated in 30-min incubations with 10 mM LiCl. In rat hemidiaphragms, prelabelled with D-myo-[2-3H]inositol, addition of alpha-agonists (epinephrine, norepinephrine, phenylephrine) induced a 5-8-fold increase of [3H]inositol monophosphate accumulation. This could be prevented by inclusion of alpha-antagonists (phentolamine, prazosin). beta-Agonists and/or beta-antagonists had no effect. Similar experiments with isolated flexor digitorum brevis muscle fibers yielded confirmatory results. Functional integrity of beta-receptor mediated processes was suggested by the beta-agonist-induced increase of glucose 6-phosphate in hemidiaphragms and cAMP in fiber preparations. The results indicate that phosphoinositide breakdown in differentiated rat skeletal muscle is, at least in part, under alpha-adrenergic control.

Tetrahydroisoquinoline alkaloids mimic direct but not receptor-mediated inhibitory effects of estrogens and phytoestrogens on testicular endocrine function. Possible significance for Leydig cell insufficiency in alcohol addiction.

Possible effects of various tetrahydroisoquinolines (TIQs) on rat testicular endocrine function were tested in vitro in order to prove whether these compounds, some of which have been claimed to accumulate in alcoholics, may be mediators of the development of Leydig cell insufficiency, a well-known side-effect of ethanol ingestion. TIQ effects on different levels of regulation of testis function were compared in vitro with estrogen effects, since both classes of compounds have structural similarities. Gonadotropin-stimulated testosterone production by testicular Leydig cells was inhibited by tetrahydropapaveroline and isosalsoline, the IC50 values (30 microM) being comparable to those of estradiol (3 microM), 2-hydroxyestradiol (10 microM), and the phytoestrogens, coumestrol (15 microM) and genistein (7 microM); salsolinol (85 microM) and salsoline (240 microM) were less effective, and salsolidine was ineffective. None of these TIQs interacted significantly with testicular estrogen receptor as analyzed by estradiol displacement. However, tetrahydropapaveroline, isosalsoline and salsolinol competitively inhibited (Ki 130-150 microM) substrate binding to cytochrome P450XVII, one key enzyme of androgen biosynthesis, with similar efficiency as the estrogens did (Ki 50-110 microM); salsoline and salsolidine were again much less effective. Since the efficient TIQ concentrations in this system are identical with those reported to generate central-nervous effects, it is concluded that certain TIQs may amplify peripheral inhibitory effects of ethanol on testicular endocrine function by their interaction with at least one enzyme of the androgen biosynthetic pathway.

Further characterization of estrogen binding to rat testis cytosol.

Binding of estradiol (E2), estriol (E3), RU16117, and moxestrol to testis cytosol from adult male rats was investigated. High-affinity binding sites were identified in the 8-9S region of sucrose density gradients; a second, high-capacity binding component in the 4S region was probably due to contamination with serum. Thermodynamic properties of the testicular estrogen binding site were quite similar to those of the uterine receptor. E2 had the highest affinity for testicular cytosol binding sites (Ka: E2 much greater than moxestrol greater than E3 greater than RU16117). Comparison of association rate (E2 greater than E3 greater than moxestrol = RU16117) and dissociation rate constants (E3 = RU16117 greater than E2 much greater than moxestrol) as well as studies in vivo revealed moxestrol as a long-acting and RU16117 as a short-acting compound. This difference may be useful for evaluation of the mediation of estrogen effects in the rat testis.

Steroid receptor pattern of human colorectal neoplasms.

Twenty-four colorectal carcinomas were assayed for estrogen and progestin receptors by sucrose gradient centrifugation. Most biopsies were either completely estrogen receptor negative or displayed low titers of specific estrogen binding. Only three tumors demonstrated moderate estrogen binding activity (10-18 fmol/mg cytosol protein). In four tumors specific progestin binding exceeded 20 fmol/mg protein. Only a minor subset of the binders sedimented at 8S. Twenty-one colorectal tumors examined for the presence of glucocorticoid receptors were found to be receptor positive, without exception. Biopsies from normal colorectal mucosa displayed minute quantities of specific 8S estrogen and progestin binding, but significant titers of specific glucocorticoid binding. Our findings support the hypothesis that estrogens and progestins are unlikely to play a major role in endocrine control of colorectal neoplasms. The role of glucocorticoids in growth control of colorectal neoplasms remains to be defined.

Gradient centrifugation analysis of steroid-hormone binding in human malignant melanoma.

Vertical tube rotor sucrose gradient centrifugation in the presence of the protease inhibitor sodium molybdate provide to give a deeper insight into the steroid hormone receptor status of human malignant melanoma (MM) as did previous studies using dextran-coated charcoal procedure only. As compared to endocrine dependent breast cancer, the oestrogen binding capacity of MM is low. Although in some biopsies fairly high concentrations of progestin binders were detected, the sedimentation properties were not receptor typic. Androgen binding was found to be negligible. In contrast, the presence of glucocorticoid receptors is a common feature of human MM. A human MM cell line was demonstrated to contain glucocorticoid receptors only.

Differential down-regulation and induction responses of testicular steroidogenic cytochromes P-450(cscc) and P-450(C17 alpha) to human choriogonadotropin.

Evidence is presented that the regulation of the cytochrome P-450(C17 alpha) of the steroid-17 alpha-monooxygenase and of the cytochrome P-450(cscc) of the cholesterol-monooxygenase by human choriogonadotropin (hCG) in vivo is mediated by differential mechanisms in the adult rat testis. An initial down-regulation of the cytochrome P-450(C17 alpha) but not of the P-450(cscc) can be demonstrated. Furthermore, induction of the cytochrome P-450(cscc) requires exposure to higher hCG doses (32% of the maximal induction rate of 43.7 pmol/(testis x d) are achieved with 4IU hCG/single dose) than induction of the P-450(C17 alpha) (59% of the maximal induction rate of 48.4 pmol/(testis x d) with 4IU hCG/single dose). Finally, induction of cytochrome P-450(cscc) starts faster after initiation of hCG treatment than induction of P-450(C17 alpha).

Estimation of kinetic parameters of androgen-synthesizing enzyme activities in superfused Leydig cells from rat testes: difference between endogenous and exogenous substrates.

Kinetic parameters of 3 beta-hydroxysteroid dehydrogenase/isomerase, steroid-17 alpha-monooxygenase, and steroid-17,20-lyase activities were estimated under steady-state conditions. Purified Leydig cells from rat testes were superfused with pregnenolone, progesterone, or 17 alpha-hydroxyprogesterone. The Km values for both the monooxygenase- and the lyase-catalyzed reactions were by factors of five to ten higher if analyzed with the exogenously added substrate (0.98 and 0.65 microM, respectively) than if calculated from endogenous substrate derived from a precursor (0.10 and 0.13 microM, respectively). This discrepancy may be explained by different substrate partition between the intra- and extracellular spaces and by different substrate concentration at the active site of the respective enzyme, depending on whether the actual substrate is of exogenous or endogenous source.

Identification and partial characterization of glucocorticoid receptors in human liver.

In normal human liver from adult males and females cytoplasmatic components which bind dexamethasone specifically and with high affinity, were demonstrated via dextran-coated charcoal assay, agar gel electrophoresis, isoelectric focusing and sucrose gradient centrifugation. The apparent dissociation constant of the dexamethasone-binder complex was found to be 1.7 +/- 0.3 x 10-(8) mol/l. The binding capacity was limited to 67.5 +/- 5.3 fmol/mg of cytosol protein. The ligand specificity for binding to these components indicated the requirement for glucocorticoids. It was shown by means of agar gel electrophoresis that the dexamethasone-binding entities migrate to the receptor region of the gel. The isoelectric point of the binding components in human liver cytosol was found to be pH 6.3. Sedimentation in sucrose gradients revealed the bulk of these components to be in the 7S region. It is concluded that the specific dexamethasone-binding entities in human liver have all the properties of glucocorticoid receptors.

Detection and partial characterization of glucocorticoid-binding components in hepatic metastases of human bronchial and gastric carcinoid tumors.

Metastases of human carcinoid tumors of bronchial and gastric origin have been analysed for glucocorticoid receptors using the fluorinated compound [3H] dexamethasone. In both tumors high concentrations of glucocorticoid-binding components were detected which based on several physicochemical characteristics were identified as glucocorticoid receptors in nature. The apparent dissociation constant of the dexamethasone-binder complex was found to be 1.8 x 10(-8) mol/l for the bronchial and 2.8 x 10(-8) mol/l for the gastric carcinoid tumor. Sedimentation in sucrose gradients disclosed the binding components to be macromolecules, sedimenting at about 7.S. By agar gel electrophoresis the molecular species binding dexamethasone were found to exhibit the same electrophoretic mobility as glucocorticoid receptors from well-known target organs. The ligand specificity for binding to these components indicated a requirement of active glucocorticoids as well as anti-glucocorticoids. A regulatory function of glucocorticoids in serotonin biosyntheses is discussed, which may be of potential clinical significance.

[Viable cells from human renal cell carcinoma: isolation procedure and analysis of hormone sensitivity (author's transl)]. / Vitale Nierenkarzinomzellen: Isolierungsmethode und Untersuchung der Hormonempfindlichkeit.

An enzymatic procedure for the isolation of metabolically active tumour cells from human renal cell carcinoma is described. The cells were suspended by a multistep incubation procedure of the tissue in the presence of collagenase (10 mg enzyme/g tumour wet weight) dissolved in a calcium-free buffer solution. About 90% of the isolated tumour cells were viable, as judged by routine trypan blue staining. Electron microscopic examination revealed tumour cells in various stages of dedifferentiation. The cells had retained their capability of protein synthesis. In short term experiments the effects of 17 beta-oestradiol and progesterone on the incorporation of [U - C] L-leucine into cellular proteins was studied; Progesterone was found to exhibit a slight tumour antianabolic or catabolic action. A 17 beta-oestradiol-dependent modulation of the rates of protein synthesis was not observed.

Further evidence for direct interaction of pyruvate dehydrogenase multienzyme complex with citric acid cycle based on nonlinearity of hill plots in presence of C2 and C4 substrate analogues.

1. Mammalian pyruvate dehydrogenase multienzyme complex (PDC) is measured with two different optical assays: (i) formation of p-nitro-acetanilid with arylamine-acetyltransferase and (ii) NAD reduction. 2. It is found that in contrast to the NAD assay system (ii) the coupled system (i) exhibits cooperativity with a Hill coefficient n = 3 over the whole range of substrate concentration. 3. The cooperative behaviour can be modified by presence of dichloroacetate (n = 2) and acetoin (n = 1----3). From additional measurements of PDC activity with toluene permeabilized mitochondria of fed and starved rats it is concluded that PDC activity in vivo is modified by changes in enzyme enzyme aggregation and interaction beside the known phosphorylation dephosphorylation mechanism.

The endocrine background of human renal cell carcinoma. III. Role of inhibitors of R 5020 binding in tumour cytosol.

Cytosol preparations obtained from 9 different human renal cell carcinomas were investigated for the eventual presence of progestin-binding inhibitors which might offer an explanation for the previous failure to demonstrate high receptor levels in the tumour tissue. The inhibitory potency of these preparations was estimated by measuring the decrease of R 5020 binding to uterine progestin receptors in the presence of tumour cytosol. Interestingly, cytosol from human renal cell carcinoma was found to contain progestin-binding inhibitors. The average inhibition of R-5020-receptor interaction amounted to 46%. In only 1 out of 9 cases, binding was completely suppressed. In order to circumvent the inhibitory reaction potentially occurring in cell-free systems, R-5020-binding studies additionally were performed in cell suspension. Out of 7 carcinomas studied using this assay system, 5 did not contain any specific progestin-binding entities. In 1 tumour, receptor-atypical non-saturable binding was observed. Only in isolated cells prepared from 1 other carcinoma, could slight indications for the presence of low concentrations of progestin receptors be detected.

The endocrine background of human renal cell carcinoma. IV. Glucocorticoid receptors as possible mediators of progestogen action.

In order to investigate whether progestins may trigger tumour regression by a mechanism involving the glucocorticoid receptor, human renal cell carcinomas obtained from 15 patients were analysed for cytoplasmic glucocorticoid-binding components, using [3H] dexamethasone. The existence of glucocorticoid binders could be demonstrated in 10 out of 15 tumours studied. The average binding capacity was calculated and found to be 7.1 fmol/mg of cytosol protein. The apparent dissociation specificity experiments clearly cell-free system amounted to 1.9 X 10(-8) mol/l. The ligand specificity experiments clearly indicated that binding to these receptors is not restricted to glucocorticoids alone. Progesterone and aldosterone turned out to be moderate competitors for dexamethasone binding. Medroxyprogesterone acetate, the compound widely used in hormone therapy of advanced renal cancer in man, was demonstrated to be one of the strongest inhibitors of [3H] dexamethasone. It is concluded that binding of medroxyprogesterone acetate to glucocorticoid receptors might represent the primary mechanism of action of the compound in causing tumour regression.

The endocrine background of human renal cell carcinoma. V. Binding of the highly potent androgen methyltrienolone (R 1881) by tumour cytosol.

The binding of methyltrienolone (R 1881, 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one), a highly active synthetic androgen, by cytosol preparations from human renal cell carcinoma was investigated. High-affinity, low-capacity binding components for R 1881 were detected in 3 out of 8 tumours analysed. The apparent dissociation constant of the R 1881-binder complexes was found to be in the range of 1.1-2.3 x 10(-9) mol/l. The number of binding sites in the positive tumours varied from 2.1 to 9.7 fmol/mg cytosol protein. Studies of binding specificity indicated a requirement for androgens. It is concluded that these binding components may be hormone receptors. If in consecutive studies this hypothesis holds true, progestins, most widely used in hormonal therapy of metastatic renal cancer, may possibly act on the tumour tissue by inhibiting the secretion of gonadotropins, thus lowering the blood levels of eventually growth-promoting.

Influence of insulin and glucose on pyruvate catabolism in perfused rat hindlimbs.

The effects of insulin and glucose on the oxidative decarboxylation of pyruvate in isolated rat hindlimbs was studied in non-recirculating perfusion with [1-14C]pyruvate. Insulin increased the calculated pyruvate decarboxylation rate in a concentration-dependent manner. At supramaximal insulin concentrations, the calculated pyruvate decarboxylation rate was increased by about 40% in perfusions with 0.15-1.5 mM-pyruvate. Glucose up to 20 mM had no effect. In the presence of insulin and low physiological pyruvate concentrations (0.15 mM), glucose increased the calculated pyruvate oxidation. This effect was abolished by high concentrations of pyruvate (1 mM). The data provide evidence that in resting perfused rat skeletal muscle insulin primarily increased the activity of the pyruvate dehydrogenase complex. The effect of glucose was due to increased intracellular pyruvate supply.

Evidence for the compartmentation of pyruvate metabolism in perfused rat skeletal muscle.

In rat hindlimbs perfused with [1-14C]pyruvate and 5 mM-dichloroacetate, the calculated apparent rate of pyruvate decarboxylation was decreased with increasing perfusate pyruvate concentrations. However, in the absence of dichloroacetate the apparent rate of decarboxylation increased under these conditions. Dichloroacetate enhanced [1-14C]pyruvate uptake, but decreased the specific radioactivity of effluent lactate. Glycogen metabolism remained unaffected. The results were not consistent with a common pyruvate pool, but provide evidence for the compartmentation of pyruvate metabolism.