Wolfgang Staib

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.

Effect of streptozotocin-induced hyperglycaemia on androgen-binding protein in rat testis and epididymis

SummaryIn adult male rats treated with streptozotocin 6 weeks before the experiments, androgen-binding protein concentration was increased in testicular tissue by 33% (p< 0.01) and reduced in epididymal tissue by 25% (p<0.005) in animals exhibiting severe hyperglycaemia as compared with animals remaining in normoglycaemia or moderate hyperglycaemia. Androgen-binding protein content was diminished in epididymal tissue by 40% (p<0.0005) but not changed in testicular tissue. If related to constant body weight, the sum of testicular and epididymal androgen-binding protein was identical in both normo- and hyperglycaemic animals. This disturbance in androgen-binding protein distribution may be the consequence of altered testicular secretion or impaired transport of androgen-binding protein from testes to epididymides.

A device for the liberation and determination of 14CO2.

A simple closed system for the serial determination of 14CO2 in small volumes of fluid samples, is described. The device consists of commercially available scintillation vials and silicone tube seals. 14CO2 is selectively liberated by citric acid and absorbed in a scintillation vial by Hyamine. Experiments on the effect of dichloroacetate on pyruvate dehydrogenase activity in rat hindlimbs perfused with [1-14C]pyruvate demonstrate the applicability of the method.

Specificity of steroid binding to testicular microsomal cytochrome P-450. Relation of steroid structure to type-I spectral responses after correction for hydrophobic association with the membrane

On the basis of the concept that steroids accumulate in the lipid phase of endoplasmic reticulum membranes and approach the active sites of steroidogenic cytochromes P-450 from a hydrophobic environment, we describe a procedure that allows calculation of spectral dissociation constants Ks for steroid interaction with testicular microsomal cytochrome P-450 after correction for hydrophobic association of ligand with the membrane. Maximal type-I spectral responses, apparent Ks, and partition into microsomal lipids were determined for 36 steroids, and corrected Ks values were derived from these primary data. Partition coefficients range from 60 to 62,000, and corrected Ks range from 60 microM to 25 mM steroid concentration in the lipid phase. Full spectral properties depend on a side-chain (1-3 carbon atoms) at the C17-position which may be hydrophobic or may bear a 20-oxo or 20 beta-hydroxy, but not a 20 alpha-hydroxy group. Binding constants are especially sensitive towards modifications of ring A structure (aromatization or 5 beta-, but not 5 alpha-reduction) and of the side-chain length. Androgens, with the exception of those bearing a 17 beta-acetoxy or 17 beta-propionyloxy group, are poorly accommodated by this cytochrome P-450.

Estimation of pyruvate decarboxylation in perfused rat skeletal muscle

By the determination of pyruvate dehydrogenase activity in tissue homogenates only limited information is gained on the actual metabolic flux. We therefore determined pyruvate decarboxylation in isolated rat hindlimbs non recirculating perfused with physiological (1-14C)pyruvate levels. On the basis of perfusate pyruvate specific activity a 14CO2 production of 15.8 +/- 0.5 nmol/min per g muscle was measured. However, by this method the actual pyruvate flux through the enzyme complex is underestimated by a factor of 7 due to the intracellular dilution of label.

Effect of human chorionic gonadotropin and estradiol in vivo on estradiol binder and cytochrome P-450 concentrations in rat testis

The effects of single administration to adult male rats in vivo of various amounts of human chorionic gonadotropin (HCG) and of single or repeated injections of estradiol on testicular cytoplasmic estradiol binder concentrations and on microsomal progesterone-binding cytochrome P-450 were compared. Half-life periods of HCG-induced loss of estradiol binder and cytochrome P-450 concentrations are identical (6 h) whereas a strong dissociation of these half-life periods are evident after chronic estradiol treatment (less than 2 h for the estradiol binder, about 35 h for cytochrome P-450). Depletion of cytoplasmic estradiol binder is not a sufficient condition for mediation of effects on cytochrome P-450 content. Rate of replenishment of microsomal cytochrome P-450 is similar after HCG or estradiol treatment. Both HCG- and estradiol-induced loss of cytochrome P-450 occur not only in Leydig cells but also in microsomes prepared from seminiferous tubules. Additional information is presented contradicting the hypothesis that loss of cytochrome P-450-dependent steroidogenic enzymes caused by HCG could be mediated by estrogens.

Oxidative determination of 14C-labeled 2-oxo acids

A simple and rapid assay for the determination of 1-14C- or U-14C-labeled 2-oxo acids is described. It is based on the selective and complete oxidation of the carboxyl group to 14CO2. Preceding purification procedures are not necessary. In rat hindlimb perfusion studies, the procedure was used to develop an indirect method for the estimation of the intracellular dilution of [1-14C]pyruvate and to determine the relationship between the transamination and decarboxylation rates of leucine in the perfused tissue by the use of tracer doses of L-[1-14C]leucine.

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 α‐agonists (epinephrine, norepinephrine, phenylephrine) induced a 5‐8‐fold increase of [3H]inositol monophosphate accumulation. This could be prevented by inclusion of α‐antagonists (phentolamine, prazosin). β‐Agonists and/or β‐antagonists had no effect. Similar experiments with isolated flexor digitorum brevis muscle fibers yielded confirmatory results. Functional integrity of β‐receptor mediated processes was suggested by the β‐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 α‐adrenergic control.

Differential down-regulation and induction responses of testicular steroidogenic cytochromes P-450(cscc) and P-450(C17α) to human choriogonadotropin

Evidence is presented that the regulation of the cytochrome P-450(C17α) of the steroid-17α-monooxygenase and of the cytochrome P-450(cscc) of the cholesterolmonooxygenase 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(C17α) but not of the P-450(cscc) can be demonstrated. Furthermore, induction of the cytochrome P-450(cscc) requires exposure to higher hCG doses (3270 of the maximal induction rate of 43.7 pmol/(testis x d) are achieved with 4 IU hCG/single dose) than induction of the P-450(C17α) (59% of the maximal induction rate of 48.4 pmol/(testis x d) with 4 IU hCG/single dose), Finally, induction ofcytochrome P-450(cscc) starts faster after initiation of hCG treatment than induction of P-450(C 17α).

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

Dichloroacetate Branched‐chain amino acid Pyruvate Perfused rat hindlimb