Hubert Clauser

ADP-dependent inhibition of sarcosomal adenine nucleotide translocase by N-ethylmaleimide

It seems presently established that ADP** induces structural modifications in mitochondria, independently of its action on the oxidophosphorylating system, hence independently of its influence on the mitochondrial energy level, which in turn is known to modify the morphology of these organelles [ 1, 2]. Energy-independent, ADP dependent structural modifications are revealed by electron microscopy [3]. They are also revealed by slight, but very rapid modifications of the turbidity of mitochondrial suspensions [4]. On the other hand, Zimmer [5] has shown that ADP induces an increase of the total -SH groups which may be alkylated in mitochondria, under nonphosphorylating conditions, due to the presence of oligomycin. In the course of investigations on the accumulation of large amounts of calcium phosphate in the presence of ADP by hog heart sarcosomes [6] the turbidimetric technique of Stoner and Sirak [4] has been used and the ADP dependent jump in optical density, first reported by these authors, has been systematically studied; it is established in the present work that this turbidimetric jump is modified by the atkylating agent NEM, which amplifies the phenomenon and inhibits its reversion by atractyloside. It is further demonstrated that the alkylation kinetics of reactive groups by NEM and the inhibition kinetics of the atractyloside induced re-

ADP and Mg2+ requirement for Ca2+ accumulation by hog heart mitochondria. Correlation with energy coupling

Abstract 1. 1. The action of ADP and Mg 2+ on the respiration-driven accumulation of calcium phosphate by hog heart mitochondria, in the presence of oligomycin, has been studied. The accumulation is stringently ADP-specific and this requirement does not depend on the substrate used. The site of action of the nucleotide is located at the inner side of the inner mitochondrial membrane. It does not imply the involvement of adenylic translocase. 2. 2. Maximal accumulation of calcium phosphate is observed, even in the absence of exogenous Mg 2+ , when ADP is present in the medium from the onset of the incubations. In the absence of ADP, small accumulations of calcium phosphate are obtained, the levels of which do not depend on the addition of Mg 2+ . 3. 3. Conversely, once the calcium phosphate accumulation has stopped due to lack of ADP, both this nucleotide and Mg 2+ are needed to reactivate the accumulation process. In this case, the extent of the reactivation is proportional to the Mg 2+ concentration. The fact that it is possible to reverse the inhibition of calcium phosphate accumulation, indicates that this is not due to permanent damage of the mitochondrial membrane. 4. 4. In the initial presence of ADP, calcium accumulation and oxygen uptake appear to be closely correlated. In its absence both parameters are independent: calcium phosphate accumulation stops, whereas oxygen uptake is maximally stimulated. 5. 5. In the absence of ADP, ruthenium red and ethyleneglycol-bis-(aminoethyl)-tetraacetic acid (EGTA) have no effect on the maximally stimulated oxygen uptake. When the nucleotide is initially present, ruthenium red and EGTA promote respiratory control immediately. Once lost, respiratory control in response to ruthenium red is restored when ADP and Mg 2+ are both present. However, with EGTA, Mg 2+ alone is active in re-establishing respiratory control. 6. 6. Exchange between the calcium accumulated and the calcium of the incubation medium has been studied under experimental conditions, which caused any net calcium accumulation to stop. The exchange is inhibited by low concentrations of ruthenium red under conditions where no effect of this inhibitor on the stimulated oxygen uptake can be detected. This contradicts the hypothesis according to which this stimulation could be due to a cyclic movement of Ca 2+ . 7. 7. In order to explain the results observed, the lack of symmetry between the respective ADP and Mg 2+ requirements is emphasized. It is postulated that magnesium is stringently required for energy coupling between calcium phosphate accumulation and oxygen uptake. The role of ADP is supposed to be a regulatory one and to consist in an increase of the affinity of the magnesium binding sites for this cation.

Study of the mitochondrial phosphate carrier in the course of calcium phosphate accumulation: A requirement for Mg2+ and ADP of its sensitivity to thiol reagents

Abstract 1. The accumulation of calcium phosphate driven by succinate oxidation is ADP-dependent. In its absence the accumulation stops after a short incubation time and the oxygen uptake is permanently stimulated. This uncoupled oxygen uptake is insensitive to the inhibitors of phosphate transport, like mersalyl and N -ethylmaleimide. When ADP plus Mg 2+ are added to the medium, or when ADP is added in the initial presence of magnesium, the inhibitory action of the thiol reagents on oxygen uptake is re-established. ADP alone or Mg 2+ alone are without any effect. 2. Phosphate/phosphate exchange has been studied, in the absence of ADP, when calcium phosphate accumulation had stopped and oxygen uptake is uncoupled. Under these conditions the exchange process becomes insensitive to thiol reagents. Sensitivity is recovered solely in the presence of ADP plus Mg 2+ . 3. When mitochondrial swelling is studied according to the method of Chappell, it also appears that the phosphate carrier loses it sensitivity to mersalyl in the absence of ADP, which confirms the data obtained with phosphate/phosphate exchange experiments. When ADP plus Mg 2+ are added (or present), together with mersalyl, the action of the thiol inhibitor is recovered. ADP and magnesium are inactive separately. EGTA plus Mg 2+ (but not EGTA plus ADP) may substitute for ADP plus Mg 2+ in this process. 4. A possible interaction between the magnesium binding site and the phosphate carrier is considered and discussed.

Préparation des hormones du lobe postérieur de l'hypophyse de boeuf I. Ocytocine

The method proposed for the purification of ocytocine is a part of a technique used for obtaining ocytocine and vasopressin simultaneously from ox hypophyses. The two hormones are extracted, on heating with dilute acetic acid, from an acetonic powder of posterior hypophyses lobes, then they are adsorbed on silica. Ocytocine is selectively eluted by dilute acetic acid on heating. It is purified by successive extractions with n-butanol and dilute acetic acid. The substance obtained after lyophilisation contains 100–200 I.U. ocytocine and 4–6 I.U. vasopressin per mg. The quantity of ocytocine obtained in this way is 40–45% of the quantity initially present in the glands. The substance thus prepared is sufficiently pure to undergo a final purification by countercurrent distribution.

Influence of oestradiol and progesterone injections on the acid-soluble phosphate fractions of the rat uterus

Abstract Various fractions of acid-soluble phosphates in the rat uterus were investigated. Oestradiol was found to increase greatly the high-energy phosphate content of the immature rats uterus. Progesterone provoked a further significant increase of the phosphocreatine-like fraction, as compared with the rat treated with oestradiol only. The influence of ovarietomy on the acid-soluble phosphate fractions of the adult rat was likewise studied. The physiological significance of the results is discussed.

Selective Effect of a Diet-Induced Decrease in the Arachidonic Acid Membrane-Phospholipid Content on in vitro Phospholipase C and Adenylate Cyclase-Mediated Pituitary Response to Angiotensin II

Young rats were fed on an essential fatty acid (EFA)-deprived diet for 6 weeks after weaning. Their pituitary was removed and adenohypophyseal cells dispersed and maintained in culture. Membrane lipids were analyzed and basal and stimulated levels of hormone secretion were measured after 4-day incubation in a culture medium containing or not 160 microM arachidonic acid 20:4n-6 (AA) in order to obtain EFA-deficient or EFA-restored pituitary cells, respectively. In EFA-deficient cells membrane phosphoglycerides (PGL) were depleted in AA and adrenic acid 22:4n-6; the deficit was overcome by incubation in the presence of AA. Depletion diversely affected PGL classes. AA was highly depleted in choline phosphoglycerides (ChoPG), only moderately depleted in serine and ethanolamine phosphoglycerides (SerPG and EtnPG) and not depleted at all in inositol phosphoglycerides, suggesting preferential preservation of AA in that class of PGL. Restoration of AA by addition of the fatty acid to the culture medium was complete for ChoPG and EtnPG and only partial for SerPG. Depressed levels of AA and adrenic acid in PGL were compensated for by a concomitant increase in 20:3n-9 and 22:3n-9. Growth hormone and prolactin (PRL) secretion was assessed by radioimmunoassay and possible effects of a membrane AA deficit on hormone regulation were tested in cells challenged by either growth hormone-releasing hormone, thyrotropin-releasing hormone, angiotensin II (AII), vasoactive intestinal peptide (VIP) or dopamine. Neither basal nor stimulated growth hormone secretion was different from controls in EFA-deficient cells. PRL modulation by VIP or dopamine was not affected either in EFA-deficient cells. In contrast, the capacity of AII, but not of thyrotropin-releasing hormone, to release PRL was markedly decreased in EFA-deprived cells. It was restored by addition of AA to the incubation medium. Parallel depression of AII-induced inositol phosphates and cAMP accumulation was also observed after EFA deficiency. When tested on membranes, the paradoxical inhibition of adenylate cyclase by AII documented by previous observations was reinforced in EFA-deficient membranes. In contrast, binding of AII was not affected by EFA deficiency. It is concluded that under our experimental conditions EFA deficiency affects selectively coupling of the AII receptor to its effectors without alteration of binding. The effect could involve changes in receptor interactions with coupling proteins.

Mécanisme de l'inactivation de l'ocytocine par le tissu utérin

Abstract Mechanism of inactivation of ocytocin by uterine tissue Ocytocin, when incubated under aerobic conditions with uterine tissue is never inactivated, whereas irreversible inactivation occurs rapidly when the incubation is performed under anaerobic conditions. The inactivation seems to proceed in two steps: the first step consists in a reduction of the disulfide bridge of ocytocin by uterine tissue; the second step is an irreversible inactivation of reduced ocytocin by an enyme system, which is liberated into the medium during incubation. This inactivation proceeds either through proteolysis or through irreversible formation of mixed disulfides. Similar experiments have been done with other organs, such as liver, kidney and diaphargm; it has been shown that ocytocin is inactivated by these tissues, but that inactivation in these cases proceeds equally with the reduced and the unreduced form of ocytocin. The physiological significance of these results is discussed.

Biosynthesis of acid mucopolysaccharides by the surviving new born rat skin. I. - Kinetics of the biosynthesis at the polymer level.

1) The amounts of individual mucopolysaccharides in the new born rat skin have been estimated and their specific rates of labelling assessed in vitro. Total and percentage amounts of these polymers agree satisfactorily with previously published data. 2) Relative rates of labelling from [U14C]-glucose have been estimated by combining column chromatography separation and electrophoresis on cellulose acetate strips. Specific radioactivities have been measured either with respect to the total uronic acid content of the fractions or with respect to their quantitative staining with Alcian Blue. The two methods agreed satisfactorily. 3) Average biosynthetic rates almost identical for hyaluronic acid and the total sulfated mucopolysaccharides. However, within the latter fraction, heparin + heparan sulfate incorporate [U14C]-glucose about 4 to 5 times more rapidly than the chondroitin sulfates. This result could not be expected from previous data obtained in vivo and is discussed with reference to a possible heterogeneity of the cell material whence the various mucopolysaccharides originate. 4) In the presence of puromycin, labelling of the sulfated mucopolysaccharides stops almost immediately, indicating a stringent requirement for protein primers. Biosynthesis of hyaluronic acid is affected only after preincubation of tissue with puromycin (one hour) and subsequent incubation of two hours with [U14C]-glucose.

The combined action of insulin and phlorizin on transport and metabolism of sugars and nucleotide turnover in the isolated rat diaphragm

The combined effects of insulin and phlorizin have been analyzed on two parameters of insulin stimulation in the surviving rat diaphragm : transport and metabolism of sugars, turnover of the phosphate groups of mononucleotides. Phlorizin (c mM) inhibits glucose transport both in the presence and absence of insulin and displays a small additional inhibitory effect on glycogen biosynthesis; with the non metabolizable glucose anlogue 3-O-methyl-D-glucose transport inhibition is demonstrated solely in the presence of insulin. No correlation is demonstrated between the rates of sugar transport, which are strongly phlorizin-sensitive, and the rates of nucleotide turnover, which show no such sensitivity.

Influence of adrenaline and hypophyseal growth hormone on the acid-soluble phosphate fractions of the rat uterus

Abstract Adrenaline injections are followed by an increase of “true” inorganic phosphate and a decrease of high-energy phosphate fractions in the rat uterus. Growth hormone injections provoke an increase of high-energy phosphates in the same organ. The relationship between these changes and oxidative phosphorylation phenomena is discussed with regard to the growth hormone- and adrenaline-promoted inhibitions of the uterine metabolism, which have been previously reported.