Fernand G. Péron

Immunologic methods in steroid determination

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Further studies on corticosteroidogenesis: III. Effect of biological substrates and electron transport dependence in rat adrenal mitochondria

Abstract The effect of the biological substrates succinate, malate, glutamate, citrate, isocitrate, α-ketoglutarate, β-hydroxybutyrate on 11β-hydroxylation of DOC in rat adrenal mitochondria has been studied. All substrates supported the conversion of DOC into corticosterone when the mitochondria were incubated at pH 7.4 in Tris buffer. The factors involved in the conversion of DOC into corticosterone were found to be independent of any observable swelling of the mitochondria and appeared to depend upon the coupling of substrate oxidation with components of the classical electron chain. When high levels of Ca2+ (11 mM) were added to the incubation system, the mitochondria swelled and exogenously added TPNH elicited a maximal transformation of DOC into corticosterone. These results are discussed in relation to current concepts implicating several factors in steroid hydroxylation reactions. Biological substrates appear to affect 11β-hydroxylation of DOC in rat adrenal mitochondria vy virtue of their oxydation involving the respiratory carriers of the classical electron chain. Evidence obtained in incubations carried out in the presence of substrates and the uncouplers 2,4-dinitrophenol and oligomycin, also implicate high energy compounds besides ATP in the 11β-hydroxylation of DOC by the non-swollen mitochondria. When mitochondria were maximally swollen by Ca2+ (11 mM), addition of exogenous TPNH brought about 11β-hydroxylation of DOC by other mechanism(s). In this latter instance passage of electrons along the newly proposed chain made up of the following components: flavoprotein, non-heme iron protein and a cytochrome component designated as cytochrome P450 is probably involved.

Further studies on corticosteroidogenesis: IV. Inhibition of utilization of biolgical substrates for corticoid synthesis by high calcium concentrations possible role of transhydrogenase in corticosteroidogenesis

Abstract The biological substrates isocitrate, succinate, α-ketoglutarate and s-hydroxybutyrate initiate the conversion of DOC into corticosterone in mitochondria incubated at pH 7.4. The bustrates are also utilized by the reconstituted homogenate (mitochondria + microsomes + supernatant fraction) and whole homogenate for the conversion of pregnenolone, progesterone or DOC into corticosterone. As is known to occur in liver mitochondria, low Ca2+ concentrations in rat adrenal mitochondria are postulated to increase oxidation of biological substrates thereby effecting an increased transformation of DOC into corticosterone. At higher levels of Ca2+ (1 mM) the biological substrates could no longer be utilized and this occurred when mitochondria were maximally swollen. When swelling of mitochondria was extensive, exogenous TPNH presumably penetrated into the mitochondria and was utilized for 11s-hydroxylation of DOC. In the non-swollen mitochondria or at low Ca2+ levels, TPNH was excluded. Whereas mitochondria alone could utilize the substrates, microsomes required the presence of the supernatant fraction. Data derived from mitochondrial, microsomal, particulate + supernatant fractions or whole homogenate incubations in the absence or presence of antimycin, ferricyanide, 2,4-dinitrophenol and Amytal indicated that the biological substrates are oxidized mainly via the classical electron or cytochrome chain by mitochondria. When exogenous TPNH was added to swollen mitochondria or when substrates were added to microsomes + supernatant fraction the flux of electrons initiated by TPNH oxidation appeared to occur through the chain designated as flavoprotein → non-heme iron-protein → cytochrome P450. The level of transhydrogenase activity in mitochondria and microsomes was assessed by the amount of steroid substrates transformed into their hydroxylated derivatives when the particulate fractions were incubated in the presence of relatively high levels of DPNH plus TPN+. Ca2+ was needed to demonstrate the activity in the mitochondria which indicated that non-swollen mitochondria are impermeable to pyridine nucleotides. In these instances, Ca2+ had little effect on the microsomes whereas at high Ca2+plus low TPN+plus high DPNH concentrations this ion appeared to inhibit the transhydrogenase reaction. Although various effects of adenosine-3′,5′-monophosphate, ATP, thyroxine and ADP plus P1plus Mg2+_ in mitochondrial and homogenate incubations were observed, these were of such a nature as to make impossible firm conclusions of a function of these substances in a possible control of the transhydrogenase enzyme. Incubations carried out with succinate in the absence or presence of thyroxine and ATP have yielded some data indicating that the transhydrogenase reaction might be implicated in bringing about an oxidation of biological substrates.

The role of calcium in the steroidogenic response of rat adrenal cells to adrenocorticotropic hormone.

Abstract 1. 1. The Ca2+ requirement for the steroidogenic effect of adrenocorticotropic hormone (ACTH) and N6, O2′-dibutyryladenosine 3′, 5′-monophosphate (dibutyryl cyclic AMP) was investigated in rat adrenal cell suspensions prepared by collagenase and trypsin treatment of adrenal sections. 2. 2. At low levels of ACTH addition, the Ca2+ requirement was absolute but at high levels the Ca2+ requirement diminished considerably. In contrast, the Ca2+ requirement was about the same for all concentrations of dibutyryl cyclic AMP tested. 3. 3. In the presence of the Ca2+ chelator ethyleneglycol-bis (β-aminoethyl ether)-N, N′-tetraacetic acid (EGTA), even very large amounts of ACTH could not initiate corticosterone synthesis. Dibutyryl cyclic AMP effect was still observed even in the presence of 5 mM EGTA, indicating that in the action of ACTH the Ca2+ requirement is greater for the events before the formation of cyclic AMP. 4. 4. In the experiments where binding of ACTH was studied by preincubating the cells with ACTH at 4°C, washing and finally incubating at 37°C to assess corticosterone production due to ACTH bound at 4°C, EGTA caused some inhibition in the binding at lower levels of the trophic hormone. However, this cannot explain the total failure of ACTH to initiate corticosterone synthesis in the presence of EGTA. In the presence of 2.5 mM Ca2+ added in the preincubation period ACTH binding was not much different than in the absence of added Ca2+. 5. 5. Our data supports the suggestion of others that Ca2+ may be crucially involved in the transmission of the signal arising from ACTH-receptor interaction to the adenyl cyclase.

19-hydroxy-11-deoxycortisol, a major steroid secreted by the adrenal gland of the Mongolian gerbil

Abstract Adrenal vein blood obtained by cannulation from the Mongolian gerbil ( Meriones unguiculatus ) was found to contain in equal amounts cortisol and 19-hydoxy-11-deoxycortisol (19-OHDC). Aldosterone and a yet unidentified steroid (G2), which is slightly less polar than cortisone, were also found. The 19-OHDC was identified by comparison with authentic 19-OHDC in classical chromatographic systems and by infrared analysis. When progesterone-7α-3H was incubated with quartered adrenal glands, the products included 19-OHDC and G2. The biological function of 19-OHDC in this desert animal has not been elucidated.

Further studies on the effect of calcium ions and corticosteroidogenesis. II. Adrenal mitochondrial swelling by calcium ions.

Summary Rat adrenal mitochondria and microsomes have been obtained in a form un-contaminated one with the other. It has been shown that Ca 2+ action on reactions converting n-deoxycorticosterone and progesterone into corticosterone, 18-hydroxy-n-deoxycorticosterone, and 11 β -hydroxyprogesterone is unequivocally at the mitochondrial level. Ca 2+ has no effect on reactions converting progesterone into 11-deoxy-corticosterone in the microsomes. The enzyme(s) or factors involved in mitochondrial 11 β -hydroxylation appear to have a much greater requirement for TPNH than those involved in microsomal 21-hydroxylation. Ca 2+ also brings about swelling of rat adrenal mitochondria incubated at 37°. The degree of swelling is related to the concentration of Ca 2+ present in the medium which in turn is related to the rate of transformation of 11-deoxycorticosterone into corticosterone. Although there is a close relationship between swelling and 11-deoxycorticosterone conversion into corticosterone, there appears to be an influence of Ca 2+ on 11 β -hydroxylation which is different and separate from the swelling effect.

Comparison of the Ca++ requirement for the steroidogenic effect of ACTH and Dibutyryl cyclic AMP in rat adrenal cell suspensions

Calcium requirement for ACTH and Dibutyryl cyclic AMP (DBCAMP) stimulation of steroidogenesis was compared in rat adrenal cell suspensions. In the absence of added calcium ACTH at low concentrations (< 1 mU/ml) was ineffective; however, the calcium requirement decreased when higher concentrations of ACTH were used. This was not the case with DBCAMP. At all levels of the nucleotide tested, the Ca++ requirement was about the same. When the cells were preincubated with EGTA, the Ca++ requirement became more pronounced for ACTH than for DBCAMP. The results indicate that the events before the formation of cyclic AMP show a greater dependence on Ca++ than the events following its formation.

Influence of metopirone on substrate-supported hydroxylation of DOC by rat adrenal mitochondria.

Abstract The inhibitory effect of metopirone on the 11β- and 18-hydroxylation of DOC by rat adrenal mitochondria has been investigated. The results indicate that inhibition may be substrate dependent which in turn might be energy linked. The substrate specificity appears to involve endogenous pyridine nucleotide cofactors, particularly NADPH, as well as electron transport associated with steroid hydroxylation. In regard to the latter, the action of metopirone seems to be related solely to the cytochrome P-450 system while the classical electron chain remains intact.

The effect of calcium ions on corticosteroidogenesis Conversion of pregnenolone and progesterone to deoxycorticosterone in rat adrenal homogenate fractions fortified with diphosphoryridine nucleotide plus glucose 6-phosphate

Abstract It has been shown conclusively for the first time that the conversion of deoxycorticosterone to corticosterone is stimulated by Ca 2+ when an enzyme preparation obtained from rat-adrenal homogenate is incubated with TPNH or a TPNH-generating system. In confirmation of previous work, Ca 2+ was also found to stimulate the conversion of endogenous precursor cholesterol into corticoid. In the absence of Ca 2+ and under the same incubation conditions, there is an accumulation of deoxycorticosterone from pregnonolone and progesterone while little or no corticoids are produced from endogenous precursor cholesterol under these circumstances. It has been concluded that Ca 2+ affects biochemical reactions taking place in or at the surface of mitochondria by activating or making available functional groups of enzymes responsible for cleavage of the cholesterol side-chain, 18-hydroxylation and 21-hydroxylation. In enzyme preparations fortified with maximal levels of Glc-6- P , biosynthesis of corticoids from endogenous cholesterol or exogenously added steroid substrates occurs at low levels of TPN + addition (final concentration = 5.9 · 10 −3 M). However, at low concentrations of TPN + , more intermediate products of the corticosteroidogenic chain like deoxycorticosterone accumulate. With maximal amounts of TPN + (final concentrations = 1.43 · 10 −1 − 2.39 · 10 −1 M) corticosterone and 18-hydroxydeoxycorticosterone are the major steroid products formed. Pregnenolone and progesterone were converted to deoxycorticosterone when incubations were carried out in the presence of DPN + + Glc-6- P . No corticosterone was formed from these substrates or from deoxycorticosterone. Ca 2+ had no effect on the conversions in these circumstances. Deoxycorticosterone production from pregnenolone or progesterone was not inhibited by additions of CN − , amobarbital and antimycin A, nor coulld inorganic phosphate, ATP, Ado-3′,5′- P or Glc-1- P replace the Glc-6- P requirement in these biosynthetic reactions. The interplay of factors responsible for the combined action of Glc-6- P + DPN + is unknown but deserves special scrutiny, since the 21-hydroxylating enzyme(s) bringing about the conversion of progesterone to deoxycorticosterone has hitherto been found to require TPNH specifically. DPN + was converted by the adrenal transhydrogenase to DPNH when DPN + was added to the enzyme preparation and incubated in the presence of TPNH. The transformation of pregnenolone to deoxycorticosterone or corticosterone was inhibited by the DPNH formed or by direct additions of DPNH to the enzyme preparation. DPNH-inhibition could be reversed by additions of relatively large amounts of TPN + . As the latter became reduced in the reverse of the aforementioned reactions, the transformation of pregnenolone to deoxycorticosterone and corticosterone again took place. A possible function of the transhydrogenase enzyme is discussed as to its possible function in adrenal physiology.

Further studies on the biosynthesis of corticoids by rat adrenal fractions and inhibition of endogenous cholesterol utilization by cobra venom

Abstract Biosynthesis of corticoids was found to occur in cellular fractions of rat-adrenal homogenates prepared in 0.25 M sucrose or 0.154 M KCl. Activity was found in the fraction sedimented at 5000 × g (called Pellet 2′) and the supernatant fraction obtained at 14800 × g of the sucrose homogenate, while the 14800 × g fraction (called Pellet 2) and 59500 × g supernatant fraction were also active when prepared from KCl-homogenates. Pellet 2 had associated with it the endogenous cholesterol used for corticoid synthesis. Cholesterol appeared to be the only steroid precursor and its disappearance during a final I -h incubation was stoichiometrically accounted by the amount of corticoids synthesized. Boiled or unboiled cobra venom ( Naja naja ) effectively inhibited corticoid biosynthesis from endogenous cholesterol in whole homogenates or combinations of Pellet 2 plus clear supernatant. An action of cobra venom on a possible specific phospholipid-cholesterol complex was postulated to explain the inhibition. This hypothesis was not substantiated, however, because the conversion of pregnenolone, progesterone and deoxycorticosterone when added as substrates was also inhibited. Since boiling of the cobra venom destroys most if not all of the enzymes except lecithinase (phosphatide acyl-hydrolase, EC 3.1.1.4) it was deduced that the active principle in boiled cobra venom is lecithinase. This enzyme is probably responsible also for most of the corticosteroid inhibiting activity in the non-boiled preparation. A present the mode of action of cobra venom or the lecithinase contained therein is unknown.