Ajai Haksar

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.

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.

Inhibition of ACTH-stimulated steroidogenesis in isolated rat adrenal cells treated with neuraminidase

The possible role of membrane sialic acid in the action of ACTH was investigated in rat adrenal cells. After treatment with neuraminidase, the cells showed a diminished steroidogenic response to ACTH while the response to cyclic AMP and dibutyryl cyclic AMP was unaffected. 11β-hydroxylation of deoxycorticosterone (DOC) was also not impaired. Dose response curves for three ACTH peptides (ACTH1–39′, ACTH1–24 and ACTH1–10) with neuraminidase treated cells suggest that sialic acid residues on the glycoproteins of the plasma membrane may either impart affinity to the plasma membrane for ACTH molecule or facilitate transmission of the signal arising from ACTH-receptor interaction to the catalytic site of adenyl cyclase.

The role of the krebs cycle in the generation of intramitochondrial reducing equivalents for the 11β-hydroxylation of deoxycorticosterone in isolated rat adrenal cells

Abstract Isolated rat adrenal cells were used to study the possible pathways of intramitochondrial NADPH generation for 11β-hydroxylation of 11-deoxycorticosterone. Pyruvate was efficiently utilized by the mitochondria as shown by evolution of 14 CO 2 from [1- 14 C]- and [2- 14 C]pyruvate. Citrate, isocitrate, succinate, and malate were not utilized by intact cells due to their inability to permeate the plasma membrane. For every mole of corticosterone formed, 1.9 and 0.8 moles of 14 CO 2 were formed from [1- 14 C]- and [2- 14 C]pyruvate, respectively, indicating that pyruvate dehydrogenase was quite active and supplied acetyl CoA to the Krebs cycle. Fluorocitrate and 2,4-dinitrophenol inhibited 11β-hydroxylation of 11-deoxycorticosterone as well as the production of 14 CO 2 from [2- 14 C]pyruvate. Comparison of data with the two inhibitors showed that for the same percentage of inhibition of 14 CO 2 production, the inhibition of 11β-hydroxylation was greater with 2,4-dinitrophenol than with fluorocitrate. It is concluded that operation of the Krebs cycle may be essential for 11β-hydroxylation to occur primarily because NADH generated by the cycle provides ATP, via the respiratory chain, as well as the substrate for the energy-linked transhydrogenase that forms NADPH. The NADPH required for 11β-hydroxylation seems to be derived to a large extent via the energy-linked transhydrogenase.

In vitro inhibition of cholesterol synthesis by pregnenolone in bovine corpus luteum

Abstract Bovine corpus luteum slices and. minces were incubated with acetate-1- 14 C in the presence of pregnenolone. There was a general inhibition in the utilization of acetate-1- 14 C as manifested, by a decreased incorporation of radioactivity into digitonin precipitable sterols, cholesterol, progesterone, and the total lipid extract of the tissue. LH which alone would stimulate the incorporation of acetate-1- 14 C into these substances was not able to reverse the pregnenolone-induced. inhibition. It is suggested that pregnenolone may have a role in the regulation of steroidogenesis in the corpus luteum.

Further studies on corticosteroidogenesis. IX. Energy-linked transhydrogenase in rat adrenal gland mitochondria.

While malonate inhibited succinate-supported 11β-hydroxylation of 11-deoxy-corticosterone in rat adrenal mitochondria, it had no effect on the utilization of α-ketoglutarate for this purpose. Addition of 5 mM Amytal in the presence of α-ketoglutarate and deoxycorticosterone led to an inhibition of respiration and a greatly diminished corticosterone production. Addition of succinate to this Amytal-blocked α-ketoglutarate system, reestablished O2 consumption and corticosterone production. Rotenone, 10 μM, had only a slight effect on succinate or α-ketoglutarate supported 11β-hydroxylation and respiration. It would appear that rotenone which is known to inhibit NADH oxidation in mitochondria from most mammalian cells at this concentration behaves differently in rat adrenal mitochondria. The findings obtained in the experiments with Amytal indicate participation of an energy-linked transhydrogenase enzyme involved in reaction(s) leading to the oxidation of NADH and generation of NADPH, which is necessary for 11β-hydroxylation of deoxycorticosterone. It is postulated that in the Amytal-inhibited systems ATP or high energy compounds generated by the aerobic oxidation of succinate over the uninhibited part of the respiratory chain serve as the source of energy for the transhydrogenase enzyme.

Sources of reducing equivalents for cytochrome P-450 mitochondrial steroid hydroxylations in rat adrenal cortex cells

Abstract Pyruvate-supported 11β-hydroxylation of 11-deoxycorticosterone (DOC) via cytochrome P-450 reductase chain in incubated rat adrenal mitochondria was maximal when either traces of oxaloacetate (OAA) or 2 mM ATP were added to the incubation medium. This showed that reducing equivalents formed at the pyruvate dehydrogenase level as well as those derived from Krebs-cycle activity were needed for maximal corticosterone (B) formation from DOC. These findings were confirmed in experiments with whole cells isolated from rat adrenals designed to show the possible pathways of intramitochondrial NADPH generation for steroid hydroxylations. Whereas citrate, isocitrate, succinate and malate were not metabolized because of their impermeability to the plasma membranes of the cells, both [1- 14 C] and [2- 14 C]-pyruvate were efficiently utilized by the mitochondria of the cells for B formation from DOC. Whereas arsenite completely inhibited the pyruvate- 14 C supported B formation, comparison of the data obtained with two inhibitors, 2,4-dinitrophenol (2,4-DNP) and fluorocitrate, showed that for the same per cent inhibition of 14 CO 2 production the inhibition of 11βJ-hydroxylation of DOC was greater with 2,4-DNP than with fluorocitrate. It is concluded that operation of the Krebs-cycle is essential for optimizing the production of reducing equivalents needed for 11β-hydroxylation to occur. It is also concluded that the rate of oxidation of pyruvate in the mitochondria of the cells and the activity of the Krebs-cycle is dependent on availability of OAA. When ATP levels were reduced by the uncoupling effect of 2,4-DNP resulting in a decreased production of OAA via ATP-requiring pyruvate carboxylase, a concomitant inhibition in production of reducing equivalent occurred which led to a lack of B production from DOC. Mitochondria isolated from the steroid-producing Snell adrenocortical carcinoma 494 cells (P 2 T) were unable to oxidize pyruvate and several Krebs-cycle substrates. Succinate and α-glycerol phosphate which both were oxidized via their respective P 2 T flavoprotein linked enzymes, supported high rates of O 2 uptake but had little effect on DOC conversion into B. Electron micrographs of P 2 T when compared to P 2 C from normal rat adrenals showed that their ultrastructure was markedly altered. The number of mitochondria per cell in the tumor tissue was also considerably lower than that found in normal adrenal cells. Many of the P 2 T were elongated, had lamillar-shaped cristae and showed a much less uniformed shape than P 2 C which are usually oval and contain packed vesicular cristae. Adrenodoxin reductase activity, cytochrome P-450 and NADP nucleotide levels in P 2 T were also lower than those in P 2 C which partly accounted for the low conversion of DOC into corticosterone. Because (1) mitochondrial flavoprotein-linked a-glycerol phosphate dehydrogenase activity in P 2 T was 10 times higher than that found in P 2 C, (2) an active NAD-linked α-glycerol phosphate dehydrogenase in the cytosol of the carcinoma cell was found and (3) pyruvate is not utilized by P 2 T or whole cancer cells, this suggested the possibility of an α-glycerol phosphate shuttle operating in the tumor cells. The purpose of the shuttle might be to provide some of the cellular ATP required in this rapidly growing cancer tissue.

Adrenocorticotropin stimulation of cyclic adenosine 3′,5′-monophosphate formation in isolated rat adrenal cells the role of membrane sialic acid

Abstract Neuraminidase treatment of isolated rat adrenal cells inhibited the cyclic AMP response to adrenocorticotropin stimulation. Greater percent inhibition was observed with the lower doses of adrenocorticotropin. Although the maximum amount of cyclic AMP produced by the neuraminidase-treated cells was similar to that obtained with untreated cells, the concentration of adrenocorticotropin required to produce it was greatly increased. The increase in adrenocorticotropin concentration to produce half-maximum amounts of cyclic AMP was 2-fold. Neuraminidase treatment caused a dose-related depletion of sialic acid from the cells with about 60% of the total sialic acid being released by 20 munits/ml of the enzyme. Stimulation of cyclic AMP formation by NaF in a particulate fraction obtained from homogenates of cells was unchanged after treatment of the cells with neuraminidase. The data implicate a role for sialic acid in the early events in the action of adrenocorticotropin on the cell membrane. Sialic acid may be involved in the interaction of the adrenocorticotropin molecule with the receptor or it may have a role in transmission of the signal arising from adrenocorticotropin-receptor interaction to the catalytic unit of adenyl cyclase.

Chlorpromazine: inhibition of ACTH and cyclic 3',5'-AMP stimulated corticosterone synthesis.

Summary Chlorpromazine inhibits the ACTH and cyclic AMP-stimulated corticosterone synthesis, in vitro , in the quartered rat adrenal glands without greatly affecting the basal production of corticosterone. Tissues exposed for 30 minutes to cyclic AMP do not show inhibition of cyclic AMP stimulation by chlorpromazine and the removal of chlorpromazine from the incubation medium restores the tissue response to cyclic AMP.

A possible mechanism for the stimulation of krebs cycle activity by ACTH in isolated rat adrenal cells

Summary Isolated rat adrenal cells, prepared by collagenase-trypsin treatment, were incubated in Krebs-Ringer phosphate buffer, pH 7.4, containing 0.5% BSA, 0.1% trypsin inhibitor and pyruvate-2- 14 C. Krebs cycle activity was estimated by measuring the 14 CO 2 evolved. ACTH caused a dose-dependent stimulation of 14 CO 2 production in addition to the well known effect on steroidogenesis. When the steroidogenic effect of ACTH was inhibited with amino-glutethimide, the effect of the hormone on 14 CO 2 production was also abolished. Aminoglutethimide, however, had no appreciable effect on the basal 14 CO 2 production. Addition of ll-deoxycorticosterone, but not corticosterone, to the cells also stimulated 14 CO 2 production. The data suggest that stimulation of Krebs cycle activity in the presence of ACTH may have been due to increased production of steroid precursor(s) of corticosterone which in turn would create an increased demand for reducing equivalents for their conversion into corticosterone.