Shiro Kominami

Rapid modulation of long-term depression and spinogenesis via synaptic estrogen receptors in hippocampal principal neurons

Rapid modulation of hippocampal synaptic plasticity by estrogen has long been a hot topic, but analysis of molecular mechanisms via synaptic estrogen receptors has been seriously difficult. Here, two types of independent synaptic plasticity, long‐term depression (LTD) and spinogenesis, were investigated, in response to 17β‐estradiol and agonists of estrogen receptors using hippocampal slices from adult male rats. Multi‐electrode investigations demonstrated that estradiol rapidly enhanced LTD not only in CA1 but also in CA3 and dentate gyrus. Dendritic spine morphology analysis demonstrated that the density of thin type spines was selectively increased in CA1 pyramidal neurons within 2 h after application of 1 nm estradiol. This enhancement of spinogenesis was completely suppressed by mitogen‐activated protein (MAP) kinase inhibitor. Only the estrogen receptor (ER) alpha agonist, (propyl‐pyrazole‐trinyl)tris‐phenol (PPT), induced the same enhancing effect as estradiol on both LTD and spinogenesis in the CA1. The ERbeta agonist, (4‐hydroxyphenyl)‐propionitrile (DPN), suppressed LTD and did not affect spinogenesis. Because the mode of synaptic modulations by estradiol was mostly the same as that by the ERalpha agonist, a search was made for synaptic ERalpha using purified RC‐19 antibody qualified using ERalpha knockout (KO) mice. Localization of ERalpha in spines of principal glutamatergic neurons was demonstrated using immunogold electron microscopy and immunohistochemistry. ERalpha was also located in nuclei, cytoplasm and presynapses.

The role of cytochrome b5 in adrenal microsomal steroidogenesis.

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17 alpha- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-450(17) alpha,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount of cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the first electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.

Purification and some properties of cytochrome P-450 specific for steroid 17α-hydroxylation and C17C20 bond cleavage from guinea pig adrenal microsomes

Abstract A novel species of cytochrome P-450 was purified to a specific content of 15 nmol/mg of protein from guinea pig adrenal microsomes. The purified preparation exhibited a single polypeptide band (Mr = 52,000 ± 1,000) when submitted to a sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both progesterone-17α-hydroxylase (1.67 nmol 17α-hydroxyprogesterone/min/nmol cytochrome P-450) and 17α-hydroxyprogesterone C17C20-lyase (0.68 nmol androstenedione/min/nmol cytochrome P-450) activities could be reconstituted upon mixing the purified cytochrome P-450 with NADPH-cytochrome P-450 reductase. The absorption spectrum of the oxidized cytochrome P-450 in the presence of progesterone had maxima at 394 and 650 nm, characteristic of a high spin form. The reduced CO complex of the cytochrome P-450 showed an absorption maximum at 448 nm.

Retinoic Acid Stimulates 17β-Estradiol and Testosterone Synthesis in Rat Hippocampal Slice Cultures

The hippocampus is essentially involved in learning and memory processes. Its functions are affected by various neuromodulators, including 17beta-estradiol, testosterone, and retinoid. Brain-synthesized steroid hormones act as autocrine and paracrine modulators. The regulatory mechanism underlying brain steroidogenesis has not been fully elucidated. Synthesis of sex steroids in the gonads is stimulated by retinoic acids. Therefore, we examined the effects of retinoic acids on estradiol and testosterone biosynthesis in the rat hippocampus. We used cultured hippocampal slices from 10- to 12-d-old male rats to investigate de novo steroidogenesis. The infant rat hippocampus possesses mRNAs for steroidogenic enzymes and retinoid receptors. Slices were used after 24 h of preculture to obtain maximal steroidogenic activity because steroidogenesis in cultured slices decreases with time. The mRNA levels for P450(17alpha), P450 aromatase and estrogen receptor-beta in the slices were increased by treatment with 9-cis-retinoic acid but not by all-trans-isomer. The magnitude of stimulation and the shape of the dose-response curve for the mRNA level for P450(17alpha) were similar to those for cellular retinoid binding protein type 2, the transcription of which is activated by retinoid X receptor signaling. 9-cis-Retinoic acid also induced a 1.7-fold increase in the protein content of P450(17alpha) and a 2-fold increase in de novo synthesis of 17beta-estradiol and testosterone. These steroids may be synthesized from a steroid precursor(s), such as pregnenolone or other steroids, or from cholesterol, as so-called neurosteroids. The stimulation of estradiol and testosterone synthesis by 9-cis-retinoic acid might be caused by activation of P450(17alpha) transcription via retinoid X receptor signaling.

The role of cytochromes P-450 in adrenal steroidogenesis

Abstract Steroid hormones from adrenal glands are synthesized from cholesterol via several monooxygenase reactions catalysed by different species of cytochrome P-450. P-450 scc and P-450 11β are located in the mitochondria and P-450 c21 and P-450 17α,lyase are located in the endoplasmic reticulum. There are different electron transfer pathways from NADPH to cytochrome P-450 in the two organelles. The metabolic intermediates move back and forth between the organelles during the synthetic process. Recently, steroidogenic electron transfer systems have been reconstituted and well characterized.

Steroidogenesis in liposomal system containing adrenal microsomal cytochrome P-450 electron transfer components

Purified adrenal microsomal P-450C21 and/or P-45017 alpha,lyase were incorporated with purified NADPH-cytochrome-P-450 reductase into liposome membranes composed of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine at a molar ratio of 5:3:1. The rate dependences of reduction of liposomal P-450C21 in the fast phase as well as progesterone hydroxylation activities of P-450C21 and P-45017 alpha,lyase on the reductase concentration in the liposome membranes suggested that electrons were delivered through random collisions between the reductase and cytochrome P-450s in the liposome membranes. A rapid exchange of the steroid metabolic intermediate between vesicles was observed in a reaction system consisting of P-450C21-proteoliposomes and P-45017 alpha,lyase-proteoliposomes. Using the combined liposomal system, it was definitely proved that androstenedione was formed from progesterone mainly by a successive hydroxylation reaction without the intermediate 17 alpha-hydroxyprogesterone leaving from P-45017 alpha,lyase. It was also found that 21-hydroxylation of 17 alpha-hydroxyprogesterone into 11-deoxycortisol was inhibited by a physiological concentration of progesterone.

Immunochemical studies on cytochrome P-450 in adrenal microsomes

An antibody was prepared against electrophoretically homogeneous cytochrome P-450C21 purified from bovine adrenal microsomes. This antibody was used to compare various cytochromes P-450 in bovine and guinea pig adrenal microsomes. In an Ouchterlony double diffusion test, a spur formation was observed between the precipitin lines of the purified bovine cytochrome P-450C21 and guinea pig adrenal microsomes against anti-cytochrome P-450C21 IgG. Anti-cytochrome P-450C21 IgG inhibited 21-hydroxylation both of bovine and guinea pig adrenal microsomes but the inhibition was much more effective in the bovine microsomes than in the guinea pig microsomes. These results suggest that the 21-hydroxylase in the guinea pig microsomes has some molecular similarities to the bovine cytochrome P-450C21 and a part of the antibodies cross-reacts with the 21-hydroxylase in the guinea pig microsomes. Anti-cytochrome P-450C21 IgG did not inhibit the activities of 17 alpha-hydroxylase and C17,20-lyase in the bovine and guinea pig microsomes but stimulated these activities. This result shows that different species of cytochrome P-450 other than cytochrome P-450C21 catalyzes the 17 alpha-hydroxylation and C17,20 bond cleavage. The stimulation of 17 alpha-hydroxylation and C17,20 bond cleavage by blocking 21-hydroxylation indicates that the electron transfer systems for various cytochromes P-450 are intimately linked in adrenal microsomes.

Adrenal cytochrome P-45011β-proteoliposomes catalyzing aldosterone synthesis: preparation and characterization

Purified cytochrome P-45011 beta from bovine adrenocortical mitochondria was successfully incorporated into the liposome membranes composed of phosphatidylcholine, phosphatidylethanolamine and cardiolipin at a molar ratio of 2:2:1. The incorporation of P-45011 beta into the liposome membranes was ascertained by the Ficoll density gradient centrifugation and the protein refractoriness to trypsin digestion. The prepared proteoliposomes containing P-45011 beta and phospholipid at a molar ratio of 1:3000 were unilamellar vesicles of about 40 nm in average diameter. The P-45011 beta embedded in the liposome membranes was found to be more stable than the detergent-solubilized form. The reconstituted system containing the P-45011 beta-proteoliposomes, adrenodoxin and NADPH-adrenodoxin reductase showed catalytic activities not only for the hydroxylation of 11-deoxycorticosterone at 11 beta- and 18-positions but also for its conversion into aldosterone with a turnover number of 2.3 nmol/min per nmol of P-45011 beta. A successive reaction without the intermediates leaving from the enzyme was suggested for the P-45011 beta-mediated conversion of 11-deoxycorticosterone to aldosterone following the result that the formation of aldosterone was linear with respect to time without the lag phase; this was confirmed by the result that radioactivity in aldosterone from 3H-labeled 11-deoxycorticosterone was scarcely decreased by the addition of unlabeled intermediates to the reactions system.

Kinetic control of steroidogenesis by steroid concentration in guinea pig adrenal microsomes

For clarification of the effects of steroid concentration on steroidogenesis of adrenal microsomes, the kinetic parameters, Km and kcat, were determined in the steady-state for progesterone and 17 alpha-hydroxyprogesterone metabolism catalyzed by P-450C21 and P-450(17 alpha lyase) in guinea pig adrenal microsomes. At a high concentration of progesterone, it was equally metabolized by P-450C21 and P-450(17 alpha lyase), while at a low concentration, it was hydroxylated at 17 alpha-position with twice higher rate than at 21-position. 17 alpha-Hydroxyprogesterone is apparently metabolized preferentially by P-450C21 at any concentration. Although the productions of deoxycortisol and androstenedione from 17 alpha-hydroxyprogesterone were strongly inhibited by progesterone, androstenedione formation from progesterone was not inhibited by a high concentration of progesterone. The addition of liposomal P-450C21 to the reaction medium containing adrenal microsomes caused a decrease in the concentration of 17 alpha-hydroxyprogesterone released into the medium in the steady state reaction, but this had no effect on the activity of androstenedione formation from high concentrations of progesterone. It thus follows that androstenedione is produced by successive monooxygenase reactions without the release of 17 alpha-hydroxyprogesterone from P-450(17 alpha lyase) at a high concentration of progesterone, which is the condition of the adrenal microsomes in vivo.

Tributyltin disturbs bovine adrenal steroidogenesis by two modes of action

Tributyltin, an environmental pollutant, affected adrenal steroid hormone biosynthesis by two modes of action. Treatment of bovine adrenal cultured cells with 10-100 nM tributyltin for 48 h suppressed cortisol and androstenedione secretion, but induced the accumulation of 17alpha-hydroxyprogesterone and deoxycortisol, indicating that the P450(C21) and P450(11beta) activities were specifically suppressed. Direct inhibition of the enzymatic activities due to tributyltin was not observed in isolated organelles of untreated cells at concentrations less than 10 microM. Western blotting experiments using specific antibodies against steroidogenic enzymes showed that treatment with 1-100 nM tributyltin caused a decrease in cellular P450(C21) and P450(11beta) protein levels, and real-time PCR experiments showed that the decrease in protein content was attributable to decreases in mRNA of the enzymes. Tributyltin at concentrations higher than 100 nM suppressed all steroid biosynthesis in the adrenal cells. This suppression was closely correlated to the decrease in steroidogenic acute regulatory protein. Since nanomolar concentrations of tributyltin disturbed steroidogenesis in mammalian cells, there is the possibility that steroid hormone synthesis in polluted wild animals is affected by this compound.