Organizing effects of sex steroids on brain aromatase activity in quail

Preoptic/hypothalamic aromatase activity (AA) is sexually differentiated in birds and mammals but the mechanisms controlling this sex difference remain unclear. We determined here (1) brain sites where AA is sexually differentiated and (2) whether this sex difference results from organizing effects of estrogens during ontogeny or activating effects of testosterone in adulthood. In the first experiment we measured AA in brain regions micropunched in adult male and female Japanese quail utilizing the novel strategy of basing the microdissections on the distribution of aromatase-immunoreactive cells. The largest sex difference was found in the medial bed nucleus of the stria terminalis (mBST) followed by the medial preoptic nucleus (POM) and the tuberal hypothalamic region. A second experiment tested the effect of embryonic treatments known to sex-reverse male copulatory behavior (i.e., estradiol benzoate [EB] or the aromatase inhibitor, Vorozole) on brain AA in gonadectomized adult males and females chronically treated as adults with testosterone. Embryonic EB demasculinized male copulatory behavior, while vorozole blocked demasculinization of behavior in females as previously demonstrated in birds. Interestingly, these treatments did not affect a measure of appetitive sexual behavior. In parallel, embryonic vorozole increased, while EB decreased AA in pooled POM and mBST, but the same effect was observed in both sexes. Together, these data indicate that the early action of estrogens demasculinizes AA. However, this organizational action of estrogens on AA does not explain the behavioral sex difference in copulatory behavior since AA is similar in testosterone-treated males and females that were or were not exposed to embryonic treatments with estrogens.     

Mechanism of the activation of arachidonic acid release by oxysterols in NRK 49F cells: Role of calcium

We previously demonstrated that oxysterols added to the culture medium of NRK 49F cells labelled with [¹⁴C] arachidonic acid potentiated arachidonic acid (AA) release and prostaglandin (PG) E₂ biosynthesis induced by the activation of these cells with fetal calf serum (FCS). In the absence of FCS, oxysterols had no effect on AA release. As phospholipase (Plase) A₂ activity is Ca²⁺-dependent, we investigated whether oxysterol potentiating effect on AA release was related to an effect of these compounds on cell Ca²⁺ concentration. In this paper, we show that the intensity of potentiation by oxysterol varies with the external cell Ca²⁺ concentration; when external Ca²⁺ is chelated by EGTA, the oxysterol effect persists, though it is decreased. The Ca²⁺ channel inhibitor nifedipine does not decrease the potentiating effect of 25-OH cholesterol, indicating that, if oxysterol favours Ca²⁺ entry into the cell, the nifedipine inhibited channel is not involved. At the usual concentration (5 μm/ml), oxysterols are not able to increase, mimmediately or after a short time of contact (90 min) the concentration of intracellular free Ca²⁺ ([Ca²⁺])_i measured by fluorescence of Quinn-2; at very high concentration of oxysterol (25 μm/ml), [Ca²⁺]_i only slightly increases (+30%). The liberation of AA induced by cell activation with the Ca²⁺ ionophore ionomycin is also potentiated by 25-OH cholesterol. All these observations are not in favour of a proper effect o oxysterols on cell Ca²⁺ level.     

Ins(1,4,5)P3 induces Ca2+ release from brain microsomes loaded either by the Ca2+ ATPase or by the Na+/Ca2+ exchanger.

In this study we investigated the release of Ca²⁺ in brain microsomes after Ca²⁺ loading by the Ca²⁺-ATPase or by the Na⁺/Ca²⁺ exchanger. The results show that in microsomes loaded with Ca²⁺ by the Ca²⁺-ATPase, Ins(1,4,5)P₃ (5 μM) release 21±2% of the total Ca²⁺ accumulated, and that in the microsomes loaded with Ca²⁺ by the Na²⁺/Ca²⁺ exchanger, Ins(1,4,5)P₃ released 28±3% of the total Ca²⁺ accumulated. These results suggest that receptors of Ins(1,4,5)P₃ may be co-localized with the Na²⁺/Ca²⁺ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P₃ receptors in the plasma membrane where the Na²⁺/Ca²⁺ exchanger is normally present, or both. We also found that Ins(1,4,5)P₃ inhibited the Ca²⁺-ATPase by 33.7%, but that it had no significant effect on the Na²⁺/Ca²⁺ exchanger.     

Relationships between rapid changes in local aromatase activity and estradiol concentrations in male and female quail brain

Estradiol-17β (E₂) synthesized in the brain plays a critical role in the activation of sexual behavior in many vertebrate species. Because E₂ concentrations depend on aromatization of testosterone, changes in aromatase enzymatic activity (AA) are often utilized as a proxy to describe E₂ concentrations. Utilizing two types of stimuli (sexual interactions and acute restraint stress) that have been demonstrated to reliably alter AA within minutes in opposite directions (sexual interactions = decrease, stress = increase), we tested in Japanese quail whether rapid changes in AA are paralleled by changes in E₂ concentrations in discrete brain areas. In males, E₂ in the pooled medial preoptic nucleus/medial portion of the bed nucleus of the stria terminalis (POM/BST) positively correlated with AA following sexual interactions. However, following acute stress, E₂ decreased significantly (approximately 2-fold) in the male POM/BST despite a significant increase in AA. In females, AA positively correlated with E₂ in both the POM/BST and mediobasal hypothalamus supporting a role for local, as opposed to ovarian, production regulating brain E₂ concentrations. In addition, correlations of individual E₂ in POM/BST and measurements of female sexual behavior suggested a role for local E₂ synthesis in female receptivity. These data demonstrate that local E₂ in the male brain changes in response to stimuli on a time course suggestive of potential non-genomic effects on brain and behavior. Overall, this study highlights the complex mechanisms regulating local E₂ concentrations including rapid stimulus-driven changes in production and stress-induced changes in catabolism.     

Mastoparan, a peptide toxin from wasp venom, stimulates glycogenolysis mediated by an increase of the cytosolic free Ca concentration but not by an increase of cAMP in rat hepatocytes

A wasp venom, mastoparan, rapidly increased the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and activated phosphorylase in rat hepatocytes in a concentration-dependent manner. Mastoparan could increase [Ca²⁺]_i even in the absence of extracellular Ca²⁺, but a larger increase was observed in the presence of extracellular Ca²⁺. Thus, mastoparan mobilized Ca²⁺ from intracellular and extracellular Ca²⁺ stores. It also activated inositol triphosphate (IP₃) accumulation, but did not stimulate cAMP production. From these results, we conclude that mastoparan activates rat hepatic glycogenolysis mediated by the accumulation of IP₃, which causes an increase of [Ca²⁺]_i but not that mediated by cAMP.     

Rapid action on neuroplasticity precedes behavioral activation by testosterone

Testosterone has been shown to increase the volume of steroid-sensitive brain nuclei in adulthood in several vertebrate species. In male Japanese quail the volume of the male-biased sexually dimorphic medial preoptic nucleus (POM), a key brain area for the control of male sexual behavior, is markedly increased by testosterone. Previous studies assessed this effect after a period of 8–14 days but the exact time course of these effects is unknown. We asked here whether testosterone-dependent POM plasticity could be observed at shorter latencies. Brains from castrated male quail were collected after 1, 2, 7 and 14 days of T treatment (CX+T) and compared to brains of untreated castrates (CX) collected after 1 or 14 days. POM volumes defined either by Nissl staining or by aromatase immunohistochemistry increased in a time-dependent fashion in CX+T subjects and almost doubled after 14 days of treatment with testosterone while no change was observed in CX birds. A significant increase in the average POM volume was detected after only one day of testosterone treatment. The optical density of Nissl and aromatase staining was also increased after one or two days of testosterone treatment. Activation of male copulatory behavior followed these morphological changes with a latency of approximately one day. This rapid neurochemical and neuroanatomical plasticity observed in the quail POM thus seems to limit the activation of male sexual behavior and offers an excellent model to analyze features of steroid-regulated brain structure and function that determine behavior expression.     

Effects of Basal [Ca]i on Calcium Handling in Ca-Overloaded Rat Cultured Heart Muscle Cells

To elucidate the relationship between intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and Ca²⁺-signalling by the sarcoplasmic reticulum (SR) in Ca²⁺-overloaded heart muscle cells, the direct effects of “basal” [Ca²⁺]_i on calcium waves were investigated by altering the membrane potential. When basal inter-calcium wave (BCW) [Ca²⁺]_i was maintained at a high level, (i) calcium waves showed more gradual and more rapidly suppressed increase in [Ca²⁺]-profile (P < 0.005), and (ii) calcium waves occurred at a significantly higher frequency and velocity (259% and 137%), than when low BCW [Ca²⁺]_i was maintained. Similar investigations on inhibition of the Na⁺-Ca²⁺ exchanger, however, showed that membrane potential did not elicit direct effects on calcium waves. These results showed that the elevation of BCW [Ca²⁺]_i per se directly influences Ca²⁺-signalling in heart muscle cells through non-equilibrated release-restoration Ca²⁺-handling by the SR.     

The function of ATP in Ca uptake by rat brain mitochondria

Ca²⁺ uptake by rat brain mitochondria was studied under different experimental conditions. The most rapid uptake of Ca²⁺ occurred in the presence of ATP, succinate and P_i. ATP alone also supported Ca²⁺ uptake. In contrast, no Ca²⁺ uptake occurred with succinate and P_i when no ATP was added. Oligomycin and atractylate completely inhibited ATP-supported Ca²⁺ uptake but produced only a partial inhibition of Ca²⁺ transport in the presence of ATP, succinate and P_i. ATP plays a dual role in its action on brain mitochondria; it can support Ca²⁺ uptake by itself and it serves a function in allowing respiration-dependent Ca²⁺ uptake to proceed. The latter role of ATP does not involve transfer of energy from the nucleotide.     

Acid phosphatases bind to the main high density lipoprotein apolipoprotein A-I

Light-dependent Ca²⁺ efflux via the Ca²⁺/H⁺ antiport in the photosynthetic purple sulfur bacterium Chromatium vinosum was inhibited by three phenothiazines: chlorpromazine; trifluoperazine and phenothiazine. The inhibitors had no effect on Ca²⁺ uptake by C. vinosum in the dark nor any effect on the light-dependent efflux of either Na⁺ or Tl⁺ catalyzed, respectively, by the C. vinosum Na⁺/H⁺ or K⁺/H⁺ antiports. Ruthenium red and LaCl₃, neither of which inhibited light-dependent Ca²⁺ efflux in C. vinosum, markedly inhibited Ca²⁺ uptake in the dark by C. vinosum cells. Ruthenium red had no effect on the uptake of either Na⁺or the K⁺ analog T1⁺ by C. vinosum cells in the dark. These results have been interpreted in terms of two separate Ca²⁺ transport systems in C. vinosum: (i) a phenothiazine-sensitive and ruthenium red, La³⁺-insensitive Ca²⁺/H⁺ antiport responsible for Ca²⁺ efflux in the light; and (ii) a ruthenium red and La³⁺-sensitive but phenothiazine-insensitive Ca²⁺ uptake system.     

Regulation of endometrial prostaglandin synthesis during early pregnancy in cattle: Effects of phospholipases and calcium In vitro

In experiment 1, endometrial explants from 3 cyclic (Day 17) cows were incubated with arachidonic acid (AA), phospholipase A-2 (PLA-2) and calcium ionophore A23187 (CaI) or control. AA (0.2 mg), PLA-2 (1 U/ml) and Cal (4 μg/ml) increased PGF and PGE secretion. In experiment 2, endometrial explants from cyclic (n = 4) and pregnant (n = 3) cows were incubated +/- Ca⁺⁺ and with either: control, AA, PLA-2, CaI, PLA-2 + CaI, or AA + CaI. PG secretion was higher in cultures with Ca⁺⁺. In presence of Ca⁺⁺, PGF secretion was lower for pregnant than cyclic endometrium. AA with Ca⁺⁺ stimulated PGF and PGE secretion, indicating that AA availability may limit PG secretion. The stimulatory effect of PLA-2 on PGF and PGE secretion was greater in pregnant than cyclic Endometrium. However, CaI inhibited the PLA-2 response of pregnant, but not cyclic endometrium. In experiment 3, endometrium (4 cyclic cows) failed to convert ³H-PGF₂ to PGE₂ or ³H-PGE₂ to PGF₂ Responsiveness of PG secretion to PLA-2, and CaI is altered by reproductive status suggesting that these factors may be involved in the differential regulation of PG production during early pregnancy in cattle.     

Biochemical characterization and novel isolation of pure estrogen receptor hormone-binding domain

Biologically active, mouse estrogen receptor hormone-binding domain (residues 313–599) overexpressed in Escherichia coli was purified to apparent homogeneity as a single component with a molecular mass of 32.831 kDa determined by electrospray ionization mass spectrometry, and was identical to the mass predicted from the amino acid sequence. The intact domain was isolated using a novel, rapid purification scheme without recourse to any chromatographic process. Pure ERhbd maintained both high affinity estradiol binding (at optimum pH 8.0) and specificity for estrogens and anti-estrogens. The steroid-binding domain sedimented as a 4S component in the presence or absence of bound [³H]estradiol and at 2S in the presence of urea. The molecular mass of the 4S steroid unoccupied ERhbd (from dynamic light scattering) was 72 kDa, suggesting that the pure, unlabelled ERhbd formed homodimers. Steroid-labelled ERhbd electrofocussed as a single, acidic component at a pI of 5.6. Binding of ERhbd to [³H]estradiol was unaffected by Ca²⁺ and Mg²⁺ ions up to 1 mM but was significantly inhibited by Zn²⁺ ions at concentrations above 10 μM, an effect reversed by EDTA.     

2, 5-Di(Tert-Butyl)-1, 4-Benzohydroquinone —A Novel Mobilizer Of The Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Pool

Isolated hepatocytes and the isolated perfused rat liver have been used to study the alterations of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) produced by 2,5-di(tert-butyl)-l.4-benzohydroquinone (tBuBHQ), a potent inhibitor of hepatic microsomal Ca²⁺ sequestration (Moore. G.A., McConkey. D.J., Kass, G.E.N., OBrien, P.J. and Orrenius, S. FEBS Lett.,224, 331-336). (1987). Addition of tBuBHQ to isolated hepatocytes caused a rapid increase in [Ca²⁺]_i which was similar in magnitude to the [Ca²⁺]_i elevation induced by the Ca²⁺ mobilizing hormone, vasopressin. In contrast with vasopressin which caused a Ca²⁺ transient, tBuBHQ elevated [Ca²⁺]_i to a new steady state that was maintained for up to 15-20min. When vasopressin was administered during the tBuBHQ-induced period of elevated [Ca²⁺]_i. [Ca²⁺]_i, rapidly returned to basal levels. Similarly, if vasopressin was administered just prior to tBuBHQ, the resultant tBuBHQ-dependent change in [Ca²⁺]_i was transient. and not sustained. The hydroquinone mobilized the same intracellular Ca²⁺ pool as inositol 1,4,5-trisphosphate. but tBuBHQ did not produce any detectable inositol polyphosphate accumulation. IBuBHQ stimulated glucose release from perifused hepatocytes. mimicking the effect of vasopressin. In the perfused liver, tBuBHQ infusion produced a single, slow and prolonged release of Ca²⁺ into the perfusate and inhibition of subsequent vasopressin-induced Ca²⁺ effluxes. Inhibition of the response to vasopressin was reversed over time, and closely correlated with the extent of inhibition of both Ca²⁺ sequestration and (Ca²⁺-Mg²⁺)-ATPase activity in microsomes isolated from the isolated perfused liver. The present results are consistent with tBuBHQ inhibiting ATP-dependent Ca²⁺ sequestration by a direct effect on the endoplasmic reticular Ca²⁺ pump, which results in net Ca²⁺ release and elevation of [Ca²⁺]_i. Furthermore. vasopressin appears to stimulate active removal of increased [Ca²⁺] from the hepatocyte cytosol by a mechanism which does not depend on reuptake of Ca²⁺ into the endoplasmic reticulum

2,5-Di(tert-butyl) -l,4-benmhydroquinone. calcium. hepatocytes. perfused liver, endoplasmic reticulum     

Aromatase: Neuromodulator in the control of behavior

Estrogens are required for both the organization of the brain in early development and adult behavior. Two approaches have been used in our laboratory to study the behavioral role of brain aromatase. First, brain metabolism of testosterone (T) has been related to behavior in the same individual using a well established neuroendocrine model, the ring dove, in which estradiol-17β (E₂) has specific effects on brain mechanisms of male behavior. Aromatase in preoptic area (POA) (a) has a high activity (V_max) and strong substrate binding affinity (K_m < 5 nM), (b) is regulated by both androgens and estrogens, and the type of regulation differs according to brain area, (c) is influenced by products of an endogenous inactivating pathway, 5β-reduction; 5β-dihydrotestosterone and other 5β-reduced metabolites appear to be non-genomic regulators of the brain aromatase. Preoptic aromatase activity is also influenced by photoperiod and socio-sexual stimuli. The codistribution of regulated aromatase activity and estrogen receptor cells is found to be T-dependent. Our second approach has been to relate the aromatase system to developmental sex differences in brain structure and behavior of the Mongolian gerbil. Neonatal gerbil aromatase is relatively active in the POA, but has a weaker T substrate-binding affinity (K_m = 30 nM) than the dove. T acting via its metabolite, E₂, masculinizes the sexually dimorphic area of the hypothalamus; the differentiating effect is asymmetric. We suggest that the regulation of the brain aromatase system may be lateralized during steroid-sensitive periods of development.     

Arrhythmogenic Ca(2+) release from cardiac myofilaments

We investigated the initiation of Ca²⁺waves underlying triggered propagated contractions (TPCs) occurring in rat cardiac trabeculae under conditions that simulate the functional non-uniformity caused by mechanical or ischemic local damage of the myocardium. A mechanical discontinuity along the trabeculae was created by exposing the preparation to a small constant flow jet of solution with a composition that reduces excitation–contraction coupling in myocytes within that segment. Force was measured and sarcomere length as well as [Ca²⁺]_i were measured regionally. When the jet-contained Caffeine, BDM or Low-[Ca²⁺], muscle-twitch force decreased and the sarcomeres in the exposed segment were stretched by shortening of the normal regions outside the jet. During relaxation the sarcomeres in the exposed segment shortened rapidly. Short trains of stimulation at 2.5 Hz reproducibly caused Ca²⁺-waves to rise from the borders exposed to the jet. Ca²⁺-waves started during force relaxation of the last stimulated twitch and propagated into segments both inside and outside of the jet. Arrhythmias, in the form of non-driven rhythmic activity, were triggered when the amplitude of the Ca²⁺-wave increased by raising [Ca²⁺]_o. The arrhythmias disappeared when the muscle uniformity was restored by turning the jet off. We have used the four state model of the cardiac cross bridge (Xb) with feedback of force development to Ca²⁺ binding by Troponin-C (TnC) and observed that the force–Ca²⁺ relationship as well as the force–sarcomere length relationship and the time course of the force and Ca²⁺ transients in cardiac muscle can be reproduced faithfully by a single effect of force on deformation of the TnC·Ca complex and thereby on the dissociation rate of Ca²⁺. Importantly, this feedback predicts that rapid decline of force in the activated sarcomere causes release of Ca²⁺ from TnC.Ca²⁺,which is sufficient to initiate arrhythmogenic Ca²⁺ release from the sarcoplasmic reticulum. These results show that non-uniform contraction can cause Ca²⁺-waves underlying TPCs, and suggest that Ca²⁺ dissociated from myofilaments plays an important role in the initiation of arrhythmogenic Ca²⁺-waves.     

Inhibition of cell growth by K channel modulators is due to interference with agonist-induced Ca release

The effects of K⁺ channel modulators, tetraethylammonium, 4-aminopyridine and diazoxide, and high extracellular K⁺ on cell growth and agonist-induced intracellular Ca²⁺ mobilization were investigated. Two human brain tumour cell lines, U-373 MG astrocytoma and SK-N-MC neuroblastoma, were used as model cellular systems. K⁺ channel modulators and increased extracellular K⁺ concentration inhibited tumour cell growth in a dose-related fashion in both cell lines. In addition, agonist (carbachol or serum)-induced intracellular Ca²⁺ mobilization was also blocked by the pretreatment of growth-inhibitory concentrations of K⁺ channel modulators and high extracellular K⁺. Thus, these results suggest that K⁺ channel modulators are effective inhibitors of brain tumour cell growth and that their growth regulation may be due to the interference with the intracellular Ca²⁺ signalling mechanisms.