Rapid Nongenomic Effects Research Articles

Low doses of fludrocortisone and hydrocortisone, alone or in combination, on vascular responsiveness to phenylephrine in healthy volunteers

A single administration of hydrocortisone has been shown to enhance the pressor response to phenylephrine in healthy volunteers and to norepinephrine in septic shock patients. Similar data do not exist for fludrocortisone. Since there continues to be disagreement about the utility of fludrocortisone in septic shock, we assessed the effects of a single administration of low doses of hydrocortisone (50 mg intravenously) and fludrocortisone (50 μg orally), given either alone or in combination, on phenylephrine mean arterial pressure and cardiac systolic and diastolic function dose-response relationships in 12 healthy male volunteers with hypo-aldosteronism induced by intravenous sodium loading. This was a placebo-controlled, randomized, double-blind, crossover study performed according to a 2 × 2 factorial design. Subjects received first a 2000 ml infusion of NaCl 0.9% during 2 h. Then fludrocortisone 50 μg (or its placebo) was administered orally and hydrocortisone 50 mg (or its placebo) was injected intravenously. At 1.5 h after treatment administration, incremental doses of phenylephrine were infused (from 0.01 to 3 μg kg(-1) min(-1)), each dose being infused during 5 min. Both fludrocortisone (P < 0.001) and hydrocortisone (P = 0.002) induced a significant decrease in pressor response to phenylephrine, their effects being additive (fludrocortisone × hydrocortisone interaction, P = 0.792). The two drugs did not induce any detectable cardiac effect. Single administrations of fludrocortisone and hydrocortisone decreased the pressor response to phenylephrine in healthy volunteers with hypo-aldosteronism. These similar effects of hydrocortisone and fludrocortisone probably express a rapid non-genomic vasodilating effect of the two steroids in the context of acute volume loading.

Neurotrophic effects of neudesin in the central nervous system

Neudesin (neuron-derived neurotrophic factor; NENF) was identified as a neurotrophic factor that is involved in neuronal differentiation and survival. It is abundantly expressed in the central nervous system, and its neurotrophic activity is exerted via the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. Neudesin is also an anorexigenic factor that suppresses food intake in the hypothalamus. It is a member of the membrane-associated progesterone receptor (MAPR) family and shares key structural motifs with the cytochrome b5-like heme/steroid-binding domain. Progesterone receptor membrane component 1 (PGRMC1), the first to be discovered among the MAPR family, binds progesterone to induce “rapid non-genomic effects” in biological responses that are unrelated to the nuclear progesterone receptors (PRs). Hence, neudesin may also be involved in the rapid non-genomic actions of progesterone. In this review, we summarize the identification, structure, and activity of neudesin in the central nervous system, and present an essential overview of the current understanding of its physiological roles and the prospect of elucidating its non-genomic progesterone effects.

Neuroprotective effect of estrogen: Role of nonsynaptic NR2B-containing NMDA receptors

Excessive activation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in the pathophysiology of chronic neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease. Some studies reported that NR2A and NR2B play different roles in the central nervous system (CNS). The NR2A subunit is primarily found in the synapses and is required for glutamate-mediated neuronal survival. On the other hand, the NR2B subunit is primarily found in the extrasynaptic sites and is required for glutamate-mediated neuronal death in both in vitro and in vivo experiments. Estrogen is a steroid hormone well known for its widespread effects such as neuroprotection in the brain. Classically, estrogen can bind to two kinds of nuclear receptors, namely, estrogen receptor α (ERα) and estrogen receptor β (ERβ), and produce physiological and neuroprotective effects. Aside from nuclear receptors, estrogen has one membrane receptor, which can either be G-protein-coupled receptor 30 (GPR30), Gq-mER, or ER-X. NMDA exposure clearly promotes NR2B subunit phosphorylation at Ser-1303 and causes neuronal cell death. GPR30 mediates rapid non-genomic effects to protect neurons against injury by inhibiting p-DAPK1 dephosphorylation, which inhibits NR2B subunit phosphorylation at Ser-1303. In addition, NMDA exposure and global ischemia activate the autophagy pathway and induce cell death, which are markedly blocked by the NR2B antagonist Ro 25-6981. Thus, NR2B signaling, autophagy induction and cell death may be closely related. Ro 25-6981 inhibits the dissociation of the NR2B-Beclin-1 signaling complex and delays autophagy in vivo, thus confirming the link between NR2B signaling and autophagy. In short, ERα, ERβ, and GPR30 are involved in the neuroprotection of estrogen in the CNS. Additional research must be conducted to reveal the mechanism of estrogen action fully and to identify better targets for the development of more effective drugs. This article is part of a Special Issue entitled 'Extrasynaptic ionotropic receptors'.

17β-estradiol modulates mitochondrial Ca2+ flux in rat caudate nucleus and brain stem

The aim of this study was to examine the rapid non-genomic effect of 17β-estradiol (E2) on Ca2+ transport in mitochondria isolated from the nerve terminals (synaptosomes) of caudate nuclei (NC) and brain stems (BS) of ovariectomised female rats. In physiological conditions no effect of E2 on Ca2+ influx into synaptosomal mitochondria through ruthenium red (RR)-sensitive uniporter was observed. However, in the presence of uncoupling agent carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone (FCCP) (1μmol/l), pre-treatment with 0.5nmol/l E2 protected mitochondrial membrane potential and consequently increased Ca2+ influx (2.3-fold in NC and 3.1-fold in BS). At the same time, 0.5nmol/l E2 by increasing the affinity of mitochondrial Na+/Ca2+ exchanger for Na+ inhibited mitochondrial Ca2+ efflux in NC and BS by about 40%. Also, the specific binding of physiological E2 concentrations (0.1–10nmol/l) to isolated synaptosomal mitochondria was detected. Using membrane impermeable E2 bound to bovine serum albumin and selective inhibitor of mitochondrial Na+/Ca2+ exchanger, we obtained that E2’s action on mitochondrial Ca2+ efflux at least partially is due to the direct effects on the mitochondrial membrane and/or Na+/Ca2+ exchanger located in inner mitochondrial membrane. Our results implicate E2 as a modulator of Ca2+ concentration in mitochondrial matrix, and ultimately in the cytosol. Given the vital role of Ca2+ in regulation of total nerve cells activity, especially energy metabolism, neurotransmission and directing the cells toward survival or cell death, the effects on mitochondrial Ca2+ transport could be one of the important modes of E2 neuromodulatory action independent of the genome.

17β-Estradiol induces vasorelaxation in a G-protein-coupled receptor 30-independent manner

17β-Estradiol (E2) exerts rapid non-genomic vascular effects through activation of its plasma membrane receptors. We tested the hypothesis that E2 induces vasorelaxation through activation of the G-protein-coupled receptor 30 (GPR30) in rat aorta. Rat aortic rings were mounted in organ baths and subjected to contraction followed by relaxation. Whether endothelium was intact or denuded, both E2 and G1, a GPR30 agonist, induced vasorelaxation in concentration-dependent manners. Although G15, a specific GPR30 antagonist, blocked G1-induced vasorelaxation, it did not block E2-induced vasorelaxation. In conclusion, 17β-estradiol induces vasorelaxation in a GPR30-independent manner in rat aorta.

The anxiolytic effect of testosterone in the rat is mediated via the androgen receptor

Endogenous and exogenous testosterone affects several behavioural traits as shown in human and animal studies. The effects of testosterone can be mediated via androgen or oestrogen receptors, but also via rapid non-genomic effects. The aim of this study was to evaluate whether a single testosterone injection has effects, mediated via the androgen receptor, on anxiety in intact male rats. We hypothesised that administration of testosterone will have an anxiolytic effect, mediated by the androgen receptor. Intact adult male Wistar rats were divided into groups: control, flutamide, testosterone and testosterone with flutamide. Testosterone and flutamide (as an androgen receptor blocker) were applied once, intramuscularly, at a dose of 5mg/kg. Twenty four hours later, rats underwent the following behavioural tests to analyse anxiety: open field test, elevated plus maze and light–dark box. Testosterone was measured in plasma to confirm elevated levels in groups that received testosterone. The levels of testosterone were 2.5–3 fold higher amongst rats administered with testosterone compared to controls. Flutamide did not affect plasma testosterone concentrations. Testosterone administration had no effect on anxiety in the open field and elevated plus maze. In the light–dark transition task, testosterone increased the time spent in the light part of the maze by 80%, an effect which was blocked by flutamide, and which was in support of our hypothesis. Flutamide-treated rats spent more time in the central square of the open field. Using the light–dark box we have shown that a single injection of testosterone decreases anxiety in adult male rats. This effect of increased testosterone was mediated via the androgen receptor as flutamide blocked the anxiolytic effect of exogenous testosterone. Treatment with flutamide blocked the effects of endogenous testosterone and had anxiolytic effects in the open field, suggesting a non-linear relationship between genomic effects of T and anxiety.

Non-genomic vasorelaxant effects of 17β-estradiol and progesterone in rat aorta are mediated by L-type Ca2+ current inhibition

The sex hormones 17β-estradiol (βES) and progesterone (PRG) induce rapid non-genomic vasodilator effects which could be protective for the cardiovascular system. The purpose of this study was to analyze the mechanisms underlying their vasodilator effect in rat aortic smooth muscle preparations. Endothelium-denuded aorta artery rings were prepared from male Wistar rats and incubated in an organ bath. The contractions of the preparation were recorded through isometric transducers. The effects of the hormones on K(+) current and L-type Ca(2+) current (LTCC) were analyzed by using the whole cell voltage-clamp technique in A7r5 cells. Both βES and PRG (1-100 μmol/L) concentration-dependently relaxed the endothelium-denuded aortic rings contracted by (-)-Bay K8644 (0.1 μmol/L) or by KCl (60 mmol/L). The IC(50) values of the two hormones were not statistically different. The K(V) channel blocker 4-aminopyridine (2 mmol/L), BK(Ca) channel blocker tetraethylammonium (1 mmol/L) and K(ATP) channel blocker glibenclamide (10 μmol/L) did not significantly modify the relaxant effect of the hormones. On the other hand, the blockage of the intracellular βES and PRG receptors with estradiol receptor antagonists ICI 182,780 (1 μmol/L) and PRG receptor antagonist mifepristone (30 μmol/L), respectively, did not significantly modify the relaxant action of the hormones. In A7r5 cells, both the hormones (1-100 μmol/L) rapidly and reversibly inhibited the basal and BAY-stimulated LTCC. However, these hormones had no effect on the basal K(+) current. The vasorelaxant effects of βES and PRG are due to the inhibition of LTCC. The K(+) channels are not involved in the effects.

The Protective Side of the Mineralocorticoid Receptor

The Protective Side of the Mineralocorticoid Receptor

Extrarenal effects of aldosterone

The renal distal tubule has been considered for a long time as the main cellular target of aldosterone, where the hormone enhances sodium reabsorption and potassium secretion. However, other cell types in nonepithelial tissues, such as the heart, the vessels, adipose tissue, and macrophages, are now also recognized as targets for aldosterone. The functions that aldosterone exerts in these nonclassical target tissues are still a matter of debate. This review will highlight the recent findings on the extrarenal effects of aldosterone. Numerous studies showed that aldosterone exerts profibrotic and proinflammatory effects, but one or more cofactors such as salt, angiotensin II, and oxidative stress are required. Moreover, inflammation and macrophage infiltration are a prerequisite to aldosterone-induced cardiac fibrosis. This underlines a key role for aldosterone and the mineralocorticoid receptor in macrophages. Inflammatory effects of aldosterone in vascular smooth muscle cells involve trafficking to lipid rafts/caveolae through receptor tyrosine kinases. Finally, a growing body of evidence indicates a prominent role of aldosterone/mineralocorticoid receptor in the metabolic syndrome, in insulin resistance, and in adipocyte biology. The idiom from Socrates, 'the more we learn, the less we know', can be applied to aldosterone with its different facets and its pleiotropic effects. There is clear evidence for rapid nongenomic effects of aldosterone, mineralocorticoid receptor-dependent and mineralocorticoid receptor-independent signaling, in the heart, the vessels, and other nonepithelial tissues, leading to inflammation, fibrosis, and progression of cardiovascular diseases including hypertension and metabolic syndrome.

Proteomic profiling of rapid non-genomic and concomitant genomic effects of acute restraint stress on rat thymocytes

In order to investigate rapid non-genomic effects of acute stress, rats were restrained for 15 min which was sufficient to activate the hypothalamus-pituitary-adrenal (HPA) axis but too short to induce massive genomic effects of cortisol. Subcellular fractions of thymocytes (cytosol, nucleus, membrane) were investigated using quantitative 2D DIGE with MALDI-TOF/TOF mass spectrometry. In total, 108 proteins with differential subcellular localizations were identified. The specificity of the changes induced by psychological stress was reflected by the prominent modulation of proteins involved in the HPA and sympathoadrenal medullar (SAM) axis such as HMGB1 and NHERF1. Intracellular trafficking was characterized by a dominant protein exodus from the cytosol. Real translocation was observed for 9 proteins with 6 that shuttled from the cytosol to the nucleus (HYOU1, HNRPF, HNRPC, STRAP, PSA1, PPA1) and 3 from the nucleus to the cytosol (HMGB1, NHERF1, PSMA1). Proteins showing subcellular reshuffling were largely involved in transcription and translation processes (39 of 108) with a significant enrichment of RNA splicing factors. Bioinformatics analysis revealed significant enrichment for protein kinase A and 14-3-3 signaling, probably reflecting real non-genomic effects. This is the first study investigating rapid effects of stress-induced HPA activation in vivo at the proteome level.

Time-dependent effects of cortisol on selective attention and emotional interference: a functional MRI study.

Acute stress is known to induce a state of hypervigilance, allowing optimal detection of threats. Although one may benefit from sensitive sensory processing, it comes at the cost of unselective attention and increased distraction by irrelevant information. Corticosteroids, released in response to stress, have been shown to profoundly influence brain function in a time-dependent manner, causing rapid non-genomic and slow genomic effects. Here, we investigated how these time-dependent effects influence the neural mechanisms underlying selective attention and the inhibition of emotional distracters in humans. Implementing a randomized, double-blind, placebo-controlled design, 65 young healthy men received 10 mg hydrocortisone either 60 min (rapid effects) or 270 min (slow effects), or placebo prior to an emotional distraction task, consisting of color-naming of either neutral or aversive words. Overall, participants responded slower to aversive compared to neutral words, indicating emotional interference with selective attention. Importantly, the rapid effects of corticosteroids increased emotional interference, which was associated with reduced amygdala inhibition to aversive words. Moreover, they induced enhanced amygdala connectivity with frontoparietal brain regions, which may reflect increased influence of the amygdala on an executive network. The slow effects of corticosteroids acted on the neural correlates of sustained attention. They decreased overall activity in the cuneus, possibly indicating reduced bottom-up attentional processing, and disrupted amygdala connectivity to the insula, potentially reducing emotional interference. Altogether, these data suggest a time-specific corticosteroid modulation of attentive processing. Whereas high circulating corticosteroid levels acutely increase emotional interference, possibly facilitating the detection of threats, a history of elevation might promote sustained attention and thereby contribute to stress-recovery of cognitive function.

Action of ANP on the nongenomic dose-dependent biphasic effect of aldosterone on NHE1 in proximal S3 segment

The rapid (2min) nongenomic effects of aldosterone (ALDO) and/or spironolactone (MR antagonist), RU 486 (GR antagonist), atrial natriuretic peptide (ANP) and dimethyl-BAPTA (BAPTA) on the intracellular pH recovery rate (pHirr) via NHE1 (basolateral Na+/H+ exchanger isoform), after the acid load induced by NH4Cl, and on the cytosolic free calcium concentration ([Ca2+]i) were investigated in the proximal S3 segment isolated from rats, by the probes BCECF-AM and FLUO-4-AM, respectively. The basal pHi was 7.15±0.008 and the basal pHirr was 0.195±0.012pHunits/min (number of tubules/number of tubular areas=16/96). Our results confirmed the rapid biphasic effect of ALDO on NHE1: ALDO (10−12M) increases the pHirr to approximately 59% of control value, and ALDO (10−6M) decreases it to approximately 49%. Spironolactone did not change these effects, but RU 486 inhibited the stimulatory effect and maintained the inhibitory effect. ANP (10−6M) or BAPTA (5×10−5M) alone had no significant effect on NHE1 but prevented both effects of ALDO on this exchanger. The basal [Ca2+]i was 104±3nM (15), and ALDO (10−12 or 10−6M) increased the basal [Ca2+]i to approximately 50% or 124%, respectively. RU 486, ANP and BAPTA decreased the [Ca2+]i and inhibited the stimulatory effect of both doses of ALDO. The results suggest the involvement of GR on the nongenomic effects of ALDO and indicate a pHirr-regulating role for [Ca2+]i that is mediated by NHE1, stimulated/impaired by ALDO, and affected by ANP or BAPTA with ALDO. The observed nongenomic hormonal interaction in the S3 segment may represent a rapid and physiologically relevant regulatory mechanism in the intact animal under conditions of volume alterations.

A retinoic acid receptor RARα pool present in membrane lipid rafts forms complexes with G protein αQ to activate p38MAPK

Retinoic acid (RA) regulates several gene programs by nuclear RA receptors (RARs) that are ligand-dependent transcriptional transregulators. The basic mechanism for switching on transcription of cognate-target genes involves RAR binding at specific response elements and a network of interactions with coregulatory protein complexes. In addition to these classical genomic effects, we recently demonstrated that RA also induces the rapid activation of the p38MAPK/MSK1 pathway, with characteristic downstream consequences on the phosphorylation of RARs and the expression of their target genes. Here, we aimed at deciphering the underlying mechanism of the rapid non-genomic effects of RA. We highlighted a novel paradigm in which a fraction of the cellular RARα pool is present in membrane lipid rafts, where it forms complexes with G protein alpha Q (Gαq) in response to RA. This rapid RA-induced formation of RARα/Gαq complexes in lipid rafts is required for the activation of p38MAPK that occurs in response to RA. Accordingly, in RA-resistant cancer cells, characterized by the absence of p38MAPK activation, RARα present in membrane lipid rafts does not associate with Gαq, pointing out the essential contribution of RARα/Gαq complexes in RA signaling.

Inhibition of mitochondrial Na+-dependent Ca2+ efflux by 17β-estradiol in the rat hippocampus

Our results, as well as those of others, have indicated that 17β-estradiol (E2) exerts its nongenomic effects in neuronal cells by affecting plasma membrane Ca(2+) flux. In neuronal cells mitochondria possess Ca(2+) buffering properties as they both sequester and release Ca(2+). The goal of this study was to examine the rapid non-genomic effect of E2 on mitochondrial Ca(2+) transport in hippocampal synaptosomes from ovariectomised rats. In addition, we aimed to determine if, and to what extent, E2 receptors participated in mitochondrial Ca(2+) transport modulation by E2 in vitro. E2-specific binding and Ca(2+) transport was monitored. At physiological E2 concentrations (0.1-1.5 nmol/L), specific E2 binding to mitochondria isolated from hippocampal synaptosomes was detected with a B(max.) and K(m) of 37.6±2.6 fmol/mg protein and 0.69±0.14 nmol/L of free E2, respectively. The main mitochondrial Ca(2+) influx mechanism is the Ruthenium Red-sensitive uniporter driven by mitochondrial membrane potential. Despite no effect of E2 on Ca(2+) influx, a physiological E2 concentration (0.5 nmol/L) protected mitochondrial membrane potential and consequently Ca(2+) influx from the uncoupling agent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (1 μmol/L). In neuronal cells the predominant mitochondrial Ca(2+) efflux mechanism is the Na(+)/Ca(2+) exchanger. E2 caused Ca(2+) efflux inhibition (by 46%) coupled with increased affinity of the Na(+)/Ca(2+) exchanger for Na(+). Using E2 receptor (ERα and ERβ) antagonists and agonists, we confirmed ERβ's involvement in E2-induced mitochondrial membrane potential protection as well as Ca(2+) efflux inhibition. In summary, our results indicate that the non-genomic neuromodulatory role of E2 in rat hippocampus is achieved by affecting mitochondrial Ca(2+) transport via, in part, mitochondrial ERβ.

Rapid non-genomic effects of corticosteroids and their role in the central stress response

In response to a stressful encounter, the brain activates a comprehensive stress system that engages the organism in an adaptive response to the threatening situation. This stress system acts on multiple peripheral tissues and feeds back to the brain; one of its key players is the family of corticosteroid hormones. Corticosteroids affect brain functioning through both delayed, genomic and rapid, non-genomic mechanisms. The latter mode of action has long been known, but it is only in recent years that the physiological basis in the brain is beginning to be unravelled. We now know that corticosteroids exert rapid, non-genomic effects on the excitability and activation of neurons in (amongst others) the hypothalamus, hippocampus, amygdala and prefrontal cortex. In addition, corticosteroids affect cognition, adaptive behaviour and neuroendocrine output within minutes. Knowledge on the identity of the receptors and secondary pathways mediating the non-genomic effects of corticosteroids on a cellular level is accumulating. Interestingly, in many cases, an essential role for the 'classical' mineralocorticoid and glucocorticoid receptors in a novel membrane-associated mechanism is found. Here, we systematically review the recent literature on non-genomic actions of corticosteroids on neuronal activity and functioning in selected limbic brain targets. Further, we discuss the relevance of these permissive effects for cognition and neuroendocrine control, and the integration of this novel mode of action into the complex balanced pattern of stress effects in the brain.

GPR30, Mineralocorticoid Receptors, and the Rapid Vascular Effects of Aldosterone

See related article, pp 442–451 It is now generally accepted that in common with other steroid hormones aldosterone has rapid nongenomic effects, in addition to those mediated via DNA-directed, RNA-mediated protein synthesis. The currently accepted physiology of aldosterone was primarily charted by nephrologists, who understandably focused on the epithelial (and genomic) effects of aldosterone on urinary electrolytes, and the homeostatic changes in aldosterone secretion in response to sodium deficiency or potassium loading. The most acute physiological stimulus to aldosterone secretion, however, is assumption of the upright posture. Given this, it is not inappropriate to seek responses, similarly acute, to this rapid change in plasma aldosterone levels. In the paper by Gros et al,1 the authors conclusively show that aldosterone at low picomolar concentrations can act rapidly via both GPR30 (originally an “orphan” G protein–coupled receptor, subsequently an erstwhile membrane estrogen receptor) and classic mineralocorticoid receptors (MR) over a range of parameters. These include extracellular signal-regulated kinase (ERK) 1/2 activation and myosin light chain phosphorylation in rat aortic vascular smooth muscle cells (VSMC) in vitro; for ERK activation, aldosterone has equivalent action via both receptors at low picomolar concentrations. The effects on GPR30 appear mineralocorticoid specific: in rat aortic endothelial cells, with MR expression below detection levels by Western blotting, aldosterone increased ERK activation with an EC50 < 10 pM, an action abrogated by the GPR30 antagonist G15. In contrast, the effect of estradiol on inhibition of ERK activation was unaffected by G15 but blocked by the estrogen receptor α (ERα) antagonist ICI-182780. The first point to be made is that these studies firmly establish GPR30 as a bona fide receptor for aldosterone, given the low picomolar concentrations used (except for myosin light chain phosphorylation, where inexplicably 10 nmol/L aldosterone was used, and Figures 8 and 9 point in …

RAPID NONGENOMIC EFFECTS OF CORTISOL ON MEMBRANE FLUIDITY AND PHOSPHORYLATION IN RAINBOW TROUT LIVER.

Event Abstract Back to Event RAPID NONGENOMIC EFFECTS OF CORTISOL ON MEMBRANE FLUIDITY AND PHOSPHORYLATION IN RAINBOW TROUT LIVER. Laura Dindia1 and Mathilakath Vijayan1* 1 University of Waterloo, Canada While rapid steroid signaling is thought to play a key role in cellular function, little is known about the nongenomic role of cortisol in hepatic tissue. Therefore, we studied the nongenomic effects of cortisol on the biophysical properties of the liver plasma membrane and investigated the effects on the phosphorylation profile of protein kinase A (PKA), protein kinase C (PKC) and AKT kinase substrate proteins. The effect of cortisol on membrane fluidity was examined by measuring fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in isolated liver plasma membrane fractions. In vitro, physiological (100 ng/ml) and pharmacological (500 and 1000 ng/ml) cortisol concentrations were shown to rapidly increase hepatic membrane fluidity. Moreover, cortisol rapidly induced phosphorylation of several PKC substrate proteins. These results indicate for the first time that stress-induced plasma cortisol levels modulates liver membrane fluidity, which may lead to rapid intracellular signaling in rainbow trout. Acknowledgements This research was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to MMV, and a NSERC Post-graduate Scholarship (PGS D) to LD.

Time-dependent effects of corticosteroids on human amygdala processing.

Acute stress is associated with a sensitized amygdala. Corticosteroids, released in response to stress, are suggested to restore homeostasis by normalizing/desensitizing brain processing in the aftermath of stress. Here, we investigated the effects of corticosteroids on amygdala processing using functional magnetic resonance imaging. Since corticosteroids exert rapid nongenomic and slow genomic effects, we administered hydrocortisone either 75 min (rapid effects) or 285 min (slow effects) before scanning in a randomized, double-blind, placebo-controlled design. Seventy-two healthy males were scanned while viewing faces morphing from a neutral facial expression into fearful or happy expressions. Imaging results revealed that hydrocortisone desensitizes amygdala responsivity rapidly, while it selectively normalizes responses to negative stimuli slowly. Psychophysiological interaction analyses suggested that this slow normalization is related to an altered coupling of the amygdala with the medial prefrontal cortex. These results reveal a temporarily fine-tuned mechanism that is critical for avoiding amygdala overshoot during stress and enabling adequate recovery thereafter.

17β-Estradiol Elevates cGMP and, via Plasma Membrane Recruitment of Protein Kinase GIα, Stimulates Ca2+ Efflux from Rat Hepatocytes

Rapid non-genomic effects of 17beta-estradiol, the principal circulating estrogen, have been observed in a wide variety of cell types. Here we investigate rapid signaling effects of 17beta-estradiol in rat hepatocytes. We show that, above a threshold concentration of 1 nm, 17beta-estradiol, but not 17alpha-estradiol, stimulates particulate guanylyl cyclase to elevate cGMP, which through activation and plasma membrane recruitment of protein kinase G isoform Ialpha, stimulates plasma membrane Ca(2+)-ATPase-mediated Ca(2+) efflux from rat hepatocytes. These effects are extremely rapid in onset and are mimicked by a membrane-impermeant 17beta-estradiol-BSA conjugate, suggesting that 17beta-estradiol acts at the extracellular face of the plasma membrane. We also show that 17beta-estradiol binds specifically to the intact hepatocyte plasma membrane through an interaction that is competed by an excess of atrial natriuretic peptide but also shows many similarities to the pharmacological characteristics of the putative gamma-adrenergic receptor. We, therefore, propose that the observed rapid signaling effects of 17beta-estradiol are mediated either through the guanylyl cyclase A receptor for atrial natriuretic peptide or through the gamma-adrenergic receptor, which is either itself a transmembrane guanylyl cyclase or activates a transmembrane guanylyl cyclase through cross-talk signaling.

Classical membrane progesterone receptors in murine mammary carcinomas: agonistic effects of progestins and RU-486 mediating rapid non-genomic effects

In this article, we demonstrate the expression of functional progesterone binding sites at the cell membrane in murine mammary carcinomas that are stimulated by progestins and inhibited by antiprogestins. Using confocal immunofluorescence, ligand binding and cell compartment-specific western blots, we were able to identify the presence of the classical progesterone receptors. Medroxyprogesterone acetate (MPA) and RU-486 (1 × 10(-11) and 1 × 10(-8) M) behaved as agonists activating extracellular signal-regulated kinases (ERKs) and progestin-regulated proteins, except for Cyclin D1 and Tissue factor which failed to increase with 1 × 10(-8) M RU-486, an experimental condition that allows PR to bind DNA. These results predicted a full agonist effect at low concentrations of RU-486. Accordingly, at concentrations lower than 1 × 10(-11) M, RU-486 increased cell proliferation in vitro. This effect was abolished by incubation with the ERK kinase inhibitor PD 98059 or by OH-tamoxifen. In vivo, at a daily dose of 1.2 μg/kg body weight RU-486 increased tumor growth, whereas at 12 mg/kg induces tumor regression. Our results indicate that low concentrations of MPA and RU-486 induce similar agonistic non-genomic effects, whereas RU-486 at higher concentrations may inhibit cell proliferation by genomic-induced effects. This suggests that RU-486 should be therapeutically administered at doses high enough to guarantee its genomic inhibitory effect.