In vivo response of some immune and endocrine variables to LPS in Eurasian perch (Perca fluviatilis, L.) and modulation of this response by two corticosteroids, cortisol and 11-deoxycorticosterone.

In fish, the endocrine system, especially corticosteroids pathway, strongly interacts with immune system. On the other hand, in vivo co-stimulation of both systems is not well documented. To better understand this interaction, we decided to evaluate the in vivo effects of both stimulation of the immune system and co-stimulation of both systems in Eurasian perch juveniles. Fish were injected either with 10mgkg(-1) LPS, or with a combination of LPS and 0.8mgkg(-1) cortisol or LPS and 0.08mgkg(-1) 11-deoxycorticosterone (DOC) and sampled 1, 3 or 7days after injection. LPS affected the immune system by increasing plasma lysozyme activity and blood neutrophils populations. During the same time-course, LPS decreased the proportion of a mixture of lymphocytes and thrombocytes in blood and TNF-α expression in spleen. Cortisol modulated the LPS-mediated response in TNF-α mRNA expression levels in spleen. Contrary to LPS alone, the association of LPS with DOC modulated the abundance of complement component 3 (C3) mRNA in spleen. On the other hand, LPS altered the corticotropic axis by decreasing mRNA expression levels of all corticosteroid receptors and of 11ß-HSD-2 in spleen. Both corticosteroids injected were not able to balance these LPS-induced suppressive effects on corticosteroid receptors and 11ß-HSD-2 expression levels in spleen. Contrary to LPS alone, the association of LPS with DOC modulated GR-1b expression in gills. These results indicated that LPS is a strong modulator of the corticosteroid receptors expression in spleen. Furthermore, we report for the first time a LPS-induced decrease of the mineralocorticoid receptor expression. Finally, corticosteroids were able to modulate the LPS-mediated response at the transcriptional level.

DOCA sensitive pendrin expression in kidney, heart, lung and thyroid tissues.

BACKGROUND/AIMS: Pendrin (SLC26A4), a transporter accomplishing anion exchange, is expressed in inner ear, thyroid gland, kidneys, lung, liver and heart. Loss or reduction of function mutations of SLC26A4 underlie Pendred syndrome, a disorder invariably leading to hearing loss with enlarged vestibular aqueducts and in some patients to hypothyroidism and goiter. Renal pendrin expression is up-regulated by mineralocorticoids such as aldosterone or deoxycorticosterone (DOCA). Little is known about the impact of mineralocorticoids on pendrin expression in extrarenal tissues. METHODS: The present study utilized RT-qPCR and Western blotting to quantify the transcript levels and protein abundance of Slc26a4 in murine kidney, thyroid, heart and lung prior to and following subcutaneous administration of 100 mg/kg DOCA. RESULTS: Slc26a4 transcript levels as compared to Gapdh transcript levels were significantly increased by DOCA treatment in kidney, heart, lung and thyroid. Accordingly pendrin protein expression was again significantly increased by DOCA treatment in kidney, heart, lung and thyroid. CONCLUSION: The observations reveal mineralocorticoid sensitivity of pendrin expression in kidney, heart, thyroid and lung.

The mislabelling of deoxycorticosterone: making sense of corticosteroid structure and function.

Over the 70 or so years since their discovery, there has been continuous interest and activity in the field of corticosteroid functions. However, despite major advances in the characterisation of receptors and coregulators, in some ways we still lack clear insight into the mechanism of receptor activation, and, in particular, the relationship between steroid hormone structure and function remains obscure. Thus, why should deoxycorticosterone (DOC) reportedly be a weak mineralocorticoid, while the addition of an 11ß-hydroxyl group produces glucocorticoid activity, yet further hydroxylation at C18 leads to the most potent mineralocorticoid, aldosterone? This review aims to show that the field has been confused by the misreading of the earlier literature and that DOC, far from being relatively inactive, in fact has a wide range of activities not shared by the other corticoids. In contrast to the accepted view, the presence of an 11ß-hydroxyl group yields, in corticosterone or cortisol, hormones with more limited functions, and also more readily regulated, by 11ß-hydroxysteroid dehydrogenase. This interpretation leads to a more systematic understanding of structure-function relationships in the corticosteroids and may assist more rational drug design.

Corticosteroid regulation of Na(+),K(+)-ATPase α1-isoform expression in Atlantic salmon gill during smolt development.

The proposed mineralocorticoid-like signalling axis in teleost fish, consisting of the hormone 11-deoxycorticosterone (DOC) and a mineralocorticoid receptor (MR), has recently challenged our conception of cortisol being the only osmoregulatory corticosteroid in teleost fish. This paper aimed at comparing the osmoregulatory role of DOC with that of cortisol during the pre-adaptive development of SW-tolerance, smoltification, in Atlantic salmon. Using an in vitro gill block incubation system, the effect of DOC and cortisol in the gill was investigated from January to September, using Na(+),K(+)-ATPase α-subunit isoforms α-1a and α-1b mRNA levels as targets for regulation by the hormones. Cortisol and DOC both conferred significant up-regulation of α-1a and α-1b mRNA levels at specific time-points during smoltification. However, the effect of cortisol and DOC on α-subunit isoforms varied seasonally between isoforms and hormones. The maximum induction of α-1a was 3- to 4-fold compared to controls whereas a 2-fold induction was observed for α-1b. The pattern and capacity of stimulation of α-1a through smoltification were similar for cortisol and DOC, whereas cortisol had an enhanced capacity to stimulate α-1b as compared to DOC. Even though there was no demonstrable change in cortisol or DOC sensitivity in the gill, the magnitude of the hormonal effects were seasonally dependent. This is the first report of DOC-induced effects on osmoregulatory targets in fish, thus indicating a role for DOC and MR signalling in osmoregulation.

Neurosteroids differentially modulate P2X ATP-gated channels through non-genomic interactions.

As neuroactive steroids modulate several ionotropic receptors, we assessed whether the ATP-gated currents elicited by P2X(4) receptors are modulated by these compounds. We transfected HEK293 cells or injected Xenopus laevis oocytes with the cDNA coding for rat P2X(4) receptor. Application of 0.1-10 microM alfaxolone potentiated within 60-s the 1 microM ATP-evoked currents with a maximal potentiation of 1.8 and 2.6-fold in HEK293 or oocytes cells respectively. Allopregnalolone or 3alpha, 21-dihydroxy-5alpha-pregnan-20-one (THDOC) also potentiated the ATP-gated currents but with a maximal effect only averaging 1.25 and 1.35-fold respectively. In contrast, 0.3-10 microM pregnanolone, but not its sulfated derivative, inhibited the ATP-gated currents; the maximal inhibition reached 40% in both cell types. THDOC, but not other neurosteroids increased significantly the tau(off) of the ATP-evoked currents, revealing another mode of neurosteroid modulation. Sexual steroids such as 17beta-estradiol or progesterone were inactive revealing explicit structural requirements. Alfaxolone or THDOC at concentrations 30- to 100-fold larger than required to modulate the receptor, gated the P2X(4) receptor eliciting ATP-like currents that were reduced with suramin or brilliant blue G, but potentiated the P2X(4) receptor more than 10-fold by 10 microM zinc. In conclusion, neurosteroids rapidly modulate via non-genomic mechanisms and with nanomolar potencies, the P2X4 receptor interacting likely at distinct modulator sites.

Multiple corticosteroid receptors in fish: from old ideas to new concepts.

The effect of corticosteroid hormones in fish are mediated through intracellular receptors that act as ligand-binding transcription factors. Many studies have been devoted to cortisol binding using radiolabeled ligand in fish and allowed characterization of a single class of high affinity binding sites in various tissues. Molecular characterization of cortisol receptors has only been initiated recently by cloning the different receptor forms: Following a isolation of a first glucocorticoid receptor (GR), a mineralocorticoid receptor (MR) was described and the presence of various GR isoforms was recently reported. Sequence comparison and phylogenetic analysis of these sequences confirm that fish possess both GR and MR and that GR gene is duplicated. The importance of these various corticosteroid receptor forms is also illustrated by analysis of their transcriptional activity. When tested in human cell lines, these receptors showed functionally distinct actions on GR-sensitive promotors, thus suggesting a more complicated corticosteroid signaling system than initially anticipated from binding studies. These results also suggest that, whereas cortisol is certainly the physiological ligand for GR, this may not be the case for MR which showed high sensitivity for deoxycorticosterone (DOC) and aldosterone. As this last hormone is probably absent in fish, these results raise the question as to whether DOC could be a physiological ligand for MR in fish. Information on DOC effect in fish is very scarce and clarification of the differential osmoregulatory roles of cortisol and DOC in fish needs ellucidation. This will require analysis of all actors of the corticosteroid signaling system at pre-receptor, receptor, and post-receptor levels.

[The role of neurosteroids in the central nervous system function]. / Znaczenie neurosteroidów w regulacji czynnosci osrodkowego ukladu nerwowego.

Neurosteroids--important modulators of the central nervous system activity--have been biochemically and functionally well characterized in recent years. Inhibitory neurosteroids are positive allosteric modulators of GABAA receptors which show anxiolytic and anticonvulsant properties, whereas negative modulators of GABAA receptors facilitate memory processes and at high doses show proconvulsant activity. Allopregnanolone is the most potent inhibitory neurosteroid, and the reduced metabolites of deoxycorticosterone and androgens have similar though weaker action. Pregnenolone, dehydroepiandrosterone and their sulfate derivatives, belong to stimulating neurosteroids, that besides the inhibitory effect on GABAA receptors enhance activity of glutamatergic NMDA receptors and sigma1 receptors which leads to an increase in acetylcholine release, in consequence, strengthening cognitive processes. Neurosteroids seem to be also involved in neuronal cell regeneration, regulation of hypothalamic-pituitary-adrenal axis activity and in the mechanism of drug dependence and depression. In contrast to well-documented beneficial effects of some neurosteroids on animal brain function, scarce clinical data do not allow to draw final conclusions about potential usefulness of these compounds in diagnosis or treatment of neurological and psychiatric disorders.

Is there a physiological role for the neurosteroid THDOC in stress-sensitive conditions?

Endogenous neurosteroids affect brain excitability during physiological states such as pregnancy and the menstrual cycle, and during conditions of acute and chronic stress. The neurosteroid allotetrahydrodeoxycorticosterone (THDOC) is an allosteric modulator of the GABA(A) receptor. Although the role of THDOC within the brain is undefined, recent studies indicate that stress induces THDOC to levels that can activate GABA(A) receptors. These results might have significant implications for human stress-sensitive conditions such as epilepsy, post-traumatic stress disorder and depression.

The effects of the neuroactive steroid 3 alpha,5 alpha-THDOC on sleep in the rat.

This vehicle-controlled study assessed the sleep effects of the naturally occurring neuroactive steroid 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-THDOC; 7.5 and 15 mg/kg), administered i.p. to rats, and compared them with those of another neuroactive steroid allopregnanolone (15 mg/kg). 3alpha,5alpha-THDOC shortened sleep latency, selectively promoted pre-REMS (a transitional state between non-REMS and REMS) and lengthened the non-REMS episodes dose-dependently. Spectral analysis of the EEG within non-REMS found significant attenuations of low-frequency activity and elevations in the spindle and higher frequency bands. The effects of 3alpha,5alpha-THDOC closely match those of allopregnanolone, indicating a common mechanism of action. Since the sleep changes produced by these steroids resemble the sleep profile of benzodiazepine hypnotics, they are probably caused by a positive allosteric modulation of GABAA receptor function.

The clinical potentials of endogenous neurosteroids.

Stress increases plasma and brain concentrations of the neurosteroids allopregnanolone and allotetrahydrodeoxycorticosterone (THDOC), which can have potent effects on GABAA receptors in the brain. Blockade of the formation of neurosteroids prevents specific biochemical and behavioral effects of stress, suggesting that those effects are dependent upon the actions of GABA(A)-receptor active neurosteroids. Recent investigations provide a better understanding of the role of endogenous neurosteroids in normal neuronal development and in the pathophysiology of brain disorders. Physiological neurosteroid fluctuations have potential implications for stress-sensitive neurological conditions such as epilepsy, infantile spasms, as well as psychiatric disorders such as schizophrenia, posttraumatic stress disorder and depression. Future studies may provide important new evidence that may not only explain acute actions of stress, but also reveal the clinical importance of neurosteroid mechanisms during chronic stress.

Stress and neuroactive steroids.

The discovery that the endogenous steroid derivatives 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone, or 3 alpha,5 alpha-TH PROG) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone, or 3 alpha,5 alpha-TH DOC) elicit marked anxiolytic and anti-stress effects and selectively facilitate gamma-aminobutyric acid (GABA)-mediated neurotransmission in the central nervous system (see Chapter 3) has provided new perspectives for our understanding of the physiology and neurobiology of stress and anxiety. Evidence indicating that various stressful conditions that downregulate GABAergic transmission and induce anxiety-like states (Biggio et al., 1990) also induce marked increases in the plasma and brain concentrations of these neuroactive steroids (Biggio et al., 1996, 2000) has led to the view that stress, neurosteroids, and the function of GABAA receptors are intimately related. Changes in the brain concentrations of neurosteroids may play an important role in the modulation of emotional state as well as in the homeostatic mechanisms that counteract the neuronal overexcitation elicited by acute stress. Indeed, neurosteroids not only interact directly with GABAA receptors but also regulate the expression of genes that encode subunits of this receptor complex. This chapter summarizes observations from our laboratories and others, suggesting that neurosteroids and GABAergic transmission are important contributors to the changes in emotional state induced by environmental stress.

Neuroactive steroids: their mechanism of action and their function in the stress response.

Steroids are usually identified as genomic regulators, yet recently a body of evidence has accumulated demonstrating specific plasma membrane effects, as well as coordinative effects, of some steroids on both membrane and intracellular receptors. The resulting rapid (<1 min) modulation of cellular activity has strongly suggested a non-genomic, and possibly modulatory, role for certain steroid compounds, and dramatic effects on membranes of excitable as well as other tissues have been demonstrated. Steroid synthesis and metabolism have been shown to exist in the CNS, and the effects have been seen in both the central and peripheral nervous systems. The major groups of neuroactive steroids, and their metabolites, have been progesterone, deoxycorticosterone, and some androgens, notably dihydroxyepiandrosterone (DHEA). These compounds show increased concentrations both in blood and in the brain following stress and they have also been associated with anxiolytic effects and antiepileptic activity. In the periphery, some of these compounds show remarkable inhibitory effects on the secretion of catecholamines and other neurotransmitters. The mechanism for the majority of the effects of these steroids is via their effect on receptor-mediated binding to ligand-gated ion channels. Activation of the GABAA receptor complex, resulting in the opening of its central chloride channel, is the major target of the neuroactive steroids, resulting in re-polarization of the plasma membrane and inhibition of further neuronal firing. The anxiolytic, anti-convulsant and sedative-hypnotic actions of these neuroactive steroids have resulted in their being used as therapeutic agents for the treatment of anxiety, epilepsy, insomnia, and possibly for the alteration of pain thresholds.

Metabolism of malate in bovine adrenocortical mitochondria studied by 13C-NMR spectroscopy.

13C-NMR spectroscopy was used to study the metabolism of [13C]malate in bovine coupled adrenocortical mitochondria. The most apparent difference between the mitochondria from steroidogenic tissues and mitochondria from other tissues is the presence, in addition to the normal respiratory chain, of a second electron-transport system responsible for steroid hydroxylation. [13C]malate was synthesized from [13C]succinate by isolated adrenocortical mitochondria. The basic functional suspension consisted of oxygenated mitochondria to which were added ADP, inorganic phosphate (Pi) and [13C]malate, both in the absence or presence of the steroid substrate, deoxycorticosterone. These mitochondria synthesized [13C]citrate and [13C]pyruvate from [13C]malate. The 13C labeling of these two metabolites demonstrated an important role of the malic enzyme and the kinetics depended on the presence of the steroid substrate; the citric acid cycle was stopped during the hydroxylation pathway. The addition of cyanide, a strong inhibitor of the respiratory chain, confirmed an increased malic enzyme activity when hydroxylation occurred, since pyruvate was trapped by formation of a cyanohydrin. The relative enzymic activities of malic enzyme and isocitrate dehydrogenase were compared, both in the absence or presence of the steroid substrate, by supplementing the basic suspension with unlabeled exogenous metabolites, such as pyruvate or oxaloacetate.

Synergist interaction between angiotensin II and DOCA on sodium and water balance in rats.

Blockade of central angiotensin receptors with the specific antagonist [Leu8]-ANG II abolished water ingestion and water and sodium excretion induced by infusion of angiotensin II (ANGII) into the lateral ventricle (LV) of rats. The antagonist reduced but did not suppress the salt appetite induced by ANGII infusion. Subcutaneous injection of deoxycorticosterone acetate (DOCA) caused increases in water and 3% NaCl ingestion and decreases in sodium excretion. When central ANGII infusion was combined with peripheral DOCA, the water intake was similar to that induced by ANGII alone and the ingestion of 3% NaCl was increased, whereas sodium excretion was inhibited. When ANGII was infused alone, a detailed temporal analysis of fluid and sodium balance showed a negative balance similar those saline controls that persisted throughout the experiment. Combined administration of ANGII and DOCA induce significant changes in water and sodium balance. Sodium and water maintained a positive balance through out the 8-h experiment. The data support an interaction of central ANGII and DOCA on sodium intake and water and sodium balance.

The anteroventral wall of the third ventricle and the angiotensinergic component of need-induced sodium intake in the rat.

Because the anteroventral wall of the third ventricle (AV3V) has been implicated in the control of sodium intake it was studied in rats with damage of the AV3V region in the sodium-replete and sodium-deplete states, and when they were treated with either pulse intracerebroventricular (pICV) injection of renin to activate brain angiotensin or daily subcutaneous injections of deoxycorticosterone (DOCA). The response of rats with AV3V damage to sodium depletion was retarded and the excess sodium intake that is induced by pICV renin was absent, but their daily need-free sodium intake and the sodium intake that is induced by DOCA were essentially normal. The results suggest that the AV3V is responsible for the angiotensinergic, but not the aldosteronergic, component of the ANG II/ALDO synergy that controls need-induced sodium appetite in the rat. The integrity of the AV3V is not necessary for daily need-free sodium intake or for its enhancement by sodium depletions, suggesting that ANG II is probably not important for these phenomena. But ANG II action within the AV3V might be important for the rapid burst of sodium intake that immediately follows a period of depletion.

Deficits in NaCl ingestion after damage to the central nucleus of the amygdala in the rat.

These studies examined the NaCl intake behaviors of rats with bilateral electrolytic lesions of the central nucleus of the amygdala (CeAX). Daily need-free intake of 3% NaCl was abolished by CeAX even in rats in which it had been enhanced preoperatively by a history of repeated sodium depletions but was slightly restored by three successive postoperative sodium depletions. CeAX rats drank water but not 3% NaCl to high doses of DOCA and to the activation of cerebral angiotensin II, and expressed small but reliable salt intake (need-induced salt intake or salt appetite) after postoperative sodium depletions. Other ingestive behaviors (water drinking, intake of food and 5% sucrose) were normal. When given decreasing concentrations of NaCl solution the CeAX rats rejected them until the concentration reached 0.2%. These findings suggest that lesions to the central nucleus of the amygdala produce a global impairment in salt intake behaviors that is possibly due to an alteration in the central processing of the salt taste signal.