Stacking anabolic androgenic steroids (AAS) during puberty in rats: a neuroendocrine and behavioral assessment

Anabolic androgenic steroid (AAS) abuse is increasing in teenagers. We examined the effects of stacked AAS in adolescent male rats. Stacking, in which multiple AAS are taken simultaneously, is commonly employed by humans. Beginning at puberty gonadally intact male rats received testosterone, nandrolone, or stanozolol. Additional groups received stacked AAS: testosterone + stanozolol, nandrolone + stanozolol, or nandrolone + testosterone. Injections continued during tests for sexual behavior, vocalizations, scent marking, partner preference, aggression and fertility. Body and reproductive tissue weights were taken. Sexual and aggressive behaviors were increased by testosterone yet inhibited by stanozolol; nandrolone had no effect. Stacking testosterone with stanozolol prevented the inhibitory effects of stanozolol. Body weight was decreased by testosterone and all stacked AAS. Cell nuclear androgen receptor binding in brain was significantly increased in nandrolone males and decreased in stanozolol males; testosterone males were slightly higher than controls. Androgen receptors in stacked groups were intermediate between individual AAS suggesting that stanozolol competed with other AAS for androgen receptors despite its low affinity. The results indicate that stacking AAS influences the effects of individual AAS on behavioral and endocrine measures, and levels of androgen receptor occupation are not directly correlated with AAS effects on behavior.     

Regulation by astrocytic ATP of synaptic transmission

Originally ascribed to having only passive roles in the CNS, astrocytes are now known to have an active role in the regulation of synaptic transmission. Neuronal activity can evoke Ca(2+) transients in astrocytes and Ca(2+) transients in astrocytes can evoke changes in neuronal activity. The excitatory neurotransmitter glutamate has been shown to mediate such bi-directional communication between astrocytes and neurons. We demonstrate here that ATP, a primary mediator of intercellular Ca(2+) signaling among astrocytes, also mediates intercellular signaling between astrocytes and neurons in hippocampal cultures. Mechanical stimulation of astrocytes evoked Ca(2+) waves mediated by the release of ATP and activation of P2 receptors. Mechanically evoked Ca(2+) waves led to decreased excitatory glutamatergic synaptic transmission in an ATP-dependent manner. Exogenous application of ATP does not affect post-synaptic glutamatergic responses but decreased pre-synaptic exocytotic events. Finally, we show that astrocytes exhibit spontaneous Ca(2+) oscillations mediated by extracellular ATP and that inhibition of these Ca(2+) responses enhanced excitatory glutamatergic transmission. We therefore conclude that ATP released from astrocytes exerts tonic and activity-dependent down-regulation of synaptic transmission via pre-synaptic mechanisms.     

Ginsenoside Re, a main phytosterol of Panax ginseng, activates cardiac potassium channels via a nongenomic pathway of sex hormones

Ginseng root is one of the most popular herbs throughout the world and is believed to be a panacea and to promote longevity. It has been used as a medicine to protect against cardiac ischemia, a major cause of death in the West. We have previously demonstrated that ginsenoside Re, a main phytosterol of Panax ginseng, inhibits Ca(2+) accumulation in mitochondria during cardiac ischemia/reperfusion, which is attributable to nitric oxide (NO)-induced Ca(2+) channel inhibition and K(+) channel activation in cardiac myocytes. In this study, we provide compelling evidence that ginsenoside Re activates endothelial NO synthase (eNOS) to release NO, resulting in activation of the slowly activating delayed rectifier K(+) current. The eNOS activation occurs via a nongenomic pathway of each of androgen receptor, estrogen receptor-alpha, and progesterone receptor, in which c-Src, phosphoinositide 3-kinase, Akt, and eNOS are sequentially activated. However, ginsenoside Re does not stimulate proliferation of androgen-responsive LNCaP cells and estrogen-responsive MCF-7 cells, implying that ginsenoside Re does not activate a genomic pathway of sex hormone receptors. Fluorescence resonance energy transfer experiments with a probe, SCCoR (single cell coactivator recruitment), indicate that the lack of genomic action is attributable to failure of coactivator recruitment. Thus, ginsenoside Re acts as a specific agonist for the nongenomic pathway of sex steroid receptors, and NO released from activated eNOS underlies cardiac K(+) channel activation and protection against ischemia-reperfusion injury.     

Regional differences in the vasorelaxing effects of testosterone and its 5-reduced metabolites in the canine vasculature

Although the vasorelaxing effects of testosterone (T) and various androgen metabolites have been observed in a variety of blood vessels and species, previous studies have not systematically compared the vasorelaxing effects of androgen metabolites in different vascular beds within the same species. Therefore, we studied the vasorelaxing effects of T and its 5-reduced metabolites (5α- and 5β-DHT) on KCl-induced contractions of the canine left coronary artery, femoral artery and saphenous vein, using standard isometric recordings. KCl contractions were inhibited by each androgen in a concentration-dependent manner from 1.8 to 310μM. Vascular sensitivity and efficacy were expressed as inhibitory concentration 50 (IC??) and maximal relaxation (R(max)), respectively. The coronary artery was significantly more sensitive to androgen-induced vasorelaxation than the saphenous vein or femoral artery. These vasorelaxing responses were unaffected by an antiandrogen (Flutamide) or the sulfhydryl reagent, N-ethylmaleimide, suggesting a nongenomic mechanism independent of signaling mediated by the androgen receptor or G proteins. Concentration-response curves were unchanged in endothelium-denuded preparations; thus, the endothelium appears to have no role in androgen-induced vasorelaxation. 5β-DHT was the most potent androgen in both coronary and femoral artery, but all three androgens were equipotent in the saphenous vein. It is concluded that: 1) significant regional differences exist in vasorelaxing effects of androgen metabolites in the canine vasculature; 2) structural differences in these androgens determine their vasorelaxing efficacy; and 3) regional differences in androgen-induced vasorelaxation may account for some of the conflicting findings reported on the vasorelaxing effects of the androgens.     

Acute and nongenomic effects of testosterone on isolated and perfused rat heart

Gonadal steroid hormones influence vascular tone and the development of hypertension. There are sex differences in the incidence of cardiovascular diseases, and great attention has been placed on the study of estrogen cardiovascular effects. However, there are only a few reports on the effects of testosterone on the vasculature. It is commonly accepted that the mechanism of the action of steroid hormones on target tissues is mediated through the binding of hormones to cytoplasmic or nuclear receptors. However, some studies indicate that steroid action can be extremely rapid and therefore unlikely to be through a genomic mechanism. The purpose of this study was to assess the effect of intravascularly confined testosterone on an isolated rat heart to demonstrate acute and possibly nongenomic effects of the steroid. Our results show that testosterone blocked the adenosine vasodilator effect and increased vascular resistance, even when its presence was restricted to the coronary vascular lumen. These effects were exerted rapidly and possibly through nongenomic mechanisms.     

Changes in intracellular Ca2+ by activation of P2 receptors in submucosal neurons in short-term cultures

Electrophysiological and Ca2+ microfluorimetric techniques were used to characterize the pharmacological profile of the P2 receptors expressed in submucosal neurons and the changes in intracellular Ca2+ associated with activation of these receptors. ATP caused a fast and slow membrane depolarizations during intracellular recordings. ATP induced a rapid inward current during whole-cell experiments. Receptors mediating the inward current and fast depolarization have the same pharmacological profile and these ATP responses were more sensitive to pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid than Basilen BlueE-3G, and potentiated by suramin. The slow depolarization was not blocked by these P2 receptor antagonists, pertussis toxin, or KT5720 (protein kinase A inhibitor). N-ethylmaleimide or protein kinase C inhibitors (staurosporine and calphostin) blocked this depolarization. ATP induced complex multi-phasic Ca2+ transients in most neurons, classified as fast, slow, or mixed fast/slow responses. In conclusion, the fast and slow Ca2+ responses were mediated by respective activation of P2X and P2Y receptors and were associated with fast and slow depolarizations, respectively.     

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 5 mg/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.     

Riluzole inhibits spontaneous Ca2+ signaling in neuroendocrine cells by activation of K+ channels and inhibition of Na+ channels

The neuroprotective drug riluzole has multiple effects on cellular signaling. We found that riluzole rapidly and reversibly inhibited spontaneous Ca2+ oscillations in both immortalized GnRH-secreting hypothalamic neurons (GT1 cells) and in the prolactin and growth-hormone-secreting GH3 cell line. At lower concentrations (100 nm-5 microM), riluzole reduced the amplitude and frequency of spontaneous Ca2+ oscillations, whereas at higher concentrations it abolished spontaneous Ca2+ signaling. Whole-cell current clamp recordings in GH3 cells revealed that riluzole decreased the action potential frequency, amplitude, and duration. Riluzole inhibited voltage-gated Na+ currents, increased iberiotoxin-sensitive voltage-gated K+ currents, and had no effect on voltage-gated Ca2+ currents in GH3 cells. Riluzole also inhibited voltage-gated Na+ currents and increased voltage-gated K+ channels in GT1 cells. The inhibitory effects of riluzole on Ca2+ signaling were blocked by pretreatment with iberiotoxin in GH3 cells, but only partially reduced by iberiotoxin in GT1 cells. These results indicate that riluzole inhibits Ca2+ signaling primarily by activation of K+ channels in GH3 cells, and also by inhibition of Na+ channels in GT1 cells. Riluzole's inhibition of spontaneous excitability and Ca2+ signaling may be involved in its multiple effects on cellular function in the nervous system.     

Detrimental effects of anabolic steroids on human endothelial cells

The aim of this study is to investigate the effects in vitro induced by androgenic anabolic steroids (AAS) (testosterone, nandrolone, androstenedione, norandrostenedione, and norandrostenediol) used illicitly in sport competitions, on the proliferation ability, apoptosis and the intracellular calcium concentration ([Ca2+]i) in human umbilical vein endothelial cells (HUVECs), selected as a prototype of a biological target system whose structure and function can be affected by steroids. For this purpose, we evaluated the proliferation inhibition by cytotoxic assay expressed as the concentration of drug inducing a 50% decrease in growth (IC50). The IC50 was reached for testosterone at 100 microM, androstenedione at 375 microM, nandrolone at 9 microM, norandrostenedione at 500 microM. The IC50 value for norandrostenediol was not reached until a concentration of 6000 microM. The apoptotic effect was evaluated by flow cytometry at IC50 for each drug. We observed that testosterone induced 31% of apoptotic cells, norandrostenedione 25%, androstenedione 15% and nandrolone 18%. We have analyzed the effects of these drugs on [Ca2+]i both in the immediate and long-term continuous presence of each compound. Our data show a statistically significant increase of [Ca2+]i in the acute condition and in long-term treated cultures, suggesting that androgen steroids modulate intracellular levels of calcium independent of incubation time or compound identity. As a whole, this study demonstrates that AAS might alter endothelial homeostasis, predisposing to the early endothelial cell activation that is responsible for vascular complications observed frequently in AAS users.     

Transduction of intracellular calcium signals through G protein-mediated activation of phospholipase C by recombinant sphingosine 1-phosphate receptors

Sphingosine 1-phosphate (S1P) increases intracellular Ca2+ concentration in many cell types, but the signaling mechanism remains uncertain. The recent identification of three closely related seven-transmembrane domain receptors for S1P, termed Edg1, H218, and Edg3, support the extracellular ligand role of S1P and allowed examination of Ca2+ responses mediated specifically by each receptor subtype. To substantiate each subtype in S1P-induced Ca2+ responses and to study the transductional mechanisms, we applied the aequorin luminescence method and the fura-2 fluorescence method in two transfected mammalian cell systems. We showed that H218 and Edg3 were capable of mediating S1P-induced mobilization of intracellular Ca2+ when transiently transfected in human TAg-Jurkat T cells. Ca2+ responses mediated by Edg1 in TAg-Jurkat cells required coexpression of the Gqi5 chimeric G protein that links Gi-coupled receptors to Gq. When H218 and Edg3 were stably expressed in rat HTC4 hepatoma cells, S1P induced Ca2+ responses with nanomolar EC50 values. Edg3, but not H218, elicited a sustained influx of extracellular Ca2+. The coincident formation of inositol phosphates and the complete inhibition of Ca2+ responses by the phospholipase C inhibitor U73122 indicated that H218 and Edg3 mobilized Ca2+ through activation of phospholipase C. Partial inhibition of Ca2+ responses and inositol phosphates formation by pertussis toxin implied that H218 and Edg3 transduce phospholipase C activation and Ca2+ responses only partially through Gi proteins. Although these results did not dismiss that S1P may function as an intracellular second messenger in other settings, they definitively proved that S1P can mobilize Ca2+ as an extracellular ligand for G protein-coupled receptors.     

N-methyl-d-aspartate receptor function in neuronal and synaptic development and signaling

The N-methyl-d-aspartate (NMDA) receptor, among the ionotropic glutamate receptors, are fundamental to integrating and transducing complex signaling in neurons. Glutamate activation of these receptors mediates intracellular signals essential to neuronal and synaptic formation and synaptic plasticity and also contribute to excitotoxic processes in several neurological disorders. The NMDA receptor signaling is mediated by the permeability to Ca2+ and by the large network of signaling and scaffolding proteins associated mostly with the large C-terminal domain of GluN2 subunits. Important studies showed that GluN2 C-terminal interactions differ in accordance with the GluN2 subtype, and this influences the type of signaling that NMDA receptor activity controls. Thus, it is not surprising that mutations in genes that codify for NMDA receptor subunits have been associated with severe neuronal diseases. We will review recent advances and explore outstanding problems in this active area of research.     

Coexpression of delta-opioid receptors with micro receptors in GH3 cells changes the functional response to micro agonists from inhibitory to excitatory

GH3 cells show spontaneous activity characterized by bursts of action potentials and oscillations in [Ca 2+]i. This activity is modulated by the activation of exogenously expressed opioid receptors. In GH3 cells expressing only micro receptors (GH3MOR cells), the micro receptor-specific ligand [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) inhibited spontaneous Ca 2+ signaling by the inhibition of voltage-gated Ca 2+ channels, activation of inward-rectifying K+ channels, and inhibition of adenylyl cyclase. In contrast, in cells expressing both micro and delta receptors (GH3MORDOR cells), DAMGO had an excitatory effect on Ca 2+ signaling that was mediated by phospholipase C and release of Ca 2+ from intracellular stores. The excitatory effect of DAMGO was also inhibited by pretreatment with pertussis toxin. Despite the excitatory effect on Ca 2+ signaling, DAMGO inhibited Ca 2+ channels and activated inward-rectifying K+ channels in GH3MORDOR cells, although to a lesser extent than in GH3MOR cells. Long-term treatment with the delta receptor-specific ligand [D-Pen2,D-Pen5]-enkephalin reduced the excitatory effect of DAMGO in the majority of GH3MORDOR cells and restored the inhibitory response to DAMGO in some cells. The inhibitory effect of somatostatin on Ca 2+ signaling was not different in GH3MORDOR versus GH3MOR cells. These results indicate that interaction between micro- and delta-opioid receptors causes a change in the functional response to micro ligands, possibly by the formation of a micro/delta heterodimer with distinct functional properties.     

Functional importance and caffeine sensitivity of ryanodine receptors in primary lymphocytes

Calcium signaling patterns are important for the specific regulation of activation and effector function in lymphocytes. Studies of [Ca2+]i regulation in lymphocytes, including the involvement of ryanodine receptors (RyR) and the importance of caffeine-sensitive pools, have been carried out mainly in lymphocyte cell lines and the presence and functional importance of these pools in primary lymphocytes has not been addressed. Here we show by confocal microscopy that caffeine caused a prompt but transitory increase of [Ca2+]i in primary lymphocytes, an effect that was inhibited by pre-treatment with ryanodine. Furthermore, the increase of [Ca2+]i in CD4+ and CD8+ MLR T lymphocytes stimulated by 5 microg/ml concanavalin A was significantly inhibited by pretreatment with caffeine. In functional studies, caffeine decreased cytotoxicity against myocyte target cells which is probably related to an altered calcium signaling in CD8+ MLR lymphocytes. Caffeine also terminated spontaneous Ca2+ oscillations and induced a rise in [Ca2+]i in CD4- and CD8- MLR lymphocytes probably of B cell origin. These results demonstrate that caffeine alters Ca2+ signaling in primary lymphocytes, and suggest that RyR, probably the skeletal muscle receptor (RyR-1) and brain receptor (RyR-3), are involved in mediating this effect. It is also possible that blocking of inositol-1,4,5-triphosphate (IP3) receptors is involved in the effects of caffeine on lymphocyte activation.     

Modulation of potassium current and calcium influx by somatostatin in rod bipolar cells isolated from the rabbit retina via sst2 receptors

Somatostatin (somatotropin release-inhibiting factor, SRIF) receptor subtypes are expressed by several retinal neurons, suggesting that SRIF acts at multiple levels of the retinal circuitry, although functional data on this issue are scarce. Of the SRIF receptors, the sst2A isoform is expressed by rod bipolar cells (RBCs) of the rabbit retina, and in isolated RBCs we studied the role of sst2 receptors in modulating both K+ current (IK) and the intracellular free [Ca2+] ([Ca2+]i) using both voltage-clamp and Ca2+-imaging techniques. SRIF and octreotide (a SRIF agonist that binds to sst2 receptors) inhibited that component of IK corresponding to the activation of large-conductance, Ca2+- and voltage-dependent K+ channels (IBK) and reduced the K+-induced [Ca2+]i accumulation, suggesting that SRIF effects on IBK may have been secondary to inhibition of Ca2+ channels. Octreotide effects on IBK or on [Ca2+]i accumulation were prevented by RBC treatment with L-Tyr8-Cyanamid 154806, a novel sst2 receptor antagonist, indicating that SRIF effects were mediated by sst2 receptor activation. The present data indicate that SRIF may modulate the information flow through second-order retinal neurons via an action predominantly at sst2 receptors, contribute to the proposition that SRIF be added to the growing list of retinal neuromodulators, and suggest that one of its possible roles in the retina is to regulate transmitter release from RBCs.     

Stress Impairs GABAergic Network Function in the Hippocampus by Activating Nongenomic Glucocorticoid Receptors and Affecting the Integrity of the Parvalbumin-Expressing Neuronal Network

Stress facilitates the development of psychiatric disorders in vulnerable individuals. It affects physiological functions of hippocampal excitatory neurons, but little is known about the impact of stress on the GABAergic network. Here, we studied the effects of stress and a synthetic glucocorticoid on hippocampal GABAergic neurotransmission and network function focusing on two perisomatic interneurons, the parvalbumin (PV)- and the cholecystokinin (CCK)-positive neurons. In acute hippocampal slices of rat, application of the potent glucocorticoid receptor (GR) agonist dexamethasone (DEX) caused a rapid increase in spontaneous inhibitory postsynaptic currents (sIPSCs) in CA1 pyramidal neurons. This effect was mediated by a nongenomic GR that evoked nitric oxide (NO) release from pyramidal neurons. Retrograde NO signaling caused the augmentation of GABA release from the interneurons and increased CCK release, which in turn further enhanced the activity of the PV-positive cells. Interestingly, chronic restraint stress also resulted in increased sIPSCs in CA1 pyramidal neurons that were Ca²⁺-dependent and an additional DEX application elicited no further effect. Concomitantly, chronic stress reduced the number of PV-immunoreactive cells and impaired rhythmic sIPSCs originating from the PV-positive neurons. In contrast, the CCK-positive neurons remained unaffected. We therefore propose that, in addition to the immediate effect, the sustained activation of nongenomic GRs during chronic stress injures the PV neuron network and results in an imbalance in perisomatic inhibition mediated by the PV and CCK interneurons. This stress-induced dysfunctional inhibitory network may in turn impair rhythmic oscillations and thus lead to cognitive deficits that are common in stress-related psychiatric disorders.