Purine nucleotides reduce superoxide production by nitric oxide synthase in a murine sepsis model

Sepsis involves a systemic inflammatory response of multiple endogenous mediators, resulting in many of the injurious and sometimes fatal physiological symptoms of the disease. This systemic activation leads to a compromised vascular response and endothelial dysfunction. Purine nucleotides interact with purinoceptors and initiate a variety of physiological processes that play an important role in maintaining cardiovascular function. The purpose of the present study was to investigate the effects of ATP on vascular function in a lipopolysaccharide (LPS) model of sepsis. LPS induced a significant increase in aortic superoxide production 16 h after injection. Addition of ATP to the organ bath incubation solution reduced superoxide production by the aortas of endotoxemic animals. Reactive Blue, an antagonist of the P2Y receptor, blocked the effect of ATP on superoxide production, and the nonselective P2Y agonist MeSATP inhibited superoxide production. Nitric oxide synthase (NOS) inhibition by L-NAME blocked vascular relaxation and reduced superoxide production in LPS-treated animals. In the presence of L-NAME there was no ATP effect on superoxide production. A vascular reactivity study showed that ATP increased maximal relaxation in LPS-treated animals compared to controls. The presence of ATP induced increases in Akt and endothelial NOS phosphorylated proteins in the aorta of septic animals. ATP reduces superoxide release resulting in an improved vasorelaxant response. Sepsis may uncouple NOS to produce superoxide. We showed that ATP through Akt pathway phosphorylated endothelial NOS and “re-couples” NOS function.

Protective mechanism of agmatine pretreatment on RGC-5 cells injured by oxidative stress

Agmatine has neuroprotective effects on retinal ganglion cells (RGCs) as well as cortical and spinal neurons. It protects RGCs from oxidative stress even when it is not present at the time of injury. As agmatine has high affinity for various cellular receptors, we assessed protective mechanisms of agmatine using transformed RGCs (RGC-5 cell line). Differentiated RGC-5 cells were pretreated with 100 μM agmatine and consecutively exposed to 1.0 mM hydrogen peroxide (H2O2). Cell viability was determined by measuring lactate dehydrogenase (LDH), and the effects of selective alpha 2-adrenergic receptor antagonist yohimbine (0-500 nM) and N-methyl-D-aspartic acid (NMDA) receptor agonist NMDA (0-100 µM) were evaluated. Agmatine’s protective effect was compared to a selective NMDA receptor antagonist MK-801. After a 16-h exposure to H2O2, the LDH assay showed cell loss greater than 50%, which was reduced to about 30% when agmatine was pretreated before injury. Yohimbine almost completely inhibited agmatine’s protective effect, but NMDA did not. In addition, MK-801 (0-100 µM) did not significantly attenuate the H2O2-induced cytotoxicity. Our results suggest that neuroprotective effects of agmatine on RGCs under oxidative stress may be mainly attributed to the alpha 2-adrenergic receptor signaling pathway.

Human monocytes tolerant to LPS retain the ability to phagocytose bacteria and generate reactive oxygen species

Tolerance to lipopolysaccharide (LPS) occurs when animals or cells exposed to LPS become hyporesponsive to a subsequent challenge with LPS. This mechanism is believed to be involved in the down-regulation of cellular responses observed in septic patients. The aim of this investigation was to evaluate LPS-induced monocyte tolerance of healthy volunteers using whole blood. The detection of intracellular IL-6, bacterial phagocytosis and reactive oxygen species (ROS) was determined by flow cytometry, using anti-IL-6-PE, heat-killed Staphylococcus aureus stained with propidium iodide and 2',7'-dichlorofluorescein diacetate, respectively. Monocytes were gated in whole blood by combining FSC and SSC parameters and CD14-positive staining. The exposure to increasing LPS concentrations resulted in lower intracellular concentration of IL-6 in monocytes after challenge. A similar effect was observed with challenge with MALP-2 (a Toll-like receptor (TLR)2/6 agonist) and killed Pseudomonas aeruginosa and S. aureus, but not with flagellin (a TLR5 agonist). LPS conditioning with 15 ng/mL resulted in a 40% reduction of IL-6 in monocytes. In contrast, phagocytosis of P. aeruginosa and S. aureus and induced ROS generation were preserved or increased in tolerant cells. The phenomenon of tolerance involves a complex regulation in which the production of IL-6 was diminished, whereas the bacterial phagocytosis and production of ROS was preserved. Decreased production of proinflammatory cytokines and preserved or increased production of ROS may be an adaptation to control the deleterious effects of inflammation while preserving antimicrobial activity.

Effects of reversible inactivation of the dorsomedial hypothalamus on panic- and anxiety-related responses in rats

The medial hypothalamus is part of a neurobiological substrate controlling defensive behavior. It has been shown that a hypothalamic nucleus, the dorsomedial hypothalamus (DMH), is involved in the regulation of escape, a defensive behavior related to panic attacks. The role played by the DMH in the organization of conditioned fear responses, however, is less clear. In the present study, we investigated the effects of reversible inactivation of the DMH with the GABA A agonist muscimol on inhibitory avoidance acquisition and escape expression by male Wistar rats (approximately 280 g in weight) tested in the elevated T-maze (ETM). In the ETM, inhibitory avoidance, a conditioned defensive response, has been associated with generalized anxiety disorder. Results showed that intra-DMH administration of the GABA A receptor agonist muscimol inhibited escape performance, suggesting an antipanic-like effect (P < 0.05), without changing inhibitory avoidance acquisition. Although a higher dose of muscimol (1.0 nmol/0.2 µL; N = 7) also altered locomotor activity in an open field when compared to control animals (0.2 µL saline; N = 13) (P < 0.05), the lower dose (0.5 nmol/0.2 µL; N = 12) of muscimol did not cause any motor impairment. These data corroborate previous evidence suggesting that the DMH is specifically involved in the modulation of escape. Dysfunction of this regulatory mechanism may be relevant in the genesis/maintenance of panic disorder.

The role of the SLAM-SAP signaling pathway in the modulation of CD4+ T cell responses

The signaling lymphocytic activation molecule (SLAM), present on the surface of hematopoietic cells, can regulate some events of the immune responses. This modulatory action is associated with the capacity of SLAM to interact with an intracytoplasmic adapter, such as SLAM-associated protein (SAP). SLAM is constitutively expressed in most of these cells, is rapidly induced after antigenic or inflammatory stimuli, and participates in the immunological synapse. Defects in the function of the SLAM-SAP pathway contribute to immunological abnormalities, resulting in autoimmune diseases, tumors of the lymphoid tissues and inadequate responses to infectious agents. Initially, the role of SLAM was investigated using an anti-SLAM monoclonal antibody (α-SLAM mAb) identified as an agonist of the SLAM-SAP pathway, which could induce the production of interferon-γ and could redirect the immune response to a T helper 1 (Th1) cell profile. However, in this review we postulate that the SLAM-SAP pathway primarily induces a Th2 response and secondarily suppresses the Th1 response.

5-HT1A autoreceptor modulation of locomotor activity induced by nitric oxide in the rat dorsal raphe nucleus

The dorsal raphe nucleus (DRN) is the origin of ascending serotonergic projections and is considered to be an important component of the brain circuit that mediates anxiety- and depression-related behaviors. A large fraction of DRN serotonin-positive neurons contain nitric oxide (NO). Disruption of NO-mediated neurotransmission in the DRN by NO synthase inhibitors produces anxiolytic- and antidepressant-like effects in rats and also induces nonspecific interference with locomotor activity. We investigated the involvement of the 5-HT1A autoreceptor in the locomotor effects induced by NO in the DRN of male Wistar rats (280-310 g, N = 9-10 per group). The NO donor 3-morpholinosylnomine hydrochloride (SIN-1, 150, and 300 nmol) and the NO scavenger S-3-carboxy-4-hydroxyphenylglycine (carboxy-PTIO, 0.1-3.0 nmol) were injected into the DRN of rats immediately before they were exposed to the open field for 10 min. To evaluate the involvement of the 5-HT1A receptor and the N-methyl-D-aspartate (NMDA) glutamate receptor in the locomotor effects of NO, animals were pretreated with the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 8 nmol), the 5-HT1A receptor antagonist N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-2-pyridinyl-cyclohexanecarboxamide maleate (WAY-100635, 0.37 nmol), and the NMDA receptor antagonist DL-2-amino-7-phosphonoheptanoic acid (AP7, 1 nmol), followed by microinjection of SIN-1 into the DRN. SIN-1 increased the distance traveled (mean ± SEM) in the open-field test (4431 ± 306.1 cm; F7,63 = 2.44, P = 0.028) and this effect was blocked by previous 8-OH-DPAT (2885 ± 490.4 cm) or AP7 (3335 ± 283.5 cm) administration (P < 0.05, Duncan test). These results indicate that 5-HT1A receptor activation and/or facilitation of glutamate neurotransmission can modulate the locomotor effects induced by NO in the DRN.

Involvement of β3-adrenergic receptors in the control of food intake in rats

This study examined the food intake changes evoked by intracerebroventricular (icv) injection of a selective agonist (BRL37344, 2 and 20 nmol) or antagonist (SR59230A, 10 and 50 nmol) of β3-adrenergic receptors in 24-h fasted rats (adult male Wistar rats, 200-350 g, N = 6/treatment). The animals were also pretreated with saline icv (SAL) or SR59230A (50 nmol) followed by BRL37344 (20 nmol) or SAL in order to determine the selectivity of the effects evoked by BRL37344 on food intake or the selectivity of the effects evoked by SR59230A on risk assessment (RA) behavior. The highest dose of BRL37344 (N = 7) decreased food intake 1 h after the treatment (6.4 ± 0.5 g in SAL-treated vs 4.2 ± 0.8 g in drug-treated rats). While both doses of SR59230A failed to affect food intake (5.1 ± 1.1 g for 10 nmol and 6.0 ± 1.8 g for 50 nmol), this treatment reduced the RA frequency (number/30 min) (4 ± 2 for SAL-treated vs 1 ± 1 for 10 nmol and 0.5 ± 1 for 50 nmol SR59230A-treated rats), an ethological parameter related to anxiety. While pretreatment with SR59230A (7.0 ± 0.5 g) abolished the hypophagia induced by BRL37344 (3.6 ± 0.9 g), BRL37344 suppressed the reduction in RA frequency caused by SR59230A. These results show that the hypophagia caused by BRL37344 is selectively mediated by β3-adrenergic receptors within the central nervous system. Moreover, they suggest the involvement of these receptors in the control of anxiety.

Participation of the GABAergic system in the anesthetic effect of Lippia alba (Mill.) N.E. Brown essential oil

The objective of this study was to identify the possible involvement of the GABAergic system in the anesthetic effect of Lippia alba essential oil (EO). We propose a new animal model using silver catfish (Rhamdia quelen) exposed to an anesthetic bath to study the mechanism of action of EO. To observe the induction and potentiation of the anesthetic effect of EO, juvenile silver catfish (9.30 ± 1.85 g; 10.15 ± 0.95 cm; N = 6) were exposed to various concentrations of L. alba EO in the presence or absence of diazepam [an agonist of high-affinity binding sites for benzodiazepinic (BDZ) sites coupled to the GABA A receptor complex]. In another experiment, fish (N = 6) were initially anesthetized with the EO and then transferred to an anesthetic-free aquarium containing flumazenil (a selective antagonist of binding sites for BDZ coupled to the GABA A receptor complex) or water to assess recovery time from the anesthesia. In this case, flumazenil was used to observe the involvement of the GABA-BDZ receptor in the EO mechanism of action. The results showed that diazepam potentiates the anesthetic effect of EO at all concentrations tested. Fish exposed to diazepam and EO showed faster recovery from anesthesia when flumazenil was added to the recovery bath (12.0 ± 0.3 and 7.2 ± 0.7, respectively) than those exposed to water (9.2 ± 0.2 and 3.5 ± 0.3, respectively). In conclusion, the results demonstrated the involvement of the GABAergic system in the anesthetic effect of L. alba EO on silver catfish.

Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of kappa group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female reproduction

Acute exposure to lead increases myocardial contractility independent of hypertension development

We studied the effects of the acute administration of small doses of lead over time on hemodynamic parameters in anesthetized rats to determine if myocardial contractility changes are dependent or not on the development of hypertension. Male Wistar rats received 320 µg/kg lead acetateiv once, and their hemodynamic parameters were measured for 2 h. Cardiac contractility was evaluated in vitro using left ventricular papillary muscles as were Na+,K+-ATPase and myosin Ca2+-ATPase activities. Lead increased left- (control: 112 ± 3.7 vs lead: 129 ± 3.2 mmHg) and right-ventricular systolic pressures (control: 28 ± 1.2vs lead: 34 ± 1.2 mmHg) significantly without modifying heart rate. Papillary muscles were exposed to 8 µM lead acetate and evaluated 60 min later. Isometric contractions increased (control: 0.546 ± 0.07 vs lead: 0.608 ± 0.06 g/mg) and time to peak tension decreased (control: 268 ± 13vs lead: 227 ± 5.58 ms), but relaxation time was unchanged. Post-pause potentiation was similar between groups (n = 6 per group), suggesting no change in sarcoplasmic reticulum activity, evaluated indirectly by this protocol. After 1-h exposure to lead acetate, the papillary muscles became hyperactive in response to a β-adrenergic agonist (10 µM isoproterenol). In addition, post-rest contractions decreased, suggesting a reduction in sarcolemmal calcium influx. The heart samples treated with 8 µM lead acetate presented increased Na+,K+-ATPase (approximately 140%, P < 0.05 for control vs lead) and myosin ATPase (approximately 30%, P < 0.05 for control vs lead) activity. Our results indicated that acute exposure to low lead concentrations produces direct positive inotropic and lusitropic effects on myocardial contractility and increases the right and left ventricular systolic pressure, thus potentially contributing to the early development of hypertension.

Myosin light chain kinase is necessary for post-shock mesenteric lymph drainage enhancement of vascular reactivity and calcium sensitivity in hemorrhagic-shocked rats

Vascular hyporeactivity is an important factor in irreversible shock, and post-shock mesenteric lymph (PSML) blockade improves vascular reactivity after hemorrhagic shock. This study explored the possible involvement of myosin light chain kinase (MLCK) in PSML-mediated vascular hyporeactivity and calcium desensitization. Rats were divided into sham (n=12), shock (n=18), and shock+drainage (n=18) groups. A hemorrhagic shock model (40±2 mmHg, 3 h) was established in the shock and shock+drainage groups. PSML drainage was performed from 1 to 3 h from start of hypotension in shock+drainage rats. Levels of phospho-MLCK (p-MLCK) were determined in superior mesenteric artery (SMA) tissue, and the vascular reactivity to norepinephrine (NE) and sensitivity to Ca2+ were observed in SMA rings in an isolated organ perfusion system. p-MLCK was significantly decreased in the shock group compared with the sham group, but increased in the shock+drainage group compared with the shock group. Substance P (1 nM), an agonist of MLCK, significantly elevated the decreased contractile response of SMA rings to both NE and Ca2+ at various concentrations. Maximum contractility (Emax) in the shock group increased with NE (from 0.179±0.038 to 0.440±0.177 g/mg, P<0.05) and Ca2+ (from 0.515±0.043 to 0.646±0.096 g/mg, P<0.05). ML-7 (0.1 nM), an inhibitor of MLCK, reduced the increased vascular response to NE and Ca2+ at various concentrations in the shock+drainage group (from 0.744±0.187 to 0.570±0.143 g/mg in Emax for NE and from 0.729±0.037 to 0.645±0.056 g/mg in Emax for Ca2+, P<0.05). We conclude that MLCK is an important contributor to PSML drainage, enhancing vascular reactivity and calcium sensitivity in rats with hemorrhagic shock.

Role of α2-adrenoceptors in the lateral parabrachial nucleus in the control of body fluid homeostasis

Central α2-adrenoceptors and the pontine lateral parabrachial nucleus (LPBN) are involved in the control of sodium and water intake. Bilateral injections of moxonidine (α2-adrenergic/imidazoline receptor agonist) or noradrenaline into the LPBN strongly increases 0.3 M NaCl intake induced by a combined treatment of furosemide plus captopril. Injection of moxonidine into the LPBN also increases hypertonic NaCl and water intake and reduces oxytocin secretion, urinary sodium, and water excreted by cell-dehydrated rats, causing a positive sodium and water balance, which suggests that moxonidine injected into the LPBN deactivates mechanisms that restrain body fluid volume expansion. Pretreatment with specific α2-adrenoceptor antagonists injected into the LPBN abolishes the behavioral and renal effects of moxonidine or noradrenaline injected into the same area, suggesting that these effects depend on activation of LPBN α2-adrenoceptors. In fluid-depleted rats, the palatability of sodium is reduced by ingestion of hypertonic NaCl, limiting intake. However, in rats treated with moxonidine injected into the LPBN, the NaCl palatability remains high, even after ingestion of significant amounts of 0.3 M NaCl. The changes in behavioral and renal responses produced by activation of α2-adrenoceptors in the LPBN are probably a consequence of reduction of oxytocin secretion and blockade of inhibitory signals that affect sodium palatability. In this review, a model is proposed to show how activation of α2-adrenoceptors in the LPBN may affect palatability and, consequently, ingestion of sodium as well as renal sodium excretion.

Ropinirole alters gene expression profiles in SH-SY5Y cells: a whole genome microarray study

Ropinirole (ROP) is a dopamine agonist that has been used as therapy for Parkinson's disease. In the present study, we aimed to detect whether gene expression was modulated by ROP in SH-SY5Y cells. SH-SY5Y cell lines were treated with 10 µM ROP for 2 h, after which total RNA was extracted for whole genome analysis. Gene expression profiling revealed that 113 genes were differentially expressed after ROP treatment compared with control cells. Further pathway analysis revealed modulation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway, with prominent upregulation of PIK3C2B. Moreover, batches of regulated genes, including PIK3C2B, were found to be located on chromosome 1. These findings were validated by quantitative RT-PCR and Western blot analysis. Our study, therefore, revealed that ROP altered gene expression in SH-SY5Y cells, and future investigation of PIK3C2B and other loci on chromosome 1 may provide long-term implications for identifying novel target genes of Parkinson's disease.

Montelukast reduces seizures in pentylenetetrazol-kindled mice

Cysteinyl leukotrienes (CysLTs) have been implicated in seizures and kindling; however, the effect of CysLT receptor antagonists on seizure frequency in kindled animals and changes in CysLT receptor expression after pentylenetetrazol (PTZ)-induced kindling have not been investigated. In this study, we evaluated whether the CysLT1 inverse agonist montelukast, and a classical anticonvulsant, phenobarbital, were able to reduce seizures in PTZ-kindled mice and alter CysLT receptor expression. Montelukast (10 mg/kg, sc) and phenobarbital (20 mg/kg, sc) increased the latency to generalized seizures in kindled mice. Montelukast increased CysLT1immunoreactivity only in non-kindled, PTZ-challenged mice. Interestingly, PTZ challenge decreased CysLT2 immunoreactivity only in kindled mice. CysLT1 antagonists appear to emerge as a promising adjunctive treatment for refractory seizures. Nevertheless, additional studies are necessary to evaluate the clinical implications of this research.

Random-start controlled ovarian stimulation for emergency fertility preservation in a patient with myelodysplastic syndrome: a case report

This study reports a case of a gonadotropin-releasing hormone agonist trigger in a young female with myelodysplastic syndrome (MDS) who underwent fertility preservation using random-start controlled ovarian stimulation. This method involves the stimulation of the ovary regardless of a patient's menstrual-cycle phase. A review of the related literature is also provided. A 17-year-old patient was diagnosed with MDS and required initiation of peripheral blood stem cell transplantation within a maximum of 3 weeks and was in the luteal phase of the menstrual cycle when the possibility of attempting preservation of fertility was presented to her. She opted for a random-start controlled ovarian stimulation with gonadotropins. With successful hemorrhagic prophylaxis, 17 oocytes were retrieved including 10 mature and 7 immature oocytes. Of the immature oocytes, 3 were successfully matured in vitro and a vitrification protocol was used to freeze the 13 mature oocytes.