Proinflammatory and vasodilator effects of nociceptin/orphanin FQ in the rat mesenteric microcirculation are mediated by histamine

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). N/OFQ causes hypotension and vasodilation, and we aimed to determine the role of histamine in inflammatory microvascular responses to N/OFQ. Male Wistar rats (220-300 g, n = 72) were anesthetized with thiopental (30 mg/kg bolus, 40-90 mg x kg(-1) x h(-1) iv), and the mesentery was prepared for fluorescent intravital microscopy using fluorescein isothiocyanate-conjugated BSA (FITC-BSA, 0.25 ml/100 g iv) or 1 microm fluorescently labeled microspheres. N/OFQ (0.6-60 nmol/kg iv) caused hypotension (SAP, baseline: 154 +/- 11 mmHg, 15 nmol/kg N/OFQ: 112 +/- 10 mmHg, P = 0.009), vasodilation (venules: 23.9 +/- 1.2 microm, 26.7 +/- 1.2 microm, P = 0.006), macromolecular leak (interstitial gray level FITC-BSA: 103.7 +/- 3.4, 123.5 +/- 11.8, P = 0.009), and leukocyte adhesion (2.0 +/- 0.9, 15.2 +/- 0.9/100 microm, P = 0.036). Microsphere velocity also decreased (venules: 1,230 +/- 370 microm/s, P = 0.037), but there were no significant changes in blood flow. Flow cytometry measured a concurrent increase in neutrophil expression of cd11b with N/OFQ vs. controls (Geo mean fluorescence: 4.19 +/- 0.13 vs. 2.06 +/- 0.38, P < 0.05). The NOP antagonist [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-101; 60 and 150 nmol/kg iv), H(1) and H(2)antagonists pyrilamine (mepyramine, 1 mg/kg iv) and ranitidine (1 mg/kg iv), and mast cell stabilizer cromolyn (1 mg x kg(-1) x min(-1)) also abolished vasodilation and macromolecular leak to N/OFQ in vivo (P < 0.05), but did not affect hypotension. Isolated mesenteric arteries (approximately 200 microm, n = 25) preconstricted with U-46619 were also mounted on a pressure myograph (60 mmHg), and both intraluminally and extraluminally administered N/OFQ (10(-5) M) caused dilation, inhibited by pyrilamine in the extraluminal but not the intraluminal (control: -6.9 +/- 3.8%; N/OFQ: 32.6 +/- 8.4%; pyrilamine: 31.5 +/- 6.8%, n = 18, P < 0.05) experiments. We conclude that, in vivo, mesenteric microvascular dilation and macromolecular leak occur via N/OFQ-NOP-mediated release of histamine from mast cells. Therefore, N/OFQ-NOP has an important role in microvascular inflammation, and this may be targeted during disease, particularly as we have proven that UFP-101 is an effective antagonist of microvascular responses in vivo.     

Reduced de novo synthesis of 5-methyltetrahydrofolate and reduced taurine levels in ethanol-treated chick brains

In previous studies, exogenous ethanol (3 mmol EtOH/kg egg) caused a 1.6-fold increase in chick brain homocysteine (HoCys) levels at 11 days of development and the mixture of 3 mmol EtOH/kg egg and 34 μmol folic acid/kg egg attenuated EtOH-induced increases in chick brain HoCys levels. Because HoCys is converted to methionine utilizing the methyl donor, 5-methyltetrahydrofolate (5-methyl THF), we studied whether exogenous ethanol (3 mmol EtOH/kg egg) or the mixture of 3 mmol EtOH/kg egg and 34 μmol 5-methyl THF/kg egg inhibited chick brain 10-formyltetrahydrofolate dehydrogenase (10-FTHF DH; EC 1.5.1.6) activities and brain N⁵, N¹⁰-methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) activities at 11 days of development. Three daily dosages of 3 mmol EtOH/kg egg (E_0–2) caused approximately a 7-fold reduction in brain 10-FTHF DH activities and approximately a 1.9-fold reduction in brain MTHFR activities as compared to controls at 11 days of development (p ≤ 0.05). Because HoCys is also removed by the transsulfuration pathway, which synthesizes taurine, we studied whether exogenous ethanol (3 mmol EtOH/kg egg) or the mixture of 3 mmol EtOH/kg egg and 34 μmol 5-methyl THF/kg egg influenced chick brain taurine levels. In EtOH-treated and EtOH and 5-methyl THF-treated embryos, brain taurine levels decreased by approximately 5.5-fold and 6.2-fold as compared to controls, respectively (p ≤ 0.05). Exogenous 5-methyl THF failed to attenuate EtOH-induced decreased brain taurine levels at 11 days of development.     

Nociceptin/orphanin FQ prevents gastric damage induced by cold-restraint stress in the rat by acting in the periphery

The influence of peripheral nociceptin/orphanin FQ (N/OFQ) on cold restraint-induced gastric mucosal damage in the rat was investigated. Exposure to cold-restraint for 3 and 4h caused the formation of hemorrhagic lesions in the glandular portion of the stomach. N/OFQ dose-dependently decreased lesion formation, in the range 0.03-1 microg/kg/h i.p. Its effect was reversed by the selective NOP receptor antagonist [Nphe(1)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-101), 30 microg/kg/h ip. The selective NOP receptor agonist [(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112), 0.01-0.3 microg/kg/h i.p., similarly reduced lesion formation. Light and scanning electron microscopy confirmed the protective activity of N/OFQ. Cold-restraint stress causes a reduction in mucus content and in adhering mucus layer, partly counteracted by N/OFQ. These results suggest that N/OFQ counteracts acute stress-induced gastric mucosal damage by interacting with NOP receptor and by influencing mucous cell activity.     

In vitro and in vivo studies on UFP-112, a novel potent and long lasting agonist selective for the nociceptin/orphanin FQ receptor

[(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112) has been designed as a novel ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) by combining into the same peptide different chemical modifications reported to increase N/OFQ potency. In vitro data obtained in the electrically stimulated mouse vas deferens demonstrated that UFP-112 behaved as a high potency (pEC(50) 9.43) full agonist at the NOP receptor. UFP-112 effects were sensitive to the NOP antagonist UFP-101 but not to naloxone and no longer evident in tissues taken from NOP(-/-) mice. In vitro half life of UFP-112 in mouse plasma and brain homogenate was 2.6- and 3.5-fold higher than that of N/OFQ. In vivo, in the mouse tail withdrawal assay, UFP-112 (1-100pmol, i.c.v.) mimicked the actions of N/OFQ producing pronociceptive effects after i.c.v. administration and antinociceptive effects when given i.t.; in both cases, UFP-112 was approximately 100-fold more potent than the natural peptide and produced longer lasting effects. UFP-112 also mimicked the hyperphagic effect of N/OFQ producing a bell shaped dose response curve with the maximum reached at 10pmol. The hyperphagic effects of N/OFQ and UFP-112 were absent in NOP(-/-) mice. Equi-effective high doses of UFP-112 (0.1nmol) and N/OFQ (10nmol) were injected i.c.v. in mice and spontaneous locomotor activity recorded for 16h. N/OFQ produced a clear inhibitory effect which lasted for 60min while UFP-112 elicited longer lasting effects (>6h). In conscious rats, UFP-112 (0.1 and 10nmol/kg, i.v.) produced a marked and sustained decrease in heart rate, blood pressure, and urinary sodium excretion and a profound increase in urine flow. Collectively, these findings demonstrate that UFP-112 behaves in vitro and in vivo as a highly potent and selective ligand able to produce full and long lasting activation of NOP receptors.     

Blockade of nociceptin/orphanin FQ transmission in rat substantia nigra reverses haloperidol-induced akinesia and normalizes nigral glutamate release

We recently showed that pharmacological blockade of nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors located in the substantia nigra stimulates the nigrostriatal dopaminergic pathway and motor behavior (Marti et al. J. Neurosci. 2004, 24, 6659-6666). To investigate whether such motor-stimulating action was dependent on functional dopaminergic transmission, the selective NOP receptor peptide antagonist [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101) was microinjected into the substantia nigra reticulata of rats made cataleptic by systemic haloperidol administration. UFP-101 reduced haloperidol-induced akinesia as measured by immobility time in the bar test. UFP-101 also induced contralateral turning in cataleptic rats. To investigate the mechanisms involved in the anti-akinetic action of UFP-101, nigral glutamate release was monitored by microdialysis technique. The anti-akinetic action of UFP-101 correlated with normalization of nigral glutamate release, previously elevated by haloperidol injection. We conclude that endogenous N/OFQ in the substantia nigra sustains akinesia generated by impaired DA transmission and subthalamic nucleus overactivation. NOP receptor antagonists may be beneficial in the symptomatic therapy of parkinsonism, via normalization of subthalamonigral glutamatergic transmission.     

Substance P N-terminal fragment SP(1-7) attenuates chronic morphine tolerance and affects dynorphin B and nociceptin in rats

The N-terminal substance P fragment SP_1-7 is known to modulate hyperalgesia and opioid withdrawal in animal models. This study examined the effects of intraperitoneal (i.p.) injections of SP_1-7 on chronic morphine tolerance and on the levels of dynorphin B (DYN B) and nociceptin/orphanin FQ (N/OFQ) in various brain areas of male Sprague-Dawley rats. Morphine tolerance was induced by subcutaneous injections of the opioid (10 mg/kg) twice daily for 7 days. SP_1-7 injected i.p. (185 nmol/kg) 30 min prior to morphine reduced the development of morphine tolerance. Immunoreactive (ir) DYN B and N/OFQ peptide levels were measured in several areas of the central nervous system. Levels of ir DYN B in rats treated with SP_1-7 and morphine were decreased in the nucleus accumbens, substantia nigra and ventral tegmental area and increased in the frontal cortex. The ir N/OFQ levels were increased in the periaqueductal gray and decreased in the nucleus accumbens. Since the concentration profiles of the two peptides were altered by SP_1-7 in the areas that are implicated in the modulation of opioid tolerance and analgesia, it is suggested that DYN B and N/OFQ systems may be involved in the effects of SP_1-7 on opioid tolerance.     

Simultaneous production of citric acid and invertase by Yarrowia lipolytica SUC+ transformants

Simultaneous production of citric acid (CA) and invertase by Yarrowia lipolytica A-101-B56-5 (SUC⁺ clone) growing from sucrose, mixture of glucose and fructose, glucose or glycerol was investigated. Among the tested substrates the highest concentration of CA was reached from glycerol (57.15 g/L) with high yield (Y_CA/S = 0.6 g/g). When sucrose was used, comparable amount of CA was secreted (45 g/L) with slightly higher yield (Y_CA/S = 0.643 g/g). In all cultures amount of isocitrate (ICA) was below 2% of total citrates. Considering invertase production, the best carbon source appeared to be sucrose (72 380 U/L). The highest yield of CA and invertase biosynthesis calculated for 1 g of biomass was obtained for cells growing from glycerol (9.9 g/g and 4325 U/g, respectively). Concentrates of extra- and intracellular invertase of the highest activity were obtained from sucrose as substrate (0.5 and 1.8 × 10⁶ U/L, respectively).     

Nociceptin/orphanin FQ prevents ethanol-induced gastric lesions in the rat

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP receptor, exerts a variety of effects on the gastrointestinal tract. The present study was aimed at evaluating the possible implication of N/OFQ in the maintenance of gastric mucosal integrity. N/OFQ was given either centrally or peripherally 30 min prior to intragastric administration (i.g.) of 1 ml/rat of ethanol (either 25% or 50%, v/v), which produces macroscopically visible gastric lesions. Intracerebroventricular (i.c.v.) injection of 2 microg/rat of N/OFQ significantly reduced lesions caused by 50% ethanol, while 1 microg/rat was enough to significantly reduce lesions caused by 25% ethanol. Intracerebroventricular injection of 5 microg/rat of the selective NOP receptor antagonist, UFP-101, completely reversed the protective effect of N/OFQ, 1 or 4 microg/rat against 25% or 50% ethanol, respectively. The intraperitoneal (i.p.) injection of N/OFQ produced a significant reduction of lesions induced by 50% ethanol, the peak effect being observed at 10 microg/kg. Intraperitoneal pretreatment with UFP-101, 120 microg/kg, completely abolished the protective effect of peripherally injected N/OFQ. Therefore, N/OFQ acts both centrally and peripherally as a protective agent against ethanol-induced gastric lesions, and its effect is mediated by NOP receptors.     

UFP-101 antagonizes the spinal antinociceptive effects of nociceptin/orphanin FQ: behavioral and electrophysiological studies in mice

Nociceptin/orphanin FQ (N/OFQ) modulates various biological functions, including nociception, via selective stimulation of the N/OFQ peptide receptor (NOP). Here we used the NOP selective antagonist UFP-101 to characterize the receptor involved in the spinal antinociceptive effects of N/OFQ evaluated in the mouse tail withdrawal assay and to investigate the mechanism underlying this action by assessing excitatory postsynaptic currents (EPSC) in laminas I and II of the mouse spinal cord dorsal horn with patch-clamp techniques. Intrathecal (i.t.) injection of N/OFQ in the range of 0.1-10 nmol produced a dose dependent antinociceptive effect, which was prevented by UFP-101, but not by naloxone. In contrast the antinociceptive effect of the mu-opioid peptide receptor agonist endomorphin-1 was blocked by naloxone but not by UFP-101. Moreover, N/OFQ and endomorphin-1 induced a significant antinociceptive effect in wild type mice while in mice knockout for the NOP receptor gene only endomorphin-1 was found to be active. In mouse spinal cord slices 1 microM N/OFQ reduced EPSC to 60+/-4% of control values. This inhibitory effect was reversed in a concentration dependent manner by UFP-101 (pA2 value 6.44). The present results demonstrate that N/OFQ-induced spinal antinociception in vivo and inhibition of spinal excitatory transmission in vitro are mediated by receptors of the NOP type.     

The gastric effects of UFP-112, a new nociceptin/orphanin receptor agonist, in physiological and pathological conditions

Nociceptin/orphanin FQ (N/OFQ), the endogenous NOP receptor ligand, centrally modulates gastric motor and secretory functions and prevents ethanol-induced gastric lesions in rats. A recently synthesized N/OFQ analog, [(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112), acts as a highly potent and selective peptide agonist for NOP receptors and produces longer-lasting in vitro and in vivo effects in mice than the natural ligand N/OFQ. In this study, we evaluated the effects of centrally (intracerebroventricularly/icv) and peripherally (intraperitoneally/ip) injected UFP-112 on gastric emptying and gastric acid secretion, and on the development of gastric mucosal lesions induced by 50% ethanol in the rat. When injected icv, it dose-dependently delayed gastric emptying of a phenol red meal (by up to 70%), decreased gastric secretion in water-loaded rats after 90 pylorus ligature, and reduced ethanol-induced gastric lesions (by up to 87%). In all three assays, UFP-112 was more effective than N/OFQ. The highly selective NOP receptor antagonist, UFP-101, decreased the efficacy of UFP-112, thus confirming that central NOP receptors mediate inhibitory control on these functional and pathological conditions in rats. Ip injected N/OFQ and UFP-112 induced non-dose-related gastric hypersecretory and antiulcer effects, which UFP-101 partially abolished. Ip N/OFQ appeared equiactive but about 30-100 times less potent than ip UFP-112 in stimulating gastric acid secretion and preventing lesion formation. When ip injected, both UFP-112 and N/OFQ left gastric emptying in rats unchanged, suggesting that peripheral NOP receptors have a role in mediating gastric hypersecretory and antiulcer effects but are not involved in regulating gastric motility. In addition, the inhibitory effects induced by this novel NOP receptor agonist lasted longer than those induced by N/OFQ. In conclusion, UFP-112 is a promising new pharmacological tool for studying the functional roles of the central and peripheral N/OFQ receptor system.     

In vivo effects of N/OFQ(1–13)NH2 and its structural analogue [ORN9]N/OFQ(1–13)NH2 on carrageenan-induced inflammation: rat-paw oedema and antioxidant status

The effects of nociceptin(1–13)NH₂ (N/OFQ(1–13)NH₂) and its structural analogue [Orn⁹]N/OFQ(1–13)NH₂ on acute carrageenan (CG)-induced peripheral inflammation and paw antioxidant status were studied. CG was injected intraplantarly in the right hind paw of rats and the volume of the inflamed paw was measured each 30 min for a period of 4h. When administered simultaneously with CG, N/OFQ(1–13)NH₂ decreased the paw volume, whereas if injected 15 min before CG it had no effect. [Orn₉]N/OFQ(1–13)NH₂ produced the opposite effects at the same time-intervals of its administration. We also investigated whether these neuropeptides influence CG-induced changes in cell antioxidant system, especially at the 4th hour of CG administration. CG alone decreased the glutathione level and superoxide dismutase activity, as measured in post-nuclear homogenate of the inflamed paw. However, CG injection increased glutathione peroxidase and glucose-6-phospate dehydrogenase activities, while the activity of glutathione reductase was unchanged. The peptides themselves did not change all measured parameters. Moreover, neither N/OFQ(1–13)NH₂ nor [Orn⁹]N/OFQ(1–13)NH₂ modified CG-induced changes in the antioxidant status, regardless of the time of their injection (simultaneously or 15 min before CG). The present results suggest that N/OFQ(1–13)NH₂ and [Orn⁹]N/OFQ(1–13)NH₂ most likely affect the neuronal inflammation, rather than act as pro- or antioxidants.     

Effect of novel nociceptin/orphanin FQ-NOP receptor ligands on ethanol drinking in alcohol-preferring msP rats

Activation of the NOP receptor by the endogenous ligand nociceptin/orphanin FQ (N/OFQ) reduces alcohol consumption in genetically selected alcohol-preferring Marchigian Sardinian (msP) rats. The present study evaluated the effect of three newly synthesized peptidergic and one brain-penetrating heterocyclic NOP receptor agonists on alcohol drinking in the two bottle choice paradigm. MsP rats were intracerebroventricularly (ICV) injected with the NOP receptor agonists OS-462 (0.5 and 1.0 μg), UFP-102 (0.25 and 1.0 μg) or UFP-112 (0.01 and 0.05 μg), or with Ro 64-6198 (0.3 and 1.0 mg/kg) given intraperitoneally (i.p.) and tested for 10% alcohol consumption. Results showed decreased alcohol consumption after treatment with all three peptidergic NOP receptor agonists (OS-462, UFP-102 and UFP-112). OS-462 (at the 1.0 μg dose) and UFP-102 (at the 0.25 μg dose) induced a significant increase in food intake as well. Surprisingly, Ro 64-6198 was ineffective at the 0.3 mg/kg dose, whereas it increased ethanol and food consumption at the 1.0 mg/kg dose. Pre-treatment with the selective μ-receptor antagonist naloxone (0.5 mg/kg, i.p.) reduced these effects of 1.0 mg/kg of Ro 64-6198. These findings confirm that activation of brain NOP receptors reduces alcohol drinking in msP rats and demonstrate that OS-462, UFP-102 and UFP-112 act as potent NOP receptor agonists. On the other hand, Ro 64-6198 increased alcohol drinking, an effect probably induced by a residual agonist activity of this compound at μ-opioid receptors. Overall, the results indicate that OS-462, UFP-102 and UFP-112 may represent valuable pharmacological tools to investigate the functional role of the brain N/OFQ system.     

Nociceptin/orphanin FQ and related peptides reduce the increase in plasma corticosterone elicited in mice by an intracerebroventricular injection

Nociceptin/orphanin FQ (=N/OFQ), the endogenous ligand of ORL1 receptor (=NOP), has been reported to induce, in rodents, after intracerebroventricular (i.c.v.) administration, anti-stress and anxiolytic effects. We have observed that the handling of mice followed by an i.c.v. injection of saline, induced a marked increase in the plasma corticosterone level (+250%) measured 30 minutes later. When N/OFQ was injected intracerebroventricularly, using a 1 microg dose, the increase in plasma corticosterone was significantly lower than in saline injected mice. N/OFQ(1-13)NH(2), known as a NOP receptor agonist, at the same 1 microg dose, also induced a lesser increase in plasma corticosterone level than a saline i.c.v. injection. The pseudopeptide [Phe(1)-psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2), defined either as an agonist or an antagonist of NOP receptor, at the 0.1 microg dose, behaved in a similar manner as N/OFQ, by decreasing the plasma corticosterone level. Finally, [Nphe(1)]N/OFQ(1-13)NH(2), although presumed to be a selective NOP receptor antagonist, also decreased the corticosterone level at the 0.1 microg dose. These observations suggest the implication of N/OFQ in the regulation of response to stress, through an action on the hypothalamo-pituitary-adrenocortical axis. Moreover, they evidence a similar effect of N/OFQ and N/OFQ(1-13)NH(2), but also of two other related peptides displaying antagonist properties on NOP receptors. These data suggest that several subtypes of N/OFQ receptors could exist.     

Whole blood,flow-chamber studies in real-time indicate a biphasic role for thymosin β-4 in platelet adhesion

Background

Thymosin beta 4 (Tβ₄) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti-inflammatory agent and promotes corneal wound healing.

Methods

In the present study, we constructed a four channel cylindrical flow chambers out of polydimethylsiloxane (PDMS) on microscope coverslips. The platelet-binding proteins–fibrinogen and collagen–were immobilized onto the middle ~ 25% of the inner cylindrical surface. The flow method introduced here was employed to determine the effect of Tβ₄, on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers.

Results

The binding data from the flow chambers indicated that the both the rate constant of platelet deposition (average: 0.026 ± 0.0015 s^− 1, corresponding to a half-life of 26.7 s) and the total number of deposited platelets were independent of the platelet binding protein and the activating agent. Our results show that low concentrations of Tβ₄ (0.2 μM to 0.5 μM) increased both the rate constant of platelet deposition by ~ 1.5-fold (i.e. half-life decreased from 26.7 s to 17.6 s) and the total number of deposited platelets by ~ 3-fold. However at higher concentrations (> 1 μM) the Tβ₄-potentiating effect was diminished to near control levels. Tβ₄ did interact with fibrinogen with an estimated K_D of ~ 126 ± 18 nM or 66 ± 20 nM under equilibrium or flow, respectively.

Conclusion

These results suggest that Tβ₄ could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tβ₄ could also bind to fibrinogen and as its concentration increased would prevent platelet–fibrinogen interactions resulting in the attenuation of platelet deposition.

General significance

This work suggests that Tβ₄ might have a dual role in platelet function.     

Blockade of the nociceptin/orphanin FQ/NOP receptor system in the rat ventrolateral periaqueductal gray potentiates DAMGO analgesia

Nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are involved in various biological functions including pain. High density of NOP receptor has been found in the ventrolateral periaqueductal gray (vlPAG), the main output pathway involved in descending pain-control system. The aim of our work was to evaluate the involvement of the N/OFQ/NOP system in the modulation of MOP analgesia in the rat vlPAG using UFP-101, a selective NOP antagonist. N/OFQ significantly blocked DAMGO (a selective MOP agonist) analgesia, while UFP-101 enhanced the effect of the opioid given at a subanalgesic dose. These results confirm our hypothesis of an antiopioid role for N/OFQ in the vlPAG.     

Effect of novel NOP receptor ligands on food intake in rats

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand for the NOP opioid receptor, stimulates feeding in rats. The present study evaluated the effect of three newly synthesized NOP receptor agonists and two NOP receptor antagonist on food intake. Freely feeding rats were tested with intracerebroventricular (ICV) injections of the NOP receptor agonists OS-500, OS-462 and OS-461. OS-500 and OS-462 evoked a hyperphagic effect more potent and far more pronounced than that of N/OFQ, while OS-461 was ineffective. OS-500 and OS-462 were also tested by intraperitoneal injection, but were unable to evoke hyperphagia following this route of administration. The NOP receptor antagonist NC-797 and UFP-101 did not modify feeding in freely feeding rats while fully antagonized the hyperphagic effect of N/OFQ. Pre-treatment with UFP-101 but not with NC-797 antagonized the hyperphagic effect of OS-462 and OS-500. The present findings indicate that OS-500, OS-462 may act as potent and long-lasting NOP receptor agonists, whereas UFP-101 and NC-797 show antagonistic properties. The higher efficacy of UFP-101 in blocking the hyperphagic effect of OS-462 and OS-500 may be linked to the better pharmacokinetic profile of this antagonist compared to NC-797. Overall, the results indicate that these compounds may represent valuable pharmacological tools to investigate the role of the brain N/OFQ system.     

Amelioration of rotenone-induced dopaminergic cell death in the striatum by oxytocin treatment

Oxytocin (OT) is essentially associated with uterine contraction during parturition and milk ejection reflex. Although several studies implicate the role of OT in anti-inflammatory, anti-oxidative and anti-apoptotic pathways, there is a lack of data with regard to the protective effects of oxytocin in neurodegenerative models such as Parkinson's disease (PD). The present study was undertaken to investigate the neuroprotective effects of oxytocin (OT) on rotenone-induced PD in rats. Twenty adult Sprague-Dawley rats were injected with rotenone (3 μg/μl in DMSO) or vehicle (1 μl DMSO) into the left substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) under stereotaxic surgery, and PD model was assessed by rotational test ten days after drug infusion. The valid PD rats were randomly divided into two groups; Group 1 (n = 7) and Group 2 (n = 7) were administered saline (1 ml/kg/day, i.p.) and oxytocin (160 μg/kg/day, i.p.) through 20 days, respectively. The effects of OT treatment were evaluated by behavioral, histological and immunohistochemical parameters. Apomorphine-induced stereotypic rotations in PD rats were significantly inhibited by OT treatment (p < 0.05). In addition, immunohistochemical studies clearly demonstrated the suppression of Bax, caspase-3, caspase-8 and elevation of Bcl-2 and tyrosine hydroxylase immunoexpression in OT-treated rats compared to saline group. Our findings suggest that oxytocin may have cytoprotective and restorative effects on dopaminergic neurons against rotenone-induced injury. The underlying mechanism may be associated with the inhibition of apoptotic pathways.     

Exploring LPS-induced sepsis in rats and mice as a model to study potential protective effects of the nociceptin/orphanin FQ system

The nociceptin receptor (NOP) and its ligand nociceptin/orphanin FQ (N/OFQ) have been shown to exert a modulatory effect on immune cells during sepsis. We evaluated the suitability of an experimental lipopolysaccharide (LPS)-induced sepsis model for studying changes in the nociceptin system. C57BL/6 mice BALB/c mice and Wistar rats were inoculated with different doses of LPS with or without a nociceptin receptor antagonist (UFP-101 or SB-612111). In C57BL/6 mice LPS 0.85 mg/kg injection produced no septic response, whereas 1.2 mg/kg produced a profound response within 5 h. In BALB/c mice, LPS 4 mg/kg produced no response, whereas 7 mg/kg resulted in a profound response within 24 h. In Wistar rats LPS 15 mg/kg caused no septic response in 6/10 animals, whereas 25 mg/kg resulted in marked lethargy before 24 h. Splenic interleukin-1β mRNA in BALB/c mice, and serum TNF-α concentrations in Wistar rats increased after LPS injection in a dose-dependent manner, but were undetectable in control animals, indicating that LPS had stimulated an inflammatory reaction. IL-1β and TNF-α concentrations in LPS-treated animals were unaffected by administration of a NOP antagonist. Similarly NOP antagonists had no effect on survival or expression of mRNA for NOP or ppN/OFQ (the N/OFQ precursor) in a variety of tissues. In these animal models, the dose–response curve for LPS was too steep to allow use in survival studies and no changes in the N/OFQ system occurred within 24 h. We conclude that LPS-inoculation in rodents is an unsuitable model for studying possible changes in the NOP-N/OFQ system in sepsis.     

Horizontal or vertical photobioreactors? How to improve microalgae photosynthetic efficiency

The productivity of a vertical outdoor photobioreactor was quantitatively assessed and compared to a horizontal reactor. Daily light cycles in southern Spain were simulated and applied to grow the microalgae Chlorella sorokiniana in a flat panel photobioreactor.The maximal irradiance around noon differs from 400 μmol photons m⁻² s⁻¹ in the vertical position to 1800 μmol photons m⁻² s⁻¹ in the horizontal position. The highest volumetric productivity was achieved in the simulated horizontal position, 4 g kg culture⁻¹ d⁻¹. The highest photosynthetic efficiency was found for the vertical simulation, 1.3 g of biomass produced per mol of PAR photons supplied, which compares favorably to the horizontal position (0.85 g mol⁻¹) and to the theoretical maximal yield (1.8 g mol⁻¹). These results prove that productivity per unit of ground area could be greatly enhanced by placing the photobioreactors vertically.     

The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation

Background

Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.

Methods

Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.

Results

More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe²⁺ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s_20,w = 17.0 S and D_20,w = 3.21 × 10⁻⁷ cm²/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s_20,w = 18.3 S and D_20,w = 3.18 × 10⁻⁷ cm²/s.

Significance

The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals.