Repeated doses of captopril induce airway hyperresponsiveness by modulating the TRPV1 receptor in rats.

Although TRPV1 receptors play an essential role in the adverse effects on the airways following captopril treatment, there is no available evidence of their involvement in treatment regimens involving repeated doses of captopril. Comparing the difference in these two treatment regimens is essential since captopril is a continuous-use medication. Thus, this study explored the role of the transient receptor potential vanilloid 1 (TRPV1) in the effects of captopril on rat airways using two treatment regimens. Airway resistance, bronchoalveolar lavage (BAL), and histological and immunohistochemical analyses were conducted in rats administered with single or repeated doses of captopril. This study showed that the hyperresponsiveness to bradykinin and capsaicin in captopril-treated rats was acute. Treatment with the selective B2 antagonist, HOE140 reduced bradykinin hyperresponsiveness and abolished capsaicin exacerbation in single-dose captopril-treated rats. Likewise, degeneration of TRPV1-positive neurones also reduced hyperresponsiveness to bradykinin. Single-dose captopril treatment increased leukocyte infiltration in the BAL when compared with the vehicle and this increase was reduced by TRPV1-positive neurone degeneration. However, when compared with the vehicle treatment, animals treated with repeated doses of captopril showed an increase in leukocyte influx as early as 1 h after the last captopril treatment, but this effect disappeared after 24 h. Additionally, an increase in TRPV1 expression occurred only in animals who received repeated captopril doses and the degeneration of TRPV1-positive neurones attenuated TRPV1 upregulation. In conclusion, these data strongly indicate that a treatment regimen involving multiple doses of captopril not only enhances sensitisation but also upregulates TRPV1 expression. Consequently, targeting TRPV1 could serve as a promising strategy to reduce the negative impact of captopril on the airways.

Role of kinin receptors in skin pigmentation.

Previous studies have shown that all kinin system is constitutively expressed in the normal and inflamed skin, with a potential role in both physiological and pathological processes. However, the understanding regarding the involvement of the kinin system in skin pigmentation and pigmentation disorders remains incomplete. In this context, the present study was designed to determine the role of kinins in the Monobenzone (MBZ)-induced vitiligo-like model. Our findings showed that MBZ induces higher local skin depigmentation in kinin receptors knockout mice (KOB1R, KOB2R and KOB1B2R) than in wild type (WT). Remarkably, lower levels of melanin content and reduced ROS generation were detected in KOB1R and KOB2R mice treated with MBZ. In addition, both KOB1R and KOB2R show increased dermal cell infiltrate in vitiligo-like skin, when compared to WT-MBZ. Additionally, lack of B1R was associated with greater skin accumulation of IL-4, IL-6, and IL-17 by MBZ, while KOB1B2R presented lower levels of TNF and IL-1. Of note, the absence of both kinin B1 and B2 receptors demonstrates a protective effect by preventing the increase in polymorphonuclear and mononuclear cell infiltrations, as well as inflammatory cytokine levels induced by MBZ. In addition, in vitro assays confirm that B1R and B2R agonists increase intracellular melanin synthesis, while bradykinin significantly enhanced extracellular melanin levels and proliferation of B16F10 cells. Our findings highlight that the lack of kinin receptors caused more severe depigmentation in the skin, as well as genetic deletion of both B1/B2 receptors seems to be linked with changes in levels of constitutive melanin levels, suggesting the involvement of kinin system in crucial skin pigmentation pathways.

Recent progress and prospects for anti-cytokine therapy in preclinical and clinical acute lung injury.

Acute respiratory distress syndrome (ARDS) is a heterogeneous cause of respiratory failure that has a rapid onset, a high mortality rate, and for which there is no effective pharmacological treatment. Current evidence supports a critical role of excessive inflammation in ARDS, resulting in several cytokines, cytokine receptors, and proteins within their downstream signalling pathways being putative therapeutic targets. However, unsuccessful trials of anti-inflammatory drugs have thus far hindered progress in the field. In recent years, the prospects of precision medicine and therapeutic targeting of cytokines coevolving into effective treatments have gained notoriety. There is an optimistic and growing understanding of ARDS subphenotypes as well as advances in treatment strategies and clinical trial design. Furthermore, large trials of anti-cytokine drugs in patients with COVID-19 have provided an unprecedented amount of information that could pave the way for therapeutic breakthroughs. While current clinical and nonclinical ARDS research suggest relatively limited potential in monotherapy with anti-cytokine drugs, combination therapy has emerged as an appealing strategy and may provide new perspectives on finding safe and effective treatments. Accurate evaluation of these drugs, however, also relies on well-founded experimental research and the implementation of biomarker-guided stratification in future trials. In this review, we provide an overview of anti-cytokine therapy for acute lung injury and ARDS, highlighting the current preclinical and clinical evidence for targeting the main cytokines individually and the therapeutic prospects for combination therapy.

Anti-proliferative and anti-inflammatory effects of the application of baclofen cream, a GABAB receptor agonist, on skin inflammation in mice.

Previous studies have demonstrated the role of γ-aminobutyric acid type B (GABAB) receptors in skin-related conditions and pain. However, most studies have focused on the main effects of GABAB on the central nervous system. Therefore, this study has aimed to determine the potential topical anti-inflammatory and anti-proliferative effects of baclofen cream in an inflammatory skin disease model. The effects of the baclofen cream were evaluated using acute and chronic models of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mouse ears. Histological and immunohistochemical evaluations were performed using an ear oedema assay. The effect of baclofen on keratinocyte proliferation was assessed in PAM212, the murine keratinocyte cell line. The results demonstrate that a single topical application of 5% baclofen, 7.5% baclofen, and 1% dexamethasone each inhibited acute TPA-induced ear oedema (58.94 ± 6.14%, 47.73 ± 11.26%, and 87.33 ± 4.59%, respectively). These results were confirmed by histological analysis. In the chronic model, baclofen (5%) and dexamethasone (1%) each inhibited ear oedema and the maximum inhibitory effect was reached at the end of the experiment (9th day of TPA application) with a percentage inhibition of 54.60 ± 6.15% for baclofen and 71.68 ± 3.45% for dexamethasone, when compared to the vehicle. These results were confirmed by histological analysis. Baclofen and dexamethasone also reduced proliferating cell nuclear antigen expression by 62.01 ± 6.65% and 70.42 ± 6.11%, respectively. However, baclofen did not inhibit keratinocyte proliferation in PAM212 cells. In conclusion, these results demonstrate that baclofen exhibits notable topical antiproliferative and anti-inflammatory properties and could be a potential therapeutic alternative for treating inflammatory and proliferative skin diseases.

Critical Pronociceptive Role of Family 2 Voltage-Gated Calcium Channels in a Novel Mouse Model of HIV-Associated Sensory Neuropathy.

Some people living with HIV present painful sensory neuropathy (HIV-SN) that is pharmacoresistant, sex-associated, and a major source of morbidity. Since the specific mechanisms underlying HIV-SN are not well understood, the aim of our study was to characterize a novel model of painful HIV-SN by combining the HIV-1 gp120 protein and the antiretroviral stavudine (d4T) in mice and to investigate the pronociceptive role of the family 2 voltage-gated calcium channel (VGCC) α1 subunit (Cav2.X channels) in such a model. HIV-SN was induced in male and female C57BL/6 mice by administration of gp120 and/or d4T and detected by a battery of behavior tests and by immunohistochemistry. The role of Cav2.X channels was assessed by the treatment with selective blockers and agonists as well as by mRNA detection. Repeated administration with gp120 and/or d4T produced long-lasting touch-evoked painful-like behaviors (starting at 6 days, reaching a maximum on day 13, and lasting up to 28 days after treatment started), with a greater intensity in female mice treated with the combination of gp120 + d4T. Moreover, gp120 + d4T treatment reduced the intraepidermal nerve fibers and well-being of female mice, without altering other behaviors. Mechanistically, gp120 + d4T treatment induced Cav2.1, 2.2, and 2.3 transcriptional increases in the dorsal root ganglion and the Cav2.X agonist-induced nociception. Accordingly, intrathecal selective Cav2.2 blockade presented longer and better efficacy in reversing the hyperalgesia induced by gp120 + d4T treatment compared with Cav2.1 or Cav2.3, but also presented the worst safety (inducing side effects at effective doses). We conclude that the family 2 calcium channels (Cav2.X) exert a critical pronociceptive role in a novel mouse model of HIV-SN.

miRNA-203b-3p Induces Acute and Chronic Pruritus through 5-HTR2B and TRPV4.

Growing evidence indicates that transient receptor potential (TRP) channels contribute to different forms of pruritus. However, the endogenous mediators that cause itch through transient receptor potential channels signaling are poorly understood. In this study, we show that genetic deletion or pharmacological antagonism of TRPV4 attenuated itch in a mouse model of psoriasis induced by topical application of imiquimod. Human psoriatic lesions showed increased expression of several microRNAs, including the miR-203b-3p, which induced a calcium ion response in rodent dorsal root ganglion neurons and scratching behavior in mice through 5-HTR2B activation and the protein kinase C‒dependent phosphorylation of TRPV4. Computer simulation revealed that the miR-203b-3p core sequence (GUUAAGAA) that causes 5-HTR2B/TRPV4-dependent itch targets the extracellular side of 5-HTR2B by interacting with a portion of the receptor pocket consistent with its activation. Overall, we reveal the unconventional pathophysiological role of an extracellular microRNA that can behave as an itch promoter through 5-HTR2B and TRPV4.

TRPV1 Contributes to Cerebral Malaria Severity and Mortality by Regulating Brain Inflammation.

Transient receptor potential vanilloid 1 (TRPV1) is a Ca+2-permeable channel expressed on neuronal and nonneuronal cells, known as an oxidative stress sensor. It plays a protective role in bacterial infection, and recent findings indicate that this receptor modulates monocyte populations in mice with malaria; however, its role in cerebral malaria progression and outcome is unclear. By using TRPV1 wild-type (WT) and knockout (KO) mice, the importance of TRPV1 to this cerebral syndrome was investigated. Infection with Plasmodium berghei ANKA decreased TRPV1 expression in the brain. Mice lacking TRPV1 were protected against Plasmodium-induced mortality and morbidity, a response that was associated with less cerebral swelling, modulation of the brain expression of endothelial tight-junction markers (junctional adhesion molecule A and claudin-5), increased oxidative stress (via inhibition of catalase activity and increased levels of H2O2, nitrotyrosine, and carbonyl residues), and diminished production of cytokines. Plasmodium load was not significantly affected by TRPV1 ablation. Repeated subcutaneous administration of the selective TRPV1 antagonist SB366791 after malaria induction increased TRPV1 expression in the brain tissue and enhanced mouse survival. These data indicate that TRPV1 channels contribute to the development and outcome of cerebral malaria.

Temporal and Regional Expression of Glucose-Dependent Insulinotropic Peptide and Its Receptor in Spinal Cord Injured Rats.

Spinal cord injury (SCI) results in loss of movement, sensibility, and autonomic control at the level of the lesion and at lower parts of the body. Several experimental strategies have been used in attempts to increase endogenous mechanisms of neuroprotection, neuroplasticity, and repair, but with limited success. It is known that glucose-dependent insulinotropic peptide (GIP) and its receptor (GIPR) can enhance synaptic plasticity, neurogenesis, and axonal outgrowth. However, their role in the injury has never been studied. The aim of this study was to evaluate the changes in expression levels of both GIP and GIPR in acute and chronic phases of SCI in rats. Following SCI (2 to 24 h after damage), the rat spinal cord showed a lesion in which the epicenter had a cavity with hemorrhage and necrosis. Furthermore, the lesion cavity also showed ballooned cells 14 and 28 days after injury. We found that SCI induced increases in GIPR expression in areas neighboring the site of injury at 6 h and 28 days after the injury. Moreover, higher GIP expression was observed in these regions on day 28. Neuronal projections from the injury epicenter showed an increase in GIP immunoreactivity 24 h and 14 and 28 days after SCI. Interestingly, GIP was also found in progenitor cells at the spinal cord canal 24 h after injury, whereas both GIP and GIPR were present in progenitor cells at the injury epicenter 14 days after in SCI animals. These results suggest that GIP and its receptor might be implicated with neurogenesis and the repair process after SCI.

TRPV1 channel inhibition contributes to the antinociceptive effects of Croton macrostachyus extract in mice.

BACKGROUND: Previous study showed that extracts from Croton macrostachyus (Euphorbiaceae) exhibit analgesic effects in acute pain models. The present study evaluates the antinociceptive properties of the methanol/methylene chloride extract (MECM) of the stem bark of this plant using mice models of persistent inflammatory and neuropathic pain, and assesses its mechanism of action. METHODS: MECM was tested on Complete Freund adjuvant (CFA)-induced persistent thermal and mechanical pain, neuropathic pain induced by partial sciatic nerve ligation (PSNL), prostaglandin E2 (PGE2)-induced acute mechanical hyperalgesia, as well as on nociception induced by capsaicin in mice. Mechanical hyperalgesia was assessed using von Frey hair in awake mice. The mechanism of action of MECM was evaluated by using glibenclamide on PGE2-induced hyperalgesia or rimonabant on capsaicin-induced pain. RESULTS: MECM administered orally at the doses of 250 and 500 mg/kg, induced long lasting and significant antihyperalgesic effects on CFA-inflammatory and PSNL-induced neuropathic pain. MECM significantly reduced the mechanical hyperalgesia induced by PGE2 either when administered preventively or therapeutically. MECM also significantly and time dependently inhibited the capsaicin-induced nociception. These effects were not affected by glibenclamide or by rimonabant. CONCLUSIONS: The present results demonstrate that the oral administration of MECM to mice resulted in long lasting antihyperalgesic activity in inflammatory and neuropathic pain as well as in acute and persistent pain. The mechanism underlying the long lasting MECM antihyperalgesic effect is currently unknown, but might be mediated, at least partially, through the modulation of TRPV1 receptors.

Phα1ß, a peptide from the venom of the spider Phoneutria nigriventer shows antinociceptive effects after continuous infusion in a neuropathic pain model in rats.

BACKGROUND: Neuropathic pain is a severe painful pathology that is difficult to treat. One option for its management is the continuous intrathecal (i.t.) infusion of ziconotide (the Conus magnus peptide ω-conotoxin MVIIA), which, in addition to being effective, produces serious adverse effects at analgesic doses. Single i.t. administration of Phα1ß, a peptide purified from the venom of the spider Phoneutria nigriventer, has antinociceptive effects with a greater therapeutic window than ziconotide in rodents. To further evaluate its analgesic potential, we investigated the antinociceptive and toxic effects of Phα1ß after single or continuous i.t. infusion in a rat model of neuropathic pain. METHODS: Adult male Wistar rats (200-300 g) bred in-house were used. Chronic constriction injury (CCI) of the sciatic nerve was used as the neuropathic pain model. Nociception was assessed by detecting mechanical hyperalgesia, considering a significant reduction in 50% paw withdrawal threshold values after CCI compared with baseline values. First, we assessed the antinociceptive effect of a single i.t. injection of Phα1ß (10, 30, or 100 pmol/site) in a model of neuropathic pain 8 days after nerve injury. In a different experiment, we delivered Phα1ß (60 pmol/µL/h) or vehicle (phosphate-buffered saline, 1.0 µL/h) through continuous infusion using an osmotic pump by spinal catheterization for 7 days in rats submitted to nerve injury. Behavioral adverse effects were evaluated after single or continuous Phα1ß i.t. administration, and histopathological analysis of spinal cord, brainstem, and encephalon was performed after continuous Phα1ß i.t. injection. RESULTS: We observed that CCI of the sciatic nerve but not sham surgery caused intense (reduction of approximately 2.5 times in mechanical withdrawal threshold) and persistent (up to 14 days) nociception in rats. The single i.t. injection of Phα1ß (30 or 100 pmol/site) reduced neuropathic nociception from 1 to 6 hours after administration, without showing detectable side effects. Similarly, the continuous infusion of Phα1ß (60 pmol/µL/h for 7 days) was also able to reverse nerve injury-induced nociception from 1 to 7 days, but did not cause either behavioral side effects or histopathological changes in the central nervous system. CONCLUSIONS: Thus, we have shown for the first time that the continuous i.t. delivery of Phα1ß produces analgesia disconnected from toxicity in a relevant model of neuropathic pain, indicating that it is an effective and safe drug with a great potential to treat pain.

Connective tissue reaction of rats to a new zinc-oxide-eugenol endodontic sealer.

The aim of this study was to evaluate the biocompatibility in rat subcutaneous connective tissue of a new zinc oxide endodontic sealer (Endomethasone N) compared to those provided by Endofill and Sealer 26. Polyethylene tubes containing the test materials were implanted into dorsal subcutaneous connective tissue of Wistar albino rats. After 7 and 42 days, the implants with the surrounding tissue were collected, fixed, and processed for histologic evaluation. Sections were evaluated for the presence of inflammatory cells (poly or monomorfonuclear), blood vessels, necrosis area, and thickness of fibrous capsule. Comparisons between groups and time-periods were performed with Kruskal-Wallis and Mann-Whitney U non-parametric tests for 5% significance level. No differences in the biocompatibility patterns among the materials for the 2 experimental periods were observed. Independently of the sealer, the tissue behavior showed a tendency to decrease the irritation effect over time. It can be concluded that all sealers are irritant, but its toxicity decreased with time. Endomethásone N showed biocompatible characteristics comparable with those provided by Endofill and Sealer 26.

Anti-inflammatory lipoxin A4 is an endogenous allosteric enhancer of CB1 cannabinoid receptor.

Allosteric modulation of G-protein-coupled receptors represents a key goal of current pharmacology. In particular, endogenous allosteric modulators might represent important targets of interventions aimed at maximizing therapeutic efficacy and reducing side effects of drugs. Here we show that the anti-inflammatory lipid lipoxin A(4) is an endogenous allosteric enhancer of the CB(1) cannabinoid receptor. Lipoxin A(4) was detected in brain tissues, did not compete for the orthosteric binding site of the CB(1) receptor (vs. (3)H-SR141716A), and did not alter endocannabinoid metabolism (as opposed to URB597 and MAFP), but it enhanced affinity of anandamide at the CB1 receptor, thereby potentiating the effects of this endocannabinoid both in vitro and in vivo. In addition, lipoxin A(4) displayed a CB(1) receptor-dependent protective effect against ß-amyloid (1-40)-induced spatial memory impairment in mice. The discovery of lipoxins as a class of endogenous allosteric modulators of CB(1) receptors may foster the therapeutic exploitation of the endocannabinoid system, in particular for the treatment of neurodegenerative disorders.

Hippocampal PKA/CREB pathway is involved in the improvement of memory induced by spermidine in rats.

Spermidine (SPD) is an endogenous polyamine that modulates N-methyl-D-aspartate (NMDA) receptor function, and has been reported to facilitate memory formation. In the current study we determined whether or not the PKA/CREB signaling pathway is involved in SPD-induced facilitation of memory of inhibitory avoidance task in adult rats. The post-training administration of the cAMP-dependent protein kinase (PKA) inhibitor, N-[2-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide [H-89, 0.5 ρmol intrahippocampal (ih)] or the antagonist of the NMDA receptor polyamine-binding site (arcaine, 0.02 nmol ih) with SPD (0.2 nmol ih) prevented memory improvement induced by SPD. Intrahippocampal administration of SPD (0.2 nmol) facilitated PKA and cAMP response element-binding protein (CREB) phosphorylation in the hippocampus 180 min, but not 30 min, after administration, and increased translocation of the catalytic subunit of PKA into the nucleus. Arcaine (0.02 nmol) and H-89 (0.5 ρmol) prevented the stimulatory effect of SPD on PKA and CREB phosphorylation. These results suggest that memory enhancement induced by the ih administration of SPD involves the PKA/CREB pathways in rats.

Kinin B(1) and B(2) receptors contribute to orofacial heat hyperalgesia induced by infraorbital nerve constriction injury in mice and rats.

Mechanisms coupled to kinin B(1) and B(2) receptors have been implicated in sensory changes associated to various models of neuropathy. The current study aimed to investigate if kinins also participate in orofacial thermal hyperalgesia induced by constriction of the infraorbital nerve (CION), a model of trigeminal neuropathic pain which displays persistent hypersensitivity to orofacial sensory stimulation, in rats and mice. Male Swiss mice (30-35g) or Wistar rats (200-250g; n=6-10 per group in both cases) underwent CION or sham surgery and were submitted repeatedly to application of heat ( approximately 50 degrees C) to the ipsilateral or contralateral snout, delivered by a heat source placed 1cm from the vibrissal pad. Decreases in latency to display head withdrawal or vigorous snout flicking were considered indicative of heat hyperalgesia. CION caused long-lasting heat hyperalgesia which started on Day 2 after surgery in both species and lasted up to Day 17 in mice and Day 10 in rats. Administration of DALBK or HOE-140 (peptidic B(1) and B(2) receptor antagonists, respectively; each at 3nmol in 10microl) onto the exposed infraorbital nerve of mice at the moment of surgery delayed the development of the thermal hyperalgesia. Systemic treatment on Day 5 (mice) or Day 4 (rats) with Des-Arg(9), Leu(8)-Bradykinin (DALBK, B(1) receptor antagonist, 0.1-1micromol/kg, i.p.) or HOE-140 (B(2) receptor antagonist, 0.001-1micromol/kg, i.p.) transiently reduced heat hyperalgesia in both species. Due to the peptidic nature of DALBK and HOE-140, it is likely that their effects reported herein resulted from blockade of peripheral kinin receptors. Thus, mechanisms operated by kinin B(1) and B(2) receptors, contribute to orofacial heat hyperalgesia induced by CION in both mice and rats. Perhaps kinin B(1) and B(2) receptor antagonists might constitute effective preventive and curative treatments for orofacial thermal hyperalgesia induced by nerve injury.

Mechanisms involved in the antinociception caused by ethanolic extract obtained from the leaves of Melissa officinalis (lemon balm) in mice.

The present study examined the antinociceptive effect of the ethanolic extract from Melissa officinalis L. and of the rosmarinic acid in chemical behavioral models of nociception and investigates some of the mechanisms underlying this effect. The extract (3-1000 mg/kg), given orally (p.o.) 1 h prior to testing, produced dose-dependent inhibition of acetic acid-induced visceral pain, with ID50 value of 241.9 mg/kg. In the formalin test, the extract (30-1000 mg/kg, p.o.) also caused significant inhibition of both, the early (neurogenic pain) and the late (inflammatory pain), phases of formalin-induced licking. The extract (10-1000 mg/kg, p.o.) also caused significant and dose-dependent inhibition of glutamate-induced pain, with ID50 value of 198.5 mg/kg. Furthermore, the rosmarinic acid (0.3-3 mg/kg), given p.o. 1 h prior, produced dose-related inhibition of glutamate-induced pain, with ID50 value of 2.64 mg/kg. The antinociception caused by the extract (100 mg/kg, p.o.) in the glutamate test was significantly attenuated by intraperitoneal (i.p.) treatment of mice with atropine (1 mg/kg), mecamylamine (2 mg/kg) or l-arginine (40 mg/kg). In contrast, the extract (100 mg/kg, p.o.) antinociception was not affected by i.p. treatment with naloxone (1 mg/kg) or D-arginine (40 mg/kg). It was also not associated with non-specific effects, such as muscle relaxation or sedation. Collectively, the present results suggest that the extract produced dose-related antinociception in several models of chemical pain through mechanisms that involved cholinergic systems (i.e. through muscarinic and nicotinic acetylcholine receptors) and the L-arginine-nitric oxide pathway. In addition, the rosmarinic acid contained in this plant appears to contribute for the antinociceptive property of the extract. Moreover, the antinociceptive action demonstrated in the present study supports, at least partly, the ethnomedical uses of this plant.

Prostaglandin E2 modulates Na+,K+-ATPase activity in rat hippocampus: implications for neurological diseases.

Prostaglandin E(2) (PGE(2)) is quantitatively one of the major prostaglandins synthesized in mammalian brain, and there is evidence that it facilitates seizures and neuronal death. However, little is known about the molecular mechanisms involved in such excitatory effects. Na(+),K(+)-ATPase is a membrane protein which plays a key role in electrolyte homeostasis maintenance and, therefore, regulates neuronal excitability. In this study, we tested the hypothesis that PGE(2) decreases Na(+),K(+)-ATPase activity, in order to shed some light on the mechanisms underlying the excitatory action of PGE(2). Na(+),K(+)-ATPase activity was determined by assessing ouabain-sensitive ATP hydrolysis. We found that incubation of adult rat hippocampal slices with PGE(2) (0.1-10 microM) for 30 min decreased Na(+),K(+)-ATPase activity in a concentration-dependent manner. However, PGE(2) did not alter Na(+),K(+)-ATPase activity if added to hippocampal homogenates. The inhibitory effect of PGE(2) on Na(+),K(+)-ATPase activity was not related to a decrease in the total or plasma membrane immunocontent of the catalytic alpha subunit of Na(+),K(+)-ATPase. We found that the inhibitory effect of PGE(2) (1 microM) on Na(+),K(+)-ATPase activity was receptor-mediated, as incubation with selective antagonists for EP1 (SC-19220, 10 microM), EP3 (L-826266, 1 microM) or EP4 (L-161982, 1 microM) receptors prevented the PGE(2)-induced decrease of Na(+),K(+)-ATPase activity. On the other hand, incubation with the selective EP2 agonist (butaprost, 0.1-10 microM) increased enzyme activity per se in a concentration-dependent manner, but did not prevent the inhibitory effect of PGE(2). Incubation with a protein kinase A (PKA) inhibitor (H-89, 1 microM) and a protein kinase C (PKC) inhibitor (GF-109203X, 300 nM) also prevented PGE(2)-induced decrease of Na(+),K(+)-ATPase activity. Accordingly, PGE(2) increased phosphorylation of Ser943 at the alpha subunit, a critical residue for regulation of enzyme activity. Importantly, we also found that PGE(2) decreases Na(+),K(+)-ATPase activity in vivo. The results presented here imply Na(+),K(+)-ATPase as a target for PGE(2)-mediated signaling, which may underlie PGE(2)-induced increase of brain excitability.

Neuromodulatory effect of creatine on extracellular action potentials in rat hippocampus: role of NMDA receptors.

The creatine (Cr) and phosphocreatine (PCr) system is essential for the buffering and transport of high-energy phosphates. Although achievements made over the last years have highlighted the important role of creatine in several neurological diseases, the adaptive processes elicited by this guanidino compound in hippocampus are poorly understood. In the present study, we showed that creatine (0.5-25mM) gradually increases the amplitude of first population spike (PS) and elicits secondary PS in stratum radiatum of the CA1 region, in hippocampal slices. Creatine also decreased the intensity of the stimulus to induce PS, when compared with hippocampal slices perfused with artificial cerebrospinal fluid (ACSF). The competitive NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid (AP5; 100microM) attenuated creatine-induced increase of amplitude of PS and appearance of secondary PS, providing pharmacological evidence of the involvement of NMDA receptors in the electrophysiological effects of creatine. Accordingly, creatine (0.01-1mM) increased [3H]MK-801 binding to hippocampal membranes by 55%, further indicating that this compound modulates NMDA receptor function. These results implicate the NMDA receptor in amplitude and population spike increase elicited by creatine in hippocampus. Furthermore, these data suggest that this guanidino compound may also play a putative role as a neuromodulator in the brain, and that at least some of its effects may be mediated by an increase in glutamatergic function.

Molecular mechanisms of topical anti-inflammatory effects of lipoxin A(4) in endotoxin-induced uveitis.

Lipoxin A(4) (LXA(4)) is a lipid mediator that plays an important role in inflammation resolution. We assessed the anti-inflammatory effect of LXA(4) on endotoxin-induced uveitis (EIU) in rats. The inflammatory cell number and levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), prostaglandin E(2) (PGE(2)), and protein, as well as expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF), in the anterior chamber of the eye were determined 24 h after lipopolysaccharide (LPS; 200 mug/paw) intradermal injection. The immunohistochemical reactivities of nuclear factor-kappaB (NF-kappaB) and c-Jun were also examined. Topical LXA(4) (1-10 ng/eye) pretreatment decreased the number of inflammatory cells and the protein leakage into the aqueous humor (AqH). In addition, topical LXA(4) (10 ng/eye) inhibited the LPS-induced production of IL-1beta, TNF-alpha, and PGE(2), and expression of COX-2 and VEGF. A decreased activation of NF-kappaB and c-Jun was also found in LXA(4)-treated eyes. It is very interesting that an anti-inflammatory effect was achieved even when LXA(4) (10 ng/eye) was applied topically after LPS challenge, as indicated by the reduction in the cellular and protein extravasations into the AqH. Moreover, topical treatment of corticosteroid prednisolone (200 mug/eye) beginning before or after LPS injection reduced all of the molecular and biochemical alterations promoted on EIU rats in an efficacy similar to that of LXA(4). Together, the present results provide clear evidence that pharmacological activation of LXA(4) signaling pathway potently reduces the EIU in rats. Therefore, LXA(4) stable analogs could represent promising agents for the management of ocular inflammatory diseases.

Synthesis and pharmacological activity of chalcones derived from 2,4,6-trimethoxyacetophenone in RAW 264.7 cells stimulated by LPS: quantitative structure-activity relationships.

Inhibition of nitric oxide (NO) production by altering the expression of induced enzymes involved is potentially an important strategy for obtaining antiinflammatory agents. In the search for hits to obtain lead compounds for new drugs of this class, 14 synthetic chalcones derived from 2,4,6-trimethoxyacetophenone were evaluated in terms of their inhibitory action, in vitro, in relation to NO production in murine macrophages of the line RAW 264.7 induced by bacterial lipopolysaccharides (LPS). All the compounds were obtained by aldolic condensation between the acetophenone and corresponding aldehydes, under basic conditions. The mean IC(50) values, calculated through dose versus inhibitory effect curves, in four independent experiments, varied between 1.34 and 27.60microM, and were compared with the positive control, compound 1400W (IC(50)=3.78microM), a highly selective inhibitor of iNOS (induced nitric oxide synthase). Eight chalcones gave mean IC(50) values less than or equal to those obtained for 1400W, which suggests that these molecules may act as inhibitors of inflammatory process. The QSAR study reveals that electron-withdrawing groups in the B-ring seem to increase the inhibition of nitrite production, mainly when in position 2. A substitution in the ortho position of the A-ring seems to be necessary for the activity.