Different mechanisms are involved in the respiratory depression and analgesia induced by neurotensin in rats

The tridecapeptide neurotensin (NT), injected intracerebroventricularly in rats, induced a naloxone-insensitive and dose-dependent analgesia (tail-flick test), the ED50 being 3.22 nmol/animal. Higher doses induced a dose-dependent respiratory depression, mostly accounted for by a reduction of frequency; the ED50 for this action was 144.6 nmol/animal. In contrast to analgesia, the respiratory depression was antagonized by naloxone, suggesting the possibility that NT and opioid systems might interact at the level of respiration-related nuclei.     

Antinociceptive effects induced by desipramine and fluoxetine are dissociated from their antidepressant or anxiolytic action in mice

This study aimed to evaluate the relationship between some aspects of experimental depression, anxiety and the antinociceptive effects of fluoxetine and desipramine in mice. Acute administration of fluoxetine and desipramine (5, 10 and 20 mg/kg, i.p.) showed significant antinociceptive effects in the hot-plate test and against the early and late phase of the mouse formalin test, dissociated from its antidepressant and anxiolytic effects as measured in the forced swimming and in the plus-maze tests, respectively. Neither fluoxetine nor desipramine, at the doses tested, produced significant effects on locomotor activity. Furthermore, both compounds were ineffective in the tail-flick phasic model of nociception. In conclusion, the results suggest that without the distinction of serotonergic and noradrenergic contributions, the acute antinociceptive effects of fluoxetine and desipramine in mice are independent of their sedative, antidepressant and anti-anxiety properties.     

ERK and p38 MAP kinase are involved in downregulation of cell surface TNF receptor 1 induced by acetoxycycloheximide

Tumor necrosis factor (TNF)-alpha activates the nuclear factor kappaB (NF-kappaB) signaling pathway. The protein synthesis inhibitor cycloheximide (CHX) and its structural derivative acetoxycycloheximide (Ac-CHX) have been recently shown to block the TNF-alpha-induced activation of NF-kappaB via ectodomain shedding of TNF receptor 1 (TNF-R1) in human lung carcinoma A549 cells. In this study, we show that ERK and p38 MAP kinase are involved in the downregulation of cell surface TNF-R1 upon exposure to Ac-CHX and the subsequent inhibition of TNF-alpha-induced NF-kappaB activation. Ac-CHX was capable of promoting the sustained activation of ERK, JNK, and p38 MAP kinase. Treatment with the MEK inhibitor U0126 and the p38 MAP kinase inhibitor SB203580, but not the JNK inhibitor SP600125, reversed the diminished expression of cell surface TNF-R1 as well as the blockade of TNF-alpha-induced IkappaBalpha degradation in Ac-CHX-treated cells. Our results indicate that Ac-CHX triggers the downregulation of cell surface TNF-R1 via the activation of ERK and p38 MAP kinase, thereby preventing activation of the NF-kappaB signaling pathway by TNF-alpha.     

Unusual profile of leukocyte recruitment in mice induced by a skin secretion of the tree frog Phyllomedusa hypochondrialis

Phyllomedusa hypochondrialis skin secretion can cause both systemic and local effects. In this study, we describe the pattern of local acute inflammatory response after P. hypochondrialis skin secretion injection. The inflammatory reaction in the mice footpad was analysed, including the leukocyte recruitment into local tissue from the peripheral blood, in a mouse model of tissue injury. We also investigated the release of the cytokines IL-1, IL-6 and TNF-α, chemokines KC and MCP-1 and the eicosanoids LTB 4 and PGE₂ in mice. The present findings support the ability of P. hypochondrialis skin secretion to induce local inflammation. In addition, these skin secretion components play a role in the initial rolling and slowing of recruited leukocytes and the transition from rolling to adhesion. Levels of the proinflammatory cytokine IL-6, chemokines KC and MCP-1 as well as the eicosanoid PGE₂ were significantly increased after injection of a skin secretion of P. hypochondrialis (0.6 μg/30 μl intraplantar), whereas no changes in other parameters were observed. Finally, the mechanisms involved in the local inflammatory process induced by P. hypochondrialis skin secretion is one of the questions of relevance related to the complex pathophysiology induced by this particular secretion and other toxins.     

T cells are involved in the development of arthritis induced by anti-type II collagen antibody

T cells play an important role in initiating autoimmune responses and maintaining synovial inflammation in rheumatoid arthritis. Although, anti-type II collagen antibody-induced arthritis (CAIA) is generally believed to be a T cell- and B cell-independent model, the detailed pathogenesis of CAIA remains unclear. In the present study, to elucidate the contribution of T cells to the pathogenesis of CAIA, we evaluated the effects of CTLA4 Ig and cyclosporin (CsA). Arthritis was induced in mice by intravenous injection of anti-type II collagen antibody followed by intraperitoneal injection of lipopolysaccharide. CTLA4 Ig was intraperitoneally administered and CsA was subcutaneously administered; then the severity of arthritis was evaluated by scoring the edema and erythema of paws and by measuring hind paw thickness. Paw samples were collected 12 days after the antibody injection, and the mRNA expression levels were analyzed by real-time quantitative polymerase chain reaction. Administration of CTLA4 Ig ameliorated the increases in arthritic score and paw thickness in the later phase, but not in the early phase of arthritis. CsA suppressed the increases in arthritic score and paw thickness in both the early and later phases of arthritis. CTLA4 Ig and CsA suppressed mRNA up-regulation of T-cell markers, CD3 and CD25, and immune response-related mediators, IFN-gamma and IL-12. They also suppressed the up-regulation of macrophage marker, F4/80, and proinflammatory cytokines, TNF-alpha, IL-1beta and IL-6. The results provide direct evidence that arthritis in this model is T-cell activation dependent.     

Purines and pyrimidines are not involved in NANC relaxant responses in the rabbit vaginal wall

1. Non-adrenergic non-cholinergic (NANC) relaxant responses were elicited by electrical field stimulation (EFS) in rabbit vaginal wall strips after treatment with guanethidine and scopolamine and raising smooth muscle tone with phenylephrine. Under these conditions treatment with NOS inhibitors revealed a non-nitrergic NANC relaxant response. The possible role of purines and pyrimidines in these non-nitrergic NANC responses was investigated. 2. Exogenous application of ATP, ADP, adenosine, UTP, or UDP (all at 0.03-10 mM) induced concentration-dependent relaxant responses. 3. Responses to exogenous application of ATP were reduced by the general P2 antagonist cibacron blue (500 micro M), but not by suramin (100 micro M) and were unaffected by L-NAME (500 micro M), omega-conotoxin GVIA (omega-CTX, 500 nM) or tetrodotoxin (TTX, 1 micro M). 4. Responses to exogenous application of adenosine were reduced by the A(2A) antagonist ZM-241385 (30 micro M). 5. ATP- and ADP-induced responses were unaffected by the G-protein inhibitor pertussis toxin (100 ng ml(-1)), whilst ADP- but not ATP-induced responses were reduced by GDPbetaS (100 micro M), which stabilizes G-proteins in their inactive state. 6. EFS-induced non-nitrergic NANC relaxant responses were unaffected by suramin, cibacron blue, ZM-241385, pertussis toxin or GDPbetaS, but were completely inhibited by TTX. 7. Exogenous application of ATP (10 mM) and adenosine (10 mM) increased intracellular cyclic adenosine-3', 5'-monophosphate (cAMP). However, non-nitrergic NANC responses were not associated with increased cAMP. Neither non-nitrergic NANC responses nor responses to ATP or adenosine were associated with increased intracellular cyclic guanosine-3', 5'-monophosphate (cGMP) concentrations. 8. These results suggest that adenosine A(2A) receptors and P2 receptors are present in the rabbit vaginal wall, but that they are not involved in non-nitrergic NANC relaxant responses.     

Reactive oxygen species are involved in the apoptosis induced by nonsteroidal anti-inflammatory drugs in cultured gastric cells

We have examined the pharmacology of kainate receptors in cultured hippocampal neurons (6-8 days in vitro (DIV)) from embryonic rats (E17). Cultured neurons were pre-treated with concanavalin A to remove kainate receptor desensitization and whole-cell voltage clamp electrophysiology employed to record inward currents in response to glutamatergic agonists and antagonists. N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor responses were blocked using MK801 (3 microM) and the 2,3-benzodiazepine, LY300168 (GYKI53655, 50 microM), respectively. Inward currents were recorded in hippocampal neurons upon application of kainate and the 2S,4R isomer of 4-methyl glutamic acid (SYM2081) with EC50 values of 3.4 +/- 0.4 microM and 1.6 +/- 0.5 microM, respectively (n = 6 cells). The GluR5 selective agonists, LY339434 (100 microM) and (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl) propionic acid (ATPA) (100 microM), did not evoke detectable inward currents in any cell responding to kainate. LY293558 and the selective GluR5 antagonist, LY382884, had weak antagonist effects on responses evoked by either kainate or (2S,4R)-4-methyl glutamate (IC50 > 300 microM). The quinoxalinedione, 2,3-dihyro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX), blocked both kainate and (2S,4R)-4-methyl glutamate-activated currents at much lower concentrations (IC50 approximately 10 microM). These results provide pharmacological evidence that ion channels comprised of GluR6 kainate receptor subunits mediate kainate receptor responses in hippocampal neurons cultured 6-8 DIV.     

Mechanisms involved in the rat peritoneal leukocyte migration induced by a Kunitz-type inhibitor isolated from Dimorphandra mollis seeds

DMTI-II is a Kunitz-type inhibitor isolated from Dimorphandra mollis seeds that causes rat inflammatory edema by mechanisms involving activation of mast cells and sensory C-fibers. The present study aimed to further explore the inflammatory mechanisms involved in DMTI-II-induced inflammation, focusing to the leukocyte migration in vivo. Male Wistar rats (250-280 g) were injected with DMTI-II (1-100 μg/cavity), and at 4-24 h thereafter the leukocyte counts in peritoneal lavage were evaluated. DMTI-II caused dose- and time-dependent accumulation of neutrophils and eosinophils. The peritoneal neutrophil influx initiated at 4 h, achieving maximal responses at 16 h after DMTI-II injection (16- and 22-fold increase, respectively). The DMTI-II-induced eosinophil recruitment was observed as early as 4 h achieving the maximal responses at 16 h (12- and 17-fold increase, respectively). The mononuclear cell number increased at 4 h and 16 h (1.5-fold and 1.6-increase, respectively). Prior treatments with dexamethasone, the cyclooxygenase (COX) inhibitors indomethacin and celecoxib, as well as the PAF receptor antagonist PCA4248 largely reduced the neutrophil and eosinophil accumulation. The selective lypoxygenase inhibitor AA861, the tachykinin NK₁ antagonist SR-140333 and the nitric oxide inhibitor L-NAME reduced only the eosinophil number. The eotaxin levels were significantly higher in DMTI-II-injected rats compared with control animals. In conclusion, DMTI-II causes an early migration of eosinophils and neutrophils by mechanisms involving COX-2- and lipoxygenase-derived metabolites, PAF, substance P and NO. The capacity of DMTI-II to recruit eosinophils at early times is likely to reflect the allergen properties of proteinase inhibitors belonging to Kunitz family.     

Different potassium channels are involved in relaxation of rat renal artery induced by P1075

The ATP-sensitive K(+) channels opener (K(ATP)CO), P1075 [N-cyano-N'-(1,1-dimethylpropyl)-N″-3-pyridylguanidine], has been shown to cause relaxation of various isolated animal and human blood vessels by opening of vascular smooth muscle ATP-sensitive K(+) (K(ATP)) channels. In addition to the well-known effect on the opening of K(ATP) channels, it has been reported that vasorelaxation induced by some of the K(ATP)COs includes some other K(+) channel subtypes. Given that there is still no information on other types of K(+) channels possibly involved in the mechanism of relaxation induced by P1075, this study was designed to examine the effects of P1075 on the rat renal artery with endothelium and with denuded endothelium and to define the contribution of different K(+) channel subtypes in the P1075 action on this blood vessel. Our results show that P1075 induced a concentration-dependent relaxation of rat renal artery rings pre-contracted by phenylephrine. Glibenclamide, a selective K(ATP) channels inhibitor, partly antagonized the relaxation of rat renal artery induced by P1075. Tetraethylammonium (TEA), a non-selective inhibitor of Ca(2+)-activated K(+) channels, as well as iberiotoxin, a most selective blocker of large-conductance Ca(2+) -activated K(+) (BK(Ca)) channels, did not abolish the effect of P1075 on rat renal artery. In contrast, a non-selective blocker of voltage-gated K(+) (K(V)) channels, 4-aminopyridine (4-AP), as well as margatoxin, a potent inhibitor of K(V)1.3 channels, caused partial inhibition of the P1075-induced relaxation of rat renal artery. In addition, in this study, P1075 relaxed contractions induced by 20 mM K(+) , but had no effect on contractions induced by 80 mM K(+). Our results showed that P1075 induced strong endothelium-independent relaxation of rat renal artery. It seems that K(ATP), 4-AP- and margatoxin-sensitive K(+) channels located in vascular smooth muscle mediated the relaxation of rat renal artery induced by P1075.     

At least three phosphorylation events induced by pemphigus vulgaris sera are pathogenically involved in keratinocyte acantholysis

Intercellular adhesion among keratinocytes is guaranteed by desmosomes. Disruption of desmosomal integrity leads to cell-cell detachment or acantholysis, as it classically occurs in pemphigus vulgaris (PV), an autoimmune blistering disease of skin and mucous membranes. While purified PV IgG seems to trigger intracellular signaling that crucially involves p38 MAPK, keratinocyte acantholysis induced by whole PV serum may recruit a number of additional signals. In this study, the Pro-Q Diamond Phosphoprotein Assay was used to investigate the overall changes in protein phosphorylation levels in an in vitro model of PV. We showed that keratinocytes exposed to whole PV sera underwent at least three early and transient phosphorylation events. Two bands with apparent molecular masses of 35 and 45 kDa were found to be phosphorylated within 1 min after incubation with PV sera. A third band of about 80 kDa reached the peak of phosphorylation level after 3 hours. Morphologic evidence of cell shrinkage and acantholysis were late events and did not correlate temporally with kinase activation, suggesting that cytoskeleton reorganization is a downstream phenomenon. Interestingly, pharmacological abrogation of PV-specific protein phosphorylation was able to inhibit the cell-cell detachment, rounding up, and redistribution of Dsg3 in keratinocytes. Thus, at least three phosphorylation events are pathogenically involved in pemphigus acantholysis.     

Different K+ channels are involved in relaxation of arterial and venous graft induced by nicorandil

The drug nicorandil is a vasodilator approved for the treatment of angina. In addition to its well-known effect on the opening of ATP-sensitive K (KATP) channels, nicorandil-induced vasorelaxation also involves the opening of Ca-activated K channels. The aim of this study was to investigate the effects of nicorandil on the isolated human internal mammary artery (HIMA) and the human saphenous vein (HSV) and to define the contribution of different K channel subtypes in the nicorandil action on these arterial and venous grafts. Our results show that nicorandil induced a concentration-dependent relaxation of HSV and HIMA rings precontracted by phenylephrine. Glibenclamide, a selective KATP channels inhibitor, partially inhibited the response to nicorandil in both HSV and HIMA. Iberiotoxin, a most selective blocker of large-conductance Ca-activated K (BKCa) channels, partly antagonized relaxation of HIMA. A nonselective blocker of voltage-gated K channels, 4-aminopyridine caused partial inhibition of the nicorandil-induced relaxation of HSV but did not antagonize relaxation of HIMA induced by nicorandil. Margatoxin, a potent inhibitor of KV1.3 channels, did not abolish the effect of nicorandil on HSV and HIMA. Our results showed that nicorandil induced strong endothelium-independent relaxation of HSV and HIMA contracted by phenylephrine. It seems that KATP and 4-aminopyridine-sensitive K channels located in the smooth muscle of HSV mediated relaxation induced by nicorandil. In addition, KATP and BKCa channels are probably involved in the nicorandil action on HIMA.     

D1 and D2 dopaminergic systems in the rat basolateral amygdala are involved in anxiogenic-like effects induced by histamine

Involvement of the dopamine receptors in the basolateral amygdala (BLA) in the effects of histamine on anxiety-like behaviors of the elevated plus maze in male Wistar rats was investigated. The results showed that bilateral intra-BLA injections of histamine (2.5, 5 and 7.5 μg/rat) induced an anxiogenic-like effect, revealed by decreases in percentage of open arm time (%OAT) and open arm entries (%OAE). Intra-BLA administration of dopamine D1 receptor agonist, SKF38393 (0.25 μg/rat), and dopamine D2 receptor agonist, quinpirole (0.03 and 0.05 μg/rat), decreased %OAT but not %OAE. Conversely, intra-BLA administration of dopamine D1 receptor antagonist, SCH23390 (0.5 and 1 μg/rat), and dopamine D2 receptor antagonist, sulpiride (0.3 and 0.5 μg/rat), increased %OAT and %OAE, suggesting an anxiolytic-like effect for both drugs. Interestingly, co-administration of a silent dose of SCH23390 or sulpiride prevented anxiogenic-like effects of SKF38393 and quinpirole, respectively. Conjoint administration of a sub-effective dose of SKF38393 (0.125 μg/rat) or quinpirole (0.01 μg/rat) along with lower doses of histamine (1 and 2.5 μg/rat) induced anxiolytic-like effects. On the other hand, intra-BLA pretreatment with a silent dose of SCH23390 (0.25 μg/rat) or sulpiride (0.1 μg/rat) prevented the anxiogenic-like effect of higher doses of histamine (5 and 7.5 μg/rat). No significant change was observed in total closed arm entries, as an index for motor activity of the animals. It can be concluded that the dopamine D1 and D2 receptors in the BLA may be involved in the anxiogenic-like effects induced by histamine.     

On the mechanisms involved in antinociception induced by diphenyl diselenide

In this study, we described the local peripheral antinociceptive activity produced by diphenyl diselenide in the formalin test as compared to ebselen, an amply studied organoselenium compound. A second objective was to evaluate, the possible mechanisms underlying the antinociceptive effect caused by diphenyl diselenide. Administration of diphenyl diselenide or ebselen produced a significant antinociceptive local effect on the late phase (15-30min) of the formalin test. As well, diphenyl diselenide and ebselen injected in the contra lateral paw produced a significant decrease in licking time on the late phase (15-30min). The mechanisms underlying the analgesic action of diphenyl diselenide seem to be unlike the activation of opioid, dopaminergic D2, muscarinic cholinergic receptors or the interaction with α(1) and α(2) adrenoceptors. Furthermore, the effect of a 5-HT(3) receptor antagonist in abolishing the antinociception induced by diphenyl diselenide suggests the involvement of serotonergic pathways.     

Interleukin-12 was not involved in promotion of T helper cell differentiation induced by theophylline

AIM:ToinvestigatetheeffectoftheophyllineonthenaiveTcelldifferentiationandtheprobableroleofinterleukin-12(IL-12).METHODS:NaivecordbloodTcellsweretreatedwiththeophylline10mg/Lfor3dafterstimulationwithPHA100mg/L.DifferentiationofTcellswasanalyzedbyflowcytometry.Theophylline10mg/LandIL-12-mAb0.025mg/Lwereaddedincordbloodmononuclearcell(CBMC)culturesprimedwithLPS1mg/LtodetectthelevelsofIL-12andIL-12P40.Thewholebloodcultureswereobtainedfromtwelvehealthvolunteerswithorwithoutadministr…     

Disturbed mitotic progression and genome segregation are involved in cell transformation mediated by nano-TiO2 long-term exposure

Titanium dioxide (TiO2) nano-particles (< 100 nm in diameter) have been of interest in a wide range of applications, such as in cosmetics and pharmaceuticals because of their low toxicity. However, recent studies have shown that TiO2 nano-particles (nano-TiO2) induce cytotoxicity and genotoxicity in various lines of cultured cells as well as tumorigenesis in animal models. The biological roles of nano-TiO2 are shown to be controversial and no comprehensive study paradigm has been developed to investigate their molecular mechanisms. In this study, we showed that short-term exposure to nano-TiO2 enhanced cell proliferation, survival, ERK signaling activation and ROS production in cultured fibroblast cells. Moreover, long-term exposure to nano-TiO2 not only increased cell survival and growth on soft agar but also the numbers of multinucleated cells and micronucleus (MN) as suggested in confocal microscopy analysis. Cell cycle phase analysis showed G2/M delay and slower cell division in long-term exposed cells. Most importantly, long-term TiO2 exposure remarkably affected mitotic progression at anaphase and telophase leading to aberrant multipolar spindles and chromatin alignment/segregation. Moreover, PLK1 was demonstrated as the target for nano-TiO2 in the regulation of mitotic progression and exit. Notably, a higher fraction of sub-G1 phase population appeared in TiO2-exposed cells after releasing from G2/M synchronization. Our results demonstrate that long-term exposure to nano-TiO2 disturbs cell cycle progression and duplicated genome segregation, leading to chromosomal instability and cell transformation.     

Endothelium-derived factors and k+ channels are involved in the vasorelaxation induced by Sida cordifolia L. in the rat superior mesenteric artery

The vasorelaxantion of the aqueous fraction of the hydroalcoholic extract of the Sida cordifolia leaves (AFSC) was evaluated in this work. In rat superior mesenteric artery, AFSC (3-1000 microg/mL) induced relaxation of phenylephrine-induced contractions. This effect was significantly attenuated after removal of the endothelium, after atropine (1 microM), L-NAME (100 microM), indomethacin (10 microM), high K+ (20 mM), tetraethylammonium (1 microM), a K(Ca) blocker, apamin (1 microM), a SK(Ca) blocker and ChTX (0.1 microM), a BK(Ca) blocker, however, it was not affected after glibenclamide (10 microM), an KATP blocker, and 4-aminopyridine (1 microM), a Kv blocker. ChTX (0.1 microM) was able to induce an additional inhibition of the vasorelaxation induced by AFSC in the presence of L-NAME plus indomethacin. The vasorelaxation induced by AFSC in the presence of L-NAME plus indomethacin plus ChTX was not different from that induced by AFSC in rings without endothelium. In conclusion, the results show that endothelium-derived factors (mainly NO, PGI2) and K+ channels (BK(Ca) and SK(Ca)) play a crucial role in the vasorelaxation induced by AFSC in the rat superior mesenteric artery.     

蛋白酶体抑制剂MG132诱导HepG2细胞凋亡及其机制研究

目的观察蛋白酶体抑制剂MG132对人肝癌细胞HepG2的致凋亡作用,并从泛素蛋白酶体途径(UPP)相关基因E1、E2和E3及天冬氨酸特异的半胱氨酸蛋白酶3(Caspase3)表达初步探讨MG132的致细胞凋亡机制。方法采用多个浓度(2、5、10μmol·L-1)的蛋白酶体抑制剂MG132处理HepG2细胞;流式细胞术检测细胞周期和细胞凋亡率,DNA琼脂糖凝胶电泳检测细胞凋亡;逆转录聚合酶链反应(RTPCR)检测UPP相关基因E1、E2、E3和凋亡相关基因Caspase3的转录水平;免疫细胞化学检测Caspase3蛋白表达。结果对照组HepG2细胞凋亡率低于5%,在2、5和10μmol·L-1MG132作用下,细胞凋亡率分别为42.9%、66.1%、72.8%,MG132诱导HepG2细胞凋亡具有量效关系;RTPCR检测发现细胞内UPP相关基因E1、E2、E3mRNA表达下降,而凋亡相关基因Caspase3mRNA表达上调;免疫组化检测Caspase3蛋白表达水平升高。结论蛋白酶体抑制剂MG132能够诱导HepG2细胞凋亡,其机制可能与MG132抑制UPP活性,使细胞内Caspase3蛋白降解减少,同时上调Caspase3基因转录,促进细胞凋亡。