Activation of protein kinase C via the T-cell receptor complex potentiates cyclic AMP responses in T-cells

Summary We have recently shown that activation of protein kinase C by tumour promoting phorbolesters, such as 4--phorbol-12,13-dibutyrate, stimulates adenosine-induced accumulation of cAMP in 7urkat cells, a human T-leukaemia line. Activating the CD3 complex associated with the T-cell receptor by means of the monoclonal antibody OKT3 caused a concentration-dependent accumulation of inositol phosphates and an increase in the phosphorylation of an endogenous protein kinase C substrate. OKT3 also mimicked the previously reported effects of protein kinase C since it potentiated the cAMP stimulation by either an adenosine analogue, NECA, or cholera toxin. Thus, our results indicate that stimulation of a receptor activating phospholipase C and protein kinase C can secondarily enhance the action of agonists that act on adenylate cyclase-coupled receptors.Abbreviations cAMP cyclic adenosine-monophosphate - NECA 5-N-ethylcarboxyamido adenosine The present study was supported by grants from The Swedish Cancer Association, The Swedish Medical Research Council (project no. 2553), by Gustaf V 80 Years Fund, by Nanna Swartz foundation and by Karolinska Institutet     

Phosphorylated derivatives of phloretin inhibit cyclic AMP accumulation in neuronal and glial tumor cells in culture

Summary The potencies of polyphloretin phosphate, di-4-phloretin phosphate, 4-phloretin phosphate and phloretin to inhibit the stimulation of cAMP accumulation by prostaglandins, isoproterenol and adenosine were studied in 2 clonal cell lines of CNS origin. The sequence of potency to inhibit PGE₁ effects was the same in neuroblastoma (N4TG3) and human astrocytoma cells (1321N1): di-4-phloretin phosphate > polyphloretin phosphate > phloretin >4-phloretin phosphate. The inhibition of PGE₁ stimulated cAMP accumulation by the most prostaglandin-specific inhibitor di-4-phloretin phosphate was rapidly established after its addition, fully reversible after a 30 min preincubation period and independent of the presence of calcium. Kinetic studies of the inhibition of PGE₁ effects by di-4-phloretin-phosphate suggest a different type of inhibition in 1321N1 and N4TG3 cells.The following abbreviations are used in this report PPP polyphloretin phosphate - MPP 4-phloretin phosphate - DPP di-4-phloretin phosphate - IBMX 3-isobutyl-methylxanthine - TCA trichloroacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylenglycol-bis (-amino ethylether) N,N-tetraacetic acid - PGE₁ Prostaglandin E₁ - ¹²⁵J-TME ScAMP (¹²⁵J) 2-O-succinyl cyclic AMP tyrosine methyl ester     

Neurochemical and behavioural evidence that dopamine D-2 receptors in striatum couple to the Ni regulatory protein and inhibition of cyclic AMP accumulation

Summary The effects of intrastriatal pertussis toxin, which inactivates the regulatory protein N_i, were investigated in various models of striatal D-2 receptor function in the rat. Using a multiple injection technique unilateral intrastriatal injections of pertussis toxin induced, after a lag phase, ipsilateral postural asymmetries which intensified upon peripheral administration of apomorphine. Injections of pertussis toxin also partially reduced the ability of a selective D-2 agonist, RU 24926 [N-n-propyl di- (3-hydroxyphenyl)-ethylamine], to inhibit cyclic AMP accumulation due to a selective D-1 agonist SKF 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine), whilst also reducing the affinity of dopamine for D-2 binding sites in striatal membranes from animals given prior intrastriatal injections of the toxin. Pertussis toxin also increased striatal dopamine metabolism, seen as a reduction in the dopamine: DOPAC (3,4-Dihydroxyphenylacetic acid) ratio, similar to that seen following intrastriatal injections of the selective D-2 antagonist (±)-sulpiride. These results suggest that pertussis toxin has dopamine D-2 antagonist-like properties in the rat striatum, consistent with the idea that striatal D-2 function may rely, in part at least, upon the regulatory protein N_i and adenylate cyclase inhibition. Send offprint requests to S. R. Nahorski at the above address     

Interactions between intracellular cyclic AMP and agonist-induced inositol phospholipid breakdown in isolated gastric mucosal cells of the rat

Summary The interactions between putative second effector mechanisms for hydrogen ion secretion were studied in isolated gastric cell preparations of the rat containing 60 - 70% parietal cells. Dibutyryl-CAMP and the compounds which increased the level of cAMP (histamine plus rolipram and forskolin plus rolipram) inhibited the carbachol-induced accumulation of [³H]inositol tris-, bis- and monophosphate. There was both a temporal and quantitative correlation between the increase in cAMP and the inhibition of the accumulation of [³H]inositol phosphates. Cimetidine attenuated the inhibitory effect of histamine on the formation of [³H]inositol phosphates. The enhancement of the accumulation of [³H]inositol phosphates by various concentrations of carbachol affected neither the basal nor the histamine-stimulated cAMP levels. In contrast to dibutyryl-cAMP, dibutyryl-cGMP did not modify the carbachol-induced formation of [³H]inositol phosphates. The biologically active phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), which activates protein kinase C, inhibited both the basal and carbachol-induced accumulation of [³H]inositol phosphates. We suggest that the inhibition of the formation of inositol trisphosphate by the increase in the intracellular level of cAMP and by the activation of protein kinase C might be intracellular negative feedback systems which prevent the overreaction of the acid-secreting parietal cells under the simultaneous influence of the physiological gastric secretagogues.Fellow of the Alexander von Humboldt Foundation, FRG Send offprint requests to U. Schwabe     

Role of guanine nucleotide-binding protein in the regulation by adenosine of cardiac potassium conductance and force of contraction. Evaluation with pertussis toxin

Summary In atrial cardiac preparations adenosine exerts a receptor-mediated negative inotropic effect due to an increased potassium conductance. Pretreatment of guinea pigs with pertussis toxin abolished the negative inotropic and action potential shortening effect of adenosine and the adenosine analogue (–)-N⁶-phenylisopropyladenosine (PIA). As pertussis toxin specifically inactivates guanine nucleotide-binding proteins involved in the signal transfer from receptor binding to specific cell functions, it is concluded that a guanine nucleotide-binding protein is involved in the regulation of the receptor-mediated change in potassium conductance and force of contraction.Parts of the results have been presented at the Joint Meeting of the Belgian, Dutch, and German Pharmacological Societies at Aachen (Böhm et al. 1985a)     

Agents that elevate cyclic AMP induce receptor phosphorylation primarily at serine 331 in HEK 293 cells overexpressing human thromboxane receptor alpha

Human embryonic kidney (HEK) 293 cells stably transfected with the His-tagged thromboxane receptor alpha (TPalpha) were used to study the phosphorylation and desensitization of the receptor induced by prostaglandin E1 (PGE1) or forskolin. These agents are known to increase the intracellular level of cyclic AMP (cAMP) and activate cAMP-dependent protein kinase (PKA). Pretreatment of cells with either agent significantly attenuated Ca2+ release induced by the agonist [1S-[1alpha,2alpha(Z),3beta(1E,3S),4alpha]]-7-[3-[3-hydroxy-4-(4-indophenoxy)-1-butenyl]-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid (I-BOP). These agents also induced concentration-dependent phosphorylation of TPalpha as demonstrated by increased 32P-labeling of the receptor from cells prelabeled with 32P(i). To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for purified PKA. It was found that only the C-terminal tail fusion protein could serve as a substrate for PKA. To identify the specific serine/threonine residues in the C-terminal tail that are involved in phosphorylation, various alanine mutants of these residues were checked for their ability to serve as substrates. Ser-331 was found to be involved in PKA-mediated phosphorylation. The S331A mutant receptor overexpressed in HEK 293 cells was not phosphorylated significantly following stimulation by PGE1 or forskolin, indicating that Ser-331 was the major site of phosphorylation. Furthermore, cells overexpressing the mutant receptor became responsive to I-BOP-induced Ca2+ mobilization even after pretreatment with PGE1 or forskolin. These results indicate that Ser-331 is the primary site responsible for the phosphorylation and desensitization of the human TPalpha induced by agents that activate PKA.     

Comparison of the effects of prostaglandins E2 and I2 on testicular nociceptor activities studied in vitro

Summary Effects of exogenous prostaglandin (PG) E₂ and PGI₂ on testicular polymodal receptor activities were compared in in vitro recordings of single- or multi-fiber discharges from canine testis-spermatic nerve preparations. PGI₂ up to 1.4×10^–6 mol/l (cumulative method) or 1.0×10^–5 mol/l (non-cumulative method) excited only weakly some of the receptors, and similar observations were made with PGE2. Both PGs applied cumulatively or non-cumulatively at concentrations above 1.4×10^–8 mol/l augmented the response to bradykinin (9.4×10^–8 mol/l) in more than half of the cases tested. The augmenting effect of PGE₂ lasted longer than that of PGI₂ both with the cumulative and the non-cumulative method. The degree of augmentation tended to increase dependent on concentration, but some cases showed no further increase or rather a decrease in augmentation by PGs at a ten times higher concentration, especially when PGs were applied cumulatively. A second challenge by PG after a short interval (2 min) did not induce augmentation. These phenomena were considered to be tachyphylaxis to PGs. Cross-tachyphylaxis to PGE₂ and PGI₂ was also observed. There was not much difference in excitatory and augmenting potencies between these two PGs, but there was a clear difference in the concentrations of the PGs necessary to induce excitation of polymodal receptors and to facilitate their response to bradykinin. Send offprint requests to T. Kumazawa at the above address     

The receptor mechanism mediating the contractile response to adenosine on lung parenchymal strips from actively sensitised,allergen-challenged Brown Norway rats

Parenchymal strips prepared from lungs removed from actively sensitised Brown Norway rats challenged with allergen show hyperresponsiveness to adenosine. The response is mast cell mediated and a preliminary pharmacological analysis suggested the involvement of a receptor (or receptors) that could not be classified as any of the known adenosine receptor subtypes. We present a further analysis of the response. Male Brown Norway (BN) rats, actively sensitised to ovalbumin (OA), were challenged intratracheally with OA and killed 3 h later to provide parenchymal strip preparations. The augmented contractile responses to adenosine were partially blocked by the 5-HT receptor antagonist, methysergide, or the A₁ receptor antagonist, DPCPX, and abolished in the presence of both antagonists. Responses to high concentrations of the A₁ receptor agonist, CPA were, like those to adenosine, augmented on tissues from allergen-challenged animals and blocked by a combination of methysergide and DPCPX. The A₃ receptor agonist, Cl-IB-MECA, did not contract the tissue, but partially blocked the response to adenosine. A combination of Cl-IB-MECA and methysergide induced a similar degree of blockade to that seen with either drug given alone. Combination of Cl-IB-MECA and/or methysergide with DPCPX abolished the response to adenosine. The effects of the A₃ receptor agonist, inosine, were augmented on tissues from allergen-challenged animals and markedly inhibited by disodium cromoglycate, methysergide or Cl-IB-MECA. Responses to adenosine were abolished when parenchymal strips were taken from rats pretreated 48 h previously with pertussis toxin. 8-SPT, CGS 15943, XAC, MRS 1754, DPCPX and theophylline, at concentrations which inhibit the A₁ A_2A and/or A_2B receptors but have negligible affinity for the rat A₃ receptor, inhibited responses to adenosine, but high concentrations were required and blockade was incomplete. MRS 1523 and MRS 1191, which are antagonists at the rat A₃ receptor, had no effect on the response to adenosine. The present results support and clarify our earlier conclusion that an atypical receptor mechanism mediates contraction of the parenchymal strip prepared from the lungs of actively sensitised BN rats challenged with allergen to adenosine. The response arises from a combined effect of adenosine on the A₁ receptor and a receptor with similarities to the A₃ receptor, but where Cl-IB-MECA behaves as an antagonist and MRS 1523 and MRS 1191 are inactive at concentrations that substantially exceed their affinities for the rat A₃ receptor.A part of this work was presented to the British Pharmacological Society in January 2003     

Effect of lysophosphatidylglycerol on several signaling molecules in OVCAR-3 human ovarian cancer cells: involvement of pertussis toxin-sensitive G-protein coupled receptor

In this study, we observed that lysophosphatidylglycerol (LPG) stimulated intracellular calcium ([Ca(2+)](i)) increase in OVCAR-3 human ovarian cancer cells. LPG-stimulated [Ca(2+)](i) increase was inhibited by U-73122 but not by U-73343, suggesting that LPG stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca(2+)](i) increase by LPG, indicating the activation of PTX-sensitive G-proteins. LPG-induced [Ca(2+)](i) increase was only observed in OVCAR-3 ovarian cancer cells and SK-OV3 ovarian cancer cells among tested several cell types. LPG also induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation in OVCAR-3 ovarian cancer cells. Pertussis toxin did not affect the LPG-induced activation of ERK and Akt phosphorylation. We also found that LPG failed to stimulate NF-kappaB-driven luciferase activity in exogenously LPA(1), LPA(2), or LPA(3)-transfected HepG2 cells. Taken together we suggest that LPG stimulates a membrane bound receptor which is different from well-known LPA receptors (LPA(1), LPA(2), and LPA(3)), resulting in at least two different signaling cascades; one involves a pertussis toxin-sensitive and phospholipase C-dependent [Ca(2+)](i) increase, and the other involves a pertussis toxin-insensitive activation of ERK and Akt in ovarian cancer cells.     

A farnesylated G-protein suppresses Akt phosphorylation in INS 832/13 cells and normal rat islets: Regulation by pertussis toxin and PGE2

Protein isoprenylation constitutes incorporation of either 15-carbon farnesyl or 20-carbon geranylgeranyl derivative of mevalonic acid onto the C-terminal cysteine, culminating in increased hydrophobicity of the modified proteins for optimal membrane anchoring and interaction with their respective effectors. Emerging evidence confirms the participatory role of prenylated proteins in pancreatic β-cell function including insulin secretion. Herein, we investigated the putative regulatory roles of protein farnesylation in cell survival signaling pathways in insulin-secreting INS 832/13 cells and normal rodent islets, specifically at the level of protein kinase-B/Akt phosphorylation induced by insulin-like growth factor [IGF-1]. Selective inhibitors of farnesylation [e.g., FTI-277 or FTI-2628] or knockdown of the β-subunit of farnesyl transferase by siRNA significantly increased Akt activation under basal and IGF-1-stimulated conditions. Consequentially, the relative abundance of phosphorylated FoxO1 and Bad were increased implicating inactivation of critical components of the cell death machinery. In addition, FTI-induced Akt activation was attenuated by the PI3-kinase inhibitor, LY294002. Exposure of INS 832/13 cells to pertussis toxin [PTx] markedly potentiated Akt phosphorylation suggesting involvement of a PTx-sensitive G-protein in this signaling axis. Furthermore, prostaglandin E₂, a known agonist of inhibitory G-proteins, significantly attenuated FTI-induced Akt phosphorylation. Taken together, our findings suggest expression of a farnesylated G-protein in INS 832/13 cells and normal rat islets, which appear to suppress Akt activation and subsequent cell survival signaling steps. Potential regulatory roles of the islet endogenous protein kinase-B inhibitory protein [Probin] in islet function are discussed.     

Prostacyclin (PGI2) decreases the cyclic AMP level in coronary arteries

Summary The effects of prostacyclin (PGI₂) on vascular tension and cAMP content were measured in isolated bovine coronary artery strips. 3 nM PGI₂ did not alter the tension but diminished the cAMP content by 56% of the control level (P<0.005). 30 and 300 nM PGI₂ diminished the tension and further reduced the cAMP content, which amounted to only 5% of the control at 300 nM PGI₂. These results are in contrast to the increase in cAMP level by PGI₂ in blood platelets and might indicate a different mechanism of action of PGI₂ in platelets and vascular tissue.     

Evidence against a role of a pertussis toxin-sensitive guanine nucleotide-binding protein in the alpha1-adrenoceptor-mediated positive inotropic effect in the heart

Summary Pertussis toxin, which specifically inactivates guanine nucleotide-binding proteins (N-proteins) involved in the signal transduction in various receptor systems, did not influence the positive inotropic effect of the alpha₁-adrenoceptor agonist phenylephrine in rat isolated left auricles. This indicates that the alpha₁-adrenoceptor-mediated positive inotropic effect does not involve a pertussis toxin-sensitive N-protein. Send offprint requests to W. Schmitz at the above addressSupported by the Deutsche Forschungsgemeinschaft     

Effects of specific prostanoid EP receptor agonists on cell proliferation and intracellular Ca concentrations in human airway smooth muscle cells

Increased airway smooth muscle mass due to cell proliferation contributes to airway hyper-responsiveness and remodeling in patients with asthma. Prostaglandin E₂ (PGE₂) inhibits proliferation of airway smooth muscle cells, but the role of prostanoid EP receptor subtypes in mechanisms involved has not been fully elucidated yet. We investigated the effects of specific prostanoid EP receptor agonists on cell proliferation and intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in human airway smooth muscle cells. Cell numbers were assessed by mitochondria-dependent reduction of 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate to formazan (WST-1 assay). RT-PCR data showed that human airway smooth muscle cells express EP2, EP3, and EP4 but not EP1 receptor mRNA. PGE₂ (1 nM–1 μM) inhibited cell proliferation induced by 5% fetal bovine serum (FBS) in a concentration-dependent manner. (16S)-9-deoxy-9β-chloro-15-deoxy-16-hydroxy-17, 17-trimethylene-19, 20-didehydro PGE₂ sodium salt (ONO-AE1-259-01; EP2 receptor agonist) and 16-(3-methoxymethyl)phenyl-ω-tetranor-3,7-dithia PGE₂ (ONO-AE1-329; EP4 receptor agonist) inhibited the 5% FBS-induced cell proliferation. ONO-AE1-259-01 and ONO-AE1-329 also significantly increased the cytosolic cAMP levels. In contrast, 11,15-O-dimethyl PGE₂ (ONO-AE-248; EP3 receptor agonist) elicited an oscillatory increase in [Ca²⁺]_i but did not affect the cell growth or cAMP levels. [(17S)-2,5-ethano-6-oxo-17,20-dimethyl PGE₁] (ONO-DI-004; EP1 receptor agonist) did not affect cell growth, cAMP levels, or [Ca²⁺]_i. In conclusion, PGE₂ inhibits FBS-induced cell proliferation mostly via EP2 and EP4 receptor activation and subsequent cAMP elevation. The EP3 receptor agonist causes an increase in [Ca²⁺]_i without affecting cell growth. There is no functional expression of the EP1 receptor. Research on prostanoid EP receptors may lead to novel therapeutic strategies for treatment of asthma.     

The effect of acteoside on histamine release and arachidonic acid release in RBL-2H3 mast cells

The effect of acteoside, a phenylpropanoid glycoside isolated from Clerodendron trichotomum Thunberg, on histamine and arachidonic acid release was investigated in RBL 2H3 cells. Histamine was dose-dependently released from RBL 2H3 cells by melittin, arachidonic acid and thapsigargin. In extracellular Ca2+-free solution, basal secretion of histamine increased by two fold. The response of histamine release to melittin and thapsigargin in Ca2+-free solution was significantly decreased, whereas the response to arachidonic acid was significantly increased as compared with those in normal solution. Acteoside inhibited histamine release induced by melittin, arachidonic acid and thapsigargin in a dose-dependent manner in the presence or absence of extracellular Ca2+. However, the inhibitory activity of acteoside was more potent in normal solution than that in Ca2+-free solution. These data suggest that inhibitory mechanism of acteoside on histamine release may be related to extracellular Ca2+. On the other hand, acteoside significantly inhibited arachidonic acid release and prostaglandin E2 production induced by 0.5 microM melittin. It is possible that acteoside may be developed as an anti-inflammatory agent.     

Inhibitory effects of azelastine on substance P-induced itch-associated response in mice

The anti-pruritic mechanisms of azelastine were studied in mice. Scratching induced by intradermal histamine was inhibited by azelastine (30 mg/kg) and chlorpheniramine (30 mg/kg). Substance P-induced scratching was dose dependently suppressed by azelastine (3-30 mg/kg), but not by chlorpheniramine (10 and 30 mg/kg). Azelastine (30 mg/kg) inhibited the substance P-induced production of leukotriene B4, but not prostaglandin E2, in the skin. Azelastine (3-30 mg/kg) suppressed scratching induced by intradermal injection of leukotriene B4. The results suggest that inhibition of the production and action of leukotriene B4, as well as an anti-histamine action, is involved in the anti-pruritic action of azelastine.     

Modulation by pertussis toxin of salbutamol- and arecoline-induced effects in the isolated heart and aorta of the rat

The modulatory effects of pertussis toxin pretreatment on responses mediated via β-adrenoceptors and muscarinic acetylcholine receptors were investigated in isolated rat hearts and aortic rings 4 days after in vivo administration of pertussis toxin. In isolated hearts, pertussis toxin increased heart weight and baseline coronary flow values but did not effect baseline left ventricular pressure values. In unpaced hearts, pertussis toxin inhibited the arecoline-induced cardiac standstill, while in paced hearts, the β₂-adrenoceptor agonist salbutamol produced a dose-dependent vasodilation with similar characteristics in pertussis toxin and control preparations. Pertussis toxin had no effect on myocardial or aortic cyclic nucleotide levels and the myocardial β-adrenoceptor density (B_max) and dissociation constant (K_d). In precontracted aortic rings, pertussis toxin had no effect on the salbutamol or arecoline induced vasorelaxation. In summary, we demonstrated a reduced cholinergic responsiveness in isolated hearts but an intact β₂-adrenoceptor pathway in isolated hearts as well as in isolated aortic rings after pertussis toxin pretreatment. In aortic rings no change in muscarinic acetylcholine receptor responsiveness occurred.     

Celecoxib simulates respiratory burst through pertussis toxin-sensitive G-protein, a possible signal for beta 2-integrin expression on human neutrophils

The superoxide anion-generating effect of celecoxib (4-[5-(4-methylpheny)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide); SC58633), a selective cyclooxygenase-2 inhibitor, on human neutrophils was evaluated in this study. Celecoxib induced superoxide anion generation in a concentration-dependent manner in human neutrophils. The EC50 value of celecoxib on superoxide anion generation was 15.5+/-2.5 microM. A NADPH oxidase inhibitor, diphenyliodonium (20 microM), and superoxide dismutase (150 U/ml) completely inhibited the free radical generation caused by celecoxib, indicating that the respiratory burst was activated by celecoxib. 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM;10 microM) and staurosporine (200 nM) completely inhibited the superoxide anion release caused by celecoxib, respectively. These data indicated that celecoxib increased superoxide anion release by increasing intracellular calcium and protein kinase C activation. Moreover, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-C)-carbazole (Go-6976; 1 microM) and 3-[1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide, methane sulfate (Ro-31-8220; 0.5 microM), specific inhibitors of conventional protein kinase C isotypes (alpha, beta(I) and beta(II)), significantly inhibited superoxide anion release caused by celecoxib. Rottlerin (5 microM), a protein kinase C delta inhibitor, did not affect the free radical generation caused by celecoxib. Celecoxib caused translocation of protein kinase C alpha, beta(I) and beta(II) from the cytosol to the cellular membrane. 2-[2-amino-3-methoxyphenyl]-4H-1-benzopyran-4-one (PD98059; 20 microM) and wortmannin (100 nM) did not decrease the superoxide anion generation caused by celecoxib, indicating that Mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3 kinase) were not involved in the respiratory burst induced by celecoxib. Pertussis toxin (2 microg/ml), a Gi-protein sensitive inhibitor, significantly inhibited superoxide anion release. Moreover, pertussis toxin significantly inhibited intracellular calcium mobilization and protein kinase C alpha, beta(I) and beta(II) translocation from the cytosol to the membrane. Celecoxib increased beta(2)-integrin expression on human neutrophils and this effect was inhibited by BAPTA/AM (10 microM), superoxide dismutase (150 U/ml), genistein (25 microM) and PD98059 (20 microM). This information indicated that intracellular calcium, superoxide anion, tyrosine kinase and MAP kinase are involved in beta(2)-integrin expression. Furthermore, BAPTA/AM, superoxide dismutase and genistein inhibited celecoxib-increased MAP kinase activity, indicating that MAP kinase is a downstream signal for beta(2)-integrin expression. In conclusion, celecoxib stimulates superoxide anion release from human neutrophils by activating pertussis toxin sensitive G-protein. An increase in intracellular calcium and protein kinase C alpha, beta(I) and beta(II) is involved in this process. Celecoxib also regulates beta(2)-integrin expression through superoxide anion release, tyrosine kinase and p42/p44 MAP kinase on human neutrophils.     

肿瘤免疫治疗新靶标PGE2受体EP4的研究进展

肿瘤免疫治疗已成为人们对抗癌症的重要手段,但响应率低仍是目前亟需解决的关键问题。大量研究表明,逆转肿瘤免疫抑制是阻断肿瘤免疫逃逸、增强和扩大免疫疗法疗效的重要策略。前列腺素E₂(PGE₂)是肿瘤微环境中的强效免疫介质,可特异性结合细胞膜上的七次跨膜蛋白EP4受体,诱导肿瘤微环境免疫抑制,驱动肿瘤免疫逃逸。特异性阻断PGE₂/EP4信号通路可有效解除肿瘤微环境免疫抑制,增强抗肿瘤免疫反应,促进肿瘤消退。本文从EP4受体的结构、信号转导、调控机制及其拮抗剂开发现状等方面阐述了EP4受体在肿瘤免疫治疗领域的新进展和新发现,并展望了新的发展方向。     

Characterization of the unique regulatory mechanisms of phorbol ester-induced polymorphonuclear leukocyte spreading in an acidified environment

In vitro studies have shown that acidic conditions impair spreading of polymorphonuclear leukocytes, which is prerequisite for activation of microbicidal functions. However, the mechanisms by which pH affects polymorphonuclear leukocytes functions remain obscure. Moreover, in vitro observations seem to contradict the fact that an acidic microenvironment often prevails at sites of inflammation where polymorphonuclear leukocytes must function for host defense. In the present study, we found three peculiar characteristics of porcine polymorphonuclear leukocyte that had been induced to spread over fibrinogen-coated surfaces by phorbol 12-myristate 13-acetate (PMA) in acidified medium. First, the PMA-induced spreading at acidic pH, but not at neutral/alkaline pH, was dependent on extracellular Ca2+. Second, the spreading at acidic pH was independent of protein kinase C (PKC), whereas that at neutral/alkaline pH was strictly PKC-dependent. Finally, the spreading at acidic pH, but not at neutral/alkaline pH, was suppressed by H2O2 produced by activated NADPH oxidase or added exogenously. As a result, polymorphonuclear leukocyte spreading at acidic pH peaked at 30 min after PMA stimulation, and declined thereafter because of negative regulation triggered by accumulated H2O2, whereas that at neutral/alkaline pH was stable for at least 90 min. The NADPH oxidase inhibitor diphenyleneiodonium or the H2O2-degradation enzyme catalase consistently stabilized the spreading at acidic pH. We conclude that PMA-stimulated polymorphonuclear leukocytes spread in an acidic environment through a mechanism different from that under neutral/alkaline conditions. This H2O2-mediated negative regulation system in an acidic environment may be crucial for avoiding tissue-damaging inflammatory actions of accumulated polymorphonuclear leukocytes in vivo.