Protein kinase C and vascular smooth muscle contractility: effects of inhibitors and down-regulation

We have compared two different methods of attenuating protein kinase C (PKC) activity in vascular smooth muscle. First, the effects of two purported PKC inhibitors, staurosporine (stauro) and H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], were examined on contractility of isolated, intact canine femoral artery. In arterial rings stauro was equipotent in relaxing contractions induced by phenylephrine (PE), phorbol-12,13-dibutyrate (PDBu) and KCl (IC50, 0.31 +/- 0.19; 0.35 +/- 0.2; and 0.34 +/- 0.16 microM). H-7, in comparison, was markedly less potent than stauro (IC50, 0.67 +/- 0.2, 2.33 +/- 0.24; and 6.5 +/- 5.5 microM for PE, PDBu and KCl, respectively). Pretreatment of tissues with 1 microM stauro suppressed tension development almost completely when PE and PDBu were the contractile agonists, and partially in K(+)-depolarized rings. H-7, in contrast, had no inhibitory effect on agonist-induced contraction. Neither basal nor K(+)-stimulated calcium influx was affected by 10 microM stauro. Second, prolonged exposure of canine carotid arterial rings to PDBu (1-100 nM for 24 hr), a means of depleting PKC from the tissue, caused dose-dependent attenuation of agonist-induced contractions. Preincubation with 100 nM PDBu caused complete inhibition of tension induced by norepinephrine (NE) and serotonin and partial inhibition of PDBu- and KCl-induced contractions. Lowering the concentration of PDBu during preincubation to 30, 10 or 1 nM reduced markedly the inhibitory effects. The inactive phorbolester 4 alpha-phorbol-12,13-didecanoate (4 alpha-PDD) had no effect on agonist-induced contractions. PKC activity was determined in rings contracted isometrically with PDBu or NE after prolonged exposure to vehicle, 4 alpha-PDD or PDBu.(ABSTRACT TRUNCATED AT 250 WORDS)     

多器官功能障碍综合征患者外周血白三烯B4与p38丝裂原活化蛋白激酶的变化

目的　探讨多器官功能障碍综合征( MODS)患者外周血白三烯B4( LTB4)与p3 8丝裂原活化蛋白激酶( p3 8MAPK)含量的变化及与MODS病情的关系。方法　对2 6例MODS患者进行病情评分,采用酶联免疫吸附法( ELISA)检测血中LTB4与p3 8MAPK含量,并与1 2例健康体检者进行对照。比较MODS患者MODS评分与外周血L TB4和p3 8MAPK的相互关系,及MODS组死亡与存活患者的MODS评分、外周血LTB4与p3 8MAPK含量。结果　MODS患者血清LTB4含量为( 92 3 .96±3 0 8.65) ng/L,明显低于健康对照组( 2 453 .3 1±40 0 .93 ) ng/L( P<0 .0 5) ;MODS患者血清中p3 8MAPK含量为( 1 93 .83±1 0 6.3 2 ) ng/L,与健康对照组( 1 2 4.3 6±84.50 ) ng/L比较差异无显著性( P>0 .0 5)。MODS组中死亡患者的血清L TB4含量为( 4 44 .98±2 0 6.3 0 ) ng/L,明显低于存活患者的( 1 3 3 4 .51±53 0 .3 5) ng/L( P<0 .0 5) ;p3 8MAPK含量为( 2 72 .0 8±2 0 7.3 7) ng/L ,明显高于存活患者( 1 1 5.59±57.99) ng/L ( P<0 .0 5)。结论　MODS晚期病理生理变化与一般炎症反应有所不同。外周血L TB4与p3 8MAPK可作为MODS病情严重程度及预后判断指标。     

脑内水通道蛋白4表达调节的研究进展

脑内水通道蛋白4（AQP4）在细胞分化不同阶段表达情况不同，内皮细胞也影响其表达分布；星形胶质细胞及其微环境的渗透压、氨浓度、氧分压、温度以及一些其他因素如激素及肽类、外源性物质铅、补体抑制剂、脂多糖等的存在都会影响AQP4表达。但脑内AQP4表达的调节机制不十分清楚，与之相关的有蛋白的相互作用、蛋白激酶C（PKC）磷酸化途径、丝裂原激活的蛋白激酶信号转导途径、钙信号途径、转录因子活化等，其中研究最多的是PKC通过磷酸化抑制AQP4的活性。     

Activation of protein kinase C sensitizes human VR1 to capsaicin and to moderate decreases in pH at physiological temperatures in Xenopus oocytes

The two-electrode voltage-clamp technique was used to evaluate the effect of protein kinase C (PKC) activation on ion current flow in Xenopus laevis oocytes injected with cRNA coding for the human vanilloid receptor (VR1). In the presence of 30 nM phorbol-12,13-dibutyrate (PDBu), current evoked by an effective concentration (EC(30)) of capsaicin (CAP) was potentiated by 638+/-117% (n=8). PDBu exhibited an EC(50) of about 17+/-3 nM for this effect (n=8). Potentiation was not observed when VR1 expressing oocytes were exposed to both 30 nM PDBu and 1 microM staurosporine. In the presence of 300 nM PDBu, the EC(50) for CAP shifted from 899+/-78 to 139+/-2 1 nM (n=11 and 5, respectively). In the presence of 30 nM PDBu, the maximal current amplitude evoked by application of CAP increased by 86+/-21% (n=10), in a staurosporine sensitive manner. Application of 1 microM PDBu alone elicited a capsazepine sensitive current within 3 min of exposure. This effect was observed in the absence of previous exposure of the oocyte to CAP and was abolished in the presence of 1 microM staurosporine. No current was elicited during a 10 min application of 300 nM PDBu, the longest interval assessed. Prior to 30 nM PDBu exposure, no current was evoked at temperature ramps from room temperature (22-23 degrees C) up to 37 degrees C at pH 6.8, 7.0, or 7.4. Following PDBu treatment, VR1 mediated current was evoked at 26 degrees C at pH 7.0. Likewise, following 30 nM PDBu treatment, current was evoked by application of pH 6.8 alone and a further increase in current amplitude was evoked by heat at 24 degrees C in a staurosporine sensitive manner. These data provide direct evidence that PKC activation can increase VR1 current evoked by candidate physiological activators, pH and heat. This observation provides an empirical foundation for explaining some types of inflammatory pain in terms of PKC activation, small decreases in tissue pH levels, and small increases in skin temperature, all of which can accompany inflammatory conditions.     

Effects of deoxycholate on human colon cancer cells: apoptosis or proliferation

BACKGROUND: Deoxycholic acid has long been attributed as a tumour promoter in the colon. It exerts its growth-related actions in a phorbol ester-like manner, by stimulating protein kinase C. The aim of this study was to investigate the effect of deoxycholic acid on proliferation and apoptosis in the colon, by exposing colon cancer cells to it in increasing concentrations. METHODS: Human colon cancer cells (Caco-2 and HT-29) were treated with deoxycholate or its two structural isomers, 3-beta-12-alpha-dihydroxy-5-beta-cholan-24-oic acid and 3-alpha-12-beta-dihydroxy-5-beta-cholan-24-oic acid. Proliferation was evaluated by cell counting, and apoptosis by estimating percentage cell survival and assessment of nuclear morphology. RESULTS: Within the concentration range of up to 20 microM, deoxycholate stimulated growth of both human colon cancer cell lines. Its growth-promoting effect was abolished after inhibition of protein kinase C. At concentrations above 100 microM, deoxycholate induced apoptosis in both cell lines. Epimers of deoxycholate were significantly less potent in stimulating growth. CONCLUSION: Low-dose deoxycholate stimulates colon cancer cell proliferation while > 100 micromol L(-1) of this secondary bile acid induces apoptosis in colon cancer cells. Deoxycholate might promote the likelihood of malignant transformation by increasing epithelial cell turnover in the colon.     

Phorbol esters and D2-dopamine receptors

4-beta-Phorbol-12,13-dibutyrate (PDBu), a powerful activator of protein kinase C (PKC), enhanced dopamine (DA) release evoked by electrical stimulation (1 Hz, 2 min) from the striatum and the prefrontal cortex of the rabbit. However, acetylcholine (ACh) release from the striatum (1 Hz, 2 min), was only enhanced slightly by PDBu. The increase in DA release induced by PDBu was reduced markedly at higher frequencies of stimulation. Sulpiride (10 microM) alone, a D2 DA-receptor antagonist, or combined with nomifensine (3 microM), a neuronal-uptake inhibitor, did not prevent PDBu-induced facilitation of DA release from prefrontal cortex or striatum. The D2 DA agonists (LY-171555, bromocriptine and apomorphine) inhibited in a concentration-dependent manner the stimulation-evoked overflow of DA and ACh from the striatum, and of DA from the prefrontal cortex. Pretreatment with PDBu antagonized the inhibitory effect of the three agonists on DA and ACh release. A reduction both in Emax and IC50 was observed in PDBu-treated slices. Removal of endogenous DA by pretreatment with reserpine and alpha-methyl-p-tyrosine, failed to prevent PDBu-induced antagonism of apomorphine effects on ACh release, indicating that the antagonism of agonist effects was not due to higher synaptic levels of endogenous DA. The inactive enantiomer of PDBu, 4-alpha-12,13-dibutyrate did not enhance DA release and failed to modify the effects of D2 agonists on DA release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple actions of the leukotriene B4 receptor antagonist SC-41930.

7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl-2H-1-benzopyran-2-carboxylic acid (SC-41930), a leukotriene B4 (LTB4) receptor antagonist with anti-inflammatory activity in animal models of colitis, was evaluated for effects on superoxide, LTB4 and prostaglandin E2 production. SC-41930 inhibited human neutrophil (PMN) superoxide generation maximally stimulated by f-Met-Leu-Phe (IC50 4 microM) and C5a (IC50 approximately 12 microM). Moreover, postreceptor stimulation of superoxide production by NaF (a G protein activator), but not by phorbol myristate acetate, was significantly inhibited by SC-41930, indicating that SC-41930 may act via attenuation of a G protein-mediated signal transduction. SC-41930 also inhibited A23187-stimulated LTB4 production (IC50 5.3 microM) in human PMN as well as LTB4 (IC50 2.1 microM) and prostaglandin E2 (IC50 2.9 microM) production in HL-60 cells. When coinjected intradermally (400 micrograms/site), SC-41930 inhibited A23187-stimulated increases in LTB4 levels in guinea pig skin. SC-41930 inhibited human synovial phospholipase A2 (IC50 72 microM), A23187-stimulated 5-hydroxy-eicosatetranoic acid production in human PMN (IC50 8.5 microM), and rat peritoneal leukotriene A4 hydrolase (IC50 20 microM), but not ram seminal vesical cyclooxygenase. The results suggest that the anti-inflammatory activity of SC-41930 could be attributed to postreceptor inhibition of inflammatory mediator production by PMN and other cells in addition to antagonism of PMN LTB4 receptors.     

3H]leukotriene B4 binding to the guinea-pig spleen membrane preparation: a rich tissue source for a high-affinity leukotriene B4 receptor site

Intact human granulocytes contain a leukotriene (LT) B4 receptor binding site, but the limited supply of these cells could adversely affect further progress of the study of this receptor. To select a tissue homogenate rich for this site, we have characterized the binding of highly specific [3H]LTB4 to guinea-pig spleen membranes and we have determined the ability of LTB4 to compete with [3H]LTB4 for binding sites in the membranes of 10 nonspleen tissues. In the spleen membrane, MgCl2 and CaCl2 enhanced [3H]LTB4 binding, but NaCl and KCl decreased it. Spleen [3H] LTB4 binding was a function of protein concentration and was rapid, reversible, stereoselective and saturable. Kinetic analyses showed that the rate constant for association and dissociation at 25 degrees C was 0.47 nM-1 min-1 and 0.099 min-1, respectively. A Scatchard plot of the data of the equilibrium experiment resulted a straight line with a dissociation constant of 1.8 nM and a density of 274 fmol/mg of protein. Moreover, the LTB4/[3H]LTB4 competition study performed at 4 or 25 degrees C revealed the inhibitory constant (Ki) of LTB4 to be in the nanomolar range. The rank order of agents competing for spleen [3H]LTB4 binding was: LTB4 (Ki = 2.8 nM) greater than 20-hydroxy-LTB4 (23 nM) greater than LTA4 (48 nM) greater than LTA4 methyl ester (0.13 microM) greater than 20-carboxy-LTB4 (greater than 6.6 microM) greater than or equal to arachidonic acid (0.15mM) = FPL-55,712 and FPL-57,231 (0.1-0.2 mM). Competition studies further indicated that felodipine, a 1,4-dihyropyridine Ca++ channel blocker, exhibited micromolar inhibition of spleen [3H]LTB4 binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific inhibition of leukotriene B4-induced neutrophil activation by LY223982.

LY223982, (E)-5-(3-carboxybenzoyl)-2-((6-(4-methoxyphenyl)-5- hexenyl)oxy)benzenepropanoic acid, is a newly discovered potent inhibitor of specific binding of leukotriene B4 (LTB4) to its receptor on human neutrophils. This study demonstrated that the compound is also a specific antagonist of LTB4-induced neutrophil activation under both in vitro and in vivo conditions. LY223982 was found to be 189-fold more effective in displacing [3H]LTB4 than 35S-N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) from their corresponding receptors on human neutrophils. The concentration inhibiting 50% of response (IC50) for displacement of [3H]LTB4 (13.2 nM) was only 6.8-fold higher than the value for nonradioactive LTB4. The compound inhibited the aggregation of guinea pig neutrophils caused by LTB4 more strongly than FMLP or platelet-activating factor. The IC50 for inhibition of LTB4-induced responses (74 nM) was 93- and > 135-fold lower than the IC50 for inhibition of the corresponding FMLP and platelet-activating factor-induced effects. LY223982 was also a potent antagonist of the aggregation of human neutrophils by LTB4 (IC50, 100 nM). Chemotaxis of human neutrophils induced by LTB4 was only modestly inhibited by the compound (IC50 = 6 microM) but it had even less effect on cell movement caused by FMLP. LY223982 inhibited transient leukopenia induced in rabbits with LTB4 (ED50, 3 mg/kg) but not with FMLP. It had no agonist activity in any of the test systems. In summary, the results indicate that LY223982 is a potent specific antagonist of LTB4-induced neutrophil activation.     

支气管哮喘豚鼠肺组织中蛋白酪氨酸激酶JAK1及白细胞介素-4的表达

目的：探讨支气管哮喘豚鼠肺组织中蛋白酪氨酸激酶JAK1及白细胞介素-4（IL-4）的表达。方法：26只健康雄性豚鼠随机分为对照组和哮喘组，卵蛋白致敏法复制哮喘模型，ELISA法检测两组豚鼠肺组织匀浆中IL-4浓度，Western blot检测蛋白酪氨酸激酶JAK1的表达。结果：组织中IL-4浓度及JAK1表达两组间比较差异均有统计学意义（t=7．23、5．46，均P〈0．01），蛋白酪氨酸激酶JAK1的表达与IL4的浓度呈正相关（r=0．77，P〈0．01）。结论：JAK1调控IL-4的表达增加，可能与哮喘的炎症反应有关     

Differential effects of phorbol ester on prefrontal cortex and striatal dopamine terminals: dependence on rate and duration of stimulation

Compared to the nigrostriatal dopamine (DA) neurons, the mesocortical DA neurons projecting to the prefrontal cortex (PFC) are able to sustain higher levels of release when driven at high stimulation frequencies. The effect of a well known activator of protein kinase C (PKC), 4-beta-phorbol-12, 13-dibutyrate (PDBu), were compared on PFC and striatal DA terminals. DA release was monitored from slices of the rabbit PFC and striatum obtained from the same animal. The PKC activator, PDBu (30-1000 nM) enhanced the stimulation-evoked release (SER) of DA from PFC and striatum. The magnitude of the facilitation of DA release produced by PDBu was much greater from the PFC than from the striatum. In the striatum, PDBu produced a bell-shaped dose-response curve, i.e., 0.03 and 1 microM PDBu enhanced SER of DA by 25%, whereas 0.1 and 0.3 microM PDBu enhanced DA release by 60 and 100%, respectively (1 Hz, 120 pulses). In the PFC, 0.03 microM enhanced the SER of DA by 70% and 1 microM by 250% (1 Hz, 120 pulses). In addition, in the PFC, PDBu enhance the basal release of DA (+65% at 1 microM); this effect was not seen in the striatum. The inactive isomer, 4-alpha-phorbol-12, 13-dibutyrate (0.03-1 microM) failed to increase the SER and the basal release of DA from PFC or striatum. The SER of DA was dependent on the rate and duration of stimulation. However, under all conditions of stimulation studied DA release from PFC was always greater than from the striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450.

Human polymorphonuclear leukocytes (PMN) not only synthesize and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation. To characterize the enzyme(s) responsible for omega-oxidation of LTB4, human PMN were disrupted by sonication and subjected to differential centrifugation to yield membrane, granule, and cytosol fractions (identified by biochemical markers). LTB4 omega-hydroxylase activity was concentrated (together with NADPH cytochrome c reductase activity) only in the membrane fraction (specific activity increased 10-fold as compared to whole sonicates, 41% recovery). Negligible activity was detected in granule or cytosol fractions. LTB4 omega-hydroxylase activity in isolated PMN membranes was linear with respect to duration of incubation and protein concentration, was maximal at pH 7.4, had a Km for LTB4 of 0.6 microM, and was dependent on oxygen and on reduced pyridine nucleotides (apparent Km for NADPH = 0.5 microM; apparent Km for NADH = 223 microM). The LTB4 omega-hydroxylase was inhibited significantly by carbon monoxide, ferricytochrome c, SKF-525A, and Triton X-100, but was not affected by alpha-naphthoflavone, azide, cyanide, catalase, and superoxide dismutase. Finally, isolated PMN membranes exhibited a carbon monoxide difference spectrum with a peak at 452 nm. Thus, we have partially purified the LTB4 omega-hydroxylase in human PMN and identified the enzyme as a membrane-associated, NADPH-dependent cytochrome P-450.     

Vasopressin-induced vasoconstriction is dependent on MAPKerk1/2 phosphorylation

To investigate the involvement of the mitogen-activated protein kinase (MAPK) family of extracellular signal-regulated kinase (ERK) 1 and 2 (MAPKerk1/2) in the vasopressin-mediated vasoconstriction in the rat aorta. Vasopressin-induced vasoconstriction was measured in isolated rat thoracic aortae in the presence or absence of MAPKerk1/2 kinase (MKKmek1/2) inhibitors. Thereafter the MAPKerk1/2 phosphorylation in the rat aorta was quantified using Western blot analysis. Vasopressin (1-300 nm) induced a concentration-dependent vasoconstriction, which could be inhibited concentration dependently by the selective MKKmek1/2 inhibitors, PD 98059 (10 and 100 microm) and U 0126 (10 and 100 microm). Western blot analysis revealed a 2.7 +/- 0.6-fold increase in the MAPKerk1/2 phosphorylation induced by vasopressin (300 nm). This phosphorylation could be dose dependently prevented by both PD 98059 (100 microm) and U 0126 (10 and 100 microm). These results indicate that vasoconstriction induced by vasopressin is partly regulated by the MAPKerk1/2 pathway.     

Protein kinase C translocation in rat stomach fundus: effects of serotonin, carbamylcholine and phorbol dibutyrate

In an effort to characterize serotonergic receptor activation in rat stomach fundus, the potential role of protein kinases, more specifically protein kinase C (PKC), in serotonin-induced contraction of rat stomach fundus was examined. Staurosporine, a potent, but nonselective, inhibitor of protein kinases, attenuated basal, membrane-bound PKC activity in rat stomach fundus (IC50 = 10 nM). Although staurosporine (3-100 nM) produced a concentration-dependent inhibition of contractions elicited by serotonin (which does not increase phosphatidylinositol hydrolysis in the fundus), carbamylcholine (an agent stimulating phosphatidylinositol hydrolysis), and phorbol 12,13-dibutyrate (PDBu; a phosphatidylinositol-independent activator of PKC translocation), it was a more potent inhibitor of contractions produced by serotonin and PDBu than by carbamylcholine. Potassium chloride-induced contractions were attenuated minimally by staurosporine. These results raised the possibility that serotonin might exert an effect on protein kinase activity by a phosphatidylinositol-independent mechanism. Focusing on PKC, serotonin's ability to translocate PKC from cytosol to membrane in rat fundus was examined. Concentrations of serotonin (0.1-10 microM) which maximally contracted rat fundus did not translocate PKC. However, PDBu (10 nM-1 microM) and carbamylcholine (0.1-10 microM) significantly increased membrane-bound PKC activity. These results: 1) demonstrate that translocation of PKC occurred in rat stomach fundus in response to some, but not all, contractile agonists; 2) are consistent with the possibility that contraction of rat stomach fundus by carbamylcholine and PDBu may be related to increased membrane-bound PKC activity; and 3) indicate that serotonin-induced contraction, although potently blocked by staurosporine, did not result from PKC translocation in the rat stomach fundus.     

Production of leukotrienes and thromboxane by resident and activated rat alveolar macrophages: a possible role of protein kinase C.

Arachidonic acid metabolism in resident rat alveolar macrophages and in those activated with complete Freund's adjuvant (CFA) was studied. Adult Sprague-Dawley rats were injected with 0.05 ml CFA, and macrophages were harvested 10 days later. Macrophages were labeled overnight with carbon 14-labeled arachidonic acid, washed, and then stimulated with calcium ionophore A23187 (IoA), phorbol myristate acetate (PMA), or zymosan for 30 minutes. Prostaglandins, thromboxane, and leukotrienes were extracted from the medium and analyzed by radioimmunoassay or radio high-pressure liquid chromatography. Cell lipids were analyzed by radio thin-layer chromatography. Medium and cell beta-glucuronidase activity and protein kinase C activity of the membrane fraction were also assayed. We found (1) lower leukotriene B4 (LTB4) production in stimulated resident macrophages when compared with resident macrophages after IoA stimulation--the suppressed LTB4 production was reversed by PMA; (2) unchanged or higher LTB4 production in activated macrophages when compared with resident macrophages after zymosan stimulation; (3) inhibition of zymosan-stimulated LTB4 production by staurosporine, a protein kinase C inhibitor, in both groups; and (4) lower diacylglycerol (DAG) production in activated macrophages when compared with resident macrophages after IoA stimulation, but not after zymosan stimulation. These results suggest that the reduced response of activated macrophages to IoA is due to decreased production of an endogenous protein kinase C activator. This hypothesis was further supported by the observation that protein kinase C activation in response to IoA was lower in activated macrophages than in resident macrophages. In contrast, zymosan stimulation resulted in higher protein kinase C activation in activated macrophages when compared with resident cells. We hypothesize that protein kinase activation is necessary for leukotriene production and that the preserved ability of zymosan to activate PKC via DAG accounts for the high leukotriene production in zymosan-activated macrophages. We also found that stimulated thromboxane production was higher in activated than resident cells, regardless of the stimulus, and that thromboxane production was not affected by staurosporine. Thus alterations of eicosanoid metabolism in immunologically activated macrophages depend on the stimulus used and the type of eicosanoid examined. Furthermore, leukotriene biosynthesis in rat alveolar macrophages may be regulated by protein kinase C.     

Establishment of a binding assay for protein kinase C isozymes using synthetic C1 peptides and development of new medicinal leads with protein kinase C isozyme and C1 domain selectivity

Conventional and novel protein kinase C (PKC) isozymes contain two cysteine-rich C1 domains (C1A and C1B), both of which are candidate phorbol-12, 13-dibutyrate (PDBu)-binding sites. We synthesized C1 peptides of 50-70 residues corresponding to all PKC isozyme C1 domains using an Fmoc solid-phase strategy. These C1 peptides were successfully folded by zinc treatment, as monitored by electrospray ionization time-of-flight mass spectrometry. We measured the K(d)'s of [3H]PDBu for all PKC C1 peptides. Most of the C1 peptides, except for delta-C1A and theta-C1A, showed strong PDBu binding affinities with K(d)'s in the nanomolar range (0.45-7.4 nM) comparable with the respective whole PKC isozymes. The resultant C1 peptide library can be used to screen for new ligands with PKC isozyme and C1 domain selectivity. Non-tumor-promoting 1-oleoyl-2-acetyl-sn-glycerol and bryostatin 1 showed relatively strong binding to all CIA peptides of novel PKCs (delta, epsilon, and eta). In contrast, the tumor promoters (-)-indolactam-V, ingenol-3-benzoate, and PDBu bound selectively to all C1B peptides of novel PKCs. The preference of tumor promoters for the domain might be related to tumorigenesis since recent investigations proposed the involvement of novel PKCs in tumor promotion in vivo using transgenic or knockout mice. Moreover, we recently have found that a new lactone analogue of benzolactams (6) shows significant selectivity in PKCeta-C1B binding.     

Leukotriene B4 receptors on guinea pig alveolar eosinophils.

The existence of receptors for LTB4 on highly purified guinea pig alveolar eosinophils was investigated. Massive infiltration of eosinophils in alveolar spaces was induced in guinea pigs by i.v. injections of Sephadex beads G50 (16 mg/kg). Alveolar eosinophils (50 x 10(6) cells) were purified to approximately 98% by Percoll continuous density gradient centrifugation. The binding studies indicated that alveolar eosinophils bind LTB4 in a saturable, reversible and specific manner. Scatchard analysis indicated the existence of high-affinity binding sites (Kd1 = 1.00 +/- 0.22 nM; Bmax1 = 966 +/- 266 sites/cell) and low-affinity binding sites (Kd2 = 62.5 +/- 8.9 nM; Bmax2 = 5557 +/- 757 sites/cell). The metabolism of LTB4 by alveolar eosinophils in binding conditions was assessed by RP-HPLC and no significant degradation of [3H]LTB4 was observed. LTB4 dose-dependently stimulated eosinophil migration in both chemokinesis and chemotaxis assays with an EC50 value of 1.30 +/- 0.14 and 18.14 +/- 1.57 nM, respectively. LTB4 caused a dose-dependent increase in the production of superoxide anion with an apparent EC50 value of 50 X 10(-9) M in our experimental conditions. LTB4 also induced a dose-dependent increase in the generation of TxA2 with an EC50 value of 46.2 X 10(-9) M. Taken together, our results demonstrated that guinea pig alveolar eosinophils express two classes of specific receptors for LTB4. The high-affinity binding sites seem associated to chemokinesis and chemotaxis whereas the low-affinity binding sites seem associated to superoxide anion production and generation of TxA2. The existence of LTB4 receptors in eosinophils could explain the presence of these cells in hypersensitivity reactions.     

SB 203580, an inhibitor of p38 mitogen-activated protein kinase, enhances constitutive apoptosis of cytokine-deprived human eosinophils.

The role of p38 mitogen-activated protein (MAP) kinase, and extracellular-regulated protein kinase -1 and -2 in regulating constitutive apoptosis and interleukin (IL)-5-induced survival of human eosinophils have been investigated. Two populations of donors were identified whose eosinophils, in the absence of exogenous cytokines, underwent apoptosis at different rates. Eosinophils were thus arbitrarily classified as either "fast"- or "slow"-dying cells, where greater or less than 15% of the cells were apoptotic at 2 days, respectively. The selective p38 MAP kinase inhibitor, SB 203580, increased constitutive eosinophil apoptosis in both populations (EC(50) approximately 2 microM) as evinced from morphological analysis, flow cytometry, and DNA laddering. The ability of SB 203580 to kill eosinophils was not due to nonspecific toxicity or through the inhibition of prostanoid or leukotriene production. Exposure of eosinophils to IL-5, at a concentration (10 pM) that enhanced survival maximally, abolished SB 203580-induced apoptosis. In contrast PD 098059, which selectively blocks MAP kinase kinase (MEK) 1, did not affect apoptosis of fast- or slow-dying eosinophils, or the enhanced survival of cells effected by IL-5. Collectively, these results suggest that: 1) the basal activity of p38 MAP kinase may regulate the survival of cytokine-deprived eosinophils through inhibition of apoptosis, 2) the enhancement of eosinophil survival effected by IL-5 is mediated by a mechanism(s) divorced from the activation of p38 MAP kinase, and 3) neither spontaneous eosinophil apoptosis nor their enhanced survival by IL-5 involves the activation of MEK-1.     

Hypertonic saline reduces lipopolysaccharide-induced mouse brain edema through inhibiting aquaporin 4 expression

Introduction

Three percent sodium chloride (NaCl) treatment has been shown to reduce brain edema and inhibited brain aquaporin 4 (AQP4) expression in bacterial meningitis induced by Escherichia coli. Lipopolysaccharide (LPS) is the main pathogenic component of E. coli. We aimed to explore the effect of 3% NaCl in mouse brain edema induced by LPS, as well as to elucidate the potential mechanisms of action.

Methods

Three percent NaCl was used to treat cerebral edema induced by LPS in mice in vivo. Brain water content, IL-1β, TNFα, immunoglobulin G (IgG), AQP4 mRNA and protein were measured in brain tissues. IL-1β, 3% NaCl and calphostin C (a specific inhibitor of protein kinase C) were used to treat the primary astrocytes in vitro. AQP4 mRNA and protein were measured in astrocytes. Differences in various groups were determined by one-way analysis of variance.

Results

Three percent NaCl attenuated the increase of brain water content, IL-1β, TNFα, IgG, AQP4 mRNA and protein in brain tissues induced by LPS. Three percent NaCl inhibited the increase of AQP4 mRNA and protein in astrocytes induced by IL-1β in vitro. Calphostin C blocked the decrease of AQP4 mRNA and protein in astrocytes induced by 3% NaCl in vitro.

Conclusions

Osmotherapy with 3% NaCl ameliorated LPS-induced cerebral edema in vivo. In addition to its osmotic force, 3% NaCl exerted anti-edema effects possibly through down-regulating the expression of proinflammatory cytokines (IL-1β and TNFα) and inhibiting the expression of AQP4 induced by proinflammatory cytokines. Three percent NaCl attenuated the expression of AQP4 through activation of protein kinase C in astrocytes.