[Effect of CKIP-1 on hepatocyte apoptosis in nonalcoholic fatty liver disease].

Objective: To explore the effect and underlying mechanism of casein kinase 2 interacting protein-1 (CKIP-1) on hepatocyte apoptosis in nonalcoholic fatty liver disease (NAFLD). Methods: Experimental study. An NAFLD cell model was established by inducing human hepatoma cell line, HepG2 cells, with oleic acid (OA). Flag-CKIP-1 expression vector and shRNA-CKIP-1 were transfected into HepG2 cells. Flow cytometry was used to detect the effect of CKIP-1 on the activity and apoptosis of NAFLD hepatocytes. The levels of apoptosis-related proteins were detected by Western blot. CKIP-1 knockout mice in C57BL/6 back-ground were fed with either standard or high-fat diet for 8 weeks. Apoptosis-related signal proteins in NAFLD hepatocytes were detected by immunohistochemistry. Results: After CKIP-1 was transfected into HepG2 cells, the degree of OA induced cell liposis was significantly reduced (P<0.05). Annexin V-FITC/PI flow cytometry showed that CKIP-1 reduced the apoptosis of steatotic hepatocytes. Overexpression of CKIP-1 could significantly inhibit the expression of caspase-3 and caspase-9 and increase the expression of Bcl-2/Bax (P<0.05). Knockdown of CKIP-1 could increase the expression of caspase-3 and caspase-9 (P<0.05). CKIP-1 knockout could further increase the expression of caspase-3 and caspase-9 in NAFLD mice (P<0.01,P<0.05), and further decrease the expression of Bcl-2/Bax (P<0.05). Conclusion: CKIP-1 inhibited the apoptosis of steatotic hepatocytes by up-regulating the expression of apoptosis inhibitor gene, Bcl-2/Bax, and affecting the proteases, caspase-3 and caspase-9.

[The value of Clermont score in detection intestinal mucosal ulcer of Crohn's disease].

Objective: To explore the value of Clermont score in the detection of intestinal mucosal ulcer in Crohn's disease (CD). Methods: From May 2015 to August 2017, 45 patients (28 males and 17 females; median age was 25 years) were confirmed to have ileocolic CD by endoscopic and pathological examinations at Nanjing General Hospital. All patients underwent MRE and DWI examinations. Based on the appearance of intestinal mucosa endoscopically, intestine segments from 45 patients were divided into three groups, namely, no ulceration group (NU), superficial ulceration group (SU), and deep ulceration group (DU). Several factors contribute to Clermont score calculation. These included the measurement of intestinal wall thickness using MRE, evaluation of intestinal wall edema and ulceration by MRE, DWI performance and ADC value of each segment. One-way ANOVA was utilized to compare the Clermont scores and ADC values of the intestine segments among the three groups. The efficacy of the Clermont scores and ADC values in evaluating intestinal mucosal ulcer in CD was verified using ROC analysis. Results: A total of 137 intestine segments were included in the study with 30 cases in NU, 37 cases in SU, and 70 cases in DU.DU had the highest Clermont score (22.5±4.5),following were SU(15.8±3.5) and NU(10.2±1.3)(F=179.935,P<0.01).The ADC values of DU ((1.34±0.17)×10(-3)mm(2)/s) was lower than NU ((2.07±0.52)×10(-3)mm(2)/s) and SU ((1.52±0.23)×10(-3) mm(2)/s) (F=83.822,P<0.01).The AUCs of using Clermont score and ADC value in differentiating deep ulcerations were 0.887 and 0.733, respectively. Conclusions: Either Clermont score or ADC value can be used to evaluate mucosal ulcer in CD. Clermont score demonstrates a better efficacy than ADC value in detecting deep ulcerations.

Analysis the effect of hyperbaric oxygen preconditioning on neuronal apoptosis, Ca2+ concentration and caspases expression after spinal cord injury in rats.

OBJECTIVE: To investigate the effects of hyperbaric oxygen preconditioning (HBO-PC) on neuronal apoptosis, Ca2+ concentration, and Caspases expression after spinal cord injury (SCI) in rats. MATERIALS AND METHODS: A total of 36 rats were randomly divided into control group (CON group), hyperbaric oxygen preconditioning group (HBO-PC group) and spinal cord injury group (SCI group), with 12 rats in each group. Rats in group HBO-PC were given HBO-PC intervention before modeling. SCI model was established by modified Allen method in group HBO-PC and group SCI. Basso-Beattie-Bresnahan (BBB) locomotor rating scale and motor evoked potential (MEP) examination were used to assess the neurological function. The expression of apoptosis gene caspase (3, 7, 8, 12) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The concentration of Ca2+ in spinal cord tissue of each group was detected. RESULTS: CON group, HBO-PC group, and SCI group were gradually diminishing in BBB score and potential value and amplitude of MEP, respectively. The differences between groups were statistically significant (p<0.05). The expressions of Caspase-3 and 7, 8 and 12 mRNA in SCI group were significantly higher than those in CON group and HBO-PC group, respectively (p<0.05). There was no significant difference between CON group and HBO-PC group (p>0.05). The concentrations of Ca2+ in the CON group, HBO-PC group and SCI group were gradually increased; differences between groups were statistically significant (p<0.05). CONCLUSIONS: HBO-PC can reduce the loss of motor function of SCI rats, which may inhibit the activation of endoplasmic reticulum pathway of neural apoptosis, and reduce the calcium overload through inhibiting the expressions of pro-apoptotic proteins (Caspase-3/7/8/12), thus reducing the cell apoptosis and protecting neurons.

Comparison of molar intrusion efficiency and bone density by CT in patients with different vertical facial morphology.

The purpose of this study is to examine the relationship between molar intrusion efficiency and bone density in patients with different vertical facial morphology. Thirty-six female patients, with overerupted upper first molars, were divided into two groups according to mandiblular plane angle (FH-MP): hyperdivergent, FH-MP>30° (G1), hypodivergent, FH-MP<22° (G2). Mini-screw implants with elastic chains were used to intrude upper first molars. Spiral CT was used to measure the intrusion degree of upper first molar and bone density, and molar intrusion efficiency was calculated as amount/duration (mm month(-1) ). In addition, each tooth was divided into three portions (cervical, furcation and apical) to measure the bone density. It was found in this study that treatment duration was 3·13 and 4·71 months in G1 and G2 and that the intrusion efficiency was 1·57 and 0·81 in G1 and G2 with significant difference (P < 0·05). There were significant differences in cervical, furcation and apical bone density between two groups (P < 0·05). The bone density was significantly reduced after molar intrusion. In addition, the bone density change was greater in G1 than in G2 (P < 0·05). It was concluded that molars were more easily to be intruded in hyperdivergent than in hypodivergent patients. The difference of bone density and bone density changes during intrusion may account for the variation of molar intrusion efficiency.

Neither single-marker nor haplotype analyses support an association between the dopamine transporter gene and heroin dependence in Han Chinese.

Much evidence suggests that dysfunction of dopamine transporter-mediated dopamine transmission may be involved in the pathophysiology of substance abuse and dependence. The aim of this study was to examine whether the dopamine transporter gene (DAT1; SLC6A3) is associated with the development of heroin dependence (HD) and whether DAT1 influences personality traits in patients with HD. Polymorphisms of DAT1 were analyzed in a case-control study of 1046 Han Chinese (615 patients and 431 controls). All participants were screened using a Chinese version of the modified Schedule of Affective Disorder and Schizophrenia-Lifetime and all patients met the criteria for HD. Furthermore, a Chinese version of the Tridimensional Personality Questionnaire (TPQ) was used to assess personality traits in the patient group and examine the association between their personality traits and DAT1 polymorphisms. Of the patient group, 271 completed the TPQ. No statistically significant differences in allele or genotype frequencies of all investigated variants between HD patients and controls were observed. In haplotype analyses, four haplotype blocks of DAT1 were not associated with the development of HD. These DAT1 polymorphisms did not influence novelty seeking and harm avoidance scores in HD patients. This study suggests that the DAT1 gene may not contribute to the risk of HD and specific personality traits in HD among the Han Chinese population.

Human gastric cells resistant to (-)-epigallocatechin gallate show cross-resistance to several environmental pollutants.

After a long-term culture in (-)-epigallocatechin gallate (EGCG, 20 microM), a major constituent of green tea, human gastric AGS cells developed 2.2-fold resistance to EGCG. The resistant AGS (AGS-R) cells were cross-resistant to several N-methylcarbamate insecticides, which are among the major control agents for pest insects in Taiwan. The AGS-R cells also showed protective effects against both the cytotoxicity and DNA damage induced by one of the mutagenic derivatives of N-methylcarbamate insecticide, N-nitroso methomyl, which is known to target the mammalian gastric tract. Therefore, acquisition of resistance by AGS cells through chronic exposure to EGCG implies that the tea-drinking habit of the Taiwanese is probably beneficial for the health of the gastric tract. In addition, AGS-R cells were cross-resistant to sodium arsenite and hydrogen peroxide, indicating that tolerance to oxidative stress might play a role in the development of resistance described in this investigation.

Decoy receptor 3 (DcR3) induces osteoclast formation from monocyte/macrophage lineage precursor cells.

Recent evidence indicates that the decoy receptor 3 (DcR3) of the TNF receptor superfamily, which initially though prevents cytokine responses of FasL, LIGHT and TL1A by binding and neutralization, can modulate monocyte function through reverse signaling. We show in this work that DcR3 can induce osteoclast formation from human monocytes, murine RAW264.7 macrophages, and bone marrow cells. DcR3-differentiated cells exhibit characteristics unique for osteoclasts, including polynuclear giant morphology, bone resorption, TRAP, CD51/61, and MMP-9 expression. Consistent with the abrogation of osteoclastogenic effect of DcR3 by TNFR-Fc, DcR3 treatment can induce osteoclastogenic cytokine TNF-alpha release through ERK and p38 MAPK signaling pathways. We conclude that DcR3 via coupling reverse signaling of ERK and p38 MAPK and stimulating TNF-alpha synthesis is a critical regulator of osteoclast formation. This action of DcR3 might play an important role in significant osteoclastic activity in osteolytic bone metastases.

Anti-SSB/La is one of the antineutrophil autoantibodies responsible for neutropenia and functional impairment of polymorphonuclear neutrophils in patients with systemic lupus erythematosus.

Decreased number and impaired functions of polymorphonuclear neutrophils (PMN) due to the presence of anti-PMN autoantibodies in the serum render patients with systemic lupus erythematosus (SLE) susceptible to bacterial infections. However, the cognate antigens and pathological mechanisms of anti-PMN autoantibodies in SLE are rarely reported in the literature. In this study, we found approximately 20% of SLE sera contained anti-PMN autoantibodies detected by human PMN-coated cellular ELISA. A membrane protein with molecular weight of 50 kDa was identified as the cognate antigen of anti-PMN in Western blot after membrane-biotinylation and streptavidin column elution. The 50 kDa molecule was proved to be SSB/La after immunoscreening, molecular cloning and nucleotide sequencing of the gene from the human leucocyte cDNA library. Human anti-SSB/La autoantibodies purified from active SLE sera passing through the recombinant SSB/La conjugated Sepharose 4B affinity column could bind and penetrate into normal human PMN. Functional analysis revealed that the anti-SSB/La autoantibodies exerted a number of potent effects on human PMN, including suppressed phagocytosis, accelerated apoptosis and enhanced IL-8 production. These in vitro results suggest that anti-SSB/La is one of the anti-PMN autoantibodies capable of penetrating into PMN and responsible for neutropenia and functional impairment of PMN in patients with SLE.

Coagulation of humic-kaolin-PACI aggregates.

Water treatment residual flocs are fractal-like aggregates made of many initial aggregates. We investigated in this study the coagulation dynamics for the humic-mineral-polyaluminium chloride (PACI) aggregates using small-angle light scattering techniques and the free-settling test. In contrast to reports in the literature, the presence of humic acid did not lead to a loose floc. Not only the time evolution of the coagulation dynamics, but also the final floc characteristics are only mildly affected by the humic acid. However, the strength of the formed floc does decline with humic acid, which leads to a turbid supernatant with high level of organics.

Observations of blanket characteristics in full-scale floc blanket clarifiers.

We monitored the changes in concentrations, zeta potentials, sizes and capillary suction times of the solids flocs in the clarified water from eight floc blanket clarifiers of PingTsan Water Works of Taiwan Water Supply Company with low (< 10 NTU) and high (> 100 NTU) turbidity raw water. For the former, one-stage coagulation-sedimentation treatment was adopted which yielded a rather unstable blanket. Complete washout was noticeable when the PACl dosage was insufficient. On the treatment of high-turbidity raw water, on the other hand, the Works adopted the combined treatment process, that is, the raw water was first coagulated and settled in a pre-sedimentation tank, afterwards, its effluent was coagulated again and clarified in the clarifiers. The resulting flocs could form a networked blanket that was relatively stable to the shock load in raw water turbidity.

Increased chromosome X, Y, and 18 nondisjunction in sperm from infertile patients that were identified as normal by strict morphology: implication for intracytoplasmic sperm injection.

OBJECTIVE: To determine the incidence of nondisjunction for chromosomes X, Y, and 18 using fluorescence in situ hybridization (FISH) on morphologically normal sperm from infertile men who are candidates for ICSI. DESIGN: After standard hematoxylin staining, sperm with normal morphology were identified using Kruger's strict morphology criteria. The location of each normal-appearing sperm was recorded using an electronic microstage locator. Slides were subsequently subjected to FISH for detection of chromosomes X, Y, and 18 (control probe). Nuclei were relocated and analyzed under the fluorescent microscope. SETTING: University-affiliated IVF and intracytoplasmic sperm injection program. PATIENT(S): Men classified as infertile on the basis of abnormal strict morphology (<4% by Kruger's criteria). For controls, normal fertile men (n=6) were also analyzed. INTERVENTION(S): Semen smears were obtained retrospectively from infertile (n=8) and fertile (n=6) men. MAIN OUTCOME MEASURE(S): Ploidy of each cell was determined according to the number of signals detected for each probe. RESULT(S): Approximately 100-150 morphologically normal sperm were identified and located in each case. Subsequent FISH analysis of these normal sperm showed aneuploidy to range from 1.8% to 5.5% in the infertile group as compared with 0 to 2.6% among the control fertile group. Statistically significant differences in the incidence of aneuploidy for the sex chromosomes as well as for all three (X, Y, and 18) chromosomes was observed. CONCLUSION(S): Although 95% to 98% of the sperm were found to be normal for X, Y, and 18, our findings show that infertile couples undergoing ICSI are likely to be at an increased risk for having a genetically abnormal conceptus as compared with the fertile controls. These results demonstrate that normal morphology is not an absolute indicator for the selection of genetically normal sperm. Hence, observed pregnancy failures among ICSI patients may in part be due to the selection of aneuploid sperm.

PKC- and ERK-dependent activation of I kappa B kinase by lipopolysaccharide in macrophages: enhancement by P2Y receptor-mediated CaMK activation.

1. Although accumulating studies have identified I kappa B kinase (IKK) to be essential for controlling NF-kappa B activity in response to several cytokines, the upstream kinases that control IKK activity are still not completely known. We have previously reported that G protein-coupled P2Y(6) receptor activation by UTP potentiates lipopolysaccharide (LPS)-induced I kappa B phosphorylation and degradation, and NF-kappa B activation in J774 macrophages. In this study, we investigated the upstream kinases for IKK activation by UTP and LPS. 2. In murine J774 macrophages, LPS-induced NF-kappa B activation was inhibited by the presence of PDTC, D609, Ro 31-8220, PD 098059 and SB 203580. 3. Accompanying NF-kappa B activation, LPS induced I kappa B degradation and IKK activation were reduced by PDTC, D609, Ro 31-8220 and PD 098059, but not by SB 203580. 4. Although UTP itself slightly induced IKK activation, this response was synergistic with LPS. BAPTA/AM and KN-93 (a calcium/calmodulin-dependent protein kinase (CaMK) inhibitor) attenuated UTP- but not LPS-stimulated IKK activity. Synergistic IKK activation between LPS and thapsigargin was further demonstrated in peritoneal macrophages. 5. LPS and UTP co-stimulation additively increased p65 NF-kappa B phosphorylation. In vitro kinase assays revealed that LPS and UTP induced extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase activation were respectively inhibited by PD098059 and SB 203580. 6. Taken together, we demonstration that Gq protein-coupled P2Y(6) receptor activation can potentiate LPS-stimulated IKK activity. While PKC and ERK participate in IKK activation by LPS and UTP, the phosphatidylinositide-phospholipase C-dependent activation of CaMK plays a major role in UTP potentiation of the LPS response.

Effects of cannabinoids on LPS-stimulated inflammatory mediator release from macrophages: involvement of eicosanoids.

Delta(9)-Tetrahydrocannabinol (Delta(9)-THC) is the major psychoactive component of marijuana and elicits pharmacological actions via cannabinoid receptors. Anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG) are endogenous ligands for cannabinoid receptors, which because of their structural similarities to arachidonic acid (AA), AEA, and 2-AG could serve as substrates for lipoxygenases and cyclooxygenases (COXs) that metabolize polyunsaturated fatty acids to potent bioactive molecules. In this study, we have compared the effects of Delta(9)-THC, AEA, 2-AG, and another cannabinoid agonist, indomethacin morpholinylamide (IMMA), on lipopolysaccharide (LPS)-induced NO, IL-6, and PGE(2) release from J774 macrophages. Delta(9)-THC, IMMA, and AEA diminish LPS-induced NO and IL-6 production in a concentration-dependent manner. 2-AG inhibits the production of IL-6 but slightly increases iNOS-dependent NO production. Delta(9)-THC and IMMA also inhibit LPS-induced PGE(2) production and COX-2 induction, while AEA and 2-AG have no effects. These discrepant results of 2-AG on iNOS and COX-2 induction might be due to its bioactive metabolites, AA and PGE(2), whose incubation cause the potentiation of both iNOS and COX-2 induction. On the contrary, the AEA metabolite, PGE(2)-ethanolamide, influences neither the LPS-induced NO nor IL-6 production. Taken together, direct cannabinoid receptor activation leads to anti-inflammatory action via inhibition of macrophage function. The endogenous cannabinoid, 2-AG, also serves as a substrate for COX-catalyzing PGE(2) production, which in turn modulates the action of CB2.

An improved and easy method for the preparation of 2,2-disubstituted 1-nitroalkenes.

Reactions of ketones 1, nitromethane 2, and catalytic amount of piperidine 3 in the presence of mercaptan 6 generate beta-nitroalkyl sulfides 7-9. At 0 degrees C and by the use of dichloromethane as solvent, beta-nitroalkyl sulfides 7-9 can be oxidized by m-chloroperoxybenzoic acid (m-CPBA) 10 to generate beta-nitroalkyl sulfoxides 11-13 and undergo elimination in carbon tetrachloride solution to produce medium to high yield of 2,2-disubstituted 1-nitroalkenes 5. The irreversibility of the synthetic mechanism not only can overcome the reversibility of the Henry reaction in the synthesis of 2,2-disubstituted 1-nitroalkenes 5 but also can generate the major products "exo-nitro olefins"5c-e when cyclic ketones 1c-e were used. Under similar conditions, medium to high yield of 5-substituted-2-nitromethyl-2-phenylthioadamantane 17 also can be prepared from the reaction of 5-substituted-2-adamantanones 15, nitromethane 2, piperidine 3, thiophenol 6a. The intermediate17 can be oxidized by m-CPBA 10 in dichloromethane solution and then undergo elimination at room temperature or can be dissolved in solvent, coated on silica gel, and then heated at 90-100 degrees C to generate 5-substituted-2-nitromethyleneadamantane 16.

Ceramide inhibits lipopolysaccharide-mediated nitric oxide synthase and cyclooxygenase-2 induction in macrophages: effects on protein kinases and transcription factors.

The goal of this study was to elucidate whether triggering the sphingomyelin pathway modulates LPS-initiated responses. For this purpose we investigated the effects of N-acetylsphingosine (C(2)-ceramide) on LPS-induced production of NO and PGE(2) in murine RAW 264.7 macrophages and explored the signaling pathways involved. We found that within a range of 10-50 microM, C(2)-ceramide inhibited LPS-elicited NO synthase and cyclooxygenase-2 induction accompanied by a reduction in NO and PGE(2) formation. By contrast, a structural analog of C(2)-ceramide that does not elicit functional activity, C(2)-dihydroceramide, did not affect the LPS response. The nuclear translocation and DNA binding study revealed that ceramide can inhibit LPS-induced NF-kappaB and AP-1 activation. The immunocomplex kinase assay indicated that IkappaB kinase activity stimulated by LPS was inhibited by ceramide, which concomitantly reduced the IkappaBalpha degradation caused by LPS within 1-6 h. In concert with the decreased cytosolic p65 protein level, LPS treatment resulted in rapid nuclear accumulation of NF-kappaB subunit p65 and its association with the cAMP-responsive element binding protein. Ceramide coaddition inhibited all the LPS responses. In addition, LPS-induced PKC and p38 mitogen-activated protein kinase activation were overcome by ceramide. In conclusion, we suggest that ceramide inhibition of LPS-mediated induction of inducible NO synthase and cyclooxygenase-2 is due to reduction of the activation of NF-kappaB and AP-1, which might result from ceramide's inhibition of LPS-stimulated IkappaB kinase, p38 mitogen-activated protein kinase, and protein kinase C.

Involvement of protein kinases in the potentiation of lipopolysaccharide-induced inflammatory mediator formation by thapsigargin in peritoneal macrophages.

We have explored the regulatory roles played by Ca2+-dependent signaling on lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-alpha), and interleukin-6 (IL-6) release in mouse peritoneal macrophages. To elevate intracellular Ca2+, we used thapsigargin (TG) and UTP. Although LPS alone cannot stimulate NO synthesis, co-addition with TG, which sustainably increased [Ca2+]i, resulted in NO release. UTP, via acting on P2Y6 receptors, can stimulate phosphoinositide (PI) turnover and transient [Ca2+]i increase, however, it did not possess the NO priming effect. LPS alone triggered the release of PGE2, TNF-alpha, and IL-6; all of which were potentiated by the presence of TG, but not of UTP. The stimulatory effect of LPS plus TG on NO release was inhibited by the presence of Ro 31-8220, Go6976, KN-93, PD 098059, or SB 203580, and abolished by BAPTA/AM and nuclear factor kappaB (NF-kappaB) inhibitor, PDTC. PGE2, TNF-alpha, and IL-6 release by LPS alone were attenuated by Ro 31-8220, Go6976, PD 098059, SB 203580, and PDTC. Using L-NAME, soluble TNF-alpha receptor, IL-6 antibody, NS-398, and indomethacin, we performed experiments to understand the cross-regulation by the four mediators. The results revealed that TNF-alpha up-regulated NO, PGE2, and IL-6 synthesis; PGE2 up-regulated NO, but down-regulated TNF-alpha synthesis; and PGE2 and IL-6 mutually up-regulated reciprocally. Taken together, murine peritoneal macrophages required a sustained [Ca2+]i increase, which proceeds after TG, but not UTP, stimulation, to enhance LPS-mediated release of inflammatory mediators, particularly for NO induction. Activation of PKC-, ERK-, and p38 MAPK-dependent signaling also are essential for LPS action. The positive regulatory interactions among these mediators might amplify the inflammatory response caused by endotoxin.

Dopamine D2 receptor gene (DRD2) is associated with alcoholism with conduct disorder.

This study examined whether there is evidence for an association between alcoholism with conduct disorder and alleles of the TaqI A and TaqI B polymorphisms, both individually and as haplotypes, at the dopamine D2 receptor gene (DRD2). We studied 182 Han Chinese subjects, including 34 alcoholics with conduct disorder, 63 alcoholics without conduct disorder, and 85 nonalcoholics. Alcohol dependence and conduct disorder were defined according to DSM-III-R criteria. Significant associations were observed between TaqI A and TaqI B at the DRD2 locus, tested individually and as haplotypes, and alcoholism with conduct disorder. Our results suggested that DRD2 might be associated with conduct disorder or a predisposition to both conduct disorder and alcoholism. However, this needs to be further investigated by examining the differences among conduct disorder with alcoholism, conduct disorder only, and controls for the TaqI A and B system at DRD2.

Diagnosis and treatment of the azoospermic patient.

With the advent of intracytoplasmic injection, the management of azoospermia has become ever more important. Gametic manipulation to produce biological offspring is not feasible unless sperm from the azoospermic male patient is obtainable. This article provides an overview of the diagnosis and treatment of azoospermia.

Basal cPLA(2) phosphorylation is sufficient for Ca(2+)-induced full activation of cPLA(2) in A549 epithelial cells.

The release of [(3)H] arachidonic acid (AA) and its connection with the triggering of the MAP kinase cascade were studied in the human A549 epithelial cell line upon stimulation with thapsigargin. Thapsigargin can increase AA release along with the increase of intracellular calcium concentration, phosphorylation, and activation of extracellular regulated kinase (ERK) and cytosolic phospholipase A(2) (cPLA(2)). Both ERK and cPLA(2) phosphorylation in response to thapsigargin were inhibited by PD 98059, a specific inhibitor of MAP kinase kinase of the ERK group (MEK), and EGTA. cPLA(2) phosphorylation was not affected by Ro 31-8220 (an inhibitor of all PKC isoforms) or LY 379196 (a PKCbeta selective inhibitor), while both of them indeed attenuated ERK activation. On the other hand, rottlerin (the selective PKCdelta inhibitor), SB 203580 (the selective p38 MAPK inhibitor), and wortmannin (the PI 3-kinase inhibitor) can affect neither cPLA(2) nor ERK phosphorylation. In A549 cells, PKC activator PMA cannot increase either the basal or thapsigargin-induced (3)H-AA release, while it can induce the phosphorylation of ERK and cPLA(2.) The PMA-induced ERK phosphorylation was inhibited by Ro 31-8220, LY 379196, rottlerin, and PD 98059, but unaffected by SB 203580 and wortmannin. Moreover, the phosphorylation by PMA was non-additive with that of thapsigargin. This implies that intracellular Ca(2+) level is the key factor for induction of cPLA(2) activity and thapsigargin-elicited ERK activation itself is substantially sufficient for cPLA(2) activation upon intracellular Ca(2+) increase.