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PubMed2004

Distinct roles of Smad pathways and p38 pathways in cartilage-specific gene expression in synovial fibroblasts.

The role of TGF-beta/bone morphogenetic protein signaling in the chondrogenic differentiation of human synovial fibroblasts (SFs) was examined with the adenovirus vector-mediated gene transduction system. Expression of constitutively active activin receptor-like kinase 3 (ALK3CA) induced chondrocyte-specific gene expression in SFs cultured in pellets or in SF pellets transplanted into nude mice, in which both the Smad and p38 pathways are essential. To analyze downstream cascades of ALK3 signaling, we utilized adenovirus vectors carrying either Smad1 to stimulate Smad pathways or constitutively active MKK6 (MKK6CA) to activate p38 pathways. Smad1 expression had a synergistic effect on ALK3CA, while activation of p38 MAP kinase pathways alone by transduction of MKK6CA accelerated terminal chondrocytic differentiation, leading to type X collagen expression and enhanced mineralization. Overexpression of Smad1 prevented MKK6CA-induced type X collagen expression and maintained type II collagen expression. In a mouse model of osteoarthritis, activated p38 expression as well as type X collagen staining was detected in osteochondrophytes and marginal synovial cells. These results suggest that SFs can be differentiated into chondrocytes via ALK3 activation and that stimulating Smad pathways and controlling p38 activation at the proper level can be a good therapeutic strategy for maintaining the healthy joint homeostasis and treating degenerative joint disorders.

Activin Receptors↗

PubMed1996

Pituitary adenylate cyclase-activating polypeptide regulates prolactin promoter activity via a protein kinase A-mediated pathway that is independent of the transcriptional pathway employed by thyrotropin-releasing hormone.

The hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), can efficiently increase cAMP levels in pituitary cells and release a number of pituitary hormones, suggesting an important physiological role for this peptide in pituitary function. Exposure of GH3 rat pituitary cells to PACAP results in increases in cellular cAMP levels, PRL promoter activity, and PRL messenger RNA levels. We have employed this system to further characterize PACAP regulation of PRL gene expression. RT-PCR analysis showed that GH3 cells express transcripts for two PACAP receptors, PACAP-R-hop1 and VIP2. As the former can couple PACAP to increases in both cAMP and inositol phosphates, we investigated whether either pathway mediates PACAP action on the PRL promoter. Our observations that TRH, but not PACAP, increases the intracellular Ca2+ concentration in GH3 cell cultures and that the optimal concentrations of TRH and PACAP have additive effects on transient expression of a PRL-CAT construct imply that the inositol trisphosphate-Ca2+ pathway is not significantly involved in PACAP action on the PRL promoter. Four kinase inhibitors exhibited similar profiles of inhibition of the activity on PRL-chloramphenicol acetyltransferase (PRL-CAT) of either the adenylyl cyclase activator forskolin (FSK) or PACAP, suggesting a transcriptional role for protein kinase A (PKA). The observations that coexpression of the dominant PKA inhibitor RAB completely blocked either FSK or PACAP action on PRL-CAT and that these actions of FSK and PACAP were completely nonadditive imply that the cAMP-PKA pathway plays a dominant role in PACAP regulation of PRL gene expression. Coexpression of low levels of KCREB, a cAMP response element (CRE)-binding protein (CREB) dominant inhibitor, partially blocked regulation of PRL-CAT activity by PACAP, but not TRH, implying that PACAP action is mediated at least in part by a CREB family member that can dimerize with CREB. The PRL promoter contains an asymmetric sequence at positions -99/-92 resembling a canonical CRE and termed here the CRE-like element (CLE). Mutation of either the left or right 4 bp of the CLE yielded a strong decrease in the response to either FSK or PACAP, but not to TRH. These data imply that PACAP and TRH employ independent pathways to regulate the PRL promoter, and that PACAP action is exerted virtually entirely via a cAMP/PKA-mediated pathway that is strongly dependent upon an intact CLE sequence and at least partially dependent upon the activity of a CREB-related protein.

Animals↗

PubMed1999

Mitogenic signaling by ATP/P2Y purinergic receptors in astrocytes: involvement of a calcium-independent protein kinase C, extracellular signal-regulated protein kinase pathway distinct from the phosphatidylinositol-specific phospholipase C/calcium pathway.

Activation of ATP/P2Y purinergic receptors stimulates proliferation of astrocytes, but the mitogenic signaling pathway linked to these G-protein-coupled receptors is unknown. We have investigated the role of extracellular signal-regulated protein kinase (ERK) in P2Y receptor-stimulated mitogenic signaling as well as the pathway that couples P2Y receptors to ERK. Downregulation of protein kinase C (PKC) in primary cultures of rat cerebral cortical astrocytes greatly reduced the ability of extracellular ATP to stimulate ERK. Because occupancy of P2Y receptors also leads to inositol phosphate formation, calcium mobilization, and PKC activation, we explored the possibility that signaling from P2Y receptors to ERK is mediated by a phosphatidylinositol-specific phospholipase C (PI-PLC)/calcium pathway. However, neither inhibition of PI-PLC nor chelation of calcium significantly reduced ATP-stimulated ERK activity. Moreover, a preferential inhibitor of calcium-dependent PKC isoforms, Gö 6976, was significantly less effective in blocking ATP-stimulated ERK activity than GF102903X, an inhibitor of both calcium-dependent and -independent PKC isoforms. Furthermore, ATP stimulated a rapid translocation of PKCdelta, a calcium-independent PKC isoform, but not PKCgamma, a calcium-dependent PKC isoform. ATP also stimulated a rapid increase in choline, and inhibition of phosphatidylcholine hydrolysis blocked ATP-evoked ERK activation. These results indicate that P2Y receptors in astrocytes are coupled independently to PI-PLC/calcium and ERK pathways and suggest that signaling from P2Y receptors to ERK involves a calcium-independent PKC isoform and hydrolysis of phosphatidylcholine by phospholipase D. In addition, we found that inhibition of ERK activation blocked extracellular ATP-stimulated DNA synthesis, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y receptors.

Animals↗

PubMed1999

The phosphatidylinositol 3'-kinase pathway is a dominant growth factor-activated cell survival pathway in LNCaP human prostate carcinoma cells.

Intracellular signaling pathways that mediate survival of prostate carcinoma (PCa) cells are poorly understood. We examined the potential role of the phosphatidylinositol 3' kinase (PI3K) pathway as a mediator of cell survival in LNCaP human PCa cells, which express a variety of properties characteristic of human prostate cancer. LNCaP cell cultures rapidly became apoptotic when treated with the specific PI3K inhibitors, wortmannin and LY294002. In contrast, apoptosis was not induced when the cells were treated with: (a) rapamycin, an inhibitor of the ribosomal S6 kinase pp70S6K, which acts downstream of PI3K; (b) PD98059, a specific inhibitor of the extracellular signal-regulated kinase/mitogen-activated protein kinase (Erk/MAPK) kinase (MEK); or (c) the antiandrogen, Casodex; or when the cells were cultured under androgen-depleted conditions. Apoptosis induced by PI3K inhibition was attenuated by: (a) dihydrotestosterone; or (b) the ErbB1 activating ligands [epidermal growth factor (EGF), transforming growth factor alpha, or heparin-binding EGF-like growth factor]. In response to ErbB1 activation by ligand, the p85 regulatory subunit of PI3K associated specifically with ErbB3 but not detectably with ErbB1. The anti-apoptotic effect of ErbB1 activation was significantly reduced when cells were treated simultaneously with wortmannin and PD98059. These data indicate that survival signals can be evoked in LNCaP cells by several distinct pathways and can be triggered by nuclear and cell-surface receptors. Constitutive signaling through the PI3K pathway is required to prevent cell death in LNCaP, whereas activation of the Erk/MAPK and androgen response pathways is not obligatory for cell survival. These results also show that survival signals, as distinguished from mitogenic signals, can be evoked in PCa cells by ErbB1 ligands known to be synthesized within the human prostate.

Androgens↗

PubMed1990

Phorbol diesters stimulate the accumulation of phosphatidate, phosphatidylethanol, and diacylglycerol in three cell types. Evidence for the indirect formation of phosphatidylcholine-derived diacylglycerol by a phospholipase D pathway and direct formation of diacylglycerol by a phospholipase C pathway.

The effect of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), on phospholipid degradation was investigated in three cell lines of dissimilar origin, Madin-Darby canine kidney cells (MDCK), rat aorta smooth muscle cells (RASM), and bovine pulmonary artery endothelial cells (BPAE). In cells prelabeled with [3H]myristic acid, which is predominantly incorporated into phosphatidylcholine (PC), TPA treatment (80 nM) in the absence or presence of ethanol (2%) in the culture medium resulted in either the rapid generation of [3H]phosphatidate (PA) or the sustained accumulation of [3H]phosphatidylethanol (PEt), respectively. Increases in [3H]PA and [3H]PEt were paralleled by quantitative decreases in cellular [3H]PC radioactivity. TPA-induced [3H]PEt formation occurred in a similar fashion, irrespective of the presence of Ca2+ in the culture medium. The experiments demonstrate that TPA elicits PC degradation by phospholipase D (PLD) in cells of diverse origin. Data from further experiments revealed a complex relationship between TPA-induced [3H]PA and [3H]diacylglycerol (DG) generation in the three cell lines that was suggestive of dual pathways for the generation of [3H]DG. Experiments to discern the pathways for TPA-induced, PC-derived DG were conducted by comparing the variation of [3H]PA and [3H]DG formation in the absence and in the presence of increasing ethanol concentrations in the culture medium. With increasing amounts of ethanol, the formation of [3H]PA decreased at the expense of [3H]PEt formation, and depending upon the pathway operable, the amount of [3H]DG formed was either decreased, indicative of indirect formation of DG via PA phosphohydrolase, or not modified, indicative of DG formation by a direct phospholipase C (PLC) pathway. Increasing the concentration of ethanol in the medium blocked TPA-induced [3H]DG generation in MDCK cells in a concentration-dependent manner, while the formation of [3H]PEt increased at the expense of [3H]PA formation. In BPAE cells the presence of ethanol likewise reduced TPA-elicited formation of DG. Conversely, in two smooth muscle cell lines, RASM and A-10, ethanol was without influence on TPA-induced formation of [3H]DG, although [3H]PEt was generated at the expense of [3H]PA. In RASM cells prelabeled with [3H]choline, TPA induced the release to the medium of [3H]choline and [3H]phosphocholine, indicative of both PLD and PLC activation. These results show that TPA elicits DG formation from PC in MDCK cells predominantly by an indirect pathway, whereas in arterial smooth muscle cells DG is formed in part by the direct action of PLC.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

[Multiple pathway tachycardias: electrophysiological properties and fulguration of accessory pathways in 2 cases].

We report two patients (male, 35 and female, 28 years old) with recurrent and refractory tachycardias referred for an electrophysiological study. EKG showed left lateral accessory pathway in both, two tachycardias with identical morphology but with different frequency and activation sequence were detected. One was orthodromic with anterograde conduction by the His-Purkinje budle and retrograde by the accessory pathway and the other with slow nodal anterograde conduction. The accessory pathway was first fulgurated with radiofrequency at the third attempt. Posteriorly, a nodal reentry tachycardia was induced in the first patient and in the second, a double nodal pathway was confirmed. In both, the slow pathway was modified using Jackman's technique, after three and four attempts respectively. After these applications, the induction of tachycardia with programmed atrial or ventricular stimulation, with or without isoproterenol, was not possible. It is concluded that radiofrequency fulguration is a reasonable therapeutic alternative for patients with multiple circuit tachycardias.

Adult↗

PubMed1997

The IRS-pathway operates distinctively from the Stat-pathway in hematopoietic cells and transduces common and distinct signals during engagement of the insulin or interferon-alpha receptors.

Binding of interferon-alpha (IFN-alpha) to its receptor on hematopoietic cells activates the signal transducers and activators of transcription (Stat)- and insulin receptor substrate (IRS)-pathways, and regulates expression of antiproliferative and antiviral activities. However, it remains unknown whether these two pathways cooperate in the generation of IFN-alpha responses or function independently, and whether IRS-proteins transduce distinct downstream signals in response to IFNs or insulin/insulin-like growth factor (IGF)-1-mediated activation. Our data show that in response to IFN-alpha treatment, IRS-1 functions selectively as a docking protein for the SH2 domains of the p85 subunit of the PI 3'-kinase, but not the SH2 domain of Grb-2 which is engaged during insulin/IGF-1 signaling. In studies with THP-1 human myelomonocytic cells and 32D mouse myeloid cells, which are IRS-defective, we found that the IFN-alpha-regulated activation of Stat-1, Stat-2, and Stat-3 does not require the function of the IRS-system. Furthermore, THP-1 cells are responsive to the protective effect of IFN-alpha against vesicular stomatitis virus. Both 32D and THP-1 cells were resistant to the growth inhibitory effect of IFN-alpha, but this effect was not reversible by expression of IRS-1 or IRS-2 alone in 32D cells. Taken altogether these data show that: (1) The IRS-system transduces common and distinct signals in response to IFN-alpha or insulin/lGF-1 stimulation of hematopoietic cells. (2) The IRS-pathway operates separately from the Stat-pathway, and its function is not essential for the generation of the antiviral effect of IFN-alpha. (3) Neither the IRS- nor the Stat-pathways alone are sufficient to mediate the antiproliferative effects of IFN-alpha in hematopoietic cells, and additional signaling elements are required.

Animals↗

PubMed2000

Binding of immune complexes to erythrocyte CR1 (CD35): difference in requirement of classical pathway components and indication of alternative pathway-mediated binding in C2-deficiency.

Deficiency of complement components within the classical pathway is associated with increased risk for immune complex disease. However, C2-deficient individuals often have a mild disease and about 50% are healthy. To study the importance of the different components for immune complex clearance, bovine serum albumin (BSA)/anti-BSA complexes were opsonized in human serum and the binding to erythrocyte complement receptor type 1 (CR1, CD35) was measured in vitro. In C2-depleted serum the complexes were opsonized and bound to CR1 but the reaction needed a longer opsonization time than in normal human serum (NHS). In contrast, serum reagent lacking C1q, C4 or C3 did not promote binding in this assay system. We also demonstrated that elevated levels of factor B could restore binding of complexes to erythrocytes in C2-depleted serum via alternative pathway activation. These results indicate that in spite of lack of a complete classical pathway, C2-deficient individuals could retain some immune complex opsonizing activity via the alternative pathway. This finding could contribute to the understanding of differences in association between complement deficiency and immune-complex disease.

Antigen-Antibody Complex↗

PubMed2001

Simple and robust method for estimation of the split between the oxidative pentose phosphate pathway and the Embden-Meyerhof-Parnas pathway in microorganisms.

The flux through the oxidative pentose phosphate (PP) pathway was estimated in Bacillus clausii, Saccharomyces cerevisiae, and Penicillium chrysogenum growing in chemostats with [1-(13)C]glucose as the limiting substrate. The flux calculations were based on a simple algebraic expression that is valid irrespective of isotope rearrangements arising from reversibilities of the reactions in the PP pathway and the upper part of the Embden-Meyerhof-Parnas pathway. The algebraically calculated fluxes were validated by comparing the results with estimates obtained using a numerical method that includes the entire central carbon metabolism. Setting the glucose uptake rate to 100, the algebraic expression yielded estimates of the PP pathway flux in B. clausii, S. cerevisiae, and P. chrysogenum of 20, 42, and 75, respectively. These results are in accordance with the results from the numerical method. The information on the labeling patterns of glucose and the proteinogenic amino acids were obtained using gas chromatography / mass spectrometry, which is a very sensitive technique, and therefore only a small amount of biomass is needed for the analysis. Furthermore, the method developed in this study is fast and readily accessible, as the calculations are based on a simple algebraic expression.

Bacillus↗

PubMed1978

Initiation of the alternative pathway of complement: recognition of activators by bound C3b and assembly of the entire pathway from six isolated proteins.

An intact alternative pathway of complement activation was assembled from six isolated proteins present at their respective physiological concentrations (C3, 1200 microgram/ml: factor B, 200 microgram/ml; factor D, 2 microgram/ml; beta1H, 560 microgram/ml; C3b inactivator, 34 microgram/ml; and native properdin, 20 microgram/ml). Initiation of the pathway required the presence of five of these proteins not including properdin. The initial C3 convertase of the system was shown to be a fluid-phase rather than a surface-bound enzyme. The ability of the pathway to discriminate between activator and nonactivator was found to reside in the bound C3b molecule. When bound to the surface of an activator through its labile binding site, C3b interacts with surface structures of the activator through another site on the molecule. This interaction results in diminished beta1H binding to C3b and thereby allows the bound C3b molecule to escape control and participate in C3 convertase formation. Thus, initiation of the alternative pathway is a two-step process, the first being non-specific and the second being discriminatory.

Binding Sites↗

PubMed2000

Induction of a futile Embden-Meyerhof-Parnas pathway in Deinococcus radiodurans by Mn: possible role of the pentose phosphate pathway in cell survival.

Statistical models were used to predict the effects of tryptone, glucose, yeast extract (TGY) and Mn on biomass formation of the highly radioresistant bacterium Deinococcus radiodurans. Results suggested that glucose had marginal effect on biomass buildup, but Mn was a significant factor for biomass formation. Mn also facilitated glucose interactions with other nutrient components. These predictions were verified by in vivo and in vitro experiments. TGY-grown cells metabolized glucose solely by the pentose phosphate pathway (PPP). Although only a fraction of glucose from the medium was transported into the cells, glucose was incorporated into the DNA efficiently after cells were exposed to UV light. The presence of glucose also enhanced the radioresistance of the culture. Mn could induce an Embden-Meyerhof-Parnas (EMP) pathway in D. radiodurans. The EMP pathway and the PPP of the Mn-treated cells oxidized glucose simultaneously at a 6:1 ratio. Although glucose was hydrolyzed rapidly by the Mn-treated cells, most glucose was released as CO(2). Mn-treated cultures retained less glucose per cell than cells grown without Mn, and still less glucose was incorporated into the DNA after cells were exposed to UV light. Mn-treated cells were also more sensitive to UV light. Results suggested that metabolites of glucose generated from the PPP enhanced the survival of D. radiodurans. Induction of the EMP pathway by Mn may deplete metabolites for DNA repair and may induce oxidative stress for the cell, leading to reduction of radioresistance.

Biomass↗

PubMed2002

Sympathoinhibitory pathway from caudal midline medulla to RVLM is independent of baroreceptor reflex pathway.

Glutamate stimulation of the caudal midline medulla (CMM) causes profound sympathoinhibition due to GABAergic inhibition of presympathetic neurons in the rostral ventrolateral medulla (RVLM). We investigated whether the sympathoinhibitory pathway from CMM to RVLM, like the central baroreceptor reflex pathway, includes a glutamatergic synapse in the caudal ventrolateral medulla (CVLM). In pentobarbital sodium-anesthetized rats, the RVLM on one side was inhibited by a muscimol microinjection. Then the response evoked by glutamate microinjections into the CMM or by baroreceptor stimulation was determined before and after 1) microinjection of the GABA receptor antagonist bicuculline into the RVLM on the other side or 2) microinjections of the glutamate receptor antagonist kynurenate bilaterally into the CVLM. Bicuculline in the RVLM greatly reduced both CMM- and baroreceptor-evoked sympathoinhibition. Compared with the effect of vehicle solution, kynurenate in the CVLM greatly reduced baroreceptor-evoked sympathoinhibition, whereas its effect on CMM-evoked sympathoinhibition was not different from that of the vehicle solution. These findings indicate that the output pathway from CMM sympathoinhibitory neurons, unlike the baroreceptor and other reflex sympathoinhibitory pathways, does not include a glutamatergic synapse in the CVLM.

Animals↗

PubMed1986

Effect of aldose reductase inhibitor (sorbinil) on integration of polyol pathway, pentose phosphate pathway, and glycolytic route in diabetic rat lens.

This study examines the effect of an aldose reductase inhibitor (sorbinil) on the flux of specifically labeled glucose through alternative pathways of metabolism in the lens of normal and diabetic rats 1 wk after the induction of diabetes with alloxan. In the diabetic rat lens, there was an apparent increase in the flux of glucose through the pentose phosphate pathway (PPP), as measured by the difference in the yields of 14CO2 from [1-14C]glucose and [6-14C]glucose [C1-C6], this value was 0.087 +/- 0.005 and 0.263 +/- 0.034 mumol X g lens-1 X h (mean + SE of 6 values) for control and diabetic rats, respectively; sorbinil treatment decreased the values to 0.065 +/- 0.008 and 0.171 +/- 0.028, respectively. With glucose tritiated on carbon 2 or 3, it has been shown that the flux of glucose through the polyol route is increased, whereas the flux through the glycolytic pathway is decreased in the diabetic rat lens; both are restored toward normal in the sorbinil-treated diabetic group. These results suggest that the dual effects of diabetes in increasing the lens content of glucose and glucose 6-phosphate and the flux of glucose in the polyol pathway will result in an increased utilization of NADPH and production of NADH, factors favoring the flow of glucose through the PPP and restricting the glycolytic route in the diabetic rat lens. The inhibition of aldose reductase by sorbinil tends to normalize the redox state of the nicotinamide nucleotides, reimposing the NADPH limitation on the PPP and increasing the availability of NAD+ for the glycolytic route.

Aldehyde Reductase↗

PubMed2002

[Usefulness of clinical pathway for community-acquired pneumonia as both an educational and a cost-management tool--an intervention study to compare the usefulness of management with a critical pathway to historical control of conventional management].

To delineate the usefulness of a clinical pathway for community-acquired pneumonia (CAP) as an educational tool as well as a cost management tool, we conducted a prospective controlled trial including a historical control group. Consecutive CAP patients classified under Category 3 of the American Thoracic Society and admitted to our hospital were evaluated. Using the clinical pathway method, 42 patients were managed between April and December 2000 as the intervention group, and 33 patients received conventional management between April and December 1999 as a historical control. For the intervention group, the clinical pathway, which was a time-task matrix formatted with consideration for guidance for disease treatment, laboratory tests, physical examinations, oxygen saturation monitoring, ambulation, diet, education for the patient and clinical outcomes, was implemented. We determined (1) educational effect, measured using reduction of delay caused by physicians; (2) quality of clinical practice, measured using the success rate of the initial antimicrobial therapy and readmission rate; and (3) economic efficacy, measured using health care cost and length of hospital stay. The delay caused by physicians was reduced by 16% in the Intervention Group (5% vs. 21%; p = 0.045). The success rates of initial antimicrobial therapy in the two groups were similar (85.7% vs. 84.8%). In the intention-to-treat set, the median value of health care cost was reduced by yen 48,055 (yen 277,460 vs. yen 325,515; p = 0.017) and the median length of a hospital stay was shortened by 3 days (8 vs. 11 days; p = 0.0007) in the Intervention Group. In conclusion, the clinical pathway had an educational effect on physicians regarding the management of hospitalized patients with community-acquired pneumonia as well as on the cost management.

Aged↗

PubMed1991

Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells: II. A pathway involving arachidonic acid release, prostaglandin synthesis, and cyclic AMP accumulation.

We have previously shown that extracellular ATP acts as a mitogen via protein kinase C (PKC)-dependent and independent pathways (Wang, D., Huang, N., Gonzalez, F.A., and Heppel, L.A. Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells. I. Involvement of protein kinase C-dependent and independent pathways in the mitogenic response of mammalian cells to extracellular ATP. J. Cell. Physiol., 1991). The present aim was to determine if metabolism of arachidonic acid, resulting in prostaglandin E2 (PGE2) synthesis and elevation of cAMP levels, plays a role in mitogenesis mediated by extracellular ATP. Addition of ATP caused a marked enhancement of cyclic AMP accumulation in 3T3, 3T6, and A431 cells. Aminophylline, an antagonist of the adenosine A2 receptor, had no effect on the accumulation of cyclic AMP elicited by ATP, while it inhibited the action of adenosine. The accumulation of cyclic AMP was concentration dependent, which corresponds to the stimulation of DNA synthesis by ATP. The maximal accumulation was achieved after 45 min, with an initial delay period of about 15 min. That the activation of arachidonic acid metabolism contributed to cyclic AMP accumulation and mitogenesis stimulated by ATP in 3T3, 3T6, and A431 cells was supported by the following observations: (a) extracellular ATP stimulated the release of [3H]arachidonic acid and PGE2 into the medium; (b) inhibition of arachidonic acid release by inhibitors of phospholipase A2 blocked PGE2 production, cyclic AMP accumulation, and DNA synthesis activated by ATP, and this inhibition could be reversed by adding exogenous arachidonic acid; (c) cyclooxygenase inhibitors, such as indomethacin and aspirin, diminished the release of PGE2 and blocked cyclic AMP accumulation as well as [3H]thymidine incorporation in response to ATP; (d) PGE2 was able to restore [3H]thymidine incorporation when added together with ATP in the presence of cyclooxygenase inhibitors; (e) pertussis toxin inhibited ATP-stimulated DNA synthesis in a time- and dose-dependent fashion as well as arachidonic acid release and PGE2 formation. Other evidence for involvement of a pertussis toxin-sensitive G protein(s) in ATP-stimulated DNA synthesis as well as in arachidonic acid release is presented. In A431 cells, the enhancement of arachidonic acid and cyclic AMP accumulation by ATP was partially blocked by PKC down-regulation, implying that the activation of PKC may represent an additional pathway in ATP-stimulated metabolism of arachidonic acid. In all of these studies, ADP and AMP-PNP, but not adenosine, were as active as ATP.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1998

Blood mononuclear cell production of TNF-alpha and IL-8: engagement of different signal transduction pathways including the p42 MAP kinase pathway.

Recent studies of human peripheral blood mononuclear cells (PBMC) stimulated with IgG subclasses have suggested that tumor necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8) production proceed along different signal transduction pathways. To investigate this possibility, inhibitors of signal transduction pathways were employed. Human PBMC were pretreated with various inhibitors before being added to IgG2-coated wells and 4-h supernatant fluids evaluated for cytokine content. The effects of various inhibitors on MAP kinase activation were determined. Inhibitors of protein tyrosine kinases, phosphatases, and phospholipase C decreased TNF-alpha and IL-8 production, suggesting that all three enzyme pathways are involved in cytokine generation. Inhibitors of G-proteins had differing effects: pertussis toxin inhibited IL-8 but not TNF-alpha production, whereas cholera toxin inhibited TNF-alpha but not IL-8 production. Pretreatment of PBMC with pertussis toxin resulted in reduced IgG2-induced calcium mobilization, whereas cholera toxin had no effect, correlating with the effects of pertussis toxin on IL-8 expression. Inhibitors of protein kinase C (PKC) completely blocked TNF-alpha generation but had no effect on IL-8 production. Gö6976, which inhibits certain isoforms of PKC, inhibited production of both IL-8 and TNF-alpha. Isoforms of PKC may have opposing effects on cytokine production. PD 98059, a compound that specifically inhibits the activation of mitogen-activated protein kinase kinase (MEK1), inhibited TNF-alpha production, but had insignificant effects on IL-8 production. Pretreatment of PBMC with either PD 98059 or genistein reduced the extent of phosphorylation of p42 MAP kinase in cells activated on contact with IgG2. These findings suggest distinct signal transduction pathways for cytokine production in PBMC stimulated with IgG2.

Calcium↗

PubMed1996

Rapid formation of advanced glycation end products by intermediate metabolites of glycolytic pathway and polyol pathway.

To clarify roles of intermediate metabolites of the glycolytic pathway and the polyol pathway in nonenzymatic glycation under physiological conditions, we incubated bovine serum albumin with intermediates of both pathways in the micromolar range as well as with 20 mmol/l glucose, and observed the formation of advanced glycation end products (AGEs). We found that triose phosphates, glyceraldehyde, and a novel polyol pathway-related metabolite, fructose 3-phosphate along with its breakdown product, 3-deoxyglucosone were extremely potent glycating agents that at nearly physiological concentrations on incubation with albumin produced substantial amounts of AGEs as early as 24 hours, while 20 mmol/l glucose afforded trace amounts of AGEs after two week incubation. The results along with the previous evidence of the increased level of intermediates in diabetic states may suggest that the intermediate metabolites rather than glucose contribute to enhanced glycation in diabetic tissues, inspite of the much lower concentrations compared with glucose.

Animals↗

PubMed2001

A CCR5/CXCR4-independent coreceptor pathway on human macrophages supports efficient SIV env-mediated fusion but not infection: implications for alternative pathways of viral entry.

Several coreceptors in addition to CCR5 and CXCR4 support immunodeficiency virus entry in transfected cells, but whether they could play a role in HIV-1 pathogenesis is uncertain. To probe whether human macrophages express potentially functional alternative entry pathways, we analyzed cell-cell fusion and infection of primary macrophage by several SIVmac Envs. All Envs fused with normal macrophages. One, SIVmac316, also fused efficiently with macrophages lacking CCR5. CCR5-independent fusion was not mediated by CXCR4 and was CD4 dependent, while CCR5-mediated fusion was partly independent of CD4. However, pseudotype virions carrying the SIVmac316 Env and HIV-1 core could not infect macrophages through the CCR5-independent pathway, although they did infect wild-type macrophages. Thus, human macrophages possess an alternative coreceptor pathway that mediates SIV Env fusion but does not support infection. Macrophage entry pathways other than CCR5 and CXCR4 may have limited potential in pathogenesis given their restricted capacity for infection despite efficient fusion.

DNA Primers↗