PACAP-38 induces neuronal differentiation of human SH-SY5Y neuroblastoma cells via cAMP-mediated activation of ERK and p38 MAP kinases.

The intracellular signaling pathways mediating the neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP) were investigated in human neuroblastoma SH-SY5Y cells. Previously, we showed that SH-SY5Y cells express the PAC(1) and VIP/PACAP receptor type 2 (VPAC(2)) receptors, and that the robust cAMP production in response to PACAP and vasoactive intestinal peptide (VIP) was mediated by PAC(1) receptors (Lutz et al. 2006). Here, we investigated the ability of PACAP-38 to differentiate SH-SY5Y cells by measuring morphological changes and the expression of neuronal markers. PACAP-38 caused a concentration-dependent increase in the number of neurite-bearing cells and an up-regulation in the expression of the neuronal proteins Bcl-2, growth-associated protein-43 (GAP-43) and choline acetyltransferase: VIP was less effective than PACAP-38 and the VPAC(2) receptor-specific agonist, Ro 25-1553, had no effect. The effects of PACAP-38 and VIP were blocked by the PAC(1) receptor antagonist, PACAP6-38. As observed with PACAP-38, the adenylyl cyclase activator, forskolin, also induced an increase in the number of neurite-bearing cells and an up-regulation in the expression of Bcl-2 and GAP-43. PACAP-induced differentiation was prevented by the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine (DDA), but not the protein kinase A (PKA) inhibitor, H89, or by siRNA-mediated knock-down of the PKA catalytic subunit. PACAP-38 and forskolin stimulated the activation of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAP; p38 MAP kinase) and c-Jun N-terminal kinase (JNK). PACAP-induced neuritogenesis was blocked by the MEK1 inhibitor PD98059 and partially by the p38 MAP kinase inhibitor SB203580. Activation of exchange protein directly activated by cAMP (Epac) partially mimicked the effects of PACAP-38, and led to the phosphorylation of ERK but not p38 MAP kinase. These results provide evidence that the neurotrophic effects of PACAP-38 on human SH-SY5Y neuroblastoma cells are mediated by the PAC(1) receptor through a cAMP-dependent but PKA-independent mechanism, and furthermore suggest that this involves Epac-dependent activation of ERK as well as activation of the p38 MAP kinase signaling pathway.

Neurotrophic actions of PACAP-38 and LIF on human neuroblastoma SH-SY5Y cells.

The neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP)-38 and leukemia inhibitory factor (LIF) were investigated in human neuroblastoma SH-SY5Y cells. Effects on differentiation were assessed through monitoring morphological changes and Western blot analysis of the expression of neuronal marker proteins. In contrast to PACAP-38, which induced a 5.5-fold increase in the number of neurite-bearing cells, LIF had no significant effect on cell morphology compared to control cells over the 4-day time course. Cells co-treated with PACAP-38+LIF showed a similar increase in neurite-bearing cells compared to those treated with PACAP-38 alone. Cell morphology was similar for PACAP-38-treated and PACAP-38+LIF-co-treated cells, with the formation of bipolar neuron-like cells with long thin neurites, topped by growth cone-like structures and varicosities. SH-SY5Y cells express tyrosine hydroxylase (TH) but only low levels of the neuronal marker proteins: Bcl-2, GAP-43 and choline acetyltransferase (ChAT). Treatment of cells with PACAP-38 induced the expression of Bcl-2, GAP-43, and ChAT but did not appear to alter the expression of TH. LIF failed to induce the expression of GAP-43 and had little effect on the expression of TH, but did induce the expression of Bcl-2 and upregulated the expression of ChAT. Co-treatment with LIF had no effect on PACAP-38-induced expression of Bcl-2, GAP-43, and ChAT. Cells differentiated for 4 days with PACAP-38 or treated with LIF also displayed increased resistance to hypoxic conditions and to treatment with H2O2 and TNFalpha. The increased resistance to hypoxic conditions for PACAP-differentiated cells was blocked by the p38 MAP kinase inhibitor, SB203580, but not by the MEK1 inhibitor, PD98059. Additionally, cell proliferation assays show that LIF, but not PACAP-38, stimulates proliferation of SH-SY5Y cells, and this observed increase by LIF is not attenuated by co-treatment with PACAP. Further investigation of the intracellular signaling pathways mediating the neurotrophic effects of PACAP on SH-SY5Y cells indicate that neither phospholipase C activation nor Ca2+/calmodulin-dependent kinase II (CAMKII) are involved.

Cytokine upregulation of proteinase-activated-receptors 2 and 4 expression mediated by p38 MAP kinase and inhibitory kappa B kinase beta in human endothelial cells.

BACKGROUND AND PURPOSE: Up-regulation of proteinase-activated receptor-2 (PAR2) is a factor in a number of disease states and we have therefore examined the signalling pathways involved in the expression of the receptor. EXPERIMENTAL APPROACH: We investigated the effects of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), trypsin and the PAR2 activating peptide, 2-furoyl(2f)-LIGKV-OH on both mRNA and functional expression of PAR2 in human umbilical vein endothelial cells (HUVECs). The effect of specific chemical inhibitors and dominant negative adenovirus constructs of the mitogen-activated protein kinase (MAPK) cascade and the nuclear factor kappa B (NF-kappaB) signalling pathway was assessed. Methods included semi-quantitative and quantitative RT-PCR, [(3)H]inositol phosphate (IP) accumulation and Ca(2+)-dependent fluorescence. KEY RESULTS: The above agonists induced both mRNA and functional expression of PAR2; PAR4 mRNA, but not that for PAR1 or PAR-3, also increased following TNFalpha treatment. Inhibition of p38 MAP kinase reduced PAR2 and PAR4 expression, whilst inhibition of MEK1/ERK/JNK was without effect. A similar dependency upon p38 MAP kinase was observed for the expression of PAR4. TNFalpha -induced enhancement of PAR2 stimulated [(3)H]-inositol phosphate accumulation (IP) and Ca(2+) signalling was abolished following SB203580 pre-treatment. Infection with adenovirus encoding dominant-negative IKKbeta (Ad.IKKbeta(+/-)) and to a lesser extent dominant-negative IKKalpha (Ad.IKKalpha(+/-)), substantially reduced both control and IL-1beta- induced expression of both PAR2 and PAR4 mRNA and enhancement of PAR2-stimulated IP accumulation and Ca(2+) mobilisation. CONCLUSIONS AND IMPLICATIONS: These data reveal for the first time the signalling events involved in the upregulation of both PAR2 and PAR4 during pro-inflammatory challenge.

The effects of cardamonin on lipopolysaccharide-induced inflammatory protein production and MAP kinase and NFkappaB signalling pathways in monocytes/macrophages.

BACKGROUND AND PURPOSE: In this study we examined the effect of the natural product cardamonin, upon lipopolysaccharide (LPS)-induced inflammatory gene expression in order to attempt to pinpoint the mechanism of action. EXPERIMENTAL APPROACHES: Cardamonin was isolated from the Greek plant A. absinthium L. Its effects were assessed on LPS-induced nitrite release and iNOS and COX-2 protein expression in two macrophage cell lines. Western blotting was used to investigate its effects on phosphorylation of the mitogen activated protein (MAP) kinases, ERK, JNK and p38 MAP kinase, and activation of the NFkappaB pathway, at the level of IkappaBalpha degradation and phosphorylation of NFkappaB. Also its effects on NFkappaB and GAS/GAF-DNA binding were assessed by EMSA. KEY RESULTS: Cardamonin concentration-dependently inhibited both NO release and iNOS expression but had no effect on COX-2 expression. It did not affect phosphorylation of the MAP kinases, degradation of IkappaBalpha or phosphorylation of NFkappaB. However, it inhibited NFkappaB DNA-binding in both LPS-stimulated cells and nuclear extracts of the cells (in vitro). It also inhibited IFNgamma-stimulated iNOS induction and GAS/GAF-DNA binding. CONCLUSIONS AND IMPLICATIONS: These results show that the inhibitory effect of cardamonin on LPS-induced iNOS induction is not mediated via effects on the initial activation of the NFkappaB or MAP kinase pathways but is due to a direct effect on transcription factor binding to DNA. However, although some selectivity in cardamonin's action is implicated by its inability to affect COX-2 expression, its exact mechanism(s) of action has yet to be identified.

Insulin and platelet-derived growth factor acutely stimulate glucose transport in 3T3-L1 fibroblasts independently of protein kinase C.

Insulin and platelet-derived growth factor (PDGF) are mitogenic for murine 3T3-L1 fibroblasts. Both these mitogens acutely stimulate glucose transport by 2-4-fold in these cells, evident within minutes of agonist exposure. The tumour promoter and protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) also stimulates glucose transport by 2-3-fold over a similar time frame, suggesting that protein kinase C may be involved in the mitogenic action of insulin and PDGF in this cell line. In an attempt to address this, we have measured intracellular sn-1,2-diacylglycerol (DAG) levels in response to insulin, PDGF and PMA. We show that PDGF and PMA induce a rapid elevation in intracellular diacylglycerol levels, but insulin was without effect. In addition, we have shown that PMA and PDGF, but not insulin, stimulate protein kinase C activity. However, depletion of protein kinase C by overnight exposure to PMA, abolished PMA-stimulated glucose transport but had no effect on insulin- and PDGF-stimulated glucose transport, suggesting that the stimulation of glucose transport by these mitogens does not involve protein kinase C. The use of the selective protein kinase C inhibitor, Roche 31-8220, which inhibited PMA-stimulated glucose transport, but was without effect on insulin- and PDGF-stimulated glucose transport further supports this conclusion. Taken together, these data argue against a role for protein kinase C in the stimulation of glucose transport in 3T3-L1 fibroblasts caused by acute exposure to insulin or PDGF.

Growth factors, mitogens, oncogenes and the regulation of glucose transport.

The erythrocyte (or HepG2/brain) type glucose transporter (GLUT 1) was the first of the family of facilitative glucose transporter proteins to be cloned [M. Mueckler et al., Science 229, 941-945, 1985]. GLUT 1 is expressed in most tissue types, all cell lines, transformed cells and tumour cells. It is thought to be responsible for "housekeeping" levels of glucose transport, i.e. the uptake of glucose required for oxidative phosphorylation. The rate of glucose transport via GLUT 1 can be regulated under conditions in which the metabolic rate must be adjusted such as cell division (mitosis and meiosis), differentiation, transformation and nutrient starvation. Here we review the recent literature on the control of glucose transport of mitogens, growth factors and oncogenes, and discuss some of the implications for the integration of cellular signalling pathways and cell growth.

Inactivation of JNK activity by mitogen-activated protein kinase phosphatase-2 in EAhy926 endothelial cells is dependent upon agonist-specific JNK translocation to the nucleus.

We have investigated the termination of agonist-stimulated mitogen-activated protein (MAP) kinase activity in EAhy926 cells by MAP kinase phosphatase-2 (MKP-2). In cells expressing either wild-type (WT) or catalytically inactive (CI)-MKP-2, there was no significant differences in TNFalpha-stimulated JNK or p38 MAP kinase activity, however hydrogen peroxide (H2O2)-stimulated JNK activity was substantially reduced in WT-MKP-2 expressing clones and enhanced in cells expressing CI-MKP-2. Consistent with these findings, we observed substantial nuclear translocation of JNK occurred in response to H2O2 but not TNFalpha. Using a phosphospecific anti-JNK antibody, we found that TNFalpha-stimulated JNK activity was associated principally with the cytosol while in response to H2O2, JNK activity was found within the nucleus. These results show that the role of MKP-2 in terminating JNK activity is determined by the translocation of JNK to the nucleus, which is under agonist-specific regulation and not a universal cellular response to stimulation.

Oestradiol-17 beta modulates PAF-evoked phospholipase D activity but not inositide-lipid hydrolysis in human endometrial cell line, HEC-1B.

Platelet-activating factor (PAF) has been shown to stimulate phospholipase D (PLD) activity in human endometrium. The effect of 17 beta-oestradiol on PAF- and 12-O-tetradecanoylphorbol 13-acetate (TPA)-evoked PLD activity assayed as an accumulation of [3H]phosphatidylbutanol was examined in [3H]myristic acid labelled in a human endometrial epithelial cell line HEC-1B. TPA stimulated PLD activity in a dose-dependent manner whereas PAF had no significant effect on PLD activity. Following 48 h pretreatment with 100 nM 17 beta-oestradiol, PAF evoked PLD activity while leaving inositol trisphosphate accumulation in myo-[2-3H] inositol-labelled HEC-1B cells unaffected. In the 17 beta-oestradiol-treated cells, TPA-stimulated PLD activity was significantly elevated at 100 nM TPA (P < 0.05) and 1 microM TPA (P < 0.05) compared to responses in the untreated cells, suggesting that 17 beta-oestradiol may upregulate PKC activity. Interestingly, following a 30 min pretreatment of HEC-1B cells with a range of 17 beta-oestradiol concentrations. TPA (10 nM) and PAF (100 nM) stimulated PLD activity. However, TPA-stimulated PLD activity levels fell 10-fold while PAF-mediated PLD activity remained elevated at 10 nM and 100 nM concentrations of 17 beta-oestradiol suggesting a different mechanism of activation. These results indicate that 17 beta-oestradiol can upregulate PAF-induced PLD activity in HEC-1B cells.

Cyclic AMP inhibitors inhibits PDGF-stimulated mitogen-activated protein kinase activity in rat aortic smooth muscle cells via inactivation of c-Raf-1 kinase and induction of MAP kinase phosphatase-1.

In rat aortic smooth muscle cells (RASMC), pretreatment with forskolin inhibited the activation of p42/44 isoforms of mitogen-activated protein kinase (MAP) kinase stimulated in response to low concentrations of PDGF (10 ng/ml). This correlated with a strong inhibition of PDGF-stimulated MEK and C-Raf-1 kinase activity. However, the effect of forskolin could be surmounted by increasing the concentration of PDGF. Under such conditions forskolin was only effective against prolonged MAP kinase activation. The ability of forskolin to inhibit the late phase of MAP kinase activity was reversed by pretreatment of the cells with cycloheximide, suggesting the involvement of a protein synthesis step. This was not due to effects upstream of MAP kinase since PDGF-stimulated MEK activation was decreased by cycloheximide, an effect potentiated by forskolin. Forskolin stimulated the induction of the dual specific phosphatase MAP kinase phosphatase-1 (MKP-1), although this effect was small relative to levels induced by PDGF and angiotensin II. However, PDGF stimulated induction of MKP-1 was abolished by the protein kinase A inhibitor H89 and this correlated with the reversal of forskolin-mediated inhibition of PDGF-stimulated MAP kinase activity. These studies implicate a role for intracellular cyclic AMP in at least two aspects of MAP kinase signaling, including both the inhibition of Raf-1 activation and the induction of MKP-1.

The role of protein kinase C in activation and termination of mitogen-activated protein kinase activity in angiotensin II-stimulated rat aortic smooth-muscle cells.

Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases activated by both tyrosine kinase and G-protein-linked receptor agonists. In rat aorta vascular smooth-muscle cells (VSMC), vasoconstrictors, angiotension II (AII), and alpha-thrombin (alpha-thr), as well as platelet-derived growth factor beta beta (PDGF) stimulated the tyrosine phosphorylation and activation of MAP kinase in a time- and concentration-dependent manner. Pre-treatment of cells with the protein kinase C (PKC) inhibitor Ro-318220, inhibited the initial increase in tyrosine phosphorylation of MAP kinase in response to vasoconstrictors, suggesting the involvement of PKC. Four isoforms of PKC were identified in VSMC by western blotting: alpha, beta, epsilon, and zeta. Downregulation of PKC alpha and PKC epsilon isoforms following chronic phorbol myristate 12, 13-acetate (PMA) pre-treatment resulted in the abolition of AII-stimulated MAP kinase activation. Selective downregulation of PKC alpha following pre-treatment with bryostatin 1 did not affect AII-stimulated MAP kinase. Preincubation of cells with Ro-318220 enhanced the activation of MAP kinase at later time points. In addition, Ro-318220 pre-treatment inhibited the induction by AII of a novel transcriptionally regulated phosphatase, MAP kinase phosphatase-1 (MKP-1). However, AII-mediated activation of MAP kinase was not prolonged by cycloheximide pre-treatment and was not maintained indefinitely by Ro-318220. These results demonstrate a specific role for the Ca(2+)-independent PKC isoform, PKC epsilon, in the activation of MAP kinase in response to vasoconstrictors, and suggest that PKC-mediated induction of MKP-1 plays no role in the termination of transiently activated MAP kinase.

Tumour necrosis factor stimulates stress-activated protein kinases and the inhibition of DNA synthesis in cultures of bovine aortic endothelial cells.

In this study, we examined the ability of tumour necrosis factor-alpha (TNF) to stimulate the mitogen-activated protein (MAP) kinase homologues p42/44 MAP kinase, c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase and its effect upon DNA synthesis in primary cultures of bovine aortic endothelial cells (BAECs). TNF strongly stimulated p38 MAP kinase and JNK activity in both a time- and concentration-dependent manner. By contrast, TNF was a very poor activator of p42/44 MAP kinase relative to the known activator of p42/44 MAP kinase in endothelial cells, adenosine triphosphate (ATP). TNF-stimulated activation of p38 MAP kinase, and MAPKAP kinase-2, a known downstream target of p38 MAP kinase, was strongly inhibited by pre-incubation with the p38 MAP kinase inhibitor SB203580, whereas the minor activation of p42/44 MAP kinase was abolished by pre-incubation of the cell with the novel MAP kinase kinase 1 inhibitor PD098059. Addition of TNF resulted in a 50-60% decrease in DNA synthesis in BAECs. Pre-incubation with PD098059 or co-incubation with ATP failed to modify the inhibitory effect of TNF upon DNA synthesis. SB203580 reduced basal DNA synthesis by approximately 50%; however, if failed to modify the inhibition mediated by TNF. These results indicate that TNF strongly activates both p38 MAP kinase, JNK and, to a minor extent, p42/p44 MAP kinase. It is likely that only one of these kinases, JNK, plays a role in the regulation of DNA synthesis in these cells.

Evidence that thrombin-stimulated DNA synthesis in pulmonary arterial fibroblasts involves phosphatidylinositol 3-kinase-dependent p70 ribosomal S6 kinase activation.

We have examined the regulation of the 70 kDa ribosomal S6 kinase (p70s6k) by the G-protein-coupled receptor agonist alpha-thrombin and the role of this signalling molecule in the mitogenic effect of thrombin in cultured bovine pulmonary arterial (PA) fibroblasts. Thrombin stimulated p70s6k activity in a time and concentration-dependent manner which was abolished by the macrolide rapamycin. The phosphatidylinositol (PI) 3-kinase inhibitor wortmannin also completely blocked p70s6k activity in response to thrombin but did not affect p70s6k activity evoked by platelet-derived growth factor (PDGF) at a concentration that abrogated PDGF-stimulated PI 3-kinase activity. Activation of p70s6k by thrombin, but not PDGF, was also inhibited (by 48.3 +/- 5.4%) by pre-incubation of cells with pertussis toxin (PTX). Downregulation of protein kinase C (PKC) alpha and epsilon isoforms by pretreatment of fibroblasts for 48 h with phorbol 12-myristate 13-acetate (PMA), markedly attenuated both thrombin and PDGF-stimulated p70s6k activation (by 74.8 +/- 4.4% and 82.3 +/- 7.9% respectively). Thrombin also strongly stimulated (over 100 fold) the incorporation of [3H]thymidine into growth arrested PA fibroblasts which was inhibited by rapamycin (by 33.6 +/- 2.0%). From these results we propose that in PA fibroblasts: 1) thrombin stimulates the activation of p70s6k in a manner consistent with an involvement of a heterotrimeric G protein of the G(i) family, a PI 3-kinase other than the PI 3-kinase involved in signalling by PDGF, and PKC. 2) a p70s6k-dependent pathway plays a role in mitogenic signalling by thrombin.

Phosphatidylinositol 3'-kinase, but not p70 ribosomal S6 kinase, is involved in membrane protein recycling: wortmannin inhibits glucose transport and downregulates cell-surface transferrin receptor numbers independently of any effect on fluid-phase endocytosis in fibroblasts.

The exposure of 3T3-L1 fibroblasts to growth factors results in a 2-to-3-fold increase in 2-deoxyglucose transport and a approximately 50% to 80% increase in cell-surface transferrin receptor levels. We sought to determine the role of phosphatidylinositol-3'-kinase and p70 ribosomal S6 kinase in these stimulations, using selective inhibitors of these enzymes. Both basal and growth factor-stimulated deoxyglucose transport are blocked by wortmannin, but with different IC50 values (65 nM vs. 15 nM, respectively), suggesting a functional difference between these two states. This is accompanied by the accumulation of glucose transporters in intracellular locations. Both basal and growth factor-stimulated cell-surface transferrin receptor levels are downregulated by wortmannin, but with identical IC50 values (approximately 15 nM). These two proteins are known to recycle between an intracellular site and the plasma membrane in these cells, thus implying a functional role for phosphatidylinositol-3'-kinase in membrane recycling. In an effort to determine whether the effect of wortmannin was selective for the protein component of this recycling, we examined fluid-phase endocytosis of radiolabeled mannitol. Wortmannin was without effect on the fluid phase accumulation of mannitol, suggesting that the effects on membrane traffic are limited to the protein component of recycling membranes. Rapamycin, an inhibitor of p70 ribosomal S6 kinase, was without effect on any of these parameters, but both rapamycin and wortmannin inhibit growth factor-stimulated p70 ribosomal S6 kinase activity. These data support an important role for phosphatidylinositol-3'-kinase, but not p70 ribosomal S6 kinase, in the regulation of membrane protein traffic. We suggest that this enzyme may be involved in sorting of membrane proteins during trafficking.

Growth factor-induced stimulation of hexose transport in 3T3-L1 adipocytes: evidence that insulin-induced translocation of GLUT4 is independent of activation of MAP kinase.

We have examined the effect of growth factors on the rate of hexose transport in 3T3-L1 adipocytes. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) were found to stimulate deoxyglucose transport by about 2-fold. The concentrations of EGF and PDGF which elicited half maximal responses were 100 and 350 pM, respectively. The increases in transport rate were acute effects; the stimulations were evident within minutes of exposure to growth factors. By contrast, insulin stimulated deoxyglucose transport approximately 16-fold over similar time periods. We have measured the appearance of both the insulin-responsive glucose transporter (GLUT4) and the erythrocyte-type glucose transporter (GLUT1) at the cell surface in response to insulin, EGF and PDGF. We show that both EGF and PDGF induce a 2-fold increase in GLUT1 at the cell surface, but both these growth factors were without effect on GLUT4 levels at the cell surface. In contrast, insulin induced a 13-fold increase in cell surface GLUT4. We further show that insulin, EGF and PDGF all activate MAP kinase as determined by a shift in electrophoretic mobility of this protein on SDS-PAGE. However, since the large translocation of GLUT4 to the cell surface is specific for insulin, we suggest that activation of MAP kinase is not the sole requisite for this process.

Stress-activated protein kinases: activation, regulation and function.

The response of cells to extracellular stimuli is mediated in part by a number of intracellular kinase and phosphatase enzymes. Within this area of research the activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinases have been extensively described and characterised as central components of the signal transduction pathways stimulated by both growth factors and G-protein-coupled receptor agonists. Signaling events mediated by these kinases are fundamental to cellular functions such as proliferation and differentiation. More recently, homologues of the p42 and p44 isoforms of MAP kinase have been described, namely the stress-activated protein kinases (SAPKs) or alternatively the c-jun N-terminal kinases (JNKs) and p38 MAP kinase (the mammalian homologue of yeast HOG1). These MAP kinase homologues are integral components of parallel MAP kinase cascades activated in response to a number of cellular stresses including inflammatory cytokines (e.g., Interleukin-1 (Il-1) and tumour necrosis factor-alpha (TNF-alpha), heat and chemical shock, bacterial endotoxin and ischaemia/cellular ATP depletion. Activation of these MAP kinase homologues mediates the transduction of extracellular signals to the nucleus and are pivotal events in the regulation of the transcription events that determine functional outcome in response to such stresses. In this review we highlight the identification and characterisation of the stress-activated MAP kinase homologues, their role as components of parallel MAP kinase pathways and the regulation of cellular responses following exposure to cellular stress.

Involvement of mitogen-activated protein kinase homologues in the regulation of lipopolysaccharide-mediated induction of cyclo-oxygenase-2 but not nitric oxide synthase in RAW 264.7 macrophages.

In RAW 264.7 macrophages lipopolysaccharide (LPS) stimulated the activation of p42 and p44 MAP kinases and their upstream activator mitogen-activated protein (MAP) kinase kinase (MAPKK), and induced the 69-kDa isoform of cyclo-oxygenase-2 (COX-2) and the 130-kDa isoform of nitric oxide synthase (iNOS). PD 098059, a specific inhibitor of the activation of MAPKK, prevented LPS-mediated activation of MAPKK (IC50 = 3.0 +/- 0.1 microM, n = 3) and p42/44 MAP kinases and substantially reduced the induction of COX-2 by approximately 40%-70%, but was without effect upon the induction of iNOS. In parallel, LPS also stimulated the activation of p38 MAP kinase and the MAPKAP kinase-2, a downstream target of p38 MAP kinase. SB 203580, a specific inhibitor of p38 MAP kinase prevented the activation of p38 MAP kinase (IC50 = 3.3 +/- 1.4 microM, n = 3) and MAPKAP kinase-2 by LPS and reduced the induction of COX-2 by approximately 50-90%, with no significant effect upon iNOS expression. These studies indicate the involvement of both the classical p42/44 MAP kinases and p38 MAP kinase in the regulation of COX-2 but not iNOS induction following exposure to LPS.

Differential regulation by protein kinase C isoforms of nitric oxide synthase induction in RAW 264.7 macrophages and rat aortic smooth muscle cells.

1. In RAW 264.7 murine macrophages and rat aortic smooth muscle (RASM) cells lipopolysaccharide (LPS) alone or in combination with interferon gamma (IFN gamma) or forskolin, respectively, stimulated the expression of the 130 kDa inducible isoform of nitric oxide synthase (iNOS) in both a time- and concentration-dependent manner. 2. Incubation with the direct activator of protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA) alone, did not result in detectable iNOS expression in either cell type. 3. Chronic PMA pretreatment resulted in significant down-regulation of alpha, beta and epsilon isforms of PKC in RAW 264.7 macrophages and corresponded to a 20-30% reduction in LPS-induced iNOS expression. In contrast, IFN gamma alone or in combination with LPS stimulated an approximate 20% and 50% potentiation, respectively. 4. Pre-incubation with PKC inhibitors (calphostin C and H-7) showed similar effects upon stimulated induction of iNOS. 5. In RASM cells chronic PMA pretreatment resulted in down-regulation of alpha and epsilon PKC isoforms and corresponded to potentiation of iNOS expression in response to LPS alone or in combination with forskolin. 6. Co-incubation of RASM cells in the presence of PMA, angiotensin II (AII) or foetal calf serum (FCS) resulted in the inhibition of iNOS expression in response to LPS alone or in combination with forskolin. 7. Differential sensitivity to PKC inhibitors (calphostin C and H-7) was observed in RASM cells and exhibited both negative and positive modulation of stimulated induction. 8. In addition the PKC inhibitor compound Ro-31-8220 abolished stimulated induction in both cell types in response to all treatments. 9. These results suggest that PKC activation is required for induction of the 130 kDa isoform of NOS in both RAW 264.7 macrophages and RASM cells. However, individual PKC isoforms regulate iNOS expression in both a positive and negative manner.

Protein kinase C and tyrosine kinase pathways regulate lipopolysaccharide-induced nitric oxide synthase activity in RAW 264.7 murine macrophages.

1. In RAW 264.7 macrophages, lipopolysaccharide (LPS) and gamma-interferon (IFN gamma) alone or in combination stimulated the induction of nitric oxide synthase (iNOS) activity and increased the expression of the 130 kDa isoform of NOS. 2. LPS-induced NOS activity was reduced by incubation with CD14 neutralising antibodies and abolished in macrophages deprived of serum. 3. LPS stimulated a small increase in protein kinase C (PKC) activity in RAW 264.7 macrophages which was dependent on the presence of serum. However, IFN gamma did not potentiate LPS-stimulated PKC activity. 4. The protein kinase C inhibitor, Ro-318220, abolished both LPS- and IFN gamma-stimulated protein kinase C activity and the induction of NOS activity. 5. LPS- and IFN gamma-induced NOS activity was reduced by the tyrosine kinase inhibitor genestein. Genestein also reduced LPS-stimulated protein kinase C activity but did not affect the response to the protein kinase C activator, tetradecanoylphorbol acetate (TPA). 6. Nicotinamide, an inhibitor of poly-ADP ribosylation, abolished LPS- and IFN gamma-induced NOS activity. 7. Brefeldin A, an inhibitor of a factor which stimulates nucleotide exchange activity on the 21 kDa ADP-ribosylation factor, ARF, reduced LPS- and IFN gamma-induced NOS activity by approximately 80%. 8. These results suggest the involvement of protein kinase C, tyrosine kinase and poly-ADP ribosylation pathways in the regulation of the induction of nitric oxide synthase in RAW 264.7 macrophages by LPS and IFN gamma.

Regulation by hypoxia of endothelin-1-stimulated phospholipase D activity in sheep pulmonary artery cultured smooth muscle cells.

1. The aim of the study was to characterize the effects of hypoxia on agonist-stimulated phospholipase D (PLD) and phospholipase C activity of sheep pulmonary artery cultured smooth muscle cells. 2. Endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT) and the protein kinase C (PKC) activator tetradecanoylphorbol acetate (TPA), stimulated a time- and concentration-dependent increase in [3H]-phosphatidylbutanol accumulation. This was abolished by pretreatment of the cells with the PKC inhibitor, Ro-318220, suggesting that agonist-stimulated phospholipase D activity is dependent upon the activation of PKC. 3. Hypoxia (PO2 20 mmHg for 30 min) stimulated basal [3H]-phosphatidylbutanol accumulation by approximately 2 fold and this activity was abolished by preincubation of the cells with 10 microM Ro-318220. 4. In cells preincubated in low O2 containing medium for 30 min, the subsequent agonist-stimulated accumulation of [3H]-phosphatidylbutanol was reduced. However, the decrease in stimulation was greater for ET-1 and 5-HT than for TPA. 5. ET-1 and TPA stimulated a time-dependent increase in protein kinase C- mediated psuedosubstrate phosphorylation. Following preincubation for 30 min in low O2 containing media, basal pseudosubstrate phosphorylation increased whilst the fold stimulation by TPA and ET-1 decreased. 6. In cells preincubated in low O2 containing medium, ET-1-stimulated [3H]-inositol phosphate accumulation was reduced by approximately 30-40%. This reduction was reversed by preincubation of the cells with Ro-318220. 7. These results suggest a role for PKC in the effects of hypoxia on PLD in pulmonary artery smooth muscle cells.

Vasopressin-stimulated [3H]-inositol phosphate and [3H]-phosphatidylbutanol accumulation in A10 vascular smooth muscle cells.

1. The characteristics of vasopressin-stimulated phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) and phosphatidylcholine (PtdCh) hydrolysis were examined in A10 vascular smooth muscle cells (VSMC), by assessing the formation of [3H]-inositol phosphates ([3H]-IP) and the accumulation of the phospholipase D (PLD) specific product, [3H]-phosphatidylbutanol ([3H]-PtdBuOH). 2. Vasopressin ([Arg8]-VP) and a number of related analogues stimulated the accumulation of [3H]-IP and [3H]-PtdBuOH with similar EC50 values, generating the same rank order of potency for each response (Arg8-VP = vasotocin = Lys8-VP much greater than oxytocin). 3. Inhibition of vasopressin-stimulated [3H]-IP and [3H]-PtdBuOH accumulation by the V1a receptor antagonists, Des-Gly9[beta-mercapto-beta,beta,-cyclopentamethylene propionyl, O-Et-Tyr2,Val4,Arg8]-vasopressin generated similar IC50 values suggesting that both these responses are mediated through the activation of a single V1a receptor subtype. 4. The onset of vasopressin-stimulated inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) mass formation preceded [3H]-PtdBuOH accumulation indicating that PtdCh hydrolysis was activated subsequent to PtdIns(4,5)P2 breakdown. 5. The protein kinase C (PKC) activator, tetradecanoylphorbol acetate (TPA) also stimulated [3H]-PtdBuOH accumulation. Preincubation with the PKC inhibitor Ro-31-8220 abolished both TPA- and vasopressin-stimulated [3H]-PtdBuOH, suggesting that the intermediate activation of protein kinase C is involved in the regulation of PLD by vasopressin. 6. Pretreatment of the A10 VSMC with Ro-31-8220 (100 microM) also potentiated vasopressin-stimulated Ins(1,4,5)P3 mass formation.Therefore stimulation of PKC may have opposing roles in the regulation of agonist activation of PLC and PLD.7. Preincubation of the cells with EGTA, verapamil, or the receptor-operated calcium channel antagonist, SK&F 96365, reduced vasopressin-stimulated [3H]-PtdBuOH accumulation by approximately 30%, suggesting that influx of calcium has a significant role to play in the regulation of vasopressinstimulated PLD activity.