ATP stimulates Na+-glucose cotransporter activity via cAMP and p38 MAPK in renal proximal tubule cells

Extracellular ATP plays an important role in the regulation of renal function. However, the effect of ATP on the Na⁺-glucose cotransporters (SGLTs) has not been elucidated in proximal tubule cells (PTCs). Therefore, this study was performed to examine the action of ATP on SGLTs and their related signal pathways in primary cultured rabbit renal PTCs. ATP increased [¹⁴C]--methyl-D-glucopyranoside (-MG) uptake in a time-dependent (>1 h) and dose-dependent (>10^–6 M) manner. ATP stimulated -MG uptake by increasing in V_max without affecting K_m. ATP-induced increase of -MG uptake was correlated with the increase in both SGLT1 and SGLT2 protein expression levels. ATP-induced stimulation of -MG uptake was blocked by suramin (nonspecific P2 receptor antagonist), RB-2 (P2Y receptor antagonist), and MRS-2179 (P2Y₁ receptor antagonist), suggesting a role for the P2Y receptor. ATP-induced stimulation of -MG uptake was blocked by pertussis toxin (PTX, a G_i protein inhibitor), SQ-22536 (an adenylate cyclase inhibitor), and PKA inhibitor amide 14-22 (PKI). ATP also increased cAMP formation, which was blocked by PTX and RB-2. However, pretreatment of adenosine deaminase did not block ATP-induced cAMP formation. In addition, ATP-induced stimulation of -MG uptake was blocked by SB-203580 (p38 MAPK inhibitor), but not by PD-98059 (p44/42 MAPK inhibitor) or SP-600125 (JNK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK. In conclusion, ATP increases -MG uptake via cAMP and p38 MAPK in renal PTCs. adenosine 5'-triphosphate; mitogen-activated protein kinase     

TNF-alpha regulates myogenesis and muscle regeneration by activating p38 MAPK

Although p38 MAPK activation is essential for myogenesis, the upstream signaling mechanism that activates p38 during myogenesis remains undefined. We recently reported that p38 activation, myogenesis, and regeneration in cardiotoxin-injured soleus muscle are impaired in TNF- receptor double-knockout (p55^–/–p75^–/–) mice. To fully evaluate the role of TNF- in myogenic activation of p38, we tried to determine whether p38 activation in differentiating myoblasts requires autocrine TNF-, and whether forced activation of p38 rescues impaired myogenesis and regeneration in the p55^–/–p75^–/– soleus. We observed an increase of TNF- release from C2C12 or mouse primary myoblasts placed in low-serum differentiation medium. A TNF--neutralizing antibody added to differentiation medium blocked p38 activation and suppressed differentiation markers myocyte enhancer factor (MEF)-2C, myogenin, p21, and myosin heavy chain in C2C12 myoblasts. Conversely, recombinant TNF- added to differentiation medium stimulated myogenesis at 0.05 ng/ml while inhibited it at 0.5 and 5 ng/ml. In addition, differentiation medium-induced p38 activation and myogenesis were compromised in primary myoblasts prepared from p55^–/–p75^–/– mice. Increased TNF- release was also seen in cardiotoxin-injured soleus over the course of regeneration. Forced activation of p38 via the constitutive activator of p38, MKK6bE, rescued impaired myogenesis and regeneration in the cardiotoxin-injured p55^–/–p75^–/– soleus. These results indicate that TNF- regulates myogenesis and muscle regeneration as a key activator of p38. myocyte enhancer factor-2C; myogenin; p21; myosin heavy chain; Akt; tumor necrosis factor-; mitogen-activated protein kinase     

Modulation of alpha7-integrin-mediated adhesion and expression by platelet-derived growth factor in vascular smooth muscle cells

We showed previously that the expression of ₇-integrin in aortic vascular smooth muscle cells (VSMC) is enhanced in a rat model of atherosclerosis. In the present study, we investigated the effects of platelet-derived growth factor (PDGF) on ₇-integrin expression and VSMC adhesion and migration. Expression of the ₇-integrin gene was determined by real-time RT-PCR, whereas protein levels were determined by fluorescence-activated cell sorting analysis. PDGF increased ₇ cell surface protein expression (12 and 24 h: 3.3 ± 0.8- and 3.6 ± 0.4-fold, P < 0.05 vs. control) and mRNA levels (24 h: 3.1-fold, P < 0.05 vs. control) in a time-dependent manner. Actinomycin D and cycloheximide attenuated PDGF-induced increases in ₇-integrin, indicating the involvement of de novo mRNA and protein synthesis. Treatment with the MAPK inhibitors PD-98059, SP-600125, and SB-203580 attenuated PDGF-induced increases in mRNA. In contrast, PD-98059 and SP-600125, but not SB-203580, attenuated PDGF-induced increases in cell surface protein levels. PDGF-treated VSMC adhered to laminin more efficiently (42 ± 6% increase, P < 0.01), and this increase was partially inhibited by anti-₇-integrin function-blocking antibody. However, PDGF did not alter migration on laminin, and there was no effect of the anti-₇-integrin function-blocking antibody on basal or PDGF-stimulated migration. Immunofluorescence imaging revealed an increase in ₇-integrin distribution along the stress fibers. Together, these observations indicate that PDGF enhances ₇-integrin expression in VSMC and promotes ₇-integrin-mediated adhesion to laminin. vascular injury; laminin; mitogen-activated protein kinase     

Effects of p38MAPK isoforms on renal mesangial cell inducible nitric oxide synthase expression

Several related isoforms of p38^MAPK have been identified and cloned in many species. Although they all contain the dual phosphorylation motif TGY, the expression of these isoforms is not ubiquitous. p38 and -2 are ubiquitously expressed, whereas p38 and - appear to have more restricted expression. Because there is evidence for selective activation by upstream kinases and selective preference for downstream substrates, the functions of these conserved proteins is still incompletely understood. We have demonstrated that the renal mesangial cell expresses the mRNA for all the isoforms of p38^MAPK, with p38 mRNA expressed at the highest level, followed by p38 and the lowest levels of expression by p382 and -. To determine the functional effects of these proteins on interleukin (IL)-1-induced inducible nitric oxide synthase (iNOS) expression, we transduced TAT-p38 chimeric proteins into renal mesangial cells and assessed the effects of wild-type and mutant p38 isoforms on ligand induced iNOS expression. We show that whereas p38 and - had minimal effects on iNOS expression, p38 and -2 significantly altered its expression. p38 mutant and p382 wild-type dose dependently inhibited IL-1-induced iNOS expression. These data suggest that p38 and 2 have reciprocal effects on iNOS expression in the mesangial cell, and these observations may have important consequences for the development of selective inhibitors targeting the p38^MAPK family of proteins. TAT proteins; p38 MAPK; inducible nitric oxide synthase; mesangial cell; interleukin-1     

Lipopolysaccharide- and gram-positive bacteria-induced cellular inflammatory responses: role of heterotrimeric Galpha(i) proteins

Heterotrimeric G_i proteins may play a role in lipopolysaccharide (LPS)-activated signaling through Toll-like receptor 4 (TLR4), leading to inflammatory mediator production. Although LPS is a TLR4 ligand, the gram-positive bacterium Staphylococcus aureus (SA) is a TLR2 ligand, and group B streptococci (GBS) are neither TLR2 nor TLR4 ligands but are MyD88 dependent. We hypothesized that genetic deletion of G_i proteins would alter mediator production induced by LPS and gram-positive bacterial stimulation. We examined genetic deletion of G_i2 or G_i1/3 protein in G_i2-knockout (G_i2–/–) or G_i1/3-knockout (G_i1/3–/–) mice. LPS-, heat-killed SA-, or GBS-induced mediator production in splenocytes or peritoneal macrophages (M) was investigated. There were significant increases in LPS-, SA-, and GBS-induced production of TNF- and IFN- in splenocytes from G_i2–/– mice compared with wild-type (WT) mice. Also, LPS-induced TNF- was increased in splenocytes from G_i1/3–/– mice. In contrast to splenocytes, LPS-, SA-, and GBS-induced TNF-, IL-10, and thromboxane B₂ (TxB₂) production was decreased in M harvested from G_i2–/– mice. Also, LPS-induced production of IL-10 and TxB₂ was decreased in M from G_i1/3–/– mice. In subsequent in vivo studies, TNF- levels after LPS challenge were significantly greater in G_i2–/– mice than in WT mice. Also, myeloperoxidase activity, a marker of tissue neutrophil infiltration, was significantly increased in the gut and lung of LPS-treated G_i2–/– mice compared with WT mice. These data suggest that G_i proteins differentially regulate murine TLR-mediated inflammatory cytokine production in a cell-specific manner in response to both LPS and gram-positive microbial stimuli. G_i protein-deficient mice; endotoxin; group B streptococci; Staphylococcus aureus; Toll-like receptors     

Salutary effects of 17beta-estradiol on T-cell signaling and cytokine production after trauma-hemorrhage are mediated primarily via estrogen receptor-alpha

Although 17-estradiol (E2) administration following trauma-hemorrhage prevents the suppression in splenocyte cytokine production, it remains unknown whether the salutary effects of 17-estradiol are mediated via estrogen receptor (ER)- or ER-. Moreover, it is unknown which signaling pathways are involved in 17-estradiol's salutary effects. Utilizing an ER-- or ER--specific agonist, we examined the role of ER- and ER- in E2-mediated restoration of T-cell cytokine production following trauma-hemorrhage. Moreover, since MAPK, NF-B, and activator protein (AP)-1 are known to regulate T-cell cytokine production, we also examined the activation of MAPK, NF-B, and AP-1. Male rats underwent trauma-hemorrhage (mean arterial pressure 40 mmHg for 90 min) and fluid resuscitation. ER- agonist propyl pyrazole triol (PPT; 5 µg/kg), ER- agonist diarylpropionitrile (DPN; 5 µg/kg), 17-estradiol (50 µg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, splenic T cells were isolated, and their IL-2 and IFN- production and MAPK, NF-B, and AP-1 activation were measured. T-cell IL-2 and IFN- production was decreased following trauma-hemorrhage, and this was accompanied with a decrease in T-cell MAPK, NF-B, and AP-1 activation. PPT or 17-estradiol administration following trauma-hemorrhage normalized those parameters, while DPN administration had no effect. Since PPT, but not DPN, administration following trauma-hemorrhage was as effective as 17-estradiol in preventing the T-cell suppression, it appears that ER- plays a predominant role in mediating the salutary effects of 17-estradiol on T cells following trauma-hemorrhage, and that such effects are likely mediated via normalization of MAPK, NF-B, and AP-1 signaling pathways. shock; MAPK; NF-B; activator protein-1; propyl pyrazole triol; diarylpropionitrile     

Ribosomal S6 kinase-1 modulates interleukin-1beta-induced persistent activation of NF-kappaB through phosphorylation of IkappaBbeta

Activation of NF-B requires the phosphorylation and degradation of its associated inhibitory proteins, IB. Previously, we reported that the extracellular signal-regulated kinase (ERK) is required for IL-1 to induce persistent activation of NF-B in cultured rat vascular smooth muscle cells (VSMCs). The present study examined the mechanism by which the ERK signaling cascade modulates the duration of NF-B activation. In cultured rat VSMCs, IL-1 activated ERK and induced degradation of both IB and IB, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-B p65. RSK1, a downstream kinase of ERK, was associated with an IB/NF-B complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with IL-1 decreased IB in the RSK1/IB/NF-B complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1-induced IB decrease without influencing ether ERK phosphorylation or the earlier IB degradation. By using recombinant wild-type and mutant IB proteins, both active ERK2 and RSK1 were found to directly phosphorylate IB, but only active RSK1 phosphorylated IB on Ser19 and Ser23, two sites known to mediate the subsequent ubiquitination and degradation. In conclusion, in the ERK signaling cascade, RSK1 is a key component that directly phosphorylates IB and contributes to the persistent activation of NF-B by IL-1. extracellular signal-regulated kinase; in vitro phosphorylation assay; recombinant proteins; small interference RNA; vascular smooth muscle cell     

Effects of the JNK inhibitor anthra[1,9-cd]pyrazol-6(2H)-one (SP-600125) on soluble guanylyl cyclase alpha1 gene regulation and cGMP synthesis

The decreased expression of the nitric oxide (NO) receptor, soluble guanylyl cyclase (sGC), occurs in response to multiple stimuli in vivo and in cell culture and correlates with various disease states such as hypertension, inflammation, and neurodegenerative disorders. The ability to understand and modulate sGC expression and cGMP levels in any of these conditions could be a valuable therapeutic tool. We demonstrate herein that the c-Jun NH₂-terminal kinase JNK II inhibitor anthra[1,9-cd]pyrazol-6(2H)-one (SP-600125) completely blocked the decreased expression of sGC₁-subunit mRNA by nerve growth factor (NGF) in PC12 cells. Inhibitors of the ERK and p38 MAPK pathways, PD-98059 and SB-203580, had no effect. SP-600125 also inhibited the NGF-mediated decrease in the expression of sGC₁ protein as well as sGC activity in PC12 cells. Other experiments revealed that decreased sGC₁ mRNA expression through a cAMP-mediated pathway, using forskolin, was not blocked by SP-600125. We also demonstrate that TNF-/IL-1 stimulation of rat fetal lung (RFL-6) fibroblast cells resulted in sGC₁ mRNA inhibition, which was blocked by SP-600125. Expression of a constitutively active JNKK2-JNK1 fusion protein in RFL-6 cells caused endogenous sGC₁ mRNA levels to decrease, while a constitutively active ERK2 protein had no effect. Collectively, these data demonstrate that SP-600125 may influence the intracellular levels of the sGC₁-subunit in certain cell types and may implicate a role for c-Jun kinase in the regulation of sGC₁ expression.     

Functional analysis of Na+/K+-ATPase isoform distribution in rat ventricular myocytes

The Na⁺/K⁺-ATPase (NKA) is the main route for Na⁺ extrusion from cardiac myocytes. Different NKA -subunit isoforms are present in the heart. NKA-1 is predominant, although there is a variable amount of NKA-2 in adult ventricular myocytes of most species. It has been proposed that NKA-2 is localized mainly in T-tubules (TT), where it could regulate local Na⁺/Ca²⁺ exchange and thus cardiac myocyte Ca²⁺. However, there is controversy as to where NKA-1 vs. NKA-2 are localized in ventricular myocytes. Here, we assess the TT vs. external sarcolemma (ESL) distribution functionally using formamide-induced detubulation of rat ventricular myocytes, NKA current (I_Pump) measurements and the different ouabain sensitivity of NKA-1 (low) and NKA-2 (high) in rat heart. Ouabain-dependent I_Pump inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-2, K_1/2 = 0.38 ± 0.16 µM) that accounts for 29.5 ± 1.3% of I_Pump and a low-affinity isoform (NKA-1, K_1/2 = 141 ± 17 µM) that accounts for 70.5% of I_Pump. Detubulation decreased cell capacitance from 164 ± 6 to 120 ± 8 pF and reduced I_Pump density from 1.24 ± 0.05 to 1.02 ± 0.05 pA/pF, indicating that the functional density of NKA is significantly higher in TT vs. ESL. In detubulated myocytes, NKA-2 accounted for only 18.2 ± 1.1% of I_Pump. Thus, 63% of I_Pump generated by NKA-2 is from the TT (although TT are only 27% of the total sarcolemma), and the NKA-2/NKA-1 ratio in TT is significantly higher than in the ESL. The functional density of NKA-2 is 4.5 times higher in the T-tubules vs. ESL, whereas NKA-1 is almost uniformly distributed between the TT and ESL. T-tubules; Na⁺/K⁺ pump current; ouabain; external sarcolemma; detubulation     

Differential effects of NF-{kappa}B and p38 MAPK inhibitors and combinations thereof on TNF-{alpha}- and IL-1{beta}-induced proinflammatory status of endothelial cells in vitro

Endothelial cells actively participate in inflammatory events by regulating leukocyte recruitment via the expression of inflammatory genes such as E-selectin, VCAM-1, ICAM-1, IL-6, IL-8, and cyclooxygenase (COX)-2. In this study we showed by real-time RT-PCR that activation of human umbilical vein endothelial cells (HUVEC) by TNF- and IL-1 differentially affected the expression of these inflammatory genes. Combined treatment with TNF- and IL-1 resulted in nonadditive, additive, and even synergistic induction of expression of VCAM-1, IL-8, and IL-6, respectively. Overexpression of dominant-negative inhibitor B protein blocking NF-B signaling confirmed a major role of this pathway in controlling both TNF-- and IL-1-induced expression of most of the genes studied. Although dexamethasone exerted limited effects at 1 µM, the thioredoxin inhibitor MOL-294, which regulates the redox state of NF-B, mainly inhibited adhesion molecule expression. Its most pronounced effect was seen on VCAM-1 mRNA levels, especially in IL-1-activated endothelium. One micromolar RWJ-67657, an inhibitor of p38 MAPK activity, diminished TNF-- and IL-1-induced expression of IL-6, IL-8, and E-selectin but had little effect on VCAM-1 and ICAM-1. Combined treatment of HUVEC with MOL-294 and RWJ-67657 resulted in significant blocking of the expression of E-selectin, IL-6, IL-8, and COX-2. The inhibitory effects were much stronger than those observed with single drug treatment. Application of combinations of drugs that affect multiple targets in activated endothelial cells may therefore be considered as a potential new therapeutic strategy to inhibit inflammatory disease activity. inflammatory gene expression; anti-inflammatory drugs; pharmacology; combination treatment     

Direct association of RhoA with specific domains of PKC-alpha

Previous studies performed at our laboratory have shown that agonist-induced contraction of smooth muscle is associated with translocation of protein kinase C (PKC)- and RhoA to the membrane and that this interaction is due to a direct protein-protein interaction. To determine the domains of PKC- involved in direct interaction with RhoA, His-tagged PKC- proteins of individual domains and different combinations of PKC- domains were used to perform in vitro binding assays with the fusion protein glutathione-S-transferase (GST)-RhoA. Coimmunoprecipitation was also performed using smooth muscle cells transfected with truncated forms of PKC- in this study. The data indicate that RhoA directly bound to full-length PKC-, both in vitro (82.57 ± 15.26% above control) and in transfected cells. RhoA bound in vitro to the C1 domain of PKC- [PKC- (C1)] (70.48 ± 20.78% above control), PKC- (C2) (72.26 ± 29.96% above control), and PKC- (C4) (90.58 ± 26.79% above control), but not to PKC- (C3) (0.64 ± 5.18% above control). RhoA bound in vitro and in transfected cells to truncated forms of PKC-, PKC- (C2, C3, and C4), and PKC- (C3 and C4) (94.09 ± 12.13% and 85.10 ± 16.16% above control, respectively), but not to PKC- (C1, C2, and C3) or to PKC- (C2 and C3) (0.47 ± 1.26% and 7.45 ± 10.76% above control, respectively). RhoA bound to PKC- (C1 and C2) (60.78 ± 13.78% above control) only in vitro, but not in transfected cells, and PKC- (C2, C3, and C4) and PKC- (C3 and C4) bound well to RhoA. These data suggest that RhoA bound to fragments that may mimic the active form of PKC-. The studies using cells transfected with truncated forms of PKC- indicate that PKC- (C1 and C2), PKC- (C1, C2, and C3), and PKC- (C2 and C3) did not associate with RhoA. Only full-length PKC-, PKC- (C2, C3, and C4), and PKC- (C3 and C4) associated with RhoA. The association increased upon stimulation with acetylcholine. These results suggest that the functional association of PKC- with RhoA may require the C4 domain. domains; histidine; fusion proteins     

Critical role of 5'-AMP-activated protein kinase in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation

5'-AMP-activated protein kinase (AMPK) functions as an energy sensor to provide metabolic adaptation under conditions of ATP depletion, such as hypoxia and inhibition of oxidative phosphorylation. Whether activation of AMPK is critical for stimulation of glucose transport in response to inhibition of oxidative phosphorylation is unknown. Here we found that treatment of Glut1-expressing Clone 9 cells with sodium azide (5 mM for 2 h) or the AMPK activator 5'-aminoimidazole-4-carboxamide-1--D-ribofuranoside (AICAR, 2 mM for 2 h) stimulated the rate of glucose transport by two- to fourfold. Use of small interference RNA (siRNA) directed against AMPK₁ or AMPK₁ + AMPK₂ (total AMPK) resulted in a significant inhibition of the glucose transport response and the content of phosphorylated AMPK₁ + phosphorylated AMPK₂ (total p-AMPK) and phosphorylated acetyl-CoA carboxylase (p-ACC) in response to azide. Transfection with siRNA directed against AMPK₂ did not affect the glucose transport response. The efficacy of transfection with siRNAs in reducing AMPK content was confirmed by Western blotting. Incubation of cells with compound C, an inhibitor of AMPK, abrogated the glucose transport response and abolished the increase in total p-AMPK in azide-treated or hypoxia-exposed cells. Simultaneous exposure to azide and AICAR did not augment the rate of transport in response to AICAR alone. There was no evidence of coimmunoprecipitation of total p-AMPK with Glut1. However, LKB1 was associated with total p-AMPK. We conclude that activation of AMPK plays both a sufficient and a necessary role in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation. small interference RNA; compound C; hypoxia     

Estrogen receptor-alpha predominantly mediates the salutary effects of 17beta-estradiol on splenic macrophages following trauma-hemorrhage

Although 17-estradiol administration following trauma-hemorrhage prevents the suppression in splenic macrophage cytokine production, it remains unknown whether the salutary effects are mediated via estrogen receptor (ER)- or ER- and which signaling pathways are involved in such 17-estradiol effects. Utilizing ER-- or ER--specific agonists, this study examined the role of ER- and ER- in 17-estradiol-mediated restoration of macrophage cytokine production following trauma-hemorrhage. In addition, since MAPK and NF-B are known to regulate macrophage cytokine production, we also examined the activation of those signaling molecules. Male rats underwent trauma-hemorrhage (mean arterial pressure of 40 mmHg for 90 min) and fluid resuscitation. The ER- agonist propyl pyrazole triol (PPT; 5 µg/kg), the ER- agonist diarylpropionitrile (DPN; 5 µg/kg), 17-estradiol (50 µg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, splenic macrophages were isolated, and their IL-6 and TNF- production and activation of MAPK and NF-B were measured. Macrophage IL-6 and TNF- production and MAPK activation were decreased, whereas NF-B activity was increased, following trauma-hemorrhage. PPT or 17-estradiol administration after trauma-hemorrhage normalized those parameters. DPN administration, on the other hand, did not normalize the above parameters. Since PPT but not DPN administration following trauma-hemorrhage was as effective as 17-estradiol in preventing the suppression in macrophage cytokine production, it appears that ER- plays the predominant role in mediating the salutary effects of 17-estradiol on macrophage cytokine production following trauma-hemorrhage and that such effects are likely mediated via normalization of MAPK but not NF-B signaling pathways. shock; mitogen-activated protein kinase; nuclear factor-B; propyl pyrazole triol; diarylpropionitrile     

Evidence for nucleotide receptor modulation of cross talk between MAP kinase and NF-kappa B signaling pathways in murine RAW 264.7 macrophages

Extracellular nucleotides such as ATP are present in abundance at sites of inflammation and tissue damage, and these agents exert a potent modulatory effect on macrophage/monocyte function via the nucleotide receptor P2X₇. In this regard, after exposure to bacterial LPS, P2X₇ activation augments expression of the inducible nitric oxide (NO) synthase and production of NO in macrophages. Because P2X₇ has been reported to stimulate certain members of the MAP kinase family (ERK1/2) and can enhance the DNA-binding activity of NF-B, we tested the hypothesis that LPS and nucleotides regulate NF-B-dependent inflammatory events via cross talk with MAPK-associated pathways. In this regard, the present studies revealed that cotreatment of macrophages with LPS and the P2X₇-selective ligand 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) results in the cooperative activation of NF-B DNA-binding activity and a sustained attenuation of levels of the NF-B inhibitory protein IB. Interestingly, a persistent reduction in IB levels is also observed when the MEK1/2 inhibitor U0126 is coadministered with LPS, suggesting that components of the MEK/ERK pathway are involved in regulating IB protein expression and/or turnover. The observation that U0126 and BzATP exhibit overlapping actions with respect to LPS-induced changes in IB levels is supported by the finding that Ras activation, which is upstream of MEK/ERK activation, is reduced upon macrophage cotreatment with BzATP and LPS compared with the effects of BzATP treatment alone. These data are consistent with the concept that the Ras/MEK/ERK pathways are involved in regulating NF-B/IB-dependent inflammatory mediator production and suggest a previously unidentified mechanism by which nucleotides can modulate LPS-induced action via cross talk between NF-B and Ras/MEK/MAPK-associated pathways. nucleotide receptors; mitogen-activated protein kinases; nuclear factor-B; monocytes/macrophages; cytokines     

Involvement of G protein betagamma-subunits in diverse signaling induced by G(i/o)-coupled receptors: study using the Xenopus oocyte expression system

We studied the functions of -subunits of G_i/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl^– currents in oocytes expressing ₂-adrenoceptor and the protein kinase A-dependent Cl^– channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [D-Ala², D-Leu⁵]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing ₂-adrenoceptor-CFTR and 5-HT_1A receptor (5-HT_1AR), -opioid receptor, or GABA_B receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT_1AR. The 5-HT-induced enhancement of G_s-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin (G_t). The 5-HT-induced enhancement was further augmented by coexpression of the G-activated form of adenylate cyclase (AC) type II but not AC type III. Thus -subunits of G_i/o protein contribute to the enhancement of G_s-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT_1AR or -opioid receptor alone. They elicited Ca²⁺-activated Cl^– currents in oocytes coexpressing these receptors with the G-activated form of phospholipase C (PLC)-2 but not with PLC-1. These currents were inhibited by pretreatment with PTX and coexpression of G_t, suggesting that -subunits of G_i/o protein activate PLC-2 and then cause intracellular Ca²⁺ mobilization. Our results indicate that -subunits of G_i/o protein participate in diverse intracellular signals, enhancement of G_s-coupled receptor-mediated responses, and intracellular Ca²⁺ mobilization. G protein-coupled receptor; cystic fibrosis transmembrane conductance regulator gene; cross talk; electrophysiology     

Sinusoidal length oscillation- and receptor-mediated mRNA expression of myosin isoforms and alpha-SM actin in airway smooth muscle

We tested the hypothesis that sinusoidal length oscillation and receptor activation interactively regulate the abundance of mRNA encoding -smooth muscle (-SM) actin and myosin isoforms in intact bovine tracheal smooth muscle. We found that sinusoidal length oscillation significantly downregulated abundance of mRNA encoding -SM actin mRNA in unstimulated tissues but not in histamine- and carbachol-activated tissues. This observation suggests antagonistic interactions between mechanical stretch and receptor-mediated signal transduction in regulating the abundance of mRNA encoding -SM actin in intact airway smooth muscle. This pattern of antagonistic interaction was also observed in cholinergic receptor activation experiments. Whereas carbachol significantly upregulated myosin heavy chain SMA isoform expression in muscle strips held at slack length, carbachol did not significantly alter SMA expression in muscle strips at sinusoidal length oscillation. Carbachol also significantly upregulated GAPDH expression in bovine tracheal smooth muscle. However, unlike SMA expression, upregulation of GAPDH expression mediated by cholinergic receptor activation appeared to be insensitive to the mechanical state of airway smooth muscle. Unlike carbachol, histamine did not significantly alter the expression of GAPDH, myosin heavy chain SMA and SMB, myosin light chain LC17a and LC17b, and -SM actin in bovine tracheal smooth muscle. U0126 (10 µM) completely inhibited carbachol-induced ERK1/2 MAPK phosphorylation but did not significantly affect carbachol-induced upregulation of GAPDH and SMA expression, suggesting that the ERK1/2 MAPK pathway was not the underlying mechanism. A potential implication of these findings is that periodic stretching of airways during respiratory cycles may modulate mRNA expression by receptor agonists in airway smooth muscle cells in vivo. asthma; carbachol; deep inspiration; gene expression; histamine     

PKC-delta and CaMKII-delta 2 mediate ATP-dependent activation of ERK1/2 in vascular smooth muscle

ATP, a purinergic receptor agonist, has been shown to be involved in vascular smooth muscle (VSM) cell DNA synthesis and cell proliferation during embryonic and postnatal development, after injury, and in atherosclerosis. One mechanism that ATP utilizes to regulate cellular function is through activation of ERK1/2. In the present study, we provide evidence that ATP-dependent activation of ERK1/2 in VSM cells utilizes specific isoforms of the multifunctional serine/threonine kinases, PKC, and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) as intermediates. Selective inhibition of PKC- activity with rottlerin, or adenoviral overexpression of kinase-negative PKC-, attenuated the ATP- and phorbol 12,13-dibutyrate (PDBu)-stimulated ERK1/2 activation. Inhibition of PKC- activity with Gö-6976, or adenoviral overexpression of kinase-negative PKC-, was ineffective. Alternatively, treatment with KN-93, a selective inhibitor of CaMKII activation, or adenoviral overexpression of kinase-negative CaMKII-₂, inhibited ATP-dependent activation of ERK1/2 but had no effect on PDBu- or PDGF-stimulated ERK1/2. In addition, adenoviral overexpression of dominant-negative ras (Ad.HA-Ras^N17) partially inhibited the ATP- and PDBu-induced activation of ERK1/2 and blocked ionomycin- and EGF-stimulated ERK1/2, and inhibition of tyrosine kinases with AG-1478, an EGFR inhibitor, or the src family kinase inhibitor PP2 attenuated ATP-stimulated ERK1/2 activation. Taken together, these data indicate that PKC- and CaMKII-₂ coordinately mediate ATP-dependent transactivation of EGF receptor, resulting in increased ERK1/2 activity in VSM cells. protein kinase C-; calcium/calmodulin-dependent protein kinase II- ₂; extracellular signal-regulated kinase 1/2; epidermal growth factor receptor transactivation; adenovirus     

Effects of alpha1D-adrenergic receptors on shedding of biologically active EGF in freshly isolated lacrimal gland epithelial cells

Transactivation of EGF receptors by G protein-coupled receptors is a well-known phenomenon. This process involves the ectodomain shedding of growth factors in the EGF family by matrix metalloproteinases. However, many of these studies employ transformed and/or cultured cells that overexpress labeled growth factors. In addition, few studies have shown that EGF itself is the growth factor that is shed and is responsible for transactivation of the EGF receptor. In this study, we show that freshly isolated, nontransformed lacrimal gland acini express two of the three known ₁-adrenergic receptors (ARs), namely, _1B- and _1D-ARs. _1D-ARs mediate phenylephrine (an ₁-adrenergic agonist)-induced protein secretion and activation of p42/p44 MAPK, because the _1D-AR inhibitor BMY-7378, but not the _1A-AR inhibitor 5-methylurapidil, inhibits these processes. Activation of p42/p44 MAPK occurs through transactivation of the EGF receptor, which is inhibited by the matrix metalloproteinase ADAM17 inhibitor TAPI-1. In addition, phenylephrine caused the shedding of EGF from freshly isolated acini into the buffer. Incubation of freshly isolated cells with conditioned buffer from cells treated with phenylephrine resulted in activation of the EGF receptor and p42/p44 MAPK. The EGF receptor inhibitor AG1478 and an EGF-neutralizing antibody blocked this activation of p42/p44 MAPK. We conclude that in freshly isolated lacrimal gland acini, ₁-adrenergic agonists activate the _1D-AR to stimulate protein secretion and the ectodomain shedding of EGF to transactivate the EGF receptor, potentially via ADAM17, which activates p42/p44 MAPK to negatively modulate protein secretion. epidermal growth factor ectodomain shedding; protein secretion; signal transduction     

RhoA regulation of NF-kappaB activation is mediated by COX-2-dependent feedback inhibition of IKK in kidney epithelial cells

Numerous studies have demonstrated a central role of renal tubular epithelial cells in the etiology of kidney injury and disease through the elaboration of inflammatory mediators. However, little is known about the cellular signaling mechanisms involved in this process. In this study we employed normal rat kidney epithelial (NRK52E) cells to identify a novel LPS-induced signaling pathway in which RhoA-mediated AP-1 activity promotes expression of cyclooxygenase-2 (COX-2) with consequent feedback inhibition of NF-B activation through IKK. Inhibition of RhoA signaling using either the RhoA kinase inhibitor Y-27632 or a dominant negative mutant of RhoA (RhoA-DN) dramatically extended the duration of p65-DNA binding, IB phosphorylation, and IKK activity following LPS treatment. Prolongation of events associated with NF-B activation was also observed in cells pretreated and/or cotransfected with the JNK inhibitor SP600125 or deletion mutants of MEKK1 (MEKK1-KD) or Jun (Jun-DN). Conversely, constitutive expression of RhoA prevented NF-B activation by LPS, and this effect was reversed by cotransfection with MEKK1-KD. In addition, we found that the RhoA/AP-1 signaling axis plays a necessary role in COX-2 expression by LPS and that this effect is independent of NF-B activation. Moreover, inhibition of COX-2 activity results in persistent p65-DNA binding, IB phosphorylation, and IKK activity, similar to that observed after prevention of RhoA/AP-1 axis signaling. These findings suggest that COX-2 links the RhoA/AP-1 signaling cascade to NF-B activation, thereby defining a novel integrated model for regulation of the inflammatory response of kidney epithelial cells to LPS and potentially other external stimuli. AP-1; cyclooxygenase-2; inflammation; lipopolysaccharide, nuclear factor-B; IB kinase     

Cholinergic receptor-mediated differential cytoskeletal recruitment of actin- and integrin-binding proteins in intact airway smooth muscle

We tested the hypothesis that cholinergic receptor stimulation recruits actin- and integrin-binding proteins from the cytoplasm to the cytoskeleton-membrane complex in intact airway smooth muscle. We stimulated bovine tracheal smooth muscle with carbachol and fractionated the tissue homogenate into pellet (P) and supernatant (S) by ultracentrifugation. In unstimulated tissues, calponin exhibited the highest basal P-to-S ratio (P/S; 2.74 ± 0.47), whereas vinculin exhibited the lowest P/S (0.52 ± 0.09). Cholinergic receptor stimulation increased P/S of the following proteins in descending order of sensitivity: -actinin > talin metavinculin > -smooth muscle actin > vinculin calponin. Carbachol induced ERK1/2 phosphorylation by 300% of basal value. U0126 (10 µM) completely inhibited carbachol-induced ERK1/2 phosphorylation but did not significantly affect the correlation between -actinin P/S and carbachol concentration. This observation indicates that cytoskeletal/membrane recruitment of -actinin is independent of ERK1/2 mitogen-activated protein kinase activation. Metavinculin and vinculin are splice variants of a single gene, but metavinculin P/S was significantly higher than vinculin P/S. Furthermore, the P/S of metavinculin but not vinculin increased significantly in response to cholinergic receptor stimulation. Calponin and -actinin both belong to the family of calponin homology (CH) domain proteins. However, unlike -actinin, the calponin P/S did not change significantly in response to cholinergic receptor stimulation. These findings indicate differential cytoskeletal/membrane recruitment of actin- and integrin-binding proteins in response to cholinergic receptor stimulation in intact airway smooth muscle. -Actinin, talin, and metavinculin appear to be key cytoskeletal proteins involved in the recruitment process. actinin; mitogen-activated protein kinase; metavinculin; vinculin