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     

TNF-alpha dilates cerebral arteries via NAD(P)H oxidase-dependent Ca2+ spark activation

Expression of TNF-, a pleiotropic cytokine, is elevated during stroke and cerebral ischemia. TNF- regulates arterial diameter, although mechanisms mediating this effect are unclear. In the present study, we tested the hypothesis that TNF- regulates the diameter of resistance-sized (150-µm diameter) cerebral arteries by modulating local and global intracellular Ca²⁺ signals in smooth muscle cells. Laser-scanning confocal imaging revealed that TNF- increased Ca²⁺ spark and Ca²⁺ wave frequency but reduced global intracellular Ca²⁺ concentration ([Ca²⁺]_i) in smooth muscle cells of intact arteries. TNF- elevated reactive oxygen species (ROS) in smooth muscle cells of intact arteries, and this increase was prevented by apocynin or diphenyleneiodonium (DPI), both of which are NAD(P)H oxidase blockers, but was unaffected by inhibitors of other ROS-generating enzymes. In voltage-clamped (–40 mV) cells, TNF- increased the frequency and amplitude of Ca²⁺ spark-induced, large-conductance, Ca²⁺-activated K⁺ (K_Ca) channel transients 1.7- and 1.4-fold, respectively. TNF--induced transient K_Ca current activation was reversed by apocynin or by Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a membrane-permeant antioxidant, and was prevented by intracellular dialysis of catalase. TNF- induced reversible and similar amplitude dilations in either endothelium-intact or endothelium-denuded pressurized (60 mmHg) cerebral arteries. MnTMPyP, thapsigargin, a sarcoplasmic reticulum Ca²⁺-ATPase blocker that inhibits Ca²⁺ sparks, and iberiotoxin, a K_Ca channel blocker, reduced TNF--induced vasodilations to between 15 and 33% of control. In summary, our data indicate that TNF- activates NAD(P)H oxidase, resulting in an increase in intracellular H₂O₂ that stimulates Ca²⁺ sparks and transient K_Ca currents, leading to a reduction in global [Ca²⁺]_i, and vasodilation. cerebrovascular circulation; ryanodine-sensitive Ca²⁺ release channel; Ca²⁺-activated K⁺ channel; reactive oxygen species; vasodilation     

Mechanical loading and injury induce human myotubes to release neutrophil chemoattractants

The purpose of this study was to 1) test the hypothesis that skeletal muscle cells (myotubes) after mechanical loading and/or injury are a source of soluble factors that promote neutrophil chemotaxis and superoxide anion (O₂^–·) production and 2) determine whether mechanical loading and/or injury causes myotubes to release cytokines that are known to influence neutrophil responses [tumor necrosis factor- (TNF-), IL-8, and transforming growth factor-1 (TGF-1)]. Human myotubes were grown in culture and exposed to either a cyclic strain (0, 5, 10, 20, or 30% strain) or a scrape injury protocol. Protocols of 5, 10, and 20% strain did not cause injury, whereas 30% strain and scrape injury caused a modest and a high degree of injury, respectively. Conditioned media from strained myotubes promoted chemotaxis of human blood neutrophils and primed them for O₂^–· production in a manner that was dependent on a threshold of strain and independent from injury. Neutrophil chemotaxis, but not priming, progressively increased with higher magnitudes of strain. Conditioned media only from scrape-injured myotubes increased O₂^–· production from neutrophils. Concentrations of IL-8 and total TGF-1 in conditioned media were reduced by mechanical loading, whereas TNF- and active TGF-1 concentrations were unaffected. In conclusion, skeletal muscle cells after mechanical loading and injury are an important source of soluble factors that differentially influence neutrophil chemotaxis and the stages of neutrophil-derived reactive oxygen species production. Neutrophil responses elicited by mechanical loading, however, did not parallel changes in the release of IL-8, TGF-1, or TNF- from skeletal muscle cells. inflammation; cytokines; exercise; free radicals     

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     

HO-2 provides endogenous protection against oxidative stress and apoptosis caused by TNF-alpha in cerebral vascular endothelial cells

Tumor necrosis factor- (TNF-) causes oxidative stress and apoptosis in a variety of cell types. Heme oxygenase (HO) degrades heme to bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator, and a vasodilator. Newborn pig cerebral microvascular endothelial cells (CMVEC) highly express constitutive HO-2. We investigated the role of HO-2 in protection against TNF--induced apoptosis in cerebral vascular endothelium. In CMVEC from mice and newborn pigs, 15 ng/ml TNF- alone, or with 10 µg/ml cycloheximide (CHX) caused apoptosis detected by nuclear translocation of p65 NF-B, caspase-3 activation, DNA fragmentation, cell-cell contact destabilization, and cell detachment. TNF- did not induce HO-1 expression in CMVEC. CMVEC from HO-2 knockout mice showed greater sensitivity to apoptosis caused by serum deprivation and TNF- than did wild-type mice. TNF- increased reactive oxygen species generation, including hydrogen peroxide and superoxide radicals, as detected by dihydrorhodamine-123 and dihydroethidium. The TNF- response was inhibited by superoxide dismutase and catalase suggesting apoptosis is oxidative stress related. Inhibition of endogenous HO-2 in newborn pig CMVEC increased oxidative stress and exaggerated apoptosis caused by serum deprivation and TNF-. In HO-1-overexpressing CMVEC (HO-1 selective induction by cobalt portophyrin), TNF- did not cause apoptosis. A CO-releasing compound, CORM-A1, and bilirubin blocked TNF--induced reactive oxygen species accumulation and apoptosis consistent with the antioxidant and antiapoptotic roles of the end products of HO activity. We conclude that HO-2 is critical for protection of cerebrovascular endothelium against apoptotic changes induced by oxidative stress and cytokine-mediated inflammation. carbon monoxide; bilirubin; vascular injury; reactive oxygen species; heme oxygenase; cycloheximide     

MYPT1 mutants demonstrate the importance of aa 888-928 for the interaction with PKGIalpha

During nitric oxide signaling, type I cGMP-dependent protein kinase (PKGI) activates myosin light chain (MLC) phosphatase through an interaction with the 130-kDa myosin targeting subunit (MYPT1), leading to dephosphorylation of 20-kDa MLC and vasodilatation. It has been suggested that the MYPT1-PKGI interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH₂-terminal LZ of PKGI (HK Surks and ME Mendelsohn. Cell Signal 15: 937–944, 2003; HK Surks et al. Science 286: 1583–1587, 1999), but we previously showed that PKGI interacts with LZ-positive (LZ+) and LZ-negative (LZ–) MYPT1 isoforms (13). Interestingly, PKGI is known to preferentially bind to RR and RK motifs (WR Dostmann et al. Proc Natl Acad Sci USA 97: 14772–14777, 2000), and there is an RK motif within the aa 888–928 sequence of MYPT1 in LZ+ and LZ– isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGI interaction, we designed four MYPT1 fragments that contained both the aa 888–928 sequence and the downstream LZ domain (MYPT1FL), lacked both the aa 888–928 sequence and the LZ domain (MYPT1TR), lacked only the aa 888–928 sequence (MYPT1SO), or lacked only the LZ domain (MYPT1TR2). Using coimmunoprecipitation, we found that only the fragments containing the aa 888–928 sequence (MYPT1FL and MYPT1TR2) were able to form a complex with PKGI in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916–917 (R⁹¹⁶K⁹¹⁷) to AA decreased binding of MYPT1 to PKGI in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R⁹¹⁶K⁹¹⁷ to E⁹¹⁶E⁹¹⁷ eliminated binding, suggesting that one factor important for the PKGI-MYPT1 interaction is the charge at aa 916–917. These results suggest that, during cGMP-mediated signaling, aa 888–928 of MYPT1 mediate the PKGI-MYPT1 interaction. myosin light chain phosphatase; nitric oxide; smooth muscle; calcium desensitization; cGMP-dependent protein kinase; cGMP     

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     

Induced focal adhesion kinase expression suppresses apoptosis by activating NF-kappaB signaling in intestinal epithelial cells

Focal adhesion kinase (FAK) integrates various extracellular and intracellular signals and is implicated in a variety of biological functions, but its exact role and downstream targeting signals in the regulation of apoptosis in intestinal epithelial cells (IECs) remains unclear. The current study tested the hypothesis that FAK has an antiapoptotic role in the IEC-6 cell line by altering NF-B signaling. Induced FAK expression by stable transfection with the wild-type (WT)-FAK gene increased FAK phosphorylation, which was associated with an increase in NF-B activity. These stable WT-FAK-transfected IECs also exhibited increased resistance to apoptosis when they were exposed to TNF- plus cycloheximide (TNF-/CHX). Specific inhibition of NF-B by the recombinant adenoviral vector containing the IB superrepressor prevented increased resistance to apoptosis in WT-FAK-transfected cells. In contrast, inactivation of FAK by ectopic expression of dominant-negative mutant of FAK (DNM-FAK) inhibited NF-B activity and increased the sensitivity to TNF-/CHX-induced apoptosis. Furthermore, induced expression of endogenous FAK by depletion of cellular polyamines increased NF-B activity and resulted in increased resistance to TNF-/CHX-induced apoptosis, both of which were prevented by overexpression of DNM-FAK. These results indicate that increased expression of FAK suppresses TNF-/CHX-induced apoptosis, at least partially, through the activation of NF-B signaling in IECs. polyamines; -difluoromethylornithine; X-linked inhibitor of apoptosis protein; IB     

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     

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     

Laminin-alpha1 globular domains 3 and 4 induce heterotrimeric G protein binding to alpha-syntrophin's PDZ domain and alter intracellular Ca2+ in muscle

-Syntrophin is a component of the dystrophin glycoprotein complex (DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with -dystroglycan, which binds to extracellular matrix laminin. Syntrophin contains two pleckstrin homology (PH) domains and one PDZ domain. Because PH domains of other proteins are known to bind the -subunits of the heterotrimeric G proteins, whether this is also a property of syntrophin was investigated. Isolated syntrophin from rabbit skeletal muscle binds bovine brain G-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, - and -dystroglycan, or dystrophin precipitate a complex with G from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of G binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of G_s and G occurs and active G_s, measured as GTP-³⁵S bound, decreases. Because intracellular Ca²⁺ is elevated in Duchenne muscular dystrophy and G_s is known to activate the dihydropyridine receptor Ca²⁺ channel, whether laminin also altered intracellular Ca²⁺ was investigated. Laminin-1 decreases active (GTP-S-bound) G_s, and the Ca²⁺ channel is inhibited by laminin-1. The laminin ₁-chain globular domains 4 and 5 region, the region bound by DGC -dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to -dystroglycan inhibits G binding by syntrophin in C₂C₁₂ myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor. Duchenne muscular dystrophy; protein G -subunit; pleckstrin homology domain     

Disruption of alpha-actinin-integrin interactions at focal adhesions renders osteoblasts susceptible to apoptosis

Maintenance of bone structural integrity depends in part on the rate of apoptosis of bone-forming osteoblasts. Because substrate adhesion is an important regulator of apoptosis, we have investigated the role of focal adhesions in regulating bone cell apoptosis. To test this, we expressed a truncated form of -actinin (ROD-GFP) that competitively displaces endogenous -actinin from focal adhesions, thus disrupting focal adhesions. Immunofluorescence and morphometric analysis of vinculin and tyrosine phosphorylation revealed that ROD-GFP expression dramatically disrupted focal adhesion organization and reduced tyrosine phosphorylation at focal adhesions. In addition, Bcl-2 protein levels were reduced in ROD-GFP-expressing cells, but caspase 3 cleavage, poly(ADP-ribose) polymerase cleavage, histone H2A.X phosphorylation, and cytotoxicity were not increased due to ROD-GFP expression alone. Increases in both ERK and Akt phosphorylation were also observed in ROD-GFP-expressing cells, although inhibition of either ERK or Akt individually or together failed to induce apoptosis. However, we did find that ROD-GFP expression sensitized, whereas -actinin-GFP expression protected, cells from TNF--induced apoptosis. Further investigation revealed that activation of TNF--induced survival signals, specifically Akt phosphorylation and NF-B activation, was inhibited in ROD-GFP-expressing cells. The reduced expression of antiapoptotic Bcl-2 and inhibited survival signaling rendered ROD-GFP-expressing cells more susceptible to TNF--induced apoptosis. Thus we conclude that -actinin plays a role in regulating cell survival through stabilization of focal adhesions and regulation of TNF--induced survival signaling. tumor necrosis factor-; survival; cytoskeleton; nuclear factor-B     

Autocrine production of prostaglandin F2alpha enhances phenotypic transformation of normal rat kidney fibroblasts

We have used normal rat kidney (NRK) fibroblasts as an in vitro model system to study cell transformation. These cells obtain a transformed phenotype upon stimulation with growth-modulating factors such as retinoic acid (RA) or transforming growth factor- (TGF-). Patch-clamp experiments showed that transformation is paralleled by a profound membrane depolarization from around –70 to –20 mV. This depolarization is caused by a compound in the medium conditioned by transformed NRK cells, which enhances intracellular Ca²⁺ levels and thereby activates Ca²⁺-dependent Cl^– channels. This compound was identified as prostaglandin F₂ (PGF₂) using electrospray ionization mass spectrometry. The active concentration in the medium conditioned by transformed NRK cells as determined using an enzyme immunoassay was 19.7 ± 2.5 nM (n = 6), compared with 1.5 ± 0.1 nM (n = 3) conditioned by nontransformed NRK cells. Externally added PGF₂ was able to trigger NRK cells that had grown to density arrest to restart their proliferation. This proliferation was inhibited when the FP receptor (i.e., natural receptor for PGF₂) was blocked by AL-8810. RA-induced phenotypic transformation of NRK cells was partially (25%) suppressed by AL-8810. Our results demonstrate that PGF₂ acts as an autocrine enhancer and paracrine inducer of cell transformation and suggest that it may play a crucial role in carcinogenesis in general. membrane potential; intracellular calcium; mass spectrometry; FP receptor     

Differential regulation of voltage-gated K+ channels by oxidized and reduced pyridine nucleotide coenzymes

The activity of the voltage-sensitive K⁺ (Kv) channels varies as a function of the intracellular redox state and metabolism, and several Kv channels act as oxygen sensors. However, the mechanisms underlying the metabolic and redox regulation of these channels remain unclear. In this study we investigated the regulation of Kv channels by pyridine nucleotides. Heterologous expression of Kv1.5 in COS-7 cells led to the appearance of noninactivating currents. Inclusion of 0.1–1 mM NAD⁺ or 0.03–0.5 mM NADP⁺ in the internal solution of the patch pipette did not affect Kv currents. However, 0.5 and 1 mM NAD⁺ and 0.1 and 0.5 mM NADP⁺ prevented inactivation of Kv currents in cells transfected with Kv1.5 and Kv1.3 and shifted the voltage dependence of activation to depolarized potentials. The Kv-dependent inactivation of Kv currents was also decreased by internal pipette perfusion of the cell with 1 mM NAD⁺. The Kv1.5-Kv1.3 currents were unaffected by the internal application of 0.1 mM NADPH or 0.1 or 1 mM NADH. Excised inside-out patches from cells expressing Kv1.5-Kv1.3 showed transient single-channel activity. The mean open time and the open probability of these currents were increased by the inclusion of 1 mM NAD⁺ in the perfusate. These results suggest that NAD(P)⁺ prevents Kv-mediated inactivation of Kv currents and provide a novel mechanism by which pyridine nucleotides could regulate specific K⁺ currents as a function of the cellular redox state [NAD(P)H-to-NAD(P)⁺ ratio]. Shaker potassium ion channels; Kv subunits; patch clamp; aldo-keto reductase; COS-7 cells     

Angiotensin II-induced MMP-2 release from endothelial cells is mediated by TNF-alpha

Angiotensin II (ANG II) has been etiologically linked to vascular disease; however, its role in the alterations of endothelial function that occur in vascular disorders is not completely understood. Matrix metalloproteinases (MMPs) and proinflammatory cytokines are involved in the pathological remodeling of blood vessels that occurs in vascular disease. In this study we evaluated the effects of ANG II on tumor necrosis factor (TNF)- and MMP-2 production in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with ANG II (0.1–10 µM) for 24 h, in the presence or absence of antagonists of ANG II type 1 (AT₁R) and type 2 (AT₂R) receptors, and the production and release of TNF- and MMP-2 were assessed. ANG II increased TNF- mRNA and protein expression and the release of bioactive TNF-. Moreover, ANG II induced MMP-2 release and reduced the secretion of tissue inhibitor of MMP (TIMP)-2 from endothelial cells. To elucidate whether endogenous TNF- could mediate the effects of ANG II on MMP-2 release, cells were pretreated with anti-TNF- neutralizing antibodies or pentoxifylline (an inhibitor of TNF- synthesis). TNF- inhibition prevented the secretion of MMP-2 induced by ANG II. Furthermore, AT₁R antagonism with candesartan prevented the formation of MMP-2 and TNF- and the reduction of TIMP-2 induced by ANG II. These results indicate that ANG II, via AT₁R, modulates the secretion of TNF- and MMP-2 from endothelial cells and that TNF- mediates the effects of ANG II on MMP-2 release. remodeling; vasoactive mediators; inflammation     

The Na(+)-K(+)-ATPase alpha2-subunit isoform modulates contractility in the perinatal mouse diaphragm

This study uses genetically altered mice to examine the contribution of the Na⁺-K⁺-ATPase ₂ catalytic subunit to resting potential, excitability, and contractility of the perinatal diaphragm. The ₂ protein is reduced by 38% in ₂-heterozygous and absent in ₂-knockout mice, and ₁-isoform is upregulated 1.9-fold in ₂-knockout. Resting potentials are depolarized by 0.8–4.0 mV in heterozygous and knockout mice. Action potential threshold, overshoot, and duration are normal. Spontaneous firing, a developmental function, is impaired in knockout diaphragm, but this does not compromise its ability to fire evoked action potential trains, the dominant mode of activation near birth. Maximum tetanic force, rate of activation, force-frequency and force-voltage relationships, and onset and magnitude of fatigue are not changed. The major phenotypic consequence of reduced ₂ content is that relaxation from contraction is 1.7-fold faster. This finding reveals a distinct cellular role of the ₂-isoform at a step after membrane excitation, which cannot be restored simply by increasing ₁ content. Na⁺/Ca²⁺ exchanger expression decreases in parallel with ₂-isoform, suggesting that Ca²⁺ extrusion is affected by the altered ₂ genotype. There are no major compensatory changes in expression of sarcoplasmic reticulum Ca²⁺-ATPase, phospholamban, or plasma membrane Ca²⁺-ATPase. These results demonstrate that the Na⁺-K⁺-ATPase ₁-isoform alone is able to maintain equilibrium K⁺ and Na⁺ gradients and to substitute for ₂-isoform in most cellular functions related to excitability and force. They further indicate that the ₂-isoform contributes significantly less at rest than expected from its proportional content but can modulate contractility during muscle contraction. Na⁺-K⁺-ATPase ₂ catalytic subunit; heterozygous mice; knockout mice; resting potential     

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     

Migration of leukocytes across an endothelium-epithelium bilayer as a model of renal interstitial inflammation

Interstitial inflammation has emerged as a key event in the development of acute renal failure. To gain better insight into the nature of these inflammatory processes, the interplay between tubular epithelial cells, endothelial cells, and neutrophils (PMN) was investigated. A coculture transmigration model was developed, composed of human dermal microvascular endothelial (HDMEC) and human renal proximal tubular cells (HK-2) cultured on opposite sides of Transwell growth supports. Correct formation of an endoepithelial bilayer was verified by light and electron microscopy. The model was used to study the effects of endotoxin (LPS), tumor necrosis factor (TNF)-, and -melanocyte-stimulating hormone (-MSH) by measuring PMN migration and cytokine release. To distinguish between individual roles of microvascular endothelial and epithelial cells in transmigration processes, migration of PMN was investigated separately in HK-2 and HDMEC monolayers. Sequential migration of PMN through endothelium and epithelium could be observed and was significantly increased after proinflammatory stimulation with either TNF- or LPS (3.5 ± 0.58 and 2.76 ± 0.64-fold vs. control, respectively). Coincubation with -MSH inhibited the transmigration of PMN through the bilayer after proinflammatory stimulation with LPS but not after TNF-. The bilayers produced significant amounts of IL-8 and IL-6 mostly released from the epithelial cells. Furthermore, -MSH decreased LPS-induced IL-6 secretion by 30% but had no significant effect on IL-8 secretion. We established a transmigration model showing sequential migration of PMN across microvascular endothelial and renal tubular epithelial cells stimulated by TNF- and LPS. Anti-inflammatory effects of -MSH in this bilayer model are demonstrated by inhibition on PMN transmigration and IL-6 secretion. coculture; polymorphonuclear neutrophil migration; HK-2; interleukin-8; interleukin-6; -melanocyte-stimulating hormone     

TNF-alpha-mediated apoptosis in vascular smooth muscle cells requires p73

Atherosclerosis, now considered an inflammatory process, is the leading cause of death in the Western world and is manifested by a variety of diseases in multiple organ systems. Because of its prevalence and associated morbidity, novel therapies directed at arresting this progressive process are urgently needed. The inflammatory mediator TNF-, which is known to contribute to apoptosis in vascular smooth muscle cells, has been shown to be intimately involved in the atherosclerotic process, being present at elevated levels in human atheroma as well as possibly being responsible for plaque rupture, a clinically devastating event. In light of our earlier finding that p73 is a proapoptotic protein in vascular smooth muscle cells, which are involved in plaque progression as well as rupture, we asked whether TNF- mediates apoptosis in these cells through p73. We now show that p73 is present in spindle-shaped cells within human atheroma, and p73, an isoform that is pivotal in both apoptosis and growth suppression, is induced in vascular smooth muscle cells in vitro by serum but not by PDGF-BB. In addition, TNF-, when added to these cells in the presence of serum-containing media, increases p73 expression and causes apoptosis in both rat and human vascular smooth muscle cells. Inhibition of p73 activity with a dominant inhibitory NH₂-terminally deleted p73 plasmid results in markedly decreased TNF--induced apoptosis. Thus p73 is likely a mediator of the apoptotic effect of TNF- in the vasculature, such that future targeting of the p73 isoforms may ultimately prove useful in novel atherosclerosis therapies. atherosclerosis; inflammation; plaque     

CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to increase plasma membrane localization and 86Rb+ uptake

The carboxy terminus (CT) of the colonic H⁺-K⁺-ATPase is required for stable assembly with the -subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HK₂, we selected 84 amino acids in the CT of the -subunit of mouse colonic H⁺-K⁺-ATPase (CT-HK₂) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HK₂ with CD63, recombinant CT-HK₂ and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HK₂ protein (but not CT-HK₁) coprecipitated with CD63, confirming stable assembly of HK₂ with CD63. In HEK-293 transfected with HK₂ plus ₁-Na⁺-K⁺-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HK₂/NK₁ and ⁸⁶Rb⁺ uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HK₂-NK₁ complex in the cell membrane. protein assembly; cell surface localization