Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2

We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC₂₀) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P₁ and S1P₂ receptors. S1P activated G_q, G₁₃, and all G_i isoforms and stimulated PLC-1, PLC-3, and Rho kinase activities. PLC- activity was partially inhibited by pertussis toxin (PTX), G or G_q antibody, PLC-1 or PLC-3 antibody, and by expression of G_q or G_i minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of G₁₃ or G_q minigene and abolished by expression of both. S1P stimulated Ca²⁺ release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC₅₀ 1 nM). Initial contraction and MLC₂₀ phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and G_q or G antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC₂₀ phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P₂ and S1P₁ involving concurrent activation of PLC-1 and PLC-3 via G_q and G_i, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca²⁺ release and MLCK-mediated MLC₂₀ phosphorylation, and 2) sustained contraction exclusively mediated by S1P₂ involving activation of RhoA via G_q and G₁₃, resulting in Rho kinase- and PKC-dependent MLC₂₀ phosphorylation. muscle contraction; signal transduction     

beta-Adrenergic receptor-stimulated apoptosis in adult cardiac myocytes involves MMP-2-mediated disruption of beta1 integrin signaling and mitochondrial pathway

Stimulation of -adrenergic receptors (-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of matrix metalloproteinase-2 (MMP-2) inhibits -AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with ₁ integrins. Herein we tested the hypothesis that MMP-2 impairs ₁ integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, whereas treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited -AR-stimulated activation of JNKs and levels of cytosolic cytochrome c. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated -AR-stimulated decreases in mitochondrial membrane potential. Overexpression of ₁ integrins using adenoviruses expressing the human _1A-integrin decreased -AR-stimulated cytochrome c release and apoptosis. Overexpression of ₁ integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the ₁ integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis. matrix metalloproteinases; focal adhesion kinase; c-Jun NH₂-terminal kinase; cytochrome c     

Matrix metalloproteinases mediate beta-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes

Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in -adrenergic receptor (-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). -AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed -AR-stimulated increases in MMP-2 protein levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited -AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with ₁-integrins after -AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of ₁-integrin signaling using laminin inhibited the increased association of MMP-2 with ₁-integrins. -AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) -AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits -AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with ₁ integrins and poly-ADP-ribose-polymerase cleavage. integrins; poly-ADP-ribose-polymerase     

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     

Rat glomerular mesangial cells require laminin-9 to migrate in response to insulin-like growth factor binding protein-5

Temporal and spatial differences in extracellular matrix play critical roles in cell proliferation, differentiation and migration. Different migratory stimuli use different substrates and receptors to achieve cell migration. To understand the mechanism of insulin-like growth factor binding protein-5 (IGFBP-5)-induced migration in mesangial cells, the roles of integrins and substrates were examined. IGFBP-5 induced an increase in mRNA expression for laminin (LN) chains lama4, lamb2, and lamc1, suggesting that LN-9 might be required for migration. Antibodies to the LN₄ and LN₂ chains, but not LN₁, blocked IGFBP-5-induced migration. Anti-sense morpholino oligonucleotide inhibition of expression of LN₄ substantially reduced expression of LN-8/9 (₄₁₁/₄₂₁, 411/421) and prevented IGFBP-5-induced migration. Anti-sense inhibition of lamb2 reduced expression of LN-9. Absence of LN-9 prevented IGFBP-5-induced migration, which was not preserved by continued expression of LN-8. The requirement for LN-9 was further supported by studies of T98G cells, which express predominantly LN-8. IGFBP-5 had little effect on migration in these cells, but increased migration when T98G cells were plated on LN-8/9. IGFBP-5-mediated mesangial cell migration was inhibited by antibodies that block attachment to ₆₁-integrins but was unaffected by antibodies and disintegrins that block binding to other integrins. Furthermore, in cells with anti-sense inhibited expression of LN-9, integrin ₆₁ was no longer detected on the cell surface. These studies suggest the specificity of mechanisms of migration induced by specific stimuli and for the first time demonstrate a unique function for LN-9 in mediating IGFBP-5-induced migration. migration; integrins; extracellular matrix     

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.     

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     

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     

Mechanotransduction by integrin is essential for IL-6 secretion from endothelial cells in response to uniaxial continuous stretch

We previously reported that uniaxial continuous stretch in human umbilical vein endothelial cells (HUVECs) induced interleukin-6 (IL-6) secretion via IB kinase (IKK)/nuclear factor-B (NF-B) activation. The aim of the present study was to clarify the upstream signaling mechanism responsible for this phenomenon. Stretch-induced IKK activation and IL-6 secretion were inhibited by application of ₅₁ integrin-inhibitory peptide (GRGDNP), phosphatidylinositol 3-kinase inhibitor (LY-294002), phospholipase C- inhibitor (U-73122), or protein kinase C inhibitor (H7). Although depletion of intra- or extracellular Ca²⁺ pool using thapsigargin (TG) or EGTA, respectively, showed little effect, a TG-EGTA mixture significantly inhibited stretch-induced IKK activation and IL-6 secretion. An increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) upon continuous stretch was observed even in the presence of TG, EGTA, or GRGDNP, but not in a solution containing the TG-EGTA mixture, indicating that both integrin activation and [Ca²⁺]_i rise are crucial factors for stretch-induced IKK activation and after IL-6 secretion in HUVECs. Furthermore, while PKC activity was inhibited by the TG-EGTA mixture, GRGDNP, LY-294002, or U-73122, PLC- activity was retarded by GRGDNP or LY-294002. These results indicate that continuous stretch-induced IL-6 secretion in HUVECs depends on outside-in signaling via integrins followed by a PI3-K-PLC--PKC-IKK-NF-B signaling cascade. Another crucial factor, [Ca²⁺]_i increase, may at least be required to activate PKC needed for NF-B activation. nuclear factor-B; phosphatidylinositol 3-kinase; phospholipase C-; protein kinase C; intracellular Ca²⁺ concentration     

Oligomycin inhibits HIF-1alpha expression in hypoxic tumor cells

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of cellular responses to reduced oxygen availability. The contribution of mitochondria in regulation of HIF-1 in hypoxic cells has received recent attention. We demonstrate that inhibition of electron transport complexes I, III, and IV diminished hypoxic HIF-1 accumulation in different tumor cell lines. Hypoxia-induced HIF-1 accumulation was not prevented by the antioxidants Trolox and N-acetyl-cysteine. Oligomycin, inhibitor of F₀F₁-ATPase, prevented hypoxia-induced HIF-1 protein accumulation and had no effect on HIF-1 induction by hypoxia-mimicking agents desferrioxamine or dimethyloxalylglycine. The inhibitory effect of mitochondrial respiratory chain inhibitors and oligomycin on hypoxic HIF-1 content was pronounced in cells exposed to hypoxia (1.5% O₂) but decreased markedly when cells were exposed to severe oxygen deprivation (anoxia). Taken together, these results do not support the role for mitochondrial reactive oxygen species in HIF-1 regulation, but rather suggest that inhibition of electron transport chain and impaired oxygen consumption affect HIF-1 accumulation in hypoxic cells indirectly via effects on prolyl hydroxylase function. hypoxia-inducible factor 1; oxygen sensing     

Novel role for alphavbeta5-integrin in retinal adhesion and its diurnal peak

_v5-Integrin is the sole integrin receptor at the retinal pigment epithelium (RPE)-photoreceptor interface and promotes RPE phagocytic signaling to the tyrosine kinase Mer tyrosine kinase (MerTK) once a day in response to circadian photoreceptor shedding. Herein we identify a novel role for _v5-integrin in permanent RPE-photoreceptor adhesion that is independent of _v5's function in retinal phagocytosis. To compare retinal adhesion of wild-type and ₅-integrin^–/– mice, we mechanically separated RPE and neural retina and quantified RPE protein and pigment retention with the neural retina. Lack of _v5-integrin with normal expression of other RPE integrins greatly weakened retinal adhesion in young mice and accelerated its age-dependent decline. Unexpectedly, the strength of wild-type retinal adhesion varied with a diurnal rhythm that peaked 3.5 h after light onset, after the completion of phagocytosis, when integrin signaling to MerTK is minimal. Permanent _v5 receptor deficiency attenuated the diurnal peak of retinal adhesion in ₅-integrin^–/– mice. These results identify _v5-integrin as the first RPE receptor that contributes to retinal adhesion, a vital mechanism for long-term photoreceptor function and viability. Furthermore, they indicate that _v5 receptors at the same apical plasma membrane domain of RPE cells fulfill two separate functions that are synchronized by different diurnal rhythms. circadian rhythm; knockout; photoreceptors; retinal pigment epithelium     

Galpha12 regulates epithelial cell junctions through Src tyrosine kinases

Regulation and assembly of the epithelial cell junctional complex involve multiple signaling mechanisms, including heterotrimeric G proteins. Recently, we demonstrated that G₁₂ binds to the tight junction scaffolding protein ZO-1 through the SH3 domain and that activated G₁₂ increases paracellular permeability in Madin-Darby canine kidney (MDCK) cells (Meyer et al. J Biol Chem 277: 24855-24858, 2002). In the present studies, we explore the effects of G₁₂ expression on tight and adherens junction proteins and examine downstream signaling pathways. By confocal microscopy, we detect disrupted tight and adherens junction proteins with increased actin stress fibers in constitutively active G₁₂ (QL₁₂)-expressing MDCK cells. The normal distribution of ZO-1 and Na-K-ATPase was altered in QL₁₂-expressing MDCK cells, consistent with loss of polarity. We found that the tyrosine kinase inhibitor genistein and the Src-specific inhibitor PP-2 reversibly abrogated the QL₁₂ phenotype on the junctional complex. Junctional protein localization was preserved in PP-2- or genistein-treated QL₁₂-expressing cells, and the increase in paracellular permeability as measured by transepithelial resistance and [³H]mannitol flux was prevented by the inhibitors. Src activity was increased in QL₁₂-expressing MDCK cells as assessed by Src autophosphorylation, and -catenin tyrosine phosphorylation was also increased, although there was no detectable increase in Rho activity. Taken together, these results indicate that G₁₂ regulates MDCK cell junctions, in part through Src tyrosine kinase pathways. G proteins; tight junction; adherens junction; Rho     

Functional analysis of the R1086H malignant hyperthermia mutation in the DHPR reveals an unexpected influence of the III-IV loop on skeletal muscle EC coupling

Malignant hyperthermia (MH) is an inherited pharmacogenetic disorder caused by mutations in the skeletal muscle ryanodine receptor (RyR1) and the dihydropyridine receptor (DHPR) _1S-subunit. We characterized the effects of an MH mutation in the DHPR cytoplasmic III-IV loop of _1S (R1086H) on DHPR-RyR1 coupling after reconstitution in dysgenic (_1S null) myotubes. Compared with wild-type _1S, caffeine-activated Ca²⁺ release occurred at approximately fivefold lower concentrations in nonexpressing and R1086H-expressing myotubes. Although maximal voltage-gated Ca²⁺ release was similar in _1S- and R1086H-expressing myotubes, the voltage dependence of Ca²⁺ release was shifted 5 mV to more negative potentials in R1086H-expressing myotubes. Our results demonstrate that _1S functions as a negative allosteric modulator of release channel activation by caffeine/voltage and that the R1086H MH mutation in the intracellular III-IV linker disrupts this negative regulatory influence. Moreover, a low caffeine concentration (2 mM) caused a similar shift in voltage dependence of Ca²⁺ release in _1S- and R1086H-expressing myotubes. Compared with _1S-expressing myotubes, maximal L channel conductance (G_max) was reduced in R1086H-expressing myotubes (_1S 130 ± 10.2, R1086H 88 ± 6.8 nS/nF; P < 0.05). The decrease in G_max did not result from a change in retrograde coupling with RyR1 as maximal conductance-charge movement ratio (G_max/Q_max) was similar in _1S- and R1086H-expressing myotubes and a similar decrease in G_max was observed for an analogous mutation engineered into the cardiac L channel (R1217H). In addition, both R1086H and R1217H DHPRs targeted normally and colocalized with RyR1 in sarcoplasmic reticulum (SR)-sarcolemmal junctions. These results indicate that the R1086H MH mutation in _1S enhances RyR1 sensitivity to activation by both endogenous (voltage sensor) and exogenous (caffeine) activators. excitation-contraction coupling; calcium channel; muscle disease     

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     

nPKC{varepsilon}, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells

Airway goblet cell mucin secretion is controlled by agonist activation of P2Y₂ purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (IP₃), diacylglycerol, Ca²⁺ and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKC, nPKC, nPKC, and nPKC; of these, only nPKC translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149–L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKC, nPKC, and nPKC had the same levels of ATPS- and phorbol-1,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKC (14.6 and 23.5%, for ATPS and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKC exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATPS-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y₂-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKC knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKC KO mice relative to its WT littermates. We conclude that nPKC is the effector isoform downstream of P2Y₂-R activation in the goblet cell secretory response. The translocation of nPKC observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology. protein kinase C; mucins; goblet cells; exocytosis; airways; epithelium; lung     

Dominant-negative effects of human P/Q-type Ca2+ channel mutations associated with episodic ataxia type 2

Episodic ataxia type 2 (EA2) is an inherited autosomal dominant disorder related to cerebellar dysfunction and is associated with mutations in the pore-forming _1A-subunits of human P/Q-type Ca²⁺ channels (Cav2.1 channels). The majority of EA2 mutations result in significant loss-of-function phenotypes. Whether EA2 mutants may display dominant-negative effects in human, however, remains controversial. To address this issue, five EA2 mutants in the long isoform of human _1A-subunits were expressed in Xenopus oocytes to explore their potential dominant-negative effects. Upon coexpressing the cRNA of _1A-WT with each _1A-mutant in molar ratios ranging from 1:1 to 1:10, the amplitude of Ba²⁺ currents through wild-type (WT)-Cav2.1 channels decreased significantly as the relative molar ratio of _1A-mutants increased, suggesting the presence of an _1A-mutant-specific suppression effect. When we coexpressed _1A-WT with proteins not known to interact with Cav2.1 channels, we observed no significant suppression effects. Furthermore, increasing the amount of auxiliary subunits resulted in partial reversal of the suppression effects in nonsense but not missense EA2 mutants. On the other hand, when we repeated the same coinjection experiments of _1A-WT and mutant using a splice variant of _1A-subunit that contained a considerably shorter COOH terminus (i.e., the short isoform), no significant dominant-negative effects were noted until we enhanced the relative molar ratio to 1:10. Altogether, these results indicate that for human WT-Cav2.1 channels comprising the long-_1A-subunit isoform, both missense and nonsense EA2 mutants indeed display prominent dominant-negative effects. channelopathy; voltage clamp; Xenopus oocytes; cerebellum; splice variants     

Activation of large-conductance, Ca2+-activated K+ channels by cannabinoids

We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca²⁺-activated K⁺ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the -subunit of the BK channel (BK-), both - and ₁-subunits (BK-₁), or both - and ₄-subunits (BK-₄) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-₁ within a similar concentration range. Because methAEA could potentiate BK-, BK-₁, and BK-₄ with similar efficacy, the -subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK- and HEK-BK-₁ did not change intracellular Ca²⁺ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB₁ antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB₂ agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed. anandamide; channel opener     

An osteoclastic protein-tyrosine phosphatase may play a role in differentiation and activity of human monocytic U-937 cell-derived, osteoclast-like cells

This study investigated if an osteoclastic protein-tyrosine phosphatase (PTP), PTP-oc, plays a role in the functional activity and differentiation of osteoclastic cells by determining the effects of overexpression of wild-type (WT)- or phosphatase-deficient (PD)-PTP-oc on bone resorption activity and differentiation of human promyelomonocytic U-937 cells, which could be induced to differentiate into "osteoclast-like" cells by phorbol ester/1,25(OH)₂D₃ treatment. U-937 cells overexpressing WT- or PD-PTP-oc were produced with a transposon-based vector. The size and depth of resorption pits created by WT-PTP-oc-overexpressing osteoclast-like cells were greater, while those by PD-PTP-oc-overexpressing osteoclast-like cells were less, than those created by control osteoclast-like cells. Overexpression of WT-PTP-oc also enhanced, while overexpression of PD-PTP-oc suppressed, their differentiation into osteoclast-like cells. Overexpression of WT-PTP-oc increased apoptosis and proliferation of U-937 cells, and overexpression of PD-PTP-oc reduced cell proliferation. Cells overexpressing WT-PTP-oc has also led to greater c-Src and NF- activation, whereas cells overexpressing PD-PTP-oc resulted in less c-Src and NF- activation. c-Src activation and NF- activation each correlated with resorption activity and differentiation into osteoclast-like cells. In summary, these results show that 1) PTP-oc regulates both the activity and the differentiation of osteoclast-like cells derived from U-937 cells; 2) PTP-oc enzymatic activity is important to these processes; 3) high PTP-oc enzymatic activity caused an increase in U-937 cell apoptosis and proliferation, leading to no significant changes in the number of viable cells; and 4) some of the PTP-oc actions are mediated in part by the c-Src and/or NF- pathways. osteoclast; resorption; nuclear factor-; c-Src     

Novel effect of NF-kappaB activation: carbonylation and nitration injury to cytoskeleton and disruption of monolayer barrier in intestinal epithelium

Using monolayers of intestinal cells, we reported that upregulation of inducible nitric oxide synthase (iNOS) is required for oxidative injury and that activation of NF-B is key to cytoskeletal instability. In the present study, we hypothesized that NF-B activation is crucial to oxidant-induced iNOS upregulation and its injurious consequences: cytoskeletal oxidation and nitration and monolayer dysfunction. Wild-type (WT) cells were pretreated with inhibitors of NF-B, with or without exposure to oxidant (H₂O₂). Other cells were transfected with an IB mutant (an inhibitor of NF-B). Relative to WT cells exposed to vehicle, oxidant exposure caused increases in IB instability, NF-B subunit activation, iNOS-related activity (NO, oxidative stress, tubulin nitration), microtubule disassembly and instability (increased monomeric and decreased polymeric tubulin), and monolayer disruption. Monolayers pretreated with NF-B inhibitors (MG-132, lactacystin) were protected against oxidation, showing decreases in all measures of the NF-B iNOS NO pathway. Dominant mutant stabilization of IB to inactivate NF-B suppressed all measures of the iNOS/NO upregulation while protecting monolayers against oxidant insult. In these mutants, we found prevention of tubulin nitration and oxidation and enhancement of cytoskeletal and monolayer stability. We concluded that 1) NF-B is required for oxidant-induced iNOS upregulation and for the consequent nitration and oxidation of cytoskeleton; 2) NF-B activation causes cytoskeletal injury following upregulation of NO-driven processes; and 3) the molecular event underlying the destabilizing effects of NF-B appears to be increases in carbonylation and nitrotyrosination of the subunit components of cytoskeleton. The ability to promote NO overproduction and cytoskeletal nitration/oxidation is a novel mechanism not previously attributed to NF-B in cells. tubulin cytoskeleton; microtubules; oxidation/nitration; inducible nitric oxide synthase/peroxynitrite; inflammatory bowel disease; Caco-2 cells; gut barrier; nuclear factor-B/IB     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A