P2X7 receptor-mediated apoptosis of human cervical epithelial cells

Normal human ectocervical epithelial (hECE) cells undergo apoptosis in culture. Baseline apoptosis could be increased by shifting cells to serum-free medium and blocked by lowering extracellular calcium. Treatment with the ATPase apyrase attenuated baseline apoptosis, suggesting that extracellular ATP and purinergic mechanisms control the apoptosis. Treatment with ATP and the P2X₇ receptor analog 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) increased apoptosis significantly, in a time- and dose-related manner. The threshold of ATP effect was 0.5 µM in hECE cells and 1 µM in CaSki cancer cells. The apoptotic effect of BzATP was additive in part to that of tumor necrosis factor (TNF)-, and it could be attenuated by lowering extracellular calcium and by treatment with the caspase-9 inhibitor Leu-Glu-His-Asp-O-methyl-fluoromethylketone (LEHD-FMK). Treatment with BzATP activated caspase-9, and, in contrast to TNF-, it had only a mild effect on caspase-8. Both BzATP and TNF- activated caspase-3, suggesting that BzATP activates predominantly the mitochondrial apoptotic pathway. Both hECE and CaSki cells secrete ATP into the extracellular fluid, and mean ATP activity in conditioned medium was 0.5 µM, which is in the range of values that suffice to activate the P2X₇ receptor. On the basis of these findings we propose a novel autocrine-paracrine mechanism of cervical cell apoptosis that operates by P2X₇ receptor control of cytosolic calcium and utilizes the mitochondrial apoptotic pathway. cervix; epithelium; ATP; 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate     

Mechanisms of caspase-1 activation by P2X7 receptor-mediated K+ release

The mechanisms underlying caspase-1 activation and IL-1 processing during inflammatory activation of monocytes and macrophages are not well defined. Here, we describe an in vitro proteolytic processing assay that allows for comparison of caspase-1 regulatory components in a cell-free system separately from the confounding issue of IL-1 secretion. Analysis of in vitro IL-1 and caspase-1 processing in lysates from unstimulated Bac1 murine macrophages indicated a slow rate of basal caspase-1 activation and proteolytic maturation of IL-1. In contrast, brief (5 min) treatment of intact macrophages with extracellular ATP (as an activator of the P2X₇ receptor) or nigericin before cell lysis markedly accelerated the in vitro processing of caspase-1 and IL-1. This acceleration of in vitro processing was strictly dependent on loss of intracellular K⁺ from the intact cells. The induction of in vitro caspase-1 activation by lysis per se or by K⁺ loss before lysis was sensitive to pretreatment of intact macrophages with the tyrphostin AG-126 or bromoenol lactone, an inhibitor of Ca²⁺-independent phospholipase A₂. Caspase-1 activation and IL-1 processing in lysates from unstimulated macrophages were also accelerated by addition of recombinant ASC, a previously identified adapter protein that directly associates with caspase-1. These data indicate that increased K⁺ efflux via P2X₇ nucleotide receptor stimulation activates AG-126- and bromoenol lactone-sensitive signaling pathways in murine macrophages that result in stably maintained signals for caspase-1 regulation in cell-free assays. AG-126; ASC; bromoenol lactone; IL-1; inflammation     

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     

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     

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     

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     

Contribution of coupling between human myometrial beta2-adrenoreceptor and the BK(Ca) channel to uterine quiescence

The ₂-adrenergic receptor (₂-AR) and the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel have been shown, separately, to be involved in mediating uterine relaxation. Our recent studies reveal that the levels of both ₂-AR and BK_Ca channel proteins in pregnant human myometrium decrease by 50% after the onset of labor. We present direct evidence in support of a structural and functional association between the ₂-AR and the BK_Ca channel in pregnant human myometrium. Localization of both proteins is predominantly plasmalemmal, with 60% of ₂-AR colocalizing with the BK_Ca channel. Coimmunoprecipitation studies indicate that BK_Ca and ₂-AR are structurally linked by direct protein-protein interactions. Functional correlation was confirmed by experiments of human myometrial contractility in which the BK_Ca channel blocker, paxilline, significantly antagonized the relaxant effect of the ₂-AR agonist ritodrine. These novel findings provide an insight into the coupling between the ₂-AR and BK_Ca channel and may have utility in the application of this signaling cascade for therapeutic potential in the management of preterm labor. ₂-adrenergic receptor; myometrium; potassium channel; preterm labor; uterine contraction     

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     

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     

Nicotinic acetylcholine receptor alpha 7 regulates cAMP signal within lipid rafts

Neuronal nicotinic acetylcholine receptors (nAChRs) are made of multiple subunits with diversified functions. The nAChR ₇-subunit has a property of high Ca²⁺ permeability and may have specific functions and localization within the plasma membrane as a signal transduction molecule. In PC-12 cells, fractionation by sucrose gradient centrifugation revealed that nAChR₇ existed in low-density, cholesterol-enriched plasma membrane microdomains known as lipid rafts where flotillin also exists. In contrast, nAChR ₅- and ₂-subunits were located in high-density fractions, out of the lipid rafts. Type 6 adenylyl cyclase (AC6), a calcium-inhibitable isoform, was also found in lipid rafts and was coimmunoprecipitated with nAChR₇. Cholesterol depletion from plasma membranes with methyl--cyclodextrin redistributed nAChR₇ and AC6 diffusely within plasma membranes. Nicotine stimulation reduced forskolin-stimulated AC activity by 35%, and this inhibition was negated by either treatment with -bungarotoxin, a specific antagonist of nAChR₇, or cholesterol depletion from plasma membranes. The effect of cholesterol depletion was negated by the addition of cholesterol. These data suggest that nAChR₇ has a specific membrane localization relative to other nAChR subunits and that lipid rafts are necessary to localize nAChR₇ with AC within plasma membranes. In addition, nAChR₇ may regulate the AC activity via Ca²⁺ within lipid rafts. cholesterol; PC-12 cells     

P2X receptors in mouse Leydig cells

ATP-activated currents were studied in Leydig cells of mice with the patch-clamp technique. Whole cell currents were rapidly activating and slowly desensitizing (55% decrement from the peak value on exposure to 100 µM ATP for 60 s), requiring 3 min of washout to recover 100% of the response. The concentration-response relationships for ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPS), and 2-methylthio-ATP (2-MeS-ATP) were described by the Hill equation with a concentration evoking 50% of maximal ATP response (K_d) of 44, 110, and 637 µM, respectively, and a Hill coefficient of 2. The order of efficacy of agonists was ATP ATPS > 2-MeS-ATP > 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). -Methylene-ATP (-MeATP), GTP, UTP, cAMP, and adenosine were ineffective. Suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) blocked the responses in a concentration-dependent manner. The ATP-activated currents were dependent on extracellular pH, being maximal at pH 6.5 and decreasing with both acidification and alkalinization (apparent dissociation constant (pK_a) of 5.9 and 7.4, respectively). The whole cell current-voltage relationship showed inward rectification and reversed near 0 mV. Experiments performed in bi-ionic conditions for measurement of reversal potentials showed that this channel is highly permeable to calcium [permeability (P)_Ca/P_Na = 5.32], but not to chloride (P_Cl/P_Na = 0.03) or N-methyl-D-glucamine (NMDG) (P_NMDG/P_Na = 0.09). Unitary currents recorded in outside-out patches had a chord conductance of 27 pS (between –90 and –50 mV) and were inward rectifying. The average current passing through the excised patch decreased with time [time constant () = 13 s], resembling desensitization of the macroscopic current. These findings indicate that the ATP receptor present in Leydig cells shows properties most similar to those of cloned homomeric P2X₂. patch clamp; single channels; ATP; desensitization     

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     

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     

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     

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     

Pathological shear stress directly regulates platelet alphaIIbbeta3 signaling

Integrin mechanotransduction is a ubiquitous biological process. Mechanical forces are transduced transmembranously by an integrin's ligand-bound extracellular domain through its -subunit's cytoplasmic domain connected to the cytoskeleton. This often culminates in the activation of tyrosine kinases directing cell responses. The delicate balance between hemostasis and thrombosis requires exquisitely fine-tuned integrin function, and balance is maintained in vivo despite that the major platelet integrin _IIb3 is continuously subjected to frictional or shearing forces generated by laminar blood flow. To test the hypothesis that platelet function is regulated by the direct effects of mechanical forces on _IIb3, we examined _IIb3/cytoskeletal interactions in human platelets exposed to shear stress in a cone-plate viscometer. We observed that -actinin, myosin heavy chain, and Syk coimmunoprecipitate with _IIb3 in resting platelets and that 120 dyn/cm² shear stress leads to their disassociation from _IIb3. Shear-induced disassociation of -actinin and myosin heavy chain from the ₃ tail is unaffected by blocking von Willebrand factor (VWF) binding to glycoprotein (Gp) Ib-IX-V but abolished by blocking VWF binding to _IIb3. Syk's disassociation from ₃ is inhibited when VWF binding to either GpIb-IX-V or _IIb3 is blocked. Shear stress-induced phosphorylation of SLP-76 and its association with tyrosine-phosphorylated adhesion and degranulation-promoting adapter protein are inhibited by blocking ligand binding to _IIb3 but not by blocking ligand binding to GpIb-IX-V. Chinese hamster ovary cells expressing _IIb3 with ₃ truncated of its cytoskeletal binding domains demonstrate diminished shear-dependent adhesion and cohesion. These results support the hypothesis that shear stress directly modulates _IIb3 function and suggest that shear-induced _IIb3-mediated signaling contributes to the regulation of platelet aggregation by directing the release of constraining cytoskeletal elements from the ₃-tail. platelets; mechanoreceptor; integrin; shear stress; signal transduction     

Differential effects of beta-arrestins on the internalization, desensitization and ERK1/2 activation downstream of protease activated receptor-2

-Arrestins-1 and 2 are known to play important roles in desensitization of membrane receptors and facilitation of signal transduction pathways. It has been previously shown that -arrestins are required for signal termination, internalization, and ERK1/2 activation downstream of protease-activated-receptor-2 (PAR-2), but it is unclear whether they are functionally redundant or mediate specific events. Here, we demonstrate that in mouse embryonic fibroblasts (MEFs) from -arrestin-1/2 knockout mice, Gq signaling by PAR-2, as measured by mobilization of intracellular Ca²⁺, is prolonged. Only expression of -arrestin-1 shortened the signal duration, whereas either -arrestin-1 or 2 was able to restore PKC-induced receptor desensitization. -arrestin-1 also mediated early, while -arrestin-2 mediated delayed, receptor internalization and membrane-associated ERK1/2 activation. While -arrestin-1 colocalized with a lysosomal marker (LAMP-1), -arrestin-2 did not, suggesting a specific role for -arrestin-1 in lysosomal receptor degradation. Together, these data suggest distinct temporal and functional roles for -arrestins in PAR-2 signaling, desensitization, and internalization. arrestins; PAR-2; protease-activated-receptor; G protein-coupled receptor; ERK1/2     

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     

Control of Na+-K+-ATPase beta 1-subunit expression: role of 3'-untranslated region

Using in vitro translation and cell transfection assays, we previously demonstrated that the Na⁺-K⁺-ATPase ₁ mRNA species containing its longest 3'-untranslated region (UTR) exhibited the lowest translational efficiency. Here, employing deletions and in vivo expression assays, using direct injection of plasmids into rat ventricular myocardium, we identified a 143-nt segment located in the distal 3'-UTR of ₁ mRNA that was associated with decreased luciferase expression; interestingly, this segment contains three AUUUA motifs. Using RNA-protein binding assays and UV cross-linking of cRNA with cytosolic proteins of rat heart, we identified an 38-kDa protein that specifically bound to the cRNA encoding the 143-nt segment of ₁ mRNA 3'-UTR. Mutation of three nucleotides located in the middle region of the 143-nt segment, which was predicted to greatly disrupt a putative stem-loop structure of the cRNA in this region, was associated with reduced binding of the mutated cRNA to the protein migrating at 38 kDa. The cRNA encoding a segment of cyclooxygenase-2 mRNA 3'-UTR containing six AUUUA sequences did not bind the protein migrating at 38 kDa and did not compete with the binding of the wild-type 143-nt ₁ cRNA to the protein. The above results suggest that the 143-nt segment in the distal segment of the 3'-UTR of ₁ mRNA may play an important role in the control of ₁-subunit expression. RNA-protein binding; AUUUA sequence; plasmid expression in heart; direct myocardial injection; cardiac expression     

Involvement of NH2 terminus of PKC-delta in binding to F-actin during activation of Calu-3 airway epithelial NKCC1

Direct binding of nonmuscle F-actin and the C2-like domain of PKC- (C2-like domain) is involved in hormone-mediated activation of epithelial Na-K-2Cl cotransporter isoform 1 (NKCC1) in a Calu-3 airway epithelial cell line. The goal of this study was to determine the site of actin binding on the 123-amino acid C2-like domain. Truncations of the C2-like domain were made by restriction digestion and confirmed by nucleotide sequencing. His₆-tagged peptides were expressed in bacteria, purified, and analyzed with a Coomassie blue stain for predicted size and either a 6xHis protein tag stain or an INDIA His₆ probe for expression of the His₆ tag. Truncated peptides were tested for competitive inhibition of binding of activated, recombinant PKC- with nonmuscle F-actin. Peptides from the NH₂-terminal region, but not the COOH-terminal region, of the C2-like domain blocked binding of activated PKC- to F-actin. The C2-like domain and three NH₂-terminal truncated peptides of 17, 83, or 108 amino acids blocked binding, with IC₅₀ values ranging from 1.2 to 2.2 nmol (6–11 µM). NH₂-terminal C2-like peptides also prevented methoxamine-stimulated NKCC1 activation and pulled down endogenous actin from Calu-3 cells. The proximal NH₂ terminus of the C2-like domain encodes a 1-sheet region. The amino acid sequence of the actin-binding domain is distinct from actin-binding domains in other PKC isotypes and actin-binding proteins. Our results indicate that F-actin likely binds to the 1-sheet region of the C2-like domain in airway epithelial cells. truncation; protein kinase C-; C2-like domain; slot blot assay; inhibitory constant; bumetanide; Na-K-2Cl cotransporter