共查询到20条相似文献,搜索用时 31 毫秒
1.
Zhou YW Oak SA Senogles SE Jarrett HW 《American journal of physiology. Cell physiology》2005,288(2):C377-C388
-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 Gs and G occurs and active Gs, measured as GTP-35S bound, decreases. Because intracellular Ca2+ is elevated in Duchenne muscular dystrophy and Gs is known to activate the dihydropyridine receptor Ca2+ channel, whether laminin also altered intracellular Ca2+ was investigated. Laminin-1 decreases active (GTP-S-bound) Gs, 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 相似文献
2.
Fan H Zingarelli B Peck OM Teti G Tempel GE Halushka PV Spicher K Boulay G Birnbaumer L Cook JA 《American journal of physiology. Cell physiology》2005,289(2):C293-C301
Heterotrimeric Gi 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 Gi proteins would alter mediator production induced by LPS and gram-positive bacterial stimulation. We examined genetic deletion of Gi2 or Gi1/3 protein in Gi2-knockout (Gi2/) or Gi1/3-knockout (Gi1/3/) 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 Gi2/ mice compared with wild-type (WT) mice. Also, LPS-induced TNF- was increased in splenocytes from Gi1/3/ mice. In contrast to splenocytes, LPS-, SA-, and GBS-induced TNF-, IL-10, and thromboxane B2 (TxB2) production was decreased in M harvested from Gi2/ mice. Also, LPS-induced production of IL-10 and TxB2 was decreased in M from Gi1/3/ mice. In subsequent in vivo studies, TNF- levels after LPS challenge were significantly greater in Gi2/ 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 Gi2/ mice compared with WT mice. These data suggest that Gi proteins differentially regulate murine TLR-mediated inflammatory cytokine production in a cell-specific manner in response to both LPS and gram-positive microbial stimuli. Gi protein-deficient mice; endotoxin; group B streptococci; Staphylococcus aureus; Toll-like receptors 相似文献
3.
Sade H Muraki K Ohya S Hatano N Imaizumi Y 《American journal of physiology. Cell physiology》2006,290(1):C77-C86
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 相似文献
4.
Radzyukevich TL Moseley AE Shelly DA Redden GA Behbehani MM Lingrel JB Paul RJ Heiny JA 《American journal of physiology. Cell physiology》2004,287(5):C1300-C1310
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 0.84.0 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 相似文献
5.
CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to increase plasma membrane localization and 86Rb+ uptake 总被引:1,自引:0,他引:1
Codina Juan; Li Jian; DuBose Thomas D. Jr. 《American journal of physiology. Cell physiology》2005,288(6):C1279
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 相似文献
6.
Functional analysis of Na+/K+-ATPase isoform distribution in rat ventricular myocytes 总被引:1,自引:0,他引:1
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 (IPump) measurements and the different ouabain sensitivity of NKA-1 (low) and NKA-2 (high) in rat heart. Ouabain-dependent IPump inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-2, K1/2 = 0.38 ± 0.16 µM) that accounts for 29.5 ± 1.3% of IPump and a low-affinity isoform (NKA-1, K1/2 = 141 ± 17 µM) that accounts for 70.5% of IPump. Detubulation decreased cell capacitance from 164 ± 6 to 120 ± 8 pF and reduced IPump 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 IPump. Thus, 63% of IPump 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 相似文献
7.
Feng S Lu X Reséndiz JC Kroll MH 《American journal of physiology. Cell physiology》2006,291(6):C1346-C1354
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 相似文献
8.
Laminin-10 and Lutheran blood group glycoproteins in adhesion of human endothelial cells 总被引:3,自引:0,他引:3
Vainionpää N Kikkawa Y Lounatmaa K Miner JH Rousselle P Virtanen I 《American journal of physiology. Cell physiology》2006,290(3):C764-C775
Laminin 5-chain, a constituent of laminins-10 and -11, is expressed in endothelial basement membranes. In this study we evaluated the roles of 5 laminins and Lutheran blood group glycoproteins (Lu), recently identified receptors of the laminin 5-chain, in the adhesion of human dermal microvascular and pulmonary artery endothelial cells. Field emission scanning electron microscopy and immunohistochemistry showed that the endothelial cells spread on laminin-10 and formed fibronectin-positive fibrillar adhesion structures. Immunoprecipitation results suggested that the cells produced fibronectin, which they could use as adhesion substratum, during the adhesion process. When the protein synthesis during the adhesion was inhibited with cycloheximide, the formation of fibrillar adhesions on laminin-10 was abolished, suggesting that laminin-10 does not stimulate the formation of any adhesion structures. Northern and Western blot analyses showed that the cells expressed Mr 78,000 and 85,000 isoforms of Lu. Quantitative cell adhesion assays showed that in the endothelial cell adhesion to laminin-10, Lu acted in concert with integrins 1 and v3, whereas in the adhesion to laminin-10/11, Lu and integrin 1 were involved. In the cells adhering to the 5 laminins, Lu and the integrins showed uniform cell surface distribution. These findings indicate that 5 laminins stimulate endothelial cell adhesion but not the formation of fibrillar or focal adhesions. Lu mediates the adhesion of human endothelial cells to 5 laminins in collaboration with integrins 1 and v3. integrin; cycloheximide 相似文献
9.
Oligomycin inhibits HIF-1alpha expression in hypoxic tumor cells 总被引:2,自引:0,他引:2
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 相似文献
10.
5'-AMP-activated protein kinase (AMPK) functions as an energy sensor to provide metabolic adaptation under conditions of ATP depletion, such as hypoxia and inhibition of oxidative phosphorylation. Whether activation of AMPK is critical for stimulation of glucose transport in response to inhibition of oxidative phosphorylation is unknown. Here we found that treatment of Glut1-expressing Clone 9 cells with sodium azide (5 mM for 2 h) or the AMPK activator 5'-aminoimidazole-4-carboxamide-1--D-ribofuranoside (AICAR, 2 mM for 2 h) stimulated the rate of glucose transport by two- to fourfold. Use of small interference RNA (siRNA) directed against AMPK1 or AMPK1 + AMPK2 (total AMPK) resulted in a significant inhibition of the glucose transport response and the content of phosphorylated AMPK1 + phosphorylated AMPK2 (total p-AMPK) and phosphorylated acetyl-CoA carboxylase (p-ACC) in response to azide. Transfection with siRNA directed against AMPK2 did not affect the glucose transport response. The efficacy of transfection with siRNAs in reducing AMPK content was confirmed by Western blotting. Incubation of cells with compound C, an inhibitor of AMPK, abrogated the glucose transport response and abolished the increase in total p-AMPK in azide-treated or hypoxia-exposed cells. Simultaneous exposure to azide and AICAR did not augment the rate of transport in response to AICAR alone. There was no evidence of coimmunoprecipitation of total p-AMPK with Glut1. However, LKB1 was associated with total p-AMPK. We conclude that activation of AMPK plays both a sufficient and a necessary role in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation. small interference RNA; compound C; hypoxia 相似文献
11.
TNF-alpha dilates cerebral arteries via NAD(P)H oxidase-dependent Ca2+ spark activation 总被引:1,自引:0,他引:1
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+ (KCa) channel transients 1.7- and 1.4-fold, respectively. TNF--induced transient KCa 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 KCa 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 KCa 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 相似文献
12.
Modulation of alpha7-integrin-mediated adhesion and expression by platelet-derived growth factor in vascular smooth muscle cells 总被引:1,自引:0,他引:1
Chao JT Martinez-Lemus LA Kaufman SJ Meininger GA Ramos KS Wilson E 《American journal of physiology. Cell physiology》2006,290(4):C972-C980
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 相似文献
13.
Uezono Y Kaibara M Murasaki O Taniyama K 《American journal of physiology. Cell physiology》2004,287(4):C885-C894
We studied the functions of -subunits of Gi/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-HT1A receptor (5-HT1AR), -opioid receptor, or GABAB 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-HT1AR. The 5-HT-induced enhancement of Gs-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin (Gt). 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 Gi/o protein contribute to the enhancement of Gs-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT1AR 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 Gt, suggesting that -subunits of Gi/o protein activate PLC-2 and then cause intracellular Ca2+ mobilization. Our results indicate that -subunits of Gi/o protein participate in diverse intracellular signals, enhancement of Gs-coupled receptor-mediated responses, and intracellular Ca2+ mobilization. G protein-coupled receptor; cystic fibrosis transmembrane conductance regulator gene; cross talk; electrophysiology 相似文献
14.
Chen L Hodges RR Funaki C Zoukhri D Gaivin RJ Perez DM Dartt DA 《American journal of physiology. Cell physiology》2006,291(5):C946-C956
Transactivation of EGF receptors by G protein-coupled receptors is a well-known phenomenon. This process involves the ectodomain shedding of growth factors in the EGF family by matrix metalloproteinases. However, many of these studies employ transformed and/or cultured cells that overexpress labeled growth factors. In addition, few studies have shown that EGF itself is the growth factor that is shed and is responsible for transactivation of the EGF receptor. In this study, we show that freshly isolated, nontransformed lacrimal gland acini express two of the three known 1-adrenergic receptors (ARs), namely, 1B- and 1D-ARs. 1D-ARs mediate phenylephrine (an 1-adrenergic agonist)-induced protein secretion and activation of p42/p44 MAPK, because the 1D-AR inhibitor BMY-7378, but not the 1A-AR inhibitor 5-methylurapidil, inhibits these processes. Activation of p42/p44 MAPK occurs through transactivation of the EGF receptor, which is inhibited by the matrix metalloproteinase ADAM17 inhibitor TAPI-1. In addition, phenylephrine caused the shedding of EGF from freshly isolated acini into the buffer. Incubation of freshly isolated cells with conditioned buffer from cells treated with phenylephrine resulted in activation of the EGF receptor and p42/p44 MAPK. The EGF receptor inhibitor AG1478 and an EGF-neutralizing antibody blocked this activation of p42/p44 MAPK. We conclude that in freshly isolated lacrimal gland acini, 1-adrenergic agonists activate the 1D-AR to stimulate protein secretion and the ectodomain shedding of EGF to transactivate the EGF receptor, potentially via ADAM17, which activates p42/p44 MAPK to negatively modulate protein secretion. epidermal growth factor ectodomain shedding; protein secretion; signal transduction 相似文献
15.
Jeng CJ Chen YT Chen YW Tang CY 《American journal of physiology. Cell physiology》2006,290(4):C1209-C1220
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 相似文献
16.
Ju JS Gitcho MA Casmaer CA Patil PB Han DG Spencer SA Fisher JS 《American journal of physiology. Cell physiology》2007,292(1):C564-C572
Data from the use of activators and inhibitors of the AMP-activated protein kinase (AMPK) suggest that AMPK increases sensitivity of glucose transport to stimulation by insulin in muscle cells. We assayed insulin action after adenoviral (Ad) transduction of constitutively active (CA; a truncated form of AMPK1) and dominant-negative (DN; which depletes endogenous AMPK) forms of AMPK (Ad-AMPK-CA and Ad-AMPK-DN, respectively) into C2C12 myotubes. Compared with control (Ad-green fluorescent protein), Ad-AMPK-CA increased the ability of insulin to stimulate glucose transport. The increased insulin action in cells expressing AMPK-CA was suppressed by compound C (an AMPK inhibitor). Exposure of cells to 5-aminoimidazole-4-carboxamide-1-D-ribofuranoside (an AMPK activator) increased insulin action in uninfected myotubes and myotubes transduced with green fluorescent protein but not in Ad-AMPK-DN-infected myotubes. In Ad-AMPK-CA-transduced cells, serine phosphorylation of insulin receptor substrate 1 was decreased at a mammalian target of rapamycin (or p70 S6 kinase) target site that has been reported to be associated with insulin resistance. These data suggest that, in myotubes, activated AMPK1 is sufficient to increase insulin action and that the presence of functional AMPK is required for 5-aminoimidazole-4-carboxamide-1,D-ribofuranoside-related increases in insulin action. compound C; AMPK; insulin sensitivity; Akt; mTOR 相似文献
17.
Weiss RG O'Connell KM Flucher BE Allen PD Grabner M Dirksen RT 《American journal of physiology. Cell physiology》2004,287(4):C1094-C1102
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 (Gmax) was reduced in R1086H-expressing myotubes (1S 130 ± 10.2, R1086H 88 ± 6.8 nS/nF; P < 0.05). The decrease in Gmax did not result from a change in retrograde coupling with RyR1 as maximal conductance-charge movement ratio (Gmax/Qmax) was similar in 1S- and R1086H-expressing myotubes and a similar decrease in Gmax 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 相似文献
18.
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 总被引:1,自引:0,他引:1
Krumenacker JS Kots A Murad F 《American journal of physiology. Cell physiology》2005,289(4):C778-C784
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. 相似文献
19.
Menon B Singh M Ross RS Johnson JN Singh K 《American journal of physiology. Cell physiology》2006,290(1):C254-C261
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 相似文献
20.
Hassouna A Matata BM Galiñanes M 《American journal of physiology. Cell physiology》2004,287(5):C1418-C1425
Protein kinase C (PKC) is involved in the process of ischemic preconditioning (IPC), although the precise mechanism is still a subject of debate. Using specific PKC inhibitors, we investigated which PKC isoforms were involved in IPC of the human atrial myocardium sections and to determine their temporal relationship to the opening of mitochondrial potassium-sensitive ATP (mitoKATP) channels. Right atrial muscles obtained from patients undergoing elective cardiac surgery were equilibrated and then randomized to receive any of the following protocols: aerobic control, 90-min simulated ischemia/120-min reoxygenation, IPC using 5-min simulated ischemia/5-min reoxygenation followed by 90-min simulated ischemia/120-min reoxygenation and finally, PKC inhibitors were added 10 min before and 10 min during IPC followed by 90-min simulated ischemia/120-min reoxygenation. The PKC isoforms inhibitors investigated were V12 peptide, GO-6976, rottlerin, and LY-333531 for PKC-, -, - and -, respectively. To investigate the relation of PKC isoforms to mitoKATP channels, PKC inhibitors found to be involved in IPC were added 10 min before and 10 min during preconditioning by diazoxide followed by 90-min simulated ischemia/120-min reoxygenation in a second experiment. Creatine kinase leakage and methylthiazoletetrazolium cell viability were measured. Phosphorylation of PKC isoforms after activation of the sample by either diazoxide or IPC was detected by using Western blot analysis and then analyzed by using Scion image software. PKC- and - inhibitors blocked IPC, whereas PKC- and - inhibitors did not. The protection elicited by diazoxide, believed to be via mitoKATP channels opening, was blocked by the inhibition of PKC- but not - isoforms. In addition, diazoxide caused increased phosphorylation of PKC- to the same extent as IPC but did not affect the phosphorylation of PKC-, a process believed to be critical in PKC activation. The results demonstrate that PKC- and - are involved in IPC of the human myocardium with PKC- being upstream and PKC- being downstream of mitoKATP channels. cardioprotection; protein kinase C isoforms 相似文献