RESUMO
Vasoconstrictor peptides, such as angiotensin II (Ang II) and endothelin-1 (ET-1), stimulate the synthesis and release of vasodilatory prostaglandins from multiple tissues and diverse cellular types. The synthesis of PGE2 and PGI2 acts as a negative feedback loop to antagonize the contractile actions of Ang II and ET-1. Inhibition of prostaglandin synthesis with nonsteroidal anti-inflammatory drugs (NSAIDs) enhances the constrictor actions of Ang II and ET-1 on the vasculature of the kidney and on the glomerulus. The enhanced production of prostaglandins, in response to constrictor peptides, is both short- and long-term. Prostaglandin synthesis is regulated at multiple steps, including: (1) phospholipase A2, which releases arachidonic acid from membrane phospholipids; (2) PGH synthase (PGHS), which converts arachidonic acid to the endoperoxides PGG2 and PGH2; and (3) PG synthases convert the endoperoxides to PGI2, PGE2, and others. ET-1 acutely activates phospholipase A2 through phosphorylation and acute increases of intracellular calcium. ET-1 also chronically enhances phospholipase A2 activity by transcriptional induction of this enzyme. There are no known acute effects of ET-1 to acutely enhance PGHS activity through posttranslational modification of the molecule. Chronically, however, cellular content of PGHS-2 can be induced through transcriptional induction of the enzyme in response to ET-1. Hence, ET-1 short- and long-term activates a modulatory feedback pathway that depends on upregulation of arachidonic acid release through phospholipase A2 and enhanced synthesis of prostaglandin endoperoxides through PGHS-2.
Assuntos
Angiotensina II/farmacologia , Endotelinas/farmacologia , Prostaglandinas/biossíntese , Vasoconstritores/farmacologia , Vasodilatadores/metabolismo , Animais , Hemodinâmica/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacosRESUMO
The action of gamma-aminobutyric acid A (GABAA) and B (GABAB)-agonists has been studied on the membrane potential of astrocytes in explant cultures of rat spinal cord by means of intracellular microelectrode recordings. The GABAB-agonists (-)-baclofen and CGP 27 492 (3-aminopropyl phosphonous acid; 10(-6) to 10(-4) M) caused a hyperpolarization of the majority of astrocytes studied. On approximately 25% of the cells, the compounds had no effect. The hyperpolarization by baclofen (10(-4) M) was reversibly antagonized by the GABAB-antagonist 5-hydroxysaclofen (10(-4) M). GABA and the GABAA-agonist muscimol (10(-4) and 10(-3) M) depolarized approximately two thirds of the glial cells tested, whereas the remaining third remained unaffected. The GABAA-antagonist bicuculline (10(-4) and 10(-3) M) only reduced the depolarization by GABA (10(-4) M) but did not completely block it. On half of the cells tested, the depolarization by GABA was not affected by bicuculline, suggesting that the glial GABAA-receptor is different from the neuronal GABAA-receptor. Our electrophysiological investigations together with recent autoradiographic binding studies strongly suggest the existence of GABAB-receptors on astrocytes whereas there is less evidence for GABAA-sites on these cells.
Assuntos
Astrócitos/efeitos dos fármacos , Baclofeno/farmacologia , Medula Espinal/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologia , Animais , Astrócitos/fisiologia , Autorradiografia , Baclofeno/análogos & derivados , Baclofeno/antagonistas & inibidores , Bicuculina/farmacologia , Eletrofisiologia , Feminino , Antagonistas GABAérgicos , Potenciais da Membrana/efeitos dos fármacos , Microeletrodos , Muscimol/farmacologia , Compostos Organofosforados/farmacologia , Gravidez , Ratos , Medula Espinal/citologiaRESUMO
To test the oxyphoretic properties and potential nephrotoxic side-effects of polymerized hemoglobin solutions, isolated rat kidneys were perfused in a recirculating system for 180 min. Group I was perfused with a substrate enriched Ringer solution containing hydroxyethylstarch (HES) to produce isoncotic conditions. In group II HES was substituted in part by UPPBHb (34 g/l) with a high portion of low molecular weight molecules (= UPPBHb1). In group III 34 g/l of UPPBHb containing an increased fraction of high molecular weight polymers (= UPPBHb2) was used. Only UPPBHb2-perfused kidneys showed a reduced renal perfusate flow (RPF, 13.3 +/- 1.1 ml/min g kw), when compared to HES-perfused controls (15.5 +/- 0.8) and UPPBHb1 (15.1 +/- 1.2). Glomerular filtration rate (GFR) was significantly higher in UPPBHb1-perfused kidneys (902 +/- 107 vs 633 +/- 55 microliters/min g kw for HES). This difference became even more pronounced in the third hour of perfusion (474 +/- 125 vs. 103 +/- 33). In contrast, UPPBHb2 produced low initial GFR levels of 385 +/- 25, which had only a minor tendency to decline with time. Parallel to GFR, absolute reabsorption of sodium (TNa) andoxygen consumption (QO2) showed values of 110 +/- 16 and 5.46 +/- 0.33 mumol/min g kw in UPPBHb1-kidneys vs 83 +/- 6 and 5.09 +/- 0.27 in controls and vs 53 +/- 4 and 3.66 +/- 0.12 in UPPBHb2-kidneys. Fractional excretion of sodium (FENa), of potassium (FEK), and of water (FEH2O) in UPPBHb1 and UPPBHb2-perfused kidneys were not significantly different from HES-perfused controls at any time of perfusion. Urinary flow rate (UFR) was similar in UPPBHb1- and HES-kidneys. Nevertheless, control kidneys tended to render oliguric during the third hour of perfusion (UFR 19.9 +/- 4.1 microliters/min g kw), whereas UPPBHb1 preserved urinary flow in a better way (83.7 +/- 32.4). UFR of UPPBHb2-kidneys was significantly reduced initially (30.2 +/- 5.1 vs. 105 +/- 33 for HES), but increased steadily up to 67 +/- 23. In the UPPBHb1 and HES group, all functional parameters determined declined dramatically within the third hour of perfusion, whereas UPPBHb2 produced functional stability. The in vivo reaction pattern of renal autoregulation was better preserved in UPPBHb-perfused kidneys than in HES-perfused controls: 74 +/- 6 vs. 59 +/- 5 vs. 42 +/- 4% (of full autoregulatory response) for UPPBHb1, UPPBHb2, and HES kidneys, respectively. Light- and electron microscopic analysis revealed major alterations only for the outer medulla of HES-kidneys.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Substitutos Sanguíneos/farmacologia , Hemoglobinas/farmacologia , Rim/fisiologia , Polímeros/farmacologia , Animais , Substitutos Sanguíneos/química , Bovinos , Taxa de Filtração Glomerular , Hemoglobinas/química , Derivados de Hidroxietil Amido/farmacologia , Rim/patologia , Masculino , Perfusão , Polímeros/química , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
The duration of extracellular signal-regulated protein kinase (ERK) activation is critical for cell signaling decisions and probably determines whether a stimulus elicits proliferation or differentiation. We studied the intracellular signals regulating sustained ERK-2 activity in glomerular mesangial cells (GMC), utilizing combination of GMC mitogens of different potency. Incubation of GMC with both endothelin-1 (ET-1) and platelet-derived growth factor BB (PDGF-BB) led to a long-lasting, monophasic increase in ERK-2 activity. In contrast, when ET-1 was administered together with epidermal growth factor (EGF), a less pronounced and shorter activation occurred. Long-term stimulation of ERK-2 was accompanied by an increase in p45 MEK activity, which again was more pronounced when ET-1 was administered together with PDGF-BB compared with EGF. In the presence of actinomycin D (Act D), an inhibitor of RNA synthesis, ERK-2 activity induced by ET-1 and PDGF-BB but not by ET-1 and EGF remained elevated more than sixfold throughout the whole incubation period of 6 h. The effect of Act D on ET-1- and PDGF-BB-induced ERK-2 activation was mimicked by the protein phosphatase inhibitor sodium orthovanadate. In addition, vanadate also unmarked an ET-1- and EGF-induced ERK-2 activity after 6 h. The serine/threonine phosphatase inhibitor okadaic acid (OA) did neither alter agonist-induced ERK-2 activity after 6 h (0.5 nM OA) nor after 10 min or 1 h (250 nM). Together these results suggest that, in GMC, long-term activation of the mitogen-activated protein kinase ERK-2 is differentially regulated, depending on the combination of agonists administered. ET-1- and PDGF-BB-induced long-term activation of ERK-2 is regulated by a vanadate-sensitive protein phosphatase(s) and by a transcriptionally regulated protein(s). In contrast, ET-1- and EGF-induced sustained ERK-2 stimulation is regulated by a vanadate-sensitive protein phosphatase(s) but not by a transcriptionally regulated protein. Agonist-specific and time-dependent stimulation of ERK-2-regulating protein phosphatases may be critical for the length of ERK-2 activation in GMC and could thus be of pathophysiological significance in glomerular diseases associated with alterations in cell proliferation or cell differentiation.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Mesângio Glomerular/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno , Fosfoproteínas Fosfatases/fisiologia , Proteínas Tirosina Quinases/metabolismo , Animais , Becaplermina , Dactinomicina/farmacologia , Combinação de Medicamentos , Endotelina-1/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Mesângio Glomerular/citologia , MAP Quinase Quinase 1 , Masculino , Proteína Quinase 1 Ativada por Mitógeno , Ácido Okadáico/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes , Fatores de Tempo , Vanadatos/farmacologiaRESUMO
The present study has been performed to evaluate hemodynamic and tubular effects of various endothelin-3 (ET-3) concentrations on the isolated perfused rat kidney. Using this experimental system we observed a profound reduction of renal perfusate flow at all ET-3 concentrations tested (50, 250 and 500 pmol/l), suggesting that the vasoconstrictive potency of ET-3 in the kidney is comparable to that described for endothelin-1 (ET-1). The effects on glomerular filtration rate (GFR) differed depending on the ET-3 dose applied. While 250 pmol/l ET-3 increased GFR by 30%, 500 pmol/l ET-3 markedly reduced GFR. 50 pmol/l ET-3 did not alter GFR although renal vascular resistance significantly increased. Infusion of 1 mumol/l N-nitro-L-arginine, a specific and potent inhibitor of nitric oxide synthesis in endothelial cells, abolished the GFR elevation induced by 250 pmol/l ET-3. In parallel with the changes of GFR we observed an increase in sodium reabsorption at 250 pmol/l and a decrease of this parameter at 500 pmol/l ET-3. Moreover, an ET-3 concentration (500 pmol/l), which induced a dramatic fall in tubular sodium load, led to an increase of fractional sodium excretion and to a decrease of renal oxygen consumption. We conclude that ET-3 is a potent vasoconstrictor in the isolated perfused rat kidney. Furthermore, it modulates GFR in a differentiated mode, depending on the concentration used. The GFR increase at 250 pmol/l ET-3 seems to be mediated by endothelium-derived nitric oxide. In addition to its glomerular action, ET-3 might also affect tubular sodium transport.
Assuntos
Endotelinas/fisiologia , Taxa de Filtração Glomerular/fisiologia , Animais , Arginina/administração & dosagem , Arginina/análogos & derivados , Hemodinâmica , Técnicas In Vitro , Masculino , Nitroarginina , Perfusão , Potássio/urina , Ratos , Ratos Sprague-Dawley , Sódio/urinaRESUMO
ET release by the renal epithelial Madin-Darby canine kidney (MDCK) cell line was investigated under isosmotic (300 mosmol/kg H2O; pH 7.4) and hyperosmotic (400, 500, or 600 mosmol/kg H2O) culture and assay conditions by the use of a specific and sensitive RIA. During isosmotic incubation, MDCK cells, which may be of collecting duct origin, secreted by far more ET into the cell culture supernatant (495.7 +/- 25.5 fmol.mg of protein-1.24 h-1) than did the proximal tubule-derived LLC-PK1 (2.42 +/- 0.20 fmol.mg of protein-1.24 h-1) and opossum kidney (3.12 +/- 0.47 fmol.mg of protein-1.24 h-1) cells. ET secretion by MDCK monolayers increased progressively within 24 h and then only slightly declined up to 48 h. Phosphoramidon (100 mumol/L) inhibited the constitutive ET synthesis in MDCK cells by 60%, indicating the participation of a phosphoramidon-sensitive ET-converting enzyme in the processing of bigET to ET in these cells. MDCK epithelia grown on filter inserts showed a clear polarity in their ET release. The baseline secretion of ET was 2.5 times higher to the basolateral than to the apical side, which might be in support of a predominantly basolateral action of the peptide. Short-term incubation of MDCK cells in hyperosmotic NaCl media for 24 h dose dependently decreased ET production. When urea was used as the solute to generate hyperosmolality, ET release by MDCK cells significantly increased. In contrast, when raffinose was added to increase osmolality to 500 mosmol/kg H2O, a decrease of ET production in a range similar to the effect of NaCl was seen.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Endotelinas/metabolismo , Rim/efeitos dos fármacos , Rafinose/farmacologia , Solução Salina Hipertônica/farmacologia , Ureia/farmacologia , Animais , Células Cultivadas , Cães , Endotelinas/biossíntese , Epitélio , Glicopeptídeos/farmacologia , Soluções Hipertônicas/farmacologia , Rim/citologia , Rim/metabolismo , Gambás , SuínosRESUMO
During formation of the neuromuscular junction, acetylcholine receptors in the endplate membrane become metabolically stabilized under neural control, their half-life increasing from about 1 day to about 10 days. The metabolic stability of the receptors is regulated by the electrical activity induced in the muscle by innervation. We report here that metabolic stabilization of endplate receptors but not of extrajunctional receptors can be induced in the absence of muscle activity if muscles are treated with the calcium ionophore A23187. Acetylcholine receptor stabilization was also induced by culturing non-stimulated muscle in elevated K+ with the Ca2+ channel activator (+)-SDZ202-791. Conversely, activity-dependent receptor stabilization is prevented in muscle stimulated in the presence of the Ca2+ channel blockers (+)-PN200-110 or D-600. Treatment of muscles with ryanodine, which induces Ca2+ release from the sarcoplasmic reticulum in the absence of activity, does not cause stabilization of junctional receptors. Evidently, muscle activity induces metabolic acetylcholine receptor stabilization by way of an influx of Ca2+ ions through dihydropyridine-sensitive Ca2+ channels in the endplate membrane, whereas Ca2+ released from the sarcoplasmic reticulum is ineffective in this developmental process.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Placa Motora/metabolismo , Músculos/fisiologia , Receptores Colinérgicos/metabolismo , Animais , Calcimicina/farmacologia , Bloqueadores dos Canais de Cálcio , Canais de Cálcio/efeitos dos fármacos , Di-Hidropiridinas/farmacologia , Estimulação Elétrica , Eletrofisiologia , Meia-Vida , Masculino , Denervação Muscular , Músculos/efeitos dos fármacos , Músculos/inervação , Técnicas de Cultura de Órgãos , Potássio/farmacologia , Ratos , Ratos Endogâmicos , Rianodina/farmacologia , Tetrodotoxina/farmacologiaRESUMO
Polymorphonuclear neutrophil granulocytes (PMNs) seem to participate in the pathogenesis of renal ischemic reperfusion injury. The kidneys from male Sprague Dawley rats were immersion-fixed after 45 min of renal artery clamping followed by reperfusion for 0, 5, 20, and 120 min, respectively. The tissue distribution of PMNs in the kidneys was studied histochemically using naphthol AS-D chloroacetate esterase as a specific marker for these cells. Neutrophil counts per unit sectional area were obtained for renal cortex, outer and inner medulla. In the cortex separate intraglomerular and peritubular counts, and in the outer medulla separate outer and inner stripe counts were made. After 120 min of reperfusion the total renal PMN counts were 488 +/- 62 (n = 4) compared with 54 +/- 4 (n = 4) per cm2 in nonischemic controls. Within 120 min of reperfusion PMN counts increased by a factor of 8 in the cortex, of 12 in the outer medulla and of 14 in the inner medulla, compared with controls. The ratio of intraglomerular against peritubular PMN counts was approximately 2 in controls, but 0.5 after a 120-min reperfusion interval. The outer stripe of the outer medulla contained only a small number of PMNs whereas PMN counts of 923 +/- 197 (n = 4) per cm2 were found in the inner stripe after 120 min reperfusion. Interestingly, there was a marked increase in PMNs in the inner stripe during the first 5 min of reperfusion but no extravasation of PMNs was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Injúria Renal Aguda/patologia , Isquemia/patologia , Rim/irrigação sanguínea , Neutrófilos , Traumatismo por Reperfusão/patologia , Injúria Renal Aguda/etiologia , Animais , Biomarcadores , Capilares/patologia , Contagem de Células , Isquemia/complicações , Rim/patologia , Córtex Renal/patologia , Medula Renal/patologia , Túbulos Renais/irrigação sanguínea , Túbulos Renais/patologia , Masculino , Naftol AS D Esterase/análise , Ratos , Ratos Sprague-DawleyRESUMO
Overexpression of a constitutively active mitogen-activated protein kinase kinase (MAPKK or MEK) induces neuronal differentiation in adrenal pheochromocytoma 12 cells but transformation in fibroblasts. In the present study, we used a constitutively active MAPK/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) mutant to investigate the function of the highly conserved MEK1-ERK2 signaling module in renal epithelial cell differentiation and proliferation. Stable expression of constitutively active MEK1 (CA-MEK1) in epithelial MDCK-C7 cells led to an increased basal and serum-stimulated ERK1 and ERK2 phosphorylation as well as ERK2 activation when compared with mock-transfected cells. In both mock-transfected and CA-MEK1-transfected MDCK-C7 cells, basal and serum-stimulated ERK1 and ERK2 phosphorylation was almost abolished by the synthetic MEK inhibitor PD098059. Increased ERK2 activation due to stable expression of CA-MEK1 in MDCK-C7 cells was associated with epithelial dedifferentiation as shown by both a dramatic alteration in cell morphology and an abolished cytokeratin expression but increased vimentin expression. In addition, we obtained a delayed and reduced serum-stimulated cell proliferation in CA-MEK1-transfected cells (4.6-fold increase in cell number/cm2 after 5 days of serum stimulation) as compared with mock-transfected controls (12.9-fold increase in cell number/cm2 after 5 days). This result was confirmed by flow cytometric DNA analysis showing that stable expression of CA-MEK1 decreased the proportion of MDCK-C7 cells moving from G0/G1 to G2/M as compared with both untransfected and mock-transfected cells. Taken together, our data demonstrate an association of increased basal and serum-stimulated activity of the MEK1-ERK2 signaling module with epithelial dedifferentiation and growth inhibition in MDCK-C7 cells. Thus, the MEK1-ERK2 signaling pathway could act as a negative regulator of epithelial differentiation thereby leading to an attenuation of MDCK-C7 cell proliferation.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Rim/citologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Antígenos de Diferenciação , Diferenciação Celular , Divisão Celular , Cães , Inibidores Enzimáticos/farmacologia , Células Epiteliais , Flavonoides/farmacologia , MAP Quinase Quinase 1 , Mesoderma/fisiologia , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
MDCK-C7 cells dedifferentiated either by transient alkaline stress (C7F cells) or by transfection with a constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 (C7caMEK1 cells) were analyzed by western blot and immunofluorescence microscopy to compare the expression of different cytokeratins, vimentin, and alpha-smooth muscle actin. Expression of all cytokeratins tested, the type II-neutral and basic cytokeratins CK5, CK7, CK8 as well as the type I-acidic keratins CK17 and CK19, was substantially reduced in dedifferentiated cell lines C7F and C7caMEK1 when compared with epithelial wild-type MDCK-C7 cells or mock-transfected MDCK-C7 cells. While vimentin expression was detected in all of the four MDCK-C7 cell lines examined, only the dedifferentiated cell lines C7F and C7caMEK1, which have been reported to express highly active ERK2, exhibited formation of alpha-smooth muscle actin-containing stress fibers. Taken together our results show that, associated with an increase in ERK2 activity, an epithelial to mesenchymal dedifferentiation occurred in both MDCK-C7F cells and caMEK1-transfected MDCK-C7 cells.
Assuntos
Actinas/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Queratinas/metabolismo , Túbulos Renais Coletores/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno , Animais , Diferenciação Celular , Linhagem Celular , Citoesqueleto/metabolismo , Cães , Células Epiteliais/ultraestrutura , Túbulos Renais Coletores/ultraestrutura , MAP Quinase Quinase 1 , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Transfecção , Vimentina/metabolismoRESUMO
We have investigated the regulation and localization of mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK) in both cytosolic and nuclear fractions of glomerular mesangial cells. p42 MAPK was localized by both immunoblot and kinase activity in both cytosol and nucleus and was rapidly activated, in both fractions, by fetal bovine serum and TPA. Downregulation of protein kinase C (PKC) by TPA inhibited stimulation of cytosolic p42 MAPK, but unexpectedly had no effect on stimulated p42 MAPK in the nucleus. Next we studied the upstream kinase p45 MAPKK by indirect immunofluorescence microscopy, Western blot analysis, and kinase specific activity. Unlike MAPK, p45 MAPKK is almost exclusively cytosolic in resting cells and kinase activity stimulated by TPA is restricted to the cytosol. Interestingly, PKC downregulation for 24 h with TPA dramatically enhanced nuclear MAPKK as assessed by all three techniques. Cytosolic stimulated MAPKK was attenuated in PKC downregulation. Collectively these results show that in mesangial cells: (i) p42 MAPK and p45 MAPKK localize in both the cytosol and the nucleus, and (ii) PKC exerts a negative effect on nuclear MAPKK activity as documented by PKC downregulation, which augments p45 MAPPK nuclear mass and activity. These results indicate that the dual regulation of these two kinases is under differential control in the cytosol and the nucleus.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Núcleo Celular/enzimologia , Citosol/enzimologia , Proteínas Quinases/metabolismo , Animais , Western Blotting , Mesângio Glomerular/citologia , Mesângio Glomerular/enzimologia , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Endothelin-1 (ET-1) stimulates vascular smooth muscle and mesangial cells to release prostaglandin E2 (PGE2), which can attenuate the vasoconstrictor and mitogenic effects of this peptide. Phospholipase A2 (PLA2)-mediated release of arachidonic acid from the sn-2 position of membrane phospholipids is thought to be one of the rate-limiting steps in prostaglandin (PG) synthesis. We evaluated the role of ET-1 to regulate gene expression, protein synthesis and enzymatic activity of cytosolic PLA2 (cPLA2), an intracellular form of the PLA2 enzyme family, in cultured rat mesangial cells using both acute and chronic incubation protocols. Acute ET-1-induced stimulation of cPLA2 activity was maximal after 10 minutes (181.1 +/- 6.84% of control), persisted for 40 minutes and did not require new protein synthesis. Heparin, a potent inhibitor of intracellular Ca2+ increase as well as mitogen-activated protein (MAP) kinase activation and cell proliferation, did not affect the rapid cPLA2 stimulation by ET-1. Chronic incubation of glomerular mesangial cells with ET-1 (1 to 24 hr) led to time- and dose-dependent increases in cPLA2 mRNA expression which was maximal after six hours, persisted up to 24 hours and which was accompanied by both cPLA2 protein formation, as assessed by Western analysis, as well as by stimulation of enzymatic activity. Inhibition of protein synthesis by cycloheximide increased basal cPLA2 mRNA accumulation in quiescent mesangial cells, and the combination of ET-1 and cycloheximide resulted in a greater induction of cPLA2 gene expression when compared to ET-1 alone. Actinomycin D treatment blocked the effect of ET-1 on cPLA2 mRNA accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Endotelinas/farmacologia , Mesângio Glomerular/efeitos dos fármacos , Mesângio Glomerular/enzimologia , Fosfolipases A/metabolismo , Animais , Benzoquinonas , Células Cultivadas , Cicloeximida/farmacologia , Citosol/enzimologia , Dactinomicina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Lactamas Macrocíclicas , Fosfolipases A/genética , Fosfolipases A2 , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Quinonas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Rifabutina/análogos & derivadosRESUMO
Overexpression of a constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 (caMEK1) in epithelial Madin-Darby canine kidney (MDCK)-C7 cells disrupts morphogenesis, induces an invasive phenotype, and is associated with a reduced rate of cell proliferation. The role of cell-cell adhesion molecules and cell cycle proteins in these processes, however, has not been investigated. We now report loss of E-cadherin expression as well as a marked reduction of beta- and alpha-catenin expression in transdifferentiated MDCK-C7 cells stably expressing caMEK1 (C7caMEK1) compared with epithelial mock-transfected MDCK-C7 (C7Mock1) cells. At least part of the remaining alpha-catenin was coimmunoprecipitated with beta-catenin, whereas no E-cadherin was detected in beta-catenin immunoprecipitates. In both cell types, the proteasome-specific protease inhibitors N-acetyl-Leu-Leu-norleucinal (ALLN) and lactacystin led to a time-dependent accumulation of beta-catenin, including the appearance of high-molecular-weight beta-catenin species. Quiescent as well as serum-stimulated C7caMEK1 cells showed a higher cyclin D expression than epithelial C7Mock1 cells. The MEK inhibitor U-0126 inhibited extracellular signal-regulated kinase phosphorylation and cyclin D expression in C7caMEK1 cells and almost abolished their already reduced cell proliferation rate. We conclude that the transdifferentiated and invasive phenotype of C7caMEK1 cells is associated with a diminished expression of proteins involved in cell-cell adhesion. Although beta-catenin expression is reduced, C7caMEK1 cells show a higher expression of U-0126-sensitive cyclin D protein.
Assuntos
Moléculas de Adesão Celular/metabolismo , Ciclinas/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Transativadores , Animais , Caderinas/metabolismo , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular/citologia , Ciclina D , Cisteína Endopeptidases/fisiologia , Proteínas do Citoesqueleto/metabolismo , Cães , Rim/citologia , Rim/metabolismo , MAP Quinase Quinase 1 , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Complexos Multienzimáticos/fisiologia , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas Serina-Treonina Quinases/genética , Transfecção , alfa Catenina , beta CateninaRESUMO
The mineralocorticoid aldosterone is essential for the adequate regulation of electrolyte homeostasis, extracellular volume and blood pressure. As a steroid hormone it influences cellular functions by genomic actions. Previously it has been shown that aldosterone can activate Na+/H+-exchange (NHE) by a rapid, nongenomic mechanism. Because (1) NHE can be regulated by ERK1/2 (extracellular signal-regulated kinase) and (2) steroids have been reported to rapidly activate ERK1/2, we tested the hypothesis that activation of NHE by aldosterone involves ERK1/2, using MDCK-C11 cells. We show that nanomolar concentrations of aldosterone induce a rapid, non-genomic activation of NHE, which is characterized by an increased affinity for H+ with minor changes in the maximum transport rate. Accordingly, aldosterone led to an increase of cytosolic steady-state pH. The aldosterone-induced activation of NHE was prevented by the two specific inhibitors of ERK1/2 activation, PD 98059 (2.5 x 10(-5) mol/l) and U0126 (10(-5) mol/l). Furthermore, in the presence of U0126 there was no aldosterone-induced increase of steady-state pH. Finally, aldosterone induced a rapid phosphorylation of ERK1/2, indicating its ability to activate ERK1/2. The data presented here support the hypothesis that the rapid activation of NHE by aldosterone at nanomolar concentrations involves ERK1/2. Thus, in certain cell types, the MAPK cascade may represent an additional pathway mediating rapid aldosterone effects.
Assuntos
Aldosterona/farmacologia , Túbulos Renais Distais/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Equilíbrio Ácido-Base/efeitos dos fármacos , Equilíbrio Ácido-Base/fisiologia , Animais , Células Cultivadas , Concentração de Íons de Hidrogênio , Túbulos Renais Distais/citologia , Proteína Quinase 3 Ativada por Mitógeno , Estimulação QuímicaRESUMO
Mitogen-activated protein kinases (MAPK) play a key role in the regulation of cellular processes such as cell growth, cell differentiation, and apoptosis. However, the specific function of single isoforms of the MAPK family in renal epithelial cell differentiation and/or proliferation has not been investigated so far. We now report stable reduction of extracellular signal-regulated kinase 1 (ERK1) protein expression and lack of serum-induced ERK1 activation in alkali-dedifferentiated Madin-Darby canine kidney-C7 focus (MDCK-C7F) cells compared with their parental epithelial MDCK-C7 cells. The changes in ERK1 protein expression and activation were accompanied by a small rise in c-jun NH2-terminal kinase 1 (JNK1) protein expression but slightly decreased basal and anisomycin-stimulated JNK1 activity. In contrast, ERK2 phosphorylation, as assessed by using an antibody which detects phosphorylated tyrosine 204 of both ERK1 and ERK2, as well as enzymatic ERK2 activity, was substantially increased in untreated and fetal calf serum-treated MDCK-C7F cells, although ERK2 protein expression remained unchanged. Differential expression and activation of ERK1, ERK2, and JNK1 were accompanied by an inhibition of serum-induced MDCK-C7F cell proliferation. Together, our results demonstrate an association between changes in the activation of certain MAPK and alkali-induced stable MDCK-C7 cell dedifferentiation. Moreover, these data provide evidence for distinct signaling functions of ERK1 and ERK2 in these cells.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Rim/citologia , Rim/enzimologia , Proteínas Quinases Ativadas por Mitógeno , Álcalis/farmacologia , Animais , Anisomicina/farmacologia , Fenômenos Fisiológicos Sanguíneos , Diferenciação Celular , Linhagem Celular , Cães , Ativação Enzimática , Células Epiteliais , Epitélio/metabolismo , Sangue Fetal , Proteínas Quinases JNK Ativadas por Mitógeno , Rim/metabolismo , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Fosforilação , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
Constitutive stimulation of the mitogen-activated protein kinase (MAPK) activator MAPK/ERK kinase (MEK) is sufficient to promote long-term events such as cell differentiation, proliferation, and transformation. To evaluate a possible mechanism for the chronic regulation of MEK and p42 MAPK, we studied the long-term effects of fetal bovine serum (FBS), the G protein-coupled receptor agonist endothelin-1 (ET-1), and the protein tyrosine kinase-coupled receptor agonist platelet-derived growth factor BB (PDGF BB) on MEK and p42 MAPK in glomerular mesangial cells (GMC). FBS, ET-1, and PDGF BB led to a time-dependent increase in MEK-1 mRNA and protein expression without altering p42 MAPK mRNA and protein levels. FBS also induced MEK-1 mRNA expression in diverse cell types, including NIH/3T3 fibroblasts, A7r5 vascular smooth muscle cells, and Chinese hamster ovary cells. In GMC, cycloheximide inhibited MEK-1 mRNA induction but stimulated p42 MAPK mRNA expression in the absence and presence of FBS, ET-1, or PDGF. The FBS-induced increase in MEK-1 mRNA was accompanied by a sustained enhancement of MEK activity, as assessed by the ability of immunoprecipitated p45 MEK to activate recombinant p42 MAPK and hence phosphorylate myelin basic protein, and p42 MAPK activity. We conclude that, in GMC, MEK-1 acts like a delayed-early gene and that it can be chronically induced at the mRNA and protein level.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Mesângio Glomerular/metabolismo , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Becaplermina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Bovinos/sangue , Bovinos/embriologia , Células Cultivadas , Endotelinas/farmacologia , Sangue Fetal/fisiologia , Mesângio Glomerular/citologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-sis , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes , Fatores de TempoRESUMO
To evaluate a possible mechanism for the chronic regulation of MAPK/ERK kinase-1 (MEK-1) and p42 mitogen-activated protein kinase (MAPK) we studied the long-term effects of the G-protein-coupled receptor agonist endothelin-1 (ET-1) and the protein tyrosine kinase-coupled receptor agonist platelet-derived growth factor BB (PDGF BB) on MEK-1 and p42 MAPK in glomerular mesangial cells (GMCs). ET-1 and PDGF BB led to a time-dependent increase in MEK-1 mRNA expression without altering p42 MAPK mRNA levels. The effect of ET-1 and PDGF BB on MEK-1 mRNA expression was maximal after 24 h (3.3-fold) or 6 h (2.9-fold). Furthermore, the effect of ET-1 and PDGF BB on MEK-1 mRNA expression was additive (4.2-fold after 6 h) and was inhibited by actinomycin D (5 micrograms/ml). Cycloheximide (10 micrograms/ml) inhibited MEK-1 mRNA induction but stimulated p42 MAPK mRNA expression in both the absence and the presence of ET-1 and/or PDGF BB. The ET-1 and PDGF BB-induced increase in MEK-1 mRNA was accompanied by sustained enhancement of both p45 MEK protein expression after 12 h and by elevation of p42 MAPK activity for up to 24 h. We conclude that, in GMCs, MEK-1 acts like a delayed-early gene, whereas p42 MAPK resembles an immediate-early gene. MEK-1 mRNA and protein levels, as well as p42 MAPK activity, can be chronically regulated by both a seven-transmembrane domain receptor-coupled peptide such as ET-1 and by an agonist binding to a receptor with intrinsic protein tyrosine kinase activity, such as PDGF BB.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/biossíntese , Endotelinas/farmacologia , Mesângio Glomerular/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Tirosina Quinases/biossíntese , RNA Mensageiro/análise , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/análise , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Cicloeximida/farmacologia , Mesângio Glomerular/enzimologia , MAP Quinase Quinase 1 , Masculino , Proteína Quinase 1 Ativada por Mitógeno , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/genética , Ratos , Ratos Sprague-DawleyRESUMO
Since it became evident that organ dysfunctions after acute hemolysis are not induced by hemoglobin per se, but by stroma-contaminated hemoglobin, solutions of ultrapure stroma-free hemoglobins were regarded to be possible substitutes for blood in transfusion medicine. We tested one of the recently developed modified bovine hemoglobins (Ultrapure polymerized bovine hemoglobin 1; UPPBHb1) in the isolated perfused rat kidney (IPRK) model, using a recirculating system. Control kidneys were perfused with a substrate-enriched Ringer solution containing hydroxyethyl starch (HES) to produce isoncotic conditions. In the experimental group HES was substituted in part by UPPBHb1 (34 g/l). For determination of functional parameters, the kidneys were perfused for 180 min. A separate set of kidneys of both groups was perfusion fixed after 80 min of perfusion which is the period of optimal function. Light and electron microscopic analysis revealed major alterations only for the outer medulla of HES kidneys. Only these suffered from a considerable extent of proximal tubular S3 damage, exhibiting condensed tubular epithelia. In the inner stripe of the outer medulla, which is the zone of greatest sensitivity to damage in the isolated perfused kidney, severe hydropic degeneration, cell detachment, and necrotic destruction of the medullary thick ascending limb were seen in the HES-perfused group, too. In the UPPBHb1 group, the medullary thick ascending limb was well preserved, and S3 showed only a minor degree of damage. UPPBHB1 kidneys were further characterized by the occurrence of intracapillary and interstitial precipitates of UPPBHb1 in inner stripe of the outer medulla and inner medulla. The glomerular filtration rate was significantly higher in UPPBHb1-perfused kidneys (870 +/- 80 vs. 630 +/- 55 microliters/min/g kidney weight for HES). Absolute reabsorption of sodium paralleled the behavior of the glomerular filtration rate. The values for renal perfusate flow and urinary flow rate did not differ significantly between both groups. Renal autoregulation was better preserved in UPPBHb1-perfused kidneys (74 +/- 6% of full autoregulatory response) than in HES-perfused controls (42 +/- 4%). Our results suggest that perfusion of isolated rat kidneys with UPPBHb1 improves kidney function and morphology, providing better oxygenation than in control kidneys. UPPBHb1 does not exert additional nephrotoxic effects on the IPRK that will exceed the noxious potential of the method itself. Thus, it must be concluded that UPPBHb1 may be an oxyphoretic blood substitute with nephroprotective characteristics when compared with nonoxyphoretic substitutes. At least, UPPBHb1 seems to be a promising candidate as oxyphoretic additive to perfusates for the IPRK model.
Assuntos
Hemoglobinas/uso terapêutico , Rim/efeitos dos fármacos , Animais , Biopolímeros , Bovinos , Hemoglobinas/química , Hemoglobinas/isolamento & purificação , Rim/ultraestrutura , Córtex Renal/efeitos dos fármacos , Testes de Função Renal , Medula Renal/efeitos dos fármacos , Masculino , Perfusão , Ratos , Ratos Sprague-DawleyRESUMO
The kidneys represent one of the main targets of ochratoxin A (OTA), a secondary fungal metabolite that is produced by certain species of Aspergillus and Penicillium. OTA has the ability to disturb Madin-Darby canine kidney (MDCK) cell pH homeostasis, leading to intracellular alkalinization and morphological alterations resembling those that occur when MDCK cells are exposed to transient alkaline stress. Because alkali-induced epithelial dedifferentiation of MDCK-C7 cells is associated with an increase in the activity of extracellular signal-regulated kinases (ERK), we performed experiments that investigated a possible role for ERK1 and ERK2 as intracellular signaling molecules mediating some of the mycotoxin's effects on renal epithelia. We studied the effects of OTA on ERK1/2 phosphorylation and activation, as well as on cell morphology by using cloned MDCK-C7 and MDCK-C11 cells. In MDCK-C7 cells, but not in MDCK-C11 cells, OTA led to a time-dependent and concentration-dependent increase in ERK1/2 phosphorylation. OTA-induced ERK1/2 phosphorylation in MDCK-C7 cells occurred at concentrations of 500 nM, started after 2 hr and was maximal after 8 hr. Furthermore, after 8 hr of incubation, 500 nM and 1 microM OTA significantly increased ERK1/2 activity in MDCK-C7 but not in MDCK-C11 cells. This OTA-stimulated ERK1/2 phosphorylation and ERK1/2 activation in MDCK-C7 cells was partially inhibited by the synthetic mitogen-activated protein kinase kinase (MKK or MEK) inhibitor PD098059. Transepithelial resistance and lactate dehydrogenase release remained unaltered after incubation in the presence of 1 microM OTA for 8 hr or of 100 nM OTA for 24 hr, so it is unlikely that these OTA effects on ERK1/2 are due to secondary toxic effects of the mycotoxin. Interestingly, OTA-induced long-term activation of ERK1/2 in MDCK-C7 cells was associated with epithelial dedifferentiation, as assessed by analysis of vectorial solute and water transport as well as cell morphology. In contrast, MDCK-C11 cells, which do not show significant increases in ERK1/2 phosphorylation and ERK1/2 activity in response to OTA, retained their epithelial phenotype under identical experimental conditions. Taken together, our data demonstrate an epithelial dedifferentiation of MDCK-C7 cells, but not of MDCK-C11 cells, after long-term incubation in the presence of OTA, a result associated with the ability of this mycotoxin to stimulate ERK1/2 in MDCK-C7 cells but not in MDCK-C11 cells. We conclude that OTA-induced activation of ERK1/2 could be an important intracellular signaling pathway that mediates some of the mycotoxin's effects on renal epithelia.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/efeitos dos fármacos , Rim/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno , Micotoxinas/toxicidade , Ocratoxinas/toxicidade , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Diferenciação Celular , Células Cultivadas , DNA/biossíntese , Cães , Ativação Enzimática/efeitos dos fármacos , Flavonoides/farmacologia , Concentração de Íons de Hidrogênio , Rim/enzimologia , L-Lactato Desidrogenase/metabolismo , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , FosforilaçãoRESUMO
1. Using conventional microelectrodes, the perforated patch clamp technique and fluorescence microscopy with fura-2, we investigated the relationship between the cell membrane potential, whole-cell currents and the free cytoplasmic Ca2+ concentration ([Ca2+]i) in response to 10 nM endothelin-1 (ET) in a rat respiratory epithelial cell line (L2). 2. Microelectrode experiments revealed that ET caused an immediate depolarization of the cell membrane potential (Vm) by 25 mV, which was unaffected by Na+ replacement with N-methyl-D-glucamine+ (NMDG+) or by omission of bath Ca2+. In contrast, ET depolarized the cells by 61 mV in the presence of low C1- (6 mM), resulting in a complete breakdown of Vm. 3. In perforated patch clamp experiments, the ET-induced whole-cell current (IET) exhibited a slight outward rectification with a reversal potential (Vrev) of -22.7 mV. IET was reduced by 85 % in low C1- (6 mM), but was unaffected by Ca2+ removal, Na+ replacement with NMDG+, pipette K+ replacement with Cs+ or 1 mM Ni2+ in the bath. 4. IET was unaffected by (+)-isradipine (100 nM), a specific L-type Ca2+ channel (L-VDCC) blocker. Transient inward Sr2+ currents through L-VDCCs were blocked by ET. 5. ET induced a biphasic Ca2+ signal, consisting of a 'peak' and a 'plateau' elevation of [Ca2+]i. Simultaneous patch clamp and fura-2 measurements revealed that IET coincided with intracellular Ca2+ release but clearly outlasted the elevation of [Ca2+]i. When the rise of [Ca2+]i was prevented by pretreatment with thapsigargin in a Ca2+-free bath, both activation time and amplitude of IET were reduced. Under these conditions, ET caused a decrease of [Ca2+]i. 6. The C1- channel blocker mefenamic acid (MFA) had a dual, concentration-dependent effect on both IET and the ET-induced 'plateau' elevation of [Ca2+]i: an increase at 10 microM, but an almost complete block at 100 microM. The effect of MFA on IET preceded the effect on [Ca2+]i. 7. The ET-induced 'plateau' [Ca2+]i fell below control values in a low-C1- (6 mM) solution. 8. These data indicate an amplifying function of intracellular Ca2+ release on an otherwise Ca2+-independent, unique C1- current by ET. Moreover, this C1- current appears to be functionally coupled with dihydropyridine (DHP)-insensitive Ca2+ entry, suggesting a modulatory role for long-lasting effects of ET.