RESUMEN
Changes in expression and activity of protein kinase C (PKC) isoforms and early transcription factors may account for alterations in cell behavior seen in diabetes. We studied the expression of PKC-beta(I) in rat glomerular mesangial cells (MCs) cultured in normal or high glucose and compared it with the temporal and spatial expression of dimeric transcription factor (NF-kappaB) p50 and p65. The results show that in unstimulated cells PKC-beta(I) and NF-kappaB p50 are distributed in the cytosol and, on stimulation, their distribution is perinuclear and they are localized to the membrane. Serum-starved MCs cultured in high-glucose medium exhibit a predominantly cytosolic localization of PKC-beta(I) and both p50 and p65 NF-kappaB. However, phorbol 12-myristate 13-acetate (PMA) stimulation of cells grown in the presence of high glucose resulted in membrane translocation of PKC-beta(I) that was associated with nuclear translocation of NF-kappaB p65, but not NF-kappaB p50. Moreover, the translocation to the nucleus for NF-kappaB p65 was significantly higher in MCs exposed to high glucose compared with those exposed to normal glucose. These observations indicate that the NF-kappaB p65, but not NF-kappaB p50, expression and translocation pattern mirrors that of PKC-beta(I), which may be one important pathway by which signaling is enhanced in the high-glucose state.
Asunto(s)
Mesangio Glomerular/enzimología , Isoenzimas/metabolismo , FN-kappa B/metabolismo , Proteína Quinasa C/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Carcinógenos/farmacología , Células Cultivadas , Nefropatías Diabéticas/metabolismo , Mesangio Glomerular/química , Mesangio Glomerular/citología , Glucosa/farmacología , Hiperglucemia/metabolismo , Isoenzimas/análisis , Masculino , FN-kappa B/análisis , Proteína Quinasa C/análisis , Proteína Quinasa C beta , Ratas , Ratas Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Glomerular mesangial cells (MCs) have been used as an in vitro model for glomerular disease. The culture conditions used for these cells vary and include the use of insulin or insulin-transferrin-selenous acid (ITS) in the growth medium. We studied the effect of ITS in the growth medium containing either normal or high glucose on the expression of protein kinase C (PKC) isoforms in MCs. In the presence of ITS in the medium, MCs expressed lower levels of both PKC isoforms in their cytosol in comparison to MCs grown in medium without ITS. Upon stimulation with PMA, both isoforms were translocated to the particulate (nucleus/cytoskeleton) compartment in MCs grown in presence of ITS. However, in the absence of ITS in the growth medium, both PKC isoforms were primarily translocated to the membrane compartment upon PMA stimulation. These results indicate that insulin in the growth medium may activate MCs resulting in translocation of PKC from the cytosol to other subcellular compartments. This effect is even evident in MCs grown in normal glucose concentration. Our data indicate that the use of ITS in growth medium and eventual interpretation from such experiments involving primary mesangial cells grown in culture needs careful evaluation.
Asunto(s)
Mesangio Glomerular/efectos de los fármacos , Insulina/farmacología , Proteína Quinasa C/efectos de los fármacos , Selenio/farmacología , Transferrina/farmacología , Animales , Western Blotting , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Citoesqueleto/enzimología , Citosol/efectos de los fármacos , Citosol/enzimología , Relación Dosis-Respuesta a Droga , Mesangio Glomerular/citología , Mesangio Glomerular/enzimología , Glucosa/farmacología , Isoenzimas/efectos de los fármacos , Isoenzimas/metabolismo , Masculino , Membranas/efectos de los fármacos , Membranas/enzimología , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Increased activation of specific protein kinase C (PKC) isoforms and increased nonenzymatic glycation of intracellular and extracellular proteins [the accumulation of advanced glycation end products (AGEs)] are major mechanistic pathways implicated in the pathogenesis of diabetic complications. Blocking PKC-beta(II) has been shown to decrease albuminuria in animal models of diabetes. To demonstrate a direct relationship between AGEs and the induction and translocation of PKC-beta(II), studies were carried out in rat neonatal mesangial cells, known to express PKC-beta(II) in association with rapid proliferation in post-natal development. Oxidative stress was studied by using the fluorescent probe dichlorfluorescein diacetate. Translocation of PKC-beta(II) was demonstrated by using immunofluorescence and Western blotting of fractionated mesangial cells. Induction of intracellular oxidative stress, increase in intracellular calcium, and cytosol to membrane PKC-beta(II) translocation (with no change in PKC-alpha) were demonstrated after exposure to AGE-rich proteins. These data support the hypothesis that AGEs cause mesangial oxidative stress and alterations in PKC-beta(II), changes that may ultimately contribute to phenotypic abnormalities associated with diabetic nephropathy.
Asunto(s)
Animales Recién Nacidos/metabolismo , Mesangio Glomerular/metabolismo , Productos Finales de Glicación Avanzada/farmacología , Isoenzimas/metabolismo , Estrés Oxidativo , Proteína Quinasa C/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Activación Enzimática/fisiología , Mesangio Glomerular/citología , Mesangio Glomerular/efectos de los fármacos , Membranas Intracelulares/metabolismo , Proteína Quinasa C beta , Ratas , Ratas Sprague-DawleyRESUMEN
Changes in activity or expression of transporters may account for alterations in cell behavior in diabetes. We sought to ascertain if mesangial cells (MC) grown in different glucose concentrations exhibit changes in activity and expression of acid-extruding transporters, the Na(+)/H(+) and Na(+)-dependent Cl(-)/HCO(-)(3) exchanger. pH(i) was determined by the use of the fluorescent pH-sensitive dye BCECF. In MCs grown in 5 mM glucose (control), the Na(+)/H(+) exchanger was responsible for 31.8 +/- 5.1% of steady-state pH(i), whereas Na(+)-dependent Cl(-)/HCO(-)(3) contributed 62.9 +/- 4.0% (n = 11). In MCs grown in high glucose for 2 wk, Na(+)/H(+) exchange contribution to acid-extrusion increased as follows: 42.3 +/- 4.6% [n = 8, 10 mM, not significant (NS)], 51.1 +/- 5.1% (n = 8, 20 mM, P < 0.01), and 64.8 +/- 5.5% (n = 7, 30 mM, P < 0.001). The Na(+)-dependent Cl(-)/HCO(-)(3) exchanger contributed less [47.0 +/- 4.6, 38.6 +/- 5.8, and 21.1 +/- 3.8%, for 10, 20, and 30 mM glucose, respectively (n > 7)]. We sought to ascertain if the magnitude of the acute stimulated response to ANG II by the Na(+)/H(+) and Na(+)-dependent Cl(-)/HCO(-)(3) exchanger is changed. Na(+)/H(+) exchanger (1.89-fold increase in 30 vs. 5 mM, P < 0.002), but not Na(+)-dependent Cl(-)/HCO(-)(3) exchange (0. 17-fold, NS), exhibited an enhanced response to ANG II (1 microM). Na(+)/H(+) exchange (NHE1) expression was significantly different (1. 72-fold) after prolonged exposure to high glucose. These results suggest that the Na(+)/H(+) exchanger, but not Na(+)-dependent Cl(-)/HCO(-)(3) exchanger, may play an early role in the response to hyperglycemia in the diabetic state.
Asunto(s)
Mesangio Glomerular/efectos de los fármacos , Glucosa/farmacología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico , Angiotensinas/farmacología , Animales , Antiportadores/metabolismo , Células Cultivadas , Antiportadores de Cloruro-Bicarbonato , Fluoresceínas , Mesangio Glomerular/metabolismo , Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Hiperglucemia/metabolismo , Masculino , Manitol/farmacología , Compuestos de Amonio Cuaternario , Ratas , Ratas Sprague-Dawley , Sodio/metabolismo , Factores de TiempoRESUMEN
Changes in activity or expression of protein kinase C (PKC), reactive oxygen products, and nitric oxide (NO) may account for the alteration in cell behavior seen in diabetes. These changes have been proposed to be part of the pathophysiology of erectile dysfunction. We sought to ascertain if corpus cavernosal vascular smooth muscle cells (CCSMC) grown in a high glucose milieu exhibit changes in the activity and expression of PKC isoforms, NO, and reactive oxygen products and to find out if these changes are prevented by alpha-tocopherol. Rat CCSMC were grown in 5, 15, and 30 mM glucose concentrations for 3, 7, and 14 days. PKC isoform expression was assayed with isoform-specific antibodies. In CCSMCs grown in 30 mM glucose for 2-wk, PKC-beta(2)-isoform was upregulated (n = 4; P < 0.01), whereas the expression of alpha-, delta-, epsilon-, and beta(1)-isoforms was unchanged. NO as measured by nitrate-to-nitrite ratio was greatly diminished at 14 days in 30 mM (n = 4; P < 0.002) compared with 5 mM glucose. Reactive oxygen products were upregulated at 14 days when they were assayed by the fluorescent probe dichlorofluorescein diacetate bis(acetoxy-methyl) (DCFH-DA) (n = 5; P < 0.01). When these same cells were exposed to alpha-tocopherol for 14 days, there was a reduction of PKC-beta(2) (57.8%; P < 0.01; n = 4) and a reduction in reactive oxygen product formation (71.1%; P < 0.001; n = 4), along with an increase in nitrate-to-nitrite ratio (43.9%; P < 0.01, n = 4). These results suggest that there may be an interrelationship between PKC, NO, and reactive oxygen product formation in CCSMC exposed to a high glucose environment.
Asunto(s)
Glucosa/antagonistas & inhibidores , Glucosa/farmacología , Óxido Nítrico/metabolismo , Proteína Quinasa C/metabolismo , Vitamina E/farmacología , Animales , Células Cultivadas , Isoenzimas/metabolismo , Masculino , Nitratos/metabolismo , Nitritos/metabolismo , Pene/citología , Pene/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismoAsunto(s)
Enfermedades Renales/tratamiento farmacológico , Riñón/efectos de los fármacos , Oligodesoxirribonucleótidos Antisentido/farmacología , Transporte Biológico , Células Epiteliales/efectos de los fármacos , Canales Iónicos/genética , Canales Iónicos/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Riñón/citología , Riñón/enzimología , Glomérulos Renales/citología , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/enzimología , Túbulos Renales/citología , Túbulos Renales/efectos de los fármacos , Oligodesoxirribonucleótidos Antisentido/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismoRESUMEN
Antisense oligodeoxynucleotides offer the potential to block the expression of specific genes with the goal of altering the phenotypic behavior of the cell. Antisense technology has attracted special interest as potential therapeutic agents for the treatment of genetic disorders, viral infections, and most recently proliferative diseases such as glomerular kidney disease. This technique has recently been used for in vitro and in vivo studies in renal cells. The use of antisense technology has been applied in vitro to help define both the normal mechanisms of specific ion transport and function and the pathobiological processes leading to glomerular proliferation and matrix formation. Most promising are the recent uses of antisense technology in vivo that have been used to treat the damaged peritoneum and alter glomerular remodeling in experimental animal models. It is hoped that widespread use of antisense will not only provide new insight into the normal regulatory behavior of the kidney cells but also allow one to develop therapeutic strategies to treat kidney disease.
Asunto(s)
Biotecnología , Riñón/citología , Oligodesoxirribonucleótidos Antisentido , Células Epiteliales , Humanos , Riñón/fisiología , Enfermedades Renales/tratamiento farmacológico , Glomérulos Renales/citología , Túbulos Renales/citología , Oligodesoxirribonucleótidos Antisentido/uso terapéuticoRESUMEN
Developmental changes in activity or expression of transporters may account for alterations in cell behavior as the nonpolarized cell matures. We sought to ascertain whether there is a maturational change in each of the major acid-base transporters in the developing mesangial cell (MC), the Na/H exchanger, Na-dependent Cl/HCO3 exchanger, and the Cl/HCO3 exchanger. Intracellular pH (pHi) was determined by the use of the fluorescent pH-sensitive dye, 2',7'-bis(2- carboxyethyl)-5(6)-carboxyfluorescein (BCECF). We assessed transporter activity by studying recovery from an acid load (NH4/NH3) in CO2/HCO3. In adult MCs, Na/H exchanger was responsible for 35.2 +/- 4.3% of steady-state pHi, whereas the Na-dependent Cl/HCO3 exchanger contributed 58.7 +/- 6.1 (n = 14). In term MCs (tMCs), from days 1-3 after birth, the Na/H exchanger contributes 62.9 +/- 7.8% (n = 11, P < 0.001 vs. adult), whereas the Na-dependent Cl/HCO3 exchanger contributes 34.0 +/- 5.7% (n = 12, P < 0.001 vs. adult), to the rate of recovery from an acid load in these cells. However, in tMCs (days 4-6), the Na/H contributes 47.2 +/- 5.9% (n = 8, P < 0.05 vs. adult), whereas the Na-dependent Cl/HCO3 exchanger contributes 48.7 +/- 7.3% (n = 13, P < 0.05 vs. adult), to the rate of recovery. tMCs (days 6-12) yielded transporter activity that was not statistically different than adult MCs (37.8 +/- 4.9 and 54.3 +/- 10.2% for Na/H and Na-dependent Cl/HCO3, respectively). The magnitude of the stimulated response to angiotensin II by Na/H and Na-dependent Cl/HCO3 exchanger in adult and tMCs is unchanged throughout development. The Na/H exchanger appears to play a greater role in pHi homeostasis earlier on in development, and this may reflect developmental needs of the maturing cell.
Asunto(s)
Equilibrio Ácido-Base , Mesangio Glomerular/metabolismo , Equilibrio Ácido-Base/efectos de los fármacos , Factores de Edad , Angiotensina II/farmacología , Animales , Animales Recién Nacidos , Bicarbonatos/metabolismo , Transporte Biológico/efectos de los fármacos , Cloruros/metabolismo , Mesangio Glomerular/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Masculino , Ratas , Ratas Sprague-Dawley , Intercambiadores de Sodio-Hidrógeno/metabolismo , Factores de TiempoRESUMEN
OBJECTIVES: To evaluate whether alterations in nitric oxide (NO) synthesis or activity contribute to age-related erectile dysfunction and to elucidate the mechanisms causing these alterations using the rabbit as our model of aging. METHODS: We compared the ability of the rabbit cavernosal smooth muscle to relax in the organ bath in response to acetylcholine (Ach, endothelium-dependent vasodilator), sodium nitroprusside (SNP, an NO donor), and A23187 (a calcium ionophore) in young (6 month old) and aged (2.5 to 3.5 year old) rabbits. In addition, the immunohistochemical expression of endothelial nitric oxide synthase (eNOS) in both young and aged rabbit cavernosal tissue was examined. Endothelial integrity was examined immunohistochemically with JC70. RESULTS: Ach-mediated relaxation of penile corporal tissue was significantly attenuated from a maximum of 68.39 +/- 6.27 (0.1 mM Ach, n = 4) in young rabbits to 39.02 +/- 4.88 (0.1 mM Ach, n = 6) in aged rabbits (P < 0.04). No statistically significant difference (P > 0.05) was noted between cavernosal relaxation to sodium nitroprusside between young rabbits (97.8%, 0.1 mM SNP, n = 5) and aged rabbits (76.1%, 0.1 mM SNP, n = 5). This suggested that the defect in the Ach-NO pathway was at the level of NO synthesis, not activity. Immunohistochemical staining for eNOS demonstrated upregulation in both the vascular endothelium and corporal smooth muscle of aged rabbit tissue compared with young rabbit cavernosal tissue (n = 5). Anatomic endothelial integrity was demonstrated in the young and aged rabbits by the presence of JC70. This suggested that the defect in the Ach-NO synthetic pathway was not at the level of eNOS and was not due to anatomic endothelial cell disruption. Finally, Ach-mediated cavernosal smooth muscle relaxation in the young rabbit was not significantly augmented (P > 0.05) in the presence of the calcium ionophore A23187 (10 microM). A23187, however, significantly augmented (P < 0.04) Ach-mediated relaxation in the aged rabbit from a maximum of 33.93 +/- 6.58 to 41.55 +/- 6.58 (10 microM Ach, n = 5). This suggested that a potential defect in the Ach-NO synthetic pathway was at the level of intracellular calcium flux and possibly at the level of the calcium-eNOS interaction. CONCLUSIONS: Endothelium-dependent relaxation is attenuated in the aging rabbit; eNOS is upregulated in the aging rabbit; and no difference is noted in response to direct NO donation between the young and aged rabbit. The endothelium is anatomically intact in both the young and aging rabbit. The calcium ionophore A23187 augmented the attenuated vasorelaxation in the aging rabbit cavernosum (although not to the levels seen in the young rabbit cavernosum) and had no effect on the young rabbit cavernosum. These data suggest that erectile dysfunction in the aging rabbit cavernosum appears to be related to endothelial dysfunction and is characterized by eNOS upregulation and aberrant intracellular calcium fluxes.
Asunto(s)
Envejecimiento/metabolismo , Disfunción Eréctil/enzimología , Óxido Nítrico Sintasa/metabolismo , Acetilcolina/farmacología , Animales , Calcimicina/farmacología , Endotelio/enzimología , Inmunohistoquímica , Ionóforos/farmacología , Masculino , Nitroprusiato/farmacología , Pene/efectos de los fármacos , Pene/fisiología , Conejos , Regulación hacia Arriba , Vasodilatadores/farmacologíaAsunto(s)
Riñón/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Animales , Expresión Génica , Humanos , Riñón/citología , Riñón/metabolismo , Enfermedades Renales/genética , Enfermedades Renales/terapia , Glomérulos Renales/citología , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/metabolismo , Túbulos Renales/citología , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genéticaRESUMEN
Cell proliferation is a predominant feature in glomerulonephritis (GN). Recent work has suggested that protein kinase C (PKC) isoforms are responsible, specifically, PKC beta II in part, for cell growth. PKC beta II is expressed during cell growth in early glomerulogenesis and inflammatory mediators of glomerular disease induce PKC beta II expression. We therefore investigated the expression of PKC beta II in kidney biopsy specimens from patients with various types of proliferative (n = 41), nonproliferative GN (n = 23), and in structurally normal kidneys (n = 15). PKC beta II immunoreactivity was exclusively found in proliferative GN whereas PKC expression was not detected in normal glomerular and in nonproliferative disease states. The consistent expression of PKC beta II in proliferative GN suggests a key signaling role for this enzyme in cell proliferation in renal disease.
Asunto(s)
Glomerulonefritis/enzimología , Proteína Quinasa C/metabolismo , Adulto , Anciano , Secuencia de Aminoácidos , División Celular , Femenino , Glomerulonefritis/patología , Humanos , Inmunohistoquímica , Riñón/enzimología , Enfermedades Renales/enzimología , Enfermedades Renales/patología , Masculino , Persona de Mediana Edad , Proteína Quinasa C/genética , Proteína Quinasa C/inmunología , Proteína Quinasa C beta , Regulación hacia ArribaRESUMEN
A midregion fragment of PTH-related protein (PTHrP), which is intensively conserved across species, has been identified as a secretory product of several different cell types, including keratinocytes and squamous carcinomas. As recent data suggest that a midregion PTHrP fragment may be biologically active, we hypothesized that midregion PTHrPs interact with unique cell surface receptors that mediate autocrine or paracrine action. Dose-dependent transient elevations in intracellular calcium ([Ca2-]i) were observed in fura-2-loaded SqCC/Y1 squamous carcinoma cells exposed to human (h) PTHrP-(67-86)NH2, [Tyr36]hPTHrP-(1-36)NH2, and hPTHrP-(1-141) at concentrations ranging from 1 pM to 1 microM. The effects of maximal stimulatory concentrations of [Tyr36]PTHrP-(1-36)NH2 and PTHrP-(67-86)NH2 on [Ca2+]i were additive. The inhibitory PTH analog, [D-Trp12,Tyr34]bovine PTH-(7-34)NH2, attenuated the [Ca2+]i response to [Tyr36]hPTHrP-(1-36)NH2, but not that to PTHrP-(67-86)NH2. These data suggest that PTHrP-(67-86)NH2 activates a different receptor pathway in SqCC/Y1 cells from the one activated by [Tyr36]hPTHrP-(1-36)NH2. Radiolabeled PTHrP-(67-86)NH2 did not bind to SqCC/Y1 cells, and PTHrP-(67-86)NH2 did not compete for binding of 125I-labeled [Tyr36]PTHrP-(1-36)NH2 to PTH/PTHrP receptors on SaOS-2 osteosarcoma cells. Activation of the phospholipase C pathway by PTHrP-(67-86)NH2 was confirmed by exposing SqCC/Y1 cells to peptide for 1 min and measuring the accumulation of inositol trisphosphates. PTHrP-(67-86)NH2 treatment (100 nM) resulted in maximal stimulation of inositol trisphosphates of 3.1 +/- 0.1-fold over the control value, with an EC50 of 1.5 +/- 1.2 nm. In contrast, PTHrP-(67-86)NH2 (0.1 nM to 1 microM) did not stimulate adenylyl cyclase in SqCC/Y1 cells despite vigorous stimulation of cAMP formation by isoproterenol (1 microM) to 66-fold over the basal value. To determine whether messenger RNA (mRNA) prepared from SqCC/Y1 cells would direct the translation of a receptor protein that mediated a [Ca2+]i response to PTHrP-(67-86)NH2, we performed expression studies in Xenopus oocytes. Fluo-3 fluorescence in Xenopus oocytes expressing SqCC/Y1 mRNA was visualized by confocal video microscopy after exposure to 1 microM PTHrP-(67-86)NH2. Clear increases in [Ca2+]i were detected in mRNA-injected, but not in sham-injected, oocytes. Finally, we examined the effect of PTHrP-(67-86)NH2 treatment on fibronectin secretion from SqCC/YN1 cells. A significant 3.5-fold increase in fibronectin secretion into conditioned medium was observed when SqCC/Y1 cells were exposed to 100 nM PTHrP-(67-86)NH2, and this effect was dose dependent, with an EC50 of 0.1 nM. We conclude that PTHrP-(67-86)NH2 activates phospholipase C-dependent pathways in SqCC/Y1 cells through a receptor distinct from that activated by PTHrP-(1-36) in the same cells. As a midregion secretory fragment of PTHrP has been partially purified from several different cell types, this receptor may have broad biological significance.
Asunto(s)
Calcio/metabolismo , Carcinoma de Células Escamosas/metabolismo , Citosol/metabolismo , Inositol 1,4,5-Trifosfato/biosíntesis , Fragmentos de Péptidos/farmacología , Proteínas/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Carcinoma de Células Escamosas/patología , AMP Cíclico/metabolismo , Fibronectinas/metabolismo , Humanos , Oocitos/metabolismo , Proteína Relacionada con la Hormona Paratiroidea , Fosfatidilinositoles/metabolismo , Receptor de Hormona Paratiroídea Tipo 1 , Receptores de Hormona Paratiroidea/metabolismo , Células Tumorales Cultivadas , XenopusRESUMEN
In vitro and in vivo data suggest a remarkable plasticity in the differentiated phenotype of intrinsic glomerular cells, which after injury express new structures and functions. We have shown that a protein kinase C (PKC) isoform, beta II, is expressed in diseased but not normal glomeruli. Since intrarenal cytokine synthesis has been implicated in the pathogenesis of progressive glomerular injury, we have hypothesized that these mediators induce a change in isoform profile. To test this hypothesis in vitro, we have determined whether platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) alter the expression or activation of PKC isoforms in cultured mesangial cells (MCs). By immunoblot and ribonuclease (RNase) protection assays, both PDGF and IL-1 induce as early as 2 h de novo synthesis of PKC-beta II. Since MCs constitutively express PKC-alpha, -beta I, and -zeta, we also determined whether IL-1 or PDGF alter the activity of these isoforms. PDGF maximally induced translocation of PKC-alpha (10 min), -beta I (90 min), -epsilon (120 min), and -zeta (120 min) from the cytosolic to the membrane fraction. IL-1, in contrast, did not alter the distribution of alpha, beta I, or epsilon at any time measured but did induce PKC-zeta translocation. These data suggest inflammatory mediators regulate PKC isoform activity in diseased glomeruli both by de novo synthesis of unexpressed isoforms and by activation of constitutively expressed PKC isoforms.
Asunto(s)
Mesangio Glomerular/metabolismo , Interleucina-1/farmacología , Isoenzimas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteína Quinasa C/metabolismo , Animales , Western Blotting , Células Cultivadas , Activación Enzimática , Mesangio Glomerular/citología , Masculino , Hibridación de Ácido Nucleico , Ratas , Ratas Sprague-Dawley , RibonucleasasRESUMEN
We tested the hypothesis that acetylcholine-induced relaxation in cavernosal tissue is the result of nitric oxide production that alters K+ conductance. In the organ bath, acetylcholine- and sodium nitroprusside-induced relaxation of corpus cavernosum were significantly attenuated by tetraethylammonium. Basal [K+]i in cavernosal smooth muscle cells was 102 +/- 11 mM using a K+-sensitive fluorescent dye. Acetylcholine produced a decrease in [K+]i to 74 +/- 10 (n > 4, P < 0.05). Tetraethylammonium pretreatment blunted the acetylcholine- and sodium nitroprusside-induced decrease in [K+]i by 82%, (n > 5, P < 0.001), respectively. L-NMMA blunted the acetylcholine- and sodium nitroprusside-induced fall in [K+]i by 93% and 83% (n > 4, P < 0.001), respectively. These data suggest that acetylcholine-mediated cavernosal smooth muscle cells relaxation occurs through nitric oxide release with activation of K+ conductance.
Asunto(s)
Arginina/análogos & derivados , Inhibidores Enzimáticos/farmacología , Relajación Muscular/efectos de los fármacos , Músculo Liso/fisiología , Óxido Nítrico/fisiología , Nitroprusiato/farmacología , Pene/fisiología , Canales de Potasio/fisiología , Vasodilatadores/farmacología , Acetilcolina/farmacología , Animales , Arginina/farmacología , Células Cultivadas , AMP Cíclico/metabolismo , Cinética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Músculo Liso/efectos de los fármacos , Óxido Nítrico Sintasa/antagonistas & inhibidores , Pene/efectos de los fármacos , Potasio/metabolismo , Canales de Potasio/efectos de los fármacos , Conejos , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacología , Factores de Tiempo , omega-N-MetilargininaRESUMEN
Endothelium-derived relaxing factor is believed to be nitric oxide (NO). Evidence suggests that it has important functions in the regulation of mesangial cell (MC) tone and possibly in inflammation. As a vasodilator, its vasorelaxant effect depends, in part, on changes in cell membrane potential. We therefore tested the hypothesis that MCs in culture produce NO that results in cell relaxation through the opening of K+ conductance pathways. The K(+)-sensitive fluorescent dye, potassium-binding benzofuran isophthalate, was used to detect rapid changes in intracellular K+ concentration ([K+]i). The membrane potential-sensitive dye, 1,3-(sulfonatopropyl)-4-([beta-di-n- butylamino)-6-naphthyl]vinyl)pyridinium betaine (Di-4-AN-EPPS), was used to detect changes in membrane potential. Basal [K+]i was 92 +/- 9 mM (n = 46). In response to sodium nitroprusside (SNP), acetylcholine (ACh), and bradykinin (BK), [K+]i decreased to 72 +/- 7 (n = 5, P < 0.05), 70 +/- 8 (n = 7, P < 0.05), and to 69 +/- 13 mM (n = 6, P < 0.05), respectively. [K+]i rapidly returned to basal level in the continued presence of all three agonists. The SNP-, ACh-, and BK-induced decrease in [K+]i was significantly blunted by Ba2+ by 85, 92, and 89%, respectively (n > 4 for each agonist examined, P < 0.0001). NG-monomethyl-L-arginine (L-NMMA) and methylene blue inhibited the [K+]i-lowering effect of ACh and BK by 94 and 85% for L-NMMA (n = 5 for each agonist, P < 0.0001) and by 67 and 72% for methylene blue (n = 5 for each agonist, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
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Arginina/análogos & derivados , Mesangio Glomerular/metabolismo , Óxido Nítrico/fisiología , Nitroprusiato/farmacología , Potasio/metabolismo , Acetilcolina/farmacología , Aminoácido Oxidorreductasas/antagonistas & inhibidores , Aminoácido Oxidorreductasas/metabolismo , Animales , Arginina/farmacología , Células Cultivadas , Citosol/efectos de los fármacos , Citosol/metabolismo , Colorantes Fluorescentes , Mesangio Glomerular/efectos de los fármacos , Cinética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Óxido Nítrico Sintasa , Compuestos de Piridinio , Ratas , omega-N-MetilargininaRESUMEN
In the pre- and postnatal period of kidney development, proliferation with subsequent functional maturation of intrinsic glomerular mesangial cells (MCs) continues within the existing framework. Recent work has suggested that PKC beta isoform is responsible for the proliferation observed during maturation. We sought to ascertain whether PKC beta isoform expression is altered during the development of the mesangium. MCs were subcultured from glomerular explants of Sprague-Dawley rat kidneys, days 1, 3, 5, 8, 15 postnatally, and adult. MCs from rat kidneys postnatally days 1-5 proliferated at a significantly greater rate than adult [ > 1.169-fold, P < 0.01] but term day 8 cells did not [ < 1.34-fold, not significant)] as assessed by [3H]thymidine incorporation. Western blot analysis using isoform specific antibodies was performed on confluent neonatal and adult MC. We observed that all neonatal and adult MC express beta I PKC (n = 8 kidneys from separate primaries for each date and adult). However, unlike adult MCs, neonatal MC express beta II in postnatal days 1-5 and none thereafter. Immunofluorescent staining of postnatal kidneys confirmed that PKC beta II is present in neonatal MC up to day 5. By day 8, staining of mesangium with PKC beta II begins to disappear and assumes a parietal epithelial pattern. In adult kidneys, there was only PKC beta II staining of the parietal epithelial cells. Our results demonstrate that differential expression of PKC beta II closely parallels the proliferative behavior of the MCs of the maturing glomerulus. Therefore, PKC beta II expression and activation may play a critical role in development.
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Mesangio Glomerular/enzimología , Mesangio Glomerular/crecimiento & desarrollo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Envejecimiento , Animales , Animales Recién Nacidos , Western Blotting , División Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Ratas , Ratas Sprague-DawleyRESUMEN
Aldosterone secretion from the adrenal glomerulosa (AG) cells is inhibited by atrial natriuretic peptide (ANP). Inasmuch as alterations in K+ conductance can modulate aldosterone secretion, the effect of ANP on intracellular K+ homeostasis was investigated. Intracellular K+ concentration ([K+]i) of AG cells was assessed by spectrofluorometry using the K(+)-sensitive dye, K(+)-binding benzofuran isophthalate. The resting value of [K+]i in AG cells was determined to be 120 +/- 1.2 mM (n = 37) in a HCO3-free, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered medium. Exposure of AG cells to ANP led to a dose-dependent, transient decrease in [K+]i, from 21 +/- 3.2% (n = 7) at 100 pM to 31 +/- 2.3% at 1 microM (n = 7). In the continued presence of ANP, a rapid recovery to near basal values of [K+]i was attained within 90 s. Measurements of membrane voltage using the potential sensitive dye 1-3(-sulfonatopropyl)-4-[beta-(-(di-n-butylamino)-6-naphthyl)vinyl ]- pyridinium betaine documented an accompanying change in membrane potential. Pretreatment of AG cells with barium (0.5 mM), tetraethylammonium (0.1 mM), charybdotoxin (100 nM), or ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (0.5 mM) blunted the ANP-induced decrease in [K+]i. ANP-(7-23), the ANP-C-receptor selective agonist, which does not elevate guanosine 3',5'-cyclic monophosphate (cGMP) did not alter [K+]i in contrast to cGMP (50 microM), which did. We conclude that ANP via the activation of the ANP A receptor alters K+ homeostasis through a Ca(2+)-activatable K(+)-conductive pathway likely to be the maxi-K channel.
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Factor Natriurético Atrial/farmacología , Canales de Potasio/fisiología , Potasio/metabolismo , Zona Glomerular/fisiología , Animales , Bario/farmacología , Bovinos , Células Cultivadas , Caribdotoxina , GMP Cíclico/metabolismo , Ácido Egtácico/farmacología , Guanilato Ciclasa/metabolismo , Homeostasis/efectos de los fármacos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Fragmentos de Péptidos/farmacología , Bloqueadores de los Canales de Potasio , Canales de Potasio/efectos de los fármacos , Receptores del Factor Natriurético Atrial/metabolismo , Venenos de Escorpión/farmacología , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacología , Zona Glomerular/efectos de los fármacosRESUMEN
We investigated the long-term effects of exposing mesangial cells (MCs), previously quiescent, to platelet-derived growth factor, arginine vasopressin (AVP), epidermal growth factor, and serotonin (5-HT). MCs were exposed to the mitogens continuously for periods of 30-70 h. For each agent, we periodically measured intracellular pH (pH(i)), activities of Na+/H+, Na(+)-dependent Cl-/HCO3-, and Cl-/HCO3- exchangers, and cell number. pH(i) and transporter activity were assessed using the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyflourescein in a population of cells attached to glass cover slips. The first two transporters were assayed at the single pH(i) of 6.6, whereas Cl-/HCO3- exchange was assayed at pH(i) 7.5. Cell number was determined using fluorescence-activated cell sorting (FACS). Incubations and assays were conducted in presence of CO2/HCO30. No agent substantially altered pH(i), which generally ranged from 7.25 to 7.35. pH(i) in AVP-treated cells tended to be approximately 0.05 lower than in time-matched controls at the longer incubation times. All agents, when assayed minutes after mitogen application, caused large increases (70-130%) in activities of all three transporters. However, transporter activity gradually declined in continuous presence of each agent, first rapidly (e.g., 2-4 h) and then more slowly. Measurements of immunoreactive extracellular AVP ([AVP]o) levels indicate that the decrease in Cl-/HCO3- exchange activity is not due to a decrease in [AVP]o. Minimum transporter activity (10-40% above baseline) was achieved approximately 18-35 h after mitogen application. Later, transporter activity slowly increased, although not to the high levels achieved minutes after mitogen addition. For each mitogen, activities of all three transporters tended to rise and fall together. FACS data showed that each agent produced a gradual but significant increase in the number of cells in each cell-cycle phase (G0/G1, S, and M). There also was an increase in the total number of cells, confirming previous [3H]thymidine incorporation data. The fraction of cells in G0/G1, S, or M phase did not correlate well with the gradual decrease in transporter activity. However, the total number of cells peaked at the time of minimal transporter activity. These data demonstrate that, when studied at the cell-population level, mitogens produce a burst in acid-base transporter activity that gradually wanes as cell number increases. Conversely, transporter activity later increases after the peak in cell number.
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Antiportadores/metabolismo , Mesangio Glomerular/metabolismo , Sustancias de Crecimiento/farmacología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Sodio/farmacología , Animales , Transporte Biológico , Antiportadores de Cloruro-Bicarbonato , ADN/análisis , Citometría de Flujo , Mesangio Glomerular/citología , Concentración de Iones de Hidrógeno , Masculino , Ratas , Factores de TiempoRESUMEN
Local use of practice guidelines requires paying close attention to the concerns of the patient within the framework of society, to the professional and educational needs of the provider, and to the realities of cost. One Veterans Affairs facility took the challenge of balancing these factors and developed their own algorithms for three cardiovascular disorders.
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Algoritmos , Atención Ambulatoria/normas , Angina de Pecho/tratamiento farmacológico , Insuficiencia Cardíaca/tratamiento farmacológico , Hospitales de Veteranos/normas , Hipertensión/tratamiento farmacológico , Guías de Práctica Clínica como Asunto , Desarrollo de Programa , Angina de Pecho/diagnóstico , Angina de Pecho/etiología , Angina de Pecho/mortalidad , Inhibidores de la Enzima Convertidora de Angiotensina/economía , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Bloqueadores de los Canales de Calcio/economía , Bloqueadores de los Canales de Calcio/uso terapéutico , Análisis Costo-Beneficio , Costos de los Medicamentos , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/mortalidad , Humanos , Hipertensión/diagnóstico , Hipertensión/etiología , Hipertensión/mortalidad , OhioRESUMEN
Parathyroid hormone-related protein (PTHrP) is widely expressed in normal adult and fetal tissues, where it acts in an autocrine/paracrine fashion, stimulates growth and differentiation, and shares early response gene characteristics. Since recovery from renal injury is associated with release of local growth factors, we examined the expression and localization of PTHrP in normal and ischemic adult rat kidney. Male Sprague-Dawley rats underwent complete bilateral renal artery occlusion for 45 min, followed by reperfusion for 15 min, and 2, 6, 24, 48, and 72 h. Renal PTHrP mRNA levels, when compared with sham-operated animals, increased twofold after ischemia, and peaked within 6 h after reperfusion. PTH receptor, beta-actin, and cyclophilin mRNA levels all decreased after ischemia. PTHrP immunohistochemical staining intensity increased in proximal tubular cells after ischemia, changing its location from diffusely cytoplasmic to subapical by 24 h after reperfusion. In addition, PTHrP localized to glomerular epithelial cells (visceral and parietal), but not to mesangial cells. PTHrP and PTH stimulated proliferation two- to threefold in cultured mesangial cells. We conclude that PTHrP mRNA and protein production are upregulated after acute renal ischemic injury, that PTHrP is present in glomerulus and in both proximal and distal tubular cells, and that PTHrP stimulates DNA synthesis in mesangial cells. The precise functions of PTHrP in normal and injured kidney remain to be defined.