RESUMEN
The human response to surgical stress is characterized by massive release of neuroendocrine hormones, provoking catabolism, thermogenesis, and hyperglycemia. Considering the possible adverse outcomes of excessive stress hormones, understanding various components of the stress response may improve management of postoperative morbidity. Leptin, initially described as an adipocyte-derived signaling factor, may also play an important role in regulating the hypothalamo-pituitary-adrenocortical axis. In phase I, plasma leptin and cortisol were measured in women before, during, and after total abdominal hysterectomy. The anesthetic technique was strictly controlled, balanced anesthesia. In phase II, plasma leptin and cortisol levels were measured in cardiac surgery patients. These subjects were anesthetized with a high dose opioid technique that blunts the intraoperative surgical stress response. In phase I, mean leptin levels did not change over the week before surgery, had a maximal decrease to 49% of baseline 2 h after surgery, and increased to just above baseline 24 h postoperatively. Cortisol was 176% of the baseline just before surgery, peaked at 2 h after surgery (383%), and remained elevated 24 h (200%) and 48 h (165%) after surgery. During the first 2 h of surgery, the decrease in leptin parallels the increase in cortisol. In phase II, high dose fentanyl limited both the cortisol increase and the leptin decrease; thus, the ratio of cortisol increase to leptin decrease was similar for the cardiac patients and the hysterectomy patients. These data indicate that leptin has a role in the surgically induced acute stress response in humans. Early in surgery the decrease in leptin parallels the increase in cortisol. This suggests a possible relationship between the neurobiology of these two systems, which could have important implications for regulation of the neuroendocrine response to surgical stress.
Asunto(s)
Puente de Arteria Coronaria , Histerectomía , Proteínas/metabolismo , Estrés Fisiológico/sangre , Adulto , Anciano , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/farmacología , Anestesia , Estudios de Cohortes , Enfermedad Coronaria/cirugía , Femenino , Fentanilo/administración & dosificación , Fentanilo/farmacología , Humanos , Hidrocortisona/sangre , Cinética , Leptina , Persona de Mediana EdadRESUMEN
Alendronate (4-amino-1-hydroxybutylidene-1,1-bisphosphonate) is a potent bisphosphonate that inhibits osteoclastic bone resorption and has proven effective for the treatment of osteoporosis. Its molecular mechanism of action, however, has not been defined precisely. Here we report that alendronate is a potent inhibitor of the protein-tyrosine-phosphatase-meg1 (PTPmeg1). Two substrates were employed in this study: fluorescein diphosphate and the phosphotyrosyl peptide src-pY527. With either substrate, alendronate was a slow binding inhibitor of PTPmeg1. Among the other bisphosphonates studied, alendronate was more potent and selective for PTPmeg1. The hydrolysis of fluorescein diphosphate by PTP epsilon and PTPmeg1 was sensitive to alendronate, with IC50 values of less than 1 microM; PTPsigma, however, under the same conditions, was inhibited by only 50% with 141 microM alendronate. Similarly, with the src-pY527 substrate, alendronate inhibition was also PTP dependent. Alendronate inhibited PTPmeg1 with an IC50 value of 23 microM, PTPsigma with an IC50 value of 2 microM, and did not inhibit PTP epsilon at concentrations up to 1 mM. The alendronate inhibition of these three PTPs and two substrates is consistent with the formation of a ternary complex comprised of enzyme, substrate, and inhibitor. PTP inhibition by hisphosphonates or vanadate was diminished by the metal chelating agent EDTA, or by the reducing agent dithiothreitol, suggesting that a metal ion and the oxidation of a cysteine residue are required for full inhibition. These observations show substrate- and enzyme-specific PTP inhibition by alendronate and support the possibility that a certain PTP(s) may be the molecular target for alendronate action.
Asunto(s)
Alendronato/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , ADN Complementario , Humanos , Proteína Tirosina Fosfatasa no Receptora Tipo 4 , Proteínas Tirosina Fosfatasas/metabolismo , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
Osteoclast activation involves attachment to the mineralized bone matrix and reorganization of the cytoskeleton, leading to polarization of the cell. Signaling molecules, PI3-kinase, rho A, and pp60c-src, were shown to be essential for osteoclastic bone resorption. In this study we have focused on the involvement of these signaling molecules in the early event of osteoclast activation: attachment, spreading, and organization of the cytoskeleton. Highly purified osteoclasts were fractionated into Triton X-100-soluble or cytosolic and Triton X-100-insoluble or cytoskeletal fractions, and the distribution of above-mentioned signaling molecules between the two fractions was examined. PI3-kinase, rho A, and pp60c-src all showed translocation to the cytoskeletal fraction upon osteoclast attachment to plastic. However, PI3-kinase and rho A, but not pp60c-src, showed further translocation of 2.4- and 3.2-fold, respectively, upon attachment of osteoclasts to bone. PI3-kinase translocation to the cytoskeleton was inhibited by either cytochalasin B or colchicine. Furthermore, treatment of osteoclasts with the PI3-kinase inhibitor wortmannin decreased its translocation, suggesting that PI3-kinase activity was needed for its translocation. Moreover, wortmannin inhibited osteoclast attachment to both bone and plastic and caused drastic changes in osteoclast morphology resulting in rounding of the cells, disappearance of F-actin structures or podosomes, and appearance of punctate or vesicular structures inside the cells. Osteoblastic MB1.8 cells and IC-21 macrophages did not show additional translocation of PI3-kinase or rho A upon attachment to bone or changes in attachment or morphology in response to wortmannin. Finally, PI3-kinase coimmunoprecipitated with alpha v beta 3 integrin from osteoclasts.
Asunto(s)
Osteoclastos/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Androstadienos/farmacología , Animales , Células de la Médula Ósea/citología , Huesos/citología , Adhesión Celular , Células Cultivadas , Citoesqueleto/enzimología , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/metabolismo , Ratones , Osteoclastos/ultraestructura , Inhibidores de las Quinasa Fosfoinosítidos-3 , Receptores de Vitronectina/metabolismo , Wortmanina , Proteína de Unión al GTP rhoARESUMEN
To gain insight into the role of the eukaryotic translation initiation factor, eIF-5A, we investigated the subcellular distribution of this protein in several cultured cell types and at different stages of the cell cycle using a highly potent monospecific polyclonal antibody to eIF-5A. Studies using indirect immunofluorescence and confocal microscopy in conjunction with subcellular fractionation demonstrate that eIF-5A is primarily localized in the cytoplasm of cells. This cytoplasmic location of eIF-5A is not significantly altered in different stages of the cell cycle and the subcellular distribution pattern of eIF-5A is not changed by viral oncogene transformation. Cell fractionation experiments identified two populations of eIF-5A in the cytoplasm, a soluble fraction and a fraction bound to internal membranes. By double immunofluorescence staining with an antibody against calnexin, a resident protein of the endoplasmic reticulum (ER), we demonstrate that the membrane-bound fraction of eIF-5A colocalizes with the ER and not with the cytoskeleton. Expression of Rev, a regulatory protein of human immunodeficiency virus type 1 (HIV-1), does not alter the subcellular distribution of endogenous eIF-5A in these cells. eIF-5A is detected in all tissues and cells examined including extracts prepared from Xenopus oocytes. Our results indicate that eIF-5A is a ubiquitous cytoplasmic protein and suggest that a site of eIF-5A function is likely to be in association with the ER.
Asunto(s)
Células 3T3/ultraestructura , Síndrome de Inmunodeficiencia Adquirida/virología , VIH-1/genética , Factores de Iniciación de Péptidos/análisis , Proteínas de Unión al ARN , Fracciones Subcelulares/química , Células 3T3/química , Animales , Especificidad de Anticuerpos , Western Blotting , Ciclo Celular/fisiología , Citoesqueleto/química , Retículo Endoplásmico/química , Expresión Génica/fisiología , Productos del Gen rev/genética , Ratones , Microscopía Confocal , Factores de Iniciación de Péptidos/inmunología , Biosíntesis de Proteínas/fisiología , Proteínas Virales de Fusión/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Acid extrusion is essential for osteoclast (OC) activity. We examined Na+ and HCO3(-)-independent H+ extrusion in rat- and mouse OCs by measuring intracellular pH (pHi) changes, with the pHi indicator BCECF (biscarboxyethyl-5-(6) carboxyfluorescein) after H+ loading with an ammonium pulse. 90% of OCs attached to glass do not possess HCO3- and Na(+)-independent H(+)-extrusion (rate of pHi recovery = 0.043 +/- 0.007 (SEM) pH U/min, n = 26). In contrast, in OCs attached to bone, the pHi recovery rate is 0.228 +/- 0.011 pHi U/min, n = 25. OCs on bone also possess a NH(4+)-permeable pathway not seen on glass. The bone-induced H+ extrusion was inhibited by salmon calcitonin (10(-8) M, for 2 h), and was not present after pretreating the bone slices with the aminobisphosphonate alendronate (ALN). At ALN levels of 0.22 nmol/mm2 bone, H+ extrusion was virtually absent 12 h after cell seeding (0.004 +/- 0.002 pH U/min) and approximately 50% inhibition was observed at 0.022 pmol ALN/mm2 bone. The Na(+)-independent H+ extrusion was not inhibited by bafilomycin A1 (up to 10(-7) M), although a bafilomycin A1 (10(-8) M)-sensitive H+ pump was present in membrane vesicles isolated from these osteoclasts. These findings indicate that Na(+)-independent acid extrusion is stimulated by osteoclast attachment to bone and is virtually absent when bone is preincubated with ALN, or when osteoclasts are treated with salmon calcitonin.
Asunto(s)
Huesos/metabolismo , Calcitonina/farmacología , Difosfonatos/farmacología , Hidrógeno/metabolismo , Osteoclastos/metabolismo , Alendronato , Animales , Adhesión Celular , Células Cultivadas , Técnicas de Cocultivo , Concentración de Iones de Hidrógeno/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Ratones , RatasRESUMEN
We evaluated the possible involvement of intracellular Ca2+ concentration ([Ca2+]i) changes in the action of alpha v beta 3-ligands, known to regulate osteoclast function. Rat osteoclasts or mouse osteoclast-like cells, as examined by microfluorimetry and fura 2, showed a transient [Ca2+]i increase when perfused with (all 0.1 microM) vitronectin, osteopontin, polypeptide echistatin, fibronectin, and Arg-Gly-Asp-Asp and Arg-Gly-Asp-Ser peptides (10(-4) M) but not with laminin, collagen I, collagen IV, or [Ala24]echistatin, in which Ala was substituted for Arg in the Arg-Gly-Asp complex. The threshold for echistatin was 10 pM, the 50% effective concentration was 1 nM, and the median [Ca2+]i increase was 420 nM above the resting level (217 +/- 22 nM) at saturating concentration of 0.1 microM. Echistatin did not cause Mn2+ influx, and 10 microM nifedipine, 10 nM omega-conotoxin, 5 mM Ni2+, or Cd2+ did not prevent [Ca2+]i change. However, extracellular Ca2+ was needed for the [Ca2+]i increase, probably enabling ligand-integrin interaction. Polyclonal and monoclonal (LM609) antibody as well as depletion of [Ca2+]i stores with 5 microM thapsigargin and Ca(2+)-free medium abolished the [Ca2+]i increase, after restoring extracellular Ca2+. Furthermore, the LM609 antibody induced a Ca2+ signal in the presence or absence of extracellular Ca2+, suggesting that the alpha v beta 3-ligand interaction is mediated at least partially by Ca2+ mobilized from intracellular stores.
Asunto(s)
Calcio/metabolismo , Integrinas/metabolismo , Membranas Intracelulares/metabolismo , Osteoclastos/metabolismo , Péptidos , Animales , Anticuerpos Monoclonales , Células Cultivadas , Sueros Inmunes , Péptidos y Proteínas de Señalización Intercelular , Ligandos , Ratones , Concentración Osmolar , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Solubilidad , Venenos de Víboras/química , Venenos de Víboras/farmacologíaRESUMEN
The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hidrógeno/metabolismo , Túbulos Renales Proximales/enzimología , Macrólidos , ATPasas de Translocación de Protón/análisis , Adenosina Trifosfato/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Antiarrítmicos/farmacología , Antibacterianos/farmacología , Antifúngicos/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Proteínas Portadoras/fisiología , Fraccionamiento Celular , Citofotometría , Electroforesis , Fluorescencia , Concentración de Iones de Hidrógeno , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/fisiología , Masculino , ATPasas de Translocación de Protón/metabolismo , ATPasas de Translocación de Protón/fisiología , Ratas , Ratas Endogámicas , Intercambiadores de Sodio-HidrógenoRESUMEN
In the presence of inhibitors for mitochondrial H+-ATPase, (Na+ + K+)- and Ca2+-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane ("ecto-ATPases"). These ATPases accept several nucleotides, are stimulated by Ca2+ and Mg2+, and are inhibited by N.N'-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brush-border and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg2+ and Mn2+, but not by Ca2+, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator. ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H+ secretion is electrogenic and requires either exit of a permeant anion (Cl-) or entry of a cation, e.g., Na+ via electrogenic Na+/D-glucose and Na+/L-phenylalanine uptake. In the presence of Na+, ATP-driven H+ efflux is stimulated by blocking the Na+/H+ exchanger with amiloride. These data prove the coexistence of Na+-coupled substrate transporters, Na+/H+ exchanger, and an ATP-driven H+ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H+ pumps with (a part of) the DCCD- and NEM- sensitive ATPases at the cytosolic side of the brush-border membrane.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Túbulos Renales Proximales/enzimología , Adenosina Trifosfatasas/antagonistas & inhibidores , Animales , Transporte Biológico Activo , Diciclohexilcarbodiimida/farmacología , Electrólitos/fisiología , Etilmaleimida/farmacología , Técnicas In Vitro , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Microvellosidades/enzimología , Microvellosidades/metabolismo , Nucleótidos/metabolismo , Octoxinol , Polietilenglicoles , Protones , RatasRESUMEN
1. Na-Pi co-transport was analysed using renal cortical and small intestinal brush-border membrane vesicles which were isolated from control (normal, heterozygotes) and rachitic piglets (homozygotes). 2. A kinetic analysis of Na-dependent initial linear uptake of Pi was performed using vesicles obtained from control animals. The results suggest similar kinetic properties for the renal and small intestinal co-transport system. (i) A sigmoidal dependence on Na concentration of Pi uptake suggests the involvement of more than one Na ion in the co-transport. (ii) Increasing Na concentration leads to an increase in the apparent affinity of the transport system for Pi and has minimal effect on the apparent Vmax (maximum velocity of uptake). (iii) Increasing pH leads to an increase in Pi transport rate. 3. The kinetic characteristics of the Na-Pi co-transport system in vesicles obtained from rachitic animals were similar to those in controls. The apparent Vmax, but not the apparent Km (Michaelis constant) for Na and Pi, is reduced in intestinal and renal brush-border membranes isolated from rachitic animals as compared to control animals. Injection of vitamin D3, three days prior to killing of rachitic litter-mates, increased the Na-Pi uptake rate in the brush-border membrane vesicles isolated from these piglets. 4. It is concluded that intestinal and renal brush-border membranes from piglets contain a similar Na-Pi co-transport system and that in vitamin-D-dependent rickets the number of operating transport units is reduced in both membranes.
Asunto(s)
Intestino Delgado/metabolismo , Riñón/metabolismo , Microvellosidades/metabolismo , Fosfatos/metabolismo , Raquitismo/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Colecalciferol/uso terapéutico , Femenino , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Raquitismo/tratamiento farmacológico , Sodio/farmacología , PorcinosRESUMEN
Na+-H+ exchange in rat and mouse renal brush-border membrane vesicles was studied by fluorescence quenching of the delta pH indicator, acridine orange. Brush-border membrane vesicles were isolated by a modified Mg/EGTA-precipitation method at low speed centrifugation (8000 X g). The enzymatic characteristics of these membrane vesicles were similar to those obtained by the original high-speed centrifugation method (Biber et al. (1981) Biochim. Biophys. Acta, 647, 169-176). The rates of Na+-H+ exchange in renal brush-border membrane vesicles from male and female rats were similar. Neither ovariectomy nor treatment of ovariectomized rats with estradiol or testosterone changed the activity of Na+-H+ exchanger. The rates of Na+-H+ exchange in the mouse were smaller than in the rat indicating the existence of species differences. Na+-H+ exchange in mouse renal brush-border membranes exhibit strong sex differences, the rates in the male being higher than in the female. Castration of male mice led to a decrease in Na+-H+ exchange to values found in females. Treatment of castrated mice with estradiol had no effect. In contrast, treatment with testosterone increased the rate of the exchanger by more than 100%. The effect of testosterone was restricted to the Vmax of the Na+-H+ exchanger, whereas the apparent Km for Na+ remained unchanged. Na+-dependent D-glucose transport in mouse renal luminal membranes exhibited also sex differences due to the potent stimulatory effect of testosterone. Therefore, Na+-H+ exchange and Na+-dependent D-glucose transport in the mouse kidney are under control of androgen hormones. This effect could be in close connection with the wellknown renotropic action of androgens in the mouse.