RESUMEN
In these studies, we have used several approaches to systematically explore the contribution of transcellular vesicular transport (transcytosis) to the blood-to-bile movement of inert fluid-phase markers of widely varying molecular weight. First, under steady-state conditions, the perfused rat liver secreted even large markers in appreciable amounts. The bile-to-plasma (B/P) ratio of these different markers, including microperoxidase (B/P ratio = 0.06; mol wt = 1,879), insulin (B/P ratio = 0.09, mol wt = 5,000), horseradish peroxidase (B/P ratio = 0.04, mol wt = 40,000), and dextran (B/P ratio = 0.09, mol wt = 70,000), exhibited no clear ordering based on size alone, and when dextrans of two different sizes (40,000 and 70,000 mol wt) were studied simultaneously, the relative amounts of the two dextran species in bile were the same as in perfusate. Taurocholate administration produced a 71% increase in bile flow but little or no (0-20%) increase in the output of horseradish peroxidase, microperoxidase, inulin, and dextran. Second, under nonsteady-state conditions in which the appearance in or disappearance from bile of selected markers was studied after their abrupt addition to or removal from perfusate, erythritol reached a B/P ratio of 1 within 2 min. Microperoxidase and dextran appeared in bile only after a lag period of approximately 12 min and then slowly approached maximal values, whereas sucrose exhibited kinetically intermediate behavior. A similar pattern was observed after removal of greater than 95% of the marker from the perfusate. Erythritol rapidly reapproached a B/P ratio of 1, whereas the B/P ratio for sucrose, dextran, and microperoxidase fell much more slowly and exceeded 1 for a full 30 min after perfusate washout. Finally, electron microscopy and fluorescence microscopy of cultured hepatocytes demonstrated the presence of horseradish peroxidase and fluorescein-dextran, respectively, in intracellular vesicles, and fractionation of perfused liver homogenates revealed that at least 35-50% of sucrose, inulin, and dextran was associated with subcellular organelles. Collectively, these observations are most compatible with a transcytosis pathway that contributes minimally to the secretion of erythritol, but accounts for a substantial fraction of sucrose secretion and virtually all (greater than 95%) of the blood-to-bile transport of microperoxidase and larger markers. These findings have important implications with respect to current concepts of canalicular bile formation as well as with respect to the conventional use of solutes such as sucrose as markers of canalicular or paracellular pathway permeability.
Asunto(s)
Bilis/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Hígado/metabolismo , Animales , Transporte Biológico , Dextranos , Eritritol/metabolismo , Fluoresceínas , Hígado/ultraestructura , Microscopía Electrónica , Peso Molecular , Perfusión , Peroxidasas/metabolismo , RatasRESUMEN
To characterize the transport mechanisms responsible for formation of canalicular bile, we have examined the effects of ion substitution on bile acid-dependent and bile acid-independent bile formation by the isolated perfused rat liver. Complete replacement of perfusate sodium with choline and lithium abolished taurocholate-induced choleresis and reduced biliary taurocholate output by greater than 70%. Partial replacement of perfusate sodium (25 of 128 mM) by choline reduced bile acid-independent bile formation by 30% and replacement of the remaining sodium (103 mM) by choline reduced bile acid-independent bile formation by an additional 64%. In contrast, replacement of the remaining sodium (103 mM) by lithium reduced bile acid-independent bile formation by only an additional 20%, while complete replacement of sodium (128 mM) by lithium reduced bile formation by only 17%, and lithium replaced sodium as the predominant biliary cation. Replacement of perfusate bicarbonate by Tricine, a zwitterionic amino acid buffer, decreased bile acid-independent bile formation by greater than or equal to 50% and decreased biliary bicarbonate output by approximately 60%, regardless of the accompanying cation. In separate experiments, replacement of sodium by lithium essentially abolished Na,K-ATPase activity measured either as ouabain-suppressible ATP hydrolysis in rat liver or kidney homogenates, or as ouabain-suppressible 86Rb uptake by cultured rat hepatocytes. These studies indicate that bile acid(taurocholate)-dependent bile formation by rat liver exhibits a specific requirement for sodium, a finding probably attributable to the role(s) of sodium in hepatic sodium-coupled taurocholate uptake and/or in maintenance of Na,K-ATPase activity. The surprising finding that bile acid-independent bile formation was substantially unaltered by complete replacement of sodium with the permeant cation lithium does not appear to be explained by Na,K-ATPase-mediated lithium transport. Although alternative interpretations exist, this observation is consistent with the hypothesis that much of basal bile acid-independent bile formation is attributable to an ion pump other than Na,K-ATPase, which directly or indirectly mediates bicarbonate transport.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Bilis/metabolismo , Hígado/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Aniones/farmacología , Bicarbonatos/farmacología , Transporte Biológico Activo , Cationes Monovalentes/farmacología , Riñón/enzimología , Hígado/enzimología , Masculino , Ratas , Tasa de Secreción/efectos de los fármacos , Sodio/fisiologíaRESUMEN
We have systematically investigated certain characteristics of the ATP-dependent proton transport mechanism of bovine brain clathrin-coated vesicles. H+ transport specific activity was shown by column chromatograpy to co-purify with coated vesicles, however, the clathrin coat is not required for vesicle acidification as H+ transport was not altered by prior removal of the clathrin coat. Acidification of the vesicle interior, measured by fluorescence quenching of acridine orange, displayed considerable anion selectively (Cl- greater than Br- much greater than NO3- much greater than gluconate, SO2-(4), HPO2-(4), mannitol; Km for Cl- congruent to 15 mM), but was relatively insensitive to cation replacement as long as Cl- was present. Acidification was unaffected by ouabain or vanadate but was inhibited by N-ethylmaleimide (IC50 less than 10 microM), dicyclohexylcarbodiimide (DCCD) (IC50 congruent to 10 microM), chlorpromazine (IC50 congruent to 15 microM), and oligomycin (IC50 congruent to 3 microM). In contrast to N-ethylmaleimide, chlorpromazine rapidly dissipated preformed pH gradients. Valinomycin stimulated H+ transport in the presence of potassium salts (gluconate much greater than NO3- greater than Cl-), and the membrane-potential-sensitive dye Oxonol V demonstrated an ATP-dependent interior-positive vesicle membrane potential which was greater in the absence of permeant anions (mannitol greater than potassium gluconate greater than KCl) and was abolished by N-ethylmaleimide, protonophores or detergent. Total vesicle-associated ouabain-insensitive ATPase activity was inhibited 64% by 1 mM N-ethylmaleimide, and correlated poorly with H+ transport, however N-ethylmaleimide-sensitive ATPase activity correlated well with proton transport (r = 0.95) in the presence of various Cl- salts and KNO3. Finally, vesicles prepared from bovine brain synaptic membranes exhibited H+ transport activity similar to that of the coated vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Encéfalo/ultraestructura , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Endosomas/enzimología , ATPasas de Translocación de Protón/metabolismo , Naranja de Acridina/metabolismo , Animales , Encéfalo/enzimología , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Bovinos , Cloruros/farmacología , Clorpromazina/farmacología , Diciclohexilcarbodiimida/farmacología , Concentración de Iones de Hidrógeno , Potenciales de la Membrana/efectos de los fármacos , Microscopía Electrónica , Peso Molecular , Ouabaína/farmacología , Valinomicina/farmacologíaRESUMEN
Lysosomes, endosomes, and a variety of other intracellular organelles are acidified by a family of unique proton pumps, termed the vacuolar H(+)-ATPases, that are evolutionarily related to bacterial membrane proton pumps and the F1-F0 H(+)-ATPases that catalyze ATP synthesis in mitochondria and chloroplasts. The electrogenic vacuolar H(+)-ATPase is responsible for generating electrical and chemical gradients across organelle membranes with the magnitude of these gradients ultimately determined by both proton pump regulatory mechanisms and, more importantly, associated ion and organic solute transporters located in vesicle membranes. Analogous to Na+, K(+)-ATPase on the cell membrane, the vacuolar proton pump not only acidifies the vesicle interior but provides a potential energy source for driving a variety of coupled transporters, many of them unique to specific organelles. Although the basic mechanism for organelle acidification is now well understood, it is already apparent that there are many differences in both the function of the proton pump and the associated transporters in different organelles and different cell types. These differences and their physiologic and pathophysiologic implications are exciting areas for future investigation.
Asunto(s)
Endosomas/metabolismo , Lisosomas/metabolismo , Animales , Humanos , Concentración de Iones de Hidrógeno , Transporte IónicoAsunto(s)
Linfocitos/metabolismo , Péptido Intestinal Vasoactivo/biosíntesis , Animales , Sistema Digestivo/metabolismo , Electrólitos/metabolismo , Humanos , Inmunidad/efectos de los fármacos , Inflamación/metabolismo , Receptores de la Hormona Gastrointestinal/fisiología , Receptores de Péptido Intestinal Vasoactivo , Péptido Intestinal Vasoactivo/análisis , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Many intracellular organelles contain a unique primary, electrogenic proton pump termed the vacuolar H(+)-ATPase. This pump, found in many endocytic, secretory, and storage vesicles in fungal, plant and animal cells, functions, in conjunction with a chloride conductance, to acidify the vesicle interior. Although remotely related to the mitochondrial ATP synthase, the vacuolar H(+)-ATPase is a distinct pump which differs in inhibitor sensitivity, subunit composition and function. The vacuolar H(+)-ATPase transports only protons, and permeable anions (chloride) are required for optimal vesicle acidification. Allosteric and regulatory effects are not yet fully understood. Vesicle acidification appears to be essential for receptor-mediated endocytosis, protein synthesis, and secretion and storage of small solutes such as neurotransmitters. A similar plasma membrane-located H(+)-ATPase may contribute to urinary acidification and cell pH regulation.
Asunto(s)
Orgánulos/enzimología , ATPasas de Translocación de Protón/metabolismo , Animales , Transporte Biológico Activo/fisiología , ATPasas de Translocación de Protón/antagonistas & inhibidores , Protones , Vacuolas/enzimologíaRESUMEN
Endocytic vesicles possess an electrogenic proton pump, and measurements of ATPase activity suggest that Cl- may stimulate proton pump activity. This study was undertaken to measure the steady-state pH, potential (delta psi), and total proton electrochemical gradients established by the rat liver multivesicular body (MVB) proton pump and to examine the effects of Cl- (0.5-140 mM) on these gradients. Radiolabeled [( 14C] methylamine and 36Cl-) and fluorescent (fluorescein isothiocyanate-conjugated low density lipoproteins) probes were used to assess internal pH (pHi) and delta psi. In the absence of ATP, pHi averaged 7.37 +/- 0.05 (extracellular pH 7.31 +/- 0.02), and delta psi ranged from -32 to -71 mV; but neither pHi nor delta psi varied consistently with [Cl-]. In the presence of ATP, pHi decreased progressively with increasing [Cl-] to a plateau value of about 5.89 at greater than or equal to 25 mM Cl-, and MVB exhibited an interior positive delta psi that was maximal at the lowest Cl- concentration (+65.5 mV) and decreased as medium Cl- increased. The total ATP-dependent proton electrochemical gradient (proton-motive force (delta p] averaged 118.0 +/- 4.3 mV and did not change in any consistent manner as [Cl-] varied almost 300-fold. However, initial rates of MVB acidification increased with increasing [Cl-]. These studies indicate that: (a) in the absence of ATP, isolated MVB exhibited a negative delta psi, probably a Donnan potential; (b) in the presence of ATP and at a [Cl-] similar to that in hepatocyte cytoplasm (25 mM), MVB pHi was 5.89, and delta psi was +9.6 mV; and (c) over the range of [Cl-] tested, the magnitudes of delta pH and delta psi were inversely related, apparently related to Cl- availability, but the ATP-dependent delta p did not vary. Therefore, it is concluded that Cl- increases the initial rate of vesicle acidification in MVB and also affects the relative chemical and electrical contributions of the steady-state proton pump-determined delta p. Cl-, however, does not alter steady-state delta p.
Asunto(s)
Cloruros/farmacología , Hígado/citología , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Cloruros/farmacocinética , Electroquímica , Endocitosis , Concentración de Iones de Hidrógeno , Hígado/efectos de los fármacos , Metilaminas/farmacocinética , RatasRESUMEN
Rat liver multivesicular bodies (MVB), as well as other hepatic subcellular organelles, are acidified by an electrogenic ATP-dependent proton pump that requires Cl- for maximal acidification (Van Dyke, R. W., Hornick, C. A., Belcher, J., Scharschmidt, B. F., and Havel, R.J. (1985) J. Biol. Chem. 260, 11021-11026), suggesting that Cl- serves as a permeable charge-compensating anion. However, we have observed that NO3- is unable to substitute for Cl-. This study was undertaken therefore to examine more closely the effects of Cl- on MVB acidification and to determine whether NO3- and other anions interact with the proton pump. ATP-dependent vesicle acidification and membrane potential (psi) were measured using the fluorescent dyes acridine orange and Oxonol V (bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol), respectively. Cl- both stimulated acidification (Km = 23.2 +/- 4.2 mM) and decreased psi (IC50 = 3.4 +/- 0.6 mM) in a concentration-dependent, nonlinear fashion. In the presence of saturating Cl- (100 mM), however, NO3- (shown to be more permeable than Cl-) and the impermeant anions SO4(2-) and PO4(2-), inhibited both ATP-dependent acidification and psi in a concentration-dependent manner. Other anions, including gluconate and HCO3-, had no effect. The inhibitory effect of NO3- was reversible. Neither SO4(2-) nor PO4(2-) appeared to block Cl- movement across the vesicle membrane as assessed by the ability of Cl- to decrease an established psi. In additional experiments, the effects of anions on relaxation of a previously established pH gradient were measured. Compared to Cl- or gluconate, NO3- had no significant effect on pH gradient relaxation, even when MVB were preloaded with NO3-, indicating that rapid cycling of NO3-/HNO3 across the MVB membrane does not occur. The organic nitrate, isosorbide dinitrate, also inhibited both acidification and psi and, similar to NO3-, had no effect on pH gradient relaxation. By contrast, NO2- potently inhibited both MVB acidification and psi but also rapidly relaxed a pre-established pH gradient, suggesting that NO2- increases MVB membrane proton permeability. Finally, MVB exhibited N-ethylmaleimide-sensitive ATPase activity that was inhibited 23.9% by NO3- (100 mM). In conclusion, although MVB are permeable to a variety of anions (Cl-, Br-, NO3-, NO2-), only Cl- and Br- support maximal rates of acidification by the proton pump.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Aniones/farmacología , Hígado/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Adenosina Trifosfatasas/análisis , Animales , Transporte Biológico/efectos de los fármacos , Cloruros/farmacología , Concentración de Iones de Hidrógeno , Dinitrato de Isosorbide/farmacología , Cinética , Hígado/ultraestructura , Masculino , Potenciales de la Membrana/efectos de los fármacos , Nitratos/farmacología , Protones , Ratas , Ratas EndogámicasRESUMEN
Both lysosomes and endosomes are acidified by an electrogenic proton pump, although studies in intact cells indicate that the steady-state internal pH (pHi) of lysosomes is more acid than that of endosomes. We undertook the present study to examine in detail the acidification mechanism of purified rat liver secondary lysosomes and to compare it with that of a population of early endosomes. Both endosomes and lysosomes exhibited ATP-dependent acidification, but proton influx rates were 2.4- to 2.7-fold greater for endosomes than for lysosomes because of differences in both buffering capacity and acidification rates, suggesting that endosomes exhibited greater numbers or rates of proton pumps. Lysosomes, however, exhibited a more acidic steady-state pHi due in part to a slower proton leak rate. Changes in medium Cl- increased acidification rates of endosomes more than lysosomes, and the lysosome ATP-dependent interior-positive membrane potential was only partially eliminated by high-Cl- medium. Permeability studies suggested that lysosomes were less permeable to Na+, Li+, and Cl- and more permeable to K+ and PO4(2-) than endosomes. Na-K-adenosine-triphosphatase did not appear to regulate acidification of either vesicle type. Endosome and lysosome acidification displayed similar inhibition profiles to N-ethylmaleimide, dicyclohexyl-carbodiimide, and vanadate, although lysosomes were somewhat more sensitive [concentration producing 50% maximal inhibition (IC50) 1 nM] to bafilomycin A1 than endosomes (IC50 7.6 nM). Oligomycin (1.5-3 microM) stimulated lysosome acidification due to shunting of membrane potential. Overall, acidification of endosomes and lysosomes was qualitatively similar but quantitatively somewhat different, possibly related to differences in the density or rate of proton pumps as well as vesicle permeability to protons, anions, and other cations.
Asunto(s)
Ácidos/metabolismo , Hígado/metabolismo , Lisosomas/metabolismo , Animales , Aniones/metabolismo , Tampones (Química) , Concentración de Iones de Hidrógeno , Bombas Iónicas/antagonistas & inhibidores , Iones , Masculino , Permeabilidad , Protones , Ratas , Ratas Sprague-DawleyRESUMEN
In prior studies, we showed that cholera (CTX) and pertussis toxins (PTX) increase rat liver endosome acidification. This study was performed to characterize the effects of these toxins and cyclic adenosine monophosphate (cAMP) on endosome ion transport, fluid-phase endocytosis (FPE), and endosome trafficking in liver. In control liver, more mature populations of endosomes acidified progressively more slowly, but both toxins and cAMP caused retention of an early endosome acidification profile in maturing endosomes. CTX caused a density shift in endosomes, and all agents increased net FPE at time points from 5 to 60 minutes. By confocal microscopy, fluorescent dextrans first appeared in small vesicles at the hepatocyte sinusoidal membrane and trafficked rapidly to the pericanalicular area, near lysosomes and the trans-Golgi network (TGN). Prolonged exposure to these agents caused redistribution of many labeled vesicles to the perinuclear region, colocalized with markers of both early (EEA1 and transferrin receptor) and late (LAMP1) endosomes. We conclude that cAMP is the common agent that disrupted normal maturation and trafficking of endosomes and increased net FPE, in part via decreased diacytosis.
Asunto(s)
Toxina del Cólera/farmacología , AMP Cíclico/farmacología , Endocitosis/efectos de los fármacos , Endosomas/fisiología , Hígado/efectos de los fármacos , Hígado/fisiología , Ácidos/metabolismo , Ácidos/farmacología , Animales , Biomarcadores , Bucladesina/farmacología , Hígado/metabolismo , Hígado/ultraestructura , Lisosomas/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Distribución Tisular/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Acidification of endocytic vesicles, driven by the vacuolar H+ pump, is affected by parallel ion transporters. Because adenosine 3',5'-cyclic monophosphate (cAMP) and heterotrimeric G proteins may alter ion transporters, I tested whether cholera and pertussis toxins affected acidification of rat liver endosomes. Fluorescein-labeled dextran-loaded "10-min" endosomes from cholera toxin-treated rats exhibited ATP-dependent rates of acidification in the presence and absence of Cl- or K+ that were approximately 60-120% (P < 0.05) faster than rates from control endosomes. This increase was greater for "older" "20-min" endosomes and less for 'early" "2-min" endosomes. Ion transport functions of 10-min and 20-min toxin-exposed endosomes were similar to those of 2-min control endosomes. Cholera toxin also increased ATP-dependent steady-state intravesicular H+ concentration by 38-218% (P < 0.05). Pertussis toxin increased endosome acidification rates by 20-54% (P < 0.05). Both toxins increased liver cAMP content, and endosomes prepared from perfused livers exposed to 0.75 mM dibutyryl cAMP exhibited similar increases in acidification rates. These studies indicate that both cholera and pertussis toxins markedly alter the function of rat liver endosomes. The mechanism is unlikely to reflect major changes in vesicle ion transporters but rather may indicate either an increase in the number of H+ pumps per endosome and/or changes in fusion, remodeling, and maturation of early endocytic vesicles in response to cAMP.
Asunto(s)
Ácidos/metabolismo , Toxina del Cólera/farmacología , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Hígado/metabolismo , Toxina del Pertussis , Factores de Virulencia de Bordetella/farmacología , Animales , AMP Cíclico/metabolismo , AMP Cíclico/fisiología , Endocitosis , Endosomas/fisiología , Técnicas In Vitro , Iones , Hígado/fisiología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Endocytic vesicles are acidified by an electrogenic vacuolar H(+)-ATPase. These studies examined whether rat liver endosomes also exhibit Na+/H+ exchange and whether this transporter alters acidification. Extravesicular Na+ caused saturable proton efflux from acidified endosomes with a Michaelis constant for Na+ of 7.6 mM, whereas an in-to-out Na+ gradient caused endosome acidification without MgATP and accelerated acidification with MgATP. Na(+)-driven proton fluxes were little altered by valinomycin or carbonyl cyanide m-chlorophenylhydrazone. Na+/H+ exchange was inhibited by Li+ but was not affected by K+, Cl-, amiloride (1 mM), or 5-(N,N-dimethyl) amiloride (0.1 mM). Na+/H+ exchange was detected in "early" but not in "late" liver endosomes or in lysosomes. These data suggest that early rat liver endosomes exhibit Na+/H+ exchange that, immediately after endosome formation, may accelerate vesicular acidification. Because of its insensitivity to amiloride, this exchanger may be a pharmacologically altered form of Na+/H+ exchanger-1 or a new isoform.
Asunto(s)
Hígado/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Ácidos/metabolismo , Animales , Endocitosis , Endosomas/metabolismo , Cinética , Litio/farmacología , Masculino , Potasio/farmacología , Preservación Biológica , Ratas , Ratas Sprague-Dawley , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidoresRESUMEN
The aim of the present study was to examine the possible role of microtubules in chloride secretion by distal rat colon stimulated by prostaglandin (PGE2) and theophylline. Distal colonic tissue from male rats was mounted in Ussing chambers, and short-circuit current (Isc) was measured to assess chloride secretion. Three microtubule inhibitors, colchicine, nocodazole, and taxol, all inhibited the stimulated Isc and reduced the 60-min integrated secretory response to PGE2 and theophylline (integral of Iscdt) by 39-52%, whereas the inactive colchicine analog lumicolchicine did not. Atropine and tetrodotoxin had no effect on stimulated chloride secretion. To confirm the source of Isc, unidirectional 22Na+ and 36Cl- fluxes were measured in tissues exposed to lumicolchicine (control) or colchicine. Control tissues absorbed both chloride [5.0 (1.1-8.6) (median and 95% confidence interval) mueq/cm2/hr] and sodium [2.8 (0.9-7.2) mueq/cm2/hr], and this net absorption was reduced by 96% and 79%, respectively, by treatment with PGE2 and theophylline due to an increase in serosal-to-mucosal chloride and sodium movement. Colchicine-treated tissues exhibited similar net basal chloride and sodium absorption that was reduced by 71% and 75%, respectively, by treatment with PGE2 and theophylline. Thus the PGE2- and theophylline-induced increase in chloride secretion was significantly reduced by colchicine (P < 0.05 by Wilcoxon rank-sum test), whereas colchicine had no effect on PGE2- and theophylline-induced changes in sodium fluxes. Furthermore, the colchicine-related changes in stimulated chloride secretion were numerically similar to colchicine-related changes in stimulated Isc.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Cloruros/fisiología , Colon/efectos de los fármacos , Dinoprostona/farmacología , Microtúbulos/efectos de los fármacos , Teofilina/farmacología , Análisis de Varianza , Animales , Colon/metabolismo , Intervalos de Confianza , Interacciones Farmacológicas , Análisis de los Mínimos Cuadrados , Masculino , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Treatment with ethinyl estradiol is known to impair bile formation, bile acid transport and Na,K-ATPase activity, to alter receptor-mediated endocytosis and transcytosis of IgA and asialoorosomucoid and to affect membrane lipid composition and fluidity. Because appropriate sorting and trafficking of asialoorosomucoid requires adequate acidification of endocytic vesicles by a lipid-sensitive electrogenic proton pump, we examined the effects of 5 days of treatment with ethinyl estradiol (5 mg/kg body wt, subcutaneously) on acidification of early endosomes prepared from male rat livers. Littermate control animals received equal volumes of the solvent propylene glycol. Pretreatment with ethinyl estradiol reduced ATP-dependent initial rates of endosome acidification by 11% to 25% when measured in potassium medium containing 0 to 140 mmol/L chloride; these differences were significant at four of six chloride concentrations tested. The proton pumps of ethinyl estradiol and propylene glycol endosomes exhibited similar Michaelis-Menten constants for MgATP (Michaelis-Menten constant of 63 and 66 mumol/L in the absence of chloride and 101 and 126 mumol/L in the presence of chloride, respectively). Acidification of ethinyl estradiol and propylene glycol endosomes changed in the same manner when various cations or anions were substituted for potassium gluconate, although the effects of ethinyl estradiol were less marked in the absence of K+. Kinetics of inhibition for ethinyl estradiol and propylene glycol endosomes were similar for the proton pump inhibitors N-ethylmaleimide (50% inhibitory concentrations of 13.5 and 18.1 mumol/L), dicyclohexylcarbodiimide (50% inhibitory concentrations of 206 and 216 mumol/L) and bafilomycin A (50% inhibitory concentrations of 11 and 6 nmol/L). Although initial rates of acidification were slower in ethinyl estradiol endosomes, ATP-dependent steady-state vesicle interior pH was the same as that of propylene glycol endosomes over a range of chloride concentrations; this appeared to be due mainly to a trend toward decreased proton leak rates in ethinyl estradiol endosomes. Overall, ethinyl estradiol treatment modestly decreased initial rates of acidification and vesicle proton leakage, perhaps because of changes in endosome lipid composition; differences in the number, density or activation state of proton pumps; or differences in endosome geometry. Because the decrease in acidification rates was small, the effects of estrogen on the efficiency of uncoupling of endocytosed ligands such as asialoorosomucoid from their receptors in early endosomes; thus the rates of sorting and distribution of ligands remain unclear.
Asunto(s)
Endocitosis/efectos de los fármacos , Etinilestradiol/farmacología , Hígado/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Cloruros/farmacología , Gluconatos/farmacología , Concentración de Iones de Hidrógeno , Cinética , Hígado/metabolismo , Hígado/ultraestructura , Masculino , Orgánulos/efectos de los fármacos , Orgánulos/metabolismo , Cloruro de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
It is apparent that proton transport plays an important role in many essential hepatocyte functions. Important unanswered issues include the location of the H+-ATPase and its role in hepatic functions, the regulators of Na+-H+ exchange, the exact role of Na+-H+ exchange in bile formation and in hepatic regeneration, and the role of bile acids such as UDCA and nor-UDCA in mediating transepithelial proton transport.
Asunto(s)
Membrana Celular/metabolismo , Organoides/metabolismo , Protones , Animales , Transporte Biológico Activo , ATPasas de Translocación de Protón/metabolismoRESUMEN
Inhibition of Na+-K+-ATPase and sodium-dependent bile acid transport has been suggested as a mechanism for the cholestasis produced by certain drugs such as chlorpromazine. We examined the effects of chlorpromazine (and in selected studies, two of its metabolites) on Na+-K+-ATPase cation pumping (ouabain-suppressible 86Rb uptake), exchangeable intracellular sodium content, membrane potential (assessed by 36Cl- distribution), and sodium-dependent transport of taurocholate and alanine in primary cultures of rat hepatocytes. Chlorpromazine (10-300 microM), 7,8-dihydroxychlorpromazine (10-300 microM), and ouabain (0.1-2 mM), but not chlorpromazine sulfoxide, produced a concentration-dependent decrease in Na+-K+-ATPase cation pumping and an increase in intracellular sodium content. Chlorpromazine (100 microM) and ouabain (0.75 mM) also modestly decreased hepatocyte membrane potential. In further studies, chlorpromazine (75 and 100 microM) and ouabain (0.1, 0.5, and 0.75 mM) decreased initial sodium-dependent uptake rates of taurocholate and alanine by 18-63%. Although the steady-state intracellular content of alanine was decreased 25-53% by both agents, chlorpromazine increased the steady-state content of taurocholate by 171% and decreased taurocholate efflux, apparently related to partitioning of taurocholate into a large, slowly turning over intracellular pool. These studies provide direct evidence that chlorpromazine inhibits Na+-K+-ATPase cation pumping in intact cells and that partial inhibition of Na+-K+-ATPase cation pumping is associated with a reduction of both the electrochemical sodium gradient and sodium-dependent solute transport. These effects of chlorpromazine may contribute to chlorpromazine-induced cholestasis in animals and humans.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Clorpromazina/farmacología , Canales Iónicos/metabolismo , Hígado/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Alanina/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Cloruros/metabolismo , Canales Iónicos/efectos de los fármacos , Cinética , Hígado/efectos de los fármacos , Ouabaína/farmacología , Ratas , Radioisótopos de Rubidio/metabolismo , Sodio/farmacología , Radioisótopos de Sodio/metabolismo , Ácido Taurocólico/metabolismoRESUMEN
Endocytic vesicles are acidified by an electrogenic proton pump and a parallel chloride conductance; however, acidification might be decreased if electrogenic transporters, such as Na,K-ATPase, that increase vesicle interior-positive membrane potential were also present. We examined this issue in early rat liver endosomes using ion substitution and inhibitors to alter Na,K-ATPase activity. These early endosomes, labeled for 2 min with the fluorescent fluid-phase marker fluorescein isothiocyanate-dextran, consistently acidified faster than endosomes similarly labeled for a 10-min period. In chloride-free media initial rates of acidification of early endosomes were faster in K+ media than in Na+ medium, although addition of K+ to Na+ or Na+ to K+ media to allow Na,K-ATPase to function did not decrease the rate of acidification. In chloride-containing media, rates were the same regardless of cation composition. The Na,K-ATPase inhibitor vanadate was prepared from orthovanadate by several methods, all of which inhibited liver ATPase activity. Two hundred mumol/L vanadate, prepared Cl(-)-free, tended to decrease rates of acidification in all media tested and these effects achieved statistical significance in Cl(-)-free media containing 150 mmol/L K+ or mixtures of Na+ and K+ and in 145 mmol/L KCl/5 mmol/L NaCl medium. Vanadate stocks pH-adjusted with hydrogen chloride increased rates of acidification in sodium gluconate buffers, probably as a result of the effects of the included Cl-. Five mmol/L ouabain (loaded into vesicles by endocytosis) and the membrane-permeable analog strophanthidin (2 mmol/L) both markedly inhibited endosome acidification, regardless of buffer ion composition. Collectively, these results suggest that Na,K-ATPase does not regulate acidification of rat liver early endocytic vesicles, that vanadate may modestly inhibit endosome acidification and that ouabain at high concentrations may inhibit acidification from the vesicle interior face.
Asunto(s)
Endocitosis , Hígado/química , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Transporte Biológico Activo , Cloruros/metabolismo , Medios de Cultivo , Dextranos , Fluoresceína-5-Isotiocianato/análogos & derivados , Gluconatos/metabolismo , Concentración de Iones de Hidrógeno , Hígado/citología , Hígado/ultraestructura , Masculino , Orgánulos/química , Orgánulos/enzimología , Ouabaína/farmacología , Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Estrofantidina/farmacología , Vanadatos/farmacologíaRESUMEN
Endocytosed ligands move through a series of progressively more acidic vesicles. These differences in pH (pHi) could reflect differences in ion transport mechanisms. Vesicles representing three stages of endocytosis, compartment for uncoupling of receptor and ligand (CURL), multivesicular bodies (MVB), and receptor recycling compartment (RRC), were studied, and all exhibited ATP-dependent electrogenic acidification that was a saturable function of medium chloride. Initial rates of acidification differed (RRC > CURL > MVB), and proton influx was similar for CURL and RRC but slower for MVB. Steady-state ATP-dependent pHi in the three vesicles was more similar. Vesicle membrane potential was substantial (+41 to +69 mV) in low-chloride medium and greatest for RRC but was low (-6 to +6 mV) in 140 mM KCl. These vesicles also exhibited -22 to -37 mV Donnan potentials. Steady-state pump-generated proton electrochemical gradients (delta mu H+) ranged from 114 to 175 mV and were greater for CURL and RRC than for MVB; however, delta mu H+ changed little over a 140-fold difference in chloride concentration. Proton leak rates were faster in CURL and RRC than in MVB, but proton efflux was similar. Finally, proton fluxes and permeabilities, calculated with regard to surface area, differed in the opposite direction (MVB > CURL > RRC). Thus, for the endocytic vesicles studied, intrinsic differences in proton flux and in vesicle geometry could be demonstrated that contributed to differences in pre-steady-state vesicle pHi.
Asunto(s)
Ácidos/metabolismo , Endocitosis , Hígado/metabolismo , Orgánulos/metabolismo , Adenosina Trifosfato/fisiología , Animales , Cloruros/farmacología , Electroquímica , Masculino , Potenciales de la Membrana , Orgánulos/fisiología , Concentración Osmolar , Permeabilidad , Protones , Ratas , Ratas Sprague-DawleyRESUMEN
Hepatic organic cation transport has been characterized in rat liver plasma membrane vesicles, using the quaternary amine tetraethylammonium (TEA) as a model substrate. Sinusoidal TEA uptake is stimulated by an inside-negative membrane potential; TEA transport across the canalicular membrane is mediated by electroneutral organic cation-H+ exchange. Substrates for these transport processes include procainamide ethobromide (PAEB) and vecuronium, cationic drugs that undergo biliary excretion. Given the apparent absence of sinusoidal transport mechanisms able to generate high hepatocyte-to-blood organic cation concentration ratios, intracellular transport of organic cations may involve sequestration and concentration within acidified organelles. Therefore, the characteristics of TEA uptake were examined in isolated rat liver lysosomes that are acidified by a well-described H(+)-adenosinetriphosphatase (ATPase). Lysosomal uptake of [14C]TEA was a time- and ATP-dependent process, reaching steady state after 30-60 min. Steady-state [14C]TEA uptake was significantly reduced by omission of ATP and by addition of monensin, conditions that alter lysosomal pH and membrane potential gradients, and by the H(+)-ATPase inhibitors, N-ethylmaleimide and bafilomycin A. ATP-dependent lysosomal [14C]TEA uptake was significantly inhibited by PAEB, vecuronium, and other organic cationic substrates of canalicular TEA/H+ exchange. These findings demonstrate that rat liver lysosomes sequester certain organic cationic drugs, most likely via organic cation/H+ exchange driven by H(+)-ATPase. Canalicular organic cation/H+ exchange may reflect, in part, the exocytic insertion of this transporter from an intracellular compartment to this membrane domain.
Asunto(s)
Hígado/ultraestructura , Lisosomas/metabolismo , Compuestos de Tetraetilamonio/metabolismo , Adenosina Trifosfato/farmacología , Animales , Bilis/metabolismo , Transporte Biológico , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Cationes , Etilmaleimida/farmacología , Concentración de Iones de Hidrógeno , Masculino , Monensina/farmacología , Procainamida/metabolismo , Ratas , Ratas Sprague-Dawley , Tetraetilamonio , Bromuro de Vecuronio/metabolismoRESUMEN
(Na,K)-ATPase is thought to maintain the transmembrane electrochemical sodium gradient which powers secondary active sodium-coupled transport of a variety of solutes including amino acids and bile acids. However, little is known regarding the effect of sodium-coupled solute transport on intracellular sodium concentration ( [Na]ic) and on (Na,K)-ATPase-mediated cation pumping in the intact cell. In order to address this question, we have measured 22Na uptake rate, steady state 22Na content, and ouabain-suppressible 86Rb uptake rate in primary cultures of adult rat hepatocytes under a variety of conditions. Compared with control conditions (sodium uptake rate = 6.00 +/- 0.40 nmol X min-1 X mg-1; [Na]ic = 11.96 +/- 0.54 mM; cation pumping = 2.53 +/- 0.18 nmol X min-1 X mg-1), cation pumping was increased by taurocholate (less than or equal to 158%), alanine (less than or equal to 246%), monensin (less than or equal to 400%), and cold exposure (less than or equal to 525%), and this increase was accompanied by increases in Na uptake and [Na]ic. In contrast, preincubation in low sodium medium decreased all three variables. These changes in cation pumping were blocked in the absence of extracellular sodium and were not accompanied by changes in ouabain-suppressible ATP hydrolysis measured in cell homogenate. An overall plot of cation pumping versus [Na]ic yielded a sigmoid-shaped curve. Values for KNa (17.8 +/- 1.4 mM) and Vmax (8.98 +/- 0.62 nmol X min-1 X mg-1) for cation pumping were estimated assuming three sodium sites per pump unit. These findings indicate that: 1) uptake of alanine and taurocholate is associated with a rapid increase in (Na,K)-ATPase cation pumping; 2) this increase probably results from an increase in pumping per pump unit rather than an increase in the total number of pump units, and it appears to be mediated via an increase in sodium influx and [Na]ic; 3) [Na]ic under control conditions is close to the apparent KNa of cation pumping, implying that substrate availability may be the mechanism whereby sodium uptake is tightly linked to (Na,K)-ATPase cation pumping in intact hepatocytes.