RESUMEN
The role of serosal bicarbonate ions (HCO3-) in protection against acid injury was investigated in rabbit esophageal mucosa mounted in Ussing chambers. Luminal acidification reduced potential difference and resistance in tissues exposed serosally to HCO3- or (unbuffered) HCO3-free solution. Whereas resistance declined similarly in both groups, potential difference declined less in HCO3- solution. After washout, HCO3-bathed tissues also had a greater increase in resistance, lower permeability to mannitol, and less histologic damage. Furthermore, as protection by HCO3- was not blocked by pretreatment with either the anion exchange blocker, 4 acetamido-4'-isothiocyanatostilbene 2-2'-disulfonic acid, or the carbonic anhydrase inhibitor, acetazolamide, and replacement of HCO3- with N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid, a buffer impermeant to cells, was protective, an extracellular site for protection by HCO3- was likely. Where in the extracellular space HCO3- buffers H+ is unclear, but the absence of change in luminal pH and the inability to prevent the acid-induced increase in permeability in HCO3-bathed tissues argue against a luminal (preepithelial) site. Also, rapid repair was not demonstrated, indicating that a luminal site for protection after surface cell damage was unlikely. We conclude that serosal HCO3- is important in esophageal protection against acid damage by buffering H+ within the intercellular compartment of the extracellular space.
Asunto(s)
Bicarbonatos/fisiología , Esófago/fisiología , Mucosa Gástrica/fisiología , Reflujo Gastroesofágico/fisiopatología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Acetazolamida/farmacología , Animales , HEPES/farmacología , Ácido Clorhídrico/farmacología , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , ConejosRESUMEN
Proton and formic acid permeabilities were measured in the in vivo microperfused rat proximal convoluted tubule by examining the effect on intracellular pH when [H] and/or [formic acid] were rapidly changed in the luminal or peritubular fluids. Apical and basolateral membrane H permeabilities were 0.52 +/- 0.07 and 0.67 +/- 0.18 cm/s, respectively. Using these permeabilities we calculate that proton backleak from the luminal fluid to cell does not contribute significantly to net proton secretion in the early proximal tubule, but may contribute in the late proximal tubule. Apical and basolateral membrane formic acid permeabilities measured at extracellular pH 6.62 were 4.6 +/- 0.5 X 10(-2) and 6.8 +/- 1.5 X 10(-2) cm/s, respectively. Control studies demonstrated that the formic acid permeabilities were not underestimated by either the simultaneous movement of formate into the cell or the efflux of formic acid across the opposite membrane. The measured apical membrane formic acid permeability is too small to support all of transcellular NaCl absorption in the rat by a mechanism that involves Na/H-Cl/formate transporters operating in parallel with formic acid nonionic diffusion.
Asunto(s)
Bicarbonatos/fisiología , Permeabilidad de la Membrana Celular , Túbulos Renales Proximales/fisiología , Cloruro de Sodio/fisiología , Sodio/fisiología , Absorción , Animales , Formiatos/fisiología , Concentración de Iones de Hidrógeno , Líquido Intracelular/fisiología , Perfusión , Protones , Ratas , Bicarbonato de SodioRESUMEN
Higher plants, algae, and cyanobacteria are known to require bicarbonate ions for electron flow from the first stable electron acceptor quinone QA to the second electron acceptor quinone QB, and to the intersystem quinone pool. It has been suggested that in Photosystem II of oxygenic photosynthesis, bicarbonate ion functions to maintain the reaction center in a proper conformation and, perhaps, to provide the protons needed to stabilize the semiquinone (QB-). In this paper, we show that bicarbonate ions do not influence the electron flow, from the quinone QA to QB and beyond, in the photosynthetic bacterium Rhodobacter sphaeroides. No measurable effect of bicarbonate depletion, obtained by competition with formate, was observed on cytochrome b-561 reduction in chromatophores; on the flash-dependent oscillation of semiquinone formation in reaction centers; on electron transfer from QA- to QB; or on either the fast or slow recovery of the oxidized primary donor (P+) which reflects the P+QA- ----PQA or the P+QB- ----PQB reaction. The lack of an observed effect in Rhodobacter sphaeroides in contrast to the effect seen in Photosystem II is suggested to be due to the amino-acid sequence differences between the reaction centers of the two systems.
Asunto(s)
Cromatóforos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Bicarbonatos/fisiología , Quinonas/metabolismo , Rhodobacter sphaeroides/metabolismo , Transporte de Electrón , Formiatos/farmacología , Modelos Químicos , Proteínas del Complejo del Centro de Reacción FotosintéticaRESUMEN
The functional importance of the "mucus-bicarbonate barrier" in protecting the gastric mucosa against injury by acid or pepsin was examined using an ex vivo gastric chamber preparation in the rat. Conditions were created such that the effectiveness of a mucus-stabilized pH gradient on the mucosal surface would be minimized. Thus solutions of hydrochloric acid of pH 1.3 or 0.8 were applied to the mucosal surface and continually stirred for 60 min. By use of an antimony pH microelectrode, these concentrations of acid were shown to dissipate the pH gradient on the mucosal surface within 5 min. The effects of addition of the mucolytic agents, pepsin or N-acetylcysteine, to both acid solutions were also assessed. Finally, the ability of the mucosa to resist injury by acid after disruption of the surface epithelium (with hypertonic saline) was examined. Exposure to the acid solutions, with or without added mucolytic agents, was without damaging effects on the mucosa, as assessed macroscopically, histologically, or by measurement of transmucosal potential difference and luminal protein concentration. Conversely, disruption of the surface epithelium rendered the mucosa significantly more susceptible to the damaging actions of acid. These results therefore demonstrate that under conditions in which a pH gradient on the mucosal surface was no longer detectable, the mucosa was resistant to injury by acid. In the undamaged rat stomach, therefore, the mucus-bicarbonate barrier may be functionally redundant.
Asunto(s)
Bicarbonatos/fisiología , Ácido Gástrico/fisiología , Mucosa Gástrica/fisiología , Acetilcisteína/farmacología , Azul Alcián/metabolismo , Animales , Epitelio/fisiología , Mucosa Gástrica/anatomía & histología , Mucosa Gástrica/efectos de los fármacos , Ácido Clorhídrico/farmacología , Concentración de Iones de Hidrógeno , Masculino , Potenciales de la Membrana , Microelectrodos , Pepsina A/farmacología , Proteínas/metabolismo , Ratas , Ratas EndogámicasRESUMEN
Internal pH (pHi) was analyzed in rat extensor digitorum longus (Edl) muscle at 30 degrees C with single-barrel liquid ion-selective electrodes. Average pHi in 284 cells was 7.197 +/- 0.006. Increases in CO2 from nominally 0 to 5% produced an acidification from which recovery took place. In different groups of cells, recovery from the 5% CO2 acidification was significantly inhibited by 100 microM 4,4' diisothiocyanatostilbene 2,2' disulfonic acid (DIDS), Cl removal, Na removal and 2 mM amiloride. Prepulsing with 20 mM NH4 in the presence of CO2/HCO3 typically reduced pHi to only about neutral, whereas 50 mM reduced pHi to 6.7-6.8. In the nominal absence of CO2/HCO3, 20 mM NH4 reduced pHi to about 6.7 from which recovery took place at about 58% of the rate seen in different cells in the presence of CO2/HCO3. In the presence of CO2/HCO3, cells prepulsed with 50 mM NH4 had fully recovered to an average pHi of 7.22 +/- 0.04 about 90 min after removal of NH4. However, 90 min after removal of 20 mM NH4 in the absence of CO2/HCO3, average pHi was significantly less (7.05 +/- 0.03). Intrinsic buffering capacity (beta i) was obtained during pulses of CO2, acetic acid or after an NH4 pulse. beta i was significantly reduced in the absence of HCO3, Cl or Na and HCO3. The data provide significant support for an important role of HCO3 in the control of pHi in fast-twitch muscle.
Asunto(s)
Bicarbonatos/fisiología , Músculos/fisiología , Animales , Bicarbonatos/metabolismo , Femenino , Concentración de Iones de Hidrógeno , Masculino , Músculos/metabolismo , Ratas , Ratas EndogámicasRESUMEN
The effect of chronic dietary acid on the apical membrane Na/H antiporter and basolateral membrane Na(HCO3)3 symporter was examined in the in vivo microperfused rat proximal tubule. Transporter activity was assayed with the epifluorescent measurement of cell pH using the intracellular, pH-sensitive fluorescent dye, (2'7')-bis(carboxyethyl)-(5,6)-carboxy-fluorescein (BCECF). BCECF was calibrated intracellularly, demonstrating similar pH-sensitivity of the dye in control and acidotic animals. In subsequent studies, lumen and peritubular capillaries were perfused to examine Na/H and Na(HCO3)3 transporter activity in the absence of contact with native fluid. The initial rate of change in cell pH (dpHi/dt) was 97, 50, and 44% faster in tubules from acidotic animals when peritubular [HCO3] was changed from 25 to 10 mM in the presence or absence of chloride, or peritubular [Na] was changed from 147 to 50 mM, respectively. dpHi/dt was 57% faster in tubules from acidotic animals when luminal [Na] was changed from 152 to 0 mM. Buffer capacities, measured using NH3/NH+4 addition, were similar in the two groups. The results demonstrate that chronic metabolic acidosis causes an adaptation in the intrinsic properties of both the apical membrane Na/H antiporter and basolateral membrane Na(HCO3)3 symporter.
Asunto(s)
Acidosis Tubular Renal/metabolismo , Adaptación Fisiológica , Bicarbonatos/fisiología , Proteínas Portadoras/fisiología , Túbulos Renales Proximales/metabolismo , Sodio/fisiología , Acidosis Tubular Renal/etiología , Acidosis Tubular Renal/fisiopatología , Animales , Membrana Basal/metabolismo , Membrana Basal/fisiopatología , Tampones (Química) , Calibración , Cloruros , Enfermedad Crónica , Líquido Intracelular/fisiología , Túbulos Renales Proximales/fisiopatología , Masculino , Protones , Ratas , Ratas Endogámicas , Simportadores de Sodio-Bicarbonato , Intercambiadores de Sodio-HidrógenoRESUMEN
During near complete hyperglycemic brain ischemia, brain lactate levels rise in excess of 16-18 mmol/K and are associated with severe brain infarction. Analyses of pHo, Pt(CO2), and total brain lactate under these circumstances suggest that H+, HCO3, and lactate become unequally distributed between cells and the interstitial space and, perhaps, even between different types of brain cells. In addition, to whatever physiological advantages it may generate, such compartmentalization may be a factor leading to cell death in brain ischemia.
Asunto(s)
Equilibrio Ácido-Base , Isquemia Encefálica/fisiopatología , Animales , Bicarbonatos/fisiología , Concentración de Iones de Hidrógeno , Hiperglucemia/fisiopatología , Lactatos/análisis , Masculino , Ratas , Ratas EndogámicasRESUMEN
Intracellular pH (pHi) in confluent monolayers of cultured bovine corneal endothelial cells was determined using the pH-dependent absorbance of intracellularly trapped 5(and 6)carboxy-4',5'-dimethylfluorescein. Steady-state pH was 7.05 +/- 0.1 in the nominal absence of bicarbonate, and 7.15 +/- 0.1 in the presence of 28 mM HCO3-/5% CO2. Following an acid load imposed by a NH4Cl prepulse, pHi was regulated in the absence of HCO3- by a Na+-dependent process inhibitable to a large extent by 1 mM amiloride and 0.1 mM dimethylamiloride. In the presence of 28 mM HCO3-/5% CO2, this regulation was still dependent on Na+, but the inhibitory potency of amiloride was less. DIDS (1 mM) partially inhibited this regulation in the presence, but not in the absence of bicarbonate. With cells pretreated with DIDS, amiloride was as effective in inhibiting recovery from acid load as in the absence of HCO3-. The presence of intracellular Cl- did not appreciably affect this recovery, which was still sensitive to DIDS in the absence of Cl-. Removal of extracellular Na+ led to a fall of pHi, which was greatly attenuated in the absence of HCO3-. This acidification was largely reduced by 1 mM DIDS, but not by amiloride. Cl removal led to an intracellular alkalinization in the presence of HCO3-. The presence of a Cl-/HCO3- exchanger was supported by demonstrating DIDS-sensitive 36Cl- uptake into confluent cell monolayers. Thus, bovine corneal endothelial cells express three processes involved in intracellular pH regulation: an amiloride-sensitive Na+/H+ antiport, a Na+-HCO3- symport and a Cl-/HCO3- exchange, the latter two being DIDS sensitive.
Asunto(s)
Equilibrio Ácido-Base , Bicarbonatos/fisiología , Citoplasma/fisiología , Endotelio Corneal/fisiología , Animales , Transporte Biológico Activo , Bovinos , Células Cultivadas , Cloruros/fisiología , Fluoresceínas , Hidrógeno/fisiología , Fotometría/instrumentación , Sodio/fisiologíaRESUMEN
The present study has been designed to test for the influence of cell swelling on the potential difference and conductive properties of the basolateral cell membrane in isolated perfused proximal tubules. During control conditions the potential difference across the basolateral cell membrane (PDbl) is -65 +/- 1 mV (n = 74). Decrease of peritubular osmolarity by 80 mosmol/l depolarizes the basolateral cell membrane by +7.8 +/- 0.5 mV (n = 42). An increase of bath potassium concentration from 5 to 20 mmol/l depolarizes the basolateral cell membrane by +25 +/- 1 mV (n = 11), an increase of bath bicarbonate concentration from 20 to 60 mmol/l hyperpolarizes the basolateral cell membrane by -3.2 +/- 0.5 mV (n = 13). A decrease of bath chloride concentration from 79.6 to 27 mmol/l hyperpolarizes the basolateral cell membrane by -1.8 +/- 0.7 mV (n = 6). During reduced bath osmolarity, the influence of altered bath potassium concentration on PDbl is decreased (delta PDbl = +16 +/- 2 mV, n = 11), the influence of altered bicarbonate concentration on PDbl is increased (delta PDbl = -6.0 +/- 0.8 mV, n = 13), and the influence of altered bath chloride concentration on PDbl is unaffected (delta PDbl = -1.8 +/- 0.6 mV, n = 6). Barium depolarizes the basolateral cell membrane to -28 +/- 2 mV (n = 16). In the presence of 1 mmol/l barium, decrease of peritubular osmolarity by 80 mosmol/l leads to a transient hyperpolarization of the basolateral cell membrane by -5.9 +/- 0.5 mV (n = 16).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Túbulos Renales Proximales/fisiología , Animales , Bicarbonatos/fisiología , Cloruros/fisiología , Soluciones Hipotónicas , Técnicas In Vitro , Potenciales de la Membrana , Ratones , Potasio/fisiologíaRESUMEN
The transmural potential difference and short-circuit current of the porcine Graafian follicle have been measured in an attempt to test whether antral fluid accumulates as a result of active transport of salt. The values obtained by mounting explants of follicle wall in Ussing chambers were close to zero and the specific electrical resistance was only 59 delta.cm2. The elemental composition of the follicular fluid was similar to that of ovarian venous plasma with the exception of follicular Na+ which was slightly more abundant. Bicarbonate concentrations were slightly lower in follicular fluids. These findings were interpreted as evidence that the follicular wall is a leaky epithelium and, therefore, any charge resulting from net ion transport will be shunted along low resistance paracellular pathways.
Asunto(s)
Folículo Ovárico/fisiología , Animales , Bicarbonatos/fisiología , Cationes/fisiología , Cloruros/fisiología , Conductividad Eléctrica , Electrofisiología , Femenino , PorcinosRESUMEN
UNLABELLED: To study the regulation of cell pH by ambient pH, carbon dioxide tension (PCO2), and bicarbonate (HCO3), cell pH was measured in the isolated, in vitro microperfused rabbit proximal convoluted tubule using the fluorescent dye (2',7')-bis-(carboxyethyl)-(5,6)-carboxyfluorescein. For the same changes in external pH, changes in [HCO3] and PCO2 affected cell pH similarly ([HCO3]: pHi/pHe = 0.67, PCO2: pHi/pHe = 0.64, NS). Isohydric changes in extracellular [HCO3] and PCO2 did not change cell pH significantly. Changes in peritubular [HCO3] elicited larger changes in cell pH than changes in luminal [HCO3], which were enhanced by peritubular 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS). The cell pH defense against acute increases and decreases in PCO2 was inhibited by sodium, but not by chloride removal. Peritubular SITS inhibited the cell pH defense against increases and decreases of PCO2, whereas luminal amiloride inhibited cell pH defense against increases in PCO2. CONCLUSIONS: (a) Steady-state cell pH changes in response to changes in extracellular [HCO3] and PCO2 are quantitatively similar for a given change in extracellular pH; (b) the rate of the basolateral Na/(HCO3)3 cotransporter is a more important determinant of cell pH than the rate of the apical membrane mechanism(s); (c) cell pH defense against acute changes in PCO2 depends on the basolateral Na/(HCO3)3 cotransporter (acid and alkaline loads) and the luminal Na/H antiporter (acid loads).
Asunto(s)
Bicarbonatos/fisiología , Dióxido de Carbono/fisiología , Concentración de Iones de Hidrógeno , Túbulos Renales Proximales/fisiología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Amilorida/farmacología , Animales , Fluoresceínas , Técnicas In Vitro , Túbulos Renales Proximales/citología , ConejosRESUMEN
The mechanism of basolateral membrane base transport was examined in the in vitro microperfused rabbit proximal convoluted tubule (PCT) in the absence and presence of ambient CO2/HCO3- by means of the microfluorometric measurement of cell pH. The buffer capacity of the cells measured using rapid NH3 washout was 42.8 +/- 5.6 mmol.liter-1.pH unit-1 in the absence and 84.6 +/- 7.3 mmol.liter-1.pH unit-1 in the presence of CO2/HCO3-. In the presence of CO2/HCO3-, lowering peritubular pH from 7.4 to 6.8 acidified the cell by 0.30 pH units and lowering peritubular Na from 147 to 0 mM acidified the cell by 0.25 pH units. Both effects were inhibited by peritubular 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS). In the absence of exogenous CO2/HCO3-, lowering peritubular pH from 7.4 to 6.8 acidified the cell by 0.25 pH units and lowering peritubular Na from 147 to 0 mM decreased cell pH by 0.20 pH units. Lowering bath pH from 7.4 to 6.8 induced a proton flux of 643 +/- 51 pmol.mm-1.min-1 in the presence of exogenous CO2/HCO3- and 223 +/- 27 pmol.mm-1.min-1 in its absence. Lowering bath Na from 147 to 0 mM induced proton fluxes of 596 +/- 77 pmol.mm-1.min-1 in its absence. The cell acidification induced by lowering bath pH or bath Na in the absence of CO2/HCO3- was inhibited by peritubular SITS or by acetazolamide, whereas peritubular amiloride had no effect. In the absence of exogenous CO2/HCO3-, cyanide blocked the cell acidification induced by bath Na removal, but was without effect in the presence of exogenous CO2/HCO3-. We reached the following conclusions. (a) The basolateral Na/base n greater than 1 cotransporter in the rabbit PCT has an absolute requirement for CO2/HCO3-. (b) In spite of this CO2 dependence, in the absence of exogenous CO2/HCO3-, metabolically produced CO2/HCO3- is sufficient to keep the transporter running at 30% of its control rate in the presence of ambient CO2/HCO3-. (c) There is no apparent amiloride-sensitive Na/H antiporter on the basolateral membrane of the rabbit PCT.
Asunto(s)
Álcalis/metabolismo , Bicarbonatos/fisiología , Dióxido de Carbono/fisiología , Proteínas Portadoras/metabolismo , Túbulos Renales Proximales/metabolismo , Sodio/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Acetazolamida/farmacología , Amilorida/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Tampones (Química) , Cianuros/farmacología , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Conejos , Intercambiadores de Sodio-HidrógenoRESUMEN
Intracellular Cl- activity (AiCl) of the bullfrog choroidal epithelium has been studied using double-barreled Cl(-)-selective microelectrodes. In bicarbonate-buffered saline, the brush-border membrane potential (VVC) was -43 mV, and AiCl was 24 mM which was twice the predicted equilibrium activity. The uphill Cl- accumulation required the presence of external Na+ and was inhibited by furosemide added to the basolateral side. Removal of HCO3- from the bath solution slightly increased AiCl. On addition of 3-isobutyl-1-methylxanthine (IBMX), VVC depolarized, and AiCl approached the equilibrium activity. It is concluded that net Cl- secretion by the choroidal epithelium is mediated by a furosemide-sensitive, Na+-coupled Cl- uptake mechanism at the basolateral border and a Cl- conductive pathway at the brush border membrane. The results suggest that intracellular cAMP either increases the Cl- conductance of the epithelial membranes and/or inhibits the NaCl co-transport mechanism.
Asunto(s)
Cloruros/metabolismo , Plexo Coroideo/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Animales , Bicarbonatos/fisiología , Transporte Biológico/efectos de los fármacos , Cloruros/fisiología , Plexo Coroideo/efectos de los fármacos , Plexo Coroideo/fisiología , Electroquímica , Epitelio/metabolismo , Epitelio/fisiología , Furosemida/farmacología , Técnicas In Vitro , Líquido Intracelular/metabolismo , Líquido Intracelular/fisiología , Potenciales de la Membrana/efectos de los fármacos , Rana catesbeiana , Sodio/fisiologíaRESUMEN
Morphological changes of S. mutans NCTC 10449S associated with growth in modified Jordan medium and FMC medium (Terleckyj et al., Infect. Immun. 11:649-655, 1975) were studied by scanning electron microscopy. The cells were bacillary in Jordan medium, but coccoid and of unequal size in FMC. Transfer of the cells from Jordan medium to FMC and vice versa reversed their shapes, as did salt exchange between these media. Morphological changes could not be ascribed to either medium pH, concentration of P, or Na+/K+ ratio. However, they were growth dependent, since the changes did not occur when the cells were suspended in salt components alone or in media supplemented with protein synthesis inhibitors. Only a high bicarbonate/K+ ratio, as in FMC, produced spherical cells, whereas cells remained bacillary in medium with a low bicarbonate/K+ ratio, as in Jordan medium. Manipulating this ratio in other media resulted in similar shape changes. Thus, the shape of S. mutans 10449S can be dictated by the ratio of bicarbonate to K+ in the growth medium.
Asunto(s)
Bicarbonatos/fisiología , Potasio/fisiología , Streptococcus mutans/ultraestructura , Medios de Cultivo , Microscopía Electrónica de Rastreo , Streptococcus mutans/fisiologíaRESUMEN
Membrane potentials and intracellular pH were measured on rat renal proximal tubular cells in vivo to test whether sodium-bicarbonate cotransport across the peritubular cell membrane accepts OH- (or H+ in opposite direction) or whether it requires the CO2, HCO3-, CO3= buffer to operate. It was found that step changes of peritubular pH in nominally HCO3(-)-free and CO2-free solutions produced qualitatively similar initial potential responses and cell pH responses as changes in peritubular HCO3- concentrations. These responses, however, were considerably smaller and they were neither reduced in Na+-free solutions nor inhibited by the stilbene derivative SITS which is known to block Na+ (HCO3-)n cotransport completely. We conclude that the cotransporter requires the CO2, HCO3-, CO3= buffer for its physiological operation but that high rates of OH- or H+ can also be transferred across the peritubular cell membrane in HCO3(-)-free solutions, probably through a separate transport system.
Asunto(s)
Bicarbonatos/metabolismo , Túbulos Renales Proximales/metabolismo , Sodio/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Animales , Bario/farmacología , Bicarbonatos/fisiología , Transporte Biológico/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Concentración de Iones de Hidrógeno , Hidróxidos/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Protones , Ratas , Sodio/fisiología , Bicarbonato de SodioRESUMEN
To examine whether Cl-coupled HCO3 transport mechanisms were present on the basolateral membrane of the mammalian proximal tubule, cell pH was measured in the microperfused rat proximal convoluted tubule using the pH-sensitive, intracellularly trapped fluorescent dye (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein. Increasing the peritubular Cl concentration from 0 to 128.6 meq/liter caused cell pH to decrease from 7.34 +/- 0.04 to 7.21 +/- 0.04 (p less than 0.001). With more acid extracellular fluid (pH 6.62), a similar increase in the peritubular Cl concentration caused cell pH to decrease by a similar amount from 6.97 +/- 0.04 to 6.84 +/- 0.05 (p less than 0.001). This effect was blocked by 1 mM SITS. To examine the Na dependence of Cl/HCO3 exchange, the above studies were repeated in the absence of luminal and peritubular Na. In alkaline Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.57 +/- 0.06 to 7.53 +/- 0.06 (p less than 0.025); in acid Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.21 +/- 0.04 to 7.19 +/- 0.04 (p less than 0.05). The effect of Cl on cell pH was smaller in the absence of luminal and peritubular Na than in its presence. To examine whether the previously described Na/(HCO3)n greater than 1 cotransporter was coupled to or dependent on Cl, the effect of lowering the peritubular Na concentration from 147 to 25 meq/liter was examined in the absence of ambient Cl. Cell pH decreased from 7.28 +/- 0.03 to 7.08 +/- 0.03, a response similar to that observed previously in the presence of Cl. The results demonstrate that Cl/HCO3 (or Cl/OH) exchange is present on the basolateral membrane. Most of Cl/HCO3 exchange is dependent on the presence of Na and may be coupled to it. The previously described Na/(HCO3)n greater than 1 cotransporter is the major basolateral membrane pathway for the coupling of Na and HCO3 and is not coupled to Cl.
Asunto(s)
Bicarbonatos/fisiología , Cloruros/fisiología , Túbulos Renales Proximales/fisiología , Sodio/fisiología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas de Transporte de Anión , Transporte Biológico , Compartimento Celular , Membrana Celular/fisiología , Electrofisiología , Concentración de Iones de Hidrógeno , Masculino , RatasRESUMEN
Proximal tubular cells of the frog (Rana esculenta) kidney were fused within an isolated tubule portion to giant cells according to the polyethylene-glycol fusion method. Cell membrane potentials (Vm) were measured while cells were superfused with various experimental solutions. Rapid concentration step-changes of different ions allowed to calculate the respective transference numbers (tion). In some experiments the specific cell membrane resistances (Rm) were evaluated by measuring Vm induced by short current pulses injected into the cell with a second electrode. The experiments reveal: i) Fused cells of the proximal tubule exhibit a Vm of -49.5 +/- 1.6 mV (n = 65). ii) Addition of glucose to the perfusate yields a transient depolarization, consistent with a rheogenic Na/glucose cotransport system. iii) In absence of organic substrates the whole cell membrane conductance is made up of K+ and HCO3-. iv) There is a positive relationship between Vm and tK+ and a negative relationship between Vm and tHCO3-. v) HCO3--induced Vm changes are attenuated or abolished when Na+ is replaced with choline+, consistent with a rheogenic Na+/HCO3- cotransport system. vi) Replacement of Na+ by choline+ depolarizes Vm and increases Rm by about 50%; addition of 3 mmol/liter Ba2+ to the Na+-free perfusate increases Rm by about 58% compared to the initial control value. vii) There is no measurable cell membrane Cl- conductance. We conclude that fused cells of proximal tubule exert both luminal and peritubular membrane properties. In absence of organic substrates the cell membrane potential is determined by the HCO3- and K+ transport systems.