RESUMEN
A stopped-flow rapid reaction apparatus was used to study the rate of pH equilibration in human red cell suspensions. Flux of OH- or H+ was determined over a wide range of extracellular pH (4-11) in CO2-free erythrocyte suspensions. In these experiments, an erythrocyte suspension at pH 7.3 is rapidly mixed with an equal volume of NaCl solution at 3.0 greater than pH greater than 11.5. The pH of the extracellular fluid of the mixture changes rapidly as OH- or H+ moves across the red cell membrane. Flux and velocity constants can be calculated from the initial dp H/dt using the known initial intra- and extracellular pH. It was found that the further the extracellular pH is from 7.3 (in either direction from 4-11), the greater the absolute valute of total OH- and/or H+ flux. Pretreatment with SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid), a potent anion exchange inhibitor, greatly reduces flux over the entire pH range, while exposure to valinomycin, a potassium ionophore, has no measurable effect. These data suggest that (i) both H+ and OH- may be moving across the red cell membrane at all pH; (ii) the species dominating pH equilibration is probably dependent on the extracellular pH, which determines the magnitude of the driving gradient for each ion; and (iii) the rapid exchange pathway of the erythrocyte membrane may be utilized for both H+ and OH- transport during CO2-free pH equilibration.
Asunto(s)
Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Concentración de Iones de Hidrógeno , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Dióxido de Carbono/sangre , Membrana Eritrocítica/efectos de los fármacos , Humanos , Cinética , Presión Parcial , Valinomicina/farmacologíaRESUMEN
The fluid and solute transport properties of pleural tissue were studied using specimens of intact visceral pleura from adult sheep lungs. After thoracotomy, a shallow incision through the pleural surface permitted 10-cm2 by 10-micrometer pieces of visceral pleura free of lung parenchyma to be peeled off the lung surface. The pleura was then mounted as a planar sheet separating 2 reservoirs of Krebs-Ringer solution. Electrical potential and resistance, hydraulic water permeability, and diffusional permeability to water and several hydrophilic solutes were measured. The results showed that (1) no spontaneous voltage difference was present across the pleura; (2) electrical resistance (27.1 omega/cm2) was very low; (3) hydraulic water permeability was extremely high (1.64 X 10(-8) ml/dyne-s); and (4) diffusional permeability was high, varying from 5.24 X 10(-4) cm/s for water to 4 X 10(-5) cm/s for hemoglobin. Blue dextran (molecular weight, 2 X 10(6) daltons) did not cross the pleura in measurable quantities. We concluded that the isolated visceral pleura of the adult sheep is an extremely "leaky" tissue that probably does not actively transport salt and water. These findings are consistent with a passive model of pleural fluid formation and reabsorption, and suggest that the transport properties of normal pleural tissue are unlikely to be responsible for any differences in composition between interstitial and pleural fluids.
Asunto(s)
Agua Corporal/metabolismo , Pleura/metabolismo , Sodio/metabolismo , Animales , Transporte Biológico , Difusión , Hemoglobinas/metabolismo , Presión Hidrostática , Técnicas In Vitro , Permeabilidad , OvinosRESUMEN
A stopped-flow rapid reaction apparatus was used to monitor changes in extracellular pH in dogfish (Mustelus canis) erythrocyte suspensions under conditions where dpH/dt was determined by the rate of HCO3-/Cl- exchange across the red cell membrane. Experiments were performed on erythrocytes suspended either in their own plasma or in elasmobranch Ringer solution over a range of temperatures from 5 to 35 degrees C. The exchange fluxes at 25 degrees C for red blood cells suspended in their own plasma (2.03 nmol/cm2-s) or in Ringer solution (2.00 nmol/cm2-s) are not significantly different and can be compared to those obtained under similar conditions for human red cell suspensions (0.910 nmol/cm2-s). The flux for dogfish erythrocytes suspended in Ringer solution was reduced by 80% after exposure of the cells to SITS. An Arrhenius plot of the exchange rate constant yielded an activation energy of about 13.2 kcal/mol. We conclude that 1) plasma has no inhibitory effect of HCO3-/Cl- exchange across the dogfish erythrocyte membrane or on activity of intraerythrocyte carbonic anhydrase, 2) HCO3-/Cl- exchange probably occurs via the same mechanism in fish and mammalian erythrocytes, and 3) the conversion of plasms HCO3- to CO2 in dogfish can be catalyzed by intraerythrocyte carbonic anhydrase.
Asunto(s)
Bicarbonatos/metabolismo , Cloruros/metabolismo , Cazón/metabolismo , Eritrocitos/metabolismo , Tiburones/metabolismo , Animales , Transporte Biológico Activo , Dióxido de Carbono/metabolismo , Anhidrasas Carbónicas/metabolismo , Eritrocitos/enzimologíaRESUMEN
Fresh human blood was diluted 1:5000 in buffered saline-sucrose solution and titrated to a pH varying from 4.5 to 10.5 with 0.1 N HCl or 0.1 N NaOH. Circular regions of the membrane of individual cells were then deformed at 25 degrees C by aspiration into a micropipette having an internal tip diameter of 0.9-1.4 micron. A membrane surface elasticity modulus, mu (dyn/cm), was computed from the relationship between length of the aspirated membrane and the deforming pressure according to a two-dimensional membrane model. Surface elasticity increases with decreasing pH and with time after the cell suspension is acidified, rising several orders of magnitude with a t1/2 of 1--5 h as pH is lowered from 7.2 to 4.6. This increase in mu is only partially reversible. pH greater than 7.2 had little effect on mu. Membrane surface elasticity is not affected by variations in external [Ca2+] over the range of 0--50 mM, tonicity of the suspension medium from 275--400 mosM, or age of 0--50 h. Addition of 50 mM NaHCO3 to the medium increases the rate of change of mu at a given pH. These results suggest that the elastic properties of the red cell membrane are largely determined by interactions among structural proteins located on the cytoplasmic surface of the membrane and that these interactions are initiated by changes in intracellular pH.