RESUMEN
To examine directly in real time the efflux of organic compounds [e. g., organic anions (OAs) such as fluorescein (FL)] across the luminal membrane of isolated, perfused renal tubules during net secretion, we devised an approach utilizing a recently developed epifluorescence microscopy system for continuous monitoring of fluorescence in the collected perfusate. To illustrate this approach, we measured the luminal efflux rate of FL in mineral oil-covered, isolated, perfused S2 segments of rabbit renal proximal tubules. The washout profile of FL showed a deviation from linearity at time 0 when plotted on a semilog scale, indicating that the luminal efflux of FL was a saturable process. We were able for the first time to determine the kinetic parameters of luminal efflux [FL concentration at one-half maximal FL efflux (K(t)(lumen)) of approximately 560 microM and maximal rate of FL efflux across the luminal membrane (J(max)(lumen)) of approximately 635 fmol. min(-1). mm(-1)]. From the present study, we conclude that the transport step for OAs across the luminal membrane of OAs is a carrier-mediated process. This approach will work to measure luminal transport in real time for any secreted organic compound that is sufficiently fluorescent to be measured with commonly available, highly sensitive optical equipment.
Asunto(s)
Colorantes Fluorescentes/farmacocinética , Túbulos Renales/metabolismo , Animales , Aniones/farmacocinética , Transporte Biológico/fisiología , Fluoresceína/farmacocinética , Técnicas In Vitro , Túbulos Renales/citología , Cinética , Microscopía Fluorescente , Perfusión , ConejosRESUMEN
To examine the role of protein kinase C (PKC) in organic anion (OA) secretion, we used epifluorescence microscopy to study steady-state transepithelial secretion of 1 microM fluorescein (FL) by isolated perfused S2 segments of rabbit renal proximal tubules. Addition of 100 nM phorbol 12-myristate 13-acetate (PMA), a known PKC activator, to the bathing medium decreased steady-state secretion of FL by approximately 30% after 25 min. This inhibition was irreversible and, indeed, increased to approximately 40% at 25 min following removal of PMA [10 microM 1,2-dioctanoyl-sn-glycerol (DOG) produced a comparable inhibition]. The inhibition produced by PMA was blocked when 100 nM of either staurosporine (ST) or bisindolylmaleimide I (BIM), both known PKC inhibitors, was added to the bath for a 20-min preexposure followed by the addition of PMA. ST or BIM alone had no significant effect on FL secretion, suggesting that the basal FL secretion rate was not under influence of PKC. Addition of 1 microM of either the peptide hormone bradykinin (BK) or the alpha(1)-receptor agonist phenylephrine (PE), both of which stimulate PKC via a ligand-receptor-PKC coupling reaction, to the bath also inhibited FL secretion by approximately 22 and approximately 27%, respectively. However, the inhibition was completely reversible after removal of BK or PE. Pretreatment of tubules with 100 nM BIM eliminated the inhibition of FL secretion produced by exposure to PE. We conclude that PKC negatively regulates the net secretion of OAs in rabbit renal proximal tubules. The data indicate that BK or catecholamines can play a physiological role in regulating OA secretion via PKC activation.
Asunto(s)
Túbulos Renales Proximales/metabolismo , Proteína Quinasa C/metabolismo , Animales , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Epitelio/efectos de los fármacos , Fluoresceína/análisis , Túbulos Renales Proximales/efectos de los fármacos , Microscopía Fluorescente/métodos , Perfusión , Proteína Quinasa C/antagonistas & inhibidores , Conejos , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
To determine the quantitative roles of the basolateral and luminal Na(+)-dicarboxylate (Na-DC) cotransporters in establishing and maintaining the alpha-ketoglutarate (alphaKG) gradient required for renal tubular secretion of organic anions, we measured net steady-state transepithelial secretion of fluorescein (FL) in real time in isolated, perfused S2 segments of rabbit renal proximal tubules. Net "basal" FL secretion in the absence of exogenous alphaKG had a K(t) of approximately 4 microM and a maximal transepithelial secretion rate (J(max)) of approximately 380 fmol. min(-1). mm(-1) (where K(t) is the FL concentration that produces one-half the J(max)). It could be almost completely inhibited by basolateral p-aminohippurate (PAH). Selective inhibition of the basolateral Na-DC cotransporter indicated that recycling via this transporter of alphaKG that had been exchanged for FL supports approximately 25% of the "basal" FL secretion. Physiological alphaKG concentrations of 10 microM in the bath or 50 microM in the perfusate stimulated net secretion of FL by approximately 30 or approximately 20%, respectively. These data indicate that the basolateral Na-DC cotransporter supports approximately 42% of the net FL secretion. The luminal and basolateral effects of physiological concentrations of alphaKG were additive, indicating that the combined function of the luminal and basolateral Na-DC cotransporters can support approximately 50% of the net FL secretion. This apparently occurs by their establishing and maintaining approximately 50% of the outwardly directed alphaKG gradient that is responsible for driving basolateral FL/alphaKG exchange. The remaining approximately 50% would be maintained by metabolic production of alphaKG in the cells.
Asunto(s)
Aniones/metabolismo , Sistemas de Computación , Transportadores de Ácidos Dicarboxílicos , Ácidos Cetoglutáricos/farmacología , Túbulos Renales Proximales/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente , Simportadores , Animales , Proteínas Portadoras/fisiología , Medios de Contraste/farmacocinética , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Fluoresceína/farmacocinética , Técnicas In Vitro , Túbulos Renales Proximales/efectos de los fármacos , Cinética , Proteínas de la Membrana/fisiología , Perfusión , Conejos , Ácido p-Aminohipúrico/farmacologíaRESUMEN
In nonperfused proximal tubules isolated from chicken long-looped mammalian-type nephrons, intracellular pH (pHi), measured with the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, was approximately 7.3 under control conditions (HEPES-buffered medium with pH 7.4 at 37 degrees C) and was reduced to approximately 7.0 in response to NH4Cl pulse. The rate of recovery of pHi from this level to the resting level was 1) significantly reduced by the removal of Na+ from the bath, 2) significantly increased by the removal of Cl- from the bath, 3) unchanged by the removal of both Na+ and Cl- from the bath, 4) significantly reduced by the addition of either ethylisopropylamiloride or DIDS to the bath, 5) significantly increased by a high bath K+ concentration, and 6) unchanged by the addition of Ba2+ to the bath. These data suggest that both Na+-coupled and Cl--coupled basolateral acid-base fluxes are involved in determining the rate of recovery of pHi after acidification. The most likely ones to be important in regulating pHi are a Na+/H+ exchanger and a Na+-coupled Cl-/HCO-3 exchanger. In birds, long-looped mammalian-type nephrons resemble short-looped transitional nephrons but differ markedly from superficial loopless reptilian-type nephrons.
Asunto(s)
Pollos/metabolismo , Hidrógeno/metabolismo , Membranas Intracelulares/metabolismo , Túbulos Renales Proximales/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Amoníaco/metabolismo , Cloruro de Amonio/farmacología , Animales , Bario/farmacología , Cloruros/farmacología , Femenino , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Túbulos Renales Proximales/efectos de los fármacos , Asa de la Nefrona/anatomía & histología , Nefronas/anatomía & histología , Nefronas/metabolismo , Permeabilidad , Sodio/farmacología , Cloruro de Sodio/farmacologíaRESUMEN
In proximal tubules isolated from chicken superficial loopless reptilian-type nephrons, intracellular pH (pHi), measured with pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, was approximately 7.1-7.2 under control conditions (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered medium with pH 7.4 at 37 degrees C), and was reduced to approximately 6.9 in response to NH4Cl pulse. The rate of recovery of pHi (control value approximately equal to 5 x 10(-3) pH U/s) from this acid level was 1) significantly decreased by removal of Na+ or both Na+ and Cl- from the bath or addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.25 mM) to the bath, 2) significantly increased by high bath K+ (75 mM), and 3) unchanged by removal of Cl- alone from the bath or addition of ethylisopropylamiloride (1 mM) or Ba2+ (5 mM) to the bath. Resting pHi was 1) significantly decreased by Na+ or simultaneous Na+ and Cl- removal, 2) significantly increased by high K+, and 3) unchanged by Cl- removal alone or addition of Ba2+. The data do not fit the concept of pHi regulation by the most commonly suggested basolateral transporters (Na+/H+ exchanger, Na(+)-dependent and Na(+)-independent Cl-/HCO3- exchangers, or Na(+)-HCO3(-)-CO3(2-) cotransporter).