RESUMEN
Dietary salt intake in domestic fowl affects epithelial transport and morphology of the lower intestine (colon and coprodeum). This study investigated lower intestinal morphology and transport activity in two wild bird species with natural diets containing either low or high salt. Tissues from rock ptarmigan (Lagopus mutus) and common murres (Uria aalge) were sampled for histology and electrophysiological analyses. The ptarmigan exists on a low salt diet, while the murre lives on a high protein and high salt diet. The ptarmigan colon and coprodeum had villi/folds and crypts and the epithelium contained absorptive epithelial cells, mitochondria-rich cells and goblet cells. The colon had significant amiloride-inhibitable Isc, 5-15 µA/cm(2), with no glucose-stimulated Isc, and no significant phloridzin inhibition. The coprodeum also had high amiloride-inhibitable Isc. This transport pattern corresponded to that of chickens on low-salt diets. However, the ptarmigan colon also had a significant lysine/leucine-stimulated Isc of 3±1.0 µA/cm(2). The short U. aalge colon was similar to that of ptarmigans, but with no villi. It demonstrated a significant lysine/leucine-stimulated Isc (11±3.5 µA/cm(2)) with no amiloride-inhibitable Isc, similar to the high-salt chicken colon, but with no Na(+)-glucose cotransport. The murre coprodeum was inert to all substances and showed high resistance (1000 Ω·cm(2)), with a multilayered squamous epithelium. Despite some variations possibly associated with dietary protein intake, we conclude that natural high and low salt diets in different avian species are associated with different lower intestinal transport patterns, providing for post-renal adjustments in ion and water excretion.
Asunto(s)
Aves/fisiología , Colon/fisiología , Ecosistema , Animales , Aves/anatomía & histología , Colon/anatomía & histología , Especificidad de la EspecieRESUMEN
In primary cell cultures of the avian (Gallus gallus) renal proximal tubule parathyroid hormone and cAMP activation generate a Cl(-)-dependent short circuit current (I(SC)) response, consistent with net transepithelial Cl(-) secretion. In this study we investigated the expression and physiological function of the Na-K-2Cl (NKCC) transporter and CFTR chloride channel, both associated with Cl(-) secretion in a variety of tissues, in these proximal tubule cells. Using both RT-PCR and immunoblotting approaches, we showed that NKCC and CFTR are expressed, both in proximal tubule primary cultures and in a proximal tubule fraction of non-cultured (native tissue) fragments. We also used electrophysiological methods to assess the functional contribution of NKCC and CFTR to forskolin-activated I(SC) responses in filter grown cultured monolayers. Bumetanide (10 µM), a specific blocker of NKCC, inhibited forskolin activated I(SC) by about 40%, suggesting that basolateral uptake of Cl(-) is partially mediated by NKCC transport. In monolayers permeabilized on the basolateral side with nystatin, forskolin activated an apical Cl(-) conductance, manifested as bidirectional diffusion currents in the presence of oppositely directed Cl(-) gradients. Under these conditions the apical conductance appeared to show some bias towards apical-to-basolateral Cl(-) current. Two selective CFTR blockers, CFTR Inhibitor 172 and GlyH-101 (both at 20 µM) inhibited the forskolin activated diffusion currents by 38-68%, with GlyH-101 having a greater effect. These data support the conclusion that avian renal proximal tubules utilize an apical CFTR Cl(-) channel to mediate cAMP-activated Cl(-) secretion.
Asunto(s)
Pollos/metabolismo , Cloruros/metabolismo , AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Túbulos Renales Proximales/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Animales , Pollos/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Expresión Génica , Cultivo Primario de Células , Simportadores de Cloruro de Sodio-Potasio/genéticaRESUMEN
Experiments were designed to characterize an active, electrogenic transport of NH(4)(+) ions across the colonic epithelium of the domestic fowl (Gallus gallus). Colonic segments were isolated and stripped of underlying muscle. The mucosal epithelia were mounted in Ussing chambers and voltage-clamped to measure the short-circuit currents (I(SC)) associated with transport. Bilateral addition of NH(4)(+) caused a dose-dependent outward current (negative I(SC)), with a Km of 34+/-8 mM and a maximal current response of 311+/-47 microA cm(-2) (12+/-2 microEq cm(-2) h(-1)). A similar effect was seen with unilateral addition of NH(4)(+) to the serosal (s) side, but not with mucosal (m) addition. Pre-treatment with 10(-4) M amiloride exposed a net outward (negative) I(SC), and serosal NH(4)(+) addition further increased this outward current with a Km of 53+/-24 mM. Decreasing the bath pH from 7.3 to 6.0 did not affect the I(SC) response to NH(4)(+). Unidirectional NH(4)(+) flux measurements revealed a net secretory flux (8.8+/-3.1 micromol cm(-2) h(-1) s-m, versus 2.6+/-1.4 micromol cm(-2) h(-1) m-s). Furthermore, the secretory flux closely matched the resulting change in I(SC) with serosal NH(4)(+), showing that the transepithelial flux of NH(4)(+) could account for the outward current response. Addition of 50 nM bafilomycin A to the mucosal solution completely eliminated serosal to mucosal NH(4)(+) transport, implicating an apical V-type H(+)-ATPase in this transport process. The I(SC) response to NH(4)(+) was partially inhibited by ouabain, a blocker of the Na(+)/K(+)-ATPase, but only minimally affected by bumetanide, an inhibitor of the serosal Na(+)-K(+)-2Cl(-) cotransporter. Active NH(4)(+) extrusion across the mucosal membrane, combined with low permeability to NH(3) in this tissue, allow for maintenance of steep ammonia gradients across the colonic epithelium and protection from ammonia toxicity. Furthermore, these studies indicate that the hen colon may be a useful new model system for the study of NH(4)(+) transport.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Pollos/metabolismo , Colon/metabolismo , Mucosa Intestinal/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Animales , Transporte Biológico/fisiología , Colon/citologíaRESUMEN
The lower intestine (colon and coprodeum) of the domestic fowl maintains a very active, transporting epithelium, with a microvillus brush border, columnar epithelial cells, and a variety of transport systems. The colon of normal or high salt-acclimated hens expresses sodium-linked glucose and amino acid cotransporters, while the coprodeum is relatively inactive. Following acclimation to low salt diets, however, both colon and coprodeum shift to a pattern of high expression of electrogenic sodium channels, and the colonic cotransporter activity is simultaneously downregulated. These changes in the transport patterns seem to be regulated, at least in part, by aldosterone. Our recent work with this tissue has focused on whether aldosterone alone can account for the low salt pattern of transport. Other work has looked at the changes in morphology and in proportions of cell types that occur during chronic acclimation to high or low salt diets, and on a cAMP-activated chloride secretion pathway. Recent findings suggesting effects of other hormones on lower intestinal transport are also presented.
Asunto(s)
Aldosterona/metabolismo , Aves/fisiología , Transporte Iónico/fisiología , Tracto Gastrointestinal Inferior/metabolismo , Aclimatación , Aldosterona/farmacología , Animales , Dieta Hiposódica , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/fisiología , Tracto Gastrointestinal Inferior/anatomía & histología , Modelos Biológicos , Porfirinas/metabolismo , Aves de Corral/metabolismo , Sodio/metabolismo , Simportadores del Cloruro de Sodio/metabolismo , Gorriones/metabolismoRESUMEN
The electrophysiological effects of parathyroid hormone (PTH) were studied in a primary cell culture model of the chick (Gallus domesticus) proximal tubule. In this model, confluent monolayers are grown on permeable filters and exhibit vectorial transport, including glucose-stimulated current. Under short-circuit conditions, PTH, at 10(-9) M, induced a positive current [short-circuit current (I(sc))] response, with an average 2-min peak response of 14.30 +/- 1.58 microA/cm(2) over the baseline I(sc,) followed by a slow decay. The PTH response was dose dependent, with a half-maximal response at 5 x 10(-9) M and maximal response at 5 x 10(-8) M. Forskolin and dibutyryl-cAMP also stimulated I(sc), as did the phosphodiesterase inhibitor IBMX. In contrast, the phorbol ester PMA inhibited baseline I(sc). The PTH response was nearly abolished by apical addition of 100 microM EIPA, an inhibitor of Na(+)/H(+) exchangers, and partially blocked by the Cl(-) channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 100 microM) and glibenclamide (300 microM). Higher doses of EIPA or NPPB alone (500 microM) were almost fully effective, with no or slight additional effects of NPPB or EIPA, respectively. The anion exchange inhibitor DIDS (100 microM) and the Na(+) channel blocker amiloride (10 microM) had no effect. Bilateral reduction of Cl(-) in the buffer, from 137 to 2.6 mM, abolished the PTH response; increasing Cl(-) concentration restored the I(sc) response, with a half-maximal effect at 50 mM. These data suggest that, in the chick proximal tubule, PTH activates both an Na(+)/H(+) exchanger and a Cl(-) channel that may be functionally linked.