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Transport in the colon of the domestic fowl switches from sodium-linked hexose and amino acid cotransport on high-salt intake to amiloride-sensitive sodium channel expression on low-salt (LS) diets. The present experiments were designed to investigate the role of aldosterone in suppression of the colonic sodium-glucose luminal cotransporter (SGLT). LS-adapted hens were resalinated with or without simultaneous aldosterone treatment. Changes in the electrophysiological responses and SGLT protein expression levels were examined at 1, 3, and 7 days of treatment. Serum aldosterone levels fell from approximately 400 pmol/l in LS-adapted hens to values below the detection limit (<44 pmol/l) after 1 day of resalination. At the same time, glucose-stimulated short circuit current (I(SC)) increased from 20.9 +/- 8.7 to 56.3 +/- 15.5 microA/cm(2), whereas amiloride-sensitive I(SC) decreased from -68.9 +/- 12.7 microA/cm(2) on LS to +0.6 +/- 12.0 microA/cm(2). Glucose-stimulated I(SC) increased further at 3 and 7 days of resalination, whereas amiloride-sensitive I(SC) remained suppressed. When resalinated birds were simultaneously treated with aldosterone, the LS pattern of high amiloride-sensitive I(SC) and low glucose-stimulated I(SC) was maintained. Immunoblotting results from the same tissues demonstrated that SGLT-like protein expression increased following resalination. Aldosterone treatment completely blocked this effect. These results demonstrate that aldosterone suppresses both activity and protein expression of hen colonic SGLT. Resalination either through decreased aldosterone or other factors may be able to activate SGLT activity independently of increases in protein expression.

Methods have been developed for producing functional, transporting monolayers of avian proximal tubule (PT) cells. A highly homogenous fraction of PT fragments was prepared by enzymatic digestion (collagenase + Dispase) of chick (3- to 5-day-old) kidneys, followed by Percoll gradient centrifugation. The PT fraction was enriched in glucose-6-phosphatase, a proximal enzyme marker, and reduced in specific activity of hexokinase, a distal marker. PT fragments were grown to confluence in serum-free media on collagen-coated permeable filter supports. Electron microscopy of confluent monolayers revealed numerous microvilli and mitochondria, central cilia, and tight junctions, all characteristic of PT cells. γ-Glutamyltranspeptidase, a proximal brush-border enzyme, showed threefold higher activity on apical than on basolateral sides of the monolayer. The electrophysiological characteristics of monolayers were investigated by voltage-clamp techniques. Monolayers displayed low transepithelial resistances (40–60 Ω ⋅ cm2), lumen-negative potentials, and baseline currents of 6–12 μA/cm2 (with or without 5 mM glucose). Both α-methyl-d-glucose (2 mM), a nonmetabolizable hexose, and phenylalanine (2 mM) significantly stimulated short-circuit current when added to the mucosal side of glucose-free monolayers. Phloridzin, a specific inhibitor of Na+-coupled glucose transport, significantly inhibited short-circuit current, as did 10−5 M amiloride. Monolayers also expressed net secretory transport of urate. This cell culture preparation may provide a useful working model for the study of avian PT transport.

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2001.-The effect of parathyroid hormone (PTH) and activation of protein kinase C (PKC) and protein kinase A (PKA) on transepithelial Pi transport was examined in monolayers of chick proximal tubule cells in primary culture (PTCs). Acute exposure of the PTCs to PTH (10 Ϫ9 M, basolateral side) significantly decreased the net reabsorption of Pi by ϳ66%. There was no effect after the addition of PTH to the luminal side. Activation of PKC by phorbol 12-myristate 13-acetate (PMA; 0.1 M) dramatically decreased net P i reabsorption by ϳ60%. Bisindolylmaleimide I (BIM; 1 M), a highly selective PKC inhibitor, prevented PMA-induced inhibition. Activation of adenylate cyclase/PKA by forskolin (10 M) mimicked the effect of PTH by significantly reducing net P i reabsorption by one-half. Addition of H-89 (10 M), a potent inhibitor of PKA, abolished forskolin-induced inhibition. PTH inhibition was blocked by either BIM or H-89. Tissue electrophysiology remained stable after all treatments. There was a decreased immunoreactivity of the luminal Na ϩ -Pi cotransporter NaPi-IIa after PTH treatment. These data indicate that PTH inhibition of Pi reabsorption in this in vitro system is mediated by PKC and PKA. primary cultures; reabsorptive flux; secretory flux; luminal sodium-inorganic phosphate cotransporter NaPi-IIa; protein kinase C; protein kinase A THE STUDY OF RENAL FUNCTION in birds and other nonmammalian vertebrates has provided valuable insights into the regulation of renal phosphate (P i ) excretion. In mammals, filtration and reabsorption provide sufficient control of P i excretion; however, in birds, which generally have lower and more variable glomerular filtration rates (46), the capacity for net tubular P i secretion has been reported (3). P i can be excreted by the avian kidney in quantities severalfold higher than the filtered load (21). Because avian kidneys possess this additional level of control for renal P i handling, comparative studies of its regulation should further our understanding of P i homeostasis.In both the avian and mammalian renal proximal tubule, P i reabsorption occurs via secondary, Na ϩ -dependent electroneutral transport across the luminal membrane [brush-border membrane (BBM)] (15, 34) coupled to an undefined exit process in the basolateral membrane (BLM) (1). In both mammals and birds, parathyroid hormone (PTH) has been found to be the major regulator of this process (28,24). PTH decreases BBM Na ϩ -P i cotransport activity in both mammals and birds and, in the latter, additionally stimulates net P i secretion (1, 34), which likely occurs through a unique Na ϩ -independent, voltage-and K ϩ -dependent transporter in the BBM (1, 35).Studies with the opossum kidney cell line (OK cells) have shown that PTH exerts its effects through activation of both protein kinase A (PKA) and protein kinase C (PKC) (7, 32), resulting in inhibition of BBM Na ϩ -P i cotransport. Other studies indicated the phosphaturic effect of PTH involves endocytic retrieval followed by lysosomal degradation of the BBM Na ϩ -P...

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