Parathyroid hormone and dibutyryl cAMP inhibit Na+/H+ exchange in renal brush border vesicles

1985 ◽  
Vol 248 (2) ◽  
pp. F212-F218 ◽  
Author(s):  
A. M. Kahn ◽  
G. M. Dolson ◽  
M. K. Hise ◽  
S. C. Bennett ◽  
E. J. Weinman
Keyword(s):  
Parathyroid Hormone ◽  
Proximal Tubule ◽  
Brush Border ◽  
Sodium Phosphate ◽  
Brush Border Membrane ◽  
Gradient Centrifugation ◽  
Membrane Vesicles ◽  
Enzymatic Digestion ◽  
Dibutyryl Camp ◽  
Ficoll Gradient

Parathyroid hormone (PTH) and cAMP inhibit sodium, water, and bicarbonate reabsorption in the proximal tubule. We wished to determine whether these agents directly inhibit proximal tubular Na+/H+ exchange. A suspension of rabbit proximal tubules was prepared by enzymatic digestion and Ficoll gradient centrifugation. Oxygen consumption at 37 degrees C was stable over 60 min, averaged 20 nmol X mg protein-1 X min-1, and was inhibited 60% by ouabain. Over 96% of cells excluded trypan blue. From this suspension, brush border membrane vesicles were isolated. The vesicles were enriched 12.7 times in alkaline phosphatase relative to a cortical homogenate and demonstrated pH gradient-stimulated, amiloride-sensitive Na+/H+ countertransport and sodium-phosphate and sodium-D-glucose cotransport. When the tubule suspension was exposed to PTH or dibutyryl cAMP, the activity of Na+/H+ countertransport in the resultant brush border vesicles was inhibited. Neither PTH nor dibutyryl cAMP affected the amiloride-insensitive component of sodium transport or sodium-phosphate or sodium-D-glucose cotransport. The effect of PTH on Na+/H+ counter-transport could not be explained by an alteration in fluidity of the brush border membrane. These experiments demonstrate that PTH and dibutyryl cAMP directly inhibit Na+/H+ countertransport in the brush border membrane of the rabbit proximal tubule.

Insulin stimulates Pi transport in brush border vesicles from proximal tubular segments

1984 ◽  
Vol 247 (5) ◽  
pp. E616-E624 ◽  
Author(s):  
M. R. Hammerman ◽  
S. Rogers ◽  
V. A. Hansen ◽  
J. R. Gavin
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Direct Action ◽  
Membrane Vesicles ◽  
Pi Transport ◽  
Glomerular Filtrate ◽  
Dog Kidney ◽  
Proximal Tubular ◽  
Altered Transport

Induction of hyperinsulinemia in dogs results in enhanced reabsorption of Pi from glomerular filtrate in the renal proximal tubule. To determine whether this may be a direct action of insulin mediated by altered transport characteristics of the proximal tubular brush border membrane, we measured Na+-dependent 32Pi transport in brush border membrane vesicles prepared from isolated proximal tubular segments originating from dog kidney that had been incubated with or without insulin. Specific high affinity binding sites for insulin were detected in proximal tubular segments. Increased initial rates (15 s) of Na+-dependent 32Pi transport were measured in brush border vesicles prepared from segments that had been incubated with insulin. This effect of insulin was concentration dependent over the range of 10(-10) to 10(-6) M insulin. These studies demonstrate the feasibility of using brush border vesicles prepared from proximal tubular segments to study solute transport. Our findings suggest that insulin-induced increased Pi reabsorption in the proximal tubule is mediated by a direct action of insulin on the proximal tubular cell, which results in increased Na+-Pi cotransport across the brush border membrane.

Parathyroid hormone-induced translocation of Na-H antiporters in rat proximal tubules

1989 ◽  
Vol 257 (4) ◽  
pp. C637-C645 ◽  
Author(s):  
C. B. Hensley ◽  
M. E. Bradley ◽  
A. K. Mircheff
Keyword(s):  
Parathyroid Hormone ◽  
Density Gradient ◽  
Brush Border ◽  
Gradient Centrifugation ◽  
Membrane Vesicles ◽  
Subcellular Fractionation ◽  
Proximal Tubules ◽  
Nylon Mesh ◽  
Sequence Of Events ◽  
Distinct Membrane

Parathyroid hormone (PTH) is believed to inhibit bicarbonate reabsorption by inhibiting Na-H antiport activity in proximal tubular brush-border membranes. The sequence of events triggered by PTH was investigated in a crude preparation of proximal tubules obtained by mechanical disruption and filtration through nylon mesh filters. Tubule samples were subjected to analytical subcellular fractionation after 2-, 5-, and 30-min treatments with 1 IU/ml PTH. These PTH-treatment intervals caused 54, 63, and 68% decreases in the Na-H antiport activity of a population of brush-border membrane vesicles that was resolved from a PTH-unresponsive brush-border population by density-gradient centrifugation. The rapid loss of Na-H antiport activity from the responsive population was accompanied by a transient increase in the Na-H antiport activity of a region of the density gradient, designated density window III, which was shown to contain two distinct membrane populations; these populations were both enriched in acid phosphatase activity, and one of them was also an important locus of galactosyltransferase activity. The increase in the Na-H antiport activity of window III accounted for 52% of the activity lost from the PTH-responsive population after 2 min, and for 43% of the activity lost after 5 min, but it was completely abolished after 25 more minutes in the presence of PTH. These observations suggest that PTH triggers a rapid translocation of Na-H antiporters from the microvillus membrane to a distinct membrane domain, where they are subsequently inactivated.

Facilitated diffusion of urate in avian brush-border membrane vesicles

2002 ◽  
Vol 283 (4) ◽  
pp. C1155-C1162 ◽  
Author(s):  
Steven M. Grassl
Keyword(s):  
Membrane Potential ◽  
Proximal Tubule ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Facilitated Diffusion ◽  
Transport Pathways ◽  
Proximal Tubule Cells ◽  
Avian Kidney

Membrane transport pathways mediating transcellular secretion of urate across the proximal tubule were investigated in brush-border membrane vesicles (BBMV) isolated from avian kidney. An inside-positive K diffusion potential induced a conductive uptake of urate to levels exceeding equilibrium. Protonophore-induced dissipation of membrane potential significantly reduced voltage-driven urate uptake. Conductive uptake of urate was inhibitor sensitive, substrate specific, and a saturable function of urate concentration. Urate uptake was trans-stimulated by urate and cis-inhibited by p-aminohippurate (PAH). Conductive uptake of PAH was cis-inhibited by urate. Urate uptake was unaffected by an outward α-ketoglutarate gradient. In the absence of a membrane potential, urate uptake was similar in the presence and absence of an imposed inside-alkaline pH gradient or an outward Cl gradient. These observations suggest a uniporter-mediated facilitated diffusion of urate as a pathway for passive efflux across the brush border membrane of urate-secreting proximal tubule cells.

Ischemic injury induces ADF relocalization to the apical domain of rat proximal tubule cells

2001 ◽  
Vol 280 (5) ◽  
pp. F886-F894 ◽  
Author(s):  
Sharon L. Ashworth ◽  
Ruben M. Sandoval ◽  
Melanie Hosford ◽  
James R. Bamburg ◽  
Bruce A. Molitoris
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Apical Membrane ◽  
Critical Role ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Actin Binding ◽  
Proximal Tubule Cell ◽  
Apical Domain

Breakdown of proximal tubule cell apical membrane microvilli is an early-occurring hallmark of ischemic acute renal failure. Intracellular mechanisms responsible for these apical membrane changes remain unknown, but it is known that actin cytoskeleton alterations play a critical role in this cellular process. Our laboratory previously demonstrated that ischemia-induced cell injury resulted in dephosphorylation and activation of the actin-binding protein, actin depolymerizing factor [(ADF); Schwartz, N, Hosford M, Sandoval RM, Wagner MC, Atkinson SJ, Bamburg J, and Molitoris BA. Am J Physiol Renal Fluid Electrolyte Physiol 276: F544–F551, 1999]. Therefore, we postulated that ischemia-induced ADF relocalization from the cytoplasm to the apical microvillar microfilament core was an early event occurring before F-actin alterations. To directly investigate this hypothesis, we examined the intracellular localization of ADF in ischemic rat cortical tissues by immunofluorescence and quantified the concentration of ADF in brush-border membrane vesicles prepared from ischemic rat kidneys by using Western blot techniques. Within 5 min of the induction of ischemia, ADF relocalized to the apical membrane region. The length of ischemia correlated with the time-related increase in ADF in isolated brush-border membrane vesicles. Finally, depolymerization of microvillar F-actin to G-actin was documented by using colocalization studies for G- and F-actin. Collectively, these data indicate that ischemia induces ADF activation and relocalization to the apical domain before microvillar destruction. These data further suggest that ADF plays a critical role in microvillar microfilament destruction and apical membrane damage during ischemia.

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