Alkaline phosphatase activity does not mediate phosphate transport in the renal-cortical brush-border membrane

We studied (1) the effect of primary modulators of phosphate transport, namely the hypophosphataemic mouse mutant (Hyp) and low-phosphorus diet, on alkaline phosphatase activity in mouse renal-cortex brush-border membrane vesicles and (2) the effect of several primary inhibitors of alkaline phosphatase on phosphate transport. Brush-border membrane vesicles from Hyp-mouse kidney had 50% loss of Na+-dependent phosphate transport, but only 18% decrease in alkaline phosphatase activity. The low-phosphorus diet effectively stimulated Na+/phosphate co-transport in brush-border membrane vesicles (+ 118%), but increased alkaline phosphatase activity only slightly (+13%). Levamisole (0.1 mM) and EDTA (1.0 mM) inhibited brush-border membrane-vesicle alkaline phosphatase activity of 82% and 93% respectively, but had no significant effect on Na+/phosphate co-transport. We conclude that alkaline phosphatase does not play a direct role in phosphate transport across the brush-border membrane of mouse kidney.

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Inhibition of alkaline phosphatase activity and d-glucose uptake in rat renal brush-border membrane vesicles by aminoglycosides

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(87)90152-0 ◽

1987 ◽

Vol 903 (1) ◽

pp. 31-36 ◽

Cited By ~ 7

Author(s):

Masayuki Takahashi ◽

Yukihiko Aramaki ◽

Asaichi Inaba ◽

Seishi Tsuchiya

Keyword(s):

Alkaline Phosphatase ◽

Glucose Uptake ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Brush Border ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Renal Brush Border Membrane ◽

Renal Brush Border

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Effects of L-bromotetramisole on phosphate transport by the proximal renal tubule: failure to demonstrate a direct involvement of alkaline phosphatase

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y82-038 ◽

1982 ◽

Vol 60 (3) ◽

pp. 276-281 ◽

Cited By ~ 11

Author(s):

Michele G. Brunette ◽

Vincent W. Dennis

Keyword(s):

Alkaline Phosphatase ◽

Brush Border ◽

Brush Border Membrane ◽

Renal Tubule ◽

Direct Action ◽

Membrane Vesicles ◽

Phosphate Transport ◽

Proximal Convoluted Tubule ◽

Brush Border Membrane Vesicles ◽

Inorganic Phosphate Reabsorption

Several recent studies have suggested that the alkaline phosphatase situated in the brush border membrane of the proximal convoluted tubule is positively related to inorganic phosphate reabsorption through this segment of the nephron. We examined this hypothesis by studying the influence of an inhibitor of alkaline phosphatase, L-bromotetramisole, upon phosphate transport through the isolated rabbit tubule, phosphate transport through brush border membrane vesicles of rats, and phosphate efflux from these vesicles. Results were compared with those obtained with D-bromotetramisole which is inactive on alkaline phosphatase.The microperfusion studies on isolated tubules did not show any significant influence of L-bromotetramisole on phosphate transport which slightly increased by 0.22 ± 0.78 pmol ∙ mm−1 ∙ min−1 (NS).The experiments performed on brush border membrane vesicles indicated that at pH 7.5, both L- and D-bromotetramisole at a concentration of 0.1 mM slightly but significantly increased phosphate input from 1800 ± 149 to 1999 ± 157 and 1982 ± 168 pmol/mg protein per 30-s incubation (P < 0.01 and P < 0.005, respectively). At pH 8.5, the same tendency was observed but was significant with the D isomer only. Finally, the effect of an increasing phosphate transport was accentuated when both isomers were tested at 1.0-mM concentration. Phosphate efflux from brush border membrane vesicles was not influenced by L- or D-bromotetramisole at 0.1 mM. At 1.0 mM, however, both isomers accelerated this release.These results do not support the hypothesis of a direct action of alkaline phosphatase upon phosphate transport through the entire proximal convoluted tubule, nor upon input or output of this electrolyte through the brush border membrane vesicles. The two forms of bromotetramisole have variable effects upon phosphate transport which are unrelated to the alkaline phosphatase activity.

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Phosphate transport and alkaline phosphatase in confluent MDCK cell monolayers

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y87-182 ◽

1987 ◽

Vol 65 (6) ◽

pp. 1151-1156 ◽

Cited By ~ 8

Author(s):

Marc Letellier ◽

Normand Brière ◽

Gérard E. Plante ◽

Claude Petitclerc

Keyword(s):

Alkaline Phosphatase ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Brush Border ◽

Mdck Cell ◽

Inorganic Phosphate ◽

Brush Border Membrane ◽

Phosphate Transport ◽

Cell Monolayers ◽

Sodium Dependent

Several studies have suggested that the brush border membrane alkaline phosphatase is involved in the renal reabsorption of inorganic phosphate along the proximal convoluted tubule. However, other studies on the influence of l(−)bromotetramisole, an inhibitor of alkaline phosphatase, upon phosphate transport into brush border membrane vesicles have failed to show an involvement of alkaline phosphatase. The present experiments were designed to demonstrate that the MDCK (Madin, Darby, canine, kidney) cell line can be used as an alternative model to study phosphate transport, to examine the effect of l(−)bromotetramisole and the role of alkaline phosphatase. MDCK cell monolayers were concomitantly used for alkaline phosphatase activity measurement and phosphate transport analysis. While alkaline phosphatase activity increases by 37-fold from day 2 to day 8 of culture, reaching a plateau at day 10, the sodium-dependent phosphate transport into the cell monolayers decreases by 2-fold during that same period. The phosphate transport was also studied in the presence of l(−)bromotetramisole at pH 8.5. The sodium-dependent phosphate uptake of inorganic phosphate is reduced by 43% in the presence of l(−)bromotetramisole at day 3 of culture but is not reduced in the 7-day-old culture. Our results suggest a participation of alkaline phosphatase upon phosphate transport in MDCK cell monolayers and indicate that this cell line constitutes a good model to study the relationship between alkaline phosphatase and phosphate transport.

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On the correlation between alkaline phosphatase and phosphate transport in rat renal brush border membrane vesicles

Pflügers Archiv - European Journal of Physiology ◽

10.1007/bf00584431 ◽

1980 ◽

Vol 384 (2) ◽

pp. 149-153 ◽

Cited By ~ 25

Author(s):

C. Storelli ◽

H. Murer

Keyword(s):

Alkaline Phosphatase ◽

Brush Border ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Phosphate Transport ◽

Brush Border Membrane Vesicles ◽

Renal Brush Border Membrane ◽

Renal Brush Border

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Effect of temperature and pH on phosphate transport through brush border membrane vesicles in rats

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y84-034 ◽

1984 ◽

Vol 62 (2) ◽

pp. 229-234 ◽

Cited By ~ 24

Author(s):

Michèle G. Brunette ◽

Richard Beliveau ◽

Meanthan Chan

Keyword(s):

Brush Border ◽

Brush Border Membrane ◽

Phosphate Uptake ◽

Membrane Vesicles ◽

Phosphate Transport ◽

Mean Value ◽

Brush Border Membrane Vesicles ◽

Effect Of Temperature ◽

Total Phosphate ◽

Renal Brush Border Membrane

The kinetics of sodium gradient dependent phosphate uptake by the renal brush border membrane vesicles of the rat have been studied under various conditions of temperature and pH. From 7 to 30 °C the Lineweaver-Burk plots are linear, and the apparent Km progressively increases from 54 to 91 μM. Above 30 °C, the apparent Km continues to increase to reach 135 μM at 40 °C, but a break is observed in the Lineweaver-Burk plots at the substrate concentration of 300 μM. The existence of this break, confirmed by the Eadie-Hofstee plot supports the hypothesis of a dual mechanism of phosphate transport, one for low concentrations of substrate with a Km of 100 μM and the other for high concentrations with a Km of approximately 240 μM. When the two components of the Eadie-Hofstee plot are analyzed according to a nonlinear regression program, these two values of Km become 70 μM and 1.18 mM, respectively. The Vmax continuously increases with temperature. However, the Arrhenius plot (In Vmax vs. 1/Tk) shows an abrupt discontinuity at 23 °C. pH experiments were performed at 35 °C. In the absence of a proton gradient, increasing the pH from 6.5 to 7.5 and 8.5 decreases the apparent Km from 341 to 167 and 94 μM, respectively. When only the divalent form of phosphate is considered as the substrate, the apparent Km does not vary anymore with the pH and remains around the mean value of 105 μM. The uniformity of the apparent Km for the total phosphate uptake, when only the divalent phosphate is considered as being the substrate, suggests that this divalent form is the only one which is transported. Whatever the substrate considered, total phosphate or divalent phosphate, the highest Vmax is obtained at pH 7.5 which probably approximates the optimum pH inside the vesicles for the phosphate uptake.

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