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2006 ◽  
Vol 291 (6) ◽  
pp. C1377-C1387 ◽  
Author(s):  
Pernille Bøttger ◽  
Susanne E. Hede ◽  
Morten Grunnet ◽  
Boy Høyer ◽  
Dan A. Klærke ◽  
...  

The general phosphate need in mammalian cells is accommodated by members of the Pitransport (PiT) family ( SLC20), which use either Na+or H+to mediate inorganic phosphate (Pi) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na+-dependent Pi(NaPi) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with32Pias a traceable Pisource. For PiT1, the Michaelis-Menten constant for Piwas determined as 322.5 ± 124.5 μM. PiT2 was analyzed for the first time and showed positive cooperativity in Piuptake with a half-maximal activity constant for Piof 163.5 ± 39.8 μM. PiT1- and PiT2-mediated Na+-dependent Piuptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na+dependency patterns. However, only PiT2 was capable of Na+-independent Pitransport at acidic pH. Study of the impact of divalent cations Ca2+and Mg2+revealed that Ca2+was important, but not critical, for NaPitransport function of PiT proteins. To gain insight into the NaPicotransport function, we analyzed PiT2 and a PiT2 Pitransport knockout mutant using22Na+as a traceable Na+source. Na+was transported by PiT2 even without Piin the uptake medium and also when Pitransport function was knocked out. This is the first time decoupling of Pifrom Na+transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E55and E575are responsible for linking Piimport to Na+transport in PiT2.


2002 ◽  
Vol 283 (2) ◽  
pp. C579-C586 ◽  
Author(s):  
Timothy D. Ardizzone ◽  
Xiao-Hong Lu ◽  
Donard S. Dwyer

The goal of these studies was to determine whether different calcium channel antagonists affect glucose transport in a neuronal cell line. Rat pheochromocytoma (PC-12) cells were treated with L-, T-, and N-type calcium channel antagonists before measurement of accumulation of 2-[3H]deoxyglucose (2-[3H]DG). The L-type channel antagonists nimodipine, nifedipine, verapamil, and diltiazem all inhibited glucose transport in a dose-dependent manner (2–150 μM) with nimodipine being the most potent and diltiazem only moderately inhibiting transport. T- and N-type channel antagonists had no effect on transport. The L-type channel agonist l-BAY K 8644 also inhibited uptake of 2-[3H]DG. The ability of these drugs to inhibit glucose transport was significantly diminished by the presence of unlabeled 2-DG in the uptake medium. Some experiments were performed in the presence of EDTA (4 mM) or in uptake buffer without calcium. The absence of calcium in the uptake medium had no effect on inhibition of glucose transport by nimodipine or verapamil. To examine the effects of these drugs on a cell model of a peripheral tissue, we studied rat L6 muscle cells. The drugs inhibited glucose transport in L6 myoblasts in a dose-dependent manner that was independent of calcium in the uptake medium. These studies suggest that the calcium channel antagonists inhibit glucose transport in cells through mechanisms other than the antagonism of calcium channels, perhaps by acting directly on glucose transporters.


2002 ◽  
Vol 283 (1) ◽  
pp. E132-E137 ◽  
Author(s):  
James A. Rillema

Inorganic phosphate is present in milk at a concentration that is severalfold higher than in maternal plasma. In cultured mammary tissues from 12- to 14-day-pregnant mice, the intracellular concentration of 32PO4 was six times higher than in the culture medium after a 4-h treatment with32PO4. Of the principal lactogenic hormones [insulin (I), cortisol (H), and prolactin (PRL)], only I and PRL (in the presence of H and I) stimulated 32PO4uptake into cultured mammary tissues; H, by itself or in the presence of I or PRL, inhibited 32PO4 uptake. All three lactogenic hormones together effected the greatest stimulation of32PO4 uptake. Similar hormone effects were observed with regard to 32PO4 incorporation into lipids and trichloroacetic acid-insoluble molecules. In a time course study, the onset of the PRL stimulation of32PO4 uptake and incorporation occurred 8–12 h after PRL addition; in dose-response studies, the PRL effect was manifested with PRL concentrations of 50 ng/ml and above. From kinetic studies, the apparent maximal velocity of PO4uptake was determined to be ∼7.7 mM · h−1 · l cell water−1; the apparent Michaelis-Menten constant was ∼3–5 mM. The PRL effect on 32PO4 uptake was abolished when sodium was absent from the uptake medium. These studies thus demonstrate a complex interaction of three hormones (I, H, and PRL) in the regulation of 32PO4 uptake and incorporation into macromolecules in cultured mouse mammary tissues.


2002 ◽  
Vol 282 (4) ◽  
pp. F599-F607 ◽  
Author(s):  
Norma Vázquez ◽  
Adriana Monroy ◽  
Elisa Dorantes ◽  
Rosario A. Muñoz-Clares ◽  
Gerardo Gamba

The purpose of the present study was to determine the major functional, pharmacological, and regulatory properties of the flounder thiazide-sensitive Na-Cl cotransporter (flTSC) to make a direct comparison with our recent characterization of the rat TSC (rTSC; Monroy A, Plata C, Hebert SC, and Gamba G. Am J Physiol Renal Physiol 279: F161–F169, 2000). When expressed in Xenopus laevis oocytes, flTSC exhibits lower affinity for Na+ than for Cl−, with apparent Michaelis-Menten constant ( K m) values of 58.2 ± 7.1 and 22.1 ± 4.2 mM, respectively. These K m values are significantly higher than those observed in rTSC. The Na+ and Cl− affinities decreased when the concentration of the counterion was lowered, suggesting that the binding of one ion increases the affinity of the transporter for the other. The effect of several thiazides on flTSC function was biphasic. Low concentrations of thiazides (10−9 to 10−7 M) resulted in activation of the cotransporter, whereas higher concentrations (10−6 to 10−4 M) were inhibitory. In rTSC, this biphasic effect was observed only with chlorthalidone. The affinity for thiazides in flTSC was lower than in rTSC, but the affinity in flTSC was not affected by the Na+ or the Cl− concentration in the uptake medium. In addition to thiazides, flTSC and rTSC were inhibited by Hg2+, with an apparent higher affinity for rTSC. Finally, flTSC function was decreased by activation of protein kinase C with phorbol esters and by hypertonicity. In summary, we have found significant regulatory, kinetic, and pharmacological differences between flTSC and rTSC orthologues.


2000 ◽  
Vol 278 (1) ◽  
pp. C207-C211 ◽  
Author(s):  
Daryl A. Scott ◽  
Lawrence P. Karniski

Pendred syndrome, characterized by congenital sensorineural hearing loss and goiter, is one of the most common forms of syndromic deafness. The gene causing Pendred syndrome ( PDS) encodes a protein designated pendrin, which is expressed in the thyroid, kidney, and fetal cochlea. Pendrin functions as an iodide and chloride transporter, but its role in the development of hearing loss and goiter is unknown. In this study, we examined the mechanism of pendrin-mediated anion transport in Xenopus laevis oocytes. Unlabeled formate added to the uptake medium inhibited pendrin-mediated 36Cl uptake in X. laevis oocytes. In addition, the uptake of [14C]formate was stimulated in oocytes injected with PDS cRNA compared with water-injected controls. These results indicate that formate is a substrate for pendrin. Furthermore, chloride stimulated the efflux of [14C]formate and formate stimulated the efflux of 36Cl in oocytes expressing pendrin, results consistent with pendrin-mediated chloride/formate exchange. These data demonstrate that pendrin is functionally similar to the renal chloride/formate exchanger, which serves as an important mechanism of chloride transport in the proximal tubule. A similar process could participate in the development of ion gradients within the inner ear.


1999 ◽  
Vol 276 (3) ◽  
pp. F359-F366 ◽  
Author(s):  
Consuelo Plata ◽  
David B. Mount ◽  
Verena Rubio ◽  
Steven C. Hebert ◽  
Gerardo Gamba

The functional properties of alternatively spliced isoforms of the mouse apical Na+-K+-2Cl−cotransporter (mBSC1) were examined, using expression in Xenopus oocytes and measurement of22Na+or86Rb+uptake. A total of six isoforms, generated by the combinatorial association of three 5′ exon cassettes (A, B, and F) with two alternative 3′ ends, are expressed in mouse thick ascending limb (TAL) [see companion article, D. B. Mount, A. Baekgaard, A. E. Hall, C. Plata, J. Xu, D. R. Beier, G. Gamba, and S. C. Hebert. Am. J. Physiol. 276 ( Renal Physiol. 45): F347–F358, 1999]. The two 3′ ends predict COOH-terminal cytoplasmic domains of 129 amino acids (the C4 COOH terminus) and 457 amino acids (the C9 terminus). The three C9 isoforms (mBSC1-A9/F9/B9) all express Na+-K+-2Cl−cotransport activity, whereas C4 isoforms are nonfunctional in Xenopus oocytes. Activation or inhibition of protein kinase A (PKA) does not affect the activity of the C9 isoforms. The coinjection of mBSC1-A4 with mBSC1-F9 reduces tracer uptake, compared with mBSC1-F9 alone, an effect of C4 isoforms that is partially reversed by the addition of cAMP-IBMX to the uptake medium. The inhibitory effect of C4 isoforms is a dose-dependent function of the alternatively spliced COOH terminus. Isoforms with a C4 COOH terminus thus exert a dominant negative effect on Na+-K+-2Cl−cotransport, a property that is reversed by the activation of PKA. This interaction between coexpressed COOH-terminal isoforms of mBSC1 may account for the regulation of Na+-K+-2Cl−cotransport in the mouse TAL by hormones that generate cAMP.


1998 ◽  
Vol 9 (11) ◽  
pp. 1973-1982 ◽  
Author(s):  
J R Welborn ◽  
C E Groves ◽  
S H Wright

Epifluorescence microscopy was used to study peritubular transport of the fluorescent mycotoxin ochratoxin A (OTA) into single proximal tubule segments of the rabbit. Initial rates of OTA uptake into S2 segments were saturable and adequately described by Michaelis-Menten kinetics, with an apparent Km of 2.2+/-0.3 microM (SEM). Several lines of evidence indicated that peritubular uptake of OTA in S2 segments was effectively limited to the "classical" organic anion transporter. First, 5 mM p-aminohippurate (PAH) cis-inhibited the uptake of 1 microM OTA into tubules by 96%. Kinetic analysis of the inhibition of OTA uptake by PAH (100 microM to 5 mM) yielded an apparent Ki of 164 microM, similar to the 100 to 200 microM range of Km values previously reported for the peritubular uptake of PAH. Second, efflux of OTA from tubules was trans-stimulated 3.2-fold by the presence of 2.5 mM PAH in the uptake medium. Third, 100 microM alpha-ketoglutarate (alphaKG) trans-stimulated the uptake rate of 1 microM OTA by 1.8-fold. Fourth, besides PAH, other organic anions effectively cis-inhibited the uptake of 1 microM OTA into tubules (inhibitor, % inhibition): 1.5 mM alphaKG, 80%; 1 mM probenecid, 100%; 1 mM piroxicam, 100%; 1 mM octanoate, 100%. In contrast, 1.5 mM tetraethylammonium, an organic cation, blocked uptake of 1 microM OTA by only 7%. The inhibition of OTA uptake into S1 and S3 segments of the proximal tubule was qualitatively similar: 5 mM PAH cis-inhibited the uptake of 1 microM OTA by approximately 95% in both S1 and S3 segments. Thus, peritubular OTA uptake into all segments of the proximal tubule appears to be dominated by its interaction with the classical organic anion transporter. The high-affinity and relatively high capacity of this pathway for OTA suggest that peritubular uptake may be a significant avenue for the entry of this toxin into proximal tubule cells.


1992 ◽  
Vol 2 (10) ◽  
pp. 1507-1515
Author(s):  
T D McKinney ◽  
M B Scheller ◽  
M Hosford ◽  
M E Lesniak ◽  
T S Haseley

In these studies, a clone of cells derived from the porcine renal epithelial line LLC-PK1 grown on porous filters was used to evaluate basolateral uptake of the organic cation tetraethylammonium (TEA). (3H) TEA (1 microM) entered cells in a saturable and time-dependent manner achieving a steady-state value at 2 to 2.5 h. Uptake was reduced by hypothermia and the metabolic inhibitors sodium azide and iodoacetate. Several other organic cations in 1 mM concentrations inhibited the majority of TEA uptake. In lower concentrations, the inhibitory potency of these was: verapamil greater than cimetidine approximately amiloride approximately quinidine greater than procainamide approximately N1-methylnicotinamide. When sodium was replaced with potassium in the uptake medium, TEA uptake was also reduced consistent with electrogenic transport. However, uptake was reduced further by 1 mM cimetidine in the presence of both NaCl and KCl buffers. TEA uptake was not significantly different when the media pH was varied from 6.0 to 8.0. In addition, results of experiments in which intracellular pH was altered with NH4Cl were not consistent with the presence of organic cation/proton exchange. TEA/TEA exchange could not be demonstrated in experiments in which cells were preloaded with 1 mM nonradioactive TEA and uptake of (3H)TEA was measured or in which nonradioactive TEA in the external medium failed to enhance efflux from cells preloaded with (3H)TEA. These results indicate that the basolateral membrane of LLC-PKc10 cells has one or more transport processes for the mediated uptake of organic cations. However, the precise mechanism(s) involved in this transport remains to be elucidated.


1990 ◽  
Vol 258 (4) ◽  
pp. C583-C588 ◽  
Author(s):  
M. Szczepanska-Konkel ◽  
A. Hoppe ◽  
J. T. Lin ◽  
T. P. Dousa

We investigated the suitability of alpha-bromophosphonoacetic acid (alpha-BrPAA) to act as a possible irreversible inhibitor of Na(+)-dependent transport of Pi across renal brush-border membrane (BBM). When added directly into the Pi uptake medium, alpha-BrPAA causes specific, competitive [apparent inhibition constant (Ki) = 0.33 mM; no change in maximum velocity (Vmax)], and reversible (by washing) inhibition of Na+ gradient [Na+o greater than Na+i]-dependent uptake of Pi by BBM vesicles (BBMV). Next, BBMV were preincubated with 5 mM alpha-BrPAA in alkaline (pH 9) medium for 30 min, then twice washed by 1:100 dilution and recentrifugation, and tested for transport and other properties. This preincubation of BBMV with alpha-BrPAA in alkaline medium resulted in a different type of inhibition [lower Vmax; no change in Michaelis constant (Km)] of the Na+ gradient-dependent uptake of 32Pi, whereas the uptakes of D-[3H]glucose and other solutes were not altered. This inhibition of Pi transport was not reversed by dilution and washing of BBMV. The BBMV Na(+)-dependent binding of [14C]phosphonoformic acid, but not of [3H]phlorizin, was decreased; activities of BBM marker enzymes were not changed. Results suggest that alpha-BrPAA binds onto the same locus on luminal surface of BBM on which Pi and Na+ bind and inhibits Na(+)-Pi cotransporter similar to phosphonoformic acid. Furthermore, after a 30-min incubation in alkaline medium, alpha-BrPAA apparently forms a more stable association with BBM in the vicinity of the Na(+)-Pi cotransporter. We thus suggest that alpha-BrPAA acts under these conditions as an apparently irreversible inhibitor of Na(+)-Pi cotransporter in BBM.(ABSTRACT TRUNCATED AT 250 WORDS)


1989 ◽  
Vol 263 (2) ◽  
pp. 333-339 ◽  
Author(s):  
S Muallem ◽  
T G Beeker

Electrically permeabilized rat pancreatic acini were used to evaluate the contributions of GTP and Ins(1,4,5) P3 to hormone-stimulated Ca2+ uptake and release from intracellular pools. Treatment of permeabilized acini with Ca2+-mobilizing hormones, GTP or GTP[S] resulted in stimulation of an ATP-dependent, VO4(2-)-sensitive Ca2+ uptake into a non-mitochondrial intracellular pool. GTP and GTP[S] also augmented the hormone-mediated stimulation of Ca2+ uptake. Including oxalate in the uptake medium increased Ca2+ uptake into this pool but did not modify the stimulation of Ca2+ uptake induced by hormones or GTP. Ins(1,4,5)P3 released all the extra Ca2+ accumulated as a result of hormone, GTP or GTP[S] stimulation. Hence, these stimuli activated the Ca2+ pump localized in the membrane of the hormone and Ins(1,4,5)P3-sensitive Ca2+ pool. Including 2,3-diphosphoglyceric acid (PGA) [an inhibitor of Ins(1,4,5)P3 hydrolysis] in the incubation medium blunted the GTP and GTP[S]-stimulated Ca2+ uptake. In the presence of PGA, the hormones inhibited Ca2+ accumulation, and GTP and GTP[S] augmented this effect. Accordingly, PGA stabilized the Ins(1,4,5)P3-evoked Ca2+ release from intracellular pools. Only in the presence of PGA was it possible to demonstrate hormonally-evoked Ca2+ release from permeabilized cells. GTP, and more importantly GTP[S], augmented the hormone-evoked Ca2+ release. Hormones and Ins(1,4,5)P3 in the presence or absence of GTP or GTP[S] released Ca2+ from the same intracellular pool. The extent of Ca2+ release caused by the combination of hormones and GTP or GTP[S] was similar to that evoked by Ins(1,4,5)P3 alone. Taken together, these results suggest that GTP or GTP[S] facilitates stimulation of phospholipase C by hormones. Such stimulation results in stimulation of protein kinase C and increased levels of Ins(1,4,5)P3 and is sufficient to explain the effects of GTP and GTP[S] on Ca2+ uptake and release from pancreatic acinar cells.


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