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J L Seifter

Publications and source records attributed to J L Seifter.

28 records · Page 2Linked to original sources
PubMed1986

Intracellular pH regulation and proton transport by rabbit renal medullary collecting duct cells. Role of plasma membrane proton adenosine triphosphatase.

Proton secretion in the renal medullary collecting duct is thought to occur via a luminal proton-ATPase. In order to determine what mechanism(s) participate in proton transport across medullary collecting duct (MCD) cells membranes, intracellular pH (pHi) regulation and proton extrusion rates were measured in freshly prepared suspensions of rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pHi was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Proton extrusion rates were measured using a pH stat. The resting pHi of MCD cells was 7.19 +/- 0.05 (SE) in a nonbicarbonate medium of pH 7.30. When cells were acidified by exposure to acetate salts or by abrupt withdrawal of ammonium chloride, they exhibited pHi recovery to the resting pHi over a 5-min time-course. Depletion of greater than 95% of cellular ATP content by poisoning with KCN in the absence of glucose inhibited pHi recovery. ATP depletion inhibited proton extrusion from MCD cells. Treatment with N-ethylmaleimide also inhibited pHi recovery. In addition, cellular ATP content was dependent on transmembrane pH gradients, suggesting that proton extrusion stimulated ATP hydrolysis. Neither removal of extracellular sodium nor addition of amiloride inhibited pHi recovery. These results provide direct evidence that a plasma membrane proton-ATPase, but not a Na+/H+ exchanger, plays a role in proton transport and pHi regulation in rabbit MCD.

Acetates↗
PubMed1986

Sodium-hydrogen exchange and glucose transport in renal microvillus membrane vesicles from rats with diabetes mellitus.

Diabetes mellitus is associated with important changes in renal hemodynamics and transport function. Disturbances in solute transport have also been characterized in nonrenal tissues during hyperglycemia and insulinopenia. The purpose of this study was to determine if diabetes is associated with adaptive changes in function of the brush-border membrane of the proximal tubule. We studied Na+ and glucose transport in rat microvillus membrane vesicles isolated from the renal cortex of streptozotocin-induced and BB/W autoimmune diabetic rats. Untreated diabetes was associated with an increase in pH-stimulated total and amiloride-sensitive 22Na+ uptake into vesicles. Insulin treatment returned vesicle 22Na+ uptake to control levels. The increased Na+/H+ exchange was shown to be a result of increased net renal acid production rather than a specific response to insulinopenia because treatment with NaHCO3 also returned 22Na+ uptake to control levels. On the other hand, Na+-glucose cotransport, which was depressed in vesicles from untreated diabetics, returned to control levels with insulin but not NaHCO3 administration. This decreased Na+-glucose cotransport was not secondary to reduction in transport sites in untreated diabetics. These results show that in diabetes mellitus, increased Na+/H+ exchange activity is not the direct result of insulinopenia. However, the diabetic state appears to alter the functioning of the luminal Na+-glucose cotransporter.

Acid-Base Equilibrium↗
PubMed1986

Intracellular pH regulation in rabbit renal medullary collecting duct cells. Role of chloride-bicarbonate exchange.

The renal medullary collecting duct (MCD) secretes protons into its lumen and HCO3 into its basolateral space. Basolateral HCO3 transport is thought to occur via Cl/HCO3 exchange. To further characterize this Cl/HCO3 exchange process, intracellular pH (pHi) regulation was monitored in freshly prepared rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pHi was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Cells were preincubated in bicarbonate-containing solutions and then abruptly diluted into bicarbonate-free media. The MCD cell pHi response to abrupt removal of CO2/HCO3 included an initial alkalinization due to rapid CO2 efflux, followed by an acidification due to HCO3 efflux and a gradual recovery to the resting pHi of 7.24 +/- 0.06 partly due to the action of a plasma membrane H+-ATPase. The initial alkalinization required a CO2/HCO3 gradient and did not occur in the presence of acetazolamide. The acidification phase required intracellular HCO3 and extracellular Cl, which was consistent with a Cl/HCO3 exchange. MCD HCO3 efflux exhibited saturable kinetics for extracellular Cl, with a Michaelis constant (Km) of 29.9 +/- 7.7 mM. HCO3 efflux also exhibited preference for halides over NO3, SCN, and gluconate, and striking sensitivity to disulfonic stilbene and acetazolamide inhibition, with an apparent K1 of 5 X 10(-7) M for DIDS. The final pHi recovery required intracellular ATP, which indicated that Cl/HCO3 and H+-ATPase activities are present in the same cells in these suspensions. The results provide direct evidence for MCD Cl/HCO3 exchange and describe some of the properties of this transport process.

Acetazolamide↗
PubMed1985

Water and urea transport in renal microvillus membrane vesicles.

Light scattering was used to measure the water and urea permeability of brush border membrane vesicles (BBMV) isolated from rabbit renal cortex. In stop-flow experiments, exposure of BBMV to a 200 mM inwardly directed mannitol gradient gave a monophasic time course of decreasing BBMV volume corresponding to an osmotic water permeability (Pf) of 1.1 +/- 0.1 X 10(-2) cm/s at 37 degrees C. The temperature dependence of Pf was biphasic with delta H = 2 kcal/mol for T less than 33 degrees C and delta H = 14 kcal/mol for T greater than 33 degrees C. A 200 mM inwardly directed urea gradient gave a biphasic time course of BBMV volume due to rapid water efflux (approximately 50 ms) followed by slower urea influx (1-5 s) with urea permeability (Purea) of 2.4 +/- 0.2 X 10(-6) cm/s. Preincubation of BBMV with increasing [urea] reversibly inhibited both urea flux (Kd = 1,200 mM) and thiourea flux (Kd = 370 mM) according to a single-site inhibition model, suggesting a saturable urea carrier. Comparison of BBMV Pf and Purea with proximal tubule transepithelial water and urea transport rates suggests that the permeability of the tubular apical membrane (BBMV) is high enough to support a transcellular route for both osmotic water and urea transport.

Animals↗
PubMed1985

Radiation inactivation studies of renal brush border water and urea transport.

Radiation inactivation was used to determine the nature and molecular weight of water and urea transport pathways in brush border membrane vesicles (BBMV) isolated from rabbit renal cortex. BBMV were frozen to -50 degrees C, irradiated with 1.5 MeV electrons, thawed, and assayed for transport or enzyme activity. The freezing process had no effect on enzyme or transport kinetics. BBMV alkaline phosphatase activity gave linear ln(activity) vs. radiation dose plots with a target size of 68 +/- 3 kDa, similar to previously reported values. Water and solute transport were measured using the stopped-flow light-scattering technique. The rates of acetamide and osmotic water transport did not depend on radiation dose (0-7 Mrad), suggesting that transport of these substances does not require a protein carrier. In contrast, urea and thiourea transport gave linear ln(activity) vs. dose curves with a target size of 125-150 kDa; 400 mM urea inhibited thiourea flux by -50% at 0 and 4.7 Mrad, showing that radiation does not affect inhibitor binding to surviving transporters. These studies suggest that BBMV urea transport requires a membrane protein, whereas osmotic water transport does not.

Acetamides↗
PubMed1984

Absence of Cl-OH exchange and NaCl cotransport in rabbit renal microvillus membrane vesicles.

Cl-transport was studied in microvillus membrane vesicles isolated from the rabbit renal cortex. Inwardly directed K+ gradients in the presence of the K+ ionophore valinomycin (Val) enhanced 10 mM 36Cl uptake 2.5-fold, confirming a Cl- conductive pathway. An inwardly directed H+ gradient (pHin 7.5, pHout 6.0) stimulated 10 mM Cl- uptake 1.5-fold compared with pHin = pHout = 6.0. However, this H+ gradient stimulation of Cl- uptake appeared secondary to the H+ diffusion potential rather than to Cl-OH exchange, as it was abolished by Val and K+in = K+out. Additional evidence against Cl- transport via anion exchange was the failure of an inwardly directed Cl- gradient to generate an inside-acid pH gradient as monitored by quenching of acridine orange fluorescence. Cl- influx was the same in the presence of inwardly directed gradients of Na+, K+, Cs+, Li+, and Rb+, arguing against NaCl cotransport. Finally, conductive Cl- transport was reduced by the inhibitors furosemide, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. These data indicate the presence of an inhibitor-sensitive, conductive mode of Cl- transport but fail to demonstrate significant pathways for Cl- OH exchange or NaCl cotransport in rabbit renal microvillus membrane vesicles.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

Cl- transport via anion exchange in Necturus renal microvillus membranes.

We investigated the mechanism of Cl- transport in microvillus membrane vesicles isolated from Necturus kidneys. Cl- influx was insensitive to changes in membrane potential induced by K+ gradients and the K+ ionophore valinomycin, arguing against conductive Cl- transport. Inward gradients of Na+ or Na+ + K+ did not stimulate initial Cl- influx, arguing against direct Na+-Cl- or Na+-K+-Cl- cotransport. External Cl-, HCO3-, and NO3- each stimulated 36Cl efflux and inhibited 36Cl uptake, indicating anion exchange. Outward HCO3- gradients but not OH- gradients stimulated 36Cl influx, consistent with Cl- -HCO3- exchange. Cl- transport via anion exchange was inhibited by furosemide, bumetanide, and disulfonic stilbenes, but not by acetazolamide. External halides stimulated 36Cl efflux (Cl- = Br- greater than I- greater than F-) but the organic anions lactate, p-aminohippurate, and urate did not. Amiloride-sensitive Na+-H+ exchange was demonstrated. Finally, in the presence of a CO2/HCO3 buffer system, imposing an inward Na+ gradient caused a time-delayed stimulation of 36Cl uptake, consistent with indirect coupling of Na+-H+ and Cl- -HCO3- exchangers. We conclude that the parallel operation of Na+-H+ and Cl- -HCO3- exchangers rather than direct cotransport may account for the Na+-coupled uphill Cl- entry previously observed in the intact proximal tubular cell of Necturus.

Amiloride↗
PubMed1984

Adaptation of Na+-H+ exchange in renal microvillus membrane vesicles. Role of dietary protein and uninephrectomy.

The ablation of renal mass and institution of a high protein diet both lead to renal cortical hypertrophy and increased glomerular filtration rate (GFR). We studied Na+ transport in rat microvillus membrane vesicles isolated from uninephrectomized or sham operated rats fed 6% (low), 24% (standard), or 40% (high) protein diets. The feeding of high protein, as compared with low protein, was associated with a 50% increase in rates of pH-stimulated 22Na+ transport in isolated vesicles from sham and uninephrectomized animals. Values for the standard protein diet were intermediate to values for high and low protein. At each level of dietary protein intake, vesicular Na+ transport was greater in the uninephrectomized than in sham rats. The high protein diet was also associated with increased vesicular 22Na+ flux inhibitable by 1 mM amiloride. Increases in total and amiloride sensitive flux were also noted in the absence of a pH gradient. Conductive Na+ and H+ transport were not altered, nor were sodium-glucose and sodium-alanine cotransport. Kinetic studies revealed evidence for an increased Vmax of Na+-H+ exchange in uninephrectomized animals fed a 40 vs. a 6% protein diet whereas Km was unchanged. Supplements of NaHCO3 in the 40% protein diet, to adjust for an increased rate of net acid excretion, did not prevent the increased rates of Na+-H+ exchange. However, treatment with actinomycin D (0.12 mg/kg) prevented the increased Na+-H+ activity as well as the increased renal mass and GFR noted 24 h after unilateral nephrectomy. Na+-H+ exchange rate was closely correlated with GFR (r = 0.961; P less than 0.005) and renal mass (r = .986; P less than 0.001). These observations provide evidence for modification of the luminal membrane Na+-H+ exchanger in response to changes in dietary protein content and nephron number.

Animals↗