Measurement of Na-K-ATPase-mediated rubidium influx in single segments of rat nephron

1990 ◽  
Vol 259 (1) ◽  
pp. F111-F121 ◽  
Author(s):  
L. Cheval ◽  
A. Doucet
Keyword(s):  
Choline Chloride ◽  
Hydrolytic Activity ◽  
Metabolic Inhibitor ◽  
Thick Ascending Limb ◽  
Intact Cells ◽  
Medullary Thick Ascending Limb ◽  
Carbonyl Cyanide ◽  
Collecting Tubule ◽  
Single Nephron ◽  
Rubidium Influx

To determine the functioning rate of Na-K-ATPase in the rat nephron, a micromethod was developed to measure the rate of rubidium uptake in single nephron segments microdissected from collagenase-treated kidneys. Because the hydrolytic activity of Na-K-ATPase displayed the same apparent affinity for K and Rb ions, whereas the Vmax elicited by K was higher than that in the presence of Rb, experiments were performed in the presence of cold Rb plus 86Rb. Before the assay, tubules were preincubated for 10 min at 37 degrees C to restore the normal transmembrane cation gradients. 86Rb uptake was measured after washing out extracellular cations by rinsing the tubules in ice-cold choline chloride solution containing Ba2+. Rb uptake increased quasi-linearly as a function of incubation time up to 30 s in the thick ascending limb, 1 min in the proximal convoluted tubule, and 5 min in the collecting tubule, and reached an equilibrium after 5–;30 min. The initial rates of Rb uptake increased in a saturable fashion as Rb concentration in the medium rose from 0.25 to 5 mM. In medullary thick ascending limb, the initial rate of Rb uptake was inhibited by greater than 90% by 2.5 mM ouabain and by 10(-5) M of the metabolic inhibitor carbonyl cyanide trifluoromethoxyphenylhydrazone. Correlation of Na-K-ATPase hydrolytic activity at Vmax and initial rates of ouabain-sensitive Rb uptake in the successive segments of nephron indicates that in intact cells the pump works at approximately 20–30% of its Vmax. Increasing intracellular Na concentration by tubule preincubation in a Rb- and K-free medium increased the initial rates of Rb intake up to the Vmax of the hydrolytic activity of the pump.

Quantitation of [3H]ouabain binding and turnover of Na-K-ATPase along the rabbit nephron

1984 ◽  
Vol 247 (1) ◽  
pp. F158-F167 ◽  
Author(s):  
G. El Mernissi ◽  
A. Doucet
Keyword(s):  
Turnover Rate ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Thick Ascending Limb ◽  
Ouabain Binding ◽  
Medullary Thick Ascending Limb ◽  
Large Numbers ◽  
Collecting Tubule ◽  
Pump Activity ◽  
Single Nephron

To determine the number of Na-K-ATPase units and the enzyme's turnover rate along the rabbit nephron, the specific binding of [3H]ouabain and the Na-K-ATPase activity were measured in single nephron segments microdissected from collagenase-treated kidneys. The highest density of Na-K-ATPase (20-30 fmol X mm-1) was found in the distal convoluted tubule and the medullary thick ascending limb. Binding was intermediate (10 fmol X mm-1) in the proximal convoluted tubule and connecting tubule, and it was lowest (2-7 fmol X mm-1) in the pars recta, the cortical thick ascending limb, and the collecting tubule. In the medullary thick ascending limb, Scatchard analysis of the specific [3H]ouabain binding indicated a dissociation constant of 1.8 microM. The pump activity was proportional to the number of catalytic units, indicating that the maximal turnover rate of Na-K-ATPase (2,000 ATP molecules per minute per ouabain binding site) was similar in the various segments of the nephron. The method developed for quantitating [3H]ouabain binding is technically simple enough to permit simultaneous measurement of the enzyme in large numbers of tubules and sufficiently sensitive to determine the number of Na-K-ATPase units in each region of the nephron.

Potassium adaptation in hypothyroidism: changes in transport adenosinetriphosphatases

1993 ◽  
Vol 264 (1) ◽  
pp. F31-F36 ◽  
Author(s):  
M. M. Salem ◽  
Y. Chen ◽  
S. K. Mujais
Keyword(s):  
Morphological Changes ◽  
Thick Ascending Limb ◽  
Adaptive Responses ◽  
Medullary Thick Ascending Limb ◽  
Potassium Adaptation ◽  
Adenosine Triphosphatases ◽  
Collecting Tubule ◽  
Pump Activity ◽  
Major Increase ◽  
K Depletion

To evaluate whether hypothyroidism alters the adaptive responses of renal transport adenosine-triphosphatases (ATPases) to modifications in dietary K content, we examined the activities of Na-K pump and H-K pump in hypothyroid rats under basal conditions and after dietary K changes. Hypothyroidism led to a decline in Na-K pump activity in all three nephron segments examined [proximal convoluted tubule from 2,333 +/- 103 to 1,099 +/- 32, medullary thick ascending limb from 4,344 +/- 119 to 1,613 +/- 61, and cortical collecting tubule (CCT) from 1,133 +/- 45 to 640 +/- 38 pmol.mm-1 x h-1; all P < 0.01 vs. euthyroid] along with morphological changes manifest in a decrease in tubule diameter. K loading led to an increase in Na-K pump activity in the CCT of both euthyroid (from 1,133 +/- 45 to 2,269 +/- 74, pmol.mm-1 x h-1, P < 0.01) and hypothyroid (from 640 +/- 38 to 1,118 +/- 67 pmol.mm-1 x h-1, P < 0.01) animals. Furthermore, in euthyroid rats, 3 wk of K depletion led to a major increase in H-K pump activity in both the CCT (from 203 +/- 14 to 331 +/- 22 pmol.mm-1 x h-1, P < 0.01) and medullary collecting tubule (MCT, from 137 +/- 9 to 210 +/- 14 pmol.mm-1 x h-1, P < 0.01). Hypothyroidism was associated with a decline in H-K pump activity in the CCT and MCT (to 94 +/- 6 and 55 +/- 5 pmol.mm-1 x h-1, respectively; both P < 0.01 vs. euthyroid).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of metabolic acidosis and alkalosis on NEM-sensitive ATPase in rat nephron segments

1992 ◽  
Vol 262 (4) ◽  
pp. F583-F590 ◽  
Author(s):  
C. Khadouri ◽  
S. Marsy ◽  
C. Barlet-Bas ◽  
L. Cheval ◽  
A. Doucet
Keyword(s):  
Metabolic Acidosis ◽  
Atpase Activity ◽  
Thick Ascending Limb ◽  
Nephron Segments ◽  
Medullary Thick Ascending Limb ◽  
Henle's Loop ◽  
Collecting Tubule ◽  
Nephron Segment ◽  
Electrogenic Proton Pump ◽  
Chloride Treatment

An N-ethylmaleimide (NEM)-sensitive adenosinetriphosphatase (ATPase) displaying the kinetic and pharmacological properties of an electrogenic proton pump has been described in the different segments of rat nephron, where it mediates part of the active tubular proton secretion. This study was therefore designed to evaluate whether changes in urinary acidification observed during metabolic acidosis or alkalosis were associated with alterations of the activity of tubular NEM-sensitive ATPase, and if so, to localize the nephron segments responsible for these changes. Within 1 wk after the onset of ammonium chloride treatment, rats developed a metabolic acidosis, and NEM-sensitive ATPase activity was markedly increased in the medullary thick ascending limb of Henle's loop and outer medullary collecting tubule, and slightly increased in the cortical collecting tubule. Conversely, treatment with sodium bicarbonate induced a metabolic alkalosis that was accompanied by decreased NEM-sensitive ATPase activity in medullary thick ascending limb and outer medullary collecting tubule. NEM-sensitive ATPase activity was not altered in any other nephron segment tested in alkalotic and acidotic rats, i.e., the proximal tubule and the cortical thick ascending limb of Henle's loop. Changes qualitatively similar were observed as soon as 3 h after the onset of NaHCO3 or NH4Cl-loading. In the medullary collecting tubule, alterations of NEM-sensitive ATPase activity are in part due to hyperaldosteronism observed in both acidotic and alkalotic rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of renal Na-K-ATPase in the rat: effect of uninephrectomy

1986 ◽  
Vol 251 (3) ◽  
pp. F506-F512 ◽  
Author(s):  
S. K. Mujais ◽  
N. A. Kurtzman
Keyword(s):  
Enzyme Activity ◽  
Adrenal Gland ◽  
Atpase Activity ◽  
Thick Ascending Limb ◽  
Medullary Thick Ascending Limb ◽  
Collecting Tubule ◽  
Pars Recta ◽  
Intact Rats ◽  
Cortical Collecting Tubule

This study has examined the temporal profile and the segmental localization along the rat nephron of the increase in Na-K-ATPase produced by uninephrectomy, and the role of the adrenal gland in the generation of the increase in enzyme activity. In adrenal-intact rats, an increase in Na-K-ATPase activity in the cortical collecting tubule (CCT) was observed at 1 wk (140 +/- 13% of sham, P less than 0.05) and sustained at 2 wk (140 +/- 8% of sham, P less than 0.05). In contrast, the enhancement of enzyme activity in the proximal convoluted tubule (PCT) was transient (at 1 wk: 164 +/- 20% of sham, P less than 0.05; and at 2 wk: 97 +/- 9% of sham, P greater than 0.5). No changes in Na-K-ATPase activity were observed in the other nephron segments studied: pars recta, medullary thick ascending limb, cortical thick ascending limb, distal convoluted tubule, and medullary collecting tubule. In adrenalectomized rats, CCT enzyme activity was lower than in adrenal-intact rats (761 +/- 84 vs. 1,984 +/- 276 pmol X mm-1 X h-1, P less than 0.001) and was not altered by uninephrectomy (849 +/- 91 pmol X mm-1 X h-1, NS). We conclude that the increase in Na-K-ATPase activity following uninephrectomy is restricted to two segments of the nephron and follows a distinctive pattern in each. In the PCT a transient enhancement in enzyme activity is observed, whereas in the CCT the increase in Na-K-ATPase is sustained and requires the presence of an intact adrenal gland.

Suspension of medullary thick ascending limb tubules from the rabbit kidney

1984 ◽  
Vol 247 (6) ◽  
pp. F955-F964 ◽  
Author(s):  
M. E. Chamberlin ◽  
A. LeFurgey ◽  
L. J. Mandel
Keyword(s):  
Oxygen Consumption ◽  
Cellular Metabolism ◽  
Isolation Procedure ◽  
Rabbit Kidney ◽  
Enzyme Assays ◽  
Thick Ascending Limb ◽  
Marker Enzyme ◽  
Direct Inhibition ◽  
Medullary Thick Ascending Limb ◽  
Carbonyl Cyanide

A procedure for isolating a suspension of tubules derived from the rabbit medullary thick ascending limb is described. The purity of the preparation was assessed by microscopy and enzyme assays and the viability of the preparation was assessed by measuring oxygen consumption. Microscopy revealed that the suspension contains 95% thick ascending limbs and that the isolation procedure preserves the structure of the epithelium except for the loss of the basement membrane. The preparation had a high activity of calcitonin-sensitive adenylate cyclase, a marker enzyme for the medullary thick ascending limb. Control oxygen consumption was considerably higher than that reported for proximal tubules in the literature, and nystatin or carbonyl cyanide p-trifluoromethoxyphenylhydrazone addition produced a more than 100% increase in oxygen consumption. Furosemide inhibited the oxygen consumption by 43% and ouabain inhibited it by 42%. Furosemide inhibited sodium chloride entry without directly affecting the Na-K-ATPase or cellular metabolism. Chloride removal depressed oxygen consumption to the same extent as furosemide, but some of this action was through direct inhibition of cellular metabolism.

Segmental synthesis and actions of prostaglandins along the nephron

1987 ◽  
Vol 253 (3) ◽  
pp. F377-F387 ◽  
Author(s):  
J. P. Bonvalet ◽  
P. Pradelles ◽  
N. Farman
Keyword(s):  
Renal Tubule ◽  
Thick Ascending Limb ◽  
Pge2 Synthesis ◽  
Complex Interactions ◽  
Quantitative Measurements ◽  
Medullary Thick Ascending Limb ◽  
Exogenous Substrate ◽  
Collecting Tubule ◽  
The Difference ◽  
Cortical Collecting Tubule

The sites of synthesis and action of prostaglandins (PGs) along the renal tubule are examined. We focused our attention on experiments performed on well-defined nephron segments, using direct quantitative measurements of prostaglandin synthesis by radio- or enzyme-immunoassay. On the other hand, we selected, among the described effects of PGs, those obtained on precisely defined tubular segments. Among PGs, PGE2 synthesis is largely predominant all along the tubule. Its main sites of synthesis are the medullary collecting tubule and, to a lesser extent, the cortical collecting tubule and the thin limb of Henle's loop. Synthesis of PGE2 is amplified approximately tenfold in the presence of an excess exogenous substrate, arachidonic acid, compared with values measured without addition of substrate. Other eicosanoids have roughly the same distribution along the tubule as PGE2. Their rate of synthesis is, however, much less than that of PGE2, approximately 20-fold lower for PGF2 alpha and 6-keto-PGF1 alpha, and 100-fold lower for thromboxane B2 (TxB2). This contrasts with glomerular PG synthesis, where the difference between the production of PGE2 and other eicosanoids is much less marked. Most studies agree that antidiuretic hormone (ADH) and kinins augment PGE2 synthesis, whereas corticosteroids decrease it, at least in the collecting tubule. Direct effects of PGE2 have been described mainly in the medullary thick ascending limb and collecting tubule. They generally consist of a decrease in transepithelial potential difference and reabsorptive rates of water and solutes, in particular sodium and chloride. However, whatever the solute or tubular segment concerned, some studies failed to find such effects. The bulk of evidence suggests that ADH and PGs interact in kidney tubular cells. It is generally accepted that PGs antagonize the hydrosmotic effects of ADH in the collecting tubule. The mechanisms underlying these complex interactions are still under discussion: they probably involve several types of receptors and pathways for ADH action, which intervene in the modulation of both PG synthesis and cyclic nucleotides, and several types of PG receptors, either stimulatory or inhibitory to adenylate cyclase.

Isolated MTAL cells produce an inhibitor of ouabain-sensitive oxygen consumption

1991 ◽  
Vol 260 (2) ◽  
pp. F210-F215 ◽  
Author(s):  
L. G. Cantley ◽  
R. Fuhro ◽  
P. Silva
Keyword(s):  
Arachidonic Acid ◽  
Oxygen Consumption ◽  
Nordihydroguaiaretic Acid ◽  
Rabbit Kidney ◽  
Thick Ascending Limb ◽  
Direct Inhibition ◽  
Intact Cells ◽  
Medullary Thick Ascending Limb ◽  
Transport Inhibitor

Dispersed medullary thick ascending limb (MTAL) cells of the rabbit kidney produce an inhibitor of transport-related oxygen consumption when incubated at 37 degrees C. Appearance of this inhibitor is enhanced by incubation with arachidonic acid. Its appearance can be prevented by either 10(-6) M nordihydroguaiaretic acid or 10(-4) M indomethacin, but not by 10(-6) M indomethacin. These results suggest that the transport inhibitor is an eicosanoid produced by a pathway other than that catalyzed by cyclooxygenase. It inhibits ouabain-sensitive respiration in intact cells treated with amphotericin, suggesting direct inhibition of the Na(+)-K(+)-ATPase.

High-affinity Ca-Mg-ATPase along the rabbit nephron

1982 ◽  
Vol 242 (4) ◽  
pp. F346-F352 ◽  
Author(s):  
A. Doucet ◽  
A. I. Katz
Keyword(s):  
Ruthenium Red ◽  
Calcium Flux ◽  
Transepithelial Transport ◽  
Mitochondrial Atpase ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Medullary Thick Ascending Limb ◽  
Collecting Tubule ◽  
Dose Dependent

Maintenance of cell calcium homeostasis and transepithelial transport of this cation require its extrusion from the cell against a steep electrochemical gradient. Because it has been proposed that a membrane Ca-ATPase activated by micromolar concentrations of Ca2+ prevailing in the cell participates in these processes, we attempted in this study to determine whether such an enzyme is present in the rabbit nephron. A magnesium-dependent ATPase, maximally activated by Ca2+ (Ca-Mg-ATPase) concentrations between 1.1 and 2.3 microM (apparent Km = 0.3-0.4 microM), was found in all segments of the nephron. Ca-Mg-ATPase (pmol.mm-1.h-1) was highest in the distal convoluted tubule (243) and cortical collecting tubule (208), intermediate in the proximal convoluted tubule (140) and medullary thick ascending limb of Henle's loop (135), and lower in the pars recta (97), cortical thick ascending limb (50), and medullary collecting tubule (51). The enzyme was insensitive to ouabain and vanadate, but was inhibited by ruthenium red in a dose-dependent manner (Ki congruent to 2.10(-6) M). Sodium azide, an inhibitor of mitochondrial ATPase, did not affect Ca-Mg-ATPase, suggesting that the enzyme was located in the plasma membrane. The Ca-Mg-ATPase activity measured in most segments of the rabbit nephron in this study appears sufficient to account in theory for the active component of the unidirectional (lumen-to-bath) calcium flux found in the corresponding region of the nephron with in vitro single tubule microperfusion techniques.

Alpha2-adrenoceptors and cellular cAMP levels in single nephron segments from the rat

1985 ◽  
Vol 249 (1) ◽  
pp. F28-F33 ◽  
Author(s):  
S. Umemura ◽  
D. Marver ◽  
D. D. Smyth ◽  
W. A. Pettinger
Keyword(s):  
Adenylate Cyclase ◽  
Phosphodiesterase Inhibitor ◽  
Thick Ascending Limb ◽  
Intact Cells ◽  
Camp Content ◽  
Nephron Segments ◽  
Pathophysiological Role ◽  
Single Nephron ◽  
Alpha2 Adrenoceptor

A functional role for the numerically predominant renal alpha2-adrenoceptors, which in other tissues inhibit adenylate cyclase, remains undefined. We therefore examined the effect of alpha2-adrenoceptor stimulation with (-)-epinephrine (E) on cell cAMP content in the isolated proximal convoluted tubule (PCT), medullary and cortical thick ascending limb of Henle, and collecting tubule (MTAL, CTAL, MCT, and CCT, respectively). Parathyroid hormone (1-34 PTH), in PCT or CTAL, or arginine vasopressin (AVP), in MTAL, CTAL, MCT, or CCT, was used to activate adenylate cyclase in intact cells from these microdissected nephron segments in the presence of 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor) and propranolol. Alpha2-Adrenoceptors were activated using varying concentrations of E (37 degrees C, 2 min). Alpha2-Adrenoceptor activation with E (5 X 10(-7) to 5 X 10(-6) M) suppressed cellular cAMP stimulation by PTH by 35% in PCT and stimulation by AVP in CCT by 50%. This suppression by E in PCT and CCT was inhibited by 5 X 10(-6) M yohimbine or 5 X 10(-7) M phentolamine but not by 5 X 10(-6) M prazosin. E also suppressed cAMP stimulated by AVP in MCT, but it did not suppress the PTH-or AVP-stimulated increase in cellular cAMP in CTAL and MTAL. These studies show that there are alpha2-adrenoceptors in the rat nephron. Activation of these alpha 2-adrenoceptors can inhibit cAMP formation stimulated by PTH in PCT and by AVP in the CCT and MCT but not in the CTAL and MTAL. A pathophysiological role of altered regulation of these receptors is yet to be described.

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