Search PubMed⌕ Search

Biomedical subjects

A Doucet

Publications and source records attributed to A Doucet.

At least 91 records · Page 5Linked to original sources
PubMed1985

Renal sodium transport in vitamin D resistant hypophosphatemic rickets.

To investigate the possible role of a Na transport defect in the pathogenesis of the phosphaturia in vitamin D resistant rickets, we studied the activity of the Na-K ATPase activity along the microdissected segments of the nephron in normal (N) and hypophosphatemic mice (Hyp), the Na uptake by renal brush border membrane (BBM), as well as the interrelationship between Na and phosphate transport through this membrane. In N mice, Na-K ATPase activity was present in decreasing order, in the distal tubule, the ascending branch of the loop of Henle, the proximal tubule, and the collecting tubule. In Hyp mice, the Na-K ATPase activity was comparable to that measured in N mice, except in the granular segment of the distal tubule where a 256% of the control activity was reproducibly observed. In N mice, Na initial uptake by BBM vesicles increased with Na concentration in the incubation medium, according to two kinetic components: one saturable, evident at low substrate concentrations and the other, nonsaturable, corresponding to a passive diffusion. The addition of 5 mM PO4 in the incubation medium did not significantly influence Na transport. In contrast, Na concentration in the incubation medium largely modified the kinetics of PO4 uptake: increasing Na concentration enhanced PO4 uptake and decreased the apparent Km. In Hyp mice, Na uptake by BBM was identical to that observed in N mice, but PO4 uptake was decreased by half. Na concentration in the incubation medium similarly influenced PO4 uptake in N and Hyp mice, and the Km values at each concentration of Na were comparable in the two series of animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

[Site and mechanism of tubular Na-K-ATPase regulation by aldosterone].

Effects of adrenalectomy and acute administration of physiologic doses of aldosterone on the activity and the number of catalytic units of Na-K-ATPase, the biochemical equivalent of the sodium pump, were evaluated on single microdissected nephron segments. After adrenalectomy, Na-K-ATPase is markedly decreased in all rabbit nephron segments, except the proximal tubule. However, aldosterone administration restores Na-K-ATPase exclusively in the collecting tubule, where mineralocorticoid receptors have been localized. Aldosterone induces both the activity and the appearance of new catalytic units with a kinetics similar to that of the antinatriuretic action of the hormone. These results suggest that Na-K-ATPase might be a primary target for aldosterone action.

Aldosterone↗
PubMed1984

Specific activity of Na-K-ATPase after adrenalectomy and hormone replacement along the rabbit nephron.

Both aldosterone and dexamethasone are known to stimulate renal Na-K-ATPase activity although their action is restricted to specific nephron segments: the collecting tubule, the target site for mineralocorticoids, and the thick ascending limb and distal convoluted tubule, the target sites for glucocorticoids. As this stimulation by corticosteroids is very fast, we attempted to establish whether it occurs through de novo synthesis of new Na-K-ATPase units or by increasing the specific activity of the Na-K-ATPase units already present. For this purpose we studied the effects of aldosterone and dexamethasone on Na-K-ATPase specific activity in microdissected nephron segments from adrenalectomized rabbits. This specific activity was determined by the ratio of ATPase activity over the apparent number of catalytic units, as measured by specific 3H ouabain binding. In the proximal tubule, neither adrenalectomy nor steroid replacement altered Na-K-ATPase activity or the apparent number of catalytic sites. In other nephron segments, adrenalectomy reduced Na-K-ATPase activity and specific 3H ouabain binding concomitantly, and therefore left this enzyme's specific activity unaltered. In the cortical and outer medullary collecting tubules, 10 micrograms/kg aldosterone simultaneously restored both the activity and apparent number of catalytic units of Na-K-ATPase to their control levels, and therefore did not modify the specific activity of the pump. Conversely, 100 micrograms/kg dexamethasone increased Na-K-ATPase activity in the thick ascending limb and distal convoluted tubule without changing the apparent number of catalytic units.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenalectomy↗
PubMed1984

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

Animals↗
PubMed1984

Stimulation of Na-K-ATPase in the rat collecting tubule by two diuretics: furosemide and amiloride.

To test whether sodium availability controls the concentration of renal Na-K-ATPase, we evaluated the effect of chronic alterations in apical membrane sodium permeability in specific nephron segments on the maximal activity of Na-K-ATPase. For this purpose Na-K-ATPase activity was determined in nephron segments microdissected from rats treated continuously for 3-8 days with either furosemide or amiloride, two diuretics known to lower the apical permeability to sodium in the thick ascending limb and the collecting tubule, respectively. Unexpectedly, Na-K-ATPase activity was decreased neither in the thick ascending limb nor in the collecting tubule after administration of either drug. In fact, both diuretics paradoxically increased the pump activity by 60-150% in the collecting tubule. This stimulation of Na-K-ATPase activity was accompanied with an enhancement of the collecting tubule diameter. Stimulation of Na-K-ATPase was identical in the collecting tubule of diuretic-treated rats receiving spironolactone. These results suggest that Na-K-ATPase maximal activity is not controlled by sodium availability or by aldosterone under these conditions and that chronic administration of furosemide or amiloride induces Na-K-ATPase activity in the collecting tubule. This effect appears to be independent of aldosterone.

Adenosine Triphosphatases↗
PubMed1983

Short-term effect of aldosterone on renal sodium transport and tubular Na-K-ATPase in the rat.

The short-term effect of one single injection of aldosterone on the renal sodium transport on one hand, and the Na-K-ATPase activity on the other hand, was studied in chronic adrenalectomized rats. Sodium transport was estimated by clearances, and Na-K-ATPase was measured in microdissected fragments of the nephron, according to our microtechnique previously described. Five to eight days after adrenalectomy, only 30% of the initial enzyme activity was recovered in the cortical collecting tubule (CCT). Administration of aldosterone completely restored the ATP-ase activity within three hours. Adrenalectomy also curtailed by 20-45% the activity of other nephron segments but aldosterone had no stimulatory effect on them. Sodium-reabsorption also increased after the hormone injection, following the same time (0.5 less than t1/2 less than 1 h) and dose dependencies (0.8 less than K1/2 less than 0.9 micrograms/kg) as those observed for the enzyme activity in the CCT. It is concluded that the short-term stimulation of Na-K-ATPase in the collecting tubule, after an acute administration of aldosterone, may be responsible for the simultaneous increase in sodium transport.

Adenosine Triphosphatases↗
PubMed1983

Short-term effects of aldosterone and dexamethasone on Na-K-ATPase along the rabbit nephron.

Both glucocorticoids and mineralcorticoids stimulate the renal Na-K-ATPase. However, the exact site of their respective action is not precisely determined and it is still unknown whether these effects are cumulative or not. We studied the effects of dexamethasone and aldosterone on Na-K-ATPase activity in microdissected nephron segments from adrenalectomized rabbits. In proximal convoluted tubule (PCT) the enzyme activity was altered neither by adrenalectomy nor by any steroid replacement. In the medullary thick ascending limb of the loop of Henle (MAL) and the distal convoluted tubule (DCT), Na-K-ATPase activity decreased by 40% after adrenalectomy, and was restored to control level three hours after administration of dexamethasone (100 micrograms/kg) but not by aldosterone (up to 10 micrograms/kg). In the cortical (CCT) and medullary (MCT) collecting tubule the enzyme activity decreased by 75% after adrenalectomy but in contrast with the MAL and the DCT, these two segments were sensitive to both dexamethasone (100 micrograms/kg) and aldosterone (10 micrograms/kg) and recovered their activities within 3 h after the hormone injection. These effects were not additive. Spironolactone (100 micrograms/kg) abolished the action of each of the two hormones on the CCT and MCT. In contrast, spironolactone did not curtail the effect of dexamethasone on MAL and DCT. These results indicate that whereas glucocorticoid action is localized in MAL, DCT, CCT and MCT, the mineralocorticoid effect is restricted to the CCT and MCT exclusively. They also suggest that, in the CCT and MCT, the two types of hormones share the same receptors.

Adenosine Triphosphatases↗
PubMed1983

Changes in tubular basolateral membrane markers after chronic DOCA treatment.

Chronic administration of DOCA to rabbits is known to increase the surface area of the basolateral membrane and the Na-K-ATPase activity of the cortical collecting tubule (CCT). We attempted to ascertain 1) whether Na-K-ATPase is the only basolateral membrane marker induced by DOCA, and 2) whether CCT is the only nephron segment affected by this steroid. We measured the activity of Na-K-ATPase and adenylate cyclase (AC) and the protein content of nephron segments microdissected from control and DOCA-treated rabbits. Morphogenic effects of DOCA, assessed by 30-60% increases in protein content, were specifically observed in the distal convoluted tubule, CCT, and medullary collecting tubule. When expressed as a function of tubular length, Na-K-ATPase activity rose from 80 to 200% in all these segments, whereas the increments in AC of 40-70%, observed in response to four different hormones, occurred only in some of them. When expressed as a function of protein content, Na-K-ATPase activity increased but AC activity remained unchanged. This study indicates that the morphogenic action resulting from chronic DOCA administration affects the entire rabbit distal nephron. During this action Na-K-ATPase is the preferentially induced enzyme.

Adenosine Triphosphatases↗
PubMed1983

Na-K-ATPase in single nephron segments of hypertension-prone rats.

The activity of renal Na-K-ATPase was compared in hypertension-prone (SBH) and hypertension-resistant (SBN) Sabra rats on regular sodium intake and 2-3 weeks after a high sodium diet. ATPase activity was determined in single nephron segments by a micromethod. The activity profile was found to be similar in the two substrains on both regimens. Following high sodium intake there was a significant increment of Na-K-ATPase activity which was limited to the medullary thick ascending limb in the two substrains. The results clearly indicate a lack of relationship between renal Na-K-ATPase activity and proneness or resistance to hypertension in this experimental model.

Animals↗
PubMed1982

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

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.

Animals↗
PubMed1981

Short-term effect of aldosterone on Na-K-ATPase in single nephron segments.

One of the theories proposed to explain the action of mineralocorticoids on target tissues attributes their effect on cation transport to stimulation of the "sodium pump." To test this hypothesis we evaluated the short-term effect of aldosterone on Na-K-ATPase in discrete nephron segments microdissected from kidneys of adrenalectomized animals. Na-K-ATPase activity measured in proximal and distal nephron segments from adrenalectomized as well as intact mice remained unchanged 1 or 3 h after a single injection of D-aldosterone (10 micrograms/100 g body wt i.p.). Similarly, no effect of aldosterone could be demonstrated in experiments in vitro, in which tubules from adrenalectomized mice and rabbits were incubated (90 min, 37 degrees C) in the presence of physiologic or pharmacologic concentrations of the hormone (3.10(-10) to 3.10(-6) M). These studies did not show an effect of aldosterone on isolated tubule Na-K-ATPase within the time interval in which this hormone alters tubular cation transport and therefore do not support the "pump theory" of mineralocorticoid action.

Adrenalectomy↗
PubMed1981

Mineralcorticoid receptors along the nephron: [3H]aldosterone binding in rabbit tubules.

To identify the site of mineralocorticoid action along the nephron, we measured the specific binding of [3H]aldosterone to nephron segments microdissected from aldosterone-deficient rabbits. Specific binding was defined as the difference between binding measured in the absence or in the presence of 2,000-fold excess of unlabeled hormone (in 10(-18) mol X cm tubule length-1 +/- SE). High specific binding capacity was found in the branched collecting tubule (108 +/- 4), the cortical collecting tubule (119 +/- 9), and the outer medullary collecting tubule (115 +/- 16), whereas specific binding was negligible in the proximal convoluted tubule (8 +/- 9), pars recta (2 +/- 6), medullary thick ascending limb (4 +/- 6), cortical thick ascending limb (6 +/- 2), and distal convoluted tubule (6 +/- 6). In cortical collecting tubules, Scatchard analysis of the specific [3H]aldosterone binding indicated a dissociation constant (KD) of 2.2 X 10(-9) M and a maximum number of binding sites of 157 X 10(-18) mol X cm tubule length-1. The steroid specificity was assessed from the competition of various steroids for [3H]aldosterone binding sites. Receptors from the cortical collecting tubule revealed the following sequence of affinities: aldosterone greater than DOCA greater than spironolactone greater than dexamethasone greater than 5 alpha-dihydrotestosterone = progesterone = 17 beta-estradiol, indicating that the binding sites in the collecting tubule are mineralocorticoid receptors. These results demonstrate significant [3H]aldosterone binding to receptors of high affinity and mineralocorticoid specificity only in the collecting tubule and suggest that this nephron segment is the target site of mineralocorticoid action in the rabbit kidney.

Adrenalectomy↗
PubMed1980

Renal potassium adaptation: Na-K-ATPase activity along the nephron after chronic potassium loading.

To identify nephron sites where renal potassium adaptation takes place, Na-K-ATPase was measured with a micromethod along the nephron of potassium-loaded mice. A possible role of aldosterone in this process was evaluated in K-loaded animals treated concurrently with pharmacologic doses of spironolactone. Animals fed a potassium-enriched diet for at least 2 wk excreted about 90% of ingested potassium in the urine, and fractional potassium clearance averaged 87 +/- 8%, compared with 13 +/- 2% in controls. Na-K-ATPase activity per millimeter tubule length increased by 225% in the cortical collecting tubule and by 177% in the medullary collecting tubule, but was not substantially affected in other nephron segments. Stimulation of Na-K-ATPase was identical in the cortical collecting tubule of K-loaded mice treated with spironolactone. These results indicate that chronic potassium loading in the mouse results in an adaptive increase potassium loading in the mouse results in an adaptive increase in Na-K-ATPase in the collecting tubule and suggest that this nephron segment is the major site of potassium adaptation in this species. This effect appears to be independent of aldosterone.

Adenosine Triphosphatases↗
PubMed1979

Determination of Na-K-ATPase activity in single segments of the mammalian nephron.

A micromethod for the determination of Na-K-ATPase in discrete segments of nephrons from rabbit, rat, and mouse kidneys is described. To facilitate tubule microdissection, the kidneys were perfused with collagenase after it had been verified that collagenase had no effect on ATPase activity. Individual tubule segments were dissected under stereomicroscopic observation, exposed to a hypotonic environment followed by rapid freezing, and incubated in 1 microliter assay medium. Enzyme activity was determined by direct measurement of labeled inorganic phosphate release by the hydrolysis of [gamma-32P]ATP and was expressed as a function of tubule length. This method is technically simple enough to permit simultaneous measurement of the enzyme in large numbers of tubules and sufficiently sensitive to determine its activity in each region of the nephron. Correlation of Na-K-ATPase activity in single tubules with functional measurements obtained in the corresponding segment of the nephron with the perfused tubule or micropuncture techniques should help define the role of this enzyme in tubular ion transport.

Adenosine Triphosphatases↗
PubMed1979

Na-K-ATPase activity along the rabbit, rat, and mouse nephron.

Na-K-ATPase activity along the rabbit, rat, and mouse nephron was determined with a micromethod that measures directly labeled phosphate released by the hydrolysis of [gamma-32P]ATP. Na-K-ATPase activity was highest in the rat, intermediate in the mouse, and lowest in the rabbit nephron. With the exception of rabbit cortical thick ascending limb, the enzyme profile was similar in the three species: Na-K-ATPase activity per millimeter tubule length was highest in the distal convoluted tubule and thick ascending limb of Henle's loop, intermediate in the proximal convoluted tubule, and lowest in the pars recta and collecting tubule. The enzyme was present in the thin limbs of Henle's loop, but its activity was very low and measurements were close to the sensitivity limit of the method. Both the absolute activity and the fraction of the total enzyme represented by Na-K-ATPase were severalfold higher than in kidney homogenates. Finally, the Na-K-ATPase activity measured in certain segments of the rat and rabbit nephron in this study seems sufficient to account in theory for the active component of the net sodium transport found in the corresponding region of the nephron with either in vivo or in vitro single tubule microperfusion techniques.

Adenosine Triphosphatases↗