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Biomedical subjects

M Paillard

Publications and source records attributed to M Paillard.

At least 37 records · Page 2Linked to original sources
PubMed1999

Sleep disorders in children with blindness.

To evaluate the frequency and type of sleep disorders seen in blind children compared with matched controls, a 42-item questionnaire was used on 156 children (77 blind children) ranging from 3 to 18 years of age. A total of 17.4% of blind children reported sleeping less than 7 hours per night on weekdays compared with 2.6% of controls, with blind children awakening much earlier. Blind children had more sleep complaints, and 13.4% of blind subjects had daily episodes of involuntary sleepiness compared with 1.3% of controls. Blindness has an impact on sleep and alertness that adds to the primary disability.

Adolescent↗
PubMed1999

Kidney cortex cells derived from SV40 transgenic mice retain intrinsic properties of polarized proximal tubule cells.

BACKGROUND: We have developed a nontransformed immortalized mice kidney cortex epithelial cell (MKCC) culture from a mouse transgenic for a recombinant plasmid adeno-SV40 (PK4). Methods and Results. After 12 months in culture, the immortalized cells had a stable homogeneous epithelial-like phenotype, expressed simian virus 40 (SV40) T-antigen, but failed to induce tumors after injection in nude mice. Epithelium exhibited polarity with an apical domain bearing many microvilli separated from lateral domains by junctional complexes with ZO1 protein. The transepithelial resistance was low. A Na-dependent glucose uptake sensitive to phlorizin and a Na-dependent phosphate uptake sensitive to arsenate were present. Western blot analysis of membrane fractions showed that anti-Na-Pi antiserum reacted with a 87 kD protein. The Na/H antiporters NHE-1, NHE-2, and NHE-3 mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR). The corresponding proteins with molecular weights of 111, 81, and 75 kD, respectively, could be detected by Western blot and were shown to be functional. Parathyroid hormone (PTH) induced a tenfold increase in cAMP and reduced the Na-dependent phosphate uptake and NHE-3 activity, as observed in proximal tubule cells. Isoforms alpha, delta, epsilon, and zeta of protein kinase C (PKC) were present in the cells. Angiotensin II (Ang II) elicited a translocation of the PKC-alpha toward the basolateral and apical domains. CONCLUSION: Thus, the MKCC culture retains the structural and functional properties of proximal tubular cells. To our knowledge, it is the first cell culture obtained from transgenic mice that exhibits the NHE-3 antiporter and type II Na-Pi cotransporter. MKCCs also display functional receptors for PTH and Ang II. Thus, MKCCs offer a powerful in vitro system to study the cellular mechanisms of ion transport regulation in proximal epithelium.

Angiotensin II↗
PubMed1999

Adaptation of NHE-3 in the rat thick ascending limb: effects of high sodium intake and metabolic alkalosis.

The present studies examined the effects of chronic NaCl administration and metabolic alkalosis on NHE-3, an apical Na+/H+ exchanger of the rat medullary thick ascending limb of Henle (MTAL). NaCl administration had no effect on NHE-3 mRNA abundance as assessed by competitive RT-PCR, as well as on NHE-3 transport activity estimated from the Na+-dependent cell pH recovery of Na+-depleted acidified MTAL cells, in the presence of 50 microM Hoe-694, which specifically blocks NHE-1 and NHE-2. Two models of metabolic alkalosis were studied, one associated with high sodium intake, i.e., NaHCO3 administration, and one not associated with high sodium intake, i.e., chloride depletion alkalosis (CDA). In both cases, the treatment induced a significant metabolic alkalosis that was associated with a decrease in NHE-3 transport activity (-27% and -25%, respectively). Negative linear relationships were observed between NHE-3 activity and plasma pH or bicarbonate concentration. NHE-3 mRNA abundance and NHE-3 protein abundance, assessed by Western blot analysis, also decreased by 35 and 25%, respectively, during NaHCO3-induced alkalosis, and by 47 and 33%, respectively, during CDA. These studies demonstrate that high sodium intake has per se no effect on MTAL NHE-3. In contrast, chronic metabolic alkalosis, regardless of whether it is associated with high sodium intake or not, leads to an appropriate adaptation of NHE-3 activity, which involves a decrease in NHE-3 protein and mRNA abundance.

Adaptation, Physiological↗
PubMed1999

Pathways for HCO-3 exit across the basolateral membrane in rat thick limbs.

We studied the pathways for HCO-3 transport in basolateral membrane vesicles (BLMV) purified from rat medullary thick ascending limbs (MTAL). An inward HCO-3 gradient in the presence of an inside-positive potential stimulated the rate of 22Na uptake minimally and did not induce a 22Na overshoot, arguing against the presence of electrogenic Na+-HCO-3 cotransport in these membranes. An inside-acid pH gradient stimulated to the same degree uptake of 86Rb+ (a K+ analog) with or without HCO-3. Conversely, applying an outward K+ gradient caused a modest intracellular pH (pHi) decrease of approximately 0.38 pH units/min, as monitored by quenching of carboxyfluorescein; its rate was unaffected by HCO-3, indicating the absence of appreciable K+-HCO-3 cotransport. On the other hand, imposing an inward Cl- gradient in the presence of HCO-3 caused a marked pHi decrease of approximately 1.68 pH units/min; its rate was inhibited by a stilbene derivative. Finally, we could not demonstrate the presence of a HCO-3/lactate exchanger in BLMV. In conclusion, the presence of significant Na+-, K+-, or lactate-linked HCO-3 transport could not be demonstrated. These and other data suggest that basolateral Cl-/HCO-3 exchange could be the major pathway for HCO-3 transport in the MTAL.

Acids↗
PubMed1999

Regulation by PKC isoforms of Na(+)/H(+) exchanger in luminal membrane vesicles isolated from cortical tubules.

The present study was designed to determine the Na/H exchanger isoforms present in luminal membrane vesicles (LMV) isolated from rat kidney cortical tubule suspensions, as well as the effects of acute phorbol ester (phorbol myristate acetate, PMA) and angiotensin II (ANG II) pretreatment of suspensions on NHE activity and protein kinase C (PKC) isoform abundance. In LMV, both NHE3 and NHE2 proteins were found by Western blot analysis, but only ethylisopropylamiloride-sensitive and almost completely Hoe-694-resistant Na/H exchange activity was observed from (22)Na uptake and thus attributed to NHE3. PMA pretreatment increased Na/H exchange activity and PKC isoforms alpha, delta, and epsilon abundance in LMV, and these effects were prevented by PKC inhibition. Low-dose ANG II (10(-11) M) pretreatment increased Na/H exchange activity and only PKC-zeta abundance in LMV, and these effects were also prevented by PKC inhibition. After high-dose ANG II (10(-7) M), Na/H exchange activity was decreased in LMV. PKC inhibition did not prevent this effect. In conclusion, the stimulating effects of PMA and low-dose ANG II are explained by the translocation of different isoforms of PKC in LMV, whereas the inhibitory effect of high-dose ANG II is not PKC dependent.

Angiotensin II↗
PubMed1999

Prevalence of sleep/wake disorders in persons with blindness.

Blind individuals are not only handicapped by their loss of vision, but are also affected because the loss of sight may have a secondary impact on functioning of their biological clock. The objective of the present study was to determine the impact of visual loss on sleep/wake disorders. A prospective 48-item questionnaire survey was distributed to blind individuals through the French Association Valentin Haüy, which serves blind persons. A control group matched by age, sex, geographical location and professional activity/non-activity was obtained from a panel of 20000 households representative of the French population, and this group also completed the questionnaire. From a potential blind population of 1500 subjects, 1073 questionnaires (71.5%) were completed and usable for analysis, and from a potential 1000 control subjects, 794 (79. 4%) of the questionnaires were returned and analysed. Criteria outlined in the Diagnostic and Statistical Manual of Mental Disorders, 4th revision, and the International Classification of Sleep/Wake Disorders (1990) were used to determine pathology. Individuals determined to be 'totally blind' and 'almost blind' (i.e. with less than 10% vision left in only one eye) presented a significantly higher occurrence of sleep/wake disorders than controls. Nocturnal sleep disruption, daytime somnolence, and (to a lesser degree) a 'free-running' condition are significantly more common in blind individuals. There is an increased use of sleeping pills, and a higher incidence of inappropriate involuntary daily naps. In conclusion, individuals with blindness report a significant curtailment of total sleep time and hence resulting daytime somnolence, which impacts on daytime activities. A 'free-running' condition is also a common sleep/wake impairment that may compound the handicap of blindness.

Adolescent↗
PubMed1998

[Primary hyperparathyroidism].

Primary hyperparathyroidism was initially regarded as a rare and severe disease. In recent years, introduction of routine screening of serum calcium has contributed to a dramatic increased rate of detection of primary hyperparathyroidism in the population, and asymptomatic forms of this disease are now the rule. Surgery remains the only curative treatment. However a medical follow-up may be justified in asymptomatic patients whose serum calcium levels are only midly elevated and whose renal and bone status are close to normal. The medical follow-up is considered to be safe only with conscientious long-term monitoring. Surgery becomes mandatory if the follow-up shows worsening hypercalcemia, bone deterioration, renal impairment, calcium stone, or increased hypercalciuria.

Female↗
PubMed1998

NH4+ as a substrate for apical and basolateral Na(+)-H+ exchangers of thick ascending limbs of rat kidney: evidence from isolated membranes.

1. We have used highly purified right-side-out luminal and basolateral membrane vesicles (LMVs and BLMVs) isolated from rat medullary thick ascending limb (MTAL) to study directly the possible roles of the LMV and BLMV Na(+)-H+ exchangers in the transport of NH4+. 2. Extravesicular NH4+ ((NH4+)o) inhibited outward H+ gradient-stimulated 22Na+ uptake in both types of vesicles. This inhibition could not be accounted for by alteration of intravesicular pH (pHi). 3. Conversely, in both plasma membrane preparations, the imposition of outward NH4+ gradients stimulated 22Na+ uptake at the acidic pHi (6.60) of MTAL cells, under conditions in which possible alterations in pHi were prevented. All NH4+ gradient-stimulated Na+ uptake was sensitive to 0.5 mM 5-(N,N-dimethyl)-amiloride. 4. The BLMV and LMV Na(+)-H+ exchangers had a similar apparent affinity for internal H+ (Hi+), with pK (-log of dissociation constant) values of 6.58 and 6.52, respectively. 5. These findings indicate that NH4+ interacts with the external and internal transport sites of the LMV and BLMV Na(+)-H+ antiporters, and that both of these exchangers can mediate the exchange of internal NH4+ ((NH4+)i) for external Na+ (Na+o) at the prevailing pHi of MTAL cells. 6. We conclude that operation of the BLMV Na(+)-H+ exchanger on the NH4(+)-Na+ mode may represent an important pathway for mediating the final step of NH4+ absorption, whereas transport of NH4+ on the apical antiporter may provide negative feedback regulation of NH4+ absorption.

Ammonia↗
PubMed1998

Long-term shake suspension and membrane vesicles of medullary thick ascending limb.

Cultured medullary thick ascending limb (MTAL) cells may lack some of the main carriers of fresh MTAL cells, such as apical Na+-K+(NH4+)-2Cl- cotransporter (BSC-1) and Na+/H+ exchanger (NHE-3). We have developed a technique to maintain rat MTALs several hours in suspension and in a good state of viability. Medullary thick ascending limbs were suspended in a 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's essential medium (HDMEM) supplemented with 25 mM HCO3- and gassed with 95% O2/5% CO2; the resulting mixture was placed in a rotary shaking water bath at 37 degrees C for 16 hours. As seen by electron microscopy, MTALs from the HDMEM-suspension retained a virtually normal tubular organization. Na+-K+(NH4+)-2Cl- cotransport activity and NHE consistent with both apical NHE-3 and basolateral NHE-1 activities were underscored both in intact cells by intracellular pH measurements and in a membrane fraction enriched in apical and basolateral membranes by 22Na+ uptake experiments. These results demonstrate that freshly harvested MTALs can be maintained in a well differentiated state for at least 16 hours; this preparation should make long-term in vitro studies of MTAL transport regulations possible.

Animals↗
PubMed1998

ANG II controls Na(+)-K+(NH4+)-2Cl- cotransport via 20-HETE and PKC in medullary thick ascending limb.

Cell pH was monitored in medullary thick ascending limbs to determine effects of ANG II on Na(+)-K+(NH4+)-2Cl- cotransport. ANG II at 10(-16) to 10(-12) M inhibited 30-50% (P < 0.005), but higher ANG II concentrations were stimulatory compared with the 10(-12) M ANG II level cotransport activity; eventually, 10(-6) M ANG II stimulated 34% cotransport activity (P < 0.003). Inhibition by 10(-12) M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase, or cytochrome P-450-dependent monooxygenase blockade; 10(-12) M ANG II had no effect additive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE). Stimulation by 10(-6) M ANG II was abolished by PLC and protein kinase C (PKC) blockade and was partially suppressed when the rise in cytosolic Ca2+ was prevented. All ANG II effects were abolished by DUP-753 (losartan) but not by PD-123319. Thus < or = 10(-12) M ANG II inhibits via 20-HETE, whereas > or = 5 x 10(-11) M ANG II stimulates via PKC Na(+)-K+(NH4+)-2Cl- cotransport; all ANG II effects involve AT1 receptors and PLC activation.

Angiotensin II↗
PubMed1998

Water and solute permeabilities of medullary thick ascending limb apical and basolateral membranes.

The medullary thick ascending limb (MTAL) reabsorbs solute without water and concentrates NH4+ in the interstitium without a favorable pH gradient, activities which require low water and NH3 permeabilities. The contributions of different apical and basolateral membrane structures to these low permeabilities are unclear. We isolated highly purified apical and basolateral MTAL plasma membranes and measured, by stopped-flow fluorometry, their permeabilities to water, urea, glycerol, protons, and NH3. Osmotic water permeability at 20 degrees C averaged 9.4 +/- 0.8 x 10(-4) cm/s for apical and 11.9 +/- 0.5 x 10(-4) cm/s for basolateral membranes. NH3 permeabilities at 20 degrees C averaged 0.0023 +/- 0.00035 and 0.0035 +/- 0.00080 cm/s for apical and basolateral membranes, respectively. These values are consistent with those obtained in isolated perfused tubules and can account for known aspects of MTAL function in vivo. Because the apical and basolateral membrane unit permeabilities are similar, the ability of the apical membrane to function as the site of barrier function arises from its very small surface area when compared with the highly redundant basolateral membrane.

Ammonia↗
PubMed1998

Functional and molecular characterization of luminal and basolateral Cl-/HCO-3 exchangers of rat thick limbs.

Cl-/HCO-3 exchange was measured in luminal (LMV) and basolateral (BLMV) membrane vesicles purified from rat medullary thick ascending limb (MTAL). Cl-/HCO-3 exchange in BLMV and LMV was inhibited by DIDS, with respective IC50 values of 3.2 +/- 0.9 and 15.2 +/- 5.2 microM, whereas Cl- conductances were DIDS insensitive. At constant external pH, BLMV 36Cl-/HCO-3 and 36Cl-/Cl- exchanges exhibited a sigmoidal pattern of activation as internal pH (pHi) increased from 6.1 to 8.0, whereas LMV 36Cl-/Cl- exchange was unchanged between pHi 6.7 and 7.8. The 165-kDa AE2 polypeptide and approximately 115-kDa AE1-related polypeptide were present only in BLMV. In contrast, AE1-related polypeptides of approximately 90 and 95 kDa were present not only in BLMV but also (in variable abundance) in LMV. We conclude that rat MTAL BLMV and LMV express distinct anion exchange activities and distinct sets of AE polypeptides. AE2 (and perhaps AE1) in BLMV likely contribute to HCO-3 absorption. In contrast, LMV exchangers may contribute to NaCl absorption via parallel coupling with the luminal Na+/H+ antiporters and/or may provide negative feedback regulation of HCO-3 absorption.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Immunolocalization of the Na+/H+ exchanger isoform NHE2 in rat kidney.

Four Na+/H+ exchangers (NHE1 to NHE4) have been detected in the kidney. Renal NHE2 expression sites have not been fully established. We have raised rabbit antisera against an oligopeptide related to the amino acids 652 to 661 of rat NHE2. Western blot analysis of plasma membrane fractions isolated from rat renal cortex showed that affinity-purified anti-NHE2 antibody detected an 85-kDa protein in apical but not in basolateral membranes. The labeling of this 85-kDa protein was specifically blocked by preincubation of the antibody with its monomeric peptide, indicating specific recognition. Indirect immunolabeling was performed on sections of paraformaldehyde-fixed rat kidney embedded in paraffin. Strong staining was seen in the apical membrane of cortical thick ascending limbs, distal convoluted tubules, and connecting tubules. Much weaker apical staining was found in medullary thick ascending limbs of Henle. In the inner medulla, some thin limbs were intensively labeled by the anti-NHE2 antibody. No staining could be detected in any segments of the proximal tubule and collecting duct.

Animals↗
PubMed1998

H+ and HCO3- transporters in the medullary thick ascending limb of the kidney: molecular mechanisms, function and regulation.

The H+ and HCO3- transporters present in the medullary thick ascending limb (MTAL) of the kidney are involved in several functions, such as transepithelial transport, defense of cell pH and cell volume. Apical H+ secretion occurs via the NHE-3 and NHE-2 isoforms of the Na+/H+ exchanger, and H(+)-ATPase. The apical Na+/H+ exchanger is responsible for most of the apical step of transepithelial HCO3- absorption and is unresponsive to cell acidification under isosmotic conditions. Basolateral HCO3- efflux mechanisms may occur via the Cl-/HCO3- exchanger and via the cotransporters K+/HCO3- (in the rat) and Na-3HCO3- (in the mouse). However, the role of each transporter in transepithelial HCO3- absorption is currently unknown. Inhibition of the basolateral Na+/H+ exchanger (NHE-1) paradoxically inhibits the apical Na+/H+ exchanger. This cross talk is independent of cell pH and may involve variations in cell volume. Arginine vasopressin (AVP) and hyperosmolality induce a differential regulation of basolateral NHE-1 and the apical Na+/H+ exchanger. They stimulate the basolateral NHE-1, and the resulting cell alkalinization probably stimulates the pHi-sensitive AE2, which restores cell volume by cellular uptake of NaCl. They also inhibit the apical Na+/H+ exchanger, which reduces net HCO3- absorption and thus may prevent interstitial fluid alkalinization. Chronic metabolic acidosis markedly increases HCO3- absorptive capacity of MTAL, by stimulating at least the synthesis of apical NHE-3 protein, as in the proximal tubule. Conversely, chronic metabolic alkalosis reduces the apical NHE-3 transport activity by decreasing the synthesis of NHE-3 protein. The paradoxical increase in HCO3- absorptive capacity of MTAL observed in the model of chronic NaHCO3-load alkalosis should be due to other factors overcoming the inhibitory effect of alkalosis on NHE-3.

Acid-Base Imbalance↗
PubMed1998

[Effect of angiotensin ii on Na+/H+ exchangers of the renal tubule].

Angiotensin II is a key element in regulating the volume of extracellular liquid. It acts indirectly through aldosterone secretion by adrenals and directly on the renal tubule too: It regulates luminal Na+/H+ antiporters (NHE3 and possibly NHE2) after binding to membrane AT1 receptors located both on the basolateral and on the apical side of the cells. The main site of Ang II action is proximal tubule, mainly the S1 segment which has a high level of AT1 receptors. Circulating Ang II concentrations (10(-12) to 10(-10) M), increased NaCl, water and NaHCO3 reabsorption via NHE3 in the proximal tubule. There is also a synthesis of Ang II within the cells of proximal tubule, which is secreted within the lumen where the physiological concentration is stable 10(-8) M, i.e. 100 to 1000 times higher than the circulating concentration. Luminal ANG II originating from kidney has a physiological autocrine function on NaCl, water and probably NaHCO3 reabsorption, since inhibiting Ang II synthesis, by conversion enzyme inhibition, or effect, by AT1 receptor antagonists, induces a reduction of proximal tubule reabsorption. The stimulatory effects of circulating and intrarenal Ang II seem to be explained by protein kinase C stimulation and possibly by a reduction of cAMP production or by a stimulation of a non-receptor tyrosine kinase. When pharmacological doses of Ang II (> 10(-8) M) are applied in the peritubular or the luminal medium of isolated microperfused proximal tubule in vitro, a paradoxical inhibition of NHE3 was observed. These effects appear to involve arachidonic acid metabolites through the cytochrome P450 pathway and possibly a rise in cytosolic free Ca++. The physiological significance of these supraphysiological effects are unknown.

Angiotensin II↗