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Biomedical subjects

E Delpire

Publications and source records attributed to E Delpire.

At least 37 records · Page 2Linked to original sources
PubMed1996

Pentagastrin selectively modulates levels of mRNAs encoding apical H/K adenosine triphosphatase and basolateral Na-K-Cl cotransporter in rat gastric fundic mucosa.

BACKGROUND: Gastrin regulates gastric acid secretion and gastric mucosal cell proliferation. We hypothesized that pentagastrin administration would affect mRNA levels of two membrane proteins that are important during stimulated states of HCl secretion, the basolateral Na-K-Cl cotransporter (BSC) and the apical H/K adenosine triphosphatase (H/K). METHODS: Two groups of Fischer rats received intraperitoneal injections of pentagastrin (2.5 or 25 micrograms/kg) every 8 hours for three doses. A third group served as controls. An additional group received pentagastrin plus the gastrin receptor antagonist (GRA) L740,093. Fundic mucosae were subjected to semiquantitative Northern analysis of mRNAs encoding H/K and BSC. The mRNA for Na/K adenosine triphosphatase (Na/K), a transport protein not involved directly in acid secretion, also was evaluated. RESULTS: Administration of pentagastrin caused dose-dependent increases in levels of mRNAs encoding H/K and BSC but had no significant effect on levels of Na/K mRNA. Administration of GRA prevented the pentagastrin-induced changes in mRNA levels for these transporters. CONCLUSIONS: Pentagastrin administration selectively up-regulates levels of mRNA encoding membrane proteins involved in acid secretion. The up-regulation of the mRNAs encoding BSC during pentagastrin stimulation indicates that regulation of basolateral Cl- movement may be as important as the regulation of apical H+ movement under stimulated states.

Acids↗
PubMed1996

The Na-(K)-Cl cotransporter family in the mammalian kidney: molecular identification and function(s).

A new solute carrier gene family, SLC12, was recently described based on the molecular identification of three electroneutral Na-(K)-Cl cotransport proteins. In mammals, these proteins are encoded by three distinct but related genes: SLC12A1, SLC12A2, SLC12A3, which are located on different chromosomes. Although the expression patterns of these three cotransport proteins differ significantly, all of them are expressed in the mammalian kidney and participate in several important aspects of renal function. This review summarizes the information learned from the molecular identification of these cotransporters, evaluates the patterns of expression within the kidney, and discusses the roles that these cotransporters play in renal physiology and pathophysiology.

Animals↗
PubMed1996

Molecular mechanisms of NaCl cotransport.

Electroneutral Na-(K)-Cl cotransporters are present in most cell types, where they play an important role in both sodium-chloride absorption and secretion and cell volume regulation. Recent advances in the molecular identification of these cotransporters have provided a new level of insight into the mechanisms of sodium-chloride-coupled cotransport and its regulation. Here we review what is known about the Na-(K)-Cl cotransporters cloned to date and what can be deduced about their structure and function and summarize recent physiological investigations of the regulation of Na-(K)-Cl cotransport. These studies represent the beginning of an exciting and rapidly expanding field examining the molecular mechanisms of sodium-chloride-coupled cotransport.

Animals↗
PubMed1995

Role of basolateral Na(+)-K(+)-Cl- cotransport in HCl secretion by amphibian gastric mucosa.

In amphibians and mammals, luminal H+ secretion by the stomach requires Cl-. It is widely accepted that a basolateral Cl-/HCO3- exchanger in the acid-secreting oxyntic cell restores the Cl- deficit resulting from apical HCl secretion. In this study, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to generate a 1,200-bp fragment specific to a basolateral isoform of the Na(+)-K(+)-Cl- cotransporter in the gastric fundus of Necturus maculosus. By Northern analysis, we observed that gastric mucosa expresses greater than fivefold higher levels of mRNA encoding this cotransporter than any other tissue in the gastrointestinal tract. Feeding results in > 100% increases in mRNA levels in acid-secreting fundic mucosa but does not alter mRNA levels in the neighboring and non-acid-secreting antral mucosa or duodenum. Flux measurements using in vitro fundic mucosae indicate that acid secretion requires Na+ in the nutrient (i.e., serosal side) perfusate, is modulated by changes in nutrient K+ levels, and is inhibited by nutrient solutions containing 50 microM bumetanide, a recognized blocker of Na(+)-K(+)-Cl- cotransport. These findings suggest that this basolateral transporter plays a dominant and previously unsuspected role in secretion of HCl across the apical membrane.

Amino Acid Sequence↗
PubMed1994

Molecular cloning and chromosome localization of a putative basolateral Na(+)-K(+)-2Cl- cotransporter from mouse inner medullary collecting duct (mIMCD-3) cells.

Electroneutral Na(+)-K(+)-2Cl- cotransporters represent one of the major routes for Cl- movement in epithelia. A secretory form of the cotransporter has been described in the basolateral membrane of a variety of epithelia from fish to mammals. We isolated a putative bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter cDNA, BSC2, from mIMCD-3 cells. Northern analysis indicates that in contrast to BSC1, the recently cloned renal-specific apical isoform of the cotransporter, BSC2 is expressed in secretory epithelia and thus appears to represent the basolateral isoform. Furthermore, BSC2 is also expressed in non-polarized cells, such as red cells and myocytes. Sequence comparison and chromosome localization demonstrate that BSC2 and BSC1 are different genes that diverged before the evolution of vertebrates.

Amino Acid Sequence↗
PubMed1994

Cell volume and K+ transport during differentiation of mouse erythroleukemia cells.

In the present study, we evaluated the changes in cell volume, water content, and K+ transport in mouse erythroleukemia (MEL) cells during the transition from proerythroblast to young reticulocyte. When MEL cells were exposed to 1.8% dimethyl sulfoxide (DMSO) for a maximum of 7 days, they synthesized hemoglobin and reduced their volume by 66% while maintaining their water content. The total protein content decreased by 50%. We therefore concluded that the volume reduction was due to a loss of cellular material, water, and osmolytes. To evaluate the changes in pump and leak pathways, we performed 86Rb uptakes in the presence or absence of selected inhibitors. In undifferentiated cells, the uptake was mainly represented by the Na-K-2Cl cotransport (51%) and by the Na(+)-K+ pump (34%). A small portion of the uptake was mediated by barium- and quinidine-sensitive K+ channels (8%) and by the furosemide-sensitive K-Cl cotransporter (5%). After 4 days in DMSO, the 86Rb uptake was reduced by 57%, mainly due to a substantial (90%) decrease in Na-K-2Cl cotransport activity. The Na(+)-independent K-Cl cotransport activity also dramatically decreased by a factor of 10. In contrast, the Na(+)-K+ pump activity did not change after 4 days in DMSO. These results demonstrate a marked reduction in the activities of inorganic ion cotransport systems as red blood cells differentiate to reticulocytes. Our study also demonstrates that a strong correlation exists between cell volume reduction and a decrease in the main inward leak pathway for K+: the Na-K-2Cl cotransporter.

Animals↗
PubMed1994

Molecular characterization of ROSIT, a renal osmotic stress-induced Na(+)-Cl(-)-organic solute cotransporter.

A gene family encoding many Na(+)- and Cl(-)-dependent organic solute cotransporters has recently been recognized. Among the cotransporters that have been characterized are those for neurotransmitters, amino acids, and organic osmolytes. Using polymerase chain reaction and mRNA derived from renal cortex, we isolated a new member of this gene family, ROSIT, a renal osmotic stress-induced transporter. The cDNA is 2,354 bp long with an open reading frame of 1,845 bp. The 615 deduced amino sequence shows ROSIT to be most clearly related to two orphan cDNAs of this family isolated from brain. Northern analysis showed the mRNA is normally expressed in renal cortex but not in brain, heart, colon, liver, stomach, or skeletal muscle. Moreover, hypernatremic rats displayed a marked increase in mRNA levels in renal cortex, renal outer medulla, and perhaps intestine. Heterologous expression of the cRNA in Xenopus laevis oocytes failed to reveal the function of this gene product when analyzed with isotope fluxes or electrophysiological measurements using a wide variety of organic solutes. Although its function remains unknown, ROSIT is likely to be involved in kidney reclamation of an organic osmolyte or osmolyte precursor required for adaptation to hypertonic stress.

Amino Acid Sequence↗
PubMed1993

An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse.

The terminal inner medullary collecting duct (IMCD) plays an important role in determining the final urinary composition. Currently, there is no continuous cell line derived from this nephron segment. We have developed a cell line derived from the terminal IMCD of mice transgenic for the early region of simian virus SV40 (large T antigen). This cell line, mIMCD-3, retains many differentiated characteristics of this nephron segment including high transepithelial resistance (1,368 +/- 172 omega.cm2), inhibition of apical-to-basal sodium flux by amiloride (41 +/- 7%) and by atrial natriuretic peptide (ANP) (40 +/- 9%), the presence of the amiloride-sensitive sodium channel as determined by Western blot analysis, and accumulation of the major organic osmolytes in response to hypertonic stress. Significantly, mIMCD-3 cells adapted readily and were able to grow in hypertonic medium supplemented with NaCl and urea up to 910 mosmol/kgH2O. These extreme osmotic conditions exist in the renal medulla in vivo but are known to be lethal to most other cells. This cell line should be highly useful for the study of the cellular adaptation to osmotic stress and the cell biology and transport physiology of this nephron segment.

Adaptation, Physiological↗
PubMed1992

Kinetics of DIDS inhibition of swelling-activated K-Cl cotransport in low K sheep erythrocytes.

The inhibitory effect of various stilbene disulfonates was examined on the swelling-activated Cl-dependent K transport (K-Cl cotransport) in low K sheep erythrocytes. Both diisothiocyanatostilbenes H2DIDS and DIDS were found to be potent inhibitors. The DIDS concentration yielding 50% inhibition (IC50) of KCl cotransport was 60 microM in the absence of external K and 3 microM at physiological K concentration. Other stilbene derivatives, such as SITS (4-acetamido-4' isothiocyanatostilbene-2,2'-disulfonic acid), were only effective in the presence of external K, whereas DNDS (4,4'-dinitrostilbene-2,2'-disulfonic acid) and ISA (4-sulfophenyl isothiocyanate) had only slight effects at a concentration of 1 mM. The augmenting effect of external K is due to a second K site, distinguishable from the K transport site by its much higher affinity. No inhibition occurred in the absence of external Cl, whether or not external Rb(K) was present. Additionally, DIDS inhibited K-Cl cotransport activated by thiol alkylation with N-ethylmaleimide (NEM) as well as by Mg depletion in the presence of A23187 and a chelator. We conclude that allosteric sites affect the stilbene binding. When these sites are saturated, changes in external K or Cl concentration do not affect the affinity for DIDS (noncompetitive inhibition).

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Erythrocyte K-Cl cotransport: properties and regulation.

Erythrocytes possess a Cl-dependent, Na-independent K transport system cotransporting K and Cl in a 1:1 stoichiometry that is membrane potential independent. This K-Cl cotransporter is stimulated by cell swelling, acidification, Mg depletion, and thiol modification. Cell shrinkage, elevation of cellular divalent ions, thiol alkylation, phosphatase inhibitors, and derivatives of certain loop diuretics and stilbenes are inhibitory. Thus regulation of K-Cl cotransport at the membrane and cytoplasmic levels is highly complex. Basal K-Cl cotransport decreases with cellular maturation, whereas its modes of stimulation and inhibition are variable between species. The physiological inactivation appears to be prevented in low-K animal erythrocytes. In certain human hemoglobinopathies, K-Cl cotransport may be the cause of cellular dehydration and volume decrease. K-Cl cotransport occurs also in nonerythroid cells, such as in epithelial and liver cells of other species. At the threshold of molecular characterization, this comprehensive review places our present understanding of the mechanisms modulating K-Cl cotransport physiologically and pathophysiologically into kinetic and thermodynamic perspectives.

Animals↗
PubMed1991

Trans effects of cellular K and Cl on ouabain-resistant Rb(K) influx in low K sheep red blood cells: further evidence for asymmetry of K-Cl cotransport [corrected].

The electroneutral K-Cl cotransport in low K+ (LK) sheep red blood cells is kinetically asymmetric and thermodynamically outward poised (Delpire and Lauf, 1991a). We have shown previously by trans-inhibition kinetics that Cl- binds prior to K+ to the outside configuration of the carrier. In the present study, we confirm that K+ and Cl- bind randomly to the cytoplasmic aspect of the transporter because K+ in the absence of Cl-, and Cl- in the absence of K+, trans-inhibit ouabain-resistant Rb+(K+) influx in these cells. In contrast to the trans-inhibition pattern observed outside, neither K+ nor Cl- trans-inhibit K+(Rb+) influx in the presence of the cotransported ion, further supporting the asymmetry of the K-Cl cotransporter.

Animals↗
PubMed1991

Kinetics of Cl-dependent K fluxes in hyposmotically swollen low K sheep erythrocytes.

A detailed kinetic study of K:Cl cotransport in hyposmotically swollen low K sheep red blood cells was carried out to characterize the nature of the outwardly poised carrier. The kinetic parameters were determined from the rate of K efflux and influx under zero-K-trans conditions in red cells with cellular K altered by the nystatin method and with different extracellular K or Rb concentrations. Although apparent affinities for efflux and influx were quite similar, the maximal velocity for K efflux was approximately two times greater than for influx. Furthermore, at thermodynamic equilibrium (i.e., when the ion product of K and Cl within the cell was equal to that outside) a temperature-dependent net K efflux was observed, approaching zero only when the external product reached approximately two times the internal product. The binding order of the ions to the transporter was asymmetric, being ordered outside (Cl binding first, followed by K) and random inside. K efflux but not influx was trans-inhibited by KCl. Trans inhibition of K efflux was used to verify the order of binding outside: trans inhibition by external Cl occurred in the absence of external K, but not vice versa. Thus K:Cl cotransport is kinetically asymmetric in hyposmotically swollen low K sheep red cells.

Animals↗
PubMed1991

Magnesium and ATP dependence of K-Cl co-transport in low K+ sheep red blood cells.

1. In low K+ (LK) sheep red blood cells, depletion of adenosine triphosphate (ATP) by glycolysis inhibition induced specific effects on ouabain-resistant Cl(-)-dependent K+ transport (K-Cl co-transport), depending on the osmolarity: stimulation in isosmotic while inhibition in hyposmotic solutions. However, these effects depended upon the presence of internal Mg2+. 2. In LK sheep red blood cells, ATP constituted nearly 90% of the Mg2+ buffering capacity. As no significant reduction of total Mg2+ was observed after ATP depletion, the overall internal Mg2+ in ATP-depleted cells exists in the free form. 3. The dependence of K+ efflux on internal Mg2+ was also directly related to the presence of ATP. In control cells, Mg2+ constituted an endogenous inhibitor, inducing a 70% inhibition of K-Cl fluxes but only 30% in ATP-depleted cells. The Cl(-)-insensitive component of K+ efflux was unaffected by the divalent cation. 4. After Mg2+ removal, the rate of K+ efflux was significantly increased at all osmolarities, between 240 mosM (swollen cells) and 440 mosM (shrunken cells). Hence, Mg(2+)-depleted LK sheep red cells lose volume sensitivity of K-Cl co-transport. 5. Internal K+ or Cl- were not required for the Mg2+ inhibition, and Mg2+ did not interfere with the internal binding sites for Cl- or K+. Hence, the sites for Mg2+ or MgATP, and for K+ and Cl- are independent of each other.

Adenosine Triphosphate↗
PubMed1991

Volume regulation in rat pheochromocytoma cultured cells submitted to hypoosmotic conditions.

The mechanisms at work in cell volume regulation have been studied in PC12 cultured cells. Results show, for the first time to our knowledge, that the volume readjustment process occurring after application of a hypoosmotic saline is sensitive to amiloride, IBMX and forskoline. The process is also inhibited by quinine hydrochloride and trifluoperazine. Volume readjustment is concomtant with a decrease in K+ and Cl- intracellular levels. The decrease in K+ level can be related to an assymetrical change in the fluxes in and out of the ion as shown by flux kinetics studies using Rb86. These results are interpreted considering that the control of the activity of the ion channel pathways associated with volume readjustment in PC12 cells may implicate the Ca(2+)-calmodulin - cAMP system.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Glycine and other amino compounds prevent chromatin precipitation at physiological ionic strength.

Glycine, proline and taurine, when present in the range 0.1-0.60 M, inhibit chromatin precipitation by sodium chloride. Histone gel electrophoresis revealed that the linker histones H1 and H5 were largely depleted from the supernatant chromatin fraction at 0.2 M NaCl, while this depletion was absent in the presence of glycine. These observations are discussed in relation with the various factors which may be involved in the precipitation process.

Adrenal Gland Neoplasms↗
PubMed1988

Relations between cell volume control, microfilaments and microtubules networks in T2 and PC12 cultured cells.

The possible relations between cell volume, microfilaments and microtubules networks have been studied in cultured mice fibrosarcoma cells of line T2 and rat pheochromocytoma cells of line PC12. The obtained results show that: 1. Changes in volume induced by application of hypo-osmotic medium are concomitant with a modification in the organization of the microfilaments network as visualized by immunocytochemistry. The microtubules lattice is not affected in these conditions. 2. Disruption of the microfilaments network by cytochalasin B causes a significant decrease in cell volume in isosmotic conditions. It also deeply affects the volume regulation response of cells swollen in hypo-osmotic media. 3. Disruption of the microtubules lattice by colchicine has no effect on volume in isosmotic conditions nor on the volume regulation that follows application of hypo-osmotic shock. The possible role of microfilaments in cell volume control is discussed.

Actin Cytoskeleton↗