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F Jaisser

Publications and source records attributed to F Jaisser.

18 recordsLinked to original sources
PubMed1996

The rat distal colon P-ATPase alpha subunit encodes a ouabain-sensitive H+, K+-ATPase.

The functional properties and the pharmacological profile of the recently cloned cDNA colonic P-ATPase alpha subunit (Crowson, M.S., and Shull, G.E. (1992) J. Biol. Chem. 267, 13740-13748) were investigated by using the Xenopus oocyte expression system. Xenopus oocytes were injected with alpha subunit cRNAs from Bufo marinus bladder or rat distal colon and/or with beta subunit cRNA from B. marinus bladder. Two days after injection, K+ uptake was measured by using 86 Rb+ as a K+ surrogate, and pH measurements were performed by means of ion-selective microelectrodes. Co-injection of alpha and beta subunit cRNAs led to a large increase in 86Rb+ uptake, an intracellular alkalinization, and an extracellular medium acidification, as compared to alpha or beta injection alone. These results indicate that the colonic P-ATPase alpha subunit, like the bladder alpha subunit, acts as a functional H+,K+-ATPase, and that co-expression of alpha and beta subunits is required for the function. External K+ activation of the 86Rb+ uptake had a K1/2 of approximately 440 microM for the bladder isoform (consistent with the previously reported value (Jaisser, F., Horisberger, J.D., Geering, K., and Rossier, B.C. (1993) J. Cell. Biol. 123, 1421-1431) and a K1/2 of approximately 730 microM for the colonic isoform. Sch28080 was ineffective to reduce 86Rb+ uptake whereas ouabain inhibited the activity expressed from rat colon alpha subunit with a Ki of 970 microM when measured at the Vmax of the enzyme. We conclude that, when expressed in Xenopus oocytes, the rat colon P-ATPase alpha subunit encodes a ouabain-sensitive H+,K+-ATPase.

Animals↗
PubMed1995

Primary structure and functional expression of the mouse and frog alpha-subunit of the gastric H(+)-K(+)-ATPase.

The H(+)-K(+)-ATPase of the gastric parietal cells is responsible for the acidification of the stomach lumen. This heterodimeric protein belongs to the family of cation-translocating P-type ATPases, which includes the closely related Na(+)-ATPase. We have cloned the alpha-subunit cDNA of the Xenopus and murine gastric H(+)-K(+)-ATPase (alpha H-K). We have expressed Xenopus and murine alpha H-K along with the previously cloned gastric H(+)-K(+)-ATPase beta-subunit of rabbit (beta H-K) in Xenopus oocytes by cRNA injection. An antibody directed against the beta H-K coimmunoprecipitates under nondenaturing conditions the alpha H-K of both species, demonstrating assembly of the alpha/beta complex. Additionally, we demonstrate the presence of K(+)-transporting H(+)-K(+)-ATPase in the plasma membrane of oocytes by 86Rb- uptake. The H(+)-K(+)-ATPase-mediated K+ uptake was inhibited by the gastric H(+)-K(+)-ATPase inhibitor Sch-28080, but not by ouabain, and shows K(+)-dependent activation (K1/2 approximately 2 mM). Furthermore, H(+)-K(+)-ATPase-expressing oocytes show a Sch-28080 inhibitable proton extrusion. Our data indicate that the expressed H(+)-K(+)-ATPase behaves functionally in oocytes as in the gastric gland.

Animals↗
PubMed1994

Phosphorylation of the Na,K-ATPase alpha-subunit by protein kinase A and C in vitro and in intact cells. Identification of a novel motif for PKC-mediated phosphorylation.

Na,K-ATPase is a potential target for regulatory phosphorylation by protein kinase A and C (PKA and PKC). To identify the phosphorylation sites, we have mutated the alpha 1-subunit of Bufo marinus in a highly conservative PKA and in 20 different PKC consensus sequences. The mutants were expressed in Xenopus oocytes and their phosphorylation capacity tested in homogenates upon stimulation of PKA or PKC. While serine 943 (Ser-943) was identified as a unique target site for PKA, none of the PKC consensus serine or threonine residues are implicated in PKC phosphorylation. Controlled trypsinolysis of phosphorylated alpha-subunits of various purified enzyme preparations and of alpha/beta complexes from oocyte homogenates revealed that PKC phosphorylation was exclusively associated with the N terminus. A fusion protein containing the first 32 amino acids of the Bufo alpha-subunit was phosphorylated in vitro and serine and threonine residues (Thr-15 and Ser-16) in this region were identified by site-directed mutagenesis as the PKC phosphorylation sites. Finally, the Bufo alpha-subunit was phosphorylated by protein kinases in transfected COS-7 cells. In intact cells, PKA stimulation induced phosphorylation exclusively on Ser-943 and PKC stimulation mainly on Thr-15 and Ser-16, which are contained in a novel PKC phosphorylation motif.

Amino Acid Sequence↗
PubMed1994

Modulation of the Na,K-pump function by beta subunit isoforms.

To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K-pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+.

Animals↗
PubMed1993

Primary sequence and functional expression of a novel beta subunit of the P-ATPase gene family.

The cortical collecting tubule (CCT) of the mammalian kidney reabsorbs sodium and potassium, processes that are mediated by Na/K-ATPase and H/K-ATPase. CCT is also an important site for proton secretion, which is driven, in part, by H/K-ATPase. Na/K-ATPase and H/K-ATPase are members of the ion-motive P-ATPase gene family. They are closely related plasma membrane proteins which consist of alpha beta heterodimers. The urinary bladder of the toad Bufo marinus is the amphibian counterpart of mammalian CCT. We have previously characterized a ouabain-resistant Na/K-ATPase [see ref. 17], from TBM cells, a clonal cell line derived from the toad bladder, which expresses transepithelial sodium transport. In the present study, we report the primary sequence and functional expression of a novel beta subunit (beta bladder = beta b1) isolated from a toad bladder epithelial cell cDNA library. The deduced polypeptide is 299 amino acids in length and has a predicted molecular mass of 33 kDa. The beta b1 protein exhibits 35% amino acid identity to the previously characterized beta 1 of B. marinus Na/K-ATPase and 39% identity with beta 3 of B. marinus Na/K-ATPase. It shares 38% identity with the mammalian beta gastric H/K-ATPase and 52% with the mammalian beta 2 Na/K-ATPase. Northern blot analysis shows that a 1.4 x 10(3)-base mRNA is expressed at a high level in bladder epithelial cells and eye and at a trace level in kidney; it is not detectable in significant amounts in the stomach, colon and small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1993

Mechanisms of urinary K+ and H+ excretion: primary structure and functional expression of a novel H,K-ATPase.

The kidney plays an essential role in regulating potassium and acid balance. A major site for these regulations is in the collecting tubule. In the present study, we report the primary sequence of a novel alpha subunit of the P-ATPase gene family, which we isolated from the urinary bladder epithelium of the toad Bufo marinus, the amphibian equivalent of the mammalian collecting tubule. The cDNA encodes a protein of 1,042 amino acids which shares approximately 67% identity with the alpha 1 subunit of the ouabain-inhibitable Na,K-ATPase and approximately 69% identity with the alpha subunit of the SCH28080-inhibitable gastric H,K-ATPase. When coexpressed in Xenopus oocytes with a beta subunit isolated from the same cDNA library, the ATPase is able to transport rubidium (a potassium surrogate) inward, and hydrogen outward, leading to alkalization of the intracellular compartment and acidification of the external medium. The novel ATPase has a unique pharmacological profile showing intermediate sensitivity to both ouabain and SCH28080. Our findings indicate that the bladder ATPase is a member of a new ion motive P-ATPase subfamily. The bladder ATPase is expressed in the urinary tract but not in the stomach or the colon. This H,K-ATPase may be one of the molecules involved in H+ and K+ homeostasis, mediating the transport of these ions across urinary epithelia and therefore regulating their urinary excretion.

Amino Acid Sequence↗
PubMed1993

Role of the transmembrane and extracytoplasmic domain of beta subunits in subunit assembly, intracellular transport, and functional expression of Na,K-pumps.

The ubiquitous Na,K- and the gastric H,K-pumps are heterodimeric plasma membrane proteins composed of an alpha and a beta subunit. The H,K-ATPase beta subunit (beta HK) can partially act as a surrogate for the Na,K-ATPase beta subunit (beta NK) in the formation of functional Na,K-pumps (Horisberger et al., 1991. J. Biol. Chem. 257:10338-10343). We have examined the role of the transmembrane and/or the ectodomain of beta NK in (a) its ER retention in the absence of concomitant synthesis of Na,K-ATPase alpha subunits (alpha NK) and (b) the functional expression of Na,K-pumps at the cell surface and their activation by external K+. We have constructed chimeric proteins between Xenopus beta NK and rabbit beta HK by exchanging their NH2-terminal plus transmembrane domain with their COOH-terminal ectodomain (beta NK/HK, beta HK/NK). We have expressed these constructs with or without coexpression of alpha NK in the Xenopus oocyte. In the absence of alpha NK, Xenopus beta NK and all chimera that contained the ectodomain of beta NK were retained in the ER while beta HK and all chimera with the ectodomain of beta HK could leave the ER suggesting that ER retention of unassembled Xenopus beta NK is mediated by a retention signal in the ectodomain. When coexpressed with alpha NK, only beta NK and beta NK/HK chimera assembled efficiently with alpha NK leading to similar high expression of functional Na,K-pumps at the cell surface that exhibited, however, a different apparent K+ affinity. beta HK or chimera with the transmembrane domain of beta HK assembled less efficiently with alpha NK leading to lower expression of functional Na,K-pumps with a different apparent K+ affinity. The data indicate that the transmembrane domain of beta NK is important for efficient assembly with alpha NK and that both the transmembrane and the ectodomain of beta subunits play a role in modulating the transport activity of Na,K-pumps.

Amino Acid Sequence↗
PubMed1993

A putative H(+)-K(+)-ATPase is selectively expressed in surface epithelial cells of rat distal colon.

Recently, a putative distal colon H(+)-K(+)-ATPase alpha-subunit has been identified and characterized (M. S. Crowson and G. E. Shull. J. Biol. Chem. 267:13740-13748, 1992). In the present study, we report the tissue and cell expression of this putative H(+)-K(+)-ATPase. The results indicate that, first, in the gut, the putative H(+)-K(+)-ATPase alpha-subunit is restricted to the distal part of the colon and is predominantly expressed in surface epithelial cells, in marked contrast to the alpha 1-subunit of Na(+)-K(+)-ATPase that is also expressed in glands. These data suggest that the H(+)-K(+)-ATPase alpha-subunit is a potential marker for terminal differentiation of distal colon. Second, in the uterus, the putative H(+)-K(+)-ATPase is restricted to the region of the myometrium between the inner and midmuscular zone that is very rich in vascular supply and nerve cells. This striking expression suggests that the H(+)-K(+)-ATPase may not be involved in the control of pH and potassium concentration of the uterine fluid but rather in distinct functions of vascular and/or nerve cells. Third, with the use of three independent and different approaches (Northern blot analysis, ribonuclease protection assay, and in situ hybridization), we were unable to detect any significant amount of H(+)-K(+)-ATPase transcripts in kidney tissue. Our data suggest that the putative distal colon H(+)-K(+)-ATPase is probably distinct from the kidney isoform. Finally, we report the sequence of a set of degenerate oligonucleotides that are useful to clone alpha-subunits of the Na(+)-K(+)-/H(+)-K(+)-ATPase gene family in different tissues and different species.

Animals↗
PubMed1993

Mutation of a conserved proline residue in the beta-subunit ectodomain prevents Na(+)-K(+)-ATPase oligomerization.

A highly conserved sequence motif (4 tyrosines and 1 proline: YYPYY) of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) beta 1-subunit ectodomain has been mutagenized to study its possible role in alpha/beta-assembly and sodium pump function. Single as well as double tyrosine mutants (tyrosine to phenylalanine: Y to F) of Xenopus laevis beta 1-subunits are able to associate with alpha 1-subunits and form functional Na-K pumps at the plasma membrane that are indistinguishable from wild-type alpha 1, beta 1-Na-K pumps (as assessed by measurements of ouabain binding, 86Rb flux, Na-K pump current, and activation by external potassium). In contrast, a single proline mutation (proline to glycine: P244G) reduced by > 90% the proper assembly and function of Na(+)-K(+)-ATPase, despite a normal rate of synthesis and core glycosylation. Our data indicate that proline-244 plays a critical role in the proper folding of the beta-subunit and its ability to associate efficiently with the alpha 1-subunit in the endoplasmic reticulum.

Animals↗
PubMed1992

The beta subunit modulates potassium activation of the Na-K pump.

We recently cloned the alpha 1 and the beta 1 and beta 3 subunits of the Na,K-ATPase of the toad Bufo marinus. To investigate possible functional differences between beta 1 and beta 3, we studied the potassium activation of Na-K pumps expressed in the oocyte of Xenopus laevis. Na-K pump activity was measured as K(+)-induced current in voltage-clamped oocytes. We could take advantage of the relative resistance to ouabain conferred by the Bufo alpha subunit to study specifically the exogenously expressed Na-K pumps after inhibition of the ouabain-sensitive endogenous Xenopus Na-K pumps. Coinjection of Bufo alpha 1 subunit cRNA with either beta 1 or beta 3 cRNAs results in the expression of functional Na-K pumps that share similar low ouabain sensitivity but differ in their K+ half activation constant (K1/2). Similar results were obtained with Xenopus alpha 1 and beta 1 or beta 3 subunits and with Bufo/Xenopus heterodimers. We conclude that some specific sequence of the beta subunit can influence the activation of the Na,K pump by extracellular K+ ions.

Animals↗
PubMed1992

Primary sequence and functional expression of a novel ouabain-resistant Na,K-ATPase. The beta subunit modulates potassium activation of the Na,K-pump.

In order to understand the molecular mechanism of ouabain resistance in the toad Bufo marinus, Na,K-ATPase alpha and beta subunits have been cloned and their functional properties tested in the Xenopus laevis oocyte expression system. According to sequence comparison between species, alpha 1, beta 1, and beta 3 isoforms were identified in a clonal toad urinary bladder cell line (TBM 18-23). The sequence of the alpha 1 isoform is characterized by two positively charged amino acids (Arg, Lys) at the N-terminal border of the H1-H2 extracellular loop and no charged amino acid at the C terminus, a pattern distinct from the ouabain-resistant rat alpha 1 isoform. The coexpression of alpha 1 beta 1 or alpha 1 beta 3 TBM subunits in the Xenopus oocyte resulted in the expression of identical maximum Na,K-pump currents with identical inhibition constant for ouabain (Ki) (alpha 1 beta 1: 53 +/- 3 microM; n = 7 vs. alpha 1 beta 3: 57 +/- 3.0 microM; n = 8) but distinct potassium half activation constant (K1/2) (alpha 1 beta 1: 0.87 +/- 0.08 mM, n = 16; alpha 1 beta 3: 1.29 +/- 0.07 mM, n = 17; p less than 0.005). We conclude that (i) the TBM alpha 1 isoform is necessary and sufficient to confer the ouabain resistant phenotype; (ii) the beta 3 or beta 1 subunit can associate with the alpha 1 equally well without affecting the ouabain-resistant phenotype; (iii) some specific sequence of the beta subunit can modulate the activation of the Na,K-pump by extracellular potassium ions.

Amino Acid Sequence↗
PubMed1991

Intestinal absorption of calcium from yogurt in lactase-deficient subjects.

Fractional intestinal absorption of calcium (FACa) was measured using radioactive calcium and 200 mg of calcium carrier provided either by yogurt or by CaCl2 in 7 lactase-deficient (L(-] and 7 normal (L(+] subjects. During the control period prior to yogurt consumption, mean calcium intake was 819 mg per day in L(-) and 931 mg per day in L(+) subjects (NS). In both groups of subjects yogurt increased FACa from 20.8 +/- 3.9% to 26.9 +/- 7.2% (P = 0.065) in L(+) subjects and from 20.2 +/- 5.6% to 23.5 +/- 6.4% (P = 0.050) in L(-) subjects. The significant increase in FACa observed in L(-) subjects indicates that yogurt, which is an autodigesting source of lactose, does not impair calcium absorption. FACa increase could reflect the lower dietary calcium intake in L(-) subjects when compared with L(+) subjects, due to avoidance of milk and non-fermented dairy products which could cause intestinal discomfort. It is concluded that yogurt is a well-tolerated and efficient source of calcium in subjects with lactase deficiency.

Adult↗
PubMed1990

Effect of cell sodium on Na+/K(+)-ATPase-dependent sodium efflux in cortical collecting tubule of rabbits under different aldosterone status.

Aldosterone increased the tubular volume in cortical collecting tubules (CCD) of rabbit kidney. It modulated the rate of cell sodium accumulation, under condition of ATPase inhibition (4 degrees C, in the absence of K+). In contrast, the relationship between Na+/K(+)-ATPase-dependent Na+ extrusion rate and intracellular Na+ concentration (Nai+) was similar in control, adrenalectomized, and aldosterone-treated adrenalectomized animals: Na+ extrusion rate increased with Nai+, up to 70 mM Nai+, and then plateaued. This indicates that aldosterone does not modify the characteristics of Nai(+)-dependent Na+ extrusion rate by the Na+/K(+)-ATPase pump in CCD.

Adrenalectomy↗
PubMed1989

Antidiuretic hormone reduces the high PGE2 synthesis in papillary collecting duct of DI rats.

PGE2 synthesis was measured along the nephron of Brattleboro (DI) rats, lacking ADH, and control LE rats, using an enzyme immunoassay. Experiments were performed in vitro, in the absence of exogenous arachidonic acid, using microdissected tubular segments. The effect of a chronic treatment of dDAVP was tested on three ADH sensitive tubular segments, medullary thick ascending limb (MTAL), medullary collecting tubule (OMCD) and papillary collecting duct (IMCD). No difference in PGE2 synthesis was present between LE and DI in glomerulus and tubular segments up to OMCD. In both strains, values were low in the proximal tubule and the loop of Henle, and gradually increased along the collecting tubule. In IMCD, PGE2 synthesis was much higher in DI (12.8 +/- 2.0 pg per 30 min per mm tubular length) than in LE (3.8 +/- 0.5, LE vs. DI p less than 0.001). In MTAL and OMCD, dDAVP treatment did not affect PGE2 synthesis. In IMCD, dDAVP reduced PGE2 synthesis to values (5.3 +/- 0.8 pg per 30 min per mm tubular length), which were not significantly different from those of LE. Neither oxytocin, which has been shown to be elevated in DI rats, nor furosemide, that reduced papillary osmolarity to values comparable to those of DI rats, were able to increase PGE2 synthesis in IMCD of LE rats. The mechanism of the increase in PGE2 synthesis in IMCD of DI rats, and of the inhibitory effect of dDAVP is yet unknown; it may participate to compensate for the lack of ADH in the Brattleboro rat.

Animals↗
PubMed1989

Effects of AVP and dDAVP on PGE2 synthesis in superfused cortical collecting tubules.

Whereas interactions between antidiuretic hormone (ADH) and prostaglandins (PGs) have been reported in the cortical collecting tubule (CCD), the precise effects of arginine vasopressin (AVP) and its analogue, 1-desamino-8-D-arginine vasopressin (dDAVP) on PGE2 synthesis remain controversial. We examined the dynamic response of PGE2 synthesis to these two analogues in isolated rabbit CCD. Microdissected CCD were superfused, and basal and hormone-induced PGE2 synthesis were determined by enzyme immunoassay. Addition of arachidonic acid (AA) steeply increased basal PGE2 synthesis, in the 0-1 microM-dose range. The presence of AA was necessary to obtain a stimulatory effect of AVP on PGE2 synthesis. AVP induced an immediate, transitory, and dose-dependent stimulation of PGE2 synthesis. A maximal effect was obtained at 10(-8) M; PGE2 synthesis was increased by approximately 150-200% over the basal synthesis. With dDAVP, a very weak response was obtained only at 10(-7) M. From these results, we conclude that PGE2 synthesis in CCD is stimulated by ADH. This effect of ADH does not depend on the V2-receptor pathway and suggests the presence of V1-receptors in CCD.

Animals↗
PubMed1989

Serum beta 2 microglobulin and extracellular fluid volume during haemodialysis.

Conflicting results have been published concerning serum beta 2 microglobulin (beta 2-M) kinetics during dialysis with a cuprophane membrane which is not permeable for the protein. We have investigated the hypothesis that the apparent increase of free serum beta 2-M could result from extracellular fluid volume (ECV) contraction. Using inulin, ECV was measured before and 1 h after a dialysis session with a sodium dialysate concentration of 145 mmol/l. Dialysis was performed either with a cuprophane or a high-flux membrane. Transcellular water shift and changes in beta 2-M concentration were calculated from total body water changes (ultrafiltration) and ECV. ECV decreased from a predialysis value of 16.6 +/- 3.51 (mean +/- SD) (24.9% bodyweight) to a postdialysis value of 11.9 +/- 2.11 (19.8% bodyweight). Ultrafiltration was only 3.1 +/- 1.01, indicating concomitant water shift from ECV to intracellular fluid space. A significant decrease in corrected beta 2-M concentration was found for high-flux membranes. However, postdialysis beta 2-M did not change significantly after dialysis with cellulosic membranes. In conclusion, the apparent increase of serum beta 2-M concentration measured during dialysis with cellulosic membranes may be explained by ECV contraction. These results have to be taken into account for any pathogenic mechanism of the beta 2-M-associated amyloidosis occurring in long-term haemodialysed patients.

Amyloidosis↗
PubMed1988

Na+-K+-ATPase-dependent sodium flux in cortical collecting tubule.

The instantaneous rate of efflux of intracellular Na was studied in rabbit isolated cortical collecting tubules (CCT) as a function of temperature and intracellular Na concentration ([Na]i). [Na]i of microdissected CCT was increased by cold and K-free exposure in the presence of 22Na and the extracellular tracer [3H] sorbitol. [Na]i rose rapidly to 40 mM at 30 min, after which it rose more slowly, reaching 120-140 mM at 6 h. Kinetics of Na efflux were studied after rapid rewarming, using a special device allowing measurements at 20-s intervals. Under control conditions, the total Na load was extruded in less than 8 min, whereas, in the presence of 10(-4) M ouabain, only 50% of the load was extruded during this period of time. Ouabain-sensitive Na efflux was first evident at 13 degrees C and gradually increased between 13 and 35 degrees C. At 37 degrees C, Na+-K+-ATPase-dependent Na efflux was dependent on [Na]i. This efflux gradually increased, from 0.05 to 0.5 peq.nl tubular volume-1.s-1 as a function of [Na]i and reached a plateau at 70 mM [Na]i. It is concluded that [Na]i is a major modulator of the pump activity in CCT; at normal levels of [Na]i, the pump is operating at only a small fraction of its total capacity.

Animals↗