scholarly journals Functional differences between flounder and rat thiazide-sensitive Na-Cl cotransporter

2002 ◽  
Vol 282 (4) ◽  
pp. F599-F607 ◽  
Author(s):  
Norma Vázquez ◽  
Adriana Monroy ◽  
Elisa Dorantes ◽  
Rosario A. Muñoz-Clares ◽  
Gerardo Gamba
Keyword(s):  
Phorbol Esters ◽  
Xenopus Laevis Oocytes ◽  
Kinase C ◽  
Functional Differences ◽  
Low Concentrations ◽  
Biphasic Effect ◽  
Uptake Medium ◽  
Regulatory Properties ◽  
Menten Constant

The purpose of the present study was to determine the major functional, pharmacological, and regulatory properties of the flounder thiazide-sensitive Na-Cl cotransporter (flTSC) to make a direct comparison with our recent characterization of the rat TSC (rTSC; Monroy A, Plata C, Hebert SC, and Gamba G. Am J Physiol Renal Physiol 279: F161–F169, 2000). When expressed in Xenopus laevis oocytes, flTSC exhibits lower affinity for Na+ than for Cl−, with apparent Michaelis-Menten constant ( K m) values of 58.2 ± 7.1 and 22.1 ± 4.2 mM, respectively. These K m values are significantly higher than those observed in rTSC. The Na+ and Cl− affinities decreased when the concentration of the counterion was lowered, suggesting that the binding of one ion increases the affinity of the transporter for the other. The effect of several thiazides on flTSC function was biphasic. Low concentrations of thiazides (10−9 to 10−7 M) resulted in activation of the cotransporter, whereas higher concentrations (10−6 to 10−4 M) were inhibitory. In rTSC, this biphasic effect was observed only with chlorthalidone. The affinity for thiazides in flTSC was lower than in rTSC, but the affinity in flTSC was not affected by the Na+ or the Cl− concentration in the uptake medium. In addition to thiazides, flTSC and rTSC were inhibited by Hg2+, with an apparent higher affinity for rTSC. Finally, flTSC function was decreased by activation of protein kinase C with phorbol esters and by hypertonicity. In summary, we have found significant regulatory, kinetic, and pharmacological differences between flTSC and rTSC orthologues.

Characterization of transport mechanisms and determinants critical for Na+-dependent Pisymport of the PiT family paralogs human PiT1 and PiT2

2006 ◽  
Vol 291 (6) ◽  
pp. C1377-C1387 ◽  
Author(s):  
Pernille Bøttger ◽  
Susanne E. Hede ◽  
Morten Grunnet ◽  
Boy Høyer ◽  
Dan A. Klærke ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Divalent Cations ◽  
Maximal Activity ◽  
Xenopus Laevis Oocytes ◽  
Transport Function ◽  
Knockout Mutant ◽  
Uptake Medium ◽  
The Impact ◽  
First Time

The general phosphate need in mammalian cells is accommodated by members of the Pitransport (PiT) family ( SLC20), which use either Na+or H+to mediate inorganic phosphate (Pi) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na+-dependent Pi(NaPi) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with32Pias a traceable Pisource. For PiT1, the Michaelis-Menten constant for Piwas determined as 322.5 ± 124.5 μM. PiT2 was analyzed for the first time and showed positive cooperativity in Piuptake with a half-maximal activity constant for Piof 163.5 ± 39.8 μM. PiT1- and PiT2-mediated Na+-dependent Piuptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na+dependency patterns. However, only PiT2 was capable of Na+-independent Pitransport at acidic pH. Study of the impact of divalent cations Ca2+and Mg2+revealed that Ca2+was important, but not critical, for NaPitransport function of PiT proteins. To gain insight into the NaPicotransport function, we analyzed PiT2 and a PiT2 Pitransport knockout mutant using22Na+as a traceable Na+source. Na+was transported by PiT2 even without Piin the uptake medium and also when Pitransport function was knocked out. This is the first time decoupling of Pifrom Na+transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E55and E575are responsible for linking Piimport to Na+transport in PiT2.

Isolation and characterization of PKC-L, a new member of the protein kinase C-related gene family specifically expressed in lung, skin, and heart

1991 ◽  
Vol 11 (1) ◽  
pp. 126-133 ◽  
Author(s):  
N Bacher ◽  
Y Zisman ◽  
E Berent ◽  
E Livneh
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Gene Family ◽  
Phorbol Esters ◽  
Structural Features ◽  
Cos Cells ◽  
Isolation And Characterization ◽  
Kinase C ◽  
Human Epidermoid Carcinoma

We have isolated and characterized a new human cDNA, coding for a protein kinase, related to the protein kinase C (PKC) gene family. Although this protein kinase shares some homologous sequences and structural features with the four members of the PKC family initially isolated (alpha, beta I, beta II, and gamma), it shows more homology with the recently described PKC-related subfamily, encoded by the cDNAs delta, epsilon, and zeta. The transcript for this gene product, termed PKC-L, is most abundant in lung tissue, less expressed in heart and skin tissue, and exhibited very low expression in brain tissue. Thus, its tissue distribution is different from that described for other mammalian members of the PKC gene family, their expression being enriched in brain tissues. PKC-L is also expressed in several human cell lines, including the human epidermoid carcinoma line A431. The ability of phorbol esters to bind to and stimulate the kinase activity of PKC-L was revealed by introducing the cDNA into COS cells.

scholarly journals A growth factor-repressible gene associated with protein kinase C-mediated inhibition of adipocyte differentiation.

1988 ◽  
Vol 107 (1) ◽  
pp. 279-286 ◽  
Author(s):  
M Navre ◽  
G M Ringold
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Phorbol Esters ◽  
Adipocyte Differentiation ◽  
Fibroblast Growth ◽  
Kinase C

The conversion of determined adipoblasts to fully differentiated adipocytes requires appropriate environmental conditions. A strict dependence on cell confluence and a facilitation by glucocorticoid hormones have previously been described. We have found that agents that are capable of activating protein kinase C, such as basic fibroblast growth factor and phorbol esters, inhibit the differentiation of the adipogenic cell line TA1 without stimulating cell growth. Here we describe the sequence and characterization of a cDNA (clone 5) that detects an RNA, the expression of which is enhanced by glucocorticoids and increasing cell density. In contrast, activators of protein kinase C including basic fibroblast growth factor, phorbol esters, and synthetic diacylglycerols inhibit clone 5 gene expression. It appears that clone 5 expression is closely linked to environmental and hormonal factors that promote the differentiation of adipogenic cells.

scholarly journals 1,2-Diacylglycerols overcome cyclic AMP-mediated inhibition of phosphatidylcholine synthesis in GH3 pituitary cells

1990 ◽  
Vol 267 (1) ◽  
pp. 17-22 ◽  
Author(s):  
R N Kolesnick
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Phorbol Esters ◽  
Pituitary Cells ◽  
Dibutyryl Cyclic Amp ◽  
Kinase C ◽  
Low Concentrations ◽  
High Concentrations ◽  
Phosphatidylcholine Synthesis

Previous studies showed that phorbol esters and thyrotropin-releasing hormone (TRH) stimulated phosphatidylcholine synthesis via protein kinase C in GH3 pituitary cells [Kolesnick (1987) J. Biol. Chem. 262, 14525-14530]. In contrast, 1,2-diacylglycerol-stimulated phosphatidylcholine synthesis appeared independent of protein kinase C. The present studies compare phosphatidylcholine synthesis stimulated by these agents with inhibition via the cyclic AMP system. The potent phorbol ester phorbol 12-myristate 13-acetate (PMA, 10 nM) increased [32P]Pi incorporation into phosphatidylcholine at 30 min to 159 +/- 6% of control. The adenylate cyclase activator cholera toxin (CT; 10 nM) and the cyclic AMP analogue dibutyryl cyclic AMP (1 mM) abolished this effect. CT similarly abolished TRH-induced phosphatidylcholine, but not phosphatidylinositol, synthesis. This is the first report of inhibiton of receptor-mediated phosphatidylcholine synthesis by the cyclic AMP system. The 1,2-diacylglycerol 1,2-dioctanoylglycerol (diC8) also stimulated concentration-dependent phosphatidylcholine synthesis. DiC8 (3 micrograms/ml) induced an effect quantitatively similar to that of maximal concentrations of PMA and TRH, whereas a maximal diC8 concentration (30 micrograms/ml) stimulated an effect 3-4-fold greater than these other agents. CT decreased the effect of diC8 (3 micrograms/ml) by 80%. Higher diC8 concentrations overcame the CT inhibition. Similar results were obtained with dibutyryl cyclic AMP. Additional differences were found between low and high concentrations of diC8. Low concentrations of diC8 failed to induce additive phosphatidylcholine synthesis with maximal concentrations of PMA, whereas high concentrations were additive. Hence, low concentrations of 1,2-diacylglycerols appear to be regulated similarly to phorbol esters, and higher concentrations appear to act via a pathway unavailable to phorbol esters.

scholarly journals Functional Differences in Ionic Regulation between Alternatively Spliced Isoforms of the Na+-Ca2+ Exchanger from Drosophila melanogaster

1998 ◽  
Vol 111 (5) ◽  
pp. 691-702 ◽  
Author(s):  
Alexander Omelchenko ◽  
Christopher Dyck ◽  
Mark Hnatowich ◽  
John Buchko ◽  
Debora A. Nicoll ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Alternative Splicing ◽  
Xenopus Laevis Oocytes ◽  
Patch Clamp Technique ◽  
Inactive State ◽  
Functional Differences ◽  
Dependent Inactivation ◽  
Current Voltage ◽  
Alternatively Spliced ◽  
Regulatory Properties

Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster. These exchangers, designated CALX1.1 and CALX1.2, differ by five amino acids in a region where alternative splicing also occurs in the mammalian Na+-Ca2+ exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na+-Ca2+ exchange currents, where pipette Ca2+o exchanges for bath Na+i, were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na+i affinities and current–voltage relationships, significant differences were observed in their Na+i- and Ca2+i-dependent regulatory properties. Both isoforms underwent Na+i-dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na+i application. We observed a two- to threefold difference in recovery rates from this inactive state and the extent of Na+i-dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na+-Ca2+ exchange activity by Ca2+i, but their responses to regulatory Ca2+i differed markedly. For both isoforms, the application of cytoplasmic Ca2+i led to a decrease in outward exchange currents. This negative regulation by Ca2+i is unique to Na+-Ca2+ exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca2+ for all other characterized Na+-Ca2+ exchangers. For CALX1.1, Ca2+i inhibited peak and steady state currents almost equally, with the extent of inhibition being ≈80%. In comparison, the effects of regulatory Ca2+i occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced (≈20–40% inhibition). For both exchangers, the effects of regulatory Ca2+i occurred by a direct mechanism and indirectly through effects on Na+i-induced inactivation. Our results show that regulatory Ca2+i decreases Na+i-induced inactivation of CALX1.2, whereas it stabilizes the Na+i-induced inactive state of CALX1.1. These effects of Ca2+i produce striking differences in regulation between CALX isoforms. Our findings indicate that alternative splicing may play a significant role in tailoring the regulatory profile of CALX isoforms and, possibly, other Na+-Ca2+ exchange proteins.

Sign in / Sign up

Export Citation Format

Share Document