scholarly journals Segmental localization of mRNAs encoding Na+-K+-ATPase α- and β-subunit isoforms in rat kidney using RT-PCR

1994 ◽  
Vol 46 (3) ◽  
pp. 627-638 ◽  
Author(s):  
William L. Clapp ◽  
Paula Bowman ◽  
Geraldine S. Shaw ◽  
Pinkal Patel ◽  
Bruce C. Kone
Keyword(s):  
Rat Kidney ◽  
Β Subunit ◽  
Rt Pcr ◽  
Subunit Isoforms

Identification of the β-subunit for nongastric H-K-ATPase in rat anterior prostate

2004 ◽  
Vol 286 (6) ◽  
pp. C1229-C1237 ◽  
Author(s):  
Nikolay B. Pestov ◽  
Tatyana V. Korneenko ◽  
Rossen Radkov ◽  
Hao Zhao ◽  
Mikhail I. Shakhparonov ◽  
...  
Keyword(s):  
Structural Organization ◽  
Anterior Lobe ◽  
Β Subunit ◽  
Rt Pcr ◽  
Α Subunit ◽  
Direct Demonstration ◽  
Subunit Isoforms ◽  
Anterior Prostate ◽  
Apical Membranes

The structural organization of nongastric H-K-ATPase, unlike that of closely related Na-K-ATPase and gastric H-K-ATPase, is not well characterized. Recently, we demonstrated that nongastric H-K-ATPase α-subunit (αng) is expressed in apical membranes of rodent prostate. Its highest level, as well as relative abundance, with respect to α1-isoform of Na-K-ATPase, was observed in anterior lobe. Here, we aimed to determine the subunit composition of nongastric H-K-ATPase through the detailed analysis of the expression of all known X-K-ATPase β-subunits in rat anterior prostate (AP). RT-PCR detects transcripts of β-subunits of Na-K-ATPase only. Measurement of absolute protein content of these three β-subunit isoforms, with the use of quantitative Western blotting of AP membrane proteins, indicates that the abundance order is β1 > β3 ≫ β2. Immunohistochemical experiments demonstrate that β1 is present predominantly in apical membranes, coinciding with αng, whereas β3 is localized in the basolateral compartment, coinciding with α1. This is the first direct demonstration of the αng-β1 colocalization in situ indicating that, in rat AP, αng associates only with β1. The existence of αng-β1 complex has been confirmed by immunoprecipitation experiments. These results indicate that β1-isoform functions as the authentic subunit of Na-K-ATPase and nongastric H-K-ATPase. Putatively, the intracellular polarization of X-K-ATPase isoforms depends on interaction with other proteins.

Na-K-ATPase isoform (alpha 3, alpha 2, alpha 1) abundance in rat kidney estimated by competitive RT-PCR and ouabain binding

1996 ◽  
Vol 271 (2) ◽  
pp. F253-F260 ◽  
Author(s):  
K. Lucking ◽  
J. M. Nielsen ◽  
P. A. Pedersen ◽  
P. L. Jorgensen
Keyword(s):  
Polymerase Chain Reaction ◽  
Rat Kidney ◽  
Sodium Reabsorption ◽  
Rt Pcr ◽  
Ouabain Binding ◽  
Chain Reaction ◽  
Competitive Polymerase Chain Reaction ◽  
High Affinity ◽  
Upper Limit ◽  
Polymerase Chain

For understanding the regulation of sodium reabsorption, it is important to know whether the alpha 2- or alpha 3-isoform of Na-K-adenosinetriphosphatase (Na-K-ATPase) is expressed in mammalian kidney in addition to the predominant alpha 1 beta 1-isozyme. Here we applied competitive polymerase chain reaction (PCR) for estimation of mRNA in parenchymal zones of rat kidney for comparison to high-affinity [3H]ouabain binding. The alpha 3-isoform mRNA was demonstrated to form 0.04-0.05% of the amount of alpha 1-isoform mRNA in the cortex, medulla, and papilla of rat kidney. The alpha 2-mRNA was demonstrated in all three zones and constituted 0.03% of the amount of alpha 1-mRNA in cortex. The abundance of the alpha 1 truncated mRNA was 0.1-0.8% of that of the alpha 1-mRNA. The upper limit for expression of Na-K-ATPase isozyme with high ouabain affinity (dissociation constant, 69-141 nM) was 0.096-0.14% of the concentration of alpha 1 beta 1-Na-K-ATPase. Thus a small but well-defined pool of alpha 2- and alpha 3-isoforms constitutes < or = 0.1% of the amount of alpha 1-isoform at both the mRNA and protein level in rat kidney.

scholarly journals Expression of β subunit isoforms of the Na+ ,K+ -ATPase is muscle type-specific

FEBS Letters ◽  
1993 ◽  
Vol 328 (3) ◽  
pp. 253-258 ◽  
Author(s):  
Harinder S. Hundal ◽  
André Marette ◽  
Toolsie Ramlal ◽  
Zhi Liu ◽  
Amira Klip
Keyword(s):  
Β Subunit ◽  
Muscle Type ◽  
Subunit Isoforms

Localization of mRNAs coding for isozymes of plasma membrane Ca(2+)-ATPase pump in rat kidney

1992 ◽  
Vol 263 (1) ◽  
pp. F7-F14 ◽  
Author(s):  
M. Magosci ◽  
M. Yamaki ◽  
J. T. Penniston ◽  
T. P. Dousa
Keyword(s):  
Plasma Membrane ◽  
Rat Kidney ◽  
Rat Plasma ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Outer Medulla ◽  
Unique Distribution ◽  
Polymerase Chain ◽  
Convoluted Tubules ◽  
Specific Mrna

We have studied localization of mRNAs coding isozymes of rat plasma membrane Ca(2+)-adenosinetriphosphatase pump (rPMCA) in the rat kidney, with use of reverse transcription (RT) with subsequent amplification by polymerase chain reaction (PCR). When zones of the kidney were separated by macrodissection, a large amount of mRNA coding isozyme rPMCA1 was found in all zones; mRNA for isozyme rPMCA2 was abundant in cortex and in outer medulla, and mRNA for isozyme rPMCA3 was prominent in outer medulla. The mRNAs were analyzed in microdissected cortical nephron segments by use of RT-PCR approach described previously [T. Moriyama, H. R. Murphy, B. M. Martin, and A. Garcia-Perez. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F1470-F1474, 1990]. We detected mRNA for isozyme rPMCA2 in microdissected distal convoluted tubules (DCT) and in cortical thick ascending limbs (CTAL) and, less consistently, also in proximal convoluted tubule and in glomeruli. The mRNA for isozyme rPMCA1 was abundant in glomeruli but was absent in all examined cortical tubular segments. Our results document that mRNAs for all three major isozymes of rPMCA are present and show a unique distribution in the three major zones of rat renal parenchyma. Specific mRNA coding for rPMCA2 was detected in cortical tubules, namely in CTAL and DCT, whereas mRNA coding isozyme rPMCA1 was found in glomeruli. We suggest that isozyme rPMCA2 might be specifically related to epithelial cells and their function, whereas rPMCA1 is probably a component of nonepithelial cells including these in glomeruli.

RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb

1995 ◽  
Vol 268 (6) ◽  
pp. F1224-F1228 ◽  
Author(s):  
P. Borensztein ◽  
M. Froissart ◽  
K. Laghmani ◽  
M. Bichara ◽  
M. Paillard
Keyword(s):  
Rat Kidney ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Pcr Analysis ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Medullary Thick Ascending Limb ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Nhe Isoforms

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

Distribution and regulation of guanylyl cyclase type B in the rat nephron

1996 ◽  
Vol 270 (2) ◽  
pp. F311-F318 ◽  
Author(s):  
A. D. Dean ◽  
V. M. Vehaskari ◽  
D. Ritter ◽  
J. E. Greenwald
Keyword(s):  
Guanylyl Cyclase ◽  
Rat Kidney ◽  
Immunofluorescent Staining ◽  
Hydration Status ◽  
Mrna Levels ◽  
Type B ◽  
Rt Pcr ◽  
Data Link ◽  
Renal Inner Medulla ◽  
Polymerase Chain

C-type natriuretic peptide (CNP) has been localized to the proximal and distal nephron. In this study, we examined the distribution and regulation of the CNP receptor, guanylyl cyclase type B (GC-B), in the rat kidney. GC-B mRNA was detected most frequently in microdissected glomeruli, thin and thick limbs of the loop of Henle, and outer and inner medullary collecting ducts by reverse transcription-polymerase chain reaction (RT-PCR). This pattern of expression is supported by immunofluorescent staining, using anti-GC-B-specific antiserum. Nearly equivalent levels of GC-B and guanylyl cyclase type A (GC-A) mRNAs were found by quantitative RT-PCR (5,662 +/- 1,622 and 5,187 +/- 1,204 molecules of cDNA/microgram total RNA, respectively; means +/- SE, n = 6). Renal inner medulla GC-B mRNA levels, but not renal CNP mRNA levels, were 3.2-fold greater in hypervolemic and 2.3-fold less in hypovolemic rats compared with euvolemic controls. Immunohistochemical staining also supports a greater GC-B expression with increased volume status. These data link hydration status and GC-B expression and suggest an additional and novel mechanism for regulating intravascular volume.

Expression of the β-subunit isoforms of the Na, K-ATpase in rat embryo tissues, inner ear and choroid plexus

1994 ◽  
Vol 81 (3) ◽  
pp. 215-222 ◽  
Author(s):  
Luis M. González-Martínez ◽  
Julio Avila ◽  
Elisa Martí ◽  
Emilia Lecuona ◽  
Pablo Martín-Vasallo
Keyword(s):  
Inner Ear ◽  
Choroid Plexus ◽  
Β Subunit ◽  
Rat Embryo ◽  
Subunit Isoforms
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