Dopamine recruits D1A receptors to Na-K-ATPase-rich caveolar plasma membranes in rat renal proximal tubules

2004 ◽  
Vol 287 (5) ◽  
pp. F921-F931 ◽  
Author(s):  
Meghna Trivedi ◽  
Vihang A. Narkar ◽  
Tahir Hussain ◽  
Mustafa F. Lokhandwala
Keyword(s):  
Plasma Membrane ◽  
Adenylyl Cyclase ◽  
Plasma Membranes ◽  
Radioligand Binding ◽  
Sch 23390 ◽  
Sucrose Density Gradient ◽  
Proximal Tubules ◽  
Sodium Reabsorption ◽  
Sprague Dawley ◽  
Renal Proximal Tubules

Activation of dopamine D1A receptors in renal proximal tubules causes inhibition of sodium transporters (Na-K-ATPase and Na/H exchanger), leading to a decrease in sodium reabsorption. In addition to being localized on the plasma membrane, D1A receptors are mainly present in intracellular compartments under basal conditions. We observed, using [3H]SCH-23390 binding and immunoblotting, that dopamine recruits D1A receptors to the plasma membrane in rat renal proximal tubules. Furthermore, radioligand binding and/or immunoblotting experiments using pharmacological modulators showed that dopamine-induced D1A receptor recruitment requires activation of cell surface D1-like receptors, activation of adenylyl cyclase, and intact endocytic vesicles with internal acidic pH. A key finding of this study was that these recruited D1A receptors were functional because they potentiated dopamine-induced [35S]GTPγS binding, cAMP accumulation, and Na-K-ATPase inhibition. Interestingly, dopamine increased immunoreactivity of D1A receptors specifically in caveolin-rich plasma membranes isolated by a sucrose density gradient. In support of this observation, coimmunoprecipitation studies showed that D1A receptors interacted with caveolin-2 in an agonist-dependent fashion. The caveolin-rich plasma membranes had a high content of the α1-subunit of Na-K-ATPase, which is a downstream target of D1A receptor signaling in proximal tubules. These results show that dopamine, via the D1-like receptor-adenylyl cyclase pathway, recruits D1A receptors to the plasma membrane. These newly recruited receptors couple to G proteins, increase cAMP, and participate in dopamine-mediated inhibition of Na-K-ATPase in proximal tubules. Moreover, dopamine-induced recruitment of D1A receptors to the caveolin-rich plasma membranes brings them in close proximity to targets such as Na-K-ATPase in proximal tubules of Sprague-Dawley rats.

scholarly journals Trafficking of Na-K-ATPase and dopamine receptor molecules induced by changes in intracellular sodium concentration of renal epithelial cells

2008 ◽  
Vol 295 (4) ◽  
pp. F1117-F1125 ◽  
Author(s):  
Angel R. Cinelli ◽  
Riad Efendiev ◽  
Carlos H. Pedemonte
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Atpase Activity ◽  
Proximal Tubule ◽  
Dopamine Receptors ◽  
Sodium Concentration ◽  
Intracellular Sodium ◽  
Proximal Tubules ◽  
Sodium Reabsorption ◽  
Intracellular Sodium Concentration

Most of the transepithelial transport of sodium in proximal tubules occurs through the coordinated action of the apical sodium/proton exchanger and the basolateral Na-K-ATPase. Hormones that regulate proximal tubule sodium excretion regulate the activities of these proteins. We have previously demonstrated that the level of intracellular sodium concentration modulates the regulation of Na-K-ATPase activity by angiotensin II and dopamine. An increase of a few millimolars in intracellular sodium concentration leads to increased Na-K-ATPase activity without a statistically significant increase in the number of plasma membrane Na-K-ATPase molecules, as determined by cell surface protein biotinylation. Using total internal reflection fluorescence, we detected an increased number of Na-K-ATPase molecules in cytosolic compartments adjacent to the plasma membrane, suggesting that the increased intracellular sodium concentration induces a movement of Na-K-ATPase molecules toward the plasma membrane. While intracellular compartments containing Na-K-ATPase molecules are very close to the plasma membrane, compartments containing type 1 dopamine receptors (D1Rs) are distributed in different parts of the cell cytosol. Fluorescence determinations indicate that an increased intracellular sodium concentration induces the increased colocalization of dopamine receptors with Na-K-ATPase molecules in the region of the plasma membrane. We propose that under in vivo conditions, in response to a sodium load in the lumen of proximal tubules, an increased level of intracellular sodium in epithelial cells is an early event that triggers the cellular response that leads to dopamine inhibition of proximal tubule sodium reabsorption.

Nephron specificity of dopamine receptor-adenylyl cyclase defect in spontaneous hypertension

1993 ◽  
Vol 264 (2) ◽  
pp. F274-F279 ◽  
Author(s):  
K. Ohbu ◽  
R. A. Felder
Keyword(s):  
Adenylyl Cyclase ◽  
Dopamine Receptor ◽  
Radioligand Binding ◽  
Spontaneously Hypertensive Rat ◽  
Collecting Duct ◽  
Spontaneous Hypertension ◽  
Sodium Reabsorption ◽  
Binding Studies ◽  
Cytoplasmic Loop ◽  
The Third

Since DA1 receptors regulate renal tubular sodium transport, it is possible that the reported defect in the coupling between the DA1 dopamine receptor and adenylyl cyclase (AC) in the proximal tubule (PT) is a mechanism for the increased sodium reabsorption in animal models of spontaneous hypertension. Because the distal nephron may participate in the increased sodium retention in the spontaneously hypertensive rat (SHR), we determined whether the defective DA1 receptor-AC coupling described in PT of SHR is also present in the cortical collecting duct (CCD). Radioligand binding studies with the DA1 antagonist 125I-Sch 23982 revealed similar dissociation constants and maximum receptor densities in the CCD from Wistar-Kyoto rats (WKY) and SHR. Fenoldopam, a DA1-selective agonist, stimulated AC activity to a similar extent in CCD from both rat groups. Therefore the defective DA1 receptor-AC coupling in SHR has nephron segment specificity, since it is present in PT but not in CCD. One of the AC-linked dopamine receptors is an intronless D1A cloned from brain, which is also present in PT. Because the coupling defect in the PT may reside in the third cytoplasmic loop (involved in G protein coupling), we compared the sequence of this segment of the cloned D1A receptor using genomic DNA. Because no differences were noted between WKY and SHR, the coupling defect in the PT is not due to a mutation at the third cytoplasmic loop of the D1A receptor.

Isolation of plasma membrane fractions from the intestinal epithelial model T84

1993 ◽  
Vol 264 (5) ◽  
pp. C1327-C1335 ◽  
Author(s):  
P. Kaoutzani ◽  
C. A. Parkos ◽  
C. Delp-Archer ◽  
J. L. Madara
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Epithelial Cell Line ◽  
Membrane Domains ◽  
Intestinal Epithelial ◽  
T84 Cells ◽  
Biochemical Analyses ◽  
Sucrose Density Gradient Sedimentation

The human intestinal epithelial cell line T84 is widely used as a model for studies of Cl- secretion and crypt cell biology. We report a fractionation approach that permits separation of purified apical and basolateral T84 plasma membrane domains. T84 cellular membranes were isolated by nitrogen cavitation and differential centrifugation from monolayers grown on permeable supports. Membranes were then fractionated by isopycnic sucrose density gradient sedimentation, and fractions were assessed, using enzymatic and Western blot techniques, for apical (alkaline phosphatase) and basolateral (Na(+)-K(+)-ATPase) plasma membrane markers and for cytosolic, lysosomal, Golgi, and mitochondrial markers. Buffer conditions were defined that permitted separation of enriched apical and basolateral markers. The validity of the selected markers for the apical and basolateral domains was verified by selective apical and basolateral surface labeling studies using trace iodinated wheat germ agglutinin or biotinylation. This approach allows for separation of apical and basolateral plasma membranes of T84 cells for biochemical analyses and should thus be of broad utility in studies of this model polarized and transporting epithelium.

scholarly journals ISOLATION OF PLASMA MEMBRANE FRAGMENTS FROM HELA CELLS

1969 ◽  
Vol 41 (2) ◽  
pp. 378-392 ◽  
Author(s):  
Charles W. Boone ◽  
Lincoln E. Ford ◽  
Howard E. Bond ◽  
Donald C. Stuart ◽  
Dianne Lorenz
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Plasma Membranes ◽  
Reductase Activity ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Plasma Membrane Vesicle ◽  
Whole Cells ◽  
Continuous Sucrose Gradient

A method for isolating plasma membrane fragments from HeLa cells is described. The procedure starts with the preparation of cell membrane "ghosts," obtained by gentle rupture of hypotonically swollen cells, evacuation of most of the cell contents by repeated washing, and isolation of the ghosts on a discontinuous sucrose density gradient. The ghosts are then treated by minimal sonication (5 sec) at pH 8.6, which causes the ghost membranes to pinch off into small vesicles but leaves any remaining larger intracellular particulates intact and separable by differential centrifugation. The ghost membrane vesicles are then subjected to isopycnic centrifugation on a 20–50% w/w continuous sucrose gradient in tris-magnesium buffer, pH 8.6. A band of morphologically homogeneous smooth vesicles, derived principally from plasma membrane, is recovered at 30–33% (peak density = 1.137). The plasma membrane fraction contained a Na-K-activated ATPase activity of 1.5 µmole Pi/hr per mg, 3% RNA, and 13.8% of the NADH-cytochrome c reductase activity of a heavier fraction from the same gradient which contained mitochondria and rough endoplasmic vesicles. The plasma membranes of viable HeLa cells were marked with 125I-labeled horse antibody and followed through the isolation procedure. The specific antibody binding of the plasma membrane vesicle fraction was increased 49-fold over that of the original whole cells.

scholarly journals Subcellular localization of transglutaminase. Effect of collagen

1988 ◽  
Vol 250 (2) ◽  
pp. 421-427 ◽  
Author(s):  
M Juprelle-Soret ◽  
S Wattiaux-De Coninck ◽  
R Wattiaux
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Collagen Type I ◽  
Type I ◽  
Nuclear Fraction ◽  
Binding Studies ◽  
Isopycnic Centrifugation

1. The subcellular distribution of transglutaminase was investigated by using the analytical approach of differential and isopycnic centrifugation as applied to three organs of the rat: liver, kidney and lung. After differential centrifugation by the method of de Duve, Pressman, Gianetto, Wattiaux & Appelmans [(1955) Biochem. J. 63, 604-617], transglutaminase is mostly recovered in the unsedimentable fraction S and the nuclear fraction N. After isopycnic centrifugation of the N fraction in a sucrose density gradient, a high proportion of the enzyme remains at the top of the gradient; a second but minor peak of activity is present in high-density regions, where a small proportion of 5′-nucleotidase, a plasma-membrane marker, is present together with a large proportion of collagen recovered in that fraction. 2. Fractions where a peak of transglutaminase was apparent in the sucrose gradient were examined by electron microscopy. The main components are large membrane sheets with extracellular matrix and free collagen fibers. 3. As these results seem to indicate that some correlation exists between particulate transglutaminase distribution and those of collagen and plasma membranes, the possible binding of transglutaminase by collagen (type I) and by purified rat liver plasma membrane was investigated. 4. The binding studies indicated that collagen is able to bind transglutaminase and to make complexes with plasma-membrane fragments whose density is higher than that of plasma-membrane fragments alone. Transglutaminase cannot be removed from such complexes by 1% Triton X-100, but can be to a relatively large extent by 0.5 M-KCl and by 50% (w/v) glycerol. 5. Such results suggest that the apparent association of transglutaminase with plasma membrane originates from binding in vitro of the cytosolic enzyme to plasma membrane bound to collagen, which takes place during homogenization of the tissue, when the soluble enzyme and extracellular components are brought together.

scholarly journals The localization of vanadium- and nitrate-sensitive ATPases in Cucumis sativus L. root cells

2014 ◽  
Vol 57 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Grażyna Kłobus
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Peak Activity ◽  
Specific Density ◽  
Cucumis Sativus L ◽  
Root Cells ◽  
Atpase Inhibitors ◽  
Cucumber Root ◽  
Distinct Separation

Distinct separation of plasma membrane and tonoplast membranes was attained by centrifugation of cucumber root microsomes in a sucrose density gradient. The fractions enriched in plasma membranes, identified on the basis of the sensitivity of ATPases to VO<sub>4</sub><sup>3-</sup> sedimented at a specific density of 1. 1463-1. 1513 g x cm<sup>-3</sup>. They did not exhibit cytochrome oxidase activity and there was only trace activity of the azide-sensitive ATPase in these fractions. The fractions enriched in tonoplast membranes, having peak activity of nitrate-sensitive ATPase, were found in the region of specific densities of 1. 1082-1.1175. The presence of vanadium-sensitive and azide-sensitive ATPases was not found in these fractions. The ATPase inhibitors, DCCD, DES and EDAC, inhibited the activity of both vanadium-sensitive and nitrate-sensitive ATPases.

Electrogenic ATP-dependent Cl- transport by plasma membrane vesicles from Aplysia intestine

1988 ◽  
Vol 254 (1) ◽  
pp. R127-R133 ◽  
Author(s):  
G. A. Gerencser
Keyword(s):  
Plasma Membrane ◽  
Transport Process ◽  
Transport Mechanism ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Differential Centrifugation ◽  
Plasma Membrane Vesicles ◽  
Cl Transport ◽  
Vesicular Membrane

A Cl--stimulated adenosinetriphosphatase (ATPase) activity and an ATP-dependent Cl- transport process were found in Aplysia enterocyte plasma membranes. In an attempt to further elucidate this transport process plasma membrane vesicles from Aplysia enterocytes were prepared utilizing differential centrifugation and sucrose density gradient techniques. Electrogenicity of the ATP-dependent Cl- transport was confirmed in three ways. First, an inwardly directed valinomycin-induced K+ diffusion potential, making the vesicle interior electrically positive, enhanced ATP-driven Cl- uptake compared with vesicles lacking the ionophore. Second, ATP plus Cl- increased intravesicular negativity measured by lipophilic triphenylmethylphosphonium distribution across the vesicular membrane. Third, both vanadate and thiocyanate inhibited the ATP plus Cl--dependent intravesicular negativity. These results are consistent with the hypothesis that the active electrogenic Cl- transport mechanism in Aplysia intestine could be a Cl--stimulated ATPase found in the enterocyte plasma membrane.

scholarly journals Organization of the phosphoinositide cycle. Assessment of inositol transferase activity in purified plasma membranes

1993 ◽  
Vol 290 (1) ◽  
pp. 179-183 ◽  
Author(s):  
O M Santiago ◽  
L I Rosenberg ◽  
M E Monaco
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Density Gradient ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Tumour Cells ◽  
Assay System ◽  
Mammary Tumour ◽  
Transferase Activity ◽  
Phosphoinositide Cycle

Experiments were carried out to determine whether or not CDP-diacylglycerol:myo-inositol 3-phosphatidyltransferase (IT) activity (EC 2.7.8.11) could be detected in purified plasma-membrane fractions from WRK-1 rat mammary tumour cells. These cells have previously been shown to have a very active phosphoinositide cycle. Sucrose-density-gradient-purified plasma membranes contained no IT activity that could not be accounted for by endoplasmic-reticulum contamination. However, we also determined that the relative amount of IT activity in endoplasmic reticulum and plasma-membrane fractions could be altered by changing the concentration of detergent in the assay system.

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