Pathways involved in PTH-induced rise in cytosolic Ca2+ concentration of rat renal proximal tubule

1995 ◽  
Vol 268 (2) ◽  
pp. F330-F337 ◽  
Author(s):  
H. Tanaka ◽  
M. Smogorzewski ◽  
M. Koss ◽  
S. G. Massry
Keyword(s):  
Proximal Tubule ◽  
G Protein ◽  
Positive Control ◽  
Renal Proximal Tubule ◽  
Channel Blockers ◽  
Proximal Tubular ◽  
The Media ◽  
Renal Proximal Tubular Cells ◽  
Renal Proximal Tubule Cells ◽  
Renal Proximal Tubular

Parathyroid hormone (PTH) raises cytosolic Ca2+ concentration ([Ca2+]i) in isolated or cultured renal proximal tubule cells. The pathways through which this action is mediated are not fully delineated. This study explored these pathways utilizing fura 2. [Ca2+]i of freshly prepared renal proximal tubular cells increased from 150 +/- 3.6 to 281 +/- 9.0 nM after the exposure to 10(-7) M angiotensin II, which served as a positive control. Both PTH-(1-84) and PTH-(1-34) produced a dose-dependent rise in [Ca2+]i. The effects of both moieties were similar up to 10(-7) M, but with higher doses the rise in [Ca2+]i with PTH-(1-84) was greater (P < 0.01) than with PTH-(1-34). This effect of the hormone occurred in the presence or absence of calcium in the media, but the rise in [Ca2+]i was significantly greater in the presence of calcium. The PTH-induced rise in [Ca2+]i was markedly inhibited by PTH antagonist [Nle8,18,Tyr34]bPTH-(7-34)-NH2 (bPTH is bovine PTH), verapamil, or nifedipine. 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, increased [Ca2+]i of cells, but its effect was less than PTH. Staurosporine abolished the TPA effect and partially inhibited that of PTH. A G protein activator raised [Ca2+]i, whereas a G protein inhibitor and pertussis toxin partially blocked the effect of PTH. Sodium or chloride channel blockers or sodium-free media did not modify the effect of PTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract 653: G-protein-coupled Receptor Kinase 4 Binds Preferentially To The Caveolin-1α Isoform In Rat Renal Proximal Tubule Cells

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Dora Bigler Wang ◽  
John J Gildea ◽  
Robin A Felder
Keyword(s):  
Proximal Tubule ◽  
G Protein ◽  
Renal Proximal Tubule ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells ◽  
G Protein Coupled ◽  
Caveolin Scaffolding Domain

Caveolins, localized in lipid rafts of plasma membranes, tether and regulate signaling complexes into functional units and have been shown to inhibit g-protein-coupled receptor kinases GRK2 and GRK5 via the caveolin scaffolding domain. In human renal proximal tubule cells (RPTC), the dopamine-1-receptor (D1R) is phosphorylated and inhibited by G protein-coupled receptor kinase 4 (GRK4). Defects in D1R coupling leads to sodium-retention and a rise in blood pressure. We showed earlier that chronic disruption of lipid rafts or Caveolin-1 (Cav1) protein expression in the kidney induces hypertension in rats by relieving the steric inhibition of Cav1 on GRK4. Here we extend these studies by examining caveolin expression in renal proximal tubule cells microdissected from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). We discovered a significant decrease in Cav1 and Cav2 expression in SHR compared to WKY (98 ±1.2% reduction; n=3, P<0.001). In order to determine if the D1R coupling defect found in these SHR renal proximal tubule cells is due to loss of either of these proteins, we made stable SHR cell lines expressing either Cav1α, Cav1β or Cav2. Using co-immunoprecipitiation and western blot analysis, we demonstrate for the first time that GRK4 and Cav1 physically interact in rat cells, and that it is the Cav1α isoform that binds predominantly to GRK4 (Cav1β, Cav2 and vector control show a 90 ±1.5%, 84±0.8% and 87±2.1% reduction, respectively; n=3, P<0.001). Similar to an effect found in mouse Cav1 knockout animals, we find that Cav2 is not stable without the co-expression of Cav1. Our data suggest that Cav1α. but not Cav1β, is important for normal D1R function by binding to and inhibiting GRK4, implicating a domain other than the caveolin scaffolding domain in this interaction. These findings might be used in designing selective GRK4 inhibitors as novel antihypertensive compounds.

scholarly journals G protein-coupled receptor 37L1 regulates renal sodium transport and blood pressure

2019 ◽  
Vol 316 (3) ◽  
pp. F506-F516 ◽  
Author(s):  
Xiaoxu Zheng ◽  
Laureano D. Asico ◽  
Xiaobo Ma ◽  
Prasad R. Konkalmatt
Keyword(s):  
Proximal Tubule ◽  
G Protein ◽  
Renal Proximal Tubule ◽  
Intracellular Sodium ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells ◽  
And Function ◽  
G Protein Coupled ◽  
The Brain

G protein-coupled receptors (GPCRs) in the kidney regulate the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Abnormalities in renal epithelial ion transport play important roles in the pathogenesis of essential hypertension. The orphan G protein-coupled receptor 37L1 (GPR37L1), also known as endothelin receptor type B-like protein (ETBR-LP2), is expressed in several regions in the brain, but its expression profile and function in peripheral tissues are poorly understood. We found that GPR37L1 mRNA expression is highest in the brain, followed by the stomach, heart, testis, and ovary, with moderate expression in the kidney, pancreas, skeletal muscle, liver, lung, and spleen. Immunofluorescence analyses revealed the expression of GPR37L1 in specific regions within some organs. In the kidney, GPR37L1 is expressed in the apical membrane of renal proximal tubule cells. In human renal proximal tubule cells, the transient expression of GPR37LI increased intracellular sodium, whereas the silencing of GPR37LI decreased intracellular sodium. Inhibition of Na+/H+ exchanger isoform 3 (NHE3) activity abrogated the GPR37L1-mediated increase in intracellular sodium. Renal-selective silencing of Gpr37l1 in mice increased urine output and sodium excretion and decreased systolic and diastolic blood pressures. The renal-selective silencing of GPR37L1 decreased the protein expression of NHE3 but not the expression of Na+-K+-ATPase or sodium-glucose cotransporter 2. Our findings show that in the kidney, GPR37L1 participates in renal proximal tubule luminal sodium transport and regulation of blood pressure by increasing the renal expression and function of NHE3 by decreasing cAMP production. The role of GPR37L1, expressed in specific cell types in organs other than the kidney, remains to be determined.

The mechanism of angiotensin II binding downregulation by high glucose in primary renal proximal tubule cells

2002 ◽  
Vol 282 (2) ◽  
pp. F228-F237 ◽  
Author(s):  
Soo Hyun Park ◽  
Ho Jae Han
Keyword(s):  
Proximal Tubule ◽  
High Glucose ◽  
Renal Proximal Tubule ◽  
Channel Blockers ◽  
Ang Ii ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Pkc Inhibitors ◽  
Renal Proximal Tubule Cells ◽  
Tgf Β1

The renin-angiotensin system plays an important role in the development of diabetic nephropathy. However, the mechanism of ANG II receptor regulation in the renal proximal tubule in the diabetic condition has not been elucidated. Thus we investigated the signal pathways involved in high-glucose-induced downregulation of ANG II binding in primary cultured renal proximal tubule cells. Twenty-five millimolar glucose, but not mannitol andl-glucose, induced downregulation of the AT1receptor (AT1R) because of a significant decline in maximal binding with no significant change in the affinity constant. Twenty-five millimolar glucose also decreased AT1R mRNA and protein levels. The 25 mM glucose-induced increase in the formation of lipid peroxides was prevented by antioxidants, protein kinase C (PKC) inhibitors, or L-type calcium channel blockers. These agents also blocked 25 mM glucose-induced downregulation of 125I-ANG II binding. In addition, 25 mM glucose increased transforming growth factor (TGF)-β1 secretion, and anti-TGF-β antibody significantly blocked 25 mM glucose-induced downregulation of 125I-ANG II binding. Furthermore, the 25 mM glucose-induced increase in TGF-β1 secretion was inhibited by PKC inhibitors, L-type calcium channel blockers, or antioxidants. In conclusion, high glucose may induce downregulation of 125I-ANG II binding via a PKC-oxidative stress-TGF-β signal cascade in primary cultured rabbit renal proximal tubule cells.

Abstract 362: G Protein-Linked Receptor Kinase 4 is Palmitoylated and Acetylated and Regulates Subcellular Localization in Human Renal Proximal Tubule Cells

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
John J Gildea ◽  
Christine L Hou ◽  
Huang Zhenyue ◽  
Robin A Felder
Keyword(s):  
Plasma Membrane ◽  
Proximal Tubule ◽  
G Protein ◽  
Renal Proximal Tubule ◽  
Receptor Kinase ◽  
Cytoplasmic Fraction ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells ◽  
First Time

G protein-linked receptor kinase 4 (GRK4) is a kinase expressed in human renal proximal tubule cells (RPTC). Three single nucleotide polymorphisms in this gene are associated with hyper-phosphorylation and inactivation of the dopamine-1 receptor (D1R), and are associated with both hypertension and salt sensitivity. GRK4 is a prime therapeutic target for hypertension so we sought to discover potential post-translational modifications that could affect both localization and function. We identified two unique posttranslational modifications of GRK4 that we hypothesize are associated with protein localization. GRK4 is found at the plasma membrane (PM), in the cytoplasm and in the nucleus. We determined that GRK4 is both acetylated and palmitoylated. Using cell fractionation and immunoprecipitation, we determined that GRK4 was found in the nucleus, but acetylated GRK4 was only found in the cytoplasmic fraction using six different human RPTC cell lines (>10 fold n=6, p<0.01). We previously reported that PM GRK4 internalized following fenoldopam (FEN, dopamine agonist) stimulation, but the internalization only occurred in normally coupled human renal proximal tubule cells (nRPTC), not uncoupled cells (uRPTC). GRK4 is palmitoylated at the C-Terminal end of the protein (cystein 563, GGCPalmLTMVP). Using the palmitic acid tracer, 17-octadecynoic acid (17-ODYA) and Huisgen’s cycloaddition reaction (Click Reaction Chemistry) and biotin-azide for detection, we determined that GRK4 is depalmitoylated in a FEN (1μM, 30 min) and cAMP (forskolin, 10 μM, 30 min.) dependent manner. The amount of palmitoylated GRK4 was decreased following treatment with FEN (28.4%±3.6, n=3, p<0.01 vs VEH) or forskolin (35.3%±6.7, n=3, p<0.05 vs VEH) in nRPTC. No significant change in GRK4 palmitoylation was observed in uRPTC. The depalmitoylation of GRK4 correlates with its change in plasma membrane localization in both nRPTC and uRPTC. In conclusion we have determined for the first time that GRK4 is acetylated and the acetylated GRK4 is found only in the cytoplasmic fraction. In addition we have determined for the first time that GRK4 is depalmitoylated in nRPTC (not in uRPTC) when stimulated with FEN and this effect was mimicked by increasing intracellular cAMP using forskolin.

The Fine Structure of Rat Renal Microbodies and Cytoplasmic Bodies Following Perfusion Fixation

1973 ◽  
Vol 31 ◽  
pp. 620-621
Author(s):  
J. M. Barrett ◽  
P. M. Heidger
Keyword(s):  
Fine Structure ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Renal Proximal Tubule ◽  
Convincing Evidence ◽  
Cytoplasmic Bodies ◽  
Rat Renal Proximal Tubule ◽  
Renal Proximal Tubule Cells ◽  
Perfusion Fixation

Microbodies have received extensive morphological and cytochemical investigation since they were first described by Rhodin in 1954. To our knowledge, however, all investigations of microbodies and cytoplasmic bodies of rat renal proximal tubule cells have employed immersion fixation. Tisher, et al. have shown convincing evidence of fine structural alteration of microbodies in rhesus monkey kidney following immersion fixation; these alterations were not encountered when in vivo intravascular perfusion was employed. In view of these studies, and the fact that techniques for perfusion fixation have been established specifically for the rat kidney by Maunsbach, it seemed desirable to employ perfusion fixation to study the fine structure and distribution of microbodies and cytoplasmic bodies within the rat renal proximal tubule.

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