Role of diacylglycerol in adrenergic-stimulated 86Rb uptake by proximal tubules

We used rat proximal tubule fragments purified by Percoll centrifugation to examine the role of diacylglycerol (DAG) in noradrenergic-stimulated Na+ reabsorption. Tubular DAG concentration and ouabain-inhibitable 86Rb uptake increased within 30 s after adding norepinephrine (NE) and remained elevated for at least 5 min. NE (1 microM) increased DAG content 17% and ouabain-inhibitable 86Rb uptake 23%. Cirazoline-stimulated 86Rb uptake was not inhibited by BaCl, quinidine, or bumetanide (1-10 microM) or by the omission of HCO3- or Cl- from the medium, but it was completely inhibited by ouabain and furosemide. Oleoyl-acetyl glycerol, L-alpha-1,2-dioctanoylglycerol, and L-alpha-1,2-dioleoylglycerol (DOG) increased total 86Rb uptake 8-11%. 12-O-tetradecanoylphorbol-13-acetate (TPA) (5 nM) increased uptake by only 4%. Staurosporine at 5 nM inhibited DOG stimulation completely, whereas 50 nM staurosporine was required to inhibit NE stimulation completely. Sphingosine inhibited DOG stimulation by 66% but did not inhibit NE stimulation. Amiloride (1 mM) completely blocked DOG stimulation. Monensin increased 86Rb uptake 31% and completely blocked the DOG effect but reduced the NE effect by only 26% (P = 0.08). In tubules from salt-loaded rats, NE did not increase DAG concentration, but NE-stimulated 86Rb uptake was reduced by only 23% (P = 0.15). Thus DAG released by NE may stimulate Na+ entry through Na(+)-H+ exchange. NE predominantly stimulates Na(+)-K(+)-adenosinetriphosphatase (ATPase) by activating a protein kinase that is insensitive to DAG and TPA and is inhibited by staurosporine but not by sphingosine. NE may also stimulate K+ efflux through a BaCl-insensitive K+ channel that is inhibited by millimolar furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)

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Role of protein kinase C in beta 2-adrenoceptor function in cultured rat proximal tubule epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.1997.273.2.f193 ◽

1997 ◽

Vol 273 (2) ◽

pp. F193-F199 ◽

Cited By ~ 2

Author(s):

H. Singh ◽

S. L. Linas

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Epithelial Cells ◽

Atpase Activity ◽

Proximal Tubule ◽

Sodium Transport ◽

Kinase C ◽

Beta 2 ◽

Rat Proximal Tubule

Renal sodium excretion is regulated by the adrenergic system. We recently demonstrated the presence of functional beta 2-adrenoceptors (beta 2-AR) in cultured rat proximal tubule epithelial cells beta 2-AR activation resulted in increases in Na-K-adenosinetriphosphatase (Na-K-ATPase) activity and transcellular sodium transport as a consequence of increased apical sodium entry. The purpose of this study was to determine the role of protein kinase C (PKC) on beta 2-AR-dependent increases in Na-K-ATPase activity and sodium transport in proximal tubules. To determine the effect of PKC on basal function, cultured rat proximal tubule cells were exposed to phorbol 12-myristate 13-acetate (PMA). PMA increased apical Na entry (+/-80%), decreased Na-K-ATPase activity (+/-25%), and prevented increases in Na-K-ATPase activity after sodium entry facilitation with monensin. Decreases in Na-K-ATPase activity were associated with decreases in sodium transport (+/-30%). To determine whether beta 2-AR function was transduced by PKC, PKC activity was measured in cells exposed to the selective beta 2-AR agonist metaproterenol. Metaproterenol caused increases in PKC activity, which were blocked by a beta 2-AR but not by a beta 1-AR-receptor antagonist. beta 2-AR-dependent increases in apical Na entry, Na-K-ATPase activity, and sodium transport were blocked by calphostin C or staurosporine. To determine whether PKC had additional effects on beta 2-AR function, cells were exposed to metaproterenol and PMA. Metaproterenol-induced increases in Na-K-ATPase activity and sodium transport were blocked by PMA. In conclusion, beta 2-AR-mediated increases in Na-K-ATPase activity and sodium flux are transduced by PKC acting through increases in apical Na entry. However, activation of PKC by phorbol esters inhibits beta 2-AR-dependent increases in Na-K-ATPase activity and sodium transport.

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Fluoride mobilizes intracellular calcium and promotes Ca2+ influx in rat proximal tubules

AJP Renal Physiology ◽

10.1152/ajprenal.1991.261.2.f318 ◽

1991 ◽

Vol 261 (2) ◽

pp. F318-F327 ◽

Cited By ~ 1

Author(s):

J. H. Dominguez ◽

J. G. Garcia ◽

J. K. Rothrock ◽

D. English ◽

C. Mann

Keyword(s):

Signal Transduction ◽

Parathyroid Hormone ◽

Proximal Tubule ◽

G Protein ◽

Pertussis Toxin ◽

Time Course ◽

Proximal Tubules ◽

Signal Transduction Pathways ◽

Pi Hydrolysis

In the renal proximal tubule, external Ca2+ ([Ca2+]o) is required for parathyroid hormone to elevate cytosolic Ca2+ ([Ca2+]i). However, other hormones increase [Ca2+]i in the absence of [Ca2+]o. These differences may arise from a diversity of signal transduction pathways acting on external and internal Ca2+ pools. However, Ca2+ influx may be necessary to expedite and maintain the rise of [Ca2+]i for a period after the initial surge. In this study, F- was used to probe the roles of intracellular Ca2+ mobilization, Ca2+ influx, and phosphoinositide (PI) hydrolysis on the surge of [Ca2+]i in rat proximal tubules. In the presence of external Ca2+; 1-20 mM F- evoked incremental rises of [Ca2+]i in tubules loaded with aequorin. Whereas 10 mM F- increased [Ca2+]i in the absence of [Ca2+]o, the time constant for the [Ca2+]i surge was increased. These findings are consistent with a role of Ca2+ influx on the effect of F- on [Ca2+]i. Indeed, 10 mM F- also enhanced the uptake of 45Ca2+, and promoted Ca2+ influx in aequorin- and fura-2-loaded, Ca(2+)-deprived tubules. In tubules, F- also activated PI hydrolysis with a time course that paralleled Ca2+ mobilization. The effect of F- on [Ca2+]i was not altered when the 39-kDa pertussis toxin substrate was inactivated with the toxin. This G protein was most likely Gi, because prostaglandin E2, an activator of Gi in tubules, dissociated the pertussis toxin-sensitive protein. The results support the notion that activation of a signal-transduction complex, the F- substrate, causes Ca2+ influx, mobilizes internal Ca2+, and activates PI hydrolysis in rat proximal tubules.(ABSTRACT TRUNCATED AT 250 WORDS)

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Inhibition of bicarbonate reabsorption in the rat proximal tubule by activation of luminal P2Y1 receptors

AJP Renal Physiology ◽

10.1152/ajprenal.00033.2004 ◽

2004 ◽

Vol 287 (4) ◽

pp. F789-F796 ◽

Cited By ~ 40

Author(s):

Matthew A. Bailey

Keyword(s):

Protein Kinase ◽

Proximal Tubule ◽

Ph Value ◽

Receptor Activation ◽

Ringer Solution ◽

Dependent Manner ◽

Bicarbonate Reabsorption ◽

Mrs 2179 ◽

Rat Proximal Tubule

The present study used a stationary microperfusion technique to investigate in vivo the effect of P2Y1 receptor activation on bicarbonate reabsorption in the rat proximal tubule. Proximal tubules were perfused with a bicarbonate Ringer solution before flow was stopped by means of an oil block. The recovery of lumen pH from the initial value (pH 8.0) to stationary values (pH ∼6.7) was recorded by a H+-sensitive microelectrode inserted downstream of the perfusion pipette and oil block. The stationary pH value and the t of pH recovery were used to calculate bicarbonate reabsorption ( JHCO3). Both EIPA and bafilomycin A1 caused significant reductions in proximal tubule JHCO3, consistent with the established contributions of Na/H exchange and H+-ATPase to proximal tubule HCO3 reabsorption. The nucleotides ADP and, to a lesser extent, ATP reduced JHCO3 but AMP and UTP were without effect. 2MeSADP, a highly selective agonist of the P2Y1 receptor, reduced JHCO3 in a dose-dependent manner. MRS-2179, a P2Y1 receptor-specific antagonist, abolished the effect of 2MeSADP, whereas theophylline, an antagonist of adenosine (P1) receptors, did not. The inhibitory action of 2MeSADP was blocked by inhibition of protein kinase C and reduced by inhibition of protein kinase A. The effects of EIPA and 2MeSADP were not additive. The data provide functional evidence for P2Y1 receptors in the apical membrane of the rat proximal tubule: receptor activation impairs acidification in this nephron segment.

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Role of Ca2+/CaMK II in Ca(2+)-induced K+ channel inhibition in rat CCD principal cell

AJP Renal Physiology ◽

10.1152/ajprenal.1995.268.2.f211 ◽

1995 ◽

Vol 268 (2) ◽

pp. F211-F219 ◽

Cited By ~ 10

Author(s):

M. Kubokawa ◽

W. Wang ◽

C. M. McNicholas ◽

G. Giebisch

Keyword(s):

Protein Kinase ◽

Collecting Duct ◽

Inhibitory Effect ◽

K Channel ◽

Channel Activity ◽

Patch Clamp Technique ◽

Pkc Inhibitor ◽

Control Value ◽

Gf 109203X

The apical low-conductance K+ channel of rat cortical collecting duct (CCD) is inhibited by increased intracellular Ca2+ concentrations. This effect has been shown to be mediated at least in part by activation of protein kinase C (PKC). In the present study, we used the patch-clamp technique to examine the role of Ca2+/calmodulin-dependent protein kinase II (CaMK II) in mediating the Ca(2+)-induced inhibitory effect. In cell-attached patches of principal cells of rat tubules, clamping of intracellular Ca2+ concentration at 400 nM by using 1 microM ionomycin reduced channel activity to 26.5% of the control value. A further reduction in channel activity, to 8.8% of the control value, was observed following the addition of phorbol 12-myristate 13-acetate (PMA), an agent known to activate PKC. Pretreatment of cells with KN-62 (CaMK II inhibitor) or GF-109203X (PKC inhibitor) attenuated the inhibitory effect of Ca2+ on K+ channel activity (83.2 and 50.7% of the control value, respectively). Even in the presence of KN-62, addition of 10 microM PMA significantly decreased channel activity to 57.2% of the control value. The Ca(2+)-induced inhibition was completely abolished by simultaneous incubation with both KN-62 and GF-109203X. In inside-out patches, addition of 20 micrograms/ml CaMK II in the presence of a PKC inhibitor reduced channel activity to 66.2% of control values. It is concluded that CaMK II is involved in mediating the Ca(2+)-induced inhibition of the activity of the apical K+ channel of rat CCD.

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Role of cytochrome P-450 arachidonate metabolites in endothelin signaling in rat proximal tubule

AJP Renal Physiology ◽

10.1152/ajprenal.0064.2001 ◽

2002 ◽

Vol 282 (1) ◽

pp. F144-F150 ◽

Cited By ~ 36

Author(s):

Bruno A. Escalante ◽

John C. McGiff ◽

Adebayo O. Oyekan

Keyword(s):

Proximal Tubule ◽

Inhibitory Action ◽

Inhibitory Effect ◽

Gas Chromatography Mass Spectrometry ◽

Arachidonate Metabolites ◽

Endogenous Release ◽

Oxide Synthase ◽

Cytochrome P 450 ◽

Rat Proximal Tubule

We examined the rat proximal tubule (PT) response to endothelin-1 (ET-1) in terms of 20-hydroxyeicosatetraenoic acid (HETE) dependency. Arachidonic acid (AA) (1 μM) decreased ouabain-sensitive 86Rb uptake from 2.1 ± 0.1 to 0.3 ± 0.08 ng Rb · 10 μg protein−1 · 2 min−1( P < 0.05); 20-HETE (1 μM) had similar effects. Dibromododecenoic acid (DBDD) (2 μM), an inhibitor of ω-hydroxylase, abolished the inhibitory action of AA on86Rb uptake whereas the PT response to 20-HETE was unaffected. ET-1 at 0.1, 1, 10, and 100 nM reduced 86Rb uptake from 2.8 ± 0.3 in control PTs to 2.4 ± 0.2, 1.7 ± 0.1, 0.67 ± 0.08, and 0.1 ± 0.03 ng Rb · 10 μg protein−1 · 2 min−1, respectively. DBDD (2 μM) abolished the inhibitory effect of ET-1 on86Rb uptake as did BMS182874 (1 μM), an ETA-selective receptor antagonist. ET-1 (100 nM) significantly increased PT 20-HETE release by ∼50%, an effect prevented by DBDD. N ω-nitro-l-arginine-methyl ester (l-NAME), given for 4 days to inhibit nitric oxide synthase (NOS), increased arterial pressure from 92 ± 12 to 140 ± 8 mmHg and increased endogenous release of 20-HETE from isolated PTs (measured by gas chromatography/mass spectrometry). Inl-NAME-treated PTs, but not in control PTs, 0.1 μM AA inhibited ouabain-sensitive 86Rb uptake by >40%; the response to AA was attenuated by DBDD. We conclude that, in the PTs, 1) 20-HETE is a second messenger for ET-1 and 2) conversion of AA to 20-HETE is augmented when NOS is inhibited.

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Genetic and genomic evidence for an important role of the Na+/H+ exchanger 3 in blood pressure regulation and angiotensin II-induced hypertension

Physiological Genomics ◽

10.1152/physiolgenomics.00122.2018 ◽

2019 ◽

Vol 51 (4) ◽

pp. 97-108 ◽

Cited By ~ 3

Author(s):

Xiao C. Li ◽

Xiaowen Zheng ◽

Xu Chen ◽

Chunling Zhao ◽

Dongmin Zhu ◽

...

Keyword(s):

Blood Pressure ◽

Angiotensin Ii ◽

Proximal Tubule ◽

Proximal Tubules ◽

Ang Ii ◽

Pressure Regulation ◽

Blood Pressure Homeostasis ◽

Induced Hypertension ◽

Basal Blood Pressure

The sodium (Na+)/hydrogen (H+) exchanger 3 (NHE3) and sodium-potassium adenosine triphosphatase (Na+/K+-ATPase) are two of the most important Na+ transporters in the proximal tubules of the kidney. On the apical membrane side, NHE3 primarily mediates the entry of Na+ into and the exit of H+ from the proximal tubules, directly and indirectly being responsible for reabsorbing ~50% of filtered Na+ in the proximal tubules of the kidney. On the basolateral membrane side, Na+/K+-ATPase serves as a powerful engine driving Na+ out of, while pumping K+ into the proximal tubules against their concentration gradients. While the roles of NHE3 and Na+/K+-ATPase in proximal tubular Na+ transport under in vitro conditions are well recognized, their respective contributions to the basal blood pressure regulation and angiotensin II (ANG II)-induced hypertension remain poorly understood. Recently, we have been fortunate to be able to use genetically modified mouse models with global, kidney- or proximal tubule-specific deletion of NHE3 to directly determine the cause and effect relationship between NHE3, basal blood pressure homeostasis, and ANG II-induced hypertension at the whole body, kidney and/or proximal tubule levels. The purpose of this article is to review the genetic and genomic evidence for an important role of NHE3 with a focus in the regulation of basal blood pressure and ANG II-induced hypertension, as we learned from studies using global, kidney- or proximal tubule-specific NHE3 knockout mice. We hypothesize that NHE3 in the proximal tubules is necessary for maintaining basal blood pressure homeostasis and the development of ANG II-induced hypertension.

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Effects of endothelin on rat renal proximal tubule Na(+)-Pi cotransport and Na+/H+ exchange

AJP Renal Physiology ◽

10.1152/ajprenal.1994.266.4.f658 ◽

1994 ◽

Vol 266 (4) ◽

pp. F658-F666 ◽

Cited By ~ 7

Author(s):

J. Guntupalli ◽

T. D. DuBose

Keyword(s):

Protein Kinase ◽

Proximal Tubule ◽

Collecting Duct ◽

Second Messengers ◽

Indirect Evidence ◽

Low Concentrations ◽

Proximal Tubular ◽

Dependent Protein ◽

Medullary Collecting Duct

Endothelin (ET), a powerful vasoconstrictive peptide, is distributed ubiquitously in various organs, including the vascular endothelium and tubules of the kidney. Although localized more abundantly to the glomerulus and inner medullary collecting duct, ET receptors have been identified in the proximal tubule. The possible effects of ET on proximal tubule transport and the potential role of second messengers in this process have not been described fully. To define the role of ET in proximal tubule transport, renal cortical slices were incubated for 3 min in the presence of various concentrations of ET. Incubation with low concentrations of ET-1 (1 x 10(-9) to 1 x 10(-11) M) within the physiological range stimulated both Na(+)-Pi cotransport and Na+/H+ exchange. Pretreatment with staurosporine (0.6 microM) for 25 min abolished completely the ET-induced effects on Na(+)-Pi cotransport and Na+/H+ exchange. Similarly, preincubation with phorbol ester 12-O-tetradecanoylphorbol-13-acetate (200 nM) also abolished the effects of ET on these transporters. Incubation with ET decreased significantly intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Intravenous administration of pertussis toxin for 2 days prevented the ET-induced decrease in cAMP and abolished the stimulatory effects of ET on Na(+)-Pi cotransport and Na+/H+ exchange. These findings provide indirect evidence that ET participates in the regulation of proximal tubular Pi and bicarbonate homeostasis. These effects of ET are mediated by activation of protein kinase C and cAMP-dependent protein kinase A.

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Angiotensin-(1–7) can interact with the rat proximal tubule AT4 receptor system

AJP Renal Physiology ◽

10.1152/ajprenal.1999.277.1.f75 ◽

1999 ◽

Vol 277 (1) ◽

pp. F75-F83 ◽

Cited By ~ 11

Author(s):

Rajash K. Handa

Keyword(s):

Proximal Tubule ◽

Rat Kidney ◽

Receptor Site ◽

Transport Processes ◽

Proximal Tubules ◽

Receptor Subtype ◽

Angiotensin Receptor ◽

Peptide Fragments ◽

Receptor System ◽

Rat Proximal Tubule

This study was undertaken to identify the non-AT1, non-AT2 angiotensin receptor that mediates the ANG-(1–7) inhibitory action on rat proximal tubule transport processes. ANG-(1–7) inhibited nystatin-stimulated, ouabain-suppressible O2consumption (Qo 2) rates in freshly isolated rat proximal tubules (reflecting reduced basolateral Na+-K+-ATPase activity). Selective angiotensin-receptor subtype antagonists revealed that AT1 and AT4 receptors mediated the response of ANG-(1–7). Receptor autoradiography of the rat kidney demonstrated a high density of AT1and AT4 receptors and no specific125I-ANG(1–7) binding sites. Competition assays in rat kidney sections indicated that ANG-(1–7) competed predominantly for the AT1 receptor site, whereas its NH2-terminal-deleted metabolite, ANG-(3–7), competed primarily for the AT4-receptor site. Metabolism of125I-ANG-(1–7) in rat proximal tubules generated peptide fragments that included ANG-(3–7), with the pentapeptide producing a concentration-dependent inhibition of nystatin-stimulated proximal tubule Qo 2that was abolished by AT4-receptor blockade. These results suggest that the generation of ANG-(3–7) from the NH2-terminal metabolism of ANG-(1–7) caused the interaction of the parent peptide with the proximal tubule AT4 receptor, which elicited a decrease in energy-dependent solute transport.

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A kinetically defined Na+/H+ antiporter within a mathematical model of the rat proximal tubule.

The Journal of General Physiology ◽

10.1085/jgp.105.5.617 ◽

1995 ◽

Vol 105 (5) ◽

pp. 617-641 ◽

Cited By ~ 28

Author(s):

A M Weinstein

Keyword(s):

Kinetic Model ◽

Proximal Tubule ◽

Membrane Vesicles ◽

Model Calculations ◽

Kinetic Formulation ◽

Luminal Membrane ◽

Least Squares Fit ◽

Velocity Effect ◽

Rat Proximal Tubule

The luminal membrane antiporter of the proximal tubule has been represented using the kinetic formulation of E. Heinz (1978. Mechanics and Engergetics of Biological Transport. Springer-Verlag, Berlin) with the assumption of equilibrium binding and 1:1 stoichiometry. Competitive binding and transport of NH+4 is included within this model. Ion affinities and permeation velocities were selected in a least-squares fit to the kinetic parameters determined experimentally in renal membrane vesicles (Aronson, P.S., M.A. Suhm, and J. Nee. 1983. Journal of Biological Chemistry. 258:6767-6771). The modifier role of internal H+ to enhance transport beyond the expected kinetics (Aronson, P.S., J. Nee, and M. A. Suhm. 1982. Nature. 299:161-163) is represented as a velocity effect of H+ binding to a single site. This kinetic formulation of the Na+/H+ antiporter was incorporated within a model of the rat proximal tubule (Weinstein, A. M. 1994. American Journal of Physiology. 267:F237-F248) as a replacement for the representation by linear nonequilibrium thermodynamics (NET). The membrane density of the antiporter was selected to yield agreement with the rate of tubular Na+ reabsorption. Simulation of 0.5 cm of tubule predicts that the activity of the Na+/H+ antiporter is the most important force for active secretion of ammonia. Model calculations of metabolic acid-base disturbances are performed and comparison is made among antiporter representations (kinetic model, kinetic model without internal modifier, and NET formulation). It is found that the ability to sharply turn off Na+/H+ exchange in cellular alkalosis substantially eliminates the cell volume increase associated with high HCO3- conditions. In the tubule model, diminished Na+/H+ exchange in alkalosis blunts the axial decrease in luminal HCO3- and thus diminishes paracellular reabsorption of Cl-. In this way, the kinetics of the Na+/H+ antiporter could act to enhance distal delivery of Na+, Cl-, and HCO3- in acute metabolic alkalosis.

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