Stimulation of Na(+)-K(+)-2Cl- cotransport in rat medullary thick ascending limb by dopamine

Dopamine receptors are present in the medullary thick ascending limb (mTAL) of Henle, but their effect on ion transport in this nephron segment has not been tested. Therefore, we studied the short-term effects of dopamine on Na(+)-K(+)-2Cl- cotransport (assessed by 100 microM bumetanide-sensitive 86Rb uptake) in rat mTAL tubular suspensions. Dopamine (1 microM) stimulated bumetanide-sensitive 86Rb uptake (72.1 +/- 10.6% vs. control, n = 5) by increasing total 86Rb uptake and by decreasing bumetanide-insensitive 86Rb uptake; this effect was concentration dependent. The dopamine-induced stimulation of Na(+)-K(+)-2Cl- cotransport activity was mimicked by calyculin A, a protein phosphatase (PP) inhibitor, and Sp isomer of adenosine 3',5'-cyclic monophosphothioate (Sp-cAMP[S]), a protein kinase A (PKA) agonist, and blocked by Rp isomer of 8-(4-chlorophenylthio)-cAMP[S] (Rp-8-CPT-cAMP[S]), a PKA inhibitor (n = 5). Dopamine did not increase the stimulatory effect of the PP inhibitor. However, the stimulatory effect of the PP inhibitor and PKA agonist was additive and approached the stimulatory effect of dopamine. The stimulatory effects of dopamine, PP inhibitor, and PKA agonist persisted even when intracellular sodium was clamped by 5 microM monensin. When K+ channels were blocked by 1 mM BaCl2, the effects of dopamine and calyculin A on the cotransport were no longer apparent, although the stimulatory effect of the PKA agonist was attenuated. We conclude that dopamine stimulates Na(+)-K(+)-2Cl- cotransport activity. This action is mediated mainly by PKA-dependent phosphorylation/dephosphorylation processes and modulated by dopamine actions on K+ channels.

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Dopamine and protein phosphatase activity in renal proximal tubules

AJP Renal Physiology ◽

10.1152/ajprenal.1995.268.2.f279 ◽

1995 ◽

Vol 268 (2) ◽

pp. F279-F284 ◽

Cited By ~ 3

Author(s):

E. Slobodyansky ◽

Y. Aoki ◽

A. K. Gaznabi ◽

D. H. Aviles ◽

R. D. Fildes ◽

...

Keyword(s):

Atpase Activity ◽

Protein Phosphatase ◽

Inhibitory Effect ◽

Proximal Tubules ◽

Thick Ascending Limb ◽

Renal Proximal Tubules ◽

Medullary Thick Ascending Limb ◽

Nephron Segment ◽

The Brain ◽

Brain Striatum

In the brain, dopamine, via protein kinase A (PKA) activation of dopamine- and cAMP-regulated phosphoprotein (DARPP-32), inhibits protein phosphatase 1 (PP1) activity and keeps Na(+)-K(+)-adenosinetriphosphatase (ATPase) in its phosphorylated inactive state. In the present study, we examined the relationship among dopamine, PP1, and Na(+)-K(+)-ATPase activities in renal proximal tubules. PP1 activity in proximal tubules was not decreased by dopamine (5 x 10(-9)-10(-4) M), fenoldopam (5 x 10(-6) M), or norepinephrine (5 x 10(-7) M). In contrast, in the medullary thick ascending limb of Henle and in the brain striatum, PP1 activity was decreased by fenoldopam (5 x 10(-6) M). We also showed that the ability of dopamine (10(-6) M) to inhibit Na(+)-K(+)-ATPase activity in proximal tubules (assessed by ouabain-sensitive 86Rb uptake) occurred in the absence or presence of a sodium clamp with 5 microM monensin. Thus the inhibitory effect of dopamine on Na(+)-K(+)-ATPase activity in proximal tubules is not regulated by PP1 activity. Tautomycin and okadaic acid by themselves, at concentrations that inhibited PP1 activity, had no effect on Na(+)-K(+)-ATPase activity in proximal tubules. The ability of a dopamine D1 agonist, fenoldopam, to inhibit PP1 activity in brain striatum and in medullary thick ascending limb, but not in proximal tubules, suggests differential organ and nephron segment regulation of PP activity.

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PO-131 Impact of short-term inhibition of PKA in Nucleus Accumbens on voluntary wheel running

Exercise Biochemistry Review ◽

10.14428/ebr.v1i4.10343 ◽

2018 ◽

Vol 1 (4) ◽

Author(s):

Xuansong Mao ◽

Kolter Grigsby ◽

Frank Booth

Keyword(s):

Gene Expression ◽

Protein Kinase ◽

Wheel Running ◽

Ex Vivo ◽

Skf 38393 ◽

Short Term ◽

Voluntary Running ◽

Kinase A ◽

Stimulation Of ◽

Voluntary Wheel

Objective Based upon a Booth lab goal of establishing molecular regulators of physical activity motivation, my current study focuses on the effects of short-term inhibition of protein kinase A (PKA) activity in the nucleus accumbens (NAc). The NAc is a brain region integral to motivated behaviors. Downstream immediate-early gene (IEG) expression from PKA has been shown to exhibit rapid responses to acute stimuli, such as voluntary wheel-running behavior. According to previous work in our lab, long-term NAc overexpression of the endogenous PKA inhibitor, Protein Kinase Inhibitor Alpha (PKIα), increased nightly running distance in rats selectively bred for low voluntary running (LVR) behavior (Mol Neurobiol 2018 Jun 21). However, paradoxically, the same PKIα overexpression failed to increase running distance in wild-type (WT) rats. It is known that chronic manipulation of the NAc PKA pathway produces different molecular (gene expression profiles) and behavioral outcomes from that of acute manipulations. Given the above, the goal of the current work is to determine how short-term inhibition of PKA in the NAc influences its downstream gene networks and the nightly voluntary running behavior in WT rats. Methods An ex vivo preparation of the NAc was utilized to determine the effects of Rp-cAMPS, a selective protein kinase A inhibitor, upon its stimulation of dopamine D1-like receptor agonist SKF 38393 on downstream gene expression level in sedentary WT female rats. Further, real-time PCR was implemented to analyze the transcriptional expression of IEGs (Homer-1, Arc, Zif268) following Rp-cAMPS administration. Results Data showed that there were no significant difference of mRNA level for Homer-1, Arc or Zif268 among the vehicle, 50uM, 100uM and 200uM Rp-cAMPS treatment groups upon the stimulation of 10uM SKF 38393. Conclusions In addition to the PKA, other protein kinases such as Ca++ activated and growth factor activated kinases have both been shown to phosphorylate CREB at Ser133, and thus, lead to activation of gene transcription. Given the above results of the ex vivo experiment, in which NAc slices were treated with multiple dosages of Rp-cAMPS concurrent with the stimulation of SKF 38393, it is possible that other protein kinase pathways could be compensating the effects of short-term inhibition of PKA and, in turn, lead to no difference of IEG expression. Further experiments will need to be performed in order to testify this hypothesis.

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Regulation of Ca(2+)-activated K+ channels by protein kinase A and phosphatase inhibitors

AJP Cell Physiology ◽

10.1152/ajpcell.1991.261.2.c387 ◽

1991 ◽

Vol 261 (2) ◽

pp. C387-C392 ◽

Cited By ~ 59

Author(s):

A. Carl ◽

J. L. Kenyon ◽

D. Uemura ◽

N. Fusetani ◽

K. M. Sanders

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Okadaic Acid ◽

Colonic Motility ◽

Proximal Colon ◽

Calyculin A ◽

Open Probability ◽

Phosphatase Inhibitors ◽

K Channels ◽

Kinase A

Many proteins including ion channels are regulated by phosphorylation. We tested the effect of 10 U/ml catalytic subunit protein kinase A on 260-pS Ca(2+)-activated K+ channels in excised inside-out membrane patches from freshly dispersed smooth muscle cells of the canine proximal colon. At +50 mV with 10(-7) M Ca2+ and -50 mV with 10(-6) M Ca2+, open probability of the channels was increased to 270 +/- 48% of control (n = 12). This increase was due to a shift in voltage-dependent activation by 13.9 +/- 3.2 mV (n = 3) to more negative potentials. Protein kinase A in the absence of ATP had no effect on channel activity (n = 3). Regulation by phosphorylation must be accompanied by dephosphorylation. We tested the effect of two potent inhibitors of protein phosphatases, calyculin A and okadaic acid. Application of 10(-9) to 10(-6) M of each inhibitor in the presence of protein kinase A further increased open probability by up to 250%. Calyculin A appeared to be less effective in increasing open probability than okadaic acid, suggesting that the phosphatase involved is neither type 1, 2A, nor 2B. Calyculin A in the absence of protein kinase A was ineffective. These data suggest that endogenous phosphatases are found in excised membrane patches and that a balance between phosphorylation and dephosphorylation may provide an important control of colonic motility.

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In vitro stimulation of Na-K-ATPase in rat thick ascending limb by dexamethasone

AJP Renal Physiology ◽

10.1152/ajprenal.1986.251.5.f851 ◽

1986 ◽

Vol 251 (5) ◽

pp. F851-F857 ◽

Cited By ~ 3

Author(s):

A. Doucet ◽

A. Hus-Citharel ◽

F. Morel

Keyword(s):

Atpase Activity ◽

Sodium Reabsorption ◽

Thick Ascending Limb ◽

Dependent Manner ◽

Short Term ◽

Medullary Thick Ascending Limb ◽

Dose Dependent ◽

Stimulation Of

Dexamethasone has been reported to stimulate Na-K-ATPase activity in the medullary thick ascending limb of adrenalectomized animals within a few hours. The present study was aimed at characterizing the mechanism of this action by investigating the stimulatory effect of the hormone in vitro. Dexamethasone (10(-8) M) added in vitro to segments of the medullary thick ascending limb of Henle's loop, which were microdissected from adrenalectomized rats, restored in a dose-dependent manner the depressed Na-K-ATPase activity within one h of incubation. This stimulation of Na-K-ATPase was inhibited by cycloheximide and actinomycin D. Dexamethasone also stimulated the component of oxidative metabolism coupled to sodium transport. These results, which confirm previous in vivo observations, demonstrate that dexamethasone-induced stimulation of Na-K-ATPase is a direct tubular action of the hormone mediated by protein synthesis. They suggest that this short-term effect of dexamethasone corresponds to the stimulation of sodium reabsorption by the dilution segment.

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