scholarly journals Glucagon stimulation of hepatic Na+-pump activity and α-subunit phosphorylation in rat hepatocytes

1996 ◽  
Vol 313 (3) ◽  
pp. 983-989 ◽  
Author(s):  
Christopher J. LYNCH ◽  
Kenneth M. McCALL ◽  
Yuk-Chow NG ◽  
Stacy A. HAZEN
Keyword(s):  
Protein Kinase ◽  
Rat Kidney ◽  
Rat Hepatocytes ◽  
Transport Activity ◽  
Isolated Rat Hepatocytes ◽  
Α Subunit ◽  
Na Pump ◽  
Pump Activity ◽  
Stimulation Of

In this study the possible role of Na+ influx, arachidonate mediators and α-subunit phosphorylation in the stimulatory response of hepatic Na+/K+-ATPase to glucagon was examined. Glucagon stimulation of ouabain-sensitive 86Rb+ uptake in freshly isolated rat hepatocytes reached maximal levels in less than 1 min after hormone addition and was half-maximal (EC50) at a concentration of 2.4(±1.3)×10-10 M. Analysis of the K+-dependence of this response indicates an effect on the apparent Vmax. for K+ with no significant change in the apparent K0.5. Unlike monensin, glucagon stimulation of Na+/K+-ATPase-mediated transport activity was not associated with an increase in 22Na+ influx. This indicates that the stimulation of Na+/K+-ATPase by glucagon is not secondary to an increase in Na+ influx. A role for arachidonate mediators in this effect also appears unlikely because neither basal nor glucagon-stimulated ouabain-sensitive 86Rb+ uptake was significantly affected by supramaximal concentrations of cyclo-oxygenase, lipoxygenase, cytochrome P-450 or phospholipase A2 inhibitors. To study the possible role of protein kinase-mediated phosphorylation in the stimulation of ouabain-sensitive 86Rb+ uptake, hepatocytes were metabolically radiolabelled with [32P]Pi. Glucagon stimulated incorporation of 32P into a 95 kDa phosphoprotein that co-migrates with Na+/K+-ATPase α-subunit immunoreactivity in two-dimensional gel electrophoresis. The α-subunit could be immunoprecipitated from detergent-solubilized particulate fractions of hepatocytes using an anti-(rat kidney Na+/K+-ATPase) serum. When hepatocytes were metabolically radiolabelled with [32P]Pi, the immunoprecipitated α-subunit contained 32P. Glucagon increased the incorporation of 32P into the immunoprecipitated subunit by 197±21% (n = 6). Similar results were observed with a rabbit anti-peptide serum (‘anti-LEAVE’ serum) prepared against an amino acid sequence in the α-subunit. The EC50 for glucagon-stimulated phosphorylation of the α-subunit (1×10-10 M) was very close to that for glucagon stimulation of ouabain-sensitive 86Rb+ uptake. In conclusion, it appears that glucagon stimulation of hepatic Na+/K+-ATPase-mediated transport activity is not secondary to increases in Na+ influx or changes in the levels of an arachidonate mediator. The data provide support for the hypothesis that glucagon stimulation of Na+-pump activity in hepatocytes may be related to protein kinase-mediated changes in the phosphorylation state of the α-subunit.

scholarly journals Stimulation of hepatocytic AMP-activated protein kinase by okadaic acid and other autophagy-suppressive toxins

2005 ◽  
Vol 386 (2) ◽  
pp. 237-244 ◽  
Author(s):  
Hamid R. SAMARI ◽  
Michael T. N. MØLLER ◽  
Lise HOLDEN ◽  
Tonje ASMYHR ◽  
Per O. SEGLEN
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Rat Hepatocytes ◽  
Calyculin A ◽  
Β Subunit ◽  
Isolated Rat Hepatocytes ◽  
Α Subunit ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

Autophagic activity in isolated rat hepatocytes is strongly suppressed by OA (okadaic acid) and other PP (protein phosphatase)-inhibitory toxins as well as by AICAR (5-aminoimidazole-4-carboxamide riboside), a direct activator of AMPK (AMP-activated protein kinase). To investigate whether AMPK is a mediator of the effects of the toxin, a phosphospecific antibody directed against the activation of phosphorylation of the AMPK α (catalytic)-subunit at Thr172 was used to assess the activation status of this enzyme. AICAR as well as all the toxins tested (OA, microcystin-LR, calyculin A, cantharidin and tautomycin) induced strong, dose-dependent AMPKα phosphorylation, correlating with AMPK activity in situ (in intact hepatocytes) as measured by the AMPK-dependent phosphorylation of acetyl-CoA carboxylase at Ser79. All treatments induced the appearance of multiple, phosphatase-sensitive, low-mobility forms of the AMPK α-subunit, consistent with phosphorylation at several sites other than Thr172. The flavonoid naringin, an effective antagonist of OA-induced autophagy suppression, inhibited the AMPK phosphorylation and mobility shifting induced by AICAR, OA or microcystin, but not the changes induced by calyculin A or cantharidin. AMPK may thus be activated both by a naringin-sensitive and a naringin-resistant mechanism, probably involving the PPs PP2A and PP1 respectively. Neither the Thr172-phosphorylating protein kinase LKB1 nor the Thr172-dephosphorylating PP, PP2C, were mobility-shifted after treatment with toxins or AICAR, whereas a slight mobility shifting of the regulatory AMPK β-subunit was indicated. Immunoblotting with a phosphospecific antibody against pSer108 at the β-subunit revealed a naringin-sensitive phosphorylation induced by OA, microcystin and AICAR and a naringin-resistant phosphorylation induced by calyculin A and cantharidin, suggesting that β-subunit phosphorylation could play a role in AMPK activation. Naringin antagonized the autophagy-suppressive effects of AICAR and OA, but not the autophagy suppression caused by cantharidin, consistent with AMPK-mediated inhibition of autophagy by toxins as well as by AICAR.

scholarly journals Calcium-mobilizing hormones and phorbol myristate acetate mediate heterologous desensitization of the hormone-sensitive hepatic Na+/K+ pump

1987 ◽  
Vol 248 (3) ◽  
pp. 807-813 ◽  
Author(s):  
C J Lynch ◽  
S B Bocckino ◽  
P F Blackmore ◽  
J H Exton
Keyword(s):  
Cyclic Amp ◽  
Rat Hepatocytes ◽  
Previous Exposure ◽  
High Concentration ◽  
Isolated Rat Hepatocytes ◽  
Pump Activity ◽  
Heterologous Desensitization ◽  
Tumour Promoters ◽  
High Concentrations ◽  
Stimulation Of

The Na+/K+ pump in rat hepatocytes is stimulated in response to Ca2+-mobilizing hormones such as [arginine]vasopressin (AVP), angiotensin II and adrenaline, as well as tumour promoters such as 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). The ability of these agents to increase cellular contents of diacylglycerol and activate protein kinase C may be necessary to observe this response. In the present work, ouabain-sensitive 86Rb+ uptake was studied in isolated rat hepatocytes to help to explain why stimulation of the Na+/K+ pump by Ca2+-mobilizing hormones and tumour promoters is not temporally sustained relative to other hormone responses. A transient stimulation (3-4 min) of the Na+/K+ pump was observed in hepatocytes exposed to high (10 nM), but not low (0.1 nM), concentrations of AVP. Experiments with the Ca2+ chelator EGTA and the Na+ ionophore monensin indicate that the rapid secondary decrease in Na+/K+-pump activity which occurs after AVP stimulation is not due to changes in cytosolic Ca2+ and Na+ concentrations. When added after the stimulation and rapid decrease in Na+/K+-pump activity induced in hepatocytes by a high concentration of AVP, a second challenge with AVP or PMA failed to stimulate the pump. Similarly, previous exposure of hepatocytes to angiotensin, adrenaline or PMA attenuated the subsequent Na+/K+-pump responses to AVP and PMA. In contrast, previous exposure to AVP had no significant effect on subsequent stimulation of the Na+/K+-pump by monensin, glucagon, forskolin or 8-p-chlorophenylthio cyclic AMP. In addition, exposure to monensin had no effect on subsequent responses to AVP and PMA. These data indicate that high concentrations of Ca2+-mobilizing hormones and PMA result in heterologous desensitization of the hepatic Na+/K+ pump to subsequent stimulation by Ca2+-mobilizing hormones and PMA, but not by cyclic-AMP-dependent agonists or monensin.

scholarly journals Is Phosphorylation of the α1 Subunit at Ser-16 Involved in the Control of Na,K-ATPase Activity by Phorbol Ester–activated Protein Kinase C?

2000 ◽  
Vol 11 (1) ◽  
pp. 39-50 ◽  
Author(s):  
Eric Féraille ◽  
Pascal Béguin ◽  
Maria-Luisa Carranza ◽  
Sandrine Gonin ◽  
Martine Rousselot ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Phorbol Ester ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Wild Type ◽  
Α1 Subunit ◽  
Stimulation Of

The α1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufoα1 subunits. In cells incubated at 37°C, phorbol 12,13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally (∼20–30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A2, and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18°C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing α1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase α1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.

The role of protein kinase C in the increased generation in isolated rat hepatocytes of the hydroxyl radical by puromycin aminonucleoside

2000 ◽  
Vol 32 (6) ◽  
pp. 487-496 ◽  
Author(s):  
Kazumasa Aoyagi ◽  
Siranoush Shahrzad ◽  
Yutaka Kuzure ◽  
Akio Koyama ◽  
Ko Nakamura ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Hydroxyl Radical ◽  
Rat Hepatocytes ◽  
Puromycin Aminonucleoside ◽  
Isolated Rat Hepatocytes ◽  
Kinase C ◽  
Isolated Rat

scholarly journals The role of cytosolic Ca2+, protein kinase C, and protein kinase A in hormonal stimulation of phospholipase D in rat hepatocytes.

1994 ◽  
Vol 269 (2) ◽  
pp. 849-859
Author(s):  
L. Gustavsson ◽  
G. Moehren ◽  
M.E. Torres-Marquez ◽  
C. Benistant ◽  
R. Rubin ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Phospholipase D ◽  
Rat Hepatocytes ◽  
Hormonal Stimulation ◽  
Kinase C ◽  
Kinase A ◽  
Stimulation Of
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