scholarly journals Regulation of ERK1/2 by ouabain and Na-K-ATPase-dependent energy utilization and AMPK activation in parotid acinar cells

2008 ◽  
Vol 295 (3) ◽  
pp. C590-C599 ◽  
Author(s):  
Stephen P. Soltoff ◽  
Lee Hedden
Keyword(s):  
Protein Kinase ◽  
Muscarinic Receptor ◽  
Growth Factor Receptor ◽  
Acinar Cells ◽  
Receptor Activation ◽  
Energy Utilization ◽  
Ampk Activation ◽  
Parotid Acinar Cells ◽  
Ampk Phosphorylation ◽  
Rat Parotid

We previously found that the phosphorylation of ERK1/2 by submaximal concentrations of the muscarinic receptor ligand carbachol was potentiated in rat parotid acinar cells exposed to ouabain, a cardiac glycoside that inhibits the Na-K-ATPase. We now report that this signaling phenomenon involves the prevention of negative regulation of extracellular signal-regulated kinase-1/2 (ERK1/2) that is normally mediated by AMP-activated protein kinase (AMPK). Carbachol increases the turnover of the ATP-consuming Na-K-ATPase, reducing intracellular ATP and promoting the phosphorylation/activation of the energy sensor AMPK. Ouabain blocks the reduction in ATP and subsequent AMPK phosphorylation, which is regulated by the AMP-to-ATP ratio. The ouabain-promoted enhancement of ERK1/2 phosphorylation was not reproduced in Par-C10 cells, an immortalized rat parotid cell line that did not respond to carbachol with an ATP reduction and that employs an upstream AMPK kinase (Ca2+/calmodulin-dependent protein kinase kinase, CaMKK) different from that (LKB1) in native cells. In native parotid cells, inhibitory effects of AMPK on ERK1/2 signaling were examined by activating AMPK with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), which is converted to an AMP mimetic but does not alter parotid ATP levels. AICAR-treated cells display increases in AMPK phosphorylation and a reduced phosphorylation of ERK1/2 subsequent to activation of muscarinic and P2X7 receptors, which promote increases in Na-K-ATPase turnover, but not upon epidermal growth factor receptor activation. These results suggest that carbachol-initiated AMPK activation can produce a negative feedback on ERK1/2 signaling in response to submaximal muscarinic receptor activation and that increases in fluid secretion can modulate receptor-initiated signaling events indirectly by producing ion transport-dependent decreases in ATP.

scholarly journals Thiol-oxidation reduces the release of amylase induced by β-adrenergic receptor activation in rat parotid acinar cells

2010 ◽  
Vol 31 (5) ◽  
pp. 293-299 ◽  
Author(s):  
Ming-Yu Guo ◽  
Keitaro Satoh ◽  
Bing Qi ◽  
Takanori Narita ◽  
Osamu Katsumata-Kato ◽  
...  
Keyword(s):  
Adrenergic Receptor ◽  
Acinar Cells ◽  
Receptor Activation ◽  
Thiol Oxidation ◽  
Parotid Acinar Cells ◽  
Rat Parotid

Phosphorylation of the salivary Na+-K+-2Cl− cotransporter

2002 ◽  
Vol 282 (4) ◽  
pp. C817-C823 ◽  
Author(s):  
Kinji Kurihara ◽  
Nobuo Nakanishi ◽  
Marilyn L. Moore-Hoon ◽  
R. James Turner
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Acinar Cells ◽  
Good Evidence ◽  
Protein Kinase Activity ◽  
Adrenergic Stimulation ◽  
Parotid Acinar Cells ◽  
Rat Parotid ◽  
Phosphopeptide Mapping ◽  
Kinase A

We studied the phosphorylation of the secretory Na+-K+-2Cl− cotransporter (NKCC1) in rat parotid acinar cells. We have previously shown that NKCC1 activity in these cells is dramatically upregulated in response to β-adrenergic stimulation and that this upregulation correlates with NKCC1 phosphorylation, possibly due to protein kinase A (PKA). We show here that when ATP is added to purified acinar basolateral membranes (BLM), NKCC1 is phosphorylated as a result of membrane-associated protein kinase activity. Additional NKCC1 phosphorylation is seen when PKA is added to BLMs, but our data indicate that this is due to an effect of PKA on endogenous membrane kinase or phosphatase activities, rather than its direct phosphorylation of NKCC1. Also, phosphopeptide mapping demonstrates that these phosphorylations do not take place at the site associated with the upregulation of NKCC1 by β-adrenergic stimulation. However, this upregulatory phosphorylation can be mimicked by the addition of cAMP to permeabilized acini, and this effect can be blocked by a specific PKA inhibitor. These latter results provide good evidence that PKA is indeed involved in the upregulatory phosphorylation of NKCC1 and suggest that an additional factor present in the acinar cell but absent from isolated membranes is required to bring about the phosphorylation.

Phosphorylation of myristoylated alanine-rich C kinase substrate is involved in the cAMP-dependent amylase release in parotid acinar cells

2009 ◽  
Vol 296 (6) ◽  
pp. G1382-G1390 ◽  
Author(s):  
Keitaro Satoh ◽  
Miwako Matsuki-Fukushima ◽  
Bing Qi ◽  
Ming-Yu Guo ◽  
Takanori Narita ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Acinar Cells ◽  
Cellular Migration ◽  
Intracellular Camp ◽  
Dependent Manner ◽  
Amylase Release ◽  
Kinase Substrate ◽  
Parotid Acinar Cells ◽  
Dependent Protein ◽  
Rat Parotid

Myristoylated alanine-rich C kinase substrate (MARCKS) is known as a major cellular substrate for protein kinase C (PKC). MARCKS has been implicated in the regulation of brain development and postnatal survival, cellular migration and adhesion, as well as phagocytosis, endocytosis, and exocytosis. The involvement of MARCKS phosphorylation in secretory function has been reported in Ca2+-mediated exocytosis. In rat parotid acinar cells, the activation of β-adrenergic receptors provokes exocytotic amylase release via accumulation of intracellular cAMP levels. Here, we studied the involvement of MARCKS phosphorylation in the cAMP-dependent amylase release in rat parotid acinar cells. MARCKS protein was detected in rat parotid acinar cells by Western blotting. The β-adrenergic agonist isoproterenol (IPR) induced MARCKS phosphorylation in a time-dependent manner. Translocation of a part of phosphorylated MARCKS from the membrane to the cytosol and enhancement of MARCKS phosphorylation at the apical membrane site induced by IPR were observed by immunohistochemistry. H89, a cAMP-dependent protein kinase (PKA) inhibitor, inhibited the IPR-induced MARCKS phosphorylation. The PKCδ inhibitor rottlerin inhibited the IPR-induced MARCKS phosphorylation and amylase release. IPR activated PKCδ, and the effects of IPR were inhibited by the PKA inhibitors. A MARCKS-related peptide partially inhibited the IPR-induced amylase release. These findings suggest that MARCKS phosphorylation via the activation of PKCδ, which is downstream of PKA activation, is involved in the cAMP-dependent amylase release in parotid acinar cells.

scholarly journals Two modes of regulation of the phospholipase C-linked substance-P receptor in rat parotid acinar cells

1988 ◽  
Vol 253 (2) ◽  
pp. 459-466 ◽  
Author(s):  
H Sugiya ◽  
J F Obie ◽  
J W Putney
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Substance P ◽  
Acinar Cells ◽  
Control Mechanisms ◽  
Dependent Manner ◽  
Parotid Acinar Cells ◽  
Kinase C ◽  
Substance P Receptor ◽  
Rat Parotid

In rat parotid acinar cells prelabelled with [3H]inositol, substance P (100 nM) induced the formation of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Ins(1,4,5)P3 reached a maximum 7 s after substance P stimulation, and thereafter decreased and reached a stable value at 60 s. When the cells were exposed to substance P for 10, 30, 60, or 300 s, washed, and re-exposed to this peptide, the formation of [3H]inositol trisphosphate (InsP3) was attenuated in a time-dependent manner. In the cells pretreated as described above, the number of [3H]substance-P-binding sites (Bmax) was also decreased. Possible role(s) of Ca2+ and protein kinase (protein kinase C) control mechanisms in regulating substance P responses were investigated. Desensitization of substance P-induced InsP3 was not affected by the Ca2+ ionophore ionomycin, nor was it dependent on Ca2+ mobilization. On the other hand, in the presence of 4 beta-phorbol 12,13-dibutyrate (PDBu) and 12-O-tetradecanoyl-4 beta-phorbol 13-acetate, known activators of protein kinase C, substance P-induced InsP3 formation was inhibited. However, PDBu had no effect on [3H]substance P binding, whether present during the assay or when cells were pretreated. The persistent desensitization of InsP3 formation induced by substance P was not affected by PDBu. These results suggest that the persistent desensitization of InsP3 formation induced by substance P is a homologous process involving down-regulation of the substance P receptor; the mechanism does not appear to involve, or to be affected by, the Ca2+ or protein kinase C signalling systems. Protein kinase C activation can, however, inhibit substance P-induced InsP3 formation, which may indicate the presence of a negative-feedback control on the substance P pathway.

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