scholarly journals Characterization of a phosphorylation event resulting in upregulation of the salivary Na+-K+-2Cl−cotransporter

1999 ◽  
Vol 277 (6) ◽  
pp. C1184-C1193 ◽  
Author(s):  
Kinji Kurihara ◽  
Marilyn L. Moore-Hoon ◽  
Masato Saitoh ◽  
R. James Turner
Keyword(s):  
Binding Sites ◽  
Acinar Cells ◽  
Close Correlation ◽  
Adrenergic Stimulation ◽  
High Affinity ◽  
Parotid Acinar Cells ◽  
Phosphorylation Event ◽  
Rat Parotid ◽  
Stimulation Of

Previous studies from our laboratory have shown a close correlation between increased Na+-K+-2Cl−cotransporter activity and increased cotransporter phosphorylation after β-adrenergic stimulation of rat parotid acinar cells. We demonstrate here that these effects are paralleled by an increase in the number of high-affinity binding sites for the cotransporter inhibitor bumetanide in membranes prepared from stimulated acini. We also show that the sensitivity of cotransporter fluxes to inhibition by bumetanide is the same in both resting and isoproterenol-stimulated cells, consistent with the hypothesis that β-adrenergic stimulation and the accompanying phosphorylation result in the activation of previously quiescent transporters rather than in a change in the properties of already active proteins. In addition, we demonstrate that the increased phosphorylation on the cotransporter resulting from β-adrenergic stimulation is localized to a 30-kDa phosphopeptide obtained by cyanogen bromide digestion. Immunoprecipitation and Western blotting experiments demonstrate that this peptide is derived from the NH2-terminal cytosolic tail of the cotransporter, which surprisingly does not contain the sole protein kinase A consensus site on the molecule.

scholarly journals β-Adrenergic stimulation alters oligosaccharide pyrophosphoryl dolichol metabolism in rat parotid acinar cells

1985 ◽  
Vol 231 (2) ◽  
pp. 431-438 ◽  
Author(s):  
S R Grant ◽  
E E Kousvelari ◽  
D K Banerjee ◽  
B J Baum
Keyword(s):  
Half Life ◽  
Specific Radioactivity ◽  
Acinar Cells ◽  
Protein Glycosylation ◽  
Significant Enhancement ◽  
Adrenergic Stimulation ◽  
Parotid Acinar Cells ◽  
Secretory Glycoproteins ◽  
Rat Parotid ◽  
Stimulation Of

beta-Adrenergic stimulation of rat parotid acinar cells markedly increases [3H]mannose incorporation into N-linked glycoproteins [Kousvelari, Grant, Banerjee, Newby & Baum (1984) Biochem. J. 222, 17-24]. More than 90% of this protein-bound [3H]mannose was preferentially incorporated into four secretory glycoproteins. The ratio of [3H]mannose/[14C]leucine present in these individual proteins was 1.7-4-fold greater with isoproterenol-treated cells than with untreated controls. In isoproterenol-stimulated cells, [3H]mannose incorporation into mannosylphosphoryl dolichol and oligosaccharide-PP-dolichol was increased 2-3-fold over that observed in unstimulated cells. Similarly, formation of mannosylated oligosaccharide-PP-dolichol was increased approx. 4-fold in microsomes prepared from isoproterenol-treated cells. Also, turnover of oligosaccharide-PP-dolichol was significantly increased (5-fold) by β-adrenergic stimulation; the half-life for oligosaccharide-PP-dolichol decreased from 6 min in control cells to 1.2 min in isoproterenol-stimulated cells. By 15 min after isoproterenol addition to acinar cells, the specific radioactivity of parotid oligosaccharide moieties increased about 3-fold over the value observed in the absence of the agonist. Taken together, these results strongly suggest that elevation of N-linked protein glycosylation in rat parotid acinar cells after β-adrenoreceptor stimulation resulted from significant enhancement in the synthesis of mannosylphosphoryl dolichol and oligosaccharide-PP-dolichol and the turnover of oligosaccharide-PP-dolichol.

Relationship between muscarinic receptor occupancy and response in rat parotid acinar cells

1993 ◽  
Vol 265 (6) ◽  
pp. G1122-G1127 ◽  
Author(s):  
Y. Dai ◽  
B. J. Baum
Keyword(s):  
Binding Sites ◽  
Equilibrium Constants ◽  
Acinar Cells ◽  
Scatchard Analysis ◽  
Parotid Glands ◽  
Response Curves ◽  
Parotid Acinar Cells ◽  
Receptor Number ◽  
Rat Parotid ◽  
Q Values

To determine whether spare muscarinic cholinergic receptors (mAChRs) exist in rat parotid acinar cells, we examined the effect of propylbenzilylcholine mustard (PBCM) on agonist (carbachol)-stimulated inositol trisphosphate (IP3) formation and on mAChR number, using l-[N-methyl-3H]scopolamine methyl chloride (NMS)-binding assays. Treatment with PBCM (1, 3, 10, 30, 50 nM) for 15 min caused a 5, 22, 60, 66, and 72% decrease, respectively, in maximal IP3 formation stimulated by carbachol as well as a large reduction in the potency of carbachol in eliciting this response. Using these data, equilibrium constants (Ka) for activation of the mAChRs by carbachol were calculated. These Ka values agreed well with Kd values of high-affinity mAChR binding sites determined from carbachol displacement of [3H]NMS binding in parotid acinar cells. Reduction in mAChR number after PBCM treatment was determined by Scatchard analysis of specific [3H]-NMS binding sites and compared with the expected reduction (q values) calculated from dose-response curves for carbachol-stimulated IP3 formation before and after PBCM treatment. PBCM (1, 3, 10, 30 nM) decreased mAChR maximal binding in cells 47.5, 68.9, 82.4, and 85.3%, respectively, which did agree with the approximately 38, 70, 90, and 92% decrease in receptor number expected from the calculated q values. Data demonstrate that PBCM irreversibly inactivates mAChRs in rat parotid cells, and the decrease in receptor number, measured directly from [3H]NMS binding or calculated from receptor theory, is greater than that observed for stimulated IP3 production. These results suggest that a modest (30-40%) population of spare receptors exists for mAChR-mediated IP3 production in rat parotid glands.

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.

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