Regulation of eicosanoid biosynthesis by phorbol ester in Madin Darby canine kidney cells

1990 ◽  
Vol 259 (4) ◽  
pp. F698-F703 ◽  
Author(s):  
D. W. Coyne ◽  
M. Mordhorst ◽  
A. R. Morrison
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Mdck Cells ◽  
Pge2 Production ◽  
Madin Darby Canine Kidney ◽  
Kinase C ◽  
Protein Kinase C Inhibitor ◽  
Peptide Agonist ◽  
Darby Canine Kidney ◽  
Canine Kidney

We assessed the effects of the peptide agonist, bradykinin (BK), and phorbol myristate acetate (PMA) on prostaglandin E2 (PGE2) production, cyclooxygenase (COX) activity and mass, and arachidonic acid (AA) release in Madin Darby canine kidney (MDCK) cells. PMA stimulated PGE2 production by increasing both AA release and the activity of COX. Using [35S]methionine labeling and immunoprecipitation, we demonstrated that the increased COX activity is due to new COX synthesis. Actinomycin D and cycloheximide blocked the PMA-stimulated COX activity but not AA release. Both PMA-stimulated AA release and COX activity were reduced by the protein kinase C inhibitor staurosporine (STP). Glucocorticoids failed to alter PMA- or BK-stimulated PGE2 production was reduced by STP, indicating BK acts in part through protein kinase C activation. BK increased PGE2 production in PMA-treated cells, suggesting a protein kinase C-independent mechanism of action as well. BK did not stimulate any change in COX activity. We conclude that in MDCK cells PMA, but not BK, can stimulate both AA release and COX synthesis. Stimulation of COX synthesis requires either prolonged activation of protein kinase C and/or an additional nonprotein kinase C-mediated effect of PMA.

scholarly journals Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells

2003 ◽  
Vol 373 (3) ◽  
pp. 917-924 ◽  
Author(s):  
Jorge CERBÓN ◽  
Rosa del Carmen LÓPEZ-SÁNCHEZ
Keyword(s):  
Cell Proliferation ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Fold Increase ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Kinase C ◽  
Pkc Activation ◽  
Darby Canine Kidney ◽  
Canine Kidney

We have investigated the effects of inhibiting sphingomyelin (SM) biosynthesis on cellular diacylglycerol (DAG) content and protein kinase C (PKC) activation during growth initiation in Madin–Darby canine kidney cells. We utilized β-chloroalanine (BCA) to inactivate serine C-palmitoyltransferase, the first enzyme in the sphingolipid biosynthesis pathway. This inactivation prevented growth, but did not affect viability. When the inhibitor was replaced with fresh culture medium, the cells continued their proliferation in a normal way. BCA (2 mM) inhibited [32P]Pi, [3H]palmitic acid and [methyl-3H]choline incorporation into SM, but did not influence the synthesis of other major phospholipids. SM synthesis and DAG generation were decreased by 51% and 47.6% respectively. Particulate PKC activity was not observed in cells incubated with BCA, in contrast with a 5-fold increase in control cells. BCA inhibited 75% of the [3H]thymidine incorporation, and the cells were arrested before the S phase of the cell cycle. Moreover, exogenous d-erythrosphingosine restored SM synthesis, DAG generation and cell proliferation. These data indicate that the contribution of DAG generated during SM synthesis plays an important role in PKC activation and cell proliferation.

scholarly journals Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover

2017 ◽  
Vol 28 (25) ◽  
pp. 3595-3608 ◽  
Author(s):  
Anastasia F. Thévenin ◽  
Rachel A. Margraf ◽  
Charles G. Fisher ◽  
Rachael M. Kells-Andrews ◽  
Matthias M. Falk
Keyword(s):  
Protein Kinase ◽  
Gap Junction ◽  
Wild Type ◽  
Madin Darby Canine Kidney ◽  
Kinase C ◽  
Zonula Occludens ◽  
Zonula Occludens 1 ◽  
Darby Canine Kidney ◽  
Canine Kidney

To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series of phosphomimetic and phosphorylation-dead mutants by mutating known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373 located in the C-terminal domain of connexin43 (Cx43) into glutamic acid (E) or alanine (A) residues. All connexin mutants were translated into stable, full-length proteins and assembled into GJs when expressed in HeLa or Madin–Darby canine kidney epithelial cells. However, mutants with S residues exchanged at positions 365, 368, and 373 exhibited a significantly altered ZO-1 interaction profile, while mutants with S residues exchanged at 255 and 279/282 did not. Unlike wild-type Cx43, in which ZO-1 binding is restricted to the periphery of GJ plaques, S365A, S365E, S368A, S368E, and S373A mutants bound ZO-1 throughout the GJ plaques, while the S373E mutant did not bind ZO-1 at all. Inability to disengage from ZO-1 correlated with increased GJ plaque size and increased connexin protein half-life, while maintaining GJ channels in an open, functional state. Quantitative clathrin-binding analyses revealed no significant alterations in clathrin-binding efficiency, suggesting that the inability to disengage from ZO-1 prevented maturation of functional into nonfunctional/endocytic channels, rather than ZO-1 interfering with GJ endocytosis directly. Collectively, our results indicate that ZO-1 binding regulates channel accrual, while disengagement from ZO-1 is critical for GJ channel closure and transitioning GJ channels for endocytosis. Intriguingly, these transitional ZO-1 binding/release and channel-aging steps are mediated by a series of hierarchical phosphorylation/dephosphorylation events at S373, S365, and S368, well-known Cx43 Akt, protein kinase A, and protein kinase C phosphorylation sites located in the vicinity of the ZO-1 binding site.

scholarly journals Acute Activation of the Renal Betaine/GABA Transporter in Response to a Decrease in Extracellular Calcium

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Nehal R. Parikh ◽  
Cherissa L. Vaughn ◽  
Lagina L. Williams ◽  
Stephen A. Kempson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Growth Medium ◽  
Mdck Cells ◽  
Gaba Transporter ◽  
Cell Monolayers ◽  
Kinase C ◽  
Dependent Inhibition ◽  
Intracellular Ca ◽  
Protein Kinase C Inhibitor

The betaine/GABA transporter (BGT1) is important for osmoprotection in kidney medullary cells. We previously reported an acute (30 min) increase in extracellular Ca2+ caused dose dependent inhibition of BGT-1 in renal MDCK cells. To determine if extracellular Ca2+ might be a local regulator of BGT-1, we have tested the response to low Ca2+ serum-free growth medium (LCM, 0.05 mM Ca2+). Chronic treatment (8–24 h) of MDCK cell monolayers completely blocked hypertonic adaptation of BGT1 and disrupted tight junctions. In contrast, acute treatment activated BGT1 transport within 30 min in MDCK cells previously adapted to hypertonic growth medium containing normal Ca2+ (1.6 mM). Activation was significant after 60–90 min and was independent of medium osmolarity. Peak transport was increased 50% in isotonic LCM and 100% in hypertonic (500 mOsm) LCM over controls. The activation was reversed by restoration of normal Ca2+. Perfusion of Fura-2-loaded MDCK cells with LCM decreased intracellular Ca2+ by 31% within 6-7 min. Inclusion of staurosporine (0.6 μM), a protein kinase C inhibitor, potentiated the action of LCM. We suggest that activation of BGT1 by LCM may be due in part to inhibition of protein kinase C.

Role of PLA2, PLC, and PLD in bradykinin-induced release of arachidonic acid in MDCK cells

1996 ◽  
Vol 271 (4) ◽  
pp. C1064-C1072 ◽  
Author(s):  
C. R. Kennedy ◽  
P. R. Proulx ◽  
R. L. Hebert
Keyword(s):  
Arachidonic Acid ◽  
Mdck Cells ◽  
Pla2 Activity ◽  
Madin Darby Canine Kidney ◽  
Cytosolic Phospholipase ◽  
Cell Extracts ◽  
Kinase C ◽  
Darby Canine Kidney ◽  
Canine Kidney

The role of cytosolic phospholipase A2 (cPLA2), phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) in the bradykinin (BK)-stimulated release of arachidonic acid (AA) was examined in Madin-Darby canine kidney (MDCK) cells. Release of AA, phosphorylcholine, choline, and phosphatidic acid (PA) or the transphosphatidylation product, phosphatidylethanol, was detected after 1 min of BK stimulation. A role for PC-PLC was confirmed with D609, which reduced BK-stimulated AA by 70%. Ethanol (EtOH), which blunts PA formation, diminished BK-stimulated AA release by 50%. Together, D609 and EtOH inhibited this release almost completely. Evidence indicated that diacylglycerol and PA can enhance PLA2 activity when added to cytosol extracts. The enzyme responsible for AA release was characterized as cPLA2, since PLA2 activity assayed in cell extracts was largely inhibited by an antibody to this enzyme. The membrane fraction PLA2 activity increased significantly in BK-stimulated cells. We conclude that BK signaling in MDCK cells is mediated by the lipid products of PC-PLC and PLD, increasing cPLA2 activity, possibly by causing perturbations in the bilayer structure of its substrate, by a direct effect on the enzyme or by activation of protein kinases such as protein kinase C.

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