Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

LLC-PK1/PKE20 cells (a continuous epithelial cell line) has two different Na/H exchange activities: Na/H-1 located in the basolateral membrane and Na/H-2 located in the apical membrane [Casavola et al. (1989) Biochem Biophys Res Commun 165:833-837; Haggerty et al. (1988) Proc Natl Acad Sci USA 86:6797-6801]. In the present report we have studied hormone regulation of these exchange activities by measuring Na-dependent recovery of pHi from an acid load (by using microspectrofluorometry and 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein) in response to activation of regulatory cascades by either pharmacological agents or by vasopressin or calcitonin. Agents leading to activation of protein kinase A (cAMP-dependent), such as forskolin (10 microM), 8-Br-cAMP (0.25 mM), and isobutylmethylxanthine (0.5 mM), inhibited Na/H-2 and Na/H-1 by an average of 49%. Stimulation of protein kinase C by a phorbol ester (phorbol 12-myristate 13-acetate, TPA, 100 nM) inhibited Na/H-2 (by an average of 48%) and stimulated Na/H-1 (by an average of 38%); these effects of TPA were also observed in the presence of forskolin (100 microM). Addition of either vasopressin (2 microM) or calcitonin (0.3 microM) onto both sides of the monolayer decreased the activity of Na/H-2 by an average of 26.3% and 27.7% respectively, and stimulated the activity of Na/H-1 by an average of 17.4% and 38.7% respectively; exposure of cells to either hormone stimulated production of cAMP and inositol trisphosphate, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Polarized expression of Na+/H+ exchange activity in LLC-PK1/PKE20 cells: II. Hormonal regulation. 131 51

Calcitonin (CT) activates both the cAMP and the protein kinase C (PKC) pathways in the kidney cell line LLC-PK1. Although CT also activates cAMP in osteoclasts, its effects on PKC in this cell type are unknown. In order to determine whether the response of osteoclasts to CT also involves the PKC pathway, the effects of activators and inhibitors of PKC on bone resorption and cell surface area were analyzed in isolated rat osteoclasts. As expected, CT inhibited in a dose-dependent manner bone resorption by rat osteoclasts cultured for 24 h on devitalized bovine bone slices and this effect could be mimicked by cAMP. The inhibitory effect of CT could however also be mimicked by phorbol-12,13-dibutyrate (PDBu) and blocked by the PKC inhibitor sphingosine, as well as by the less specific inhibitors H7 and H8, none of which had detectable effects in the absence of CT. No changes in the number of attached osteoclasts were observed under any of these conditions. These results indicate that CT activates PKC in osteoclasts and that this activation, like the activation of cAMP-dependent protein kinase, leads to an inhibition of bone resorption. Quantitative time-lapse videomicroscopy showed that the CT-induced retraction of osteoclasts also involved activation of the PKC pathway and could therefore be induced by phorbol esters. In contrast, (Bu)2 cAMP (1-200 microM) failed to induce rapid cell retraction. It is concluded that, in osteoclasts, CT receptors are coupled to both the cAMP-dependent protein kinase and the PKC pathways. Although these two second messengers can have additive inhibitory effects on bone resorption, only activation of the PKC pathway induces rapid cell retraction. These two effects of calcitonin on osteoclasts are therefore independent and may be functionally unrelated.
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PMID:Differential effects of the 3',5'-cyclic adenosine monophosphate and protein kinase C pathways on the response of isolated rat osteoclasts to calcitonin. 132 63

Calcium uptake by the endoplasmic reticulum (ER) is important for cellular calcium homeostasis, yet its regulation in nonmuscle cells is poorly understood. We reported that Ca2+ uptake by a light fraction of canine renal cortical ER (LER) is stimulated by protein kinase C in vitro. Here we describe conditions in vivo that stimulate renal cortical LER Ca2+ uptake. Thirty minutes after contralateral nephrectomy in the dog, 45Ca2+ uptake into renal cortical LER was increased 42% above control LER. There was no difference in LER Ca2+ uptake 24 hours after uninephrectomy. Acute denervation did not reproduce the increase in LER 45Ca2+ uptake seen at 30 minutes after uninephrectomy, nor did prior thyroparathyroidectomy abolish it. Forty-eight hours after thyroparathyroidectomy, 45Ca2+ uptake activity into renal cortical LER was decreased approximately sevenfold. In a proximal tubular cell line (LLC-PK1), 30-minute incubation with 12-O-tetradecanoylphorbol-13-acetate doubled 45Ca2+ uptake into a nonmitochondrial pool. Pretreatment with epidermal growth factor halved ER Ca2+ uptake, whereas insulin-like growth factor and growth hormone, alone or in combination, had no effect. Our data suggest that Ca2+ uptake into renal cortical ER is stimulated acutely during compensatory renal growth, perhaps through protein kinase C, and is stimulated chronically by parathyroid hormone.
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PMID:Ca2+ uptake by endoplasmic reticulum of renal cortex. II. Effects of uninephrectomy and parathyroidectomy. 139 76

Although several cytokines have been demonstrated to exert pleiotropic responses, there is little information on cytokine regulation of renal tubular epithelial cell function. In the present studies, we find that both T cell-derived (tumor necrosis factor-beta and interleukins 2 and 3) and monocyte/macrophage derived (tumor necrosis factor alpha and interleukin 1 beta) cytokines promote basal, arginine vasopressin- and forskolin-stimulated adenylate cyclase activity in cultured LLC-PK1 cells. No effect of TNF, IL-1 beta, and IL-2 to stimulate protein kinase C activity was observed. TNF-beta, IL-1 beta and IL-2 also modestly stimulated 3H release from 3H-arachidonic acid labeled cells. Mepacrine, a phospholipase A inhibitor, prevented TNF-beta stimulation of 3H release from 3H-arachidonic acid labeled cells and TNF-beta potentiation of adenylate cyclase activity. TNF-beta potentiation of adenylate cyclase activity and stimulation of 3H release from 3H arachidonic acid labeled cells was not prevented by pertussis toxin. These results demonstrate that several cytokines can stimulate adenylate cyclase activity while not affecting protein kinase C activity in cultured renal tubular epithelial cells. The effect of TNF-beta to stimulate adenylate cyclase appears to occur independent of pertussis toxin-sensitive substrate and may involve activation of phospholipase A.
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PMID:Cytokine regulation of adenylate cyclase activity in LLC-PK1 cells. 140 34

We recently reported that mild hypoxia in LLC-PK1 cells, grown in standard fashion under a still layer of overlying medium at 5% CO2/18% O2 environment, result in decreased oxidative metabolism and impaired differentiated functions in comparison to adequately oxygenated cultures maintained either under a higher oxygen (36% O2) environment or conditions of continuous rocking of the media fluid. In the present study, subcellular distribution of a regulatory enzyme protein kinase C (PKC) was examined between hypoxic still and normoxic rocked LLC-PK1 cells. Subconfluent cultures of hypoxic LLC-PK1 cells exhibited significantly lower and predominantly membrane-bound PKC activity in comparison to mostly cytosolic localization of this enzyme in normoxic rocked cells. One hour of exposure of adequately oxygenated-rocked LLC-PK1 cells with the phorbol ester TPA, a dedifferentiating agent that did not effect the cell ATP content, resulted in significant inhibition of dome formation and sodium-dependent glucose transport activity, a partial loss of pH-responsive ammoniagenesis, and almost complete translocation of protein kinase C activity from cytosol to the membrane pool; all of which resembles the behavior of hypoxic still cultured cells. In addition, acute re-oxygenation of hypoxic still cultures by rocking the media fluid for one hour resulted in an increase in cell ATP content to the cellular levels of ATP observed in normoxic rocked cells. However, all the parameters of differentiation were unaffected by re-oxygenation. These studies support the notion that hypoxia can act in some primary fashion, independent of its effects on energy metabolism, to impair cellular differentiation in LLC-PK1 cells. They also raise the possibility that activation of protein kinase C may act as an important mediator in this process.
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PMID:Hypoxia-mediated impaired differentiation by LLC-PK1 cells: evidence based on the protein kinase C profile. 145 99

In previous experiments we have shown that acute (30 minutes) exposure to phorbol esters or other protein kinase C activators causes increased transepithelial permeability, specifically by the increased paracellular permeability through tight junctions. However, the role of protein kinase C activators in carcinogenesis is predicted upon a chronic exposure of an effective dose at frequent intervals for a prolonged period of time. We therefore sought to determine the effect of chronic phorbol ester exposure on transepithelial permeability by exposing cells of the polar renal epithelial cell line, LLC-PK1, to phorbol esters for time periods as long as 16 weeks. The following changes ensued: (1) after the initial drop in transepithelial resistance due to phorbol ester exposure, i.e., an increase in transepithelial permeability (in the acute phase of exposure), an adaptive response occurs as transepithelial resistances in chronically exposed cultures recover to approximately 50% of control values, (2) the cell sheets in chronically exposed cultures lose their acute responsiveness of transepithelial permeability to phorbol ester exposure, (3) cell sheet architecture changes as cells occasionally multilayer and actual polyp-like cell masses appear at high frequency, and (4) cytosolic protein kinase C activity decreases to 50% of control level with acute exposure and then is further decreased to less than 1% of control level in chronically treated cells; membrane-associated PKC activity is not as sharply decreased. The possible role of transepithelial permeability in carcinogenesis and the value of chronically treated epithelial cell cultures as a model for two-stage carcinogenesis are discussed.
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PMID:Effects of acute vs. chronic phorbol ester exposure on transepithelial permeability and epithelial morphology. 161 21

Subconfluent cultures of LLC-PK1 cells were incubated for 1 h in Krebs-Henseleit buffer (KHB) of pH 7.4 or 6.8 to investigate the signal transduction events associated with prostaglandin F2 alpha (PGF2 alpha) inhibition of ammonia metabolism. Exposure of these cultures to PGF2 alpha (0.1 ng/ml) inhibited the acute low pH stimulation of ammonia production and to a lesser degree alanine formation in a manner analogous to the response exhibited with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Pretreatment with an inhibitor of protein kinase C [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, i.e., H-7] or utilization of cultures with downregulated protein kinase C activity abolished the inhibitory response to PGF2 alpha. Exposure to PGF2 alpha for 10 min in KHB of pH 6.8 resulted in an activation of protein kinase C, as demonstrated by a significant increase in membrane-bound enzyme activity. Incubation of the cells with PGF2 alpha in KHB of pH 6.8 also resulted in a significant increase in inositol trisphosphate formation. Treatment of the cultures with verapamil in calcium-containing medium or removal of calcium from the incubating medium resulted in a significant loss of the PGF2 alpha inhibitory response on both ammonia and alanine production. Furthermore, under conditions of calcium-free incubation, PGF2 alpha had no significant effect on protein kinase C activity. Because both PGF2 alpha- and TPA-induced inhibition of ammoniagenic response to acute acidosis was prevented by amiloride, the underlying mechanism may involve protein kinase C-mediated changes in intracellular pH. These results indicate that the activation of protein kinase C plays a key role in mediating PGF2 alpha inhibition of ammoniagenesis.
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PMID:Signal transduction events whereby PGF2 alpha inhibits the ammoniagenic response to acute acidosis. 162 19

The present studies examined effects of ATP depletion and calmodulin antagonism on stimulation of Na(+)-H+ exchange by cytosolic acidification in renal epithelial cells (LLC-PK1). ATP depletion significantly inhibited both amiloride-sensitive 22Na+ uptake (P less than 0.001; n = 12) and Na(+)-dependent intracellular pH (pHi) recovery in 2',7'-bis (carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester (BCECF/AM)-loaded cells. Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) and calmidazolium, both caused a concentration-dependent inhibition of Na(+)-H+ exchange activity. The W-7-induced inhibition of Na(+)-H+ exchange occurred in cells incubated for 24 h with phorbol 12-myristate 13-acetate, indicating that the effect of W-7 was not mediated by protein kinase C inhibition. Both W-7 and ATP depletion shifted the pHi dependence of the antiporter, and ATP depletion also reduced the maximal activity. In LLC-PK1/CL4 cells grown on permeable filters, W-7 inhibited the cytosolic acidification-stimulated basolateral exchanger by 54 +/- 5% (P less than 0.005; n = 7) and, in contrast, stimulated the apical exchanger by 28 +/- 13% (P less than 0.05; n = 6). ATP depletion significantly inhibited apical Na(+)-H+ exchange. These results suggest that an ATP-Ca(2+)-calmodulin-dependent process is involved in regulation of Na(+)-H+ exchange in LLC-PK1 cells. A Ca(2+)-calmodulin-dependent process activated the amiloride-sensitive basolateral Na(+)-H+ exchanger and inhibited the amiloride-resistant apical antiporter. Phosphorylation of these two Na(+)-H+ exchangers or regulatory proteins by a Ca(2+)-calmodulin-dependent protein kinase may mediate this differential regulation.
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PMID:Regulation of Na(+)-H+ exchange by ATP depletion and calmodulin antagonism in renal epithelial cells. 165 80

Exposure of intact LLC-PK1 cells to the phorbol ester 4-phorbol 12-myristate 13-acetate (PMA) increases basal, arginine vasopressin-stimulated, and forskolin-stimulated adenylate cyclase activity in LLC-PK1 membranes. This observation suggests that protein kinase C can increase adenosine 3',5'-cyclic monophosphate (cAMP) in LLC-PK1 cells. To determine whether cAMP regulates protein kinase C activity in LLC-PK1 cells, intact cells were exposed to either forskolin or to soluble cAMP analogues. Acute (5 and 30 min) exposure to either forskolin or cAMP analogues increases protein kinase C activity as observed by two different methods for measuring protein kinase C. Acute exposure to PMA translocates protein kinase C from a soluble to a particulate cell fraction, whereas acute exposure to cAMP increases both soluble and particulate forms of protein kinase C. Longer exposure (18 h) to PMA results in a loss of protein kinase C activity, whereas 18-h exposure to cAMP results in a further increase in protein kinase C activity. The effect of cAMP but not of PMA to stimulate protein kinase C activity can be attenuated by the pro-R diastereoisomer of adenosine 3',5'-cyclic phosphorothioate, suggesting a protein kinase A-mediated effect. These results suggest the presence of a monodirectional mode of signal transduction system interaction in LLC-PK1 cells in which protein kinase C and protein kinase A can potentiate each other.
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PMID:cAMP stimulates protein kinase C activity in cultured renal LLC-PK1 cells. 166 Oct 84

Research on palytoxin focuses on its action as a tumor promoter and its ionophoretic action in cell membranes. The first property is unusual because palytoxin is not a protein kinase C activator. The second property is remarkable in that it may require interaction with the Na(+)-K(+)-adenosinetriphosphatase (ATPase). Our studies here with palytoxin exposure to the LLC-PK1 renal epithelial cells have yielded the following results: 1) unlike protein kinase C-activating tumor promoters (tetradecanoylphorbol 12,13-acetate or teleocidin), palytoxin does not produce a specific effect on the tight junctions between epithelia; 2) palytoxin instead produces an irreversible cytotoxic effect characterized by a pronounced cell swelling associated with sharply elevated levels of intracellular Na+ and decreased levels of intracellular K+; 3) these fluctuations in intracellular Na+ and K+ levels are explained by marked elevations in the membrane flux of 22Na+ and 86Rb+; 4) the electrophysiological reflection of these altered ion fluxes is a pronounced depolarization of the cell sheet if palytoxin is presented to the basolateral cell surface and a pronounced hyperpolarization (due to sharply elevated apical Na+ flux and transepithelial short-circuit current) if palytoxin is administered apically; 5) the apical effect of palytoxin can be blocked by apical ouabain; and 6) this apical effect of palytoxin decreases as a function of the age of the cell sheet. This first report of palytoxin action in a polar epithelial cell system provides additional evidence for palytoxin effects being mediated by contact with the Na(+)-K(+)-ATPase. It also adds to a growing literature suggesting the existence of Na(+)-K(+)-ATPase in the apical cell surface of epithelia under certain conditions.
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PMID:Effects of apical vs. basolateral palytoxin on LLC-PK1 renal epithelia. 167 41


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