scholarly journals The role of protein kinase C in carbachol-induced and of cAMP-dependent protein kinase in isoproterenol-induced secretion in primary cultured guinea pig parotid acinar cells

1996 ◽  
Vol 314 (1) ◽  
pp. 181-187 ◽  
Author(s):  
Karsten MÖLLER ◽  
Diane BENZ ◽  
Dominique PERRIN ◽  
Hans-Dieter SÖLING
Keyword(s):  
Protein Kinase ◽  
Guinea Pig ◽  
Acinar Cells ◽  
Down Regulation ◽  
Dependent Protein Kinase ◽  
Parotid Acinar Cells ◽  
Kinase C ◽  
Dependent Protein ◽  
External Calcium ◽  
Stimulation Of

Stimulation of secretion by muscarinic agonists in guinea pig parotid or pancreatic acini is accompanied by a translocation of protein kinase C (PKC) from the cytosol to the particulate fraction [Machado-De Domenech and Söling (1987) Biochem. J. 242, 749–754] and by a PKC-mediated phosphorylation of the ribosomal protein S6 [Padel and Söling (1985) Eur. J. Biochem. 151, 1–10]. In order to decide whether PKC is directly involved in the secretory process, the effect of down regulation of PKC by phorbol 12-myristate 13-acetate (PMA) was studied in primary cultured guinea pig parotid acinar cells. These cells secrete in response to carbachol and isoproterenol. Only the carbachol response is associated with an increase in cytosolic calcium. Carbachol plus isoproterenol lead to an over-additive stimulation of secretion, an effect which depends completely on the presence of external calcium. Down regulation of PKC by about 90% did not significantly affect carbachol-induced exocytosis, whereas isoproterenol-stimulated secretion was almost doubled. The secretory response to permeable cAMP analogues was also enhanced in PKC-down-regulated acini, indicating a post-receptor effect. The increased response to isoproterenol was also observed in the absence of external calcium. The isoproterenol effect was significantly inhibited by the relatively specific cAMP-dependent protein kinase inhibitor H-89, which had only a minor effect on carbachol-induced exocytosis. Although down regulation of total PKC by up to 90% did not significantly affect the secretory response to carbachol, RO 31-8220, a relatively specific inhibitor of PKC, abolished carbachol-induced secretion in normal as well as in PMA-down-regulated cells. This indicates that a PKC isoform resistant to down regulation by PMA is involved in carbachol- but not in cAMP-mediated secretion.

ANP stimulates phospholipase C in cultured RIMCT cells: roles of protein kinases and G protein

1991 ◽  
Vol 260 (4) ◽  
pp. F590-F595 ◽  
Author(s):  
T. Berl ◽  
J. Mansour ◽  
I. Teitelbaum
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinases ◽  
G Protein ◽  
Pertussis Toxin ◽  
Cyclic Nucleotide ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Dependent Protein ◽  
Stimulation Of

We examined the possibility that, in addition to stimulation of guanylate cyclase (GC), atrial natriuretic peptide (ANP) also activates phospholipase C (PLC) in cultured rat inner medullary collecting tubule (RIMCT) cells. ANP (10(-12)M) causes marked release of inositol trisphosphate (IP3) at a concentration that does not stimulate GC. Concentrations of ANP that stimulate GC (greater than or equal to 10(-10) M) result in attenuated IP3 release. Similarly, exogenous dibutyryl guanosine 3',5'-cyclic monophosphate (10(-6) M) markedly inhibits the response to 10(-10) M ANP. Inhibition of cyclic nucleotide-dependent protein kinase by H 8, but not inhibition of protein kinase C by H 7, restores the response to 10(-8) and 10(-6) M ANP. Therefore, activation of cyclic nucleotide-dependent protein kinase inhibits ANP-stimulated PLC activity. Activation of protein kinase C by phorbol 12-myristate-13-acetate (PMA) decreases ANP-stimulated IP3 production. Pretreatment with H 7, but not H 8, prevents inhibition by PMA. To explore a potential role for G proteins, we examined the effect of guanine nucleotide analogues on ANP-stimulated IP3 production in saponin-permeabilized cells. ANP-stimulated IP3 production is enhanced by GTP gamma S and is inhibited by GDP beta S. Similarly, preincubation with pertussis toxin prevents ANP-stimulated IP3 release. We conclude that ANP stimulates PLC in RIMCT cells via a pertussis toxin-sensitive G protein. Stimulation of PLC is inhibited on activation of either cyclic nucleotide or Ca2+-phospholipid dependent protein kinases.

scholarly journals Involvement of Ca2+/calmodulin-dependent protein kinase II in the activation of carnitine palmitoyltransferase I by okadaic acid in rat hepatocytes

1997 ◽  
Vol 321 (1) ◽  
pp. 211-216 ◽  
Author(s):  
Guillermo VELASCO ◽  
Manuel GUZMÁN ◽  
Victor A. ZAMMIT ◽  
Math J. H. GEELEN
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Specific Inhibitor ◽  
Rat Hepatocytes ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Dependent Protein ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I ◽  
Stimulation Of

The present work was undertaken to study the mechanism by which okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, stimulates carnitine palmitoyltransferase I (CPT-I) in isolated rat hepatocytes [Guzmán, Kolodziej, Caldwell, Costorphine and Zammit (1994) Biochem. J. 300, 693–699]. The OA-induced stimulation of CPT-I was abolished by the general protein kinase inhibitor K-252a as well as by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CM-PKII). However, neither the protein kinase C-specific inhibitor bisindolylmaleimide nor the protein kinase A/protein kinase C inhibitor H-7 was able to prevent the OA-induced stimulation of CPT-I. Hepatocyte-shrinkage-induced stimulation of CPT-I as well as OA-induced hepatocyte shrinkage was prevented by KN-62. KN-62 also antagonized the OA-enhanced release of lactate dehydrogenase from digitonin-permeabilized hepatocytes. Exposure of 32P-labelled hepatocytes to OA increased the degree of phosphorylation of Ca2+/CM-PKII, as immunoprecipitated by a monoclonal antibody raised against the α-subunit of rat brain kinase. This effect of OA was also antagonized by KN-62. The results thus indicate that the OA-dependent stimulation of CPT-I may be mediated (at least in part) by increased phosphorylation and subsequent activation of Ca2+/CM-PKII.

Calcium-activated, phospholipid-dependent protein kinase in pancreatic acinar cells

1985 ◽  
Vol 248 (6) ◽  
pp. G692-G701 ◽  
Author(s):  
M. Noguchi ◽  
H. Adachi ◽  
J. D. Gardner ◽  
R. T. Jensen
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Calcium Dependent ◽  
Kinase C ◽  
Dependent Protein

In the present study we partially purified calcium-activated, phospholipid-dependent protein kinase (protein kinase C) from pancreatic acinar cells of the guinea pig using diethylaminoethylcellulose and Sephadex G-150 chromatography and characterized the dependence of the enzyme on calcium, phospholipids, diacylglycerol (diolein), and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). The enriched preparation of protein kinase C contained no cyclic nucleotide-dependent or calcium-dependent, calmodulin-dependent protein kinase activity. The values of Km for H1-histone and ATP were 0.74 +/- 0.22 and 13.1 +/- 3.2 microM, respectively. Pancreatic protein kinase C demonstrated an absolute requirement for calcium and phospholipid for its activation, and diolein or TPA increased the affinity of the enzyme for calcium by 10-fold. With phosphatidylserine the calcium concentration that caused a half-maximal activation (Ka) was 74 +/- 17 microM, whereas with phosphatidylserine and diolein or TPA the Ka for calcium was 7.9 +/- 1.6 or 6.8 +/- 1.3 microM, respectively. Adding phosphatidylethanolamine and phosphatidylserine decreased the Ka for calcium to 2.0 +/- 0.9 microM with diolein and to 0.7 +/- 0.4 microM with TPA. Activation of protein kinase C by TPA and diolein was identical with calcium concentrations greater than 1 microM, but at low calcium concentrations (less than 1 microM) in the presence of phospholipids, maximally effective concentrations of diolein caused only 55% of the activation seen with TPA. In addition to TPA, other phorbol esters such as phorbol dibutyrate and phorbol diacetate, but not phorbol itself, activated protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

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