scholarly journals Neurotensin stimulates inositol trisphosphate-mediated calcium mobilization but not protein kinase C activation in HT29 cells. Involvement of a G-protein

1989 ◽  
Vol 264 (3) ◽  
pp. 871-878 ◽  
Author(s):  
J C Bozou ◽  
N Rochet ◽  
I Magnaldo ◽  
J P Vincent ◽  
P Kitabgi
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
G Proteins ◽  
Binding Sites ◽  
Inositol Phosphate ◽  
Calcium Mobilization ◽  
Scatchard Analysis ◽  
High Affinity ◽  
Ht29 Cells ◽  
Kinase C

It has previously been shown that neurotensin binds to high-affinity receptors in the adenocarcinoma HT29 cell line, and that receptor occupancy leads to inositol phosphate formation. The present study was designed to investigate further the effects of neurotensin on calcium mobilization and protein kinase C (PKC) activation in HT29 cells, and to assess the role of GTP-binding proteins (G-proteins) in the neurotensin response. Direct measurements of cytosolic Ca2+ variations using the fluorescent indicator quin 2 showed that neurotensin (0.1-1 microM) elicited Ca2+ transients in HT29 cells. These transients occurred after the neurotensin-stimulated formation of Ins(1,4,5)P3, as measured by means of a specific radioreceptor assay. In addition, the peptide induced a decrease in the 45Ca2+ content of cells previously equilibrated with this isotope. The peptide effect was rapid, long-lasting and concentration-dependent, with an EC50 of 2 nM. Phorbol 12-myristate 13-acetate (PMA) inhibited by 50% the neurotensin effects on both intracellular Ca2+ and inositol phosphate levels. The inhibition by PMA was abolished in PKC-depleted cells. Pertussis toxin had no effect on either the Ca2+ or inositol phosphate responses to neurotensin. Epidermal growth factor (EGF) receptors which are present in HT29 cells have been shown to be down-regulated through phosphorylation by PKC in a variety of systems. Here, PMA markedly (70-80%) inhibited EGF binding to HT29 cells. Scatchard analysis revealed that PMA abolished the high-affinity component of EGF binding, an effect that was totally reversed in PKC-depleted cells. In contrast, neurotensin slightly (10-20%) inhibited EGF binding to HT29 cells, and its effect was only partly reversed by PKC depletion. Neurotensin had no detectable effect on sn-1,2-diacylglycerol levels in HT29 cells, as measured by a specific and sensitive enzymic assay. In membranes prepared from HT29 cells, monoiodo[125I-Tyr3]neurotensin bound to a single population of receptors with a dissociation constant of 0.27 nM. Sodium and GTP inhibited neurotensin binding in a concentration-dependent manner. Maximal inhibition reached 80% with Na+ and 35% with GTP.IC50 values were 20 mM and 0.2 microM for Na+ and GTP respectively. Li+ and K+ were less effective than Na+ and the effects of GTP were shared by GDP and guanosine-5′-[beta gamma- imido]triphosphate but not by ATP. Scatchard analysis of binding data indicated that Na+ and GTP converted the high-affinity neurotensin-binding sites into lower affinity binding sites. The properties of the effects of Na+ and GTP on neurotensin-receptor interactions are characteristic of those receptors which interact with G-proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Effects of phorbol esters and secretagogues on nitrobenzylthioinosine binding to nucleoside transporters and nucleoside uptake in cultured chromaffin cells

1991 ◽  
Vol 279 (3) ◽  
pp. 651-655 ◽  
Author(s):  
E G Delicado ◽  
R P Sen ◽  
M T Miras-Portugal
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Sites ◽  
Chromaffin Cells ◽  
Phorbol Esters ◽  
Binding Studies ◽  
High Affinity ◽  
Kinase C ◽  
Adenosine Uptake ◽  
Protein Kinase C Activators

Secretagogues inhibited adenosine uptake in chromaffin cells without causing apparent changes in the uptake affinity. The inhibition caused by carbachol, nicotine and acetylcholine reached 50%. This inhibition was reproduced by the action of protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA; 100 nM), phorbol 12,13-dibutyrate (PDBu; 100 nM), dicaproin (10 micrograms/ml) and tricaprylin (10 micrograms/ml), with inhibitions of Vmax. of 18, 20, 37 and 47% respectively. No changes in the affinity of uptake were observed with these effectors. Down-regulation of protein kinase C by phorbol esters decreased the inhibitory effects of carbachol on adenosine uptake. Binding studies with nitrobenzylthioinosine (NBTI) showed a similar decrease in the number of transporters when chromaffin cells were treated with the same effectors used for the uptake studies. The high-affinity dissociation constants showed minor changes with respect to the control. The ratio between maximal uptake capacity and the transporter number per cell was not significantly modified by the action of secretagogues or direct effectors of protein kinase C. The number of high-affinity binding sites for NBTI was decreased in cellular homogenates by the direct action of protein kinase C activators, with staurosporine able to reverse this action. Protein kinase C from bovine brain in the presence of ATP and effectors, decreased the number of high-affinity NBTI-binding sites in purified chromaffin cell plasma membranes. These data suggest the possibility of a molecular modification at the transporter level.

scholarly journals Activation of insulin-epidermal growth factor (EGF) receptor chimerae regulates EGF receptor binding affinity.

1992 ◽  
Vol 116 (3) ◽  
pp. 627-633 ◽  
Author(s):  
S Tartare ◽  
R Ballotti ◽  
R Lammers ◽  
C Filloux ◽  
A Chauvel ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Receptor Binding ◽  
Egf Receptor ◽  
Scatchard Analysis ◽  
Receptor Kinase ◽  
Egf Receptors ◽  
High Affinity ◽  
Kinase C

Cell surface tyrosine kinase receptors are subject to a rapid activation by their ligand, which is followed by secondary regulatory processes. The IHE2 cell line is a unique model system to study the regulation of EGF binding to EGF receptors after activation of the EGF receptor kinase. IHE2 cells express both a chimeric insulin-EGF receptor kinase (IER) and a kinase-deficient EGF receptor (HER K721A). We have previously reported that IER is an insulin-responsive EGF receptor tyrosine kinase that activates one or several serine/threonine kinases, which in turn phosphorylate(s) the unoccupied HER K721A. In this article we show that insulin through IER activation induces a decrease in 125I-EGF binding to IHE2 cells. Scatchard analysis indicates that, as for TPA, the effect of insulin can be accounted for by a loss of the high affinity binding of EGF to HER K721A. Since this receptor transmodulation persists in protein kinase C downregulated IHE2 cells, it is likely to be due to a mechanism independent of protein kinase C activation. Using an in vitro system of 125I-EGF binding to transmodulated IHE2 membranes, we illustrate that the inhibition of EGF binding induced by IER activation is related to the phosphorylation state of HER K721A. Further, studies with phosphatase 2A, or at a temperature (4 degrees C) where only IER is functional, strongly suggest that the loss of high affinity EGF binding is related to the serine/threonine phosphorylation of HER K721A after IER activation. Our results provide evidence for a "homologous desensitization" of EGF receptor binding after activation of the EGF receptor kinase of the IER receptor.

scholarly journals Scrape-loaded p21ras down-regulates agonist-stimulated inositol phosphate production by a mechanism involving protein kinase C

1989 ◽  
Vol 260 (1) ◽  
pp. 157-161 ◽  
Author(s):  
B D Price ◽  
J D H Morris ◽  
C J Marshall ◽  
A Hall
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Inositol Phosphate ◽  
Platelet Derived Growth Factor ◽  
3T3 Cells ◽  
Ras Protein ◽  
Kinase C ◽  
Inositol Phosphate Production ◽  
Ligand Stimulation ◽  
Scrape Loading

The effect of scrape-loaded [Val-12]p21ras on agonist-stimulated phosphatidylinositol 4,5-bisphosphate (PIP2) turnover in Swiss-3T3 cells was studied. Previously [Morris, Price, Lloyd, Marshall & Hall (1989) Oncogene 4, 27-31] we demonstrated that [Val-12]p21ras activates protein kinase C within 10 min of scrape loading. Here, we show that [Val-12]p21ras inhibits bombesin and platelet-derived growth factor-stimulated PIP2 breakdown 1.5-4 h after scrape loading. This effect persisted for at least 18 h and could be mimicked in control cells by activation of protein kinase C with 12-O-tetradecanoyl 13-acetate (TPA) 15 min prior to ligand stimulation. When protein kinase C was down-regulated by chronic TPA treatment, [Val-12]p21ras was no longer able to inhibit agonist-stimulated inositol phosphate production. These results indicate that changes in inositol phosphate levels caused by ras protein are probably due to activation of protein kinase C and not to an interaction of ras with phospholipase C.

scholarly journals Regulated CD44 Cleavage under the Control of Protein Kinase C, Calcium Influx, and the Rho Family of Small G Proteins

1999 ◽  
Vol 274 (36) ◽  
pp. 25525-25534 ◽  
Author(s):  
Isamu Okamoto ◽  
Yoshiaki Kawano ◽  
Mitsuhiro Matsumoto ◽  
Moritaka Suga ◽  
Kozo Kaibuchi ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
G Proteins ◽  
Calcium Influx ◽  
Kinase C ◽  
Rho Family ◽  
Small G Proteins

scholarly journals Calcium mobilization and protein kinase C activation are required for cholecystokinin stimulation of pancreatic cholesterol esterase secretion

1995 ◽  
Vol 306 (2) ◽  
pp. 605-608 ◽  
Author(s):  
J Brodt-Eppley ◽  
D Y Hui
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Digestive Enzyme ◽  
Calcium Mobilization ◽  
Cholesterol Esterase ◽  
Ionophore A23187 ◽  
Prolonged Incubation ◽  
Ar42j Cells ◽  
Kinase C ◽  
Stimulation Of

The bile salt-stimulated cholesterol esterase is a digestive enzyme synthesized by the acinar cells of the pancreas. Previous results have shown that cholesterol esterase biosynthesis and secretion in the AR42J pancreatoma cells could be increased 3-5-fold by intestinal hormones such as cholecystokinin (CCK). The purpose of the current study is to explore the signalling mechanism by which CCK stimulation of AR42J cells results in increased biosynthesis and secretion of the cholesterol esterase. The results showed that the CCK-induced cholesterol esterase secretion could be mimicked by addition of the Ca2+ ionophore A23187 or by transient incubation of AR42J cells with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). Cholesterol esterase stimulation by CCK, A23187 and PMA could be abolished by the calcium chelator BAPTA or by specific protein kinase C inhibitors such as chelerythrine. Additionally, prolonged incubation of AR42J cells with PMA to reduce the protein kinase C level, also reduced CCK-stimulated cholesterol esterase secretion to a level similar to that observed in control cells. Taken together, these data suggested that CCK activation of cholesterol esterase secretion may be mediated by a Ca(2+)-dependent protein kinase C pathway, requiring increases in calcium mobilization and activation of protein kinase C.

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