Neurotensin stimulates inositol trisphosphate - mediated calcium mobilization but not protein kinase C activation in HT 29 cells

نویسنده

  • Jean-Pierre VINCENT
چکیده

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 ,LM) 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 45Ca21 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 ofEGF binding, an effect that was totally reversed in PKC-depleted cells. In contrast, neurotensin slightly (10-20 0%) 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["251-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 /tM 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'-[/Jy-imido]triphosphate but not by ATP. Scatchard analysis of binding data indicated that Na+ and GTP converted the high-affinity neurotensinbinding 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. Altogether, our data show that neurotensin receptors in HT29 cells activate phospholipase C through a pertussis-toxin-insensitive G-protein. This activation leads to an increase in inositol phosphate levels, and this in turn results in Ca21 mobilization. The other branch of the bifurcating phospholipase C pathway, i.e. the activation of PKC by diacylglycerol, does not seem to be significantly operated by neurotensin. The physiological implications of these findings are discussed.

برای دانلود رایگان متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Long lasting inhibition of adenylyl cyclase selectively mediated by inositol 1,4,5-trisphosphate-evoked calcium release.

In A7r5 smooth muscle cells, vasopressin stimulates release of Ca2+ from intracellular stores and Ca2+ entry, and it inhibits adenylyl cyclase (AC) activity. Inhibition of AC is prevented by inhibition of phospholipase C or when the increase in cytosolic [Ca2+] is prevented by the Ca2+ buffer, BAPTA. It is unaffected by pertussis toxin, inhibition of protein kinase C, or L-type Ca2+ channels or...

متن کامل

Angiotensin II stimulation of vascular smooth muscle phosphoinositide metabolism. State of the art lecture.

Phosphoinositide hydrolysis is an integral step in the activation of vascular smooth muscle by angiotensin II. Sequential phospholipase C-mediated hydrolysis of the polyphosphoinositides and phosphatidylinositol in cultured vascular smooth muscle cells stimulated with angiotensin II results in a coordinated series of biochemical events: a transient formation of inositol trisphosphate associated...

متن کامل

Angiotensin II Stimulation of Vascular Smooth Muscle Phosphoinositide Metabolism

Phosphoinositide hydrolysis is an integral step in the activation of vascular smooth muscle by angiotensin II. Sequential phospholipase C-mediated hydrolysis of the polyphosphoinositides and phosphatidylinositol in cultured vascular smooth muscle cells stimulated with angiotensin II results in a coordinated series of biochemical events: a transient formation of inositol trisphosphate associated...

متن کامل

5-Hydroxytryptamine induces phospholipase C-mediated hydrolysis of phosphoinositides through 5-hydroxytryptamine-2 receptors in cultured fetal mouse ventricular myocytes.

5-Hydroxytryptamine (5-HT) stimulates the rate and force of cardiac contraction. However, the molecular mechanisms of 5-HT actions on the heart are unknown. We examined effects of 5-HT on phospholipase C-mediated hydrolysis of phosphoinositides and its regulation in cultured fetal mouse ventricular myocytes labeled with [3H]inositol. Accumulation of inositol monophosphate, inositol bisphosphate...

متن کامل

Protein kinase C activation inhibits receptor- evoked inositol trisphosphate formation and induction of cytosolic calcium oscillations by decreasing the affinity-state of the cholecysto- kinin receptor in pancreatic acinar cells

Digital-imaging microscopy of Fura-2-loaded pancreatic acinar cells revealed that the C-terminal octapeptide of cholecystokinin (CCKe) dose-dependently recruited 94% of freshly isolated acinar cells in terms of receptor-evoked Ca2+ mobilization. Maximal and half-maximal cell-recruitment were reached with 0.1 nM and 16.8 pM CCKe, respectively. The upstroke of the dose-recruitment curve consisted...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

عنوان ژورنال:

دوره   شماره 

صفحات  -

تاریخ انتشار 2005