Elsevier

Cellular Signalling

Volume 9, Issue 7, November 1997, Pages 505-512
Cellular Signalling

Angiotensin II-Induced Inositol Phosphate Generation is Mediated Through Tyrosine Kinase Pathways in Cardiomyocytes

https://doi.org/10.1016/S0898-6568(97)00008-9Get rights and content

Abstract

The objective of this study was to determine whether the G-protein-linked angiotensin II receptor mediated inositol phosphate production involves a tyrosine phosphorylation (tyr phos) dependent pathway in the heart. Cardiomyocytes, in culture, from 7-day-old chick embryonic hearts were incubated with myo [3H] inositol for 18–24 h. Cells were incubated with LiCl to inhibit inositol 1-phosphate phosphatase and allow accumulation of inositol phosphates with angiotensin II (ang II) treatment. Inositol fractions were separated on column chromatography. Ang II produced significant (p < 0.01) increases of InsP1, InsP2, and InsP3, within 1 min of treatment of cardiomyocytes. Tyrosine kinase inhibition with genistein significantly (p < 0.05) reduced ang II induced inositol phosphate production. This did not occur with the analogue diazdien that is a very weak inhibitor of tyrosine kinase. The ability of ang II to induce tyr phos was demonstrated in whole cell lysates of cardiomyocytes immunoprecipitation with anti-P-Tyr antibodies. Genistein blunted this action of ang II. The rapid activation of a tyr phos dependent pathway by ang II was demonstrated by the similar time course of tyr phos of two different cardiac proteins, 70 and 195 kDa, and peak inositol phosphate production. Tyr phos of these cardiac proteins was mediated predominantly but not exclusively through the AT1 ang II receptor subtype as it was completely blocked by the AT1 antagonist losartan, while the AT2 receptor antagonist PD123319 blunted ang II-induced tyr phos. These results demonstrate a novel role for a tyr phos dependent pathway in the heart for ang II-induced inositol phosphate production and strengthens the concept of the interaction of G-protein coupled receptors with tyrosine kinases.

Introduction

The intracellular signal transduction mechanisms whereby angiotensin II (ang II), produces its cellular effects continue to be an area of intense interest. AngII stimulates multiple distinct signalling pathways including production of inositol 1,4,5-triphosphate (InsP3) 1, 2, 3, 4, 5, 6, 7. InsP3 is an important mediator of intracellular calcium and through this mechanism, it regulates many cellular processes [8]that are especially important in the heart [9]. Although much has been learned about the regulation of ligand-activated transmembrane tyrosine kinases linked to receptors for epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin, there is comparatively little known about the potential involvement of tyrosine kinases in the G-protein coupled receptor for ang II. Ang II induces protein tyrosine phosphorylation in rat liver epithelial cells [10], rat mesangial cells [11], and vascular smooth muscle cells (VSMC) 12, 13, 14, 15, 16, 17, 18, 19, 20. In VSMC, ang II induces tyrosine phosphorylation of a number of proteins including MAP kinases, p120 Ras-GAP and p190 Rho-Gap through tyrosine kinases 12, 14, 20. Ang II stimulates the tyrosine phosphorylation of pp125FAK and paxillin (75 kDa), both components of actin-associated focal adhesions sites in VSMC 15, 16, 17. However, there have been few studies of ang II-induced tyrosine phosphorylation in the heart 21, 22, 23. The ability of ang II to induce tyrosine phosphorylation in cardiac and non cardiac tissues, led us to explore the potential for a relationship between ang II-induced tyrosine phosphorylation and the more well known and described ang II activation of the phosphoinositide pathway in the heart.

Section snippets

Isolation of Chick Embryonic Cardiac Cells

Monolayer culture of 7-d embryonic chick ventricular cells were prepared using previously described methods [24]. Fertilized White Leghorn eggs were incubated in an automatic incubator for 7 d at 37.8°C and 87% humidity. Hearts were then removed, under sterile conditions; ventricles were isolated from atria and cut into 0.5 mm fragments under a dissecting microscope in a solution of balanced salts (DMS8) with the following composition (mM): NaCl 170, KC1 5.4, NAH2PO4 4.3, Na2HPO4 1, dextrose 5.

Ang II-induced InsP3 Generation

To determine whether ang II induces accumulation of InsP1, InsP2, and InsP3, cardiac myocytes, preincubated with [3H] inositol, were stimulated with ang II in the presence of lithium chloride, an inhibitor of inositol phosphatase. First, the time course of ang II induced phosphoinositide hydrolysis was examined. Ang II increased InsP1, Ins P2, and InsP3 (Fig. 1) within 1 min that were sustained for 5 min and returned to basal levels by 10 min. The kinetics of InsP3 production suggest

Discussion

The present study shows, for the first time in the heart, that ang II-induced phosphoinositol generation is mediated, at least in part, through a pathway involving tyrosine phosphorylation. We, first, established that ang II produced significant increases in the levels of InsP1, InsP2, and InsP3 in chick embryonic cardiomyocytes. This was evident within 1 min of ang II treatment consistent with a role for inositol phosphates in cardiac signalling. Data in neonatal rat cardiac myocytes is

Acknowledgements

Supported in part by a grant-in aid from the Heart and Stroke Foundation of British Columbia and the Yukon.

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