ET_A receptor-mediated Ca²⁺ mobilisation in H9c2 cardiac cells

Abstract

Expression and pharmacological properties of endothelin receptors (ETRs) were investigated in H9c2 cardiomyoblasts. The mechanism of receptor-mediated modulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) was examined by measuring fluorescence increase of Fluo-3-loaded cells with flow cytometry. Binding assays showed that [${}^{125}\text{I}$]endothelin-1 (ET-1) bound to a single class of high affinity binding sites in cardiomyoblast membranes. Endothelin-3 (ET-3) displaced bound [${}^{125}\text{I}$]ET-1 in a biphasic manner, in contrast to an ET_B-selective agonist, IRL-1620, that was ineffective. The ET_B-selective antagonist, BQ-788, inhibited [${}^{125}\text{I}$]ET-1 binding in a monophasic manner and with low potency. An ET_A-selective antagonist, BQ-123, competed [${}^{125}\text{I}$]ET-1 binding in a monophasic manner. This antagonist was found to be 13-fold more potent than BQ-788. Immunoblotting analysis using anti-ET_A and -ET_B antibodies confirmed a predominant expression of the ET_A receptor. ET-1 induced a concentration-dependent increase of Fluo-3 fluorescence in cardiomyoblasts resuspended in buffer containing 1 mM CaCl₂. Treatment of cells with antagonists, PD-145065 and BQ-123, or a phospholipase C-β inhibitor, U-73122, abolished ET-1-mediated increases in fluorescence. The close structural analogue of U-73122, U-73343, caused a minimal effect on the concentration–response curve of ET-1. ET-3 produced no major increase of Fluo-3 fluorescence. Removal of extracellular Ca²⁺ resulted in a shift to the right of the ET-1 concentration–response curve. Both the L-type voltage-operated Ca²⁺ channel blocker, nifedipine, and the ryanodine receptor inhibitor, dantrolene, reduced the efficacy of ET-1. Two protein kinase C inhibitors reduced both potency and efficacy of ET-1. Our results demonstrate that ET_A receptors are expressed and functionally coupled to rise of [Ca²⁺]_i in H9c2 cardiomyoblasts. ET-1-induced [Ca²⁺]_i increase is triggered by Ca²⁺ release from intracellular inositol 1,4,5-trisphosphate-gated stores; plasma membrane Ca²⁺ channels and ryanodine receptors participate in sustaining the Ca²⁺ response. Regulation of channel opening by protein kinase C is also involved in the process of [Ca²⁺]_i increase.

Introduction

ET-1, a 21-residue peptide, is recognised as a potent vasoconstrictor [1]. In humans three distinct genes encode for three endothelin (ET) isopeptides, ET-1, ET-2 and ET-3 [2]. Of these isopeptides, ET-1 is the only form constitutively released by ET cells, but many other cells are now known to have the ability to produce ETs (for reviews see [3], [4], [5]). Besides modulating the smooth muscle tone, ETs also stimulate cell proliferation in all tissues. All biological effects are elicited by binding and activation of specific cell surface receptors (endothelin receptors (ETRs)), which belong to the large family of G protein-coupled receptors. Pharmacological studies and molecular cloning [5] have revealed the existence of at least two ETR subtypes with different cell distribution and roles in regulating the vascular tone, termed ET_A and ET_B receptors [3]. ETR activation leads to second messenger generation through a variety of signal transduction pathways, including activation of phospholipase C-β (PLC) [5], A₂ and D [5], [6], [7], inhibition or activation of adenylyl cyclase [5], activation of both Ca²⁺-permeable non-selective cation channels [5], [8] and L-type voltage-operated Ca²⁺ channels (VOCCs) [3], [5] and regulation of Na⁺/H⁺ exchange activity [5].

ETs have direct effects on cardiac tissue [6] that synthesises, stores and releases ET-1 [9]. Both binding studies [9] and in situ hybridisation studies [8] have shown a wide expression of ET_A and ET_B receptors in human atrial and ventricular myocardium. However, homogenous populations of right atrial [10] or left [11] ventricular cardiomyocytes demonstrated a high proportion of ET_A receptor binding sites (86–90%) than that found in tissue preparations. In adult and neonatal rat ventricular cardiomyocytes a homogenous population of ET_A receptors coupled to multiple effector pathways (i.e. activation of PLC and inhibition of adenylyl cyclase) has been described [12], [13].

The clonal cardiac cell line H9c2 derived from embryonic rat heart has been used as an experimental model to study L-type VOCCs with cardiac-specific characteristics [14] and the ontogenic expression of these channels [15]. When cultured in the presence of a low foetal bovine serum (FBS) concentration, H9c2 cardiomyoblasts differentiate to myotubes expressing both cardiac and skeletal L-type VOCCs [15] and ryanodine receptor (RyR) Ca²⁺ release channels [16]. These cardiomyoblasts also express V₁ receptors resulting in vasopressin-induced mobilisation of Ca²⁺ from intracellular stores, activation of PLC, PLA₂ and the p42 MAP kinase [17], [18], [19]. In addition, vasopressin stimulates H9c2 cell hypertrophy [20]. Thus, this cardiac cell line is a particularly valuable tool for the investigation of receptor-mediated modulation of [Ca²⁺]_i during cell growth and differentiation.

The present study was undertaken in H9c2 cadiomyoblasts in order to dissect the signal transduction pathways activated by ET-1, a major hypertrophic agent for cardiomyocytes. Here, we present evidence that the ET_A receptor subtype is preferentially expressed in this cell line and functionally coupled to an increase of [Ca²⁺]_i. Receptor-mediated [Ca²⁺]_i variations are known to be triggered and sustained by Ca²⁺ release from intracellular inositol 1,4,5-trisphosphate (IP₃)-sensitive stores, but the participation of plasma membrane Ca²⁺ channels and RyRs is also obvious in this event. In addition, protein kinase C (PKC) is involved in modulating Ca²⁺ channel opening. These findings demonstrate the presence of a functional intact signal transduction machinery which couples ET_A receptors to elevation of [Ca²⁺]_i in rat cardiomyoblasts and highlight integrated responses of intracellular and plasma membrane Ca²⁺ channels. The H9c2 cell line may represent a useful model to study ETR expression and coupling to signal transduction pathways during differentiation and hypertrophic responses. The importance of this cellular model also arises from the consideration that the cardiac ET-1/ET_A receptor system is upregulated in left ventricular hypertrophy [21], [22].