Angiotensin II-stimulated cortisol secretion is mediated by phospholipase D

Abstract

Angiotensin II (Ang-II) regulates a variety of cellular functions including cortisol secretion. In the present report, we demonstrate that Ang-II activates phospholipase D (PLD) in zona fasciculata (ZF) cells of bovine adrenal glands, and that this effect is associated to the stimulation of cortisol secretion by this hormone. PLD activation was dependent upon extracellular Ca²⁺, and was blocked by inhibition of protein kinase C (PKC). Using the reverse transcription–polymerase chain reaction technique, we demonstrated that ZF cells express both PLD-1 and PLD-2 isozymes. Primary alcohols, which attenuate the formation of phosphatidate (the product of PLD), and cell-permeable ceramides, which inhibit PLD potently, blocked Ang-II-stimulated cortisol secretion. Furthermore, propranolol or chlorpromazine, which are potent inhibitors of phosphatidate phosphohydrolase (PAP) (the enzyme that produces diacylglycerol from phosphatidate), also blocked cortisol secretion. These data suggest that the PLD/PAP pathway plays an important role in the regulation of cortisol secretion by Ang-II in ZF cells.

Introduction

The regulation of cortisol secretion has been a subject of intensive investigation for many years. However, the mechanisms or signaling pathways that are involved in agonist-stimulated cortisol secretion are not completely understood. In the zona fasciculata (ZF) of the adrenal cortex, the secretion of cortisol is mainly controlled by pituitary corticotrophin (ACTH). The interaction of ACTH with specific receptors at the plasma membrane of cells causes activation of adenylyl cyclase and subsequent generation of cAMP. The effect of ACTH on stimulating cortisol secretion can be reproduced by cAMP analogues, or by forskolin, a direct activator of adenylyl cyclase (Nishikawa et al., 1996). ZF cells secrete cortisol in response to a variety of other stimuli, including angiotensin II (Ang-II) (Bird et al., 1989, Finn et al., 1988; Hadjian et al., 1984a, Hadjian et al., 1984b), the principal biologically active hormone of the renin–angiotensin system (Peach, 1977). Ang-II can elicit its biological actions by interacting with two distinct receptor subtypes, AT₁ and AT₂, which are both seven transmembrane spanning G protein-coupled receptors. Pharmacologically these receptors can be distinguished according to inhibition by specific antagonists. AT₁ receptors are selectively antagonized by biphenylimidazoles such as losartan, whereas tetrahydroimidazopyridines such as PD 123319 specifically inhibit AT₂ receptors (Touyz and Berry, 2002). Most of the physiological actions of Ang-II are mediated via the AT₁ receptor. The functional roles of the AT₂ receptor in bovine and human cells is unclear, but it has been implicated in tissue remodeling, growth and development (Jung et al., 1998, Touyz and Berry, 2002). Ang-II acts through stimulation of phosphoinositide specific phospholipase C (PI-PLC) thereby causing the formation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate, two important second messengers leading to stimulation of protein kinase C (PKC) and intracellular Ca²⁺ mobilization, respectively (Bird et al., 1991, Exton, 1998, Exton, 1999). Ang-II also causes a rise in intracellular Ca²⁺ by allowing entry of this cation from the extracellular milieu. Both DAG and Ca²⁺ are important signals that are involved in the regulation of steroid secretion, including aldosterone (Bollag et al., 1990). DAG can also be formed by the combined actions of phospholipase D (PLD) and phosphatidate phosphohydrolase (PAP) (Brindley et al., 1997, Liscovitch, 1992, Liscovitch and Cantley, 1994, Liscovitch et al., 1999). Interestingly, it was reported previously that PLD might play an important role in the stimulation of aldosterone secretion by Ang-II in zona glomerulosa cells (Bollag et al., 1990, Bollag et al., 2002, Jung et al., 1998, Zheng and Bollag, 2003), and we have recently demonstrated that sphingosine-1-phosphate is capable of stimulating both cortisol secretion and PLD, suggesting an involvement of this enzyme activity in this process (Rábano et al., 2003).

The present work was undertaken to determine whether PLD might be involved in the stimulation of cortisol secretion by Ang-II. Our results indicate that PLD is an important component in the cascade of events leading to the stimulation of cortisol secretion by Ang-II in ZF cells.