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]AA in response to the purinergic agonists was still observed; this response was not affected by AACOCF 3 and completely blocked by bromoenol lactone. ATP and Bz-ATP stimulated a calcium-independent secretion of kallikrein, which could be blocked by BEL but which was enhanced by AACOCF 3 . It is concluded that the P2X 7 receptor in ductal cells is coupled to kallikrein secretion through a calcium-dependent cPLA 2 and a calcium-independent iPLA 2 .ATP plays an important role as an extracellular agonist that mediates its various effects by acting on specific membrane P2 receptor subtypes (1, 2). P2 receptors comprise receptors of the ligand-gated ion channel type, as well as of the G-proteinlinked superfamily, termed P2X and P2Y, respectively (3). At present, seven genes for P2X receptors have been cloned (4 -8), but their physiological significance has not been fully established yet. One of the most recently cloned P2X receptors (the P2X 7 ) has a pharmacological profile typical of the receptor previously termed P 2Z (9) with the photoactivable analog of ATP, Bz-ATP, 1 the most potent agonist. The P 2Z receptor induces the formation of pores when exposed to concentrations of extracellular ATP in the 100 M to 1 mM range (Refs. 10 -12, but see also Ref. 13). In contrast to other P2X receptors, the P2X 7 receptor has a long COOH-terminal intracellular chain, which is by itself not responsible for the lytic properties of this receptor (14) but probably induces the formation of a second messenger involved in the lysis (12). In summary, the P2X 7 (P 2Z )-receptors share with the other P2X receptors the ability to open a non-selective channel and with P 2Z receptors the induction of cell lysis by repeated applications of the agonist (8).Since the pioneering work of Gallacher (15), ATP has been recognized as a major non-adrenergic non-cholinergic stimulus of saliva secretion. Salivation, like other exocrine secretions, occurs in two steps; (a) acinar cells secrete an isotonic plasmalike fluid, and (b) the electrolyte composition of this primary secretion is modified during its transfer to the mouth by the ductal tree (16). The ducts reabsorb Na ϩ and Cl Ϫ and secrete K ϩ and HCO 3 Ϫ (17). The study of these two phases of the secretory process has been facilitated by the description of an improved technique to separate ducts and acini (18). It could be observed that extracellular ATP increased the intracellular

We show here for the ¢rst time that sphingosine-1-phosphate (Sph-1-P) stimulates cortisol secretion in zona fasciculata cells of bovine adrenal glands. This e¡ect was dependent upon protein kinase C (PKC) and extracellular Ca 2+ , and was inhibited by pertussis toxin. Sph-1-P activated phospholipase D (PLD) through a pertussis toxin-sensitive mechanism, also involving extracellular Ca 2+ and PKC. Primary alcohols, which attenuate formation of phosphatidic acid (the product of PLD), and cell-permeable ceramides, which inhibit PLD, blocked Sph-1-P-induced cortisol secretion. In conclusion, Sph-1-P stimulates cortisol secretion through a mechanism involving Gi/o protein-coupled receptors, extracellular Ca 2+ , PKC and PLD.

We reported recently that sphingosine-1-phosphate (S1P) is a novel regulator of aldosterone secretion in zona glomerulosa cells of adrenal glands and that phospholipase D (PLD) is implicated in this process. We now show that S1P causes the phosphorylation of protein kinase B (PKB) and extracellularly regulated kinases 1/2 (ERK 1/2), which is an indication of their activation, in these cells. These effects are probably mediated through the interaction of S1P with the Gi protein-coupled receptors S1P1/3, as pretreatment with pertussis toxin or with the S1P1/3 antagonist VPC 23019 completely abolished the phosphorylation of these kinases. Inhibitors of phosphatidylinositol 3-kinase (PI3K) or mitogen-activated protein kinase kinase (MEK) blocked S1P-stimulated aldosterone secretion. This inhibition was only partial when the cells were incubated independently with inhibitors of each pathway. However, aldosterone output was completely blocked when the cells were pretreated with LY 294002 and PD 98059 simultaneously. These inhibitors also blocked PLD activation, which indicates that this enzyme is downstream of PI3K and MEK in this system. We propose a working model for S1P in which stimulation of the PI3K/PKB and MEK/ERK pathways leads to the stimulation of PLD and aldosterone secretion.-Brizuela, L., M. Rábano, P. Gangoiti, N. Narbona, J. M. Macarulla, M. Trueba, and A. Gómez-Muñ oz. Sphingosine-1-phosphate stimulates aldosterone secretion through a mechanism involving the PI3K/PKB and MEK/ERK 1/2 pathways. J. Lipid Res.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid capable of regulating critical physiological and pathological functions. Here, we report for the first time that S1P stimulates aldosterone secretion in cells of the zona glomerulosa of the adrenal gland. Regulation of aldosterone secretion is important because this hormone controls electrolyte and fluid balance and is implicated in cardiovascular homeostasis. S1P-stimulated aldosterone secretion was dependent upon the protein kinase C (PKC) isoforms a and d and extracellular Ca 21 , and it was inhibited by pertussis toxin (PTX). S1P activated phospholipase D (PLD) through a PTX-sensitive mechanism, also involving PKC a and d and extracellular Ca 21 . Primary alcohols, which attenuate the formation of phosphatidic acid (the product of PLD), and cellpermeable ceramides, which inhibit PLD activity, blocked S1P-stimulated aldosterone secretion. Furthermore, propranolol, chlorpromazine, and sphingosine, which are potent inhibitors of phosphatidate phosphohydrolase (PAP) (the enzyme that produces diacylglycerol from phosphatidate), also blocked aldosterone secretion. These data suggest that the PLD/PAP pathway plays a crucial role in the regulation of aldosterone secretion by S1P and that Gi protein-coupled receptors, extracellular Ca 21 , and the PKC isoforms a and d are all important components in the cascade of events controlling this process.-Brizuela, L., M. Rábano, A. Peña, P. Gangoiti, J. M. Macarulla, M. Trueba, and A. Gómez-Muñoz. Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion.