Ouabain-induced enhancement of rat mast cells response Modulation by protein phosphorylation and intracellular pH J. Lago a , A. Alfonso a , M.R. Vieytes b , L.M. Botana a, * a Departamento de Farmacologõ Âa, Facultad de Veterinaria, 27002 Lugo, Spain b Departamento de Fisiologõ Âa, Facultad de Veterinaria, 27002 Lugo, Spain Received 14 November 2000; accepted 12 March 2001 Abstract The digitalic glicoside ouabain induces potentiation of rat mast cell histamine release in response to several stimuli, which is mediated by Na + /Ca 2+ exchanger. In this work, we studied the effect of ouabain on cytosolic calcium, intracellular pH and histamine release with Ca 2+ ionophore A23187 in conditions designed to maximize ouabain-induced potentiation of rat mast cells response. The effect of protein kinase C PKC), cAMP and phosphatase inhibition was also tested. Ouabain induced an enhancement in histamine release, cytosolic calcium and intracellular pH. The adenylate cyclase activator forskolin reduced the effect of ouabain on histamine release and intracellular pH, but enhanced the effect on cytosolic calcium. PKC activator PMA enhanced the effect of ouabain on histamine release and cytosolic calcium, without affecting intracellular pH. A PKC inhibitor, GF-109203X, reduced ouabain-induced enhancement of histamine release and intracellular pH, but increased the enhancement on cytosolic calcium. Finally, inhibition of protein phosphatases 1 and 2A with okadaic acid, increased the effect of ouabain on histamine release and intracellular pH, but reduced cytosolic calcium in presence of ouabain. This result suggest that ouabain-induced potentiation of rat mast cell histamine release with A23187 is modulated by kinases, and this modulation may be carried out by changes in intracellular alkalinization. However, the mechanism underlying cellular alkalinization remains to be elucidated. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Mast cells; Histamine release; Intracellular calcium; Intracellular pH 1. Introduction Rat mast cells are nonexcitable cells widely used as a model to study exocytosis and they play an important role in asthma, allergies and inflammatory response. They respond to an ample range of immunological and nonimmunological stimuli through a fast exocytosis, releasing histamine and other inflammatory mediators. The role of calcium in rat mast cell exocytosis has been largely studied, however, little is known about the phys- iology of monovalent ions in this cellular model. Mast cells lack specific sodium channels [1], but they are endowed of a Na + /H + exchanger [2] and a Na + /K + ATPase, [3], that controls the resting membrane potential [4,5]. Mast cells also have a HCO 3 À -dependent alkalin- izing mechanism [6]. Preincubation of rat mast cells with the Na + /K + ATPase inhibitor ouabain enhances histamine release elicited by several stimuli, such us 48/80 [3,7,8] or antigen [8,9]. This enhancement of the mast cell response had been proposed to be due to the existence of a Na + /Ca 2+ exchanger in the rat mast cell [3,7]. Recently, the existence of a Na + /Ca 2+ exchanger in the rat mast cell and its active role in the regulation of calcium levels and cellular response has been proved [10,11]. We have found that the response of mast cells to the calcium ionophore A23187 is enhanced by ouabain pretreatment. This enhancement is maximal when sodium-loaded cells are challenged in a sodium-free medium [11]. The pretreatment of ouabain in a medium with sodium and the transfer thereafter to a sodium-free medium has been previously used to study the Na + /Ca 2+ exchanger [12,13]. In these conditions, the transmembrane sodium 0898-6568/01/$ ± see front matter D 2001 Elsevier Science Inc. All rights reserved. PII:S0898-656801)00169-3 Abbreviations: [Ca 2+ ] i , intracellular Ca 2+ concentration; pH i , intra- cellular pH; Ou, ouabain; FK, forskolin; OKA, okadaic acid * Corresponding author. Tel.: +34-982-252-242; fax: +34-982-252- 242. E-mail address: ffbotana@lugo.usc.es L.M. Botana). Cellular Signalling 13 2001) 515 ± 524