HCO 3 - I ons I ncrease Mas t Cell Sensi t ivi t y t o Thapsigargin- I nduced Ca 2+ En t ry Natalia Vilarin ˜o,* L. A. De la Rosa,* Mercedes R. Vieytes,† and Luis M. Botana* ,1 *Departamento de Farmacologı ´aand †Departamento de Fisiologı ´a, Facultadde Veterinaria, Universidad Santiago de Compostela, 27002 Lugo, Spain Received December 7, 2000 In rat mast cells Ca 2 entry is modified by the pres- ence or absence of other ions in theexternal medium. HCO 3  ions, which modify mast cell degranulation, seemed to modulate the Ca 2 entry elicited by the in- tracellular Ca 2 -ATPase inhibitorthapsigargin. In this work westudied the regulation of the Ca 2 entry by HCO 3  andits relationship with exocytosis. The Ca 2 entrywas activated by thapsigargin and Ca 2 in mast cells bathed by a HCO 3  -buffered mediumor a HCO 3  - free medium. Both Ca 2 entry and exocytosis wereen- hanced by the presence of HCO 3  ions. Nondegranu- lated mast cellsshowed a low Ca 2 entry either in the presence or absence of HCO 3  . Thus, mast cells with a high [Ca 2 ] i increase in a HCO 3  -buffered medium un- dergo degranulation. In thesame cells a second Ca 2 entrywassignificantly higherthan thefirst Ca 2 entry in a HCO 3  -free medium, while in a HCO 3  -buffered me- dium thefirst and second Ca 2 entries reached similar [Ca 2 ] i levels. Although the second Ca 2 entry is high in a HCO 3  -free medium, degranulation isstill low. Our results demonstrate that HCO 3  ions increase the ca- pacitative Ca 2 entry and the sensitivity of mast cells to intracellular Ca 2 in orderto induce degranulation. © 2001 Academic Press Key Words: mast cell; thapsigargin; bicarbonate; cal- cium entry. Stimulation of mast cells elicits an intracellular Ca 2+ signal. Thissignal consists of a depletion of intracellu- lar Ca 2+ stores which activates a capacitative Ca 2+ entry (1, 2). In rat mast cells this Ca 2+ entry was characterized years ago and named I CRAC (calcium release-activated calcium current)(3). In rat mast cells some stimuli, such as intracellular Ca 2+ -ATPase inhib- itor thapsigargin, require the presence of Ca 2+ ions in theexternal medium, and therefore an entry of Ca 2+ ions into the cytosol, to promote theexocytosis of pre- formed mediators (4, 5). The presence of other ions in theexternal medium has been demonstrated to modu- late the size of the Ca 2+ entry in mast cells, as it happens with Cl - and K + ions (6, 7, 2). Our previous work suggests thatthe capacitative Ca 2+ entry is also regulated by the presence of HCO 3 - ions in theexternal medium (8). HCO 3 - also modifies the response of mast cells to several stimuli (8). In this work we reportthe effect of HCO 3 - on the Ca 2+ entry and the relationship between modulation of the Ca 2+ entry by HCO 3 - and mast cell exocytosis. In order to do so, the Ca 2+ entry mechanism was activated by depleting intracellular Ca 2+ stores with thapsigargin (9). METHODS Chemicals and solutions. Fura-2/AM and BAPTA-AM (1,2-bis- (-2-aminophenoxy)ethane-N-N-N'-N'-tetraacetic acid acetoxymeth- ylester) was purchased from Molecular Probes (Leiden, The Nether- lands). Thapsigargin was from Alexis (La ¨ ufelfingen, Switzerland). Percoll was from Pharmacia (Barcelona, Spain) and orthophthal- dialdehyde from Merck (Madrid, Spain). All other chemicals were from standard commercial sources and reagent grade of the highest purity. The composition of the HCO 3 - -buffered medium was (in mM): NaCl, 119; KCl, 5.94; CaCl 2 , 1; MgSO 4 , 1.2; NaHCO 3 , 22.85 and NaH 2 PO 4 , 1.2. Glucose (1 mg/ml) was added to the solution and the pH was adjusted to 7.30 by bubbling 5% CO 2 . Mast cell purification was performed with a HCO 3 - -buffered solution containing BSA 1 mg/ml. The composition of the HCO 3 - -free medium was (in mM): NaCl, 119; KCl, 5.94; CaCl 2 ,1; MgSO 4 , 1.2; NaH 2 PO 4 , 12.49 and Na 2 HPO 4 , 7.7. Glucose (1 mg/ml) was added to the medium and the pH was adjusted to 7.30 with NaOH. Mast cell isolation and purification. Mast cells were obtained from pleural andperitoneal cavities of Sprague–Dawley rats. Isola- tion andpurification through Percoll was performed as described (10). Briefly, rats were blooded and 10 ml of the purification solution were introduced in the peritoneal cavity through a small hole. After a smooth massage, the solution was recovered with mast cellssus- pended in it. The pleural cavity lavage was performed with 5 ml of the solution in the same way. Total cells collected from one rat were centrifuged at 900 rpm for 4 min and suspended in a final volume of 1 ml. Purification was carried through 4 ml of 95% isotonic Percoll at Abbreviations used: I CRAC , calcium release-activated calcium cur- rent;[Ca 2+ ] i , intracellular calcium concentration. 1 To whom correspondence should be addressed at Department de Farmacologı ´a, Facultad de Veterinaria, Universidad Santiago de Compostela, 27002 Lugo, Spain. Fax: 34 982 252 242. E-mail: ffbotana@lugo.usc.es. Biochemical and Biophysical Research Communications 280, 518 –521 (2001) doi:10.1006/bbrc.2000.4152, available online at http://www.idealibrary.com on 518 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.