Characterization of Ca2+ mobilization in the human submandibular duct cell line A253.
The regulation of Ca2+ mobilization in the human submandibular duct cell line A253 was investigated by monitoring cytosolic free Ca2+ concentrations ([Ca2+]i) using the Ca(2+)-sensitive fluorescent indicator fura-2 and by measuring inositol 1,4,5-triphosphate (IP3) formation. An increase in [Ca2+]i was elicited by ATP, isoproterenol (IPR), or vasoactive intestinal polypeptide (VIP), but not by acetylcholine, norepinephrine, or substance P, suggesting that Ca2+ mobilization is regulated by P2-purinergic, beta 2-adrenergic, and VIP receptors. 1,4,5-IP3 formation was significantly increased by ATP but not by the other agonists. Exposure of the cells to a membrane permeable cAMP analog, dibutyryl-cAMP, or to the adenylate cyclase activator forskolin induced a smaller increase in [Ca2+]i, indicating that the IPR-induced Ca2+ release is not mediated by cyclic AMP. Inhibition of the endoplasmic Ca(2+)-ATPase with thapsigargin (TG) in Ca(2+)-free medium induced a 207% increase in [Ca2+]i, and a subsequent exposure to ATP caused a further increase in [Ca2+]i of 104%. Similarly, TG exposure after ATP induced a further Ca2+ release, suggesting that the TG-sensitive store and the IP3-sensitive store do not overlap. Similar results were observed by sequential exposure to TG and IPR or to ATP and IPR. Ca2+ influx across the plasma membrane was enhanced after ATP or TG, but not after IPR. Our findings show a unique pattern of Ca2+ mobilization in the A253 cell line: (i) Ca2+ mobilization is regulated by P2-purinergic, beta 2-adrenergic, and VIP receptors; (ii) Ca2+ release is mediated by 1,4,5-IP3 and probably by an unknown mediator; (iii) TG, P2-, and beta 2-agonists discharge separate Ca2+ stores; and (iv) ATP and TG, but not IPR, regulate Ca2+ influx.
Zhang
G H
GH
Department of Pediatrics, University of Texas Health Science Center at San Antonio 78284, USA.
Helmke
R J
RJ
Martinez
J R
JR
D000255:Adenosine Triphosphate / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D013363:Submandibular Gland / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology
D003994:Bucladesine; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D007545:Isoproterenol; D002118:Calcium