Mobilization of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores supports bradykinin- and muscarinic-evoked release of [3H] noradrenaline from SH-SY5Y cells.
The human neuroblastoma cell line SH-SY5Y, maintained at confluence for 14 days, released [3H]-noradrenaline ([3H]NA) when stimulated with either the muscarinic receptor agonist methacholine or bradykinin. The major fraction of release was rapid, occurring in < 10 s, whereas nicotine-evoked release was slower. When the extracellular [Ca2+]e) was buffered to approximately 50-100 nM, release evoked by nicotine was abolished, whereas that in response to methacholine or bradykinin was reduced by approximately 50% with EC50 values of -5.46 +/- 0.05 M and -7.46 +/- 0.06 M (log 10), respectively. Methacholine and bradykinin also produced rapid elevations of both inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and intracellular free [Ca2+] ([Ca2+]i). These elevations were reduced at low [Ca2+]e and under these conditions the EC50 values for peak elevation of [Ca2+]i were -6.00 +/- 0.14 M for methacholine and -7.95 +/- 0.34 M for bradykinin (n = 3 for all EC50 determinations). At low [Ca2+]e, depletion of nonmitochondrial intracellular Ca2+ stores with the Ca(2+)-ATPase inhibitor thapsigargin produced a transient small elevation of [Ca2+]i and a minor release of [3H]NA. At low [Ca2+]e, thapsigargin abolished elevation of [Ca2+]i in response to methacholine and bradykinin and completely inhibited their stimulation of [3H]NA release. It is proposed, therefore, that Ca2+ release from Ins (1,4,5)P3-sensitive stores is a major trigger of methacholine- and bradykinin-evoked [3H]NA release in SH-SY5Y cells.
Purkiss
J R
JR
Department of Cell Physiology and Pharmacology, University of Leicester, England.
Nahorski
S R
SR
Willars
G B
GB
D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D005728:Ganglia, Sympathetic / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D016210:Methacholine Chloride / Q000494:pharmacology; D009447:Neuroblastoma; D009538:Nicotine / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014407:Tumor Cells, Cultured
D013729:Terpenes; D016210:Methacholine Chloride; D019284:Thapsigargin; D009538:Nicotine; D015544:Inositol 1,4,5-Trisphosphate; D001920:Bradykinin; D002118:Calcium; D009638:Norepinephrine