Characterization of the neuropeptide Y-induced intracellular calcium release in human erythroleukemic cells.
Human erythroleukemic (HEL) cells, loaded with fura-2, respond to neuropeptide Y (NPY) with a fast and transient increase in intracellular calcium. The Y1 receptor-specific agonist (Leu-31,Pro-34)-NPY is 4-fold more potent and the carboxyl-terminal fragment NPY13-36 is 150-fold less potent than NPY. Thus, it is concluded that the response is mediated through the activation of a Y1 type of NPY receptor. HEL cells do not respond to a second addition of NPY but do respond to a further addition of alpha-thrombin (alpha-T). However, in a calcium-free medium, prior stimulation with NPY largely inhibits a subsequent response to alpha-T. Moreover, prior stimulation with alpha-T in the absence of external calcium completely prevents the response to the addition of NPY, indicating a common effector pathway. The latter is further reinforced by using thapsigargin (TG), which has been shown to deplete the Inositol 1,4,5-trisphosphate-dependent calcium pool in other systems. HEL cells preincubated with TG in calcium-free medium fail to respond to either NPY or alpha-T. Likewise, prior stimulation with NPY or alpha-T in calcium-free medium significantly inhibits the response to TG. Preincubation of cells with phorbol esters strongly inhibits the NPY-induced release of intracellular Ca2+ in HEL cells, an effect that is partially prevented by preincubation of the cells with H7, a protein kinase C inhibitor. However, neither the homologous nor the apparent heterologous desensitization of the NPY receptor can be prevented by H7. It is concluded that NPY releases intracellular Ca2+ from an inositol 1,4,5-trisphosphate-sensitive calcium pool, which is restored by external calcium, and that NPY receptor desensitization is protein kinase C independent.
Daniels
A J
AJ
Division of Pharmacology, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Matthews
J E
JE
Humberto Viveros
O
O
Lazarowski
E R
ER
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002118:Calcium / Q000378:metabolism*; D004789:Enzyme Activation; D004912:Erythrocytes / Q000378:metabolism; D016257:Fura-2; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D004915:Leukemia, Erythroblastic, Acute; D009478:Neuropeptide Y / Q000502:physiology*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014407:Tumor Cells, Cultured
D007546:Isoquinolines; D009478:Neuropeptide Y; D010879:Piperazines; D013729:Terpenes; D019284:Thapsigargin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D002118:Calcium; D016257:Fura-2