Neuropeptide Y Y-2 receptor and somatostatin sst(2) receptor coupling to mobilization of intracellular calcium in SH-SY5Y human neuroblastoma cells

1 In this study we have investigated neuropeptide Y (NPY) and somatostatin (SRIF) receptor-mediated elevation of intracellular Ca2+ concentration ([Ca2+](i)) in the human neuroblastoma cell line SH-SY5Y. 2 The Ca2+-sensitive dye fura 2 was used to measure [Ca2+](i) in confluent monolayers of SH-SY5Y cells. Neither NPY (30-100 nM) nor SRIF (100 nM) elevated [Ca2+](i) when applied alone. However, when either NPY (300 pM-1 mu M) or SRIF (300 pM-1 mu M) was applied in the presence of the cholinoceptor agonist carbachol (1 mu M or 100 mu M) they evoked an elevation of [Ca2+](i) above that caused by carbachol alone. 3 The elevation of [Ca2+](i) by NPY was independent of the concentration of carbachol. In the presence of 1 mu M or 100 mu M carbachol NPY elevated [Ca2+](i) with a pEC(50) of 7.80 and 7.86 respectively. 4 In the presence of 1 mu M carbachol the NPY Y-2 selective agonist peptide YY(3-36) (PYY(3-36)) elevated [Ca2+](i) with a pEC(50) of 7.94, the NPY Y-1 selective agonist [Leu(31),Pro(34)]-NPY also elevated [Ca2+](i) when applied in the presence of carbachol, but only at concentrations >300 nM. The rank order of potency, PYY(3-36) greater than or equal to NPY >> [Leu(31),Pro(34)]-NPY indicates that an NPY Y-2-like receptor is involved in the elevation of [Ca2+](i). 5 In the presence of 1 mu M carbachol, SRIF elevated [Ca2+](i) with a pEC(50) of 8.24. The sst(2) receptor-preferring analogue BIM-23027 (c[N-Me-Ala-Tyr-D-Trp-Lys-Abu-Phe]) elevated [Ca2+](i) with a pEC(50) of 8.63, and the sst(5)-receptor preferring analogue L-362855 (c[Aha-Phe-Trp-D-Trp-Lys-Thr-Phe]) elevated [Ca2+](i) with a pEC(50) of approximately 6.1. Application of the sst, receptor-preferring analogue BIM-23056 (D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-D-Nal-NH2, 1 mu M) to SH-SY5Y cells in the presence of carbachol neither elevated [Ca2+](i) nor affected the elevations of [Ca2+](i) caused by a subsequent coapplication of SRIF. The rank order of potency, BIM-23026 greater than or equal to SRIF >> L-362855 >>> BIM-23026 suggests that an sst(2)-like receptor is involved in the elevation of [Ca2+](i). 6 Block of carbachol activation of muscarinic receptors with atropine (1 mu M) abolished the elevation of [Ca2+](i) by the SRIF and NPY. 7 Muscarinic receptor activation, not a rise in [Ca2+](i), was required to reveal the NPY or SRIF response. The Ca2+ channel activator maitotoxin (2 ng ml(-1)) also elevated [Ca2+](i) but subsequent application of either NPY or SRIF in the presence of maitotoxin caused no further changes in [Ca2+](i). 8 The elevations of [Ca2+](i) by NPY and SRIF were abolished by pretreatment of the cells with pertussis toxin (200 ng ml(-1), 16 h). This treatment did not significantly affect the response of the cells to carbachol. 9 NPY and SRIF appeared to elevate [Ca2+](i) by mobilizing Ca2+ from intracellular stores. Both NPY and SRIF continued to elevate [Ca2+](i) when applied in nominally Ca2+-free external buffer. Thapsigargin (100 nM), an agent which discharges intracellular Ca2+ stores, also blocked the NPY and SRIF elevations of [Ca2+](i). 10 delta-Opioid receptor agonists applied in the presence of carbachol also elevate [Ca2+](i) in SH-SY5Y cells. When NPY (30 nM) or SRIF (100 nM) was applied together with a maximally effective concentration of the delta-opioid receptor agonist DPDPE ([D-Pen(2,5)]-enkephalin) (1 mu M), the resulting elevations of [Ca2+](i) were not greater than those caused by application of DPDPE alone. 11 Thus, in SH-SY5Y cells, NPY and SRIF can mobilize Ca2+ from intracellular stores via activation of NPY Y-2 and sst(2)-like receptors, respectively. Neither NPY nor SRIF elevated [Ca2+](i) when applied alone. The requirements for the elevations of [Ca2+](i) by NPY and SRIF are the same as those for delta- and mu-opioid receptor and nociceptin receptor mobilization of [Ca2+](i) in SH-SY5Y cells.