Neuropeptide Y Y2 receptor and somatostatin sst2 receptor coupling to mobilization of intracellular calcium in SH-SY5Y human neuroblastoma cells

Abstract

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 μM) or SRIF (300 pM-1 μM) was applied in the presence of the cholinoceptor agonist carbachol(1 μM or 100 μM) 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 μM or 100 μM carbachol NPY elevated [Ca2+](i) with a pEC50 of 7.80 and 7.86 respectively. 4. In the presence of 1 μM carbachol the NPY Y2 selective agonist peptide YY(3-36) (PYY(3-36)) elevated [Ca2+](i) with a pEC50 of 7.94, the NPY Y1 selective agonist [Leu31,Pro34]-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) ≤ NPY >> [Leu31,pro34]-NPY indicates that an NPY Y2-like receptor is involved in the elevation of [Ca2+](i). 5. In the presence of 1 μM carbachol, SRIF elevated [Ca2+](i) with a pEC50 Of 8.24. The sst2 receptor-preferring analogue BIM-23027 (c[N-Me-Ala-Tyr-D-Trp-Lys-Abu-Phe]) elevated [Ca2+](i) with a pEC50 Of 8.63, and the sst5-receptor preferring analogue L-362855 (c[Aha-Phe-Trp-D-Trp-Lys-Thr-Phe]) elevated [Ca2+](i) with a pEC50 of approximately 6.1. Application of the sst3 receptor-preferring analogue BIM-23056 (D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-D-Nal-NH2, 1 μM) 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 ≤ SRIF >> L-362855 >>> BIM-23026 suggests that an sst2-like receptor is involved in the elevation of [Ca2+](i). 6. Block of carbachol activation of muscarinic receptors with atropine (1 μM) 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 mi-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. δ-Opioid receptor agonists applied in the presence of carbachol also elevate [Ca2+](i) in SH-SY5Y cells. When NPY (30nM) or SRIF (100nM) was applied together with a maximally effective concentration of the δ-opioid receptor agonist DPDPE ([D-Pen2,5]-enkephalin) (1 μM), 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 Y2 and sst2-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 δ- and μ-opioid receptor and nociceptin receptor mobilization of [Ca2+](i) in SH-SY5Y cells.