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

Authors


Author for correspondence at present address: Department of Pharmacology D06, The University of Sydney, NSW 2006, Australia.

Abstract

  • 1In 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.
  • 2The Ca2+-sensitive dye fura 2 was used to measure [Ca2+]i in confluent monolayers of SH-SY5Y cells. Neither NPY (30–100 mi) nor SRIF (100 nM) elevated [Ca2+]i when applied alone. However, when either NPY (300 pM-1 μM) or SRIF (300pM-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.
  • 3The 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.
  • 4In 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.
  • 5In 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.
  • 6Block of carbachol activation of muscarinic receptors with atropine (1 μM) abolished the elevation of [Ca2+]i by the SRIF and NPY.
  • 7Muscarinic 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.
  • 8The 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.
  • 9NPY 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.
  • 10δ-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 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.
  • 11Thus, 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.

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