Neuropeptide Y Y2 receptor and somatostatin sst2 receptor coupling to mobilization of intracellular calcium in SH‐SY5Y human neuroblastoma cells
Authors: Connor Mark , Yeo Alison , Henderson Graeme 
Published: August 2006

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.
The 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.
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.
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.
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.
Block of carbachol activation of muscarinic receptors with atropine (1 μM) abolished the elevation of [Ca2+]i by the SRIF and NPY.
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.
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.
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.
δ‐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.
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.