xestospongin-a and Neuroblastoma

Somatostatin receptors are members of the G-protein-coupled receptor superfamily and exert their principal effects by coupling to inhibitory G-proteins. We used fura-2-based digital calcium imaging and assayed for [3H]inositol phosphates (IPs) to study the effects of somatostatin on intracellular calcium signaling in neuroblastomaxglioma NG108-15 cells. Both somatostatin-14 and octreotide induced concentration-dependent increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). Thirty-four percent of the cells responded to treatment with 100 nM somatostatin-14. Somatostatin-induced responses were not blocked by the removal of extracellular calcium; instead, they were abolished by pretreatment with 100 nM thapsigargin, an agent that depletes and prevents refilling of intracellular Ca(2+) stores. Pretreatment with the inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist xestospongin C (10 microM) for 20 min inhibited markedly the somatostatin-induced response. Somatostatin (100 nM) increased [3H]IPs formation. U73122 (1 microM), an inhibitor of phospholipase C (PLC), completely blocked the somatostatin-induced [Ca(2+)](i) increases and the formation of [3H]IPs. Pretreatment with pertussis toxin (PTX, 200 ng/ml) for 24 h blocked the somatostatin-induced responses. Thus, we conclude that activation of endogenous somatostatin receptors in NG108-15 cells induces the release of calcium from IP(3)-sensitive intracellular stores through PTX-sensitive G-protein-coupled PLC.

Topics: Animals; Calcium; Cell Line; Estrenes; Glioma; GTP-Binding Proteins; Image Processing, Computer-Assisted; Inosine Triphosphate; Macrocyclic Compounds; Mice; Neuroblastoma; Octreotide; Oxazoles; Pertussis Toxin; Phosphodiesterase Inhibitors; Pyrrolidinones; Receptors, Somatostatin; Somatostatin; Tumor Cells, Cultured; Type C Phospholipases

Neuronal nicotinic acetylcholine receptors (nAChR) can regulate several neuronal processes through Ca2+-dependent mechanisms. The versatility of nAChR-mediated responses presumably reflects the spatial and temporal characteristics of local changes in intracellular Ca2+ arising from a variety of sources. The aim of this study was to analyse the components of nicotine-evoked Ca2+ signals in SH-SY5Y cells, by monitoring fluorescence changes in cells loaded with fluo-3 AM. Nicotine (30 microm) generated a rapid elevation in cytoplasmic Ca2+ that was partially and additively inhibited (40%) by alpha7 and alpha3beta2* nAChR subtype selective antagonists; alpha3beta4* nAChR probably account for the remaining response (60%). A substantial blockade (80%) by CdCl2 (100 microm) indicates that voltage-operated Ca2+ channels (VOCC) mediate most of the nicotine-evoked response, although the alpha7 selective antagonist alpha-bungarotoxin (40 nm) further decreased the CdCl2- resistant component. The elevation of intracellular Ca2+ levels provoked by nicotine was sustained for at least 10 min and required the persistent activation of nAChR throughout the response. Intracellular Ca2+ stores were implicated in both the initial and sustained nicotine-evoked Ca2+ responses, by the blockade observed after ryanodine (30 microm) and the inositoltriphosphate (IP3)-receptor antagonist, xestospongin-c (10 microm). Thus, nAChR subtypes are differentially coupled to specific sources of Ca2+: activation of nAChR induces a sustained elevation of intracellular Ca2+ levels which is highly dependent on the activation of VOCC, and also involves Ca2+ release from ryanodine and IP3-dependent intracellular stores. Moreover, the alpha7, but not alpha3beta2* nAChR, are responsible for a fraction of the VOCC-independent nicotine-evoked Ca2+ increase that appears to be functionally coupled to ryanodine sensitive Ca2+ stores.

Topics: Aniline Compounds; Calcium; Calcium Channels; Calcium Signaling; Enzyme Inhibitors; Fluorescent Dyes; Humans; Intracellular Fluid; Macrocyclic Compounds; Mecamylamine; Neuroblastoma; Neurons; Nicotine; Nicotinic Agonists; Nicotinic Antagonists; Oxazoles; Receptors, Nicotinic; Ryanodine; Tumor Cells, Cultured; Xanthenes