Inhibition of Ca(2+)-sensitive K+ currents in NG 108-15 cells by substance P and related tachykinins.

1. The whole-cell patch-clamp technique was used to investigate the actions of substance P and other agonists at neurokinin (NK) receptors on voltage-gated K+ and Ca+ channel currents in undifferentiated mouse neuroblastoma x rat glioma NG 108-15 cells. 2. Both substance P (0.3-30 microM) and the NK1 receptor selective agonist GR73632 (10 nM-10 microM) caused concentration-dependent inhibition of K+ currents. GR64349 and senktide (agonists at NK2 and NK3 receptors respectively) also inhibited K+ currents, but only at concentrations which were several orders of magnitude greater than GR73632, suggesting that current inhibition was mediated via NK1 receptors. 3. Substance P and GR73632 were without effect on residual K+ currents recorded in the presence of extracellular Co2+ (4 mM) to abolish the Ca(2+)-sensitive component (IKca) of the K+ current. Ca2+ channel currents, recorded with either Ba2+ or Ca2+ as charge carrier, were unaffected by NK1, NK2 and NK3 receptor ligands. 4. Iontophoretic application of GR73632 produced a current-dependent reduction of K+ currents. In the presence of the non-peptide NK1 antagonists, CP-99,994 and RP67580, and the peptide antagonist, GR82334, the current-response relationship was reversibly shifted to the right. This indicates that the response is mediated by NK1 receptors. 5. Our results indicate that activation of NK1 receptors leads to the selective inhibition of IKca in undifferentiated NG 108-15 cells.

Increased perinuclear Ca2+ activity evoked by metabotropic glutamate receptor activation in rat hippocampal neurones.

1. The effect of metabotropic glutamate receptor activation on intracellular Ca2+ activity (alpha Cai) of rat hippocampal pyramidal neurones in vitro was examined using ratiometric confocal laser scanning microscopy with the Ca(2+)-sensitive fluorescent probe indo-1 AM. 2. Metabotropic receptors were selectively activated with 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD; 100 microM) in the presence of D-2-amino 5-phosphonovaleric acid (D-APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and CdCl2. Most pyramidal neurones (77/84) responded with an elevation in Ca2+ activity, maximal after 3-5 min. Fluorescence ratio responses were concentration dependent (EC50 approximately 10 microM) and were blocked by prior application of the antagonist (RS)-4-carboxy-3-hydroxyphenylglycine (RS-CHPG, 300 microM). 3. Responses to 1S,3R-ACPD (100 microM) also caused acidification of the neurones, from estimated control pH 7.2 to pH 6.6 (measured with the pH-sensitive dye SNAFL-calcein). The correction factor for indo-1 determination of Ca2+ was estimated to be x 1.4. 4. Elevations in alpha Cai were greater within the perinuclear region (> 1000 nM), than in the cytoplasm (approximately 200 nM). This region was devoid of staining by the endoplasmic reticulum staining dye 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)). 5. It is concluded that activation of metabotropic receptors in immature rat hippocampal pyramidal neurones leads to a large increase in perinuclear Ca2+ which would be well positioned to interact with the genome.