Studies of the control of intracellular Ca"+"+ in acutely dissociated rat hippocampal neurons

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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 Ca2+-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 µM) 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 µM) and were blocked by prior application of the antagonist (RS)-4-carboxy-3-hydroxyphenylglycine (RS-CHPG, 300 µM). 3. Responses to 1S,3R-ACPD (100 µM) 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 (&gt; 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.<br/

Activation of nicotinic acetylcholine-receptors expressed in quail fibroblasts: effects on intracellular calcium

1. The aim of these experiments was to determine the ability of the muscle-type nicotinic acetylcholine receptor (AChR) stably expressed in quail fibroblasts (QF18 cells) to elevate intracellular calcium ([Ca2+]i) upon activation. Ratiometric confocal microscopy, with the calcium-sensitive fluorescent dye Indo-1 was used. 2. Application of the nicotine agonist, suberyldicholine (SDC), to the transfected QF18 cells caused an increase in [Ca2+]i. Control [Ca2+]i levels in QF18 cells were found to be 164 +/- 22 nM (mean +/- s.e. mean; n = 40 cells) rising to 600 +/- 81 nM on addition of SDC (10 microM; n = 15 cells), whereas no increase in [Ca2+]i was seen in non-transfected control QT6 fibroblasts (before: 128 +/- 9 nM, n = 40; after; 113 +/- 13 nM, n = 15). 3. The increase in [Ca2+]i caused by application of SDC was dose-dependent, with an EC50 value of 12.7 +/- 5.9 microM (n = 14). 4. The responses to SDC in QF18 cells were blocked by prior application of alpha-bungarotoxin (200 nM), by the addition of Ca2+ (100 microM), by removal of Na+ ions from the extracellular solution, or by the voltage-sensitive calcium channel blockers nifedipine and omega-conotoxin, which act with IC50 values of 100 nM and 100 pM respectively. 5. We conclude that activation of the nicotinic AChRs leads to a Na(+)-dependent depolarization and hence activation of endogenous voltage-sensitive Ca2+ channels in the plasma membrane and an increase in [Ca2+]i. There is no significant entry of Ca2+ through the nicotinic receptor itself

The effect of the neuropeptide substance P on desensitization of ATP receptors of PC12 cells

1 Patch clamp recording (whole cell configuration) was employed to investigate the modulatory action of substance P on inward currents elicited by adenosine 5'-triphosphate (ATP, focally applied via a pressure pipette) from phaechromocytoma (PC12) cells usually held at -70 mV. 2 Bath-applied substance P (0.2-20 mu M) had no effect on baseline membrane current but reversibly reduced ATP peak currents in a concentration-dependent fashion. The depressant effect was not associated with a change in the ATP current reversal potential. 3 Equiamplitude peak responses induced by 50 mu M or 5 mM ATP were differentially affected by substance P which preferentially reduced currents evoked by 5 mM ATP. In the presence of substance P a conditioning pulse of ATP evoked a stronger depression to subsequent test pulses of the same agonist. 4 Combined patch clamping and confocal laser imaging of intracellular Ca2+ ([Ca2+](i)) of single PC12 cells showed that substance P (applied by a pressure pipette) pet se had no effect on [Ca2+](i) or current baseline, although it reduced the inward current and associated [Ca2+](i) rise elicited by ATP. 5 These results are interpreted as due to facilitation by substance P of desensitization of ATP-gated P-2X2 receptors of PC12 cells. It is proposed that the novel modulation by this peptide of ATP responses may serve as a model for further studies aimed at elucidating the action of substance P on purinergic neurotransmission