Increase of intracellular Ca²⁺ by P2Y but not P2X receptors in cultured cortical multipolar neurons of the rat

Abstract

The expression and functionality of P2X/P2Y receptor subtypes in multipolar nonpyramidal neurons of mixed cortical cell cultures were investigated by means of immunocytochemistry and fura-2 microfluorimetry. The morphological studies revealed that most of the neurons are immunoreactive for GABA and express a range of P2X/P2Y receptors, predominantly of the P2X(2,4,6) and P2Y(1,2) subtypes. P2X(1) and P2X(7) receptor immunoreactivity (IR) was found on thin axon-like processes and presynaptic structures, respectively. Application of ATP caused a small concentration-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in most investigated neurons, whereas only about the half of these cells responded to 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP), ADPbetaS, 2MeSADP, or 2MeSATP and even fewer cells to UTP. In contrast, alpha,beta-meATP, UDP, and UDP-glucose failed to produce any [Ca2+]i signaling. The response to ATP itself was inhibited by pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Reactive Blue 2, 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS2179), and suramin (300 microM) as well as by a cyclopiazonic acid-induced depletion of intracellular Ca2+ stores. A Ca2+-free external medium tended to decrease the ATP-induced [Ca2+]i transients, although this action did not reach statistical significance. Various blockers of voltage-sensitive Ca2+ channels and the gap junction inhibitor carbenoxolone did not interfere with the effect of ATP, whereas a combination of the ionotropic glutamate receptor antagonists D(-)-2-amino-5-phosphonopentanoic acid (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) decreased it. Cross-desensitization experiments between ADPbetaS or UTP and ATP suggested that ATP acts on the one hand via P2Y(1,2) receptors and on the other hand by additional signaling mechanisms. These mechanisms may involve the release of glutamate (which in consequence activates ionotropic glutamate receptors) and the entry of Ca2+ via store-operated Ca2+ channels. Evidence for the presence of functional P2X receptors, in particular P2X(7), remains elusive.

Release, neuronal effects and removal of extracellular β‐nicotinamide adenine dinucleotide (β‐NAD+) in the rat brain

“…β‐NAD + failed to elicit Ca 2+ transients in the absence of extracellular Ca 2+ , implying that the rapid neuronal responses are due to influx of Ca 2+ from the extracellular space. These results differ from a recent report in rat cultured neurons (Fischer et al. , 2009), which has demonstrated more modest Ca 2+ increases following much longer exposures (10 s) to lower P2 receptor agonist concentrations (i.e.…”

P2X7 receptor-pannexin 1 interaction mediates extracellular alpha-synuclein-induced ATP release in neuroblastoma SH-SY5Y cells

“…Previously, the linkage between P2X7 receptor and Panx1 was also considered to be necessary for P2X7-mediated generation of ROS and proinflammatory cytokines in macrophages [ 89 , 90 ]. On the other hand, neurons also express both P2X7 and Panx1, but their functionality remains controversial [ 91 – 93 ]. Our current studies have shown that inhibition of Panx1 dampens extracellular ATP levels; however, it does not inhibit P2X7 receptor activity.…”

P2X7 receptor inhibition ameliorates dendritic spine pathology and social behavioral deficits in Rett syndrome mice

“…Next, we tested whether the beneficial effects of blocking P2X7Rs in Mecp2 308/Y mice can be achieved by primarily inhibiting P2X7Rs in peripheral tissues (e.g., blood, brain's perivascular spaces and meninges). Brilliant Blue G (BBG) is a U.S. Food and Drug Administration-approved drug and has been shown to block P2X7R-mediated increase in membrane permeability and macroscopic currents in cell cultures and spinal cord slices 21,46 . Previous studies have shown that BBG has low penetrance to the CNS parenchyma (brain to plasma ratiõ 0.01).…”