Autoinhibition of transmitter release from PC12 cells and sympathetic neurons through a P2Y₁₂ receptor‐mediated inhibition of voltage‐gated Ca²⁺ channels

Abstract

Although feedback inhibition of noradrenaline release by coreleased nucleotides is a well known phenomenon, it remained unclear which P2 receptor subtypes and associated signalling cascades may be involved. In the rat pheochromocytoma cell line PC12, 2-methylthio-ADP reduced noradrenaline release triggered by K+ depolarization more potently than ADP and ATP, whereas UDP or UTP failed to do so. The inhibition by ADP was abolished by pertussis toxin and antagonized by reactive blue 2, 2-methylthio-AMP, and AR-C69931MX, but not by suramin. AR-C69931MX acted as a competitive antagonist with an apparent affinity of 2 nm, but did not alter noradrenaline release, when PC12 cells were continuously superfused. However, when the superfusion was halted during K+ depolarization, release was significantly reduced and this inhibition was attenuated by AR-C69931MX, thus revealing ongoing autoinhibition. Rises in cellular cyclic AMP did not alter depolarization-evoked release nor its reduction by ADP, even though the nucleotide did inhibit cyclic AMP accumulation. ADP and the direct Ca2+ channel blocker Cd2+ inhibited voltage-activated Ca2+ currents, but not ATP-induced currents, and both agents reduced K+-evoked, but not ATP-evoked, release. Hence, if voltage-gated Ca2+ channels do not contribute to stimulation-evoked release, ADP fails to exert its inhibitory action. In primary cultures of rat sympathetic neurons, ADP also reduced Ca2+ currents and K+-evoked noradrenaline release, and AR-C69931MX acted again as competitive antagonist with an apparent affinity of 3 nm. These results show that P2Y12 receptors mediate an autoinhibition of transmitter release from PC12 cells and sympathetic neurons through an inhibition of voltage-gated Ca2+ channels.

P2Y 2 receptor activates nerve growth factor/TrkA signaling to enhance neuronal differentiation

“…Nucleotides in the nervous system are stored in, and released from, synaptic vesicles and glial cells at mM concentrations, thereby affecting neurotransmitter release and glial calcium wave propagation, respectively (11,12). ATP has been proposed as an activitydependent signaling molecule that regulates glial-glial and glialneuron communication (12,13).…”

Facilitation of transmitter release from rat sympathetic neurons via presynaptic P2Y1 receptors

“…3 H]-noradrenaline release, and this is prevented by 2-methylthio-AMP, an antagonist at P2Y12 and P2Y13 receptors (Queiroz et al, 2003) Likewise, in PC12 cells and rat superior cervical ganglion (SCG) neurons, inhibitory presynaptic P2Y receptors were blocked by 2-methylthio-AMP and by the P2Y12 antagonist cangrelor (Kulick & von Kugelgen, 2002;Lechner et al, 2004). More recently, P2Y12 together with P2Y1 receptors were shown to mediate autoinhibition in sympathetically innervated tissues (Quintas et al, 2009).…”

“…ADP, at a concentration of 100 mM, has been found to reduce noradrenaline release from rat SCG neurons triggered by 30 mM K + ; however, in neurons treated with pertussis toxin to prevent the signalling via inhibitory G-proteins, the nucleotide tended to enhance stimulation-evoked release (Lechner et al, 2004). Therefore, the effect of ADP was investigated in SCG cultures treated with pertussis toxin (100 ng·mL -1 ) for 24 h and labelled with [ 3 H]-noradrenaline.…”

“…To reveal whether the facilitation by ADP may also occur in neurons with functional Gi/o proteins, experiments were repeated in cultures not treated with PTX; 100 mM ADP caused a significant reduction of electrically-evoked tritium overflow in these cultures ( Figure 3A), as described previously where this inhibition of noradrenaline release was suggested to involve P2Y12 receptors (Lechner et al, 2004). Therefore, experiments were performed in the presence of the P2Y12 antagonist cangrelor (Jacobson and Boeynaems, 2010).…”

“…[ 3 H]-noradrenaline uptake and superfusion of SCG neurons and PC12 cells was performed as described previously (Lechner et al, 2004). The plastic discs with dissociated neurons or PC12 cells were incubated in 0.05 mM ,5,6-3 H]-noradrenaline, specific activity 1.369 TBq·mmol -1 ; Perkin Elmer, Vienna, Austria) in culture medium supplemented with 1 mM ascorbic acid at 37°C for 1 h. Thereafter, the culture discs were transferred to small chambers and superfused with a buffer containing (mM) NaCl (120), KCl (3.0), CaCl2 (2.0), MgCl2 (2.0), glucose (20), HEPES (10), fumaric acid (0.5), Na-pyruvate (5.0), ascorbic acid (0.57) and desipramine (0.001), adjusted to pH 7.4 with NaOH.…”