P2Y Receptors in Noradrenergic Cells

ATP is known to act as a co-transmitter to noradrenaline in the central and peripheral nervous system. Extracellular adenine and uridine nucleotides exert their actions on either neurons or non-neural cells via membrane receptors known as P2 receptors. This family comprises two groups: P2X receptors are ATP-gated cation channels and P2Y receptors are G protein-coupled receptors. While P2X receptors are known to be involved in fast purinergic neurotransmission, the functional roles of neuronal P2Y receptors are still poorly understood. Transmitter release from central and peripheral noradrenergic neurons is known to be controlled by autoinhibitory alpha2- adrenoceptors (Starke, 1987; Boehm, 1999), the functional and pharmacological properties of which have been characterized in detail. In addition, noradrenergic neurons have been reported to be equipped with P2 receptors which also mediate an autoinhibitory regulation of transmitter release (e.g. Fuder and Muth, 1993; von Kügelgen et al, 1993 and 1995), but the characteristics of these receptors remained elusive. In previous experiments, we found that ATP released from sympathetic neurons in cell culture mediates positive as well as negative feedback modulation of noradrenaline release. The facilitatory effect was mediated by a P2X2 receptor, whereas the receptor mediating the inhibitory effect remained unknown (Boehm, 1999). The current project will characterize the receptor mediating the autoinhibitory action of ATP. In preliminary experiments, an adenine nucleotide-sensitive P2Y receptor that inhibited (i) voltage-activated Ca2+ channels, (ii) adenylyl cyclase activity, and (iii) depolarisation-evoked catecholamine secretion was detected in PC12 cells which are ontogenetically related to sympathetic neurons. Other effector systems, such as the phospholipase C-dependent generation of inositolphosphates or voltage-gated M-type K+ channels were not found to be regulated by this receptor. In sympathetic neurons, we were also able to detect the inhibitory effect of adenine nucleotides on voltage-activated Ca2+ channels, although PC12 cells and sympathetic neurons show a differing pattern of P2Y receptor expression, with P2Y1 receptors being expressed in the neurons only. The other preliminary results confirm that the inhibitory receptor is not a P2Y1 subtype, nor any of the other P2Y receptors described in the rat (P2Y2,-4, or -6), all of which are expressed in PC12 cells and sympathetic neurons. We therefore assume that it is an as yet unidentified P2Y receptor subtype that mediates the autonihibitory modulation of noradrenaline release. The experiments outlined in the proposal aim at the molecular, pharmacological and functional characterization of this inhibitory P2Y receptor, on one hand, and at a correlation between observed cellular nucleotide effects and involved P2Y receptor subtypes, on the other hand. The results will add to the current knowledge as to how