Most cells express more than one receptor plus degrading enzymes for adenine nucleotides or nucleosides, and cellular responses to purines are rarely compatible with the actions of single receptors. Therefore, these receptors are viewed as components of a combinatorial receptor web rather than self-dependent entities, but it remained unclear to what extent they can associate with each other to form signalling units. P2Y 1 , P2Y 2 , P2Y 12 , P2Y 13 , P2X 2 , A 1 , A 2A receptors and NTPDase1 and -2 were expressed as fluorescent fusion proteins which were targeted to membranes and signalled like the unlabelled counterparts. When tested by FRET microscopy, all the G protein-coupled receptors proved able to form heterooligomers with each other, and P2Y 1 , P2Y 13 , A 1 , A 2A , and P2X 2 receptors also formed homooligomers. P2Y receptors did not associate with P2X, but G protein-coupled receptors formed heterooligomers with NTPDase1, but not with NTPDase2. The specificity of prototypic interactions (P2Y 1 /P2Y 1 , A 2A / P2Y 1 , A 2A /P2Y 12 ) was corroborated by FRET competition or co-immunoprecipitation. These results demonstrate that G protein-coupled purine receptors associate with each other and with NTPDase1 in a highly promiscuous manner. Thus, purinergic signalling is not only determined by the expression of receptors and enzymes but also by their direct interaction within a previously unrecognized multifarious membrane network.

Most cells express more than one receptor plus degrading enzymes for adenine nucleotides or nucleosides, and cellular responses to purines are rarely compatible with the actions of single receptors. Therefore, these receptors are viewed as components of a combinatorial receptor web rather than self-dependent entities, but it remained unclear to what extent they can associate with each other to form signalling units. P2Y(1), P2Y(2), P2Y(12), P2Y(13), P2X(2), A(1), A(2A) receptors and NTPDase1 and -2 were expressed as fluorescent fusion proteins which were targeted to membranes and signalled like the unlabelled counterparts. When tested by FRET microscopy, all the G protein-coupled receptors proved able to form heterooligomers with each other, and P2Y(1), P2Y(12), P2Y(13), A(1), A(2A), and P2X(2) receptors also formed homooligomers. P2Y receptors did not associate with P2X, but G protein-coupled receptors formed heterooligomers with NTPDase1, but not NTPDase2. The specificity of prototypic interactions (P2Y(1)/P2Y(1), A(2A)/P2Y(1), A(2A)/P2Y(12)) was corroborated by FRET competition or co-immunoprecipitation. These results demonstrate that G protein-coupled purine receptors associate with each other and with NTPDase1 in a highly promiscuous manner. Thus, purinergic signalling is not only determined by the expression of receptors and enzymes but also by their direct interaction within a previously unrecognized multifarious membrane network.

Background and purpose: M2, M3 and/or M4 muscarinic acetylcholine receptors have been reported to mediate presynaptic inhibition in sympathetic neurons. M1 receptors mediate an inhibition of Kv7, CaV1 and CaV2.2 channels. These effects cause increases and decreases in transmitter release, respectively, but presynaptic M1 receptors are generally considered facilitatory. Here, we searched for inhibitory presynaptic M1 receptors. Experimental approach: In primary cultures of rat superior cervical ganglion neurons, Ca 2+ currents were recorded via the perforated patch-clamp technique, and the release of [ 3 H]-noradrenaline was determined. Key results: The muscarinic agonist oxotremorine M (OxoM) transiently enhanced 3 H outflow and reduced electrically evoked release, once the stimulant effect had faded. The stimulant effect was enhanced by pertussis toxin (PTX) and was abolished by blocking M1 receptors, by opening Kv7 channels and by preventing action potential propagation. The inhibitory effect was not altered by preventing action potentials or by opening Kv7 channels, but was reduced by PTX and w-conotoxin GVIA. The inhibition remaining after PTX treatment was abolished by blockage of M1 receptors or inhibition of phospholipase C. When [ 3 H]-noradrenaline release was triggered independently of voltage-activated Ca 2+ channels (VACCs), OxoM failed to cause any inhibition. The inhibition of Ca 2+ currents by OxoM was also reduced by w-conotoxin and PTX and was abolished by M1 antagonism in PTX-treated neurons. Conclusions and implications:These results demonstrate that M1, in addition to M2, M3 and M4, receptors mediate presynaptic inhibition in sympathetic neurons using phospholipase C to close VACCs.

Through inhibitory and excitatory effects on sympathetic neurons, B2 bradykinin receptors contribute to protective and noxious cardiovascular mechanisms. Presynaptic inhibition of sympathetic transmitter release involves an inhibition of CaV2 channels, neuronal excitation an inhibition of KV7 channels. To investigate which of these mechanisms prevail over time, the respective currents were determined. The inhibition of Ca2+ currents by bradykinin reached a maximum of 50%, started to fade within the first minute, and became attenuated significantly after ≥ 4 min. The inhibition of K+ currents reached a maximum of 85%, started to fade after > 3 min, and became attenuated significantly after ≥ 7 min. Blocking Ca2+‐independent protein kinase C (PKC) enhanced the inhibition of Ca2+ currents by bradykinin and delayed its fading, left the inhibition of K+ currents and its fading unaltered, and enhanced the reduction of noradrenaline release and slowed its fading. Conversely, direct activation of PKC abolished the inhibition of noradrenaline release and largely attenuated the inhibition of Ca2+ currents. These results show that the inhibitory effects of bradykinin in sympathetic neurons are outweighed over time by its excitatory actions because of more rapid, PKC‐dependent fading of the inhibitory response.