ABSTRACT
Through inhibitory and excitatory effects on sympathetic neurons, B(2) bradykinin receptors contribute to protective and noxious cardiovascular mechanisms. Presynaptic inhibition of sympathetic transmitter release involves an inhibition of Ca(V)2 channels, neuronal excitation an inhibition of K(V)7 channels. To investigate which of these mechanisms prevail over time, the respective currents were determined. The inhibition of Ca(2+) currents by bradykinin reached a maximum of 50%, started to fade within the first minute, and became attenuated significantly after > or = 4 min. The inhibition of K(+) currents reached a maximum of 85%, started to fade after > 3 min, and became attenuated significantly after > or = 7 min. Blocking Ca(2+)-independent protein kinase C (PKC) enhanced the inhibition of Ca(2+) 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 Ca(2+) 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.
Subject(s)
Neural Inhibition/physiology , Neurons/enzymology , Protein Kinase C/physiology , Receptor, Bradykinin B2/metabolism , Superior Cervical Ganglion/cytology , Adrenergic beta-Antagonists/pharmacology , Animals , Animals, Newborn , Bradykinin/analogs & derivatives , Bradykinin/pharmacology , Calcium/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Indoles/pharmacology , Maleimides/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Neural Inhibition/drug effects , Neurons/drug effects , Norepinephrine/metabolism , Patch-Clamp Techniques/methods , Rats , Rats, Sprague-Dawley , Sodium Channel Blockers/pharmacology , Statistics, Nonparametric , Tetradecanoylphorbol Acetate/analogs & derivatives , Tetradecanoylphorbol Acetate/pharmacology , Tetrodotoxin/pharmacology , Time Factors , Tritium/metabolismABSTRACT
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.
