guanosine-diphosphate and endomorphin-1

P-type calcium channels play a key role in the synaptic transmission between mammalian central neurons since a major part of calcium entering pre-synaptic terminals is delivered via these channels. Using conventional whole-cell patch clamp techniques we have studied the effect of mu-opioids on P-type calcium channels in acutely isolated Purkinje neurons from rat cerebellum. The selective mu-opioid agonist DAMGO (10nM) produced a small, but consistent facilitation of current through P-type calcium channels (10+/-1%, n=27, p<0.001). The effect of DAMGO was rapid (less than 10s) and fully reversible. This effect was both concentration and voltage-dependent. The EC(50) for the effect of DAMGO was 1.3+/-0.4nM and the saturating concentration was 100nM. The endogenous selective agonist of mu-opioid receptors, endomorphin-1 demonstrated similar action. Intracellular perfusion of Purkinje neurons with GTPgammaS (0.5mM) or GDPbetaS (0.5mM), as well as strong depolarizing pre-pulses (+50mV), did not eliminate facilitatory action of DAMGO on P-channels indicating that this effect is not mediated by G-proteins. Furthermore, the effect of DAMGO was preserved in the presence of a non-specific inhibitor of PKA and PKC (H7, 10microM) inside the cell. DAMGO-induced facilitation of P-current was almost completely abolished by the selective mu-opioid antagonist CTOP (100nM). These observations indicate that mu-type opioid receptors modulate P-type calcium channels in Purkinje neurons via G-protein-independent mechanism.

Topics: Animals; Calcium Channels, P-Type; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Oligopeptides; Protein Kinase C; Purkinje Cells; Rats; Receptors, Opioid, mu; Somatostatin; Thionucleotides

Endomorphin-1 is a peptide whose binding selectivity suggests a role as an endogenous ligand at mu-opioid receptors. In the present study, the effect of endomorphin-1 on mu receptor-coupled G proteins was compared with that of the mu agonist DAMGO by using agonist-stimulated [35S]GTPgammaS binding in rat brain. [35S]GTPgammaS autoradiography revealed a similar localization of endomorphin-1- and DAMGO-stimulated [35S]GTPgammaS binding in areas including thalamus, caudate-putamen, amygdala, periaqueductal gray, parabrachial nucleus, and nucleus tractus solitarius. Naloxone blocked endomorphin-1-stimulated labeling in all regions examined. Although the distribution of endomorphin-1-stimulated [35S]GTPgammaS binding resembled that of DAMGO, the magnitude of endomorphin-1-stimulated binding was significantly lower than that produced by DAMGO. Concentration-effect curves of endomorphin-1 and DAMGO in thalamic membranes confirmed that endomorphin-1 produced only 70% of DAMGO-stimulated [35S]GTPgammaS binding. Differences in maximal stimulation of [35S]GTPgammaS binding between DAMGO and endomorphin-1 were magnified by increasing GDP concentrations, and saturation analysis of net endomorphin-1-stimulated [35S]GTPgammaS binding revealed a lower apparent Bmax value than that obtained with DAMGO. Endomorphin-1 also partially antagonized DAMGO stimulation of [35S]GTPgammaS binding. These results demonstrate that endomorphin-1 is a partial agonist for G protein activation at the mu-opioid receptor in brain.

Topics: Animals; Autoradiography; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Male; Naloxone; Narcotic Antagonists; Oligopeptides; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Sulfur Radioisotopes