guanosine-triphosphate and naltrindole

1. A novel selective nociceptin/orphanin FQ (N/OFQ) peptide receptor antagonist, 1-[(3R,4R)-1-cyclooctylmethyl]-3-hydroxymethyl-4-piperidyl)-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (CompB), inhibited specific binding of [(3)H]N/OFQ to crude membranes from the rat brain and spinal cord in a concentration-dependent manner and their K(i) values were 7.11 and 4.02 nM, respectively. Rosenthal analysis indicated that there was a significant increase in the K(d) value for [(3)H]N/OFQ binding in the brain and spinal cord in the presence of CompB (10 nM). 2. There was a dose-dependent increase in K(d) values for [(3)H]N/OFQ binding in the brain and spinal cord following i.v. injection of CompB at relatively low doses (0.69-6.88 micro mol kg(-1)), compared with the control values. In the spinal cord, enhancement with each dose was constantly greater and the duration of enhancement (6.88 micro mol kg(-1)) was significantly longer. 3. The degree of increase in K(d) values for [(3)H]N/OFQ binding after i.v. injection of CompB (6.88 micro mol kg(-1)) was significantly larger in the lumbar region of the spinal cord compared to other regions. 4. CompB (0.1, 0.3 micro M) shifted the concentration-effect curves of N/OFQ-stimulated [(35)S]GTPgammaS binding in the brain and spinal cord to the right. 5. The i.v. injection of CompB (6.88 micro mol kg(-1)) significantly suppressed the N/OFQ-stimulated [(35)S]GTPgammaS binding in the rat spinal cord and shifted the concentration-effect curve to the right, while it produced little inhibitory effect in the brain. The present study has shown that CompB may exhibit pharmacological effects through a predominant blockade of N/OFQ peptide receptors in the spinal cord under in vivo conditions.

Topics: Animals; Benzimidazoles; Binding, Competitive; Brain; Dose-Response Relationship, Drug; Guanosine Triphosphate; Injections, Intravenous; Male; Naltrexone; Narcotic Antagonists; Nociceptin Receptor; Opioid Peptides; Peptide Fragments; Piperidines; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Spinal Cord

To assess the relative capacity of the human delta opioid receptor to activate closely related G proteins, fusion proteins were constructed in which the alpha-subunits of either G(i1) or G(o1), containing point mutations to render them insensitive to the actions of pertussis toxin, were linked in-frame with the C-terminus of the receptor. Following transient and stable expression in HEK 293 cells, both constructs bound the antagonist [(3)H]naltrindole with high affinity. D-ala(2),D-leu(5) Enkephalin effectively inhibited forskolin-stimulated adenylyl cyclase activity in intact cells in a concentration-dependent, but pertussis toxin-insensitive, manner. The high-affinity GTPase activity of both constructs was also stimulated by D-ala(2),D-leu(5) enkephalin with similar potency. However, enzyme kinetic analysis of agonist stimulation of GTPase activity demonstrated that the GTP turnover number produced in response to D-ala(2),D-leu(5) enkephalin was more than three times greater for G(i1)alpha than for G(o1)alpha. As the effect of agonist in both cases was to increase V:(max) without increasing the observed K:(m) for GTP, this is consistent with receptor promoting greater guanine nucleotide exchange, and thus activation, of G(i1)alpha compared with G(o1)alpha. An equivalent fusion protein between the human mu opioid receptor-1 and G(i1)alpha produced a similar D-ala(2),D-leu(5) enkephalin-induced GTP turnover number as the delta opioid receptor-G(i1)alpha fusion construct, consistent with agonist occupation of these two opioid receptor subtypes being equally efficiently coupled to activation of G(i1)alpha.

Topics: Adenylyl Cyclases; Cell Line; Colforsin; Diprenorphine; Enkephalin, Leucine-2-Alanine; GTP Phosphohydrolases; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Humans; Kinetics; Naltrexone; Polymerase Chain Reaction; Receptors, Opioid, delta; Recombinant Fusion Proteins; Recombinant Proteins; Transfection

The effect of (D-Ala, D-Leu) enkephalin (DADLE) on the binding of GTP in hippocampal preparations was studied. It was observed that treatment of hippocampal slices with 10(-5) -5 x 10(-5) M DADLE followed by the preparation of membrane fractions reduced the binding of 35S-GTP-gamma-S. There was no change in the affinity of the binding. This decrease of 35S-GTP-gamma-S binding was reversed when 5 x 10(-5) M naltrindole was included. The effect was not observed when the membrane fractions were incubated with DADLE. Photoaffinity labeling with the use of 32P P3-(4-azidoanilido)-P1 5'-GTP followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the incorporation of radioactivity into molecular mass of the 43 kDa and 33-34 kDa proteins. 32P Photolabeling of both the 43 kDa and 33-34 kDa bands decreased following treatment of hippocampal slices with 10(-4) M DADLE. These results suggested that DADLE reduces the GDP-GTP exchange in hippocampal membranes.

Topics: Affinity Labels; Animals; Autoradiography; Azides; Electrophoresis, Polyacrylamide Gel; Enkephalin, Leucine-2-Alanine; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hippocampus; In Vitro Techniques; Indoles; Male; Membranes; Morphinans; Naltrexone; Rats; Rats, Sprague-Dawley