In vitro and ex vivo effects of a selective nociceptin/orphanin FQ (N/OFQ) peptide receptor antagonist, CompB, on specific binding of [3H]N/OFQ and [35S]GTPgammaS in rat brain and spinal cord

Abstract

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.

Figure 1

Rosenthal plots of specific [³H]N/OFQ binding in the brain and spinal cord of rats in the absence (control) and presence of CompB. Specific binding of [³H]N/OFQ (0.01–0.6 nM) in rat tissues was measured in the absence and presence of CompB (10 nM). Values are means of four rats.

Figure 2

Rosenthal plots of specific [³H]N/OFQ binding in the brain and spinal cord of vehicle-(control) and CompB-administered rats. Rats received CompB (2.29 and 6.88 μmol kg⁻¹ i.v.), and were killed at 0.5 h after the administration. Each point represents the mean of six control- and four or five CompB-treated rats.

Figure 3

Concentration–effect curves of N/OFQ-stimulated [³⁵S]GTPγS binding in the brain and spinal cord of rats in the absence (control) and presence of CompB. The ordinate is per cent stimulation over basal [³⁵S]GTPγS binding in the absence of N/OFQ. The abscissa is −logarithmic molar concentrations of N/OFQ (0.03–3 μM). Each point represents the mean±s.e.m. of six control and four or five CompB-treated rats. Asterisks show a significant difference from the control values, *P<0.05, **P<0.01, ***P<0.001.

Figure 4

Concentration–effect curves of N/OFQ-stimulated [³⁵S]GTPγS binding in the brain and spinal cord of vehicle- (control) and CompB-administered rats. Rats received CompB (6.88 μmol kg⁻¹, i.v.), and were killed at 0.5 h after the administration. The ordinate is per cent stimulation over basal [³⁵S]GTPγS binding in the absence of N/OFQ. The abscissa is −logarithmic molar concentrations of N/OFQ (0.03–3 μM). Each point represents mean±s.e.m. of four rats. Asterisks show a significant difference from the control values, **P<0.01.