[Phe1psi(CH2-NH)Gly2]nociceptin-(1 - 13)-NH2 activation of an inward rectifier as a partial agonist of ORL1 receptors in rat periaqueductal gray

Abstract

1. [Phe1psi(CH2-NH)Gly2]nociceptin-(1 - 13)-NH2 (Phepsi), a tridecapeptide analogue of orphanin FQ/nociceptin (OFQ/N), was introduced as a competitive antagonist of opioid receptor-like orphan receptor (ORL1) in guinea-pig ileum and mouse vas deferens preparations in vitro but was recently found to act as an agonist in vivo. 2. In the periaqueductal gray, a site enriched with both OFQ/N and ORL1 and involved in OFQ/N-induced hyperalgesia and anti-analgesia, the effects of Phepsi and OFQ/N on the membrane current were studied using whole cell patch clamp recording technique in rat brain slices. 3. OFQ/N (0.01 - 1 microM) activated an inwardly rectifying type of K+ channels in ventrolateral neurons of PAG. Phepsi (0.03 - 1 microM), like OFQ/N, also activated this inward rectifier but had only 30% efficacy of OFQ/N. 4 At maximal effective concentration (1 microM), Phepsi reversed the increment of K+ conductance induced by OFQ/N (300 nM) by 46%. On the other hand, Phepsi also prevented the effect of OFQ/N if pretreated before OFQ/N. 5 It is suggested that Phepsi acts as a partial agonist of ORL1 that mediates the activation of inwardly rectifying K+ channels in ventrolateral neurons of rat periaqueductal gray.

Figure 1

Both OFQ/N and Pheψ activated an inwardly rectifying type of K⁺ channels. Cell was held at −70 mV and step to −140 mV for 100 ms followed by a ramp depolarization from −140 to −60 mV at a rate of 0.2 mV ms⁻¹ (inset). I-V curves of membrane currents evoked by ramp commands before and after treatment with 300 nM OFQ/N (Aa) or 1 μM Pheψ (Ba). The I-V curve of peptide-elicited current was obtained by subtracting the control current from that in the presence of OFQ/N (Ab) or Pheψ (Bb). Both I-V curves show an inward rectification and reverse polarity at −94 mV (A) and −88 mV (B), which are close to the presumed K⁺ equilibrium potential (−91 mV).

Figure 2

Concentration-response curves of outward currents induced by OFQ/N and Pheψ. Cells were held at −70 mV. The changes of holding currents induced by OFQ/N and Pheψ were plotted against the peptide concentrations. The curves were fitted based on the equation I=Imax/[1+(EC₅₀/D)ⁿ], where I represents the outward current, Imax the maximal current, D the concentration of peptide and n the Hill coefficient. The EC₅₀ and n obtained from the curves are, respectively, 54±12 nM and 0.98 for OFQ/N and are 77±10 nM and 0.79 for Pheψ. Figures in the parenthesis are the number of cells tested.

Figure 3

Interactions between OFQ/N and Pheψ in eliciting the inwardly rectifying K⁺ conductance. Membrane currents were evoked by the same protocol as in Figure 1. (A) Cell was treated with 300 nM OFQ/N followed by further treatment with 1 μM Pheψ. (B) Cell was treated with 1 μM Pheψ followed by further treatment with 300 nM OFQ/N. The cell in (B) showed frequent spontaneous excitatory postsynaptic currents. Note that Pheψ partially reversed the effect of OFQ/N (A) and was unable to completely prevent its effect (B).