Pharmacological profile of NOP receptors coupled with calcium signaling via the chimeric protein Gαqi5

Abstract

In this study, the Galpha(qi5) protein was used to force the human nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor to signal through the Ca(2+) pathway in CHO cells. [Ca(2+)](i) levels were monitored using the fluorometer FlexStation II and the Ca(2+) dye Fluo 4 AM. Concentration response curves were generated with a panel of full and partial agonists, while NOP antagonists were assessed in inhibition-response curves. The following rank order of potency of antagonists was measured: SB - 612111 > J - 113397 = Trap - 101 > or = UFP - 101 > [Nphe1]N/OF Q(1 - 13)NH2 >> naloxone, which is superimposable to literature findings. The rank order of potency of full and partial agonists is also similar to that obtained in previous studies with the exception of a panel of ligands (UFP-112, Ro 64-6198, ZP120, UFP-113) whose potency was relatively low in the Galpha(qi5)-NOP receptor calcium assay. Interestingly, these NOP ligands are characterized by slow kinetic of interaction with the NOP receptor, as demonstrated by bioassay experiments. These results demonstrated that the FlexStation II-Galpha(qi5)-NOP receptor calcium assay represents an adequate and useful screening for NOP receptor ligands, particularly for antagonists.

Nociceptin/Orphanin FQ Receptor Structure, Signaling, Ligands, Functions, and Interactions with Opioid Systems

“…This basic in vitro pharmacological profile has been confirmed in several studies using different assays (Dautzenberg et al, 2001;Hashiba et al, 2002;McDonald et al, 2003a;McLeod et al, 2004;Camarda et al, 2009 (Gehlert et al, 2006). However, other results underlined some differences in Ro 64-6198 versus N/OFQ in vitro actions.…”

“…Each of these compounds is characterized by a slow kinetics of activation of the NOP receptor, as suggested by bioassay experiments in isolated tissues (Rizzi et al, 2002c;Calo' et al, 2011;Guerrini et al, 2014). It is possible that the rapid kinetics that characterize the calcium transient response may be incompatible with the slow kinetics of the ligand receptor interaction (for a detailed discussion of this topic see Camarda et al, 2009;Rizzi et al, 2014). In vivo, however, ZP120 displayed very high potency and long duration of action in locomotor activity and tail withdrawal experiments in mice , effects that were no longer present in NOP(2/2) mice (Fischetti et al, 2009a).…”

Acute and subchronic antinociceptive effects of nociceptin/orphanin FQ receptor agonists infused by intrathecal route in rats

“…The spinal antinociceptive action of the NOP partial agonist UFP-113 (Arduin et al, 2007;Camarda et al, 2009) has been evaluated in the present research for the first time. Acute i.t.…”

Pharmacological studies on the NOP and opioid receptor agonist PWT2-[Dmt1]N/OFQ(1-13)

“…CHO cells stably coexpressing either human recombinant NOP, mu or kappa receptor along with the C-terminally modified Gα qi5 chimeric protein and cells co-expressing the delta receptor and the Gα qG66Di5 chimeric protein were generated as previously described (Camarda and Calo, 2013; Camarda et al, 2009). Cells were cultured in culture medium consisting of Dulbecco’s modified Eagle’s medium (DMEM)/HAMS F12 (1:1) supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/ml), streptomycin (100 μg/ml), L-glutamine (2 mM); fungizone (1 μg/ml), geneticin (G418; 200 μg/ml) and hygromycin B (100 μg/ml).…”