Reciprocal regulation of agonist and inverse agonist signaling efficacy upon short-term treatment of the human delta-opioid receptor with an inverse agonist.

Rapid regulation of receptor signaling by agonist ligands is widely accepted, whereas short-term adaptation to inverse agonists has been little documented. In the present study, guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding and cAMP accumulation assays were used to assess the consequences of 30-min exposure to the inverse agonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI174864) (1 microM) on delta-opioid receptor signaling efficacy. ICI174864 pretreatment increased maximal effect (E(max)) for the partial agonist Tyr-1,2,3,4-tetrahydroisoquinoline-Phe-Phe-OH (TIPP) at the two levels of the signaling cascade, whereas E(max) values for more efficacious agonists like (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80) and bremazocine were increased in [(35)S]GTPgammaS binding but not in cAMP accumulation assays. Pre-exposure to ICI174864 also induced a shift to the left in dose-response curves for bremazocine and TIPP. On the other hand, E(max) for the inverse agonist H-Tyr-TicPsi[CH(2)NH]Cha-Phe-OH was reduced in both assays, but no changes in potency were observed. For the weaker inverse agonist naloxone, E(max) in [(35)S]GTPgammaS binding was drastically modified because the drug turned from inverse agonist to agonist after ICI174864 pretreatment. Likewise, ICI174864 turned from inverse agonist to agonist when tested in cAMP accumulation assays. In both cases, inversion of efficacy was concomitant with marked increase in potency for agonist effects. Together with functional changes, short-term treatment with ICI174864 reduced basal receptor phosphorylation and increased immunoreactivity for Galpha(i3) in membrane preparations. Functional consequences of ICI174864 pretreatment were simulated in the cubic ternary complex model by increasing receptor/G protein coupling or G protein amount available for interaction with the receptor. Taken together, these data show that inverse agonists may induce rapid regulation in receptor signaling efficacy.

Allotopic antagonism of the non-peptide atrial natriuretic peptide (ANP) antagonist HS-142-1 on natriuretic peptide receptor NPR-A.

The microbial polysaccharide HS-142-1 has been documented as an antagonist of natriuretic peptides. It inhibits activation and peptide binding to both guanylate receptors natriuretic peptide receptor (NPR)-A and NPR-B, but has no effect on the non-cyclase receptor NPR-C. At first sight the effect of HS-142-1 on peptide binding appears to be surmountable, suggesting that it might be competitive despite its chemically divergent nature. We explored its mode of action on wild-type NPR-A (WT), on a disulphide-bridged constitutively active mutant (C423S) and on truncated mutants lacking either their cytoplasmic domain (DeltaKC) or both the cytoplasmic and the transmembrane domains (ECD). On the WT, HS-142-1 inhibited atrial natriuretic peptide (ANP) binding with a pK value of 6.51 +/- 0.07 (K(d)=0.31 microM). It displayed a similar effect on the C423S mutant (pK=6.31 +/- 0.11), indicating that its action might not be due to interference with receptor dimerization. HS-142-1 also inhibited ANP binding to DeltaKC with a pK of 7.05 +/- 0.05 (K(d)=0.089 microM), but it was inactive on ANP binding to ECD at a concentration of 10(-4) M, suggesting that the antagonism was not competitive at the peptide-binding site located on the ECD and that the transmembrane domain might be required. HS-142-1 also enhanced dissociation of NPR-A-bound (125)I-ANP in the presence of excess unlabelled ANP, implying an allotopic (allosteric) mode of action for the antagonist.