On the occasion of the thirtieth anniversary of the journal Acta Stomatologica Croatica

Introduction: Opioid receptors are currently classified as Mu (\u3bc), Delta (\u3b4), Kappa (\u3ba) plus the opioid related nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). Despite compelling evidence for interactions and benefits of targeting more than one receptor type in producing analgesia, clinical ligands are Mu agonists. In this study we have designed a Mu-NOP agonist named DeNo. The Mu agonist component is provided by dermorphin, a peptide isolated from the skin of Phyllomedusa frogs and the NOP component by the endogenous agonist N/OFQ. Methods: We have assessed receptor binding profile of DeNo and compared with dermorphin and N/OFQ. In a series of functional screens we have assessed the ability to (i) increase Ca2+ in cells coexpressing recombinant receptors and a the chimeric protein G\u3b1qi5, (ii) stimulate the binding of GTP\u3b3[35S], (iii) inhibit cAMP formation, (iv) activate MAPKinase, (v) stimulate receptor-G protein and arrestin interaction using BRET, (vi) electrically stimulated guinea pig ileum (gpI) assay and (vii) ability to produce analgesia via the intrathecal route in rats. Results: DeNo bound to Mu (pKi; 9.55) and NOP (pKi; 10.22) and with reasonable selectivity. This translated to increased Ca2+ in G\u3b1qi5 expressing cells (pEC50 Mu 7.17; NOP 9.69), increased binding of GTP\u3b3[35S] (pEC50 Mu 7.70; NOP 9.50) and receptor-G protein interaction in BRET (pEC50 Mu 8.01; NOP 9.02). cAMP formation was inhibited and arrestin was activated (pEC50 Mu 6.36; NOP 8.19). For MAPK DeNo activated p38 and ERK1/2 at Mu but only ERK1/2 at NOP. In the gpI DeNO inhibited electrically-evoked contractions (pEC50 8.63) that was sensitive to both Mu and NOP antagonists. DeNo was antinociceptive in rats. Conclusion: Collectively these data validate the strategy used to create a novel bivalent Mu-NOP peptide agonist by combining dermorphin (Mu) and N/OFQ (NOP). This molecule behaves essentially as the parent compounds in vitro. In the antonocicoeptive assays employed in this study DeNo displays only weak antinociceptive properties

The mechanism of constitutive activity in delta and mu opioid receptors

In this thesis I developed an assay based on BRET, in which receptor is tagged at the C-terminal with a luminescent donor and the G subunit of G protein is tagged at the N-terminal with a fluorescent acceptor. The extent of receptor-G protein coupling can be measured as change of the RET signal in membranes prepared from cells expressing the two tagged proteins. In this assay, the addition of GDP to the membranes can inhibit ligand-induced activity and abolish spontaneous receptor activity. Thus, the BRET signal in the presence of GDP marks the level of “zero coupling” between receptor and G protein, and the net difference between absence and presence of the guanine nucleotide is a precise measure of the constitutive activation. Ligands that enhance the BRET signal above the basal level are positive agonists, whereas those inhibiting the basal signal are inverse agonist

Novel signaling pathways in Nephrogenic Syndrome of Inappropriate Antidiuresis: Functional implication of site-specific AQP2 phosphorylation

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a rare X-linked disease caused by gain-of-functionmutations of arginine vasopressin receptor 2 (V2R). Patients with NSIAD are characterized by the inability to excrete a free water load and by inappropriately increased urinary osmolality despite very low levels of plasma vasopressin, resulting in euvolaemic hyponatraemia. To dissect the signalling downstream V2R constitutively active variants, Flp-In T-REx Madin-Darby canine kidney (FTM) cells, stably transfected with V2R mutants (R137L, R137C and F229V) and AQP2-wt or non-phosphorylatable AQP2-S269A/AQP2-S256A, were used as cellular models. All three activating V2R mutations presented constitutive plasma membrane expression of AQP2-wt and significantly higher basal water permeability. In addition, V2R-R137L/C showed significantly higher activity of Rho-associated kinase (ROCK), a serine/threonine kinase previously suggested to be involved in S269-AQP2 phosphorylation downstream of these V2R mutants. Interestingly, FTM cells expressing V2R-R137L/C mutants and AQP2-S269A showed a significant reduction in AQP2 membrane abundance and a significant reduction in ROCK activity, indicating the crucial importance of S269-AQP2 phosphorylation in the gain-of-function phenotype. Conversely, V2R-R137L/C mutants retained the gain-of-function phenotype when AQP2-S256A was co-expressed. In contrast, cells expressing the F229V mutant and the non-phosphorylatable AQP2-S256A had a significant reduction in AQP2 membrane abundance along with a significant reduction in basal osmotic water permeability, indicating a crucial role of Ser256 for this mutant. These data indicate that the constitutive AQP2 trafficking associated with the gain-of-function V2R-R137L/C mutants causing NSIAD is protein kinase A independent and requires an intact Ser269 in AQP2 under the control of ROCK phosphorylation

Crosstalk between β2- and α2-Adrenergic Receptors in the Regulation of B16F10 Melanoma Cell Proliferation

Adrenergic receptors (AR) belong to the G protein-coupled receptor superfamily and regulate migration and proliferation in various cell types. The objective of this study was to evaluate whether β-AR stimulation affects the antiproliferative action of α2-AR agonists on B16F10 cells and, if so, to determine the relative contribution of β-AR subtypes. Using pharmacological approaches, evaluation of Ki-67 expression by flow cytometry and luciferase-based cAMP assay, we found that treatment with isoproterenol, a β-AR agonist, increased cAMP levels in B16F10 melanoma cells without affecting cell proliferation. Propranolol inhibited the cAMP response to isoproterenol. In addition, stimulation of α2-ARs with agonists such as clonidine, a well-known antihypertensive drug, decreased cancer cell proliferation. This effect on cell proliferation was suppressed by treatment with isoproterenol. In turn, the suppressive effects of isoproterenol were abolished by the treatment with either ICI 118,551, a β2-AR antagonist, or propranolol, suggesting that isoproterenol effects are mainly mediated by the β2-AR stimulation. We conclude that the crosstalk between the β2-AR and α2-AR signaling pathways regulates the proliferative activity of B16F10 cells and may therefore represent a therapeutic target for melanoma therapy

β-arrestin 2 recruitment measured using BRET.

<p>Membrane extracts taken from HEK-293 and SH-SY5Y cells stably expressing NOP-RLuc and Mu-RLuc respectively together with β-arrestin 2-RGFP were used. Concentration response curves to N/OFQ, dermorphin and DeNo for receptor/β-arrestin 2 interaction in cells expressing the NOP (panel A) and Mu (panel B) receptors. Data are the mean ± SEM of at least 4 experiments performed in duplicate.</p

Displacement Binding Profiles.

<p>Displacement Binding Profiles.</p

Chemical structure of DeNo.

<p>Chemical structure of DeNo.</p

β-arrestin Recruitment.

<p>β-arrestin Recruitment.</p

The Activation of ERK1/2 and p38.

<p>A) The activity of phosphorylated p38 compared to total p38 at CHO_hMu caused by dermorphin (1μM) and DeNo (1μM). B) The activity of phosphorylated ERK1/2 compared to total ERK1/2 at CHO_hMu caused by dermorphin (1μM) and DeNo (1μM). C) The activity of phosphorylated ERK1/2 at CHO_hNOP caused by N/OFQ (1μM) and DeNo (1μM). All data are after a 15 minute incubation period. Data are mean (±SEM) for n = 5. *p<0.05; according to ANOVA followed by Dunnett’s test for multiple comparison. Representative blots are shown above mean data.</p

GTPγS Functional Activity.

<p>GTPγS Functional Activity.</p