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Antagonistic effects of [Nphe(1)]nociceptin(1-13)NH2 on nociceptin receptor mediated inhibition of cAMP formation in Chinese hamster ovary cells stably expressing the recombinant human nociceptin receptor
No full textNociceptin/orphanin FQ (NC) is the endogenous ligand for the nociceptin receptor (NCR) which is negatively coupled
to adenylyl cyclase to inhibit the formation of cAMP. In this study we describe the inhibitory action of the novel NC
analogue, [Nphe1]nociceptin(1±13)NH2 on cAMP formation in Chinese hamster ovary cells expressing the human NCR.
NC, NC(1±13)NH2, the pseudopeptides [Phe1c(CH2±NH)Gly2]NC(1±17)NH2 and [Phe1c(CH2±NH)Gly2]NC(1±13)NH2, the
hexapeptide, acetyl-Arg-Tyr-Tyr-Arg-Trp-Lys-NH2 and buprenorphine all produced a concentration dependent inhibition
of forskolin stimulated cAMP formation. This inhibition was competitively reversed by [Nphe1]NC(1±13)NH2 with essentially
identical pA2 values (6.12±6.48). [Nphe1]NC(1±13)NH2 showed per se a negligible residual agonist activity (a,0.15)
UFP-101, a high affinity antagonist for the nociceptin/orphanin FQ receptor: radioligand and GTPgamma35S binding studies
No full textAntagonistic effects of [Nphe1]nociceptin(1-13)NH2 on nociceptin receptor mediated inhibition of cAMP formation in Chinese ovary cells stably expressing the recombinant human nociceptin receptor
No full textNociceptin/orphanin FQ (NC) is the endogenous ligand for the nociceptin receptor (NCR) which is negatively coupled to adenylyl cyclase to inhibit the formation of cAMP. In this study we describe the inhibitory action of the novel NC analogue, [Nphe1]nociceptin(1-13)NH2 on cAMP formation in Chinese hamster ovary cells expressing the human NCR. NC, NC(1-13)NH2, the pseudopeptides [Phe1psi(CH2-NH)Gly2]NC(1-17)NH2 and [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2, the hexapeptide, acetyl-Arg-Tyr-Tyr-Arg-Trp-Lys-NH2 and buprenorphine all produced a concentration dependent inhibition of forskolin stimulated cAMP formation. This inhibition was competitively reversed by [Nphe1]NC(1-13)NH2 with essentially identical pA2 values (6.12-6.48). [Nphe1]NC(1-13)NH2 showed per se a negligible residual agonist activity (alpha < 0.15)
UFP-101, a high affinity antagonist for the nociceptin/orphanin FQ receptor: radioligand and GTP gamma S-35 binding studies
No full textStudies of the pharmacology of nociceptin/orphanin
FQ (N/OFQ) and its receptor (NOP) have been
hampered by the lack of a range of high potency antagonists.
In this study we have examined the effects of a
novel N/OFQ analogue [Nphe1,Arg14,Lys15]N/OFQ NH2
hereafter referred to as UFP-101. [3H]N/OFQ competition
binding and GTPγ35S binding assays were performed using
CHO cells expressing the human NOP receptor (CHOhNOP).
UFP-101 (pKi of 10.14±0.09) and a range of NOP selective
agonists displaced [3H]N/OFQ binding with the following
rank order of affinity: [Arg14,Lys15]N/OFQ>[(pF)
Phe4]N/OFQ(1–13)NH2>N/OFQ(1–13)NH2>UFP-101>N/
OFQ>Ro64–6198>[Nphe1]N/OFQ(1–13)NH2. N/OFQ, N/
OFQ(1–13)NH2, [(pF)Phe4]N/OFQ(1-13)NH2, [Arg14,Lys15]
N/OFQ and Ro64–6198 also produced a concentration dependent
(pEC50 values of 8.75±0.11, 9.28±0.15, 9.69±0.04,
9.12±0.11 and 8.09±0.07 respectively) and saturable stimulation
of GTPγ35S binding and all were full agonists.
UFP-101 did not stimulate GTPγ35S binding per se, but
produced a concentration dependent and parallel rightward
shift in the concentration response curves to all agonists.
UFP-101 yielded pA2 values in the range 8.4–9.0.
For comparison a pA2 for [Nphe1]N/OFQ(1–13)NH2 (the
template for UFP-101) against N/OFQ of 7.33±0.08 was
obtained. Slope factors for the Schild regression lines were
~1 indicating competitivity. When UFP-101 is compared
with its template molecule [Nphe1]N/OFQ(1–13)NH2,
Arg14,Lys15 substitution produced ~1 log greater potency.
We suggest that due to its high potency UFP-101 should
prove a further useful tool in the evaluation of the N/OFQNOP
receptor system
Effects of chronic nociceptin/orphanin FQ exposure on cAMP accumulation and receptor density in Chinese, hamster ovary cells expressing human nociceptin/orphanin FQ receptors
No full textNociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the Gi-coupled N/OFQ receptor (NOP). We have examined the effects of
chronic exposure of Chinese hamster ovary cells expressing the recombinant human NOP receptor (CHOhNOP) to 1 nM N/OFQ for up to 48 h
in the absence and presence of the NOP selective antagonist [Nphe1]N/OFQ (1–13)NH2 ([Nphe1]). Then, either a concentration–response
curve for N/OFQ inhibition of cAMP formation was constructed or the cells were homogenized and membrane receptor density was
determined using [125I]Y14N/OFQ. There was a time-dependent reduction in pEC50 (without a change in maximum) for N/OFQ with
significant differences observed following > 24 h of exposure (control pEC50f9.5; 48 h pretreatmentf8.7). In cells co-exposed to N/
OFQ+[Nphe1] for 48 h, there was no reduction in pEC50. There was a compensatory (f2.5-fold), [Nphe1]-sensitive increase in cAMP mass
in cells exposed to N/OFQ for 24–48 h. N/OFQ pretreatment also resulted in a time-dependent [Nphe1]-sensitive loss of cell surface
receptors. At 48 h, Bmax was reduced from f2.0 to f1.3 pmol mg1 protein without a change in pKd for N/OFQ. There was a positive
correlation between pEC50 for cAMP inhibition and Bmax. The lack of effect on maximum cAMP response probably results from receptor
overexpression and the creation of a receptor reserve
The effects of nociceptin peptide (N/OFQ)-receptor (NOP) system activation in the airways.
No full textThe effect of guanethidine and local anaesthetics on the electrically stimulated mouse vas deferens
Full text linkComplex regional pain syndrome is often treated with the sympatholytic guanethidine and a local anesthetic in a Bier's block. The efficacy of this treatment has been questioned. Because local anesthetics inhibit the norepinephrine uptake transporter, we hypothesized that this variable efficacy results from the local inhibiting the uptake of guanethidine. In this study, we tested this hypothesis by using a sympathetically innervated mouse vas deferens preparation. Organ bath-mounted mouse vasa deferentia were electrically stimulated in the absence and presence of guanethidine, prilocaine, procaine, and cocaine in various combinations. Prilocaine (1 mM) induced an immediate inhibition of twitch response (maximum 100% after 2 min) that fully reversed after washing. Guanethidine (3 microM) also inhibited twitching by 95% +/- 3% in 15 min, but this effect was only partially reversed after 1 h of washing (33% +/- 12% of control). When prilocaine and guanethidine were added in combination, a reversal of 80% +/- 13% (at 1 h) was observed. Procaine (300 micro M) produced a transient increase (152% +/- 14%) in response. When co-incubated with guanethidine (3 microM), the twitch was reduced to 24% +/- 4% of control and was reversed to 77% +/- 7% after 1 h. Cocaine (30 microM) inhibited the twitch response to 53% +/- 8%, which was fully reversed by 1 h of washing. When co-incubated with guanethidine, the response was reduced to 39% +/- 6% of control and was reversed to 86% +/- 10% after 1 h. In all cases, the reversal produced by the combination was significantly more intense (P < 0.05) than that produced by guanethidine alone. Local anesthetics reduce the sympatholytic actions of guanethidine, and this may explain the variable efficacy of guanethidine in the treatment of complex regional pain syndrome
Effects of [(pF)Phe(4)]nociceptin/orphanin FQ-(1-13)NH2 on GTP gamma S-35 binding and cAMP formation in Chinese hamster ovary cells expressing the human nociceptin/orphanin FQ receptor
No full textNociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ receptor (NOP). In this study using Chinese hamster ovary
(CHO) cells expressing the human NOP (CHOhNOP) and GTPg35S binding and cAMP inhibition assays, we have characterised a novel N/
OFQ ligand, [( pF)Phe4]N/OFQ-(1–13)NH2, ([( pF)Phe4]). [( pF)Phe4] was produced by insertion of a fluorine atom into the para position of
the phenyl ring of Phe4 of the truncated N/OFQ peptide N/OFQ-(1–13)NH2. In CHOhNOP membranes [( pF)Phe4] and N/OFQ-(1–13)NH2
stimulated GTPg35S binding with pEC50 (meanFS.E.M.) values of 9.55F0.01 and 8.94F0.5 ( P < 0.05), respectively. In whole CHOhNOP
cells [( pF)Phe4] and N/OFQ-(1–13)NH2 inhibited forskolin stimulated cAMP formation with pEC50 values of 10.19F0.06 and 9.60F0.04,
respectively ( P < 0.05). [( pF)Phe4] was more potent (f4 fold) than N/OFQ-(1–13)NH2. In both assays, the effects of [( pF)Phe4] and N/
OFQ-(1–13)NH2 were pertussis toxin sensitive and reversed by the NOP antagonists J-113397 (pA2/pKB values 7.89–8.53) and III-BTD
(pA2/pKB values 7.27–7.96). [( pF)Phe4] is therefore a potent full agonist at NOP receptors that will be useful as pharmacological tool for
defining the role of N/OFQ–NOP system in health and disease
Nociceptin/orphanin FQ inhibits ischaemia-induced glutamate efflux from rat cerebrocortical slices
No full textProinflammatory and vasodilator effects of nociceptin/orphanin FQ in the rat mesenteric microcirculation are mediated by histamine
No full textNociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). N/OFQ causes hypotension and vasodilation, and we aimed to determine the role of histamine in inflammatory microvascular responses to N/OFQ. Male Wistar rats (220-300 g, n = 72) were anesthetized with thiopental (30 mg/kg bolus, 40-90 mg x kg(-1) x h(-1) iv), and the mesentery was prepared for fluorescent intravital microscopy using fluorescein isothiocyanate-conjugated BSA (FITC-BSA, 0.25 ml/100 g iv) or 1 microm fluorescently labeled microspheres. N/OFQ (0.6-60 nmol/kg iv) caused hypotension (SAP, baseline: 154 +/- 11 mmHg, 15 nmol/kg N/OFQ: 112 +/- 10 mmHg, P = 0.009), vasodilation (venules: 23.9 +/- 1.2 microm, 26.7 +/- 1.2 microm, P = 0.006), macromolecular leak (interstitial gray level FITC-BSA: 103.7 +/- 3.4, 123.5 +/- 11.8, P = 0.009), and leukocyte adhesion (2.0 +/- 0.9, 15.2 +/- 0.9/100 microm, P = 0.036). Microsphere velocity also decreased (venules: 1,230 +/- 370 microm/s, P = 0.037), but there were no significant changes in blood flow. Flow cytometry measured a concurrent increase in neutrophil expression of cd11b with N/OFQ vs. controls (Geo mean fluorescence: 4.19 +/- 0.13 vs. 2.06 +/- 0.38, P < 0.05). The NOP antagonist [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-101; 60 and 150 nmol/kg iv), H(1) and H(2)antagonists pyrilamine (mepyramine, 1 mg/kg iv) and ranitidine (1 mg/kg iv), and mast cell stabilizer cromolyn (1 mg x kg(-1) x min(-1)) also abolished vasodilation and macromolecular leak to N/OFQ in vivo (P < 0.05), but did not affect hypotension. Isolated mesenteric arteries (approximately 200 microm, n = 25) preconstricted with U-46619 were also mounted on a pressure myograph (60 mmHg), and both intraluminally and extraluminally administered N/OFQ (10(-5) M) caused dilation, inhibited by pyrilamine in the extraluminal but not the intraluminal (control: -6.9 +/- 3.8%; N/OFQ: 32.6 +/- 8.4%; pyrilamine: 31.5 +/- 6.8%, n = 18, P < 0.05) experiments. We conclude that, in vivo, mesenteric microvascular dilation and macromolecular leak occur via N/OFQ-NOP-mediated release of histamine from mast cells. Therefore, N/OFQ-NOP has an important role in microvascular inflammation, and this may be targeted during disease, particularly as we have proven that UFP-101 is an effective antagonist of microvascular responses in vivo
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