1,720,985 research outputs found
The 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
Partial agonist behaviour depends upon the level of nociceptin/orphanin FQ receptor expression - studies using the ecdysone inducible mammalian expression system
Full text link(1) Partial agonism is primarily dependent upon receptor density and coupling efficiency. As these parameters are tissue/model dependent, intrinsic activity in different tissues can vary. We have utilised the ecdysone-inducible expression system containing the human nociceptin/orphanin FQ (N/OFQ) peptide receptor (hNOP) expressed in Chinese hamster ovary cells (CHOINDhNOP) to examine the activity of a range of partial agonists in receptor binding, GTPgamma35S binding and inhibition of adenylyl cyclase studies. (2) Incubation of CHOINDhNOP cells with ponasterone A (PON) induced hNOP expression ([leucyl-3H]N/OFQ binding) of 24, 68, 191 and 1101 fmol mg-1 protein at 1, 2, 5 and 10 microm PON, respectively. At 191 fmol mg-1, protein hNOP pharmacology was identical to that reported for other traditional expression systems. (3) pEC50 values for GTPgamma35S binding ranged from 7.23 to 7.72 (2-10 microm PON) for the partial agonist [Phe1psi(CH2-NH)Gly2]N/OFQ(1-13)-NH2 ([F/G]N/OFQ(1-13)-NH2) and 8.12-8.60 (1-10 microm PON) for N/OFQ(1-13)-NH2 and Emax values (stimulation factor relative to basal) ranged from 1.51 to 3.21 (2-10 microm PON) for [F/G]N/OFQ(1-13)-NH2 and 1.28-6.95 (1-10 microm) for N/OFQ(1-13)-NH2. Intrinsic activity of [F/G]N/OFQ(1-13)-NH2 relative to N/OFQ(1-13)-NH2 was 0.3-0.5. [F/G]N/OFQ(1-13)-NH2 did not stimulate GTPgamma35S binding at 1 microm PON, but competitively antagonised the effects of N/OFQ(1-13)-NH2 with a pKB=7.62. (4) pEC50 values for cAMP inhibition ranged from 8.26 to 8.32 (2-10 microm PON) for [F/G]N/OFQ(1-13)-NH2 and 9.42-10.35 for N/OFQ(1-13)-NH2 and Emax values (% inhibition) ranged from 19.6 to 83.2 for [F/G]N/OFQ(1-13)-NH2 and 40.9-86.0 for N/OFQ(1-13)-NH2. The intrinsic activity of [F/G]N/OFQ(1-13)-NH2 relative to N/OFQ(1-13)-NH2 was 0.48-0.97. (5) In the same cellular environment with receptor density as the only variable, we show that the profile of [F/G]N/OFQ(1-13)-NH2 can be manipulated to encompass full and partial agonism along with antagonism
Effects of nociceptin and endomorphin 1 on the electrically stimulated human vas deferens
No full textAims To examine the effects of nociceptin (NC) and endomorphin 1 (EM1) on
electrical ®eld stimulation (EFS)-induced contractions of the human vas deferens
(hVD).
Methods Concentration-response curves to NC and EM1 were constructed in the
absence and in presence of peptidase inhibitors (PI). In some experiments a NC
receptor antagonist, [Phe1y(CH2-NH)Gly2]NC(1±13)NH2 [F/G], 10 mM) or naloxone
(1 mM) were included.
Results All data are mean(95%CI). In the presence of PI, NC inhibited twitches
(Emax=67(44,90)%; pEC50=7.28(6.95,7.61)). NC inhibition was sensitive to [F/G].
EM1 also inhibited twitches both in the absence (Emax=82(73,91)% pEC50=7.07
(6.92,7.22)) and presence (Emax=83(76,90)%; pEC50=7.00(6.91, 7.09)) of PI. EM1
inhibition was sensitive to naloxone.
Conclusions These data suggest that hVD express NC and opioid receptors that
inhibit neurogenic contractions
In vitro pharmacological characterisation of a novel cyclic nociceptin/orphanin FQ analogue c[Cys(7,10)]N/OFQ(1-13)NH2
No full textNociceptin/orphanin FQ (N/OFQ) is the endogenous 17 amino acid peptide ligand for the Gi-protein-coupled N/OFQ receptor (NOP). In an attempt to improve the metabolic stability of N/OFQ, we have produced a truncated cyclic analogue with cysteine residues at positions 7 and 10, c[Cys7,10]N/OFQ(1-13)NH2 (c[Cys7,10]). c[Cys7,10], the template N/OFQ(1-13)NH2 and N/OFQ displaced the binding of [3H]N/OFQ to Chinese hamster ovary cells expressing recombinant human NOP (CHOhNOP) with pK i values of 9.98, 9.83 and 9.18, respectively. In addition, c[Cys7,10], N/OFQ(1-13)NH2 and N/OFQ stimulated the binding of guanosine triphosphate gamma [35S] to CHOhNOP cells with pEC50/E max (stimulation factor) of 9.16/5.5, 9.11/4.9 and 8.35/5.5, respectively. c[Cys7,10], N/OFQ(1-13)NH2 and N/OFQ inhibited forskolin-stimulated cyclic adenosine monophosphate (cAMP) formation with pEC50 values of 10.08, 10.11 and 9.78, respectively. All ligands produced complete inhibition of cAMP formation. In both functional assays, c[Cys7,10] was a full agonist. In a series of metabolism experiments, incubation of 1 nM c[Cys7,10], N/OFQ(1-13)NH2 and N/OFQ with a rat brain homogenate produced a time-dependent loss of peptide that was greatest for the native peptide N/OFQ. Amidation in N/OFQ(1-13)NH2 produced some metabolic protection, but this was not significantly improved by further inclusion of c[Cys7,10]. In summary, c[Cys7,10] is a high-affinity, high-potency full agonist of the NOP receptor. However, we were unable to demonstrate clear metabolic protection
Effects of nociceptin and endomorphin 1 on the electrically stimulated human vas deferens
No full textSimultaneous targeting of multiple opioid receptors: a strategy to improve side-effect profile
No full textOpioid receptors are currently classified as mu (mu: mOP), delta (delta: dOP), kappa (kappa: kOP) with a fourth related non-classical opioid receptor for nociceptin/orphainin FQ, NOP. Morphine is the current gold standard analgesic acting at MOP receptors but produces a range of variably troublesome side-effects, in particular tolerance. There is now good laboratory evidence to suggest that blocking DOP while activating MOP produces analgesia (or antinociception) without the development of tolerance. Simultaneous targeting of MOP and DOP can be accomplished by: (i) co-administering two selective drugs, (ii) administering one non-selective drug, or (iii) designing a single drug that specifically targets both receptors; a bivalent ligand. Bivalent ligands generally contain two active centres or pharmacophores that are variably separated by a chemical spacer and there are several interesting examples in the literature. For example linking the MOP agonist oxymorphone to the DOP antagonist naltrindole produces a MOP/DOP bivalent ligand that should produce analgesia with reduced tolerance. The type of response/selectivity produced depends on the pharmacophore combination (e.g. oxymorphone and naltrindole as above) and the space between them. Production and evaluation of bivalent ligands is an emerging field in drug design and for anaesthesia, analgesics that are designed not to be highly selective morphine-like (MOP) ligands represents a new avenue for the production of useful drugs for chronic (and in particular cancer) pain
Assessment of the activity of a novel nociceptin/orphanin FQ analogue at recombinant human nociceptin/orphanin FQ receptors expressed in Chinese hamster ovary cells
No full textThe neuropeptide nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the nociceptin receptor (NOP). In an attempt to identify
high potency NOP agonists for use in the brain we have compared the activity of a novel N/OFQ analogue [Phe1C(CH2-O)Gly2]N/OFQ(1-
13)NH2 ([F/G-O]) with the existing [Phe1C(CH2-NH)Gly2]N/OFQ(1-13)NH2 ([F/G]). Both peptides are modified between the first two Nterminal
amino acids and are further compared with the agonist template N/OFQ(1-13)NH2 in [3H]N/OFQ binding, GTPg[35S] binding and
cAMP inhibition studies using Chinese hamster ovary cells expressing the recombinant human NOP. All peptides displaced [3H]N/OFQ,
stimulated GTPg[35S] binding and inhibited cAMP formation. In [3H]N/OFQ binding and GTPg[35S] binding the rank order affinity and
potency was N/OFQ(1-13)NH2 . [F/G-O] . [F/G]. In GTPg[35S] binding [F/G] was a clear partial agonist with intrinsic activity (Emax
stimulation factor, mean ^ SEM, n 1⁄4 4) of 7.75 ^ 1.02 compared with N/OFQ(1-13)NH2 of 11.13 ^ 1.76. The efficacy of [F/G-O]
(10.17 ^ 1.88) approached that of the full agonist N/OFQ(1-13)NH2. Downstream, at the level of cAMP formation, all peptides were full
agonists with the following rank order potency: N/OFQ(1-13)NH2 . [F/G-O] 1⁄4 [F/G]. The enhanced potency and intrinsic activity of the
novel [F/G-O] modification makes this an interesting peptide for further in vivo analysis
Characterization and comparison of novel ligands for the nociceptin/orphanin FQ receptor
No full textStudies of nociceptin/orphanin FQ (NC) have
been hampered by the paucity of available ligands with
activity at the nociceptin receptor (NCR). In this study we
have compared the agonist profile of NC and a novel
NCR agonist, Ro65-6570, in a series of radioligand binding
studies and effects on forskolin-stimulated cAMP formation
in Chinese hamster ovary (CHO) cells expressing
the recombinant human NCR (CHOhNCR). In addition, we
report the effects of three antagonists, [Nphe1]NC(1–
13)NH2, J-113397 and III-BTD, on these responses. In radioligand
binding studies Ro65-6570, [Nphe1]NC(1–
13)NH2, J-113397 and III-BTD displaced [3H]NC with
similar pKi values (8.4–8.8). This compares with a pKD
of 10.2 for NC in a direct saturation experiment.
[Nphe1]NC(1–13)NH2 and J-113397 showed at least
100-fold selectivity over classical opioid receptors. Both
NC and Ro65-6570 produced a concentration-dependent
inhibition of cAMP formation with pEC50 values of 9.56±
0.06 and 8.68±0.04, respectively. Maximum inhibition
achieved was 100%. [Nphe1]NC(1–13)NH2, J-113397 and
III-BTD produced a parallel rightward shift in the concentration-
response curves to both NC and Ro65-6570 with
pKB values of ~6.5, ~7.5 and ~7.7, respectively. Importantly,
all three antagonists were devoid of residual agonist
activity. Collectively, these data indicate the value
of Ro65-6570, [Nphe1]NC(1–13)NH2, J-113397 and
III-BTD in studies of the physiological role played by
NC. However, due to the relatively poor selectivity of
Ro65-6570 and III-BTD caution should be exercised
when using tissues that co-express μ-opioid receptors
Cell and tissue responses of a range of Urotensin II analogs at cloned and native urotensin II receptors. Evidence for coupling promiscuity
No full textUrotensin II (U-II) is the peptide ligand for the
G-protein-coupled U-II receptor (UT). U-II has been
dubbed “the most potent vasoconstrictor identified to
date”. However, in vivo studies with this system are
hampered by the paucity of available ligands. Here, we
characterise Chinese hamster ovary (CHO) cells expressing
the human UT receptor in the following assays; (1) [125I]UII
binding, (2) GTPγ[35S] binding, (3) cAMP formation,
and (4) intracellular Ca2+. We assess activity of 9 U-II
analogues using these paradigms and examine their ability
to contract isolated rat aorta. CHOhUT cells bound [125I]UII
with a Bmax and Kd of 1,110±70 fmol/mg protein and
742 pM, respectively. hU-II stimulated GTPγ[35S] binding
(pEC50 8.38), optimal at low (0.1 μM) GDP concentrations.
The hU-II GTPγ[35S] response was partially PTx
sensitive and there was a potent (pEC50 9.23) low efficacy
(∼20% inhibition) coupling to adenylyl cyclase. In
CHOhUT cells hU-II stimulates calcium release from
intracellular stores (pEC50 8.80) and calcium influx in a
PTx-insensitive manner. In our structure-activity relationship
study most ligands acted as full agonists. However,
urantide behaved as a partial agonist (pEC50 7.67/pKB
7.55) in GTPγ[35S] binding, a full agonist (pEC50 8.11) for
increases in intracellular Ca2+ and a competitive antagonist
in the rat aorta bioassay (pKB 8.59). Collectively, these data
show promiscuity at high expression and indicate the need
for careful multi-assay evaluation of novel U-II analogues.
Further modification of urantide, in order to eliminate
residual agonist activity and to identify novel ligands for in
vivo cardiovascular studies are clearly warranted
- …
