Receptor selectivity of Met-enkephalin-Arg6-Phe7, an endogenous opioid peptide, in cerebral cortex of human and rat (original) (raw)
Related papers
Life Sciences, 1999
3H]Met-enkephalin-Arg6-Phe7 (MERF) has been shown to label opioid (~2 and S) and sigma2 sites in rat and frog brain membrane preparations, and no specific binding to ~1 opioid receptors could be established (refs. 6 and 8). In this study the binding was examined in rat cerebellar membranes which are relatively rich in x2-sites, and in guinea pig cerebellar preparations where ~1 opioid receptors are almost exclusively present. In accordance with our previous results, [3HjMERF binding could not be displaced in guinea pig cerebellar membranes neither with U-69,593 nor with naloxone or levorphanol suggesting no interaction with opioid sites, nevertheless a & of 2.8 nM was calculated in cold saturation experiments. In rat cerebellar membrane fractions about the half of the specific [3H]MERF binding sites was inhibited by opiate alkaloids such as naloxone, ethylketocyclazocine, or bremazocine. This portion of the heptapeptide binding sites was stereoselective as demonstrated by the difference in the affinities of the enantiomeric compounds levorphanol and dextrorphan, therefore it would represent an opioid site. In both tissues (-)N-allyl-normetazocine (SKF-10,047), which is also considered as sigma2 ligand, displayed the highest affinities. Among opioid peptides gendorphin and dynorphin(i.i3, showed the highest potencies, displacing [3H]MERF also from its non-opioid sites. It was concluded therefore that [3H]MERF does not bind to ~1 sites, and besides rcz-opioid sites substantial binding to peptide preferring non-opioid sites, and/or sigma2 receptors also occurs.
Neuropeptides, 1994
The modulation by morphine of the spinal release of met-enkephalin-like material (MELM) was investigated in anaesthetized rats whose intrathecal space was perfused with an artificial CSF (ACSF). Morphine (10 PM in the ACSF), as well as a CL-(DAGO, 10 PM) or 6 opioid receptor agonist (DTLET, 10 FM), significantly decreased the outflow of MELM. The effects of morphine and DTLET were prevented by the 6 antagonist, naltrindole (10 PM), but not by naloxone (10 PM). Conversely, naloxone, but not naltrindole, prevented the inhibitory effect of DAGO. Although neither the rcl agonist, U 50488H (10 PM), nor the rcl antagonist, norbinaltorphimine (10 PM), exerted on their own any significant effect, norbinaltorphimine enhanced the inhibitory action of morphine. In contrast to the inhibition induced by morphine (with or without naloxone) which was preventable by 10 PM naltrindole, the inhibition of MELM release by morphine plus norbinaltorphimine was only partly reduced by naltrindole. Thus, concomitant stimulation of p, 6 and K, receptors might account for the apparent 6 opioid receptor-dependent inhibition of MELM release by morphine. Indeed, its potential inhibitory effect through the stimulation of p receptors (normally prevented by the concomitant stimulation of K, receptors) becomes efficient only when rc, receptors are blocked.
Journal of Neurochemistry, 2006
Noradrenaline (NA) release and its modulation via presynaptic opioid receptors were studied in rabbit hippocampal slices, which were preincubated with [3H]NA, continuously superfused in the presence of 30 p M cocaine and stimulated electrically. The evoked rclease of ['HINA was strongly reduced by the preferential K-agonists ethylketocyclazocine, dynorphin A,-,,, dynorphin A, trcins-3,4-dichloro-N-methyl-N-[2-( I-pyrrolidinyl)-cyclohexyl]-benzeneacetamide (U-50,488), and ( -)-5,9-dimethyl-2'-OH-2-tetrahydrofurfuryl-6,7-benzomorphan [(-)-MR 20341, whereas ( + ) -M R 2035 [the (+)-enantiomer of (-)-MR 20341 was ineffective. In contrast, the preferential F-agonists Leu-enkephalin, Met-enkephalin, and D-Ala,-il-Leu,-enkephalin (DADLE) a s well as the p-agonists morphine, normorphine, D-AlaZ-Gly-01,-enkephalin ( D A G O ) , and P-casomorphin,_, amide (morphiceptin) were much less potent. However, in similar experiments on rat hippocampal slices DAGO (1 pM) was much more potent than ethylketocyclazocine (1 kA4) or DADLE (1 pA4). ( -)-N-(3-furylmethyl)-a-noretazocine [( -)-MR 22661, 1 p M , a preferential K-antago-nist, antagonized the effect of ethylketocyclazocinc more potently than ( -)-naloxone or ( + )-MR 2267 [the ( + )-enantiomer of (-)-MR 22661. Given alone, (-)-MR 2266 slightly and (+)-MR 2267 ( 1 pM each) greatly enhanced NA release, apparently due to a,-adrenoceptor blockade since their effects were completely abolished in the presence of yohimbine (0.1 p M ) . The effects of DADLE ( 1 phf) and DAGO ( 1 pA4) were also antagonized by ( -)-MR 2266 (0.
Non-opioid actions of opioid peptides
Life Sciences, 2004
Beside the well known actions of opioid peptides on A-, y-and n-opioid receptors, increasing amount of pharmacological and biochemical evidence has recently been published about non-opioid actions of various opioid peptides. These effects are not abolished by naloxone treatments. Such non-opioid effects are observed both in nervous tissues and in the cellular elements of the immune system. Peptides exhibiting non-opioid effects include h-endorphin, dynorphin A, nociceptin/OFQ, endomorphins, hemorphins and a number of Proenkephalin A derived peptides, such as Met-enkephalin, Met-enkephalin-Arg-Phe (MERF) and bovine adrenal medullary peptide (BAM22). Non-opioid actions are exerted through different neuronal receptors, e.g., dynorphin hyperalgesia through NMDA receptor, Met-enkephalin induced regulation of cell growth through zeta (~) receptors, pain modulation by nociceptin through ORL-1 or NOP receptors, while BAM22 acts through sensory neuron specific G protein-coupled receptors (SNSR). We have investigated Met-enkephalin-Arg-Phe (MERF) and its analogues by the means of direct and indirect radioligand binding assays. It has been found that in addition to n 2 and y-opioid receptors, MERF can act also through j 2-or probably via FMRF-NH 2 receptors in rat cerebellum. A role of functionally assembling heterodimer receptors in mediating the non-conventional actions of these peptide ligands can not be excluded as well.
Neuroscience Letters, 1992
The possible opioid control through ~,/t and x receptors of the spinal release of Met-enkephalin-like material (MELM) was investigated in halothane-anaesthetized rats. The intrathecal perfusion of the 6 agonist DTLET (10 pM) or the/1 agonist DAGO (10 #M) resulted in a marked inhibition of MELM release, which could be prevented by the selective antagonists naltrindole and naloxone, respectively. Although the ~c agonist U 50488 H (10/~M) was inactive per se, it completely suppressed the inhibitory effect of DAGO, without affecting that of DTLET. As the selective x antagonist norbinaltorphimine blocked the action of U 50488 H, it can be concluded that x receptors modulate the ,u-(but not the 6-) mediated feed back control of spinal enkephalinergic neurones.
Irreversible labelling of rat brain opioid receptors by enkephalin chloromethyl ketones
Neuropeptides, 1986
Chloromethyl ketone derivatives of leucine enkephalin (LE), D-Ala2-Leu5-enkephalin (DALE) and D-Ala2-D-Leu5-enkephalin (DADLE) were synthesized. They all show high affinity for rat brain opioid binding sites. Preincubation of the membrane fraction with enkephalin chloromethyl ketones causes a significant inhibition of /3H/-naloxone binding which cannot be reversed by extensive washing. It was found that the irreversible inhibition is selective for the high affinity (KD less than 1 nM) /3H/-naloxone binding site (putative mu-1 site). The irreversible blockade of opioid binding was partially protected by opiate alkaloids and opioid peptides, suggesting that non-specific labelling also occurs. Affinity of enkephalin chloromethyl ketones toward the mu sites is greater than that of the parent compounds. It was also found that the covalent inhibition of mu sites (/3H/-dihydromorphine and /3H/-DAGO binding) is more effective than that of delta sites (/3H/-DALE binding). We conclude that these chloromethyl ketone derivatives can be used as affinity labels for the opioid receptors, allowing us to study the structure of the mu receptor subtype.
Canadian Journal of Physiology and Pharmacology, 1989
TIBERI, M., and MAGNAN , J . 1989 Pharmacological characterization of the binding of [3~] bremazocine in guinea-pig brain: evidence for multiplicity of the K-opioid receptors. Can. J. Physiol. Phmacol. 67: 1336-1344 In guinea-pig brain, [3~]bremazocine has a binding capacity of 27.2 pmoVg wet tissue, which is statistically different from that of [3~]ethylketazocine (14.7 pmoyg wet tissue) or the sum of the individual binding capacities of p-, 8-, and K-selective ligands (15.0 pmollg wet tissue). Saturation studies of [3~]bremazocine performed in the presence of unlabelled p-, 8-, and K-blockers still reveal a homogeneous population of binding sites.
Brain Research, 1986
While the distribution of opioid receptors can be differentiated in the rat central nervous system, their precise localization has remained controversial. due. in part. to the previous lack of selective ligands and insensitive assaying conditions. The present study analyzed this issue further by examining the receptor selectivity of ['HIDAGO (Tyr-o-Ala-Gly-MePhe-Gly-ol). ['HIDPDPE (2-o-penicillamine-S-u-penicillamine-enkephalin), ["HIDSLET (Tyr-uSer -Gly-Phe-Leu-Thr) and ["H](-)b remazocine. and their suitability in autoradiographically labelling selective subpopulations of opioid receptors in rat brain. The results from saturation. competition. and autoradiographic experiments indicated that the three opioid receptor subtypes can be differentiated in the rat brain and that ['HI-DAGO and ["HIDPDPE selectively labelled ," and b binding sites, respectively. In contrast, ['HIDSLET was found to be relatively non-selective. and labelled both /(and b sites. [ZH]Bremazocine was similarly non-selective in the absence of p and b ligands and labelled all three opioid receptor subtypes. However, in the presence of 100 nM DAGO and DPDPE, concentrations sufficient to saturate the /! and 6 sites, ['Hlbremazocine did label x sites selectively. The high affinity ['Hlbremazocine binding sites showed a unique distribution with relatively dense x labelling in the hypothalamus and median eminence. areas with extremely low p and b binding. These results point to the selectivity, under appropriate conditions, of [3H]DAG0, [3H]DPDPE and [3H]bremazocine and provide evidence for the differential distribution ofp, 6, and x opioid receptors in rat brain.
Peptides, 1998
peptide receptor studies. 9. Identification of a novel non--non-␦-like opioid peptide binding site in rat brain. PEPTIDES 19(6) 1079 -1090, 1998. Quantitative binding studies resolved two high-affinity [ 3 H][D-Ala 2 ,D-Leu 5 ]enkephalin binding sites in rat brain membranes depleted of binding sites by pretreatment with the irreversible agent BIT. -The two binding sites had lower (␦ ncx-2 , Ki ϭ 96.6 nM) and higher (␦ ncx-1 , Ki ϭ 1.55 nM) affinity for DPDPE. The ligand-selectivity profile of the ␦ ncx-1 site was that of a classic ␦ binding site. The ligand-selectivity profile of the ␦ ncx-2 site was neitheror ␦-like. The Ki values of selected agents for the ␦ ncx-2 site were: [pCl]DPDPE (3.9 nM), DPLPE (140 nM), and DAMGO (2.6 nM). Under these assay conditions, [ 3 H][D-Ala 2 ,D-Leu 5 ]enkephalin binding to the cells expressing the cloned receptor is very low and pretreatment of cell membranes with BIT almost completely inhibits [ 3 H]DAMGO and [ 3 H][D-Ala 2 ,D-Leu 5 ]enkephalin binding. Intracerebroventricular administration of antisense DNA to the cloned delta receptor selectively decreased [ 3 H][D-Ala 2 ,D-Leu 5 ]enkephalin binding to the ␦ ncx-1 site. Administration of buprenorphine to rats 24 h prior to preparation of membranes differentially affected , ␦ ncx-1 , and ␦ ncx-2 binding sites. Viewed collectively, these studies have identified a novel non--non-␦-like binding site in rat brain.