High Affinity Opioid Binding Sites Research Articles

Predominance of delta-opioid-binding sites in the porcine enteric nervous system.

The antidiarrheal and constipating actions of opioids are mediated in part by enteric neurons, which lie within the wall of the small intestine and colon, but the differential expression of specific, high-affinity opioid-binding sites in ganglionated plexuses within functionally distinct intestinal segments has not been examined. We determined the saturation binding characteristics under Na+-free conditions of the nonselective opioid receptor (OPR) ligand [3H][(5alpha,7alpha)-17-(cyclopropylmethyl)-4,5-epoxy-18,19-dihydro-3-hydroxy-6-methoxy-alpha, alpha-dimethyl-6,14-ethenomorphinan-7-methanol] (diprenorphine) and the respective delta-, kappa-, and mu-OPR ligands [3H]naltrindole, D-(5alpha,7alpha,8beta)-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxoaspiro-(4,5)dec-8-yl]benzeneacetamide ([3H]U-69,593), and [3H][D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) in neuronal membranes isolated from myenteric and submucosal plexuses of porcine small intestine and colon. Naloxone-displaceable [3H]diprenorphine-binding sites (KD values ranging from 0.2-0.5 nM and Bmax = 50-95 fmol/mg of protein) were found in both subregions from all gut segments examined. High-affinity [3H]naltrindole sites (KD = 60-140 pmol) were at highest densities (approximately 60 fmol/mg of protein) in submucosal plexus of the ileum and distal colon myenteric plexus and were at lowest densities (8-9 fmol/mg of protein) in the submucosal plexuses of cecum and distal colon. [3H]U-69,593 sites (KD = 0.3-4 nM) were present only in the myenteric plexuses of all segments examined, with highest densities in cecum and proximal colon (44-47 fmol/mg of protein). [3H]DAMGO-binding sites were expressed at relatively low densities in the enteric plexuses of all gut regions. These results indicate that delta-OPRs predominate in the porcine enteric nervous system with a more circumscribed expression of kappa- and mu-OPRs.

3H]dynorphin 1–8 binding sites in frog ( Rana esculenta) brain membranes

Opioid binding sites specific for [ 3H]dynorphin 1–8 were characterized in the particulate membrane fraction of frog ( Rana esculenta) brain. The degradation of the radioligand during the assay was prevented by the use of a broad spectrum of peptidase inhibitors. The binding of [ 3H]dynorphin 1–8 to frog brain membranes was stereoselective, reversible, saturable, and displaceable by a series of opioid ligands including dynorphin 1–13, bremazocine, levorphanol and naloxone. The specific binding of [ 3H]dynorphin 1–8 can be significantly inhibited by Na + ions and/or guanine nucleotides confirming the agonist property of the ligand in vitro. A single set of high affinity opioid binding sites with a K d ≈ 7.5 nM is present in the membranes. The maximum density of binding sites (B max ≈ 1.1 pmol [ 3H]dynorphin 1–8 per mg protein) was considerably higher than such sites in guinea-pig brain. In addition, comparison with binding of tritiated opioid peptides selective for the μ- and σ-types of opioid receptor showed that in the frog brain most of the sites labelled by [ 3H]dynorphin 1–8 are κ-sites and that this is a rich source of such sites.

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.

Cholecystokinin-8 suppressed 3H-etorphine binding to rat brain opiate receptors

Radio receptor assay (RRA) was adopted to analyse the influence of CCK-8 on 3H-etorphine binding to opiate receptors in rat brain synaptosomal membranes (P2). In the competition experiment CCK-8 (1pM to 1 μM) suppressed the binding of 3H-etorphine. This effect was completely reversed by proglumide at 1 μM. Rosenthal analysis for saturation revealed two populations of 3H-etorphine binding sites. CCK-8 (1pM to 1μM) inhibited 3H-etorphine binding to the high affinity sites by an increase in Kd (up to +235%) and decrease in Bmax (up to −80%) without significant changes in the Kd and Bmax of the low affinity sites. This effect of CCK-8 (10nM) was also completely reversed by proglumide at 1 μM. Unsulfated CCK-8 (100pM to 1 μM) produced only a slight increase in Kd of the high affinity sites (+64%) without affecting Bmax. The results suggest that CCK-8 might be capable of suppressing the high affinity opioid binding sites via the activation of CCK receptor.

Characterization of high affinity opioid binding sites in rat periaqueductal gray P 2 membrane

The periaqueductal gray (PAG) region of the midbrain has been implicated in both stimulation produced and opioid induced analgesia. In the present study the opioid binding characteristics of the PAG were examined with an in vitro radioligand binding technique. [ 3H]Ethylketocyclazocine (EKC), 2 nM, was useed as a ligand to nonselevtively labels μ, δ, and κ binding sites in PAG enriched P 2 membrane. The μ selective ligand [D-Ala 2, N-methylPhe 4, Glyol 5]enkephalin (DAGO) competed with [ 3H]EKC for more than one population of binding sites with both high and low affinity. In contrast the δ selective ligand [D-Pen 2, D,-Pen 5]enkephalin (DPDPE) and the κ selective ligand trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide, methane sulfonate, hydrate (U50,488H) each competed with [ 3H]EKC for a single population of binding of sites with low affinity. DPDPE and U50,488H also competed with 2 nM [ 3H]DAGO for a single population of binding sites with similar low affinity. DAGO and not DPDPE competed with 2 nM [ 3H][D-Ala 2,D-Leu 5]enkephalin (DADLE) with high affinity. 2 nM [ 3H]DPDPE did not substantially label PAG enriched P 2 membrane, and 1 nM DAGO competed with all specific [ 3H]DPDPE binding which was observed. These binding data are consistent with the presence of a single population of μ selective high affinity binding sites in PAG enriched P 2 membrane to which δ ligands and κ ligands have low affinity.

Binding of the non-selective opioid [ 3H]etorphine in the human fetal central nervous system

The existence of specific, high-affinity opioid binding sites was demonstrated in 21-week-old human fetuses with the use of tritiated etorphine, a non-selective opioid ligand. Binding capacities measured in the brain, the cerebellum and the spinal cord give values of 2.83, 3.71 and 5.42 pmol/g of tissue, K d values do not vary from one region to the other and are respectively 0.29, 0.35 and 0.33 nM. These results are compared with the characteristics of opioid binding sites found in adults.

Hormonal regulation of β-endorphin in the testis

It is well established that β-endorphin has a regulatory influence on the reproductive function at the level of the hypothalamic-pituitary axis. However, recent immunohistochemical evidence demonstrated that β-endorphin is also present in the Leydig cells of fetal, neonatal and adult mice and hamsters. In addition, β-endorphin synthesis was localized in the Leydig cells of adult rats, leading to the hypothesis of a direct function of the peptide in the reproductive organs. Our interest was to investigate the role of β-endorphin at testicular level. We have demonstrated the presence of high-affinity opioid binding sites ( K d in the nanomolar range) in tubular homogenates and Sertoli cells in culture of adult (50 days) and immature (18 days post-natal) rat testes. Also, chronic β-endorphin treatment of the Sertoli cells significantly inhibited basal and FSH-stimulated androgen-binding protein production, this effect being prevented by the universal opiate antagonist naloxone. No opiate binding was observed on Leydig cell cultures. Furthermore, we have demonstrated that acute or chronic β-endorphin treatment does not affect testosterone production by Leydig cells in virto, consistent with the absence of receptors on these cells. On the other hand, fetal Leydig cells (21 days fetal life) in culture produced considerable amounts of β-endorphin. Also, fetal Leydig cells represented a preferred in virto system to study β-endorphin release since in adult cell culture a marked degradation of the peptide was detected (> 50%). β-endorphin accumulation for 3 and 5 days was markedly increased by inhibitors of steroid biosynthesis (1.5-fold); a significant reduction by GnRH at both days (by 50 − 30%) was observed, while by dexamethasone the reduction was only noted after 5 days of treatment (by 50%). Acute stimulation (3 h) of control cells with hCG enhanced by 10–12-fold the β-endorphin secretion. The hormone stimulation of β-endorphin production was not mediated by testosterone. On the contrary, inhibition of Leydig cells steroid biosynthesis markedly increased basal and hCG-stimulated β-endorphin production (150–200%), suggesting autocrine negative modulation of Leydig cell β-endorphin by androgen and/or its metabolites. In contrast, dexamethasone reduced basal and hCG-stimulated β-endorphin production (by 50%). Altogether these findings indicate that β-endorphin produced within the Leydig cells may behave as a paracrine inhibitor of the Sertoli cell function and demonstrate that the peptide production is under direct control by gonadotropins and is modulated by steroids.

Effects of the affinity ligands 14-β-chloroacetylnaltrexone and 14-β-bromoacetamidomorphine on [ 3H]-dihydromorphine binding sites in rat brain

The aim of the present study was to examine the inhibitory effects in vitro of the affinity ligands 14-β-chloroacetylnaltrexone (CAN) and 14-β-bromoacetamidomorphine (BAM) to characterize the pharmacological specificity of the ligands for high and low affinity opioid binding sites. Rat brain membranes were incubated with 2.0 μM BAM or CAN, or their parent compounds (morphine and naltrexone, respectively) at 37° for 45 min, and the membranes were washed extensively to remove the unbound ligand. The specific binding of 0.3 nM [ 3H]dihydromorphine ([ 3H]DHM) was reduced 32 ±7% in membranes treated with CAN and BAM, whereas specific binding in preparations treated with morphine and naltrexone was not significantly different from controls. An increased affinity of BAM and CAN relative to morphine and naltrexone could not account for the observed irreversible inhibition, since the relative affinity of CAN was similar to that of naltrexone and that of BAM was 10-fold less active than morphine. Saturation binding assays revealed that the affinity ligands selectively abolished a high affinity binding site ( K d = 0.3 nM, B max 95 moles/mg protein), which comprised approximately one-third of the total number of sites. The affinity of the remaining site ( K d = 4.0 nM) was not altered significantly. The results indicate that the inhibition caused by the affinity ligands is irreversible and represents inactivation of high affinity opioid binding sites in a relatively selective manner.