3H]ohmefentanyl preferentially binds to mu-opioid receptors but also labels sigma-sites in rat brain sections.

Ohmefentanyl has been shown to be 6300 times more potent than morphine for analgesia. The receptor binding characteristics and distribution of [3H]ohmefentanyl in rat brain sections are presented. [3H]Ohmefentanyl bound with high affinity to opioid receptors in a saturable manner (Kd = 0.95 +/- 0.08 nM, Bmax = 337 +/- 14 fmol/mg protein). We used various currently available specific mu, delta and kappa ligands to show that [3H]ohmefentanyl has a high selectivity for the mu opioid receptor. However, [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO) was unable to completely inhibit [3H]ohmefentanyl specific binding, while complete inhibition was observed with fentanyl derivatives and the benzomorphan derivative, ethylketocyclazocine. This remaining 20% DAGO-inaccessible [3H]ohmefentanyl specific binding did not correspond to either mu1, delta or kappa sites. Haloperidol and 1,3-di-o-tolylguanidine were able to inhibit DAGO-inaccessible [3H]ohmefentanyl specific binding, suggesting that [3H]ohmefentanyl might also bind to haloperidol-sensitive sigma sites. The topographical distribution of [3H]ohmefentanyl found by autoradiography was generally similar to that of [3H]DAGO. However, in agreement with the biochemical results, quantitative analysis revealed additional sites in several rat brain regions, the greatest discrepancies with [3H]DAGO distribution being observed in cerebellum, central grey, hippocampal formation and locus coeruleus. Finally, our results suggest that this capacity of binding to both mu and sigma sites is shared by various fentanyl derivatives.     

The neutral endopeptidase-24.11 inhibitor SCH 34826 does not change opioid binding but reduces D1 dopamine receptors in rat brain.

The effect of repeated administration of the neutral endopeptidase-24.11 (NEP) inhibitor SCH 34826 on the kinetic properties of opioid and dopamine binding in the rat cerebral cortex and striatum was investigated. SCH 34826, given at 100 and 300 mg/kg orally twice a day for 14 days, did not alter either Bmax or Kd for the mu, delta, or kappa opioid receptor type in the cortex, as measured by studying binding parameters for the mu-selective ligand [3H][D-Ala2, Me-Phe4,Gly(ol)5]enkephalin (DAGO), the delta-selective ligand [3H][D-Pen2,D-Pen5]enkephalin (DPDPE) and the kappa ligand [3H]ethylketazocine (EKC). SCH 34826 reduced significantly the number of D1 dopamine receptors labeled with [3H]SCH 23390 in the striatum (Bmax was 90 and 84% of controls at 100 and 300 mg/kg, respectively). The number of D2 receptors, measured by [3H]spiperone binding was unaltered. The Kd values for both receptor types were not affected. The data demonstrate that chronic inhibition of enkephalin degradation by SCH 34826 does not alter opioid receptors, whereas it reduces the number of D1 receptors. These findings provide further support for the role of opioids in modulating central dopaminergic systems. As a reduction in the number of D1 receptors is an effect common to antidepressant treatments, the antidepressant potential of NEP inhibitors should be investigated.     

Computer modeling of subtypes of kappa opioid receptors in adrenal medulla

The existence of multiple subtypes of kappa opioid receptors in brain and adrenal medulla, has been controversial. We have characterized opioid receptors in frozen membranes from bovine adrenal medulla by use of objective mathematical modeling. [3H]Etorphine, [3H]ethylketocyclazocine (EKC) and [3H][D-Ala2,D-Leu5]enkephalin (DADL) were utilized as labeled ligands. Self- and cross-displacement curves were constructed using the three corresponding unlabeled ligands in the presence or absence of increasing concentrations of DADL. Results indicated: (1) each of the three ligands studied individually showed the presence of heterogeneity of binding sites; (2) sites labeled by etorphine were heterogeneous: 84% of etorphine binding was displaceable by 10(-4) M DADL, while the remaining 16% was DADL non-suppressible; (3) 75% of the binding of EKC was displaceable by DADL while 25% was non-suppressible; (4) mathematical modeling showed the presence of three subtypes of kappa binding sites (a) kappa 1, showing slight selectivity for EKC relative to etorphine; (b) kappa 2, with Kd approximately equal to 1 nM for etorphine, and sufficiently high affinity for DADL (Kd approximately equal to 150 nM) so that it is suppressible by 100 microM DADL; and (c) K3, with no measurable affinity for DADL and a 27-fold selectivity for etorphine relative to EKC. The three subtypes of kappa sites were present at concentrations of 7.4, 75, and 55 fmol/mg protein, respectively. The relative affinities of a series of kappa agonists for the etorphine-binding sites were characterized. The present studies confirm the existence of three subtypes of kappa opioid receptors in bovine adrenal medulla, and indicate the utility of mathematical modeling for characterization of complex receptor systems.     

Characteristics of 5-HT3 binding sites in NG108-15, NCB-20 neuroblastoma cells and rat cerebral cortex using [3H]-quipazine and [3H]-GR65630 binding.

1. The biochemical and pharmacological properties of 5-HT3 receptors in homogenates of NG108-15 and NCB-20 neuroblastoma cells and rat cerebral cortex have been ascertained by the use of [3H]-quipazine and [3H]-GR65630 binding. 2. In NG108-15 and NCB-20 cell homogenates, [3H]-quipazine bound to a single class of high affinity (NG108-15: Kd = 6.2 +/- 1.1 nM, n = 4; NCB-20: Kd = 3.0 +/- 0.9 nM, n = 4; means +/- s.e.means) saturable (NG108-15: Bmax = 1340 +/- 220 fmol mg-1 protein; NCB-20: Bmax = 2300 +/- 200 fmol mg-1 protein) binding sites. In rat cortical homogenates, [3H]-quipazine bound to two populations of binding sites in the absence of the 5-hydroxytryptamine (5-HT) uptake inhibitor, paroxetine (Kd1 = 1.6 +/- 0.5 nM, Bmax1 = 75 +/- 14 fmol mg-1 protein; Kd2 = 500 +/- 300 nM, Bmax2 = 1840 +/- 1040 fmol mg-1 protein, n = 3), and to a single class of high affinity binding sites (Kd = 2.0 +/- 0.5 nM, n = 3; Bmax = 73 +/- 6 fmol mg-1 protein) in the presence of paroxetine. The high affinity (nanomolar) component probably represented 5-HT3 binding sites and the low affinity component represented 5-HT uptake sites. 3. [3H]-paroxetine bound with high affinity (Kd = 0.02 +/- 0.003 nM, n = 3) to a site in rat cortical homogenates in a saturable (Bmax = 323 +/- 45 fmol mg-1 protein, n = 3) and reversible manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mu-, delta- and kappa-opioid receptor-mediated inhibition of neurotransmitter release and adenylate cyclase activity in rat brain slices: studies with fentanyl isothiocyanate

We investigated the effects of [D-Ala2,D-Leu5]enkephalin (DADLE). [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO), [D-Pen2,D-Pen5]enkephalin (DPDPE) (0.01-1 microM) and bremazocine (0.001-0.3 microM) on the electrically evoked release of radiolabelled neurotransmitters and on the dopamine (DA)-stimulated cyclic AMP efflux from superfused rat brain slices. The differential inhibitory effects of these agonists on the evoked neurotransmitter release indicate that the opioid receptors mediating presynaptic inhibition of [3H]noradrenaline (NA, cortex), [14C]acetylcholine (ACh, striatum) and [3H]DA (striatum) release represent mu, delta and kappa receptors, respectively. In agreement with this classification, preincubation (60 min) of the slices with the delta-opioid receptor-selective irreversible ligand, fentanyl isothiocyanate (FIT, 0.01-1 microM), antagonized the inhibitory effects of DADLE and DPDPE on striatal [14C]ACh release only. On the other hand, the D-1 DA receptor-stimulated cyclic AMP efflux from striatal slices appeared to be inhibited by activation of mu as well as of delta receptors. In this case, the reversible mu antagonist, naloxone (0.1 microM), fully antagonized the inhibitory effect of the mu agonist, DAGO, without changing the effect of the delta agonist DPDPE but was ineffective as an antagonist in slices pretreated with FIT (1 microM). The inhibitory effect of DAGO on the electrically evoked [3H]NA release was antagonized by naloxone whether the receptors were irreversibly blocked by FIT or not. These data not only further support the existence of independent presynaptic mu-, delta- and kappa-opioid receptors in rat brain but also evidence strongly that mu and delta receptors mediating the inhibition of DA-sensitive adenylate cyclase could share a common binding site (for naloxone and FIT) and, therefore, may represent constituents of a functional opioid receptor complex.     

Evaluation of delta receptor mediation of supraspinal opioid analgesia by in vivo protection against the beta-funaltrexamine antagonist effect

The involvement of delta opioid receptors in supraspinal analgesia was investigated. With this aim, opioids that produced analgesia in the tail immersion test were administered i.c.v. to mice a few minutes before the irreversible antagonist, beta-funaltrexamine (beta-FNA). Protection of the respective analgesic effects from beta-FNA blockade was obtained when evaluated 24 h later. Moreover, mu ligands protected the analgesia evoked by ED50s of morphine, [D-Ala2,N-Me-Phe4,Met-(o)5-ol]enkephalin (FK 33-824), [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAGO) and human beta-endorphin at doses (ED50s) lower than those required for delta ligands (approximately ED90s) to reach a similar protection. delta Preferential ligands effectively protected the analgesia induced by ED50s of [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Thr2,Leu5]enkephalin-Thr6 (DTLET) and [D-Pen2,D-Pen5]enkephalin (DPDPE) from the beta-FNA-deteriorating effect. FK 33-824 and DAGO also provided good protection of the analgesia elicited by these delta ligands whereas morphine protected much less. Binding studies after i.c.v. injection of beta-FNA showed that its alkylating effect on opioid receptors was restricted to periventricular areas. In PAG, where the mu/delta receptor ratio is about 10, [3H]DADLE specific binding was still present after ED50s of DPDPE, DAGO, morphine and DADLE as protecting agents. [3H]Dihydromorphine [( 3H]DHM) binding was well protected by ED90s of morphine and DAGO, and to a lesser extent by DPDPE and DADLE. These results suggest that delta ligands, after binding to delta receptors, also need to act upon mu receptors to produce high levels of supraspinal analgesia in the tail immersion test.     

Morphine tolerance increases mu-noncompetitive delta binding sites

In light of more recent knowledge concerning endogenous opioid peptides and their multiple opiate receptors, we reevaluated the effects of morphine tolerance on opiate receptor binding parameters. Rats were implanted with morphine or placebo pellets, and [3H][D-Ala2,D-Leu5]enkephalin ([3H]DADL) was used to label brain membranes. Utilizing the technique of binding surface analysis, we observed a selective 47% up-regulation of lower affinity [3H]DADL binding sites (mu-noncompetitive delta binding sites) in morphine pelleted rats. To corroborate these results, we treated brain membranes with the site directed alkylating agent FIT (N-phenyl-N-[1-(2-p-isothiocyanato)phenyl-ethyl)-4-piperidinyl] propanamide), which results in membranes highly enriched with the lower affinity [3H]DADL binding site. Scatchard plots of [3H]DADL binding to FIT-treated membranes also revealed that chronic morphine treatment produced a 60-65% up-regulation of the mu-noncompetitive delta binding site. These data indicate that chronic morphine alters a selective subpopulation of opiate receptors that may play a role in the mechanisms of opiate tolerance and physical dependence.     

Changes in [3H]-PK 11195 and [3H]-8-OH-DPAT binding following forebrain ischaemia in the gerbil.

1. A high density of [3H]-PK 11195 binding sites was present in gerbil cortical membranes (Bmax [3H]-PK 11195 1360 +/- 71 fmol mg-1 protein) in comparison to rat cortical membranes (254 +/- 21 fmol mg-1 protein). This effect was species-specific as similar findings were obtained with hippocampal membranes (Bmax 1430 +/- 111 fmol mg-1 protein in gerbil, compared to 196 +/- 31 in rat). 2. RO 5-4864, also a peripheral type benzodiazepine compound, displayed low affinity for the [3H]-PK 11195 site in the gerbil (pKi 6.57 +/- 0.02 and 6.70 +/- 0.12 in hippocampus and cortex respectively) compared to rat (pKi 8.16 +/- 0.07 and 8.48 +/- 0.02). Central benzodiazepine compounds, diazepam and flunitrazepam, also displayed this trend. 3. RO 5-4864 displaced [3H]-PK 11195 binding from gerbil and rat cortical membranes through a competitive interaction with Hill slopes close to unity. In both tissues, saturation isotherms of [3H]-PK 11195 binding indicated that the presence of RO 5-4864 caused changes in Kd without any effect on Bmax. In kinetic experiments, the presence of RO 5-4864 failed to modify the rate of dissociation of [3H]-PK 11195 from equilibrium in both rat and gerbil cortical membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The electrically stimulated release of [3H]noradrenaline from nucleus tractus solitarii slices in vitro is modulated via mu-opioid receptors.

The electrically stimulated release of [3H]noradrenaline ([3H]NA) from slices of the nucleus tractus solitarii (NTS) from the rat in vitro was inhibited by the alpha 2-adrenoceptor agonist, clonidine, in a concentration-dependent manner and enhanced by the alpha 2-adrenoceptor antagonist, yohimbine. Phenylephrine, isoprenaline, carbachol, quinpirole and SKF 38393, all at 10(-6) M, did not affect the stimulus-evoked release of [3H]NA. The opioid peptides, alpha- and gamma-endorphin, did not have a significant effect on the stimulus-evoked release of [3H]NA; however, beta-endorphin reduced it in a concentration-dependent manner. [Leu5]Enkephalin also reduced [3H]NA release, but higher concentrations were necessary. The selective delta opioid receptor agonists, [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 (DSTBULET), as well as the selective kappa opioid receptor agonist, U-69593, were not effective. The selective mu opioid receptor agonist, [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO), concentration dependently reduced the stimulus-evoked release of [3H]NA to the same extent as beta-endorphin did. Naloxone, while having no effect on stimulus-evoked [3H]NA release, antagonized the effect of DAGO. These results corroborate that the release of NA from noradrenergic terminals in the NTS region of the medulla oblongata of the rat is modulated via alpha 2-adrenoceptors and suggest that the release of NA in the NTS in rats is also modulated via mu opioid receptors.     

Multiple opiate binding sites in the central nervous system of the rabbit. Large predominance of a mu subtype in the cerebellum and characterization of a kappa subtype in the thalamus

We have compared the binding characteristics of [3H]etorphine, a nonselective mu-, delta-, and kappa-opiate agonist, with those of [3H]Tyr-D-Ala-Gly-MePhe-NH(CH2)2OH ([3H]DAGO), a selective mu-agonist, in rabbit cerebellar and thalamic membranes. We have also examined the ability of various unlabeled opioid ligands to compete with the binding of [3H]etorphine in the two preparations. In cerebellar membranes, [3H]DAGO(Kd = 0.7 nM) labels slightly fewer sites than does [3H]etorphine (Kd = 0.06 nM): 0.18 versus 0.24 pmole/mg of protein. In addition, competition studies indicate that up to 75% of the [3H]etorphine binding sites in this preparation display (a) high apparent affinity for unlabeled DAGO and (b) higher apparent affinity for morphine, the prototypical mu-agonist, than for Tyr-D-Ala-Gly-Phe-D-Leu (DADL), a delta-agonist. Together, these results suggest that the rabbit cerebellum contains a very high proportion (0.7-0.8) of mu-opiate binding sites. In thalamic membranes, [3H]DAGO (Kd = 1.1 nM) labels considerably fewer sites than does [3H]etorphine (Kd = 0.08 nM): 0.09 versus 0.27 pmole/mg of protein. In this preparation, the competition curves of DAGO and of DADL resolve binding of [3H]etorphine into two components. The first component accounts for 40-50% of total binding and reflects the interaction of [3H]etorphine with mu-opiate binding sites. The second component (up to 50% of total binding) is unaffected in the presence of DADL at concentrations (1-10 microM) that rule out binding of [3H]etorphine to mu- and delta-opiate binding sites. It disappears readily in the presence of very low concentrations (Ki less than 1 nM) of benzomorphan opiates (bremazocine, cyclazocine, and ethylketocyclazocine) yet it is relatively insensitive to inhibition by mu- and delta-agonists. This second component may therefore reflect [3H]etorphine's interaction with a kappa-opiate binding site. The kappa-opiate binding site is assayed for as that site which binds [3H]etorphine (0.5 nM) in the presence of either DADL (2 microM) or 10 microM of another enkephalin: Tyr-D-Ser-Gly-Phe-Leu-Thr. We find that, in the rabbit central nervous system, the thalamus, followed by frontal cortex and caudate nucleus, shows the highest content of kappa-opiate binding sites.     

Binding in vivo of selective mu and delta opioid receptor agonists: opioid receptor occupancy by endogenous enkephalins

The in vivo binding properties of cerebral mu and delta opioid receptors were investigated in mice after the intrastriatal injection of [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGO) or [3H][D-Thr2,Leu5]enkephalyl-Thr (DTLET). Both peptides exhibited similar diffusion kinetics in the brain and 30-40% of [3H]DAGO or [3H]DTLET was shown to be present in the tissue 15 min after injection when maximal binding was observed. The specific binding of both agonists, defined as the fraction of the radioactivity bound to brain membranes which was displaced by 10 nmol of cold ligand, was reversible, saturable and displayed a pharmacological profile similar to that found in in vitro experiments. At doses producing a similar analgesic effect in the hot-plate test in mice, DTLET occupied 64% of delta sites and DAGO 15% of mu sites. However, because of the residual cross-reactivity of DTLET for mu sites, it appeared that both ligands occupied a similar number of mu receptors at their ED50 values, thus supporting a preferential involvement of mu opioid binding sites in the supraspinal pain control. [Met5]enkephalin inhibited the in vivo binding of both agonists only when the peptide was protected from degradation by the co-administration of a mixed inhibitor of enkephalin degrading enzymes RB38A (N[3(R)(hydroxyaminocarbonyl)-2-benzyl-1-oxopropyl]- L-phenylalanine). Unlike thiorphan, 5 nmol RB38A alone was able to inhibit [3H]DAGO binding by 60%. This result is the first direct demonstration of the existence of an in vivo tonic control of mu opioid receptor occupation by endogenous opioid peptides.     

Characterization of high affinity opioid binding sites in rat periaqueductal gray P2 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 used as a tracer ligand to nonselectively label mu, delta, and kappa binding sites in PAG enriched P2 membrane. The mu selective ligand [D-Ala2,N-methylPhe4,Glyol5]enkephalin (DAGO) competed with [3H]EKC for more than one population of binding sites with both high and low affinity. In contrast the delta selective ligand [D-Pen2,D-Pen5]enkephalin (DPDPE) and the kappa 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 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-Ala2,D-Leu5]enkephalin (DADLE) with high affinity. 2 nM [3H]DPDPE did not substantially label PAG enriched P2 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 mu selective high affinity binding sites in PAG enriched P2 membrane to which delta ligands and kappa ligands have low affinity.     

Comparative pharmacological properties and functional coupling of mu and delta opioid receptor sites in human neuroblastoma SH-SY5Y cells

The characteristics of mu and delta opioid receptor sites present in human neuroblastoma SH-SY5Y cells were investigated using [D-Ala2-N-methyl-Phe4-Gly-(01)5]enkephalin (DAGO) and [2-D-penicillamine, 5-D-penicillamine]enkephalin (DPDPE), which are the most selective radioligands available for mu and delta sites, respectively. Scatchard analysis of the saturation isotherms revealed high affinity binding to a single class of sites for both [3H]DAGO (mu) and [3H]DPDPE (delta). [3H]DAGO labeled twice the number of sites compared to the binding capacity of [3H]DPDPE, yielding a mu/delta ratio of 2:1. Selective suppression of [3H]diprenorphine binding by specific opioid "blocking" ligands also showed a predominance of mu receptors, representing 65-70% of the total opioid sites. Competition binding studies carried out with a series of opiates and opioid peptides displayed higher potencies of mu- and delta-selective ligands in displacing the specific binding of [3H]DAGO and [3H]DPDPE, respectively. The [3H]diprenorphine/agonist competition curves were biphasic, indicating the high and low affinity states of mu and delta receptor sites in SH-SY5Y cells. Guanine nucleotide and sodium had differential effects on the agonist affinity and the proportion of high affinity states of mu and delta receptors. The mu and delta receptor sites were shown to be functionally coupled to adenylate cyclase. All of these data support the independent existence of mu and delta receptor types in human neuroblastoma cells. SH-SY5Y cells, therefore, represent a suitable model for investigating opioid-mediated responses in nerve cell populations.     

Selective cardiorespiratory effects mediated by mu opioid receptors in the nucleus ambiguus

The respiratory and cardiovascular effects of the highly selective mu opioid agonist, D-Ala2, MePhe4, Gly- ol5 enkephalin ( DAGO ) and the relatively selective delta agonist, D-Leu5 enkephalin (DADL) were compared following injection (0.1 microliter) into the nucleus ambiguus (NA) of spontaneously-breathing and artificially-respired, pentobarbital-anesthetized rats. In non-ventilated animals, the opioids elicited dose-related (3 X 10(-11) -3 X 10(-9) M), naloxone-reversible depression of respiratory rate (RR) without altering the tidal volume. Mean arterial pressure (MAP) was unchanged at small doses and decreased at the largest dose; heart rate (HR) was unchanged. In artificially-respired animals, both peptides elicited dose-related, naloxone-reversible increases in mean arterial pressure and heart rate; DAGO was significantly more potent than DADL (P less than 0.01). Given the relative potency and selectivity of the opioids tested, these findings are consistent with the conclusion that mu receptors may selectively mediate the respiratory and cardiovascular actions of opioids in an important brain stem cardiorespiratory center in the rat. Moreover, these data indicate the importance of respiratory effects on the cardiovascular activity of centrally administered opioids.     

Identification of drug receptors in porcine dental pulp by the radioligand binding assay

1. The acetylcholine (ACh), histamine and serotonin (5-HT) receptors in porcine dental pulp were characterized by the radioligand binding assay. 2. For [3H]nicotine binding site, Kd was 8.06 +/- 1.65 nM and Bmax was 270.83 +/- 32.68 fmol/mg protein. 3. For [3H]QNB binding site, Kd was 1.04 +/- 0.14 nM and Bmax was 24.83 +/- 3.09 fmol/mg protein. 4. For [3H]histamine binding site, Kd was 1.22 +/- 0.1 nM and Bmax was 283.15 +/- 33.1 fmol/mg protein. 5. For [3H]5-HT binding site, Kd was 1.41 +/- 0.1 nM and Bmax was 53.1 +/- 3.4 fmol/mg protein. 6. These findings indicate that the specific receptors for ACh, histamine and 5-HT are present in the porcine dental pulp, and that the ACh receptor is predominantly nicotinic.     

Opioid binding sites in the guinea pig and rat kidney: radioligand homogenate binding and autoradiography.

The specific binding of the selective mu-, delta-, and kappa-opioid ligands [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin ([3H] DAGOL), [3H][D-Pen2,D-Pen5]enkephalin ([3H]DPDPE), and [3H]U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of mu- and kappa-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that [3H]DPDPE bound with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the delta-opioid binding site. Autoradiography experiments demonstrated that specific [3H]DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the delta-opioid binding site in medulla homogenates, using agonist ([3H]DPDPE) and antagonist ([3H]diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the delta-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the delta binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.     

Imidazoline receptors in rat liver cells: a novel receptor or a subtype of alpha 2-adrenoceptors?

An imidazoline/guanidine receptor has been characterized in rat liver cells. Binding of [3H]idazoxan, a selective benzodioxan antagonist, to imidazoline receptor on intact fresh hepatocytes (Bmax = 801 +/- 23 fmol/mg protein, Kd = 11 +/- 0.8 nM) and to liver membranes (Bmax = 400 +/- 38 fmol/mg protein, Kd = 10 +/- 2 nM) was saturable at 4 degrees C within 3.5 h and at 30 degrees C within 30 min, respectively. Rat lung membranes had more imidazoline sites (Bmax = 578 +/- 30 fmol/mg protein, Kd = 14 +/- 1.4 nM) than alpha 2-adrenoceptors (Bmax = 175.0 +/- 20.0 fmol/mg protein, Kd = 4.8 +/- 2.0 nM). We also screened other tissues for imidazoline sites; the ratio of adrenoceptors to total sites labeled with [3H]idazoxan displaced by cirazoline was lower in rat lung compared to rat brain and human platelets. The imidazoline receptor has common pharmacological properties with alpha 2-adrenoceptors, although it is not a subtype of the adrenoceptor, since it bound neither the endogenous agonists norepinephrine and epinephrine, nor the selective alpha 2-antagonists yohimbine and phentolamine. All guanidine type alpha 2-adrenoceptor drugs (e.g. guanbenz, guanoxan) and imidazolines (e.g., UK-14,304, naphazoline) competed with high affinity for the liver imidazoline receptor. The lack of effect by Gpp(NH)p, a non-hydrolysable GTP analogue, on the affinity of guanidine- and imidazoline-type ligands for liver imidazoline receptors suggests that the mode of action of these drugs at imidazoline receptors is different than at conventional alpha 2-adrenoceptors. Ionic changes were considered as a possible mechanism underlying the alpha 2-adrenoceptor effects in various cells. Opening of K+ channels by alpha 2-adrenoceptors agonists is a pathway which might be shared by imidazoline-type agonists at imidazoline sites. Indeed, 4-aminopyridine, a K+ channel blocker, inhibited the specific binding of [3H]idazoxan to liver cells with an IC50 of 0.34 +/- 0.07 mM a concentration which is effective in blocking K+ channels in neuronal cells. Similarly, Cs+ and NH4+ effectively interfered with [3H]idazoxan binding, suggesting a possible coupling of imidazoline sites to K+ gating. The endogenous ligand clonidine-displacing substance (CDS), which was isolated from bovine brain and which binds to alpha 2-adrenoceptors in brain membranes and human platelets competed with idazoxan at rat liver imidazoline receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specific binding sites for [3H]Ro 5-4864 in rat prostate and seminal vesicle.

1. The peripheral-type benzodiazepine receptor was characterized in rat prostate and seminal vesicle using [3H]Ro 5-4864 as radioligand. 2. The affinity of this radioligand for this receptor was higher in rat prostate (KD = 4.36 +/- 1.02 nM) than in seminal vesicle (KD = 8.45 +/- 1.34 nM). 3. The density of binding sites obtained in these two tissues was Bmax = 4164 +/- 873 fmol/mg in prostate and 5978 +/- 1022 fmol/mg in seminal vesicle. 4. The [3H]Ro 5-4864 binding was inhibited non-competitively by atractyloside and alpha,beta-methylene-ATP, suggesting a modulation by the ADP/ATP mitochondrial carrier. 5. Flutamide was able to displace bound [3H]Ro 5-4864 with an IC50 similar to protoporphyrin IX.     

Protection by opioid ligands against modification of the opioid receptor by a carbodiimide.

Opioid receptors in membranes prepared from guinea-pig cerebellum were modified irreversibly by treatment with a water soluble carbodiimide, 1-ethyl,3-(3-dimethylaminoethyl)carbodiimide (EDAC). This decreased the number of [3H]bremazocine binding sites (Bmax reduced from 140 to 100 fmol/mg by 1 mM EDAC) without changing their affinity. When the EDAC concentration used was sufficient (500 mM) to inactivate almost all of the opioid receptors, the modification was partly prevented by inclusion of high concentrations (100 microM) of opioid agonists ([D-Ala2, MePhe4, Glyol5]-enkephalin, [D-Ala2, D-Leu5]-enkephalin,(+)-trans-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl]benzo(b)thiophene-4-acetamide hydrochloride), although they exhibited equal efficacy irrespective of their mu, delta or kappa type selectivity. However, almost all of the opioid binding sites were protected when a guanine nucleotide analogue (GppNHP, 100 microM) was also included with the agonists during carbodiimide treatment.     

Opiate receptor subtypes in the nucleus tractus solitarii of the cat: the effect of vagal section

The distribution of mu, delta and kappa opioid receptors in the lower brainstem of the cat has been determined autoradiographically by studying the binding of tritiated [D-Ala2,MePhe4,Glyol5][tyrosyl-3,5-3H]enkephalin (DAGO), [D-Pen2,D-Pen5][tyrosyl-3,5-(n)-3H]enkephalin (DPDPE) and [9-3H]ethylketazocine (EKC), respectively. General opiate receptor binding was established using [3H]naloxone (NX). High densities of [3H]NX and DAGO binding sites were found most prominently in the nucleus tractus solitarii. There was no DPDPE and very weak EKC binding within this nucleus, although both these ligands bound to the cerebellum. The effect of unilateral vagotomy on receptor density was examined. Sectioning the cervical vagus had no effect on the density of mu receptors in the brainstem. Sectioning the vagus, accompanied by nodose ganglion excision, led to a marked depletion of mu receptors which was restricted to dorsal and medial regions of the ipsilateral nucleus tractus solitarii at, and rostral to, the obex. These results suggest that mu opiate receptors are located presynaptically on vagal afferents terminating within a restricted region of the nucleus tractus solitarii.