Treatment with Antisense Oligodeoxynucleotide to a Conserved Sequence of Opioid Receptors Inhibits Antinociceptive Effects of Delta Subtype Selective Ligands

Abstract

Previous work has suggested the existence of subtypes of the delta opioid receptor (DOR) which have been termed δ₁ and δ₂. [D-Ala², Glu⁴]deltorphin has been suggested to selectively elicit antinociception via the δ₂ receptor while [D-Pen², D-Pen⁵]enkephalin (DPDPE) is thought to act via the δ₁ receptor. Treatment with an antisense oligodeoxynucleotide (oligo) directed towards the N-terminal portion of the cloned DOR has been demonstrated to selectively inhibit the antinociceptive actions of [D-Ala², Glu⁴]deltorphin, but not of DPDPE, suggesting that the cloned DOR corresponds to that pharmacologically defined as δ₂. Here, an antisense oligo (or a mismatch sequence) was designed to target a conserved region of the cloned μ δ and opioid receptor. These oligos were employed in order to determine whether the antinociceptive effects of [DAla², Glu⁴]deltorphin, as well as DPDPE, could be inhibited. The data indicate that the antinociceptive actions of both ligands were inhibited by treatment with this antisense, but not with the mismatch oligo. Taken together, the results of the treatments with oligos directed towards the N-terminal portion of the cloned DOR and with that directed to the conserved region of the opioid receptors suggest that (a) DPDPE effects are mediated by a subtype of the DOR which shares a domain common to the cloned opioid receptors, and (b) the N-terminal region differs between these putative DOR subtypes.     

δ antagonist and κ agonist activity of naltriben: Evidence for differential κ interaction with the δ1 and δ2 opioid receptor subtypes

The selective δ₂ receptor antagonist Naltriben (NTB) has played an important role in the identification of subtypes of the δ opioid receptor, termed δ₁ and δ₂, and their role in antinociception. However, the majority of these studies have been conducted in the mouse. The present study determined the opioid receptor selectivity of subcutaneously (s.c.) administered NTB in the rat. Five minute pretreatment with 1 mg/kg s.c. NTB antagonized the increase in TFL produced by i.t. administration of equieffective doses of the δ₂ receptor agonist [D-Ala², Glu⁴]deltorphin (DELT) or the δ₁ receptor agonist [D-Pen², D-Pen⁵]enkephalin (DPDPE), but did not antagonize the μ receptor agonist [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO). These data confirm previous reports that NTB is a selective δ opioid receptor antagonist. However, this dose of NTB antagonized DELT and DPDPE to an equivalent extent, suggesting that its selectivity for the δ₂ receptor is not maintained after s.c. administration in the rat. A lower dose of NTB (0.56 mg/kg s.c.) was ineffective. When the dose of NTB was increased to 3 mg/kg s.c. the antagonism of DELT and of DPDPE was unexpectedly lost. Pretreatment with the κ receptor antagonist nor-binaltorphimine (nor-BNI) partially restored the antagonism of DELT, but not DPDPE by this dose of NTB and did not modify the antagonism of DAMGO by NTB. These data suggest that high doses of NTB have κ receptor agonist-like activity and support the proposal that κ opioid agonists diminish the actions of δ receptor antagonists. They also suggest that nor-BNI-sensitive κ opioid receptors interact with δ₂, but not δ₁ opioid receptors in the spinal cord.     

Streptozotocin-induced diabetes selectively enhances antinociception mediated by δ1-but not δ2-opioid receptors

We assessed the effect of diabetes on antinociception produced by intracerebroventricular injection of δ-opioid receptor agonists [D-Pen^2,5]enkephalin (DPDPE) and [D-Ala²]deltorphin II. The antinociceptive effect of DPDPE (10 nmol), administered i.c.v., was significantly greater in diabetic mice than in non-diabetic mice. The antinociceptive effect of i.c.v. DPDPE was significantly reduced in both diabetic and non-diabetic mice following pretreatment with 7-benzylidenenaltrexone (BNTX), a selective δ₁-opioid receptor antagonist, but not with naltriben (NTB), a selective δ₂- opioid receptor antagonist. There were no significant differences in the anticiceptive effect of [D-Ala²]deltorphin II (3 nmol, i.c.v.) in diabetic and non-diabetic mice. Furthermore, the antinociceptive effect of i.c.v. [D-Ala²]deltorphin II was significantly reduced in both diabetic and non-diabetic mice following pretreatment with NTB, but not with BNTX. In conclusion, mice with diabetes are selectively hyper-responsive to supraspinal δ₁-opioid receptor-mediated antinociception, but are normally responsive to activation of δ₂-opiod receptors.     

Assessment of stereoselectivity of trimethylphenylalanine analogues of delta-opioid [D-Pen(2),D-Pen(5)]-enkephalin

[D-Pen(2),D-Pen(5)]-Enkephalin (DPDPE) is an enzymatically stable delta-opioid receptor-selective peptide, which was modified by the trimethylation of the Phe(4) residue to give beta-methyl-2', 6'-dimethylphenylalanine (TMP), resulting in four conformations : (2R,3S)-beta-Phe-DPDPE, (2R,3R)-beta-Phe-DPDPE, (2R, 3S)-beta-Phe-DPDPE, and (2S,3R)-beta-Phe-DPDPE. Synthesis was by solid-phase techniques using enantiomerically pure amino acids to give the four optically pure diastereoisomer peptides. The potency and selectivity (delta- versus mu-opioid receptor) were evaluated by radioreceptor binding in rat brain, with a mu/delta ratio decrease for all TMP conformations, compared with the parent compound (DPDPE). Octanol/buffer distribution analysis showed enhanced lipophilicity of all TMP forms, with a sixfold enhancement associated with (2S,3S)-TMP. In situ vascular perfusion in anesthetized rats showed a 1.6-fold (p < 0.01) increase in the ratio of brain uptake for (2S,3S)-TMP and a 1.5-fold (p < 0.01) decrease in uptake for (2R,3R)-TMP. Saturability of (2S,3S)-TMP was shown (p < 0.01) against 100 microM unlabeled DPDPE, showing a shared nondiffusionary transport system. P-glycoprotein affinity was shown in situ for the parent and (2S,3S)-TMP (p < 0.01). Protein binding capacity of the TMP compounds in rat plasma and in situ mammalian bovine serum albumin-Ringer showed (2R,3S)-TMP and (2S,3R)-TMP with the lowest degree of protein binding (p < 0.01), and (2S,3S)-TMP and (2R,3R)-TMP with comparable affinities to DPDPE. Analgesia, via intravenous administration, showed significantly reduced (p < 0.01) end effect and time course for (2R,3R)-TMP, (2R,3S)-TMP, and (2S, 3R)-TMP as compared with DPDPE. These results demonstrate that topographical modification in a conformationally restricted peptide can significantly modulate potency and receptor selectivity, binding capacity, enzymatic stability, lipophilicity, P-glycoprotein affinity, and blood-brain barrier permeability, resulting in a change of bioavailability, and thereby provides insight for future peptide drug design.     

Selective inhibition of [D-ALA2, GLU4]deltrophin antinociception by supraspinal,but not spinal,administration of an antisense oligodeoxynucleotide to an opioid delta receptor

Evidence in vivo has suggested the existence of subtypes of the δ opioid receptor (DOR), which have been termed δ₁ and δ₂. These proposed DOR subtypes are thought to be activated by [D-Pen², D- Pen⁵]enkephalin (DPDPE, δ₁) and [D-Ala², Glu⁴]deltorphin (δ₂). Recent work in which an antisense oligodeoxynucleotide (oligo) to a cloned DOR was administered by the intrathecal (i.th.) route has demonstrated a reduction in the antinociceptive actions of both i.th. DPDPE and [D-Ala², Glu⁴]deltorphin, but not of [D-Ala², NMPhe⁴, Gly-ol]enkephalin (DAMGO, μ agonist) in mice. The present investigation has extended these observations by administering the same DOR antisense oligo sequence by the intracerebroventricular (i.c.v.) route and evaluating the antinociceptive actions of i.c.v. agonist selective for δ, μ and κ receptors. I.th. treatment with DOR antisense oligo, but not mismatch oligo, significantly inhibited the antinociceptive actions of both i.th. DPDPE and [D-Ala², Glu⁴deltorphin but not of i.th. DAMGO or U69, 593 (κ agonist), confirming previous data. In contrast, i.c.v. DOR antisense oligo, but not mismatch oligo, seletively inhibited the anitinociceptive response to i.c.v. [D-Ala², Glu⁴]deltorphin without altering the antinociceptive actions of i.c.v. DPDPE, DAMGO or U69,593. The data suggest that the cloned DOR corresponds to that pharmacologically classified as δ₂ and further, suggest that this δ receptor subtype may play a major role in eliciting spinal δ-mediated antinociception.     

A comprehensive study on the putative δ-opioid receptor (sub)types using the highly selective δ-antagonist, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH

The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH and see if putative δ-opioid receptor subtypes can be distinguished. Analgesic latencies were assessed in mouse tail-flick assays after intrathecal administration. In vitro receptor autoradiography, binding and ligand-stimulated [³⁵S]GTPγS functional assays were performed in the presence of putative δ₁-(DPDPE: agonist, BNTX: antagonist), δ₂-(agonist: deltorphin II, Ile^5,6-deltorphin II, antagonist: naltriben) and μ-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60%, but did not antagonize δ₂- and μ-agonist induced analgesia. The radiolabeled form identified binding sites with K_D = 0.18 nM and receptor densities of 102.7 fmol/mg protein in mouse brain membranes. The binding site distribution of the [³H]Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH agreed well with that of [³H]Ile^5,6-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH displayed 2.49 ± 0.06 and 0.30 ± 0.01 nM potency against DPDPE and deltorphin II in the [³⁵S]GTPγS functional assay, respectively. The rank order of potency of putative δ₁- and δ₂-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human δ-opioid receptors. Deletion of the DOR-1 gene resulted in no residual binding of the radioligand and no significant DPDPE effect on G-protein activation. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH is a highly potent and δ-opioid specific antagonist both in vivo and in vitro. However, the putative δ₁- and δ₂-opioid receptors could not be unequivocally distinguished in vitro.     

Characterization of the bioactive form of linear peptide antagonists at the δ-opioid receptor

The stereochemical requirements for δ-opioid receptor binding of a series of linear peptide antagonists with a novel conformationally restricted Phe analogue (Tic) as a second residue were examined by using a variety of computational chemistry methods. The δ-opioid receptor analogues with significant affinity, Tyr-Tic-NH₂ (TI-NH₂), Tyr-Tic-Phe-OH (TIP), Tyr-Tic-Phe-NH₂(TIP-NH₂), Tyr-Tic-Phe-Phe-OH (TIPP), Tyr-Tic-Phe-Phe-NH₂) (TIPP-NH₂), and the low affinity δ-opioid peptides Tyr-Pro-Phe-Pro-NH₂ (morphiceptin) and Tyr-Phe-Phe-Phe-NH₂ (TPPP-NH₂), were included in this study. The conformational profiles of these peptides were obtained by consecutive cycles of high and low temperature molecular dynamic simulations, coupled to molecular mechanical energy minimization carried out until no new conformational minima were obtained. Comparing the results for TPPP-NH₂ and TIPP-NH₂, the presence of the conformationally restricted Tic residue did not greatly reduce the number of unique low energy conformations, but did allow low energy conformers involving cis bonds between the first two residues. The conformational libraries of these peptides were examined for their ability to satisfy the three key ligand components for receptor recognition already identified by previous studies of high affinity cyclic (Tyr¹-D -Pen²-Gly³-Phe⁴-D -Pen⁵) enkephalin (DPDPE) type agonists: a protonated amine group, an aromatic ring, and a lipophilic moiety in a specific geometric arrangement. Two types of conformations common to the five high δ-opioid affinity L -Tic analogues were found that satisfied these requirements, one with a cis and the other with a trans peptide bond between the Tyr¹ and Tic² residues. Moreover, both the Tic² and Phe³ residues could mimic the hydrophobic interactions with the receptor of the Phe⁴ moiety in the cyclic DPDPE type agonists, consistent with the appreciable affinity of both di-and tripeptides. The low δ-opioid receptor affinity of morphiceptin can be understood as the result of conformational preferences that prevent the fulfillment of this pharmacophore for recognition. © 1996 John Wiley & Sons, Inc.     

Solution Conformation of [D-Pen2,D-Pen5]enkephalin in Water: A NMR and Molecular Dynamics Study

The solution conformation of [D -Pen²,D -Pen⁵] enkephalin (DPDPE), a highly potent δ-selective opioid agonist, was examined by means of NMR, molecular mechanics and molecular dynamics methods. The structural information in the solvent water was obtained employing one- and two-dimensional methods of ¹H and ¹³C-NMR spectroscopy. Based on the distance geometry technique using the ROE data as input, 400 conformers were obtained and considered in the structure analysis. Alternatively, about 2000 conformers were stochastically generated and related to the NMR data after energy minimization. The structure analysis provides one conformer in agreement with all NMR data, which belongs to the lowest energy conformation group. This structure may serve as a reference conformer for DPDPE analogues synthesized with the aim of activity increase.     

Development of a model for the δ-opioid receptor pharmacophore: 3. Comparison of the cyclic tetrapeptide Tyr-c[D-Cys-Phe-D-Pen] OH with other conformationally constrained δ-receptor selective ligands

We have previously proposed a model of the δ-opioid receptor bound conformation for the cyclic tetrapeptide, Tyr-c[D -Cys-Phe-D -Pen]OH (JOM-13) based on its conformational analysis and from conformation-affinity relationships observed for its analogues with modified first and third residues. To further verify the model, it is compared here with results of conformational and structure-activity studies for other known conformationally constrained δ-selective ligands: the cyclic pentapeptide agonist, Tyr-c[D -Pen-Gly-Phe-D -Phe]OH (DPDPE); the peptide antagonist, Tyr-Tic-Phe-PheOH (TIPP); the alkaloid agonist, 7-spiroindanyloxymorphone (SIOM); and the related alkaloid antagonist, oxymorphindole (OMI). A candidate δ-bound conformer is identified for DPDPE that provides spatial overlap of the functionally important N-terminal N⁺₃ and C-terminal COO⁻ groups and the aromatic rings of the Tyr and Phe residues in both cyclic peptides. It is shown that all δ-selective ligands considered have similar arrangements of their pharmacophoric elements, i.e., the tyramine moiety and a second aromatic ring (i.e., the rings of Phe³, Phe⁴, and Tic² residues in JOM-13, DPDPE, and TIPP, respectively; the indole ring system in OMI, and the indanyl ring system in SIOM). The second aromatic rings, while occupying similar regions of space throughout the analogues considered, have different orientations in agonists and antagonists, but identical orientations in peptide and alkaloid ligands with the same agonistic or antagonistic properties. These results agree with the previously proposed binding model for JOM-13, are consistent with the view that δ-opioid agonists and antagonists share the same binding site, and support the hypothesis of a similar mode of binding for opioid peptides and alkaloids. © 1996 John Wiley & Sons, Inc.     

Effects of a novel opioid peptide antagonist on rat bladder motility in vivo

The agonist, and opioid antagonist, effects of intracerebroventricularly (ICV) given D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH₂ (CTP), a cyclic analogue of somatostatin octapeptide, were evaluated using the micturition reflex of the anesthetized rat as the endpoint. Antagonist effects were evaluated against equieffective doses of selective mu [D-Ala²,NMPhe⁴,Gly-ol]enkephalin (DAGO) and delta [D-Pen²,D-Pen⁵] enkephalin (DPDPE) opioid agonists. At low ICV doses, CTP preferentially antagonized DPDPE rather than DAGO; increasing the dose of CTP further effectively antagonized both mu and delta agonists, while even higher doses showed an agonist effect alone which was not blocked by adrenergic, cholinergic or opioid antagonists. Selective opioid antagonist doses of CTP failed to block the inhibition of the micturition reflex produced by pentobarbital. Possible residual somatostatin like properties of CTP were tested by using somatostatin as a possible antagonist of equieffective doses of DPDPE and DAGO; somatostatin did not antagonize these agonists. Repeated exposure to CTP resulted in the development of acute tolerance to the agonist effect, and also prevented the inhibition of the reflex by high doses of somatostatin, with the converse experiment showing a similar pattern; thus, repeated somatostatin resulted in tolerance and subsequent cross-tolerance to the agonist effects of CTP. In animals tolerant to somatostatin, CTP nevertheless behaved as an opioid antagonist. The present results indicate that CTP possesses opioid antagonist properties in vivo which are pharmacological in nature but nevertheless retains residual somatostatin-like activity at higher doses.     

Topographic analysis of the S7 binding subsite of the tachykinin neurokinin-1 receptor

Conformationally and configurationally restricted rotameric probes of phenylalanine have been incorporated in the sequence of substance P (SP)—Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂—for analyzing the binding pockets of Phe⁷ (S₇) and Phe⁸ (S₈), in the neurokinin-1 receptor. These analogues of phenylalanine are (2S, 3R)- and (2S, 3S)-indanylglycines, E- and Z-α, β-dehydrophenylalanines, and 2(S)-α, β-cyclopropylphenylalanines [Δ^E Phe, Δ^ZPhe, ▿^E2(S)Phe, and ▿^Z2(S)Phe]. Binding data obtained with either conformationally (Ing diastereoisomers) or configurationally (Δ^EPhe, Δ^ZPhe) probes have unveiled large differences in the binding potencies of these rotameric probes. With the support of nmr data and energy calculations done on these SP-substituted analogues, we attempt to answer questions inherent to such study. First, none of these six probes prevents the formation of bioactive conformation(s) of the backbone of SP. Second, both diastereoisomers (S, S) and (S, R) of indanylglycine preferentially adopt, in the sequence of SP, the gauche (−) and trans side-chain orientations, respectively, as previously postulated from energy calculations with model peptides. However, in solution, the difference in energy between these rotamers included in the sequence of SP, compared to model peptides, is smaller since the other rotamer can be detected in [(2S, 3R) Ing⁷]SP. Finally, from this study we can hypothesize that the large variations observed in the affinities of Phe⁷ substituted analogues of SP must come from steric hindrance in the S₇ binding site, which drastically restricts the space filling around the C_α (SINGLE BOND) C_β bond of residue 7. © 1996 John Wiley & Sons, Inc.     

Enkephalin analogs containing 4,4-difluoro-2-aminobutyric acid: Synthesis and fluorine effect on the biological activity

Analogs of Met-enkephalin and [d -Pen², d -Pen⁵]enkephalin (DPDPE) containing the partially fluorinated amino acid 4,4-difluoro-2-aminobutyric acid (DFAB) in the 2- or 3-position of the peptide sequence were synthesized and their opioid activities and receptor selectivities were determined in vitro. The linear fluorinated [d -DFAB², Met⁵-NH₂]enkephalin showed μ and δ agonist potencies comparable to those of natural [Leu⁵]enkephalin. The partially fluorinated DPDPE analogs behaved differently as compared with their non-fluorinated correlates. While l -amino acid substitution in position 3 of DPDPE usually resulted in higher δ agonist potency than d -amino acid substitution, [d -DFAB³]DPDPE turned out to be a more potent δ agonist than [l -DFAB³]DPDPE. Furthermore, [d -DFAB³]DPDPE showed over 100-fold higher δ agonist potency than [d -Abu³]DPDPE (Abu=2-aminobutyric acid), indicating that the fluorine substituents interact favorably with a δ opioid receptor subsite. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

1H-NMR studies on substance P hexapeptide analogs

¹H-nmr studies of [pGlu⁶]SP_6–11, [gpGlu⁶,mPhe⁷]SP_6–11, and [pGlu⁶,N-CH₃Phe⁷]SP_6–11 in DMSO-d₆ reveal characteristic chemical shifts, ³J_NH-αCH, temperature dependence, as well as deuterium exchange half-times. Marked similarities are revealed for the two first analogs, whereas the N-methylated analog is clearly different. Possible conformations are considered.     

Preliminary ligand binding data for subtypes of the delta opioid receptor in rat brain membranes

Delta opioid binding sites were assayed using [³H][D-ala²,D-leu⁵]enkephalin and rat brain membranes depleted of μ binding sites with the site-directed acylating agent, 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5 -isothiocyanatobenzimidazole-HCI. [D-Pen², D-Pen⁵]enkephalin (DPDPE), [D-Pen²,L-Pen⁵]enkephalin, [D-Ala²]deltorphin-I and [D-Ala²]deltorphin-II inhibition curves were characterized by slope factors (Hill coefficients) less than 1. The low slope factor of DPDPE persisted in the presence of 50 μM 5'-guanylyimidodiphosphate in the assay. Quantitative analysis of [D-ala²,D-leu⁵]enkephalin, DPDPE and [D-Ala²]deltorphin-I binding surfaces resolved two binding sites. Whereas [D-ala²,D-leu⁵]enkephalin had equal affinity for both sites, DPDPE and [D-Ala²]deltorphin-I had high affinity for the high capacity binding site, and low affinity for the low capacity binding site. These data support pharmacological studies demonstrating δ receptor subtyes which mediate antinociception.     

Syntheses and biological evaluation of analogs of luteinizing hormone-releasing hormone (LH-RH) modified in position 2, 3, 4 or 5

Syntheses by the conventional methods as well as the chemical, physical and biological properties are described of the following analogs of the LH-releasing hormone (LH-RH): [Leu³]-LH-RH, [Phe³]-LH-RH, [Trp²] [His³]-LH-RH, Des-Trp³-LH-RH, Des-His²-[Phe⁵]-LH-RH, [Ala⁴]-LH-RH, [Phe⁵]-LH-RH and [Ala⁴] [Phe⁵]-LH-RH. $\text{In vivo}$ assays showed that [Leu³]-LH-RH did not release LH in doses as high as 5 – 25 μg, having less than 0.0008% of LH-RH activity, while [Phe³]-LH-RH had 0.43% of the LH-RH activity of natural LH-RH. The LH-RH activities of [Trp²] [His³]-LH-RH, Des-Trp³-LH-RH and Des-His²-[Phe⁵]-LH-RH were extremely low. On the other hand, [Ala⁴]-LH-RH, [Phe⁵]-LH-RH and [Ala⁴] [Phe⁵]-LH-RH had significant LH-RH activity. The structure-activity relationship of LH-RH is discussed on the basis of these findings.     

Investigating the effects of opioid drugs on electrocortical activity using wavelet transform

 Fetal electrocortical activity (ECoG) is characterized by two distinct patterns: HVSA (high voltage, slow activity) and LVFA (low voltage, fast activity). Using the wavelet transform (WT), we recently reported that the frequency characteristics of these two ECoG patterns undergo significant maturational changes prior to birth (Akay et al. 1994a). We now report that fetal ECoG can also be significantly affected by pharmacological agents. In this paper, we compared the effects of two opioid drugs (morphine and [D-Pen², D-Pen⁵]enkephalin, DPDPE) on fetal ECoG, using the chronically instrumented fetal lamb model. Morphine was infused intravenously (i.v.) at 2.5 mg/h, while DPDPE was infused into the lateral cerebroventricle (i.c.v.) at 30 μg/h. The ECoG was analyzed using WT. We performed multiresolution decomposition for four sets of parameters D _{2 j} where −1<j<−4. The four series WTs represent the detail signal bandwidths: (1) 16–32 Hz, (2) 8–16 Hz, (3) 4–8 Hz, (4) 2–4 Hz. The data were subjected to statistical analysis using the Kolmogorov–Smirnov (KS) test. Both morphine and DPDPE resulted in a significant increase in power in the first wavelet band, while power was reduced in the second, third and fourth wavelet bands. In addition, both drugs resulted in a disruption of the normal cyclic pattern between the two ECoG patterns. There was a difference in the time course of action between morphine and DPDPE. This is the first occasion in which continuous ECoG has been subjected to rigorous statistical analysis. The results suggest that the WT-KS method is most suitable for quantitating changes in the ECoG induced by pharmacological agents. Received: 21 January 1994/Accepted in revised form: 15 September 1994     

Modulation of Binding at Opioid Receptors by Monoand Divalent Cations and by Cholinergic Compounds

Abstract

In membrane suspensions from guinea-pig brain, NaCl, LiCl, NH₄Cl and KCl, inhibit the equilibrium binding (25°C) of the selective μ-agonist [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin, the selective δ-agonist [³H]-[D-Pen²,D-Pen⁵]enkephalin and the selective δ-agonist [³H]-dynorphin A (1-9). Choline chloride inhibits the binding of the μ- and δ-agonists but not of the δ-agonist; the choline derivative, methacholine, inhibits also the binding of the δ-agonist. Binding of the δ-agonist is potentiated by CaCl₂, MgCl₂ and MnCl₂; these salts inhibit binding of the δ-agonist. As far as binding of the μ-agonist is concerned, MgCl₂ and MnCl₂ may potentiate or inhibit whereas CaCl₂ is only inhibitory. The binding of the μ-antagonist [³H]-naloxone is potentiated by NaCl; while the threshold of inhibition by LiCl is increased there is no potentiation. In membrane suspensions of the rabbit cerebellum about 80% of the opioid binding sites are of the μ-type; the binding of the μ-agonist [³H]-[D-Ala², MePhe⁴, Gly-ol⁵]enkephalin is inhibited by NaCl, LiCl, KCl and choline chloride whereas that of the μ-antagonists [³H]-naloxone and [³H]-(-)-bremazocine is potentiated at low concentrations but inhibited at higher concentrations of NaCl. In membranes of the guinea-pig cerebellum about 80% of the opioid binding sites are of the δ-type; they are particularly effective for assays of K-receptors when the selective K-agonist [³H]-dynorphin A (1-9) is used as ligand.     

The conversion of (24S)-24-ethylcholesta-5,22,25-trien-3β-ol into poriferasterol,both in vivo and with a cell-free homogenate of the alga Trebouxia sp.

[6-³H₁] (24S)-24-Ethylcholesta-5,22,25-trien-3β-ol added to the growth medium of a culture of Trebouxia sp. 213/3 was efficiently taken-up by the cells and converted into (24R)-24-ethylcholesta-5,22-dien-3β-ol (poriferasterol) which is one of the major sterols of this alga. A cell-free homogenate was obtained from Trebouxia which catalysed the NADPH-dependent reduction of [6-³H₁] (24S)-24-ethylcholesta-5,22,25-trien-3β-ol to yield poriferasterol. The δ²⁵-sterol reductase was found to be mainly localized in the microsomal fraction of the homogenate.     

Proton magnetic resonance studies of 9-(β-d-xylofuranosyl)adenine 3′,5′-cyclic monophosphate and 9-(β-d-arabinofuranosyl)adenine 2′,5′-cyclic monophosphate

A detailed ¹H 220-MHz n.m.r. study of 9-(β-d-xylofuranosyl)adenine 3′,5′-cyclic monophosphate (3′,5′-xylo-cAMP, 1) and 9-(?-d-arabinofuranosyl)adenine 2′,5′-cyclic monophosphate (2′,5′-ara-cAMP, 2) in D₂O solution is described. The sugar-ring conformations in 1 and 2 are shown to be ³E and ²E, respectively, and the phosphate rings are in a chair form. An unusual ⁴J_P,H coupling of 2.4 Hz is observed between H-4′ and phosphorus in 1 and a vicinal J_P,H of 30.8 Hz between H-5′ and phosphorus in 2. This latter coupling verifies a similar value found previously in the ara-cytidine analog of 2. A comparison of the conformational properties of cyclic nucleotides having fused phosphate and sugar rings has been made, together with an assessment of the use of the Karplus constants in such ring-systems.