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.     

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.     

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.     

Effects of morphine-3-glucuronide on the antinociceptive activity of peptide and nonpeptide opioid receptor agonists in mice

The effects of morphine-3-glucuronide (M3G), a metabolite of morphine, were determined on the antinociceptive actions, as measured by the tail flick test, of morphine, a μ-opioid receptor agonist, of U-50,488H, a κ-opioid receptor agonist, of [ -Pen², -Pen⁵]enkephalin (DPDPE), a δ₁-opioid receptor agonist, and of [ -Ala²,Glu⁴]deltorphin II (deltorphin II), a δ₂-opioid receptor agonist in mice. Morphine administered ICV (2.5 μg/mouse) or SC (10 mg/kg), U-50,488H (25 mg/kg, IP), DPDPE (15 μg/mouse; ICV), and deltorphin II (15 μg/mouse, ICV) produced antinociception in mice. Intraperitoneal or ICV injections of M3G did not produce any effect on the tail flick latency nor did it affect the antinociception-induced by morphine, U-50,488H, DPDPE, or deltorphin II. Previously M3G has been shown to antagonize the antinociceptive effects of morphine in the rat. It is concluded that in the mouse, M3G neither produces hyperalgesia nor modifies the actions of μ-, κ-, δ₁-, or δ₂-opioid receptor agonists.     

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.     

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.     

Up-regulation of Brain N-Methyl- -aspartate Receptors Following Multiple Intracerebro- ventricular Injections of [ -Pen, -Pen] Enkephalin and [ -Ala, Glu]deltorphin II in Mice

Bhargava, H. N., S. Kumar and J. T. Bian. Up-regulation of brain N-methyl- -aspartate receptors following multiple intracerebroventricular injections of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II in mice. Peptides 18(10) 1609–1613, 1997.—The effects of chronic administration of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II, the selective agonists of the δ₁- and δ₂-opioid receptors, on the binding of [³H]MK-801, a noncompetitive antagonist of the N-methyl- -aspartate receptor, were determined in several brain regions of the mouse. Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [ -Pen², -Pen⁵]enkephalin or [ -Ala², Glu⁴]deltorphin II (20 μg/mouse) twice a day for 4 days. Vehicle injected mice served as controls. Previously we have shown that the above treatment results in the development of tolerance to their analgesic activity. The binding of [³H]MK-801 was determined in brain regions (cortex, midbrain, pons and medulla, hippocampus, striatum, hypothalamus and amygdala). At 5 nM concentration, the binding of [³H]MK-801 was increased in cerebral cortex, hippocampus, and pons and medulla of [ -Pen², -Pen⁵]enkephalin treated mice. In [ -Ala², Glu⁴]deltorphin II treated mice, the binding of [³H]MK-801 was increased in cerebral cortex and hippocampus. The changes in the binding were due to increases in the B_max value of [³H]MK-801. It is concluded that tolerance to δ₁- and δ₂-opioid receptor agonists is associated with up-regulation of brain N-methyl- -aspartate receptors, however, some brain areas affected differ with the two treatments. The results are consistent with the recent observation from this laboratory that N-methyl- -aspartate receptors antagonists block tolerance to the analgesic action of δ₁- and δ₂-opioid receptor agonists.     

Radioligands for Probing Opioid Receptors

Abstract

The three endogenous opioid precursors of almost 30000 Da are pro-opiocortin, proenkephalin and prodynorphin. Pro-opiocortin contains β-endorphin, melanotropins and ACTH. Proenkephalin yields one [Leu⁵] enkephalin, three [Met⁵] enkephalins, one [Met⁵] enkephalyl-Arg-Arg-Val-NH₂ (metorphamide or adrenorphin), one [Met⁵] enkephalyl-Arg-Gly-Leu and one [Met⁵] enkephalyl-Arg-Phe. [Leu⁵] enkephalin is common to all fragments of prodynorphin; its carboxyl extension by Arg-Lys leads to α- and β-neo-endorphin and its carboxyl extension by Arg-Arg gives two dynorphins A and B of 17 and 13 amino acids, respectively. Another endogenous peptide is dynorphin A (1-8). The three main opioid binding sites are μ, δ and ?. Their analysis has been facilitated by the synthesis of analogues of peptides and non-peptide compounds, which have selective agonist or antagonist action at only one site. The various physiological roles of the three types of the opiate receptor have so far not been sufficiently investigated.     

Single residue modifications of the delta opioid receptor selective peptide, [D-Pen2,D-Pen5]-enkephalin (DPDPE). Correlation of pharmacological effects with structural and conformational features

Six analogs of the highly delta opioid receptor selective, conformationally restricted, cyclic peptide [D-Pen2,D-Pen5]enkephalin, Tyr-D-Pen-Gly-Phe-D-PenOH (DPDPE), were synthesized and evaluated for opioid activity in rat brain receptor binding and mouse vas deferens (MVD) smooth muscle assays. All analogs were single amino acid modifications of DPDPE and employed amino acid substitutions of known effects in linear enkephalin analogs. The effect on binding affinity and MVD potency of each modification within the DPDPE structural framework was consistent with the previous reports on similarly substituted linear analogs. Conformational features of four of the modified DPDPE analogs were examined by 1H NMR spectroscopy and compared with DPDPE. From these studies it was concluded that the observed pharmacological differences with DPDPE displayed by diallyltyrosine1-DPDPE ([DAT1]DPDPE) and phenylglycine4-DPDPE ([Pgl4]DPDPE) are due to structural and/or conformational differences localized near the substituted amino acid. The observed enhanced mu receptor binding affinity of the carboxamide terminal DPDPE-NH2 appears to be founded solely upon electronic differences, the NMR data suggesting indistinguishable conformations. The observation that the alpha-aminoisobutyric acid substituted analog [Aib3]DPDPE displays similar in vitro opioid behavior as DPDPE while apparently assuming a significantly different solution conformation suggests that further detailed conformational analysis of this analog will aid the elucidation of the key structural and conformational features required for action at the delta opioid receptor.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.     

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-Opioids Stimulate Inositol 1,4,5-Trisphosphate Formation, and so Mobilize Ca2+ from Intracellular Stores, in Undifferentiated NG108-15 Cells

Abstract: δ-Opioids mobilize Ca²⁺ from intracellular stores in undifferentiated NG108-15 cells, but the mechanism involved remains unclear. Therefore, we examined the effect of [d -Pen^2,5]enkephalin on inositol 1,4,5-trisphosphate formation in these cells. [d -Pen^2,5]enkephalin caused a dose-dependent (EC₅₀ = 3.1 nM) increase in inositol 1,4,5-trisphosphate formation (measured using a specific radioreceptor mass assay), which peaked (25.7 ± 1.2 pmol/mg of protein with 1 µM, n = 9) at 30 s and returned to basal levels (10.6 ± 0.9 pmol/mg of protein, n = 9) within 4–5 min. This response was fully naloxone (1 µM) reversible and pertussis toxin (100 ng/ml for 24 h) sensitive. Preincubation with Ni²⁺ (2.5 mM) or nifedipine (1 µM) had no effect on the [d -Pen^2,5]enkephalin (1 µM)-induced inositol 1,4,5-trisphosphate response, and K⁺ (80 mM) was unable to stimulate inositol 1,4,5-trisphosphate formation, indicating Ca²⁺ influx-induced activation of phospholipase C is not involved. Preincubation with the protein kinase C inhibitor Ro 31-8220 (1 µM) enhanced, whereas acute exposure to phorbol 12,13-dibutyrate (1 µM) abolished, the [d -Pen^2,5]enkephalin (0.1 µM)-induced inositol 1,4,5-trisphosphate response, suggesting protein kinase C exerts an autoinhibitory feedback action. [d -Pen^2,5]Enkephalin also dose-dependently (EC₅₀ = 2.8 nM) increased the intracellular [Ca²⁺], which was maximal (24 nM increase with 1 µM, n = 5) at 30 s. This close temporal and dose-response relationship strongly suggests that δ-opioid receptor-mediated increases in intracellular [Ca²⁺] results from inositol 1,4,5-trisphosphate-induced Ca²⁺ release from intracellular stores, in undifferentiated NG108-15 cells.     

Influence of peptidase inhibitors on the apparent agonist potency of delta selective opioid peptides in vitro.

Several peptides of diverse structure, reported to possess high affinity and selectivity for the delta opioid receptor, were studied using the mouse isolated vas deferens preparation to determine the effect of peptidase inhibition on their apparent potency. The peptides evaluated included [Leu5] enkephalin, the cyclic enkephalin analogs [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Pen2,p-F-Phe4,D-Pen5]enkephalin (F-DPDPE), the linear enkephalin analogs [D-Ala2,D-Leu5]enkephalin (DADLE) and [D-Ser2(O-tBu), Leu5,Thr6]enkephalin (DSTBULET), and the naturally occurring amphibian peptides Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (dermenkephalin), Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (deltorphin I) and Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 (deltorphin II). Concentration-response curves were determined for each peptide in the absence and presence of a combination of the peptidase-inhibiting agents bacitracin, bestatin, and captopril. A wide range of potencies was observed, both in the control state and in the presence of peptidase inhibition. The synthetic enkephalin analogs demonstrated small increases in potency with peptidase inhibition (no increase in the case of DPDPE), whereas the naturally occurring peptides were markedly increased in potency, up to as much as 123-fold for dermenkephalin. In the presence of peptidase inhibition, deltorphin II was the most potent peptide tested (IC50 = 1.13 x 10(-10) molar), and as such is the most potent delta opioid agonist reported to date. Stability to metabolism must be considered in the design and evaluation of in vitro experiments using peptides of this type.     

Interaction of opioid peptides and other drugs with multiple δncx binding sites in rat brain: Further evidence for heterogeneity

Recent pharmacological data strongly support the hypothesis of δ receptor subtypes as mediators of both supraspinal and spinal antinociception (δ₁ and δ₂ receptors). In vitro ligand binding data, which are fully supportive of the in vivo data, are still lacking. A previous study indicated that [³H][ -Ala², -Leu⁵]enkephalin labels two binding sites in membranes depleted of μ binding sites by pretreatment with the site-directed acylating agent, 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimidazole-HCI (BIT). The main goal of the present study was to develop a ligand-selectivity profile of the two δ_ncx binding sites. The data indicated that naltrindole and oxymorphindole were relatively selective for site 1 (20-fold). [ -Ser²,Thr⁶]Enkephalin and deltorphin-II were only 2.7-fold and 2.2-fold selective for site 1. [ -Pen², -Pen⁵]Enkephalin and deltorphin-I were 80-fold and 38-fold selective for site 2.3-Iodo-Tyr- -Ala-Gly-Phe- -Leu was 52-fold selective for site 1. Morphine had moderate affinity for site 1 (K_i = 16 nM), and was about 11-fold selective for site 1. Thus, of the 10 drugs studied, only DPDPE and DELT-I were selective for site 2. Viewed collectively with other data, it is likely that the δ₁ receptor and the δ_ncx binding site are synonymous.     

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.     

Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice

Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.     

Sensitive analysis of [ -Pen]enkephalin in rat serum by capillary electrophoresis and laser-induced fluorescence detection

A highly sensitive analytical method based on capillary zone electrophoresis (CZE) coupled with a laser-induced fluorescence (LIF) detector was explored for the analysis of [ -Pen^2,5]enkephalin (DPDPE) in rat serum. DPDPE and the internal standard Phe-Leu-Glu-Glu-Ile (P9396) were extracted from serum samples with C₁₈ solid-phase extraction disk cartridges, followed by derivatization with tetramethylrhodamine-5-isothiocyanate (TRITC) isomer G before introduction onto the capillary column. Complete resolution of DPDPE and the internal standard from other serum components was achieved within 20 min on a 140 cm×50 μm I.D. capillary column with borate buffer (25 mM, pH 8.3). With the current method, it is possible to detect 1.3E-18 mol of DPDPE on column. The results suggest that CZE-LIF is a promising method for the sensitive and specific quantitation of therapeutic peptides in biological matrices.     

Influence of the Hypothalamic Arcuate Nucleus on Intraocular Pressure and the Role of Opioid Peptides

Background

An opioid peptide neuron/humoral feedback regulation might be involved in changes of intraocular pressure (IOP). The aims of this study are to investigate the effects of arcuate nucleus (ARC) and opioid peptides on intraocular pressure (IOP).

Methods

Fifty-four healthy purebred New Zealand white rabbits (108eyes) were randomly divided into 4 groups, including control group, electrical stimulation group, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) group, and [D-Pen 2, D-Pen5]- enkephalin (DPDPE) group. Bilateral IOP was measured after unilateral electrical stimulation of the ARC or unilateral microinjection into the ARC of the selective μ-opioid receptor agonist DAMGO or the selective δ opioid receptor agonist DPDPE, both alone and after pre-administration of either the non-selective opioid receptor antagonist naloxone or saline.

Results

Both electrical stimulation in ARC and micro-injection either <mu> or <delta> opioid receptor agonists, DAMGO or DPDPE, respectively, caused a significant bilateral reduction in IOP (P<0.05) which was more pronounced in the ipsilateral than in the contralateral eye. Pretreatment with naloxone prevented some, but not all IOP reductions.

Conclusion

The ARC takes part in the negative regulation of IOP, an action that may involve opioid neurons.     

Leucine enkephalin-tyrosinase reaction products — Identification and biological activity

Leucine enkephalin (1 mM) was reacted with mushroom tyrosinase under reductive conditions (ascorbic acid, 50 mM). Reaction products were isolated by high-performance liquid chromatography and identified using electrospray ionization mass spectrometry. The products of the reaction were found to be hydroxylated at the Tyr¹ moiety of the peptide. The major product was a monohydroxylated derivative of leucine enkephalin ([HO-Tyr¹]LE) and the minor product of the reaction was a dihydroxylated derivative ([(HO)₂-Tyr¹]LE). The affinity of [HO-Tyr¹]LE to receptors in rat brain homogenate was compared to that of leucine enkephalin itself. Hydroxylation of LE was found to decrease receptor affinity to both μ and δ opioid receptor sites by a factor of about 20.     

Cyclic enkephalin analogs containing a cystine bridge

Two conformationally constrained enkephalin analogs were synthesized by substitution of cysteines in positions 2 and 5 and oxidative disulfide bond formation. In the guinea pig ileum assay the obtained cyclic analogs, [D-Cys²-L-Cys⁵]enkephalinamide and [D-Cys²-D-Cys⁵]enkephalinamide, showed potency ratios of 37.9 ± 0.8 and 73.3 ± 0.9, respectively, relative to [Met⁵]enkephalin. The extremely high potency of the analogs was shown to be a consequence of the conformational restrictions introduced by cyclization. Rat brain membrane binding studies with [³H]naloxone and [³H](D-Ala², D-Leu⁵)enkephalin as radiolabels revealed a moderate preference of both analogs for μ-receptors over δ-receptors. Furthermore, the cystine-containing analogs were shown to be highly resistant to enzymatic degradation.