Enhanced analgesic activity of D-Ala2 enkephalinamides following D-isomer substitutions at position five

The analgesic potency of D-Ala², Met⁵- and D-Ala², Leu⁵-enkephalinamide was increased following substitution of the D-isomers at position five. The substituted analogs were 2.5 times as potent as morphine in producing analgesia in rats when administered intraventricularly and 0.2 as potent as morphine when administered intravenously. D-Ala², D-Leu⁵-enkephalinamide produced naloxone-reversible analgesia by the intravenous route in monkeys. The enhanced systemic activity following D-isomer substitutions at position five appeared to be due to some factor other than increased stability to peptidase degradation at the C-terminus.     

In vitro cyclic AMP-mediated lipolytic activity of endorphins, enkephalins and naloxone

The maximum lipolytic activity (L_max) of β-endorphin is two and one half times that of Leu⁵-enkephalin and twice that of Met⁵-enkephalin, D-Ala², Met⁵-enkephalinamide, α-endorphin and γ-endorphin in the rabbit adipocyte. D-Met², Pro⁵-enkephalin-amide, however, has an L_max 1.6 times greater than that of Met⁵-enkephalin. The potencies (A₅₀) of Met⁵-enkephalin and its analogs and that of Leu⁵-enkephalin lie between 1.4 and 3 μM. The A₅₀ values for α-endorphin, β-endorphin and γ-endorphin are significantly less (1.2 × 10^?1 μM). Naloxone acts as an agonist in this system (A₅₀ = 2.5 μM; L_max 1.4 × Met⁵-enkephalin). All of the peptides and naloxone stimulated adenylate cyclase activity.     

A quantitative analysis of the shaking behavior induced in rats by β-endorphin and [D-Ala2, Met5] enkephalinamide

β-Endorphin (5–80 μg) or [D-Ala², Met⁵] enkephalinamide (DALA) (5–40 μg) was administered intracerebroventricularly to rats. With both opioid peptides, there was no direct relationship between log dose and mean number of wet-dog shakes (WDS) that occured during the following 15 min. When the results were analyzed quantally, the dose of DALA that caused 50% of the rats to shake at least twice was 8.6 μg (4.9–15 μg). β-Endorphin had such poor efficacy that an ED 50 could not be obtained. Morphine (1 and 5 mg/kg, s.c.) antagonized shaking caused by the optimal dose of DALA (20 μg). Naloxone (0.1–10 mg/kg, s.c.) attenuated both DALA- and β-endorphin-induced WDS in a dose-related manner. This latter result differentiates shaking associated with opioid peptides from that caused by thyrotropin releasing hormone (TRH), another endogenous stimulant of WDS in rats. There was no cross-tolerance between RX 336-M (7,8-dihydro-5′,6′-dimethylylohex-5′-eno-1′,2′,8′,14 codeinone), a novel shake inducing agent, and β-endorphin. This finding again differentiates β-endorphin-induced shaking from that caused by TRH and also from that associated with several exogenous stimulants of WDS.     

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.     

A comparison of the analgesic and behavioral effects of [D-Ala2] Met-enkepha-linamide and morphine in the mesencephalic reticular formation of rats.

[D-Ala²]Met-enkephalinamide (DALA) injected intracerebrally (IC) at low doses into specific sites of the mesencephalic reticular formation (MRF), produced a profound, long-lasting analgesia that was blocked by naloxone, a specific opiate antagonist. Morphine was only half as potent as DALA because morphine, injected IC at similar sites in the MRF, yielded a comparable analgesia only when injected at twice the dose. The analgesic effects of morphine were also antagonized by naloxene. Both DALA and morphine produced specific behavioral effects. Naloxone blocked the behavioral effects of DALA, but not those produced by morphine.     

Enkephalin analogues and naloxone modulate the release of growth hormone and prolactin - evidence for regulation by an endogenous opioid peptide in brain

Met⁵-enkephalin amide, D-Ala²-Met⁵-enkephalin amide, D-Ala²-Leu⁵-enkephalin amide, morphine sulfate and naloxone hydrochloride were examined for their effects on growth hormone and prolactin release $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. Subcutaneous injection of D-Ala²-Met⁵ enkephalin amide^a, D-Ala²-Leu⁵ enkephalin amide^b and morphine sulfate, but not Met⁵-enkephalin and amide^c, resulted in significant elevations in the serum growth hormone and prolactin of immature female rats. Naloxone blocked the hormone-stimulatory effect of the opioid receptor agonists and when administered alone significantly reduced serum growth hormone and prolactin concentrations. None of the drugs demonstrated a direct action on anterior pituitary tissue growth hormone or prolactin release $\text{in}$$\text{vitro}$.     

Differential effects of GTP and cations on binding of labeled dimeric and monomeric enkephalins to neuroblastoma-glioma cell delta opiate receptors

Binding of radio-labeled enkephalin monomers [D-Ala²,Met⁵]Enkephalin Amide (DAMEA) and [D-Ala²,D-Leu⁵]Enkephalin (DADLE) and a dimer of [D-Ala²,Leu] Enkephalin Amide (DPE₂) to neuroblastoma-glioma (NG108-15) cells was examined in the presence and absence of GTP and/or cations. We found that: (1) binding occurs to a single class of homogeneous and non-interacting membane sites; (2) the affinity of the enkephalin dimer is reduced 50% in the presence of Mn²⁺ and 65% in the presence of both Mn²⁺ and GTP; (3) GTP alone either increases or does not change affinity of DPE₂; (4) Na⁺ and GTP significantly decrease the affinities of monomers, but not that of the dimer; and (5) a higher concentration (0.1 mM) of GTP increases the binding of DPE₂ but significantly decreases binding of monomers. Conclusion: Changes in binding of a dimeric enkephalin by Na⁺, Mn²⁺ and GTP are significantly and qualitatively different than those occurring for monomers.     

Comparison of the behavioral effects of β-endorphin and enkephalin analogs

The behavioral effects of β-endorphin, enkephalin analogs, morphine and etorphine were briefly compared. In the tail-flick test in mice and in the wet shake test in rats, β-endorphin and D-Ala²-D-Leu⁵-enkephalin had equal antinociceptive activity; D-Ala² -Met-enkephalinamide and D-Leu⁵-enkephalin were less active. The order of activity of the enkephalin analogs and opiate alkaloids for stimulating locomotor activity in mice paralleled their analgesic activities; β-endorphin, however, had only minimal stimulatory actions. Morphine sulfate, 50 μg injected into the periaqueductal gray, produced hyperactivity but this effect was not observed with etorphine or opioid peptides. By contrast, “wet dog” shakes was observed with the opioid peptides but not with either opiate alkaloid. These heterogenous behavioral responses, which were all antagonized by naloxone, indicate that multiple types of receptors mediate the effects of opiates in the central nervous system.     

Enkephalin dimers: Regulation of cyclic AMP levels in NG108-15 cells

Intracellular cyclic AMP levels were determined for dimeric and monomeric enkephalins interacting with PGE₁-stimulated NG108-15 cells. The dimeric pentapeptide enkephalin (DPE₂), [D-Ala², Leu⁵ -NH-CH₂]₂, displaying very high affinity (K = 4.2 ± 0.3 nM^?1) for the δ-opiate receptor, inhibited cyclic AMP production by 70%. Its IC₅₀-value was between 0.1 and 0.2 nM, similar to that of the potent δ-agonist [D-Ala², D-Leu⁵] enkephalin (DADLE) with K = 1.0 ± 0.1 nM^?1. [D-Ala², Leu⁵] enkephalin amide (DALEA), which is the monomer of DPE₂, showed an IC₅₀ = 4 nM. The dimeric tetrapeptide enkephalin (DTE₁₂), [D-Ala², des-Leu⁵-NH-(CH₂)₆]₂ and its monomer [D-Ala², desLeu⁵] enkephalin amide (DAPEA) showed IC₅₀ = 2 and 20 nM, respectively. These results indicate that the DPE₂ and DTE₁₂ enkephalin dimers are potent δ-agonists.     

The Met-enkephalin analog D-Ala2-Met-enkephalinamide decreases the adrenocortical response to ACTH in dispersed rat adrenal cells

The effects of the Met-enkephalin analog D-Ala²-Met-enkephalinamide (DALA) on basal and ACTH-stimulated corticosterone secretion from dispersed adrenal cells were investigated. Low doses (10^?10 and 10^?12 M) of DALA resulted in no apparent alteration in the response to ACTH (8×10^?9, 3.2×10^?8 or 1.6×10^?7 M). High doses of DALA (10^?8 and 10^?6 M) produced a decline in the steroidogenic response to ACTH. The opiate receptor antagonist naloxone (10^?4–10^?10 M) did not influence the basal production of corticosterone or the stimulating action exerted by ACTH. However, the presence of naloxone reversed the blocking action on corticosterone production that was exerted by DALA. These findings indicate that enkephalins may decrease adrenocortical responsiveness to ACTH.     

Some analogs of luteinizing hormone releasing hormone (LH-RH) having intense ovulation-inducing activity

Five new analogs of luteinizing hormone releasing hormone (LH-RH), des-Gly¹⁰-[Ala⁶]-LH-RH-ethylamide, des-Gly¹⁰-[D-Ala⁶]-LH-RH-ethylamide, des-Gly¹⁰-[α-aminoisobutyric acid⁶]-LH-RH-ethylamide, des-Gly¹⁰-[Phe⁵, D-Ala⁶]-LH-RH-ethylamide and des-Gly¹⁰-[Ile⁵, D-Ala⁶]-LH-RH-ethylamide were synthesized and evaluated for the ovulation-inducing activity in the rat, and it was found that the analogs, des-Gly¹⁰-[D-Ala⁶]-LH-RH-ethylamide and des-Gly¹⁰-[Phe⁵, D-Ala⁶]-LH-RH-ethylamide, were 50 times or more active than the original molecule.     

Naloxone blocks the effect of 5-hydroxy-tryptophan on passive avoidance learning in rats: Implication of endogenous opioid peptides

The synthetic opioid peptide D-Ala²-met-enkephalinamide (DALA) impairs retention performance of rats on a one-trial passive avoidance task. The same effect is observed after administration of 5-hydroxy-tryptophan (5-HTP). Both drugs appear to impair learning by disrupting memory consolidation since similar effects are observed when they are given either peripherally before the acquisition test or intracerebroventricularly immediately after. A moderate dose of naloxone prevents the amnesia produced by DALA or 5-HTP. However, the opiate antagonist does not block the behavioral syndrome induced by clorgyline and 5-HTP. Moreover, bacitracin, a peptidase inhibitor, potentiates the effect of 5-HTP on the passive avoidance task. The results suggest that endogenous opioid peptides are involved in the amnesic effect of 5-HTP.     

Identification of Mammalian Brain and Spinal Cord Opioid Receptor by Photoaffinity Labeling

Abstract

A radioiodinated photoreactive enkephal in derivative, ¹²⁵I(D-Ala² p-N₃-Phe⁴-Met⁵) enkephalin, was used to photoaffinity label the opioid receptor from the membranes of four mammalian brains (without cerebellum) and spinal cords. These included the cat, rabbit, guinea pig and mouse. The photolabeled membranes were analyzed by sodium dodecyl sulfate gel electrophoresis. A 43,000-daltons protein was specifically photolabeled in all the membranes tested, as the specific labeling of this protein was inhibited in the presence of 14.5 uM of (D-Ala² Met⁵) enkephalin. These data suggest that the 43,000-daltons protein is a binding protein of the opioid receptor in the different mammalian neural tissues.     

Inhibition of copulatory behavior in male rats by D-ALA2-Met-Enkephalinamide

D-Ala²-Met-Enkephalinamide (DALA), a synthetic analog of met-enkephaline resistant to enzymatic degradation, was injected intraventricularly to sexually experienced male rats paired with receptive females. A dose of DALA of 6 μg which did not influence spontaneous motor activity, completely suppressed the copulatory behavior of all animals tested. A dose of 3 μg significantly increase mounting and intromission latencies, but did not influence other measures of the copulatory behavior. The effect of DALA was prevented by naloxone (1 mg/Kg), a specific inhibitor of opioid receptors. The results suggest that enkephalins may play a role in the regulation of copulatory behavior.     

Decrease of cyclic GMP in cerebellum by intrastriatal D-Ala2-met-enkephalinamide.

The bilateral intrastriatal injection of D-Ala²-Met-Enkephalinamide (DALA) at doses of ranging from 12 to 50 μg decreased cyclic-GMP content in the cerebellum and produced catalepsy. These effects were prevented by naltrexone, an opiate receptor antagonist but not by apomorphine, a dopamine agonist. The bilateral injection of DALA in the cerebellum, and substantia nigra neither decreased cerebellar cyclic GMP content nor produced catalepsy. The bilateral injection of DALA (20 ug) into the ventromedial thalamic nuclei caused marked catalepsy but failed to decrease cerebellar cyclic GMP. The results suggest that the effect of DALA on cGMP can be differentiated from the cataleptic response and that it is mediated by an action on opioid receptors located in the striatum, beyond DA receptors.     

Conformational energy studies on N-methylated analogs of thyrotropin releasing hormone, enkephalin, and luteinizing hormone-releasing hormone

Empirical conformational energy calculations have been carried out for N-methyl derivatives of alanine and phenylalanine dipeptide models and N-methyl-substituted active analogs of three biologically active peptides, namely thyrotropin-releasing hormone (TRH), enkephalin (ENK), and luteinizing hormone-releasing hormone (LHRH). The isoenergetic contour maps and the local dipeptide minima obtained, when the peptide bond (ω) preceding the N-methylated residue is in the trans configuration show that (1) N-methylation constricts the conformational freedom of both the ith and (i + 1)th residues; (2), the lowest energy position for both residues occurs around ? = ?135° ± 5° and ψ = 75° ± 5°, and (3) the α_L conformational state is the second lowest energy state for the (i + 1)th residue, whereas for the ith residue the C₅ (extended) conformation is second lowest in energy. When the peptide bond (ω_i) is in the cis configuration the ith residue is energetically forbidden in the range ? = 0° to 180° and ψ = ?180° to +180°. Conformations of low energy for ω_i = 0° are found to be similar to those obtained for the trans peptide bond. In all the model systems (irrespective of cis or trans), the α_R conformational state is energetically very high. Significant deviations from planarity are found for the peptide bond when the amide hydrogen is replaced by a methyl group. Two low-energy conformers are found for [(N-Me)His²]TRH. These conformers differ only in the ? and ψ values at the (N-Me)His² residue. Among the different low-energy conformers found for each of the ENK analogs [D -Ala²,(N-Me)Phe⁴, Met⁵]ENK amide and [D -Ala²,(N-Me)Met⁵]ENK amide, one low-energy conformer was found to be common for both analogs with respect to the side-chain orientations. The stability of the low-energy structures is discussed in the light of the activity of other analogs. Two low-energy conformers were found for [(N-Me)Leu⁷]LHRH. These conformations differ in the types of bend around the positions 6 and 7 of LHRH. One bend type is eliminated when the active analog [D -Ala⁶,(M-Me)Leu⁷]LHRH is considered.     

Analgesic activity of intracerebroventricular administration of morphiceptin and β-casomorphins: Correlation with the morphine (μ) receptor binding affinity

Analgesic activities of morphiceptin, β-casomorphins, [D-Ala², D-Leu⁵] enkephalin and Sandoz peptide, FK 33–824, were examined by intracerebroventricular administration in rats. Their relative potencies $\text{in vivo}$ were compared with their receptor binding activities. The receptor binding affinities were determined from the competition curves against [³H]naloxone binding in the absence and presence of sodium ions for morphine (μ) receptors and against ¹²⁵I-[D-Ala², D-Leu⁵] enkephalin binding for enkephalin (δ) receptors. A good correlation between analgesic activity and morphine (μ) receptor but not enkephin (δ) receptor binding affinity was obtained. These data extend the hypothesis that morphine (δ) receptors mediate the major portion of the analgesic activity of opioids.     

Electrophysiological effects of enkephalin analogs in rat cortex.

The microiontophoretic potencies of methionine-enkephalin (ME) analogs with similar charges but different potencies in the $\text{in vitro}$ assays were studied with respect to spontaneous and evoked activity of rat cerebral cortex neurons (CX). ME slowed discharge in 65% of the 62 CX tested; activity evoked by acetylcholine (ACh) or glutamate, as well as spontaneous discharge, was affected. The vast majority of responsive cells were deeper than 500 μ and excited by ACh. Antidromic spikes were elicited in many of these neurons by pyramidal tract stimulation. The electrophysiological effects of D-Ala² ME amide, a more potent analog $\text{in vitro}$, was similar to ME when tested on the same parietal CX, however, D-Ala² ME amide was much more potent than ME on frontal CX. On the other hand N-acetyl D-Ala²-Lys³ ME amide, which has low potency $\text{in vitro}$, had little effect on CX which were readily depressed by equivalent doses of ME or the D-Ala² analog. The depressions induced by ME or the D-Ala² derivative were readily and reversibly antagonized by prior iontophoretic administration of naloxone. It is concluded that opiate receptor interactions may play an important role in electrophysiological responses to iontophoretically administered ME and that the deeper cholineceptive cortical cells, possibly pyramidal neurons, may be preferentially affected.     

Radioimmunoassay of methionine5-enkephalin sulphoxide: Phylogenetic and anatomical distribution

A sensitive and specific radioimmunoassay (RIA) for the oxidised form of methionine⁵-enkephalin (Met⁵-Enk), Met⁵-Enk sulphoxide (Met⁵-Enk-S), has been developed. Antisera were raised in rabbits against Met⁵-Enk coupled to carrier proteins with glutaraldehyde or carbodiimide. Displacement of (¹²⁵I) Met⁵-Enk bound to antiserum by Met⁵-Enk was poor, but Met⁵-Enk-S displayed good displacement suggesting that the Met⁵-Enk immunogen was oxidised to Met⁵-Enk-S and that the antisera were formed against this compound. The sensitivity of the RIA for Met⁵-Enk-S was 0.02 pmole/tube using the most sensitive antiserum. The antisera showed negligible cross-reactivity with leucine⁵-enkephalin and with both native and oxidised endorphins. Cross-reactivity was between 15% and 28% with the fragment Met⁵-Enk (2–5) sulphoxide and between 9% and 25% with D-Ala²-Met⁵-Enk sulphoxide. The antisera showed<0.01% cross-reactivity with other Met⁵-Enk fragments and naturally occurring neuropeptides. Tissue extracts were oxidised with hydrogen peroxide prior to assay. Met⁵-Enk-S immunoreactivity (IMR) was detected in brain, pituitary gland, pancreas, and intestine extracts of the rat, chicken, toad and teleost, and in cerebral-suboesophageal ganglion extracts of the snail. All tissue extracts showed parallelism in serial dilution to synthetic mammalian Met⁵-Enk-S, suggesting possible immunological identity. The results indicate that spontaneous oxidation of Met⁵-Enk immunogen occurs such that antisera are produced against the sulphoxide analogue of Met⁵-Enk, and may account for the relative insensitivity of some published RIAs using Met⁵-Enk standard. Our findings demonstrate a wide phylogenetic and anatomical distribution of Met⁵-Enk IMR.     

Radioimmunoassay of methionine-enkephalin sulphoxide: Phylogenetic and anatomical distribution

A sensitive and specific radioimmunoassay (RIA) for the oxidised form of methionine⁵-enkephalin (Met⁵-Enk), Met⁵-Enk sulphoxide (Met⁵-Enk-S), has been developed. Antisera were raised in rabbits against Met⁵-Enk coupled to carrier proteins with glutaraldehyde or carbodiimide. Displacement of (¹²⁵I) Met⁵-Enk bound to antiserum by Met⁵-Enk was poor, but Met⁵-Enk-S displayed good displacement suggesting that the Met⁵-Enk immunogen was oxidised to Met⁵-Enk-S and that the antisera were formed against this compound. The sensitivity of the RIA for Met⁵-Enk-S was 0.02 pmole/tube using the most sensitive antiserum. The antisera showed negligible cross-reactivity with leucine⁵-enkephalin and with both native and oxidised endorphins. Cross-reactivity was between 15% and 28% with the fragment Met⁵-Enk (2–5) sulphoxide and between 9% and 25% with D-Ala²-Met⁵-Enk sulphoxide. The antisera showed<0.01% cross-reactivity with other Met⁵-Enk fragments and naturally occurring neuropeptides. Tissue extracts were oxidised with hydrogen peroxide prior to assay. Met⁵-Enk-S immunoreactivity (IMR) was detected in brain, pituitary gland, pancreas, and intestine extracts of the rat, chicken, toad and teleost, and in cerebral-suboesophageal ganglion extracts of the snail. All tissue extracts showed parallelism in serial dilution to synthetic mammalian Met⁵-Enk-S, suggesting possible immunological identity. The results indicate that spontaneous oxidation of Met⁵-Enk immunogen occurs such that antisera are produced against the sulphoxide analogue of Met⁵-Enk, and may account for the relative insensitivity of some published RIAs using Met⁵-Enk standard. Our findings demonstrate a wide phylogenetic and anatomical distribution of Met⁵-Enk IMR.