D-Ala2,D-Leu5]enkephalin Research Articles

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

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

Novel Inhibitors of Enkephalin-Degrading Enzymes I: Inhibitors of Enkephalinase by Penicillins

Several penicillins have been found to have pro-antinociceptive properties and also to be enkephalinase (neutral endopeptidase-24.11) inhibitors, carfecillin being the most potent. Carfecillin i.c.v. (but not i.p.) had significant antinociceptive activity in the mouse tail immersion test and completely suppressed abdominal constrictions (acetic acid) in mice (IC50 = 23 micrograms/animal). In combination with (D-Ala2-D-leu5)-enkephalin (DADL) i.c.v. in the abdominal constriction test the complete protection observed was reversed by the opioid receptor antagonist naltrexone. Carfecillin was a competitive inhibitor of enkephalinase from mouse brain striata (IC50 = 207 + 57 nM, cf thiorphan 10.6 +/- 1.9 nM) but did not inhibit other known enkephalin- degrading enzymes. Carfecillin provides a new lead structure for the development of more potent enkephalinase inhibitors.

Quantitative autoradiographic distribution of meptazinol-sensitive binding sites in rat brain.

1. Meptazinol is an interesting opioid-producing naloxone-reversible analgesia with few cardiovascular and respiratory effects. Recent studies indicate that mu 1 opioid receptors mediate meptazinol analgesia. Using a computerized autoradiographic subtraction technique, we have examined the regional distribution of meptazinol-sensitive [3H][D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) binding and compared this with the distribution of mu 1 binding determined by competition with low [D-Ala2,D-Leu5]enkephalin (DADL) concentrations. 2. Meptazinol and DADL lowered [3H]DAGO to similar extents in most brain regions studied. The greatest levels of inhibition were observed in the periaqueductal gray, interpeduncular nucleus, thalamus, hypothalamus, and hippocampus. Low levels of inhibition were found in the temporal and frontal cortex. The correlation between the inhibition of [3H]DAGO binding by meptazinol and that by DADL was high (r = 0.83), consistent with the binding of meptazinol to mu 1 sites.

Decrease in δ-opioid receptor density in rat brain after chronic [ d-Ala 2, d-Leu 5]enkephalin treatment

Chronic treatment of Sprague-Dawley rats with [ d-Ala 2, d-Leu 5]enkephalin (DADLE) resulted in the development of tolerance to the antinociceptive effect of this opioid peptide. When opioid receptor binding was measured, time-dependent decreases in [ 3H]diprenorphine binding to the P 2 membranes prepared from the cortex, midbrain and striatum were observed. Scatchard analysis of the saturation binding data revealed a decrease in B max values and no change in the K d values of [ 3H]diprenorphine binding to these brain regions, indicative of down-regulation of the receptor. This reduction in the opioid receptor binding activities could be demonstrated to be due to the DADLE effect on the δ-opioid receptors in these brain regions. When [ 3H]DADLE binding was carried out in the presence of morphiceptin, a significant reduction in the δ-opioid receptor binding was observed in all brain areas tested. μ-Opioid receptor binding decrease was observed only in the striatum after 5 days of DADLE treatment. Additionally, the onset of δ-opioid receptor decrease in the midbrain area was rapid, within 6 h of the initiation of the chronic DADLE treatment. Thus, analogous to previous observations in which chronic etorphine treatment preferentially reduced μ-opioid receptor binding, chronic DADLE treatment preferentially reduced δ-opioid receptor binding activity.

The μ opiate receptor is responsible for descending pain inhibition originating in the periaqueductal gray region of the rat brain

Opiate agonists exhibiting selectivity for μ, κ, σ and δ opiate receptors were microinjected into the periaqueductal gray region (PAG) of the brain of rats to determine the receptor subtype(s) associated with the initiation of descending pain inhibition. The spinally organized, heat nociceptive tail-flick reflex was used to detect analgesia. Only morphine (μ) and [D-Ala 2, D-Leu 5]enkephalin (DADLE) ( δ> μ) produced analgesia. However, both drugs appeared to be acting through the μ (morphine) receptor, since: (1) the action of DADLE was not inhibited by δ receptor antagonists, (2) a more highly selective δ agonist [D-Pen 2, D-Pen 5]enkephalin was ineffective and (3) agonists selective at other non-μ receptor sites (ethylketocyclazocine and U50,488H for κ; n-allylnormetazocine for σ) were also ineffective. It appeared that DADLE might be acting as a partial agonist at the morphine receptor in the PAG. The peptide was an agonist with low efficacy, and when a maximally effective dose of the peptide was administered simultaneously with morphine antagonism was observed. Ethylketocyclazocine and n-allynormetazocine were also found to antagonize morphine, an observation that is consistent with the suggestion that they may act as μ receptor antagonists in addition to their agonistic action at κ and σ receptors, respectively. Thus, μ receptors appear to be responsible for the spinopetal analgesia from the PAG of the rat.

β-Endorphin-(1–27) antagonizes β-endorphin- but not morphine-, D-PEN 2- D-PEN 2-enkephalin- and U50, 488H-induced analgesia in mice

β-Endorphin-(1–27), administered intraventricularly has been previously reported to block the analgesia induced by β-endorphin injected intraventricularly. The present study was to determine if the blocking effect of β-endorphin-(1–27) was specific to β-endorphin which stimulates epsilon receptors, but not to other opioids with activity at different opioid receptors. The antagonistic effects of β-endorphin-(1–27) on the analgesia induced by β-endorphin (epsilon-opioid receptor agonist), d-Ala 2-NMePhe 4-Gly-ol-enkephalin (DAGO) and morphine, (mu-opioid receptor agonists), d-Pen 2- d-Pen 5-enkephalin (DPDPE) and d-Ala 2- d-Leu 5-enkephalin(DADLE) (delta-opioid receptor agonists) and U-50, 488H (kappa-opioid receptor agonist) were studied. β-Endorphin-(1–27) injected intraventricularly, at doses which, when injected alone did not produce analgesia, antagonized the analgesia induced by β-endorphin given intraventricularly. However, the analgesia induced by DAGO, morphine, DPDPE, DADLE and U-50,488H given intraventricularly was not antagonized by β-endorphin-(1–27). The data suggest that β-endorphin-(1–27) selectively blocks the analgesia induced by the stimulation of epsilon receptors but not by the stimulation of mu, delta, and kappa receptors. The results support the previously proposed hypothesis that β-endorphin produces its analgesia by stimulating specific epsilon receptors.

μ-, δ- and κ-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-Ala 2, D-Leu 5]enkephalin (DADLE). [D-Ala 2, MePhe 4,Gly-ol 5]enkephalin (DAGO), [D-Pen 2,D-Pen 5]enkephalin (DPDPE) (0.01–1 μM) and bremazocine (0.001–03 μM) 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]acethylcholine (ACh, striatum) and [ 3H]DA (striatum) release represent μ, δ and κ receptors, respectively. In agreement with this classification, preincubation (60 min) of the slices with the δ-opioid receptor-selective irreversibel ligand, fentanyl isothiocyanate (FIT, 0.01–1 μM), 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 μ as well as of δ receptors. In this case, the reversible μ antagonist, naloxone (0.1 μM), fully antagonized the inhibitory effect of the μ agonist, DAGO, without changing the effect of the δ agonist DPDPE but was ineffective as an antagonist in slices pretreated with FIT (1 μM). 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 μ-, δ- and κ-opioid receptors in rat brain but also evidence strongly that μ and δ 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.

Depressor and bradycardic effects induced by spinal subarachnoid injection of D-Ala 2-D-Leu 5-enkephalin in rats

The effects of D-Ala 2-D-Leu 5-enkephalin (DADLE), a specific delta receptor agonist, on spinal control of cardiovascular function, were investigated by its intrathecal (i.th) injection into the spinal subarachnoid space at the T-9 level. In chloralose-anesthetized rats, DADLE (17.5, 35 and 70 nmol, i.th) caused dose-dependent hypotension and bradycardia. The mean maximal hypotension by 70 nmol of DADLE was −45 ± 7 mmHg, with a bradycardia of −79 ± 15 beats/min. These inhibitory cardiovascular effects were antagonized by the opiate antagonist naloxone (50 nmol, i.th.) given prior to DADLE. Intrathecal injection of DADLE also decreased splanchnic sympathetic nerve discharge (−46 ± 5%). DADLE (70 nmol) given i.v. did not cause significant changes in mean arterial pressure (MAP) and heart rate (HR). Neither bilateral vagotomy nor pretreatment with atropine (0.2 mg/kg, i.v.) prevented the BP and HR effects of intrathecal injection on DADLE at a dose of 35 nmol. DADLE at this dose failed to produce significant alteration in the frequency of respiration and blood PaO 2, PaCO 2 and blood pH. In conscious rats, 140 nmol of DADLE (i.th.) did not produce any consistent changes in MAP and HR. These data suggest that intrathecal injection of DADLE inhibits central sympathetic activity, possibly at a spinal locus.

Opioids excite rather than inhibit sensory neurons after chronic opioid exposure of spinal cord-ganglion cultures

Tests were carried out to determine if the tolerance that develops in dorsal-horn network responses of mouse dorsal root ganglion (DRG)-spinal cord explants after chronic exposure to opioids could be accounted for by alterations in the excitability and pharmacologic properties of the afferent DRG cells. Intracellular recordings were made from DRG neurons in organotypic DRG-cord explants after chronic treatment with 1μM d-Ala 2- d-Leu 5-enkephalin (DADLEP) for>4days in vitro. Acute application of 10 μM DADLE shortened the duration of the Ca 2+ component of the somatic action potential (APD) in only 5% of the treated neurons (4 out of 79 cells), in contrast to about 50% of the cells in naive explants (36 out of 74). Thus many DRG neuron perikarya became tolerant to the APD-shortening effects of DADLE. Furthermore, 77% of the treated DRG cells (61 out of 79) showed prolongation of the APD in response to an acute increase in DADLE concentration vs 34% in naive explants (25 out of 74). However, when the DADLE responsivity tests were carried out in the presence of multiple K + channel blockers, only 20% of the treated DRG neurons showed APD prolongation (3 out of 15 cells), whereas 73% showed APD-shortening responses (11 out of 15 cells). The results suggest that: (1) DADLE-induced APD prolongation of the treated DRG neurons is mediated by opioid receptor subtypes that decrease a voltage-sensitive K + conductance; (2) the DADLE-induced APD-shortening effects which are unmasked during more complete K + channel blockade are mediated by opioid-receptor subtypes in the same neuron that reduce a voltage-sensitive Ca 2+ conductance (resembling κ receptors). DRG neurons did not become tolerant to either of these two opioid effects after chronic exposure to DADLE. Opioid shortening of the APD of DRG neuron perikarya has been generally accepted to be a model of opioid inhibition of calcium infkux and transmitter release at presynaptic DRG terminals 6,52,53,65,75,76. It is postulated that the opioid-induced APD prolongation observed in the present study provides evidence that opioids can also evoke direct excitatory eeffects on neurons. The enhancement of DADLE-induced excitatory responses and attenuation of DADLE-induced inhibitory responses of DGR neurons after chronic exposure to this opioid show striking similarities to the effects of forskolin or pertussis toxin treatment. These in vitro studies may provide clues to compensatory mechanisms underlying physiologic expression of tolerance to opioid analgesic effects in primary afferent synaptic networks.

PD117302: a selective agonist for the kappa-opioid receptor.

1. A new nonpeptide kappa-opioid compound, a cyclohexyl benzeneacetamide derivative (PD117302), has been synthesized and its affinity for the different types of opioid receptor determined. The ability of PD117302 to modify the activity of the electrically-stimulated guinea-pig ileum and rabbit vas deferens has also been evaluated. 2. In binding studies using guinea-pig brain homogenates, unlabelled PD117302 had a high affinity (Ki = 3.7 nM) at [3H]-etorphine labelled kappa sites and a low affinity at [3H]-[D-Ala2, MePhe4, glyol5]-enkephalin ([3H]-DAGOL) labelled mu sites (Ki = 408 nM) and [3H]-SKF 10047 labelled sigma sites (Ki = 1.8 microM). In bioassay studies, PD117302 was a potent agonist, producing a maximum inhibition of the electrically-evoked contractions of the guinea-pig ileum (IC50 = 1.1 nM) and rabbit vas deferens (IC50 = 45 nM) which was naloxone-reversible. 3. In guinea-pig brain, [3H]-PD117302 bound to a high-affinity opioid binding site with a KD of 2.7 nM and a Bmax of 3.4 pmol g-1 wet weight. The Bmax was found to be less than 50% of the Bmax values for [3H]-etorphine and [3H]-bremazocine suggesting that [3H]-PD117302 may be a specific ligand for a subtype of kappa receptor. [3H]-PD117302 also bound with micromolar affinity to a non-opioid binding site. 4. Kinetic studies found that [3H]-PD117302-specific binding to the high affinity site was saturable, reaching equilibrium within 20 min at 4 degrees C, and reversible, with a half-life of dissociation of 3.9 min. 5. Several unlabelled compounds with high affinities for the ['H]-etorphine labelled K binding site, had comparable affinities when competing for the ['H]-PDl 17302-specific high affinity binding site. In contrast, DAGOL, [D-Ala', D-Leu'] enkephalin (DADLE) and [D-Pen2, D-Pen'] enkephalin (DPDPE) had no significant effect on ['H]-PD 1 17302 binding, suggesting minimal interaction with mu and delta binding sites. 6. In autoradiography studies ['H]-PD1 17302 binding sites were found throughout the brain with the greatest density in the striatum, cerebral cortex (layers V-VI), substantia nigra, and the molecular layer of the cerebellum. Lowest levels were found in the granular layer of the cerebellum, thalamus and cerebral cortex (layers I - IV).

Differential sensitivity of opioid-induced feeding to naloxone and naloxonazine.

The high-affinity mu-1 opioid binding site has been implicated in some opioid responses (e.g., supraspinal analgesia) but not others (e.g., respiratory depression) by comparing the actions of naloxone, a short-acting, non-selective antagonist, and naloxonazine, an irreversible and selective mu-1 antagonist. The mu-1 site has been implicated in the opioid component modulating free feeding and deprivation-induced feeding, but not glucoprivic feeding. The present study compared naloxone and naloxonazine antagonism of hyperphagia induced by morphine, ethylketocyclazocine (EKC), dynorphin and d-ala2,d-leu5-enkephalin (DADL) in rats. Morphine produced a dose-dependent (0.01-5 mg/kg) hyperphagia in mildly food-deprived rats that was blocked by naloxone (0.01-10 mg/kg). Naloxonazine (10 mg/kg) shifted the morphine hyperphagia dose-response curve to the right. These effects could not be fully accounted for by the intrinsic hypophagic properties of these antagonists. EKC produced a dose-dependent (0.5-5 mg/kg) hyperphagia which was blocked by naloxone (10 mg/kg) only at low effective EKC doses. Naloxonazine (10 mg/kg) failed to affect EKC hyperphagia. Naloxone, but not naloxonazine also blocked dynorphin and DADL hyperphagia. These results indicate that feeding induced by opiate and opioid agonists are differentially mediated by the mu-1 and other opioid binding sites; these data contrast with the modulation by the mu-1 site of the supraspinal analgesia induced by each of these agonists.

Temperature dependence of the binding of μ, δ and κ agonists to the opiate receptors in guinea-pig brain

Membrane preparations from guinea-pig brain were used to study the effects of temperature on the interaction of [ 3H][D-Ala 2-MePhe-Gly-ol 5] (DAGO), a selective μ agonist, [ 3H][D-Ala 2, D-Leu 5]enkephalin (DADLE), a nearly selective δ agonist and (±)[ 3H]ethylketazocine (EKC), a selective κ agonist when binding was measured in the presence of an excess of DAGO and DADLE. Binding was endothermic ( ΔH 0 = 10–17 kJ mol −1) and thus driven by the large increase in entropy ( ΔS 0 = 200–230 J mol −1K −1) for all ligands under examination. Van't Hoff plot for DAGO and DADLE showed a breakdown beyond 303 K which could have been due to lower in vitro stability of the proteins to conformational changes in the receptor proteins and/or to phase changes in the membranes. The present results do not agree with the hypothesis that agonist binding is associated with very small or negative ΔS 0 (in opposition to antagonists) in view of the information transfer involved (the biological effect).

Opioid receptor desensitization in NG 108-15 cells: Differential effects of a full and a partial agonist on the opioid-dependent GTPase

Opioid-receptor binding and the opioid-mediated stimulation of low K m GTPase and inhibition of adenylate cyclase were studied in membranes derived from NG 108-15 cells pretreated with either the opioid peptide [ d-Ala 2, d-Leu 5]enkephalin (DADLE) or morphine. Pretreatment with DADLE resulted in a concentration-dependent loss of responsiveness of GTPase to the peptide; this effect was entirely accounted for by a reduction in the maximal stimulation produced acutely by DADLE, without changes in the ec 50, of the peptide, indicating a non-competitive type of desensitization. The degree of desensitization of GTPase was similar after one and 24 hr of pretreatment with DADLE, indicating that the process occurs rapidly. In contrast, morphine, which was 70–80% as potent as DADLE in stimulating GTPase and inhibiting adenylate cyclase in acute conditions, induced only a minimal desensitization of the opioid-GTPase system and, in contrast to DADLE, did not desensitize adenylate cyclase. Pretreatment with DADLE for one hour led to a decrease in opioid receptor density which was quantitatively similar to the degree of desensitization of GTPase: both these effects of DADLE were antagonized to a similar extent when morphine was also present in the pretreatment. Thus, desensitization of the opioid-stimulated GTPase appears to be correlated with down-regulation of the opioid receptor. Moreover, these findings suggest that partial agonists cannot induce this process.

Synthesis and evaluation of melphalan-containing N,N-dialkylenkephalin analogues as irreversible antagonists of the delta opioid receptor.

N,N-Dialkylated leucine enkephalin analogues containing melphalan (Mel) in place of Phe4 were synthesized as potentially irreversible antagonists of the delta opioid receptor. These compounds, along with the corresponding Phe4 peptides, were tested for both agonist and antagonist activity in the GPI and MVD smooth muscle preparations. All but two of the eight compounds showed antagonist activity at 1 microM against [D-Ala2,D-Leu5]enkephalin (DADLE) in the MVD when tested under reversible conditions; in all cases the Mel4 peptide had lower activity against DADLE than did the corresponding Phe4 peptide. At higher concentrations (10 microM) the two active Mel4 analogues, (benzyl)2Tyr-Gly-Gly-Mel-Leu (2a) and (allyl)2Tyr-Aib-Aib-Mel-Leu (3a), both showed weak irreversible antagonism at the delta receptor. Compound 2a was a selective irreversible delta opioid antagonist while 3a was an irreversible antagonist at both the mu and delta opioid receptors.

Exogenous opiates: their local mechanisms of action in the canine small intestine and stomach.

Methionine-enkephalin (Met-Enk) and dynorphin were injected intra-arterially into the distal stomach and small intestine of the anesthetized dog during quiescence and phasic activity initiated by field stimulation or intra-arterially administered motilin. The effects of morphine, [D-Ala2, N-Me-Phe4, Met(O)5-ol]enkephalin (Met-ol), and [D-Ala2, D-Leu5]enkephalin (DADLE) were also examined. Opiates had no effect on the quiescent stomach but produced inhibition of phasic contractions (potencies: dynorphin greater than Met-Enk greater than Met-ol greater than DADLE greater than morphine). In the quiescent small intestine, low doses of each opiate, except dynorphin, produced excitation (potencies: Met-Enk congruent to Met-ol congruent to DADLE greater than morphine). Excitation apparently occurred via release of acetylcholine and a partially naloxone-sensitive direct stimulation of smooth muscle. During phasic activity all opiates produced inhibition of phasic muscular contractions (relative potencies as in the stomach). Inhibition by dynorphin in both the stomach and small intestine was shown to occur via naloxone-sensitive inhibition of release of acetylcholine from nerves. Thus inhibition by opiates appears common to the stomach and small intestine, and excitation is a function of opiate action only in the small intestine. Dynorphin may be a natural neuropeptide causing inhibition, and Met-Enk may be a natural neuropeptide causing excitation.

Presynaptic opioid receptors in the portal vein of the rabbit

The effects of opioids on sympathetic neuroeffector transmission were studied in superfused strips of the portal vein of the rabbit. In the first series of experiments, the tissue was stimulated electrically every 10 min with 10 pulses at 8 Hz and a current strength of 200 mA. The contractions evoked were concentration dependently inhibited by the slightly κ-selective agonist ethylketocyclazocine and by the δ-selective agonists [D-Ala 2,D-Leu 5]enkephalin (DADLE) and [D-Pen 2,D-Pen 5]enkephalin (DPDPLE). The μ-selective agonist [D-Ala 2,NMePhe 4, Gly-ol 5]enkephalin (DAGO) had no effect. The estimated EC 50 values for ethylketocyclazocine and DADLE were 5.5 · 10 −8 and 2.9 · 10 −8 mol/1, respectively. Naloxone shifted the concentration-response curves of ethylketocyclazocine and DADLE to the right; the estimated K b values were (1.7 ± 0.4) · 10 −8mol/l and (2.9 ± 0.8) · 10 −8mol/l, respectively. The δ-selective antagonist ICI 174864 antagonized only the inhibitory effect of DADLE but not that of ethylketocyclazocine. In the second series of experiments, the tissue was preincubated with [ 2H]noradrenaline then superfused and stimulated electrically three times for 3 min at 1 Hz and a current strength of 100 mA. Ethylketocyclazocine and DADLE inhibited the evoked overflow of tritium. The inhibition produced by ethylketocyclazocine was antagonized by naloxone. Our results show for the first time that opioid agonists inhibit noradrenaline release and hence, neurogenic vasoconstriction in a vein. As in other cardiovascular tissues of the rabbit, the presynaptic opioid receptors are of the κ- and δ- but not the μ-type.

Photoinactivation of the μ opioid receptor using a novel synthetic morphiceptin analog

The benzophenone chromophore has been incorporated into a synthetic amino acid (p-benzoyl-L-phenylalanine; L-Bpa) to produce a chemically stable photoaffinity probe. L-Bpa was found to retain the photochemical reactivity of benzophenone. To test the utility of this synthetic amino acid as a photo-reactive probe for receptors, a tetrapeptide analog of morphiceptin was made as a model peptide in which the C-terminal prolinamide was replaced by L-Bpa amide. The affinity of the μ opioid receptor for this peptide is comparable to that for the parent compound, morphiceptin. Irradiation of the peptide-receptor complex reduced the subsequent binding of [ 3H]naloxone and virtually eliminated that of [ 3H]Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAGO). Binding studies with [ 3H]naloxone indicated that both the affinity and the capacity were reduced. Competition studies with [ 3H]D-Ala 2-D-Leu 5-enkephalin (DADLE) and naloxone indicated selective inactivation of a μ type opioid receptor.

The effects of long-term treatment of NG108-15 cells with penta- and tetrapeptide enkephalin dimers on opioid receptor binding and cyclic AMP (cAMP) levels.

1. The effects of chronic treatment with a dimeric or monomeric penta- or tetrapeptide enkephalin analogue on binding and cyclic AMP (cAMP) accumulation in NG108-15 cells have been studied. 2. When the cells were cultured in the presence of 1 mumol of a pentapeptide analogue (dimer or monomer) for up to 96 hr, binding was reduced by greater than or equal to 90%. 3. In contrast, in the presence of 1 mumol of a tetrapeptide analogue (dimer or monomer), binding was reduced by only less than or equal to 30%. 4. The analogues had varying effects on regulation of cAMP formation. Desensitization, indicated by impaired opioid-mediated inhibition of prostaglandin E1 (PGE1)-stimulated cAMP accumulation, was clearly apparent only for cells pretreated with [D-Ala2,D-Leu5]enkephalin (DADLE), while cells pretreated with [D-Ala2,Leu5-NH-CH2-]2 (DPE2) showed minor impairment. 5. Thus, ligand dimerization appeared to have a modulating effect on regulation of adenylate cyclase activity but not to affect opioid-induced down-regulation.

Actions of opioids on the dorsal root potential of the isolated spinal cord preparation of the neonate rat

The effects of opioids were studied on the dorsal root-dorsal root evoked potential (DRP) of the neonatal hemisected spinal cord of the rat in vitro. Applications of [ d-Ala 2,Met 5]enkephalinamide (DAME), [ d-Ala 2, d-Leu 5]enkephalin (DADL), Leu 5 enkephalin. Met 5 enkephalin, Dynorphin 1–9 and normorphine produced dose-dependent depressions of the dorsal root potential. The depressant effects of these agents were antagonised by naloxone but not by the highly selective delta-opioid receptor antagonist ICI 174864. Compounds acting on the kappa opioid receptor had only weak and inconsistent inhibitory effects on the dorsal root potential. Morphine had antagonist properties on the dorsal root potential, as did the kappa agonists ethylketocyclazocine and bremazocine, but not U50488 and tifluadom. The depressant actions of opioids on the dorsal root potential thus appeared to be mediated by actions on the mu receptor type. However, only mu agonists with relatively high intrinsic activity were able to reduce the dorsal root potential. Other mu agonists, including morphine, acted as antagonists. Actions on receptors for the delta and kappa agonists, in contrast, appeared to have little influence on this response. The antagonist actions of certain kappa agonists were probably due to their high affinity for, but low efficacy at mu receptors.

Modulation of bradykinin-induced inositol trisphosphate release in a novel neuroblastoma x dorsal root ganglion sensory neuron cell line (F-11).

In the mouse neuroblastoma x dorsal root ganglion hybrid cell line F-11, bradykinin receptor stimulation induced the release of inositol-1,4,5-trisphosphate (IP3) and inositol-1,4-bisphosphate (IP2). Maximal stimulation of [2-3H]IP3 and [2-3H]IP2 release by bradykinin in the absence of LiCl occurred at 7 (or less) and 15 s, respectively, with average levels of 5.7-(IP3) and 3.4-(IP2) fold of control values. The EC50 for bradykinin was 33 +/- 5 nM. IP3 and IP2 concentrations returned to basal levels approximately 1 min after bradykinin addition. Bradykinin-induced IP3 release was blocked by several novel bradykinin analogues. In particular, [D-Arg0]-Hyp3-Thi5,8-[D-Phe7]-bradykinin [Hyp, hydroxyproline; Thi, beta-(2-thienyl)-L-alanine] blocked IP3 production in a dose-dependent fashion. Several of these analogues alone showed little or no agonist activity. The bradykinin receptor may be coupled to phospholipase C via a GTP-sensitive protein (Gi or Go), as preincubation for 18-20 h with pertussis toxin decreased IP3 concentrations by 45%. Bradykinin is also known to modulate the concentrations of other second messengers in neurons, increasing the concentrations of Ca2+, diacylglycerol (DG), and cyclic GMP and decreasing the concentration of cyclic AMP. These second messengers modulated bradykinin-dependent IP3 release to varying degrees. A23187, a Ca2+ ionophore, produced a 37% decrease in IP3 concentration. 12-O-Tetradecanoylphorbol-13-acetate, which mimics the effects of DG and activates protein kinase C, inhibited IP3 release by 80%. Dibutyryl cyclic GMP produced little or no inhibition of IP3. [D-Ala2,D-Leu5]Enkephalin (DADLE), an opioid peptide that decreases cyclic AMP concentrations, likewise had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)