dynorphins and naloxonazine

Endomorphin analogs containing unnatural amino acids have demonstrated potent analgesic effects in our previous studies. In the present study, the differences in antinociception and the mechanisms thereof for analogs 1-3 administered intracerebroventricularly and intrathecally were explored. All analogs at different routes of administration produced potent analgesia compared to the parent peptide endomorphin-1. Multiple antagonists and antibodies were used to explore the mechanisms of action of these analogs, and it was inferred that analogs 1-3 stimulated the μ opioid receptor to induce antinociception. Moreover, the antibody data suggested that analog 2 may induce the release of immunoreactive [Leu

Topics: Analgesics; Animals; Chronic Pain; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Methionine; Enkephalins; Injections, Intraventricular; Mice; Naloxone; Narcotic Antagonists; Oligopeptides; Receptors, Opioid, mu; Spinal Cord

To study the pharmacological profile and inhibition of smooth muscle contraction by YFa and its analogs in conjunction with their receptor selectivity.. The effects of YFa and its analogs (D-Ala2) YFa, Y (D-Ala2) GFMKKKFMRF amide and Des-Phe-YGGFMKKKFMR amide in guinea pig ileum (GPI) and mouse vas deferens (MVD) motility were studied using an isolated tissue organ bath system, and morphine and DynA (1-13) served as controls. Acetylcholine was used for muscle stimulation. The observations were validated by specific antagonist pretreatment experiments using naloxonazine, naltrindole and norbinaltorphimine norBNI.. YFa did not demonstrate significant inhibition of GPI muscle contraction as compared with morphine (15% vs 62%, P = 0.0002), but moderate inhibition of MVD muscle contraction, indicating the role of κ opioid receptors in the contraction. A moderate inhibition of GPI muscles by (Des-Phe) YFa revealed the role of anti-opiate receptors in the smooth muscle contraction. (D-Ala-2) YFa showed significant inhibition of smooth muscle contraction, indicating the involvement of mainly δ receptors in MVD contraction. These results were supported by specific antagonist pretreatment assays.. YFa revealed its side-effect-free analgesic properties with regard to arrest of gastrointestinal transit. The study provides evidences for the involvement of κ and anti-opioid receptors in smooth muscle contraction.

Topics: Acetylcholine; Analgesics, Opioid; Animals; Cholinergic Agonists; Dynorphins; Enkephalin, Methionine; Guinea Pigs; Ileum; Male; Mice; Morphine; Muscle Contraction; Muscle, Smooth; Naloxone; Naltrexone; Narcotic Antagonists; Neurotransmitter Agents; Oligopeptides; Receptors, Opioid; Vas Deferens

It has been demonstrated that the antinociception induced by i.t. or i.c.v. administration of endomorphins is mediated through mu-opioid receptors. Moreover, though endomorphins do not have appreciable affinity for kappa-opioid receptors, pretreatment with the kappa-opioid receptor antagonist nor-binaltorphimine markedly blocks the antinociception induced by i.c.v.- or i.t.-injected endomorphin-2, but not endomorphin-1. These evidences propose the hypothesis that endomorphin-2 may initially stimulate the mu-opioid receptors, which subsequently induces the release of dynorphins acting on kappa-opioid receptors to produce antinociception. The present study was performed to determine whether the release of dynorphins by i.c.v.-administered endomorphin-2 is mediated through mu-opioid receptors for producing antinociception. Intracerebroventricular pretreatment with an antiserum against dynorphin A, but not dynorphin B or alpha-neo-endorphin, and s.c. pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine dose-dependently attenuated the antinociception induced by i.c.v.-administered endomorphin-2, but not endomorphin-1 and DAMGO. The attenuation of endomorphin-2-induced antinociception by pretreatment with antiserum against dynorphin A or nor-binaltorphimine was dose-dependently eliminated by additional s.c. pretreatment with a selective mu-opioid receptor antagonist beta-funaltrexamine or a selective mu1-opioid receptor antagonist naloxonazine at ultra low doses, which are inactive against micro-opioid receptor agonists in antinociception, suggesting that endomorphin-2 stimulates distinct subclass of micro1-opioid receptor that induces the release of dynorphin A acting on kappa-opioid receptors in the brain. It concludes that the antinociception induced by supraspinally administered endomorphin-2 is in part mediated through the release of endogenous kappa-opioid peptide dynorphin A, which is caused by the stimulation of distinct subclass of micro1-opioid receptor.

Topics: Analgesics; Animals; Dynorphins; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Immune Sera; Injections, Intraventricular; Male; Mice; Naloxone; Naltrexone; Oligopeptides; Protein Precursors; Receptors, Opioid, kappa; Receptors, Opioid, mu

[Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH(2); Dmt = 2',6'-dimethyltyrosine) is a dermorphin analog that shows high affinity and selectivity for the mu opioid receptor. The intrathecal potency of [Dmt(1)]DALDA far exceeded its affinity at mu receptors and suggests that other mechanisms must be involved in its action in the spinal cord. The affinity and selectivity of [Dmt(1)]DALDA was determined using cell membranes expressing cloned human mu, delta, and kappa opioid receptors. Competitive displacement binding with [(3)H][Dmt(1)]DALDA, [(3)H]DPDPE (H-Tyr-d-Pen-Gly-Phe-d-Pen), and [(3)H]U69,593 [(5alpha,7alpha,8beta)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4.5]dec-8-yl)-benzeneacetamide] revealed K(i) of 156 +/- 26 pM for mu opioid receptor (MOR), 1.67 +/- 0.04 microM for delta opioid receptor (DOR), and K(i) of 4.4 +/- 1.7 nM for kappa opioid receptor (KOR), respectively. [Dmt(1)]DALDA increased guanosine 5'-O-(3-[(35)S]thiotriphosphate) binding in MOR, DOR, and KOR membranes, with EC(50) being 17 (8.8-33) nM, 2 (1.2-3.2) microM, and 124 (15-1000) nM, respectively. Intrathecal [Dmt(1)]DALDA inhibited the tail-flick response in mice with ED(50) = 1.22 (0.59-2.34) pmol. Intrathecal administration of an antiserum against dynorphin A(1-17) or [Met(5)]enkephalin significantly attenuated the response to i.t. [Dmt(1)]DALDA, resulting in ED(50) of 6.2 (3.6-12.6) pmol and 6.6 (3.5-19.6) pmol, respectively. Neither antisera had any effect on the response to i.t. morphine. Intracerebroventricular (i.c.v.) [Dmt(1)]DALDA was not affected by previous i.c.v. administration of anti-Dyn or anti-ME. Pretreatment with norbinaltorphimine or naltriben also attenuated the antinociceptive response to i.t., but not i.c.v., [Dmt(1)]DALDA. These data suggest that i.t. [Dmt(1)]DALDA causes the release of dynorphin and [Met(5)]enkephalin-like substances that act at kappa and delta receptors, respectively, to contribute to the extraordinary potency of [Dmt(1)]DALDA.

Topics: Analgesics, Opioid; Animals; Antibodies, Blocking; Cloning, Molecular; Dynorphins; Endorphins; Enkephalin, Methionine; Guanosine 5'-O-(3-Thiotriphosphate); Hot Temperature; Injections, Intraventricular; Injections, Spinal; Male; Mice; Naloxone; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain Measurement; Radioligand Assay; Reaction Time; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

Two highly selective mu-opioid receptor agonists, endomorphin-1 and endomorphin-2, have been identified and postulated to be endogenous ligands for mu-opioid receptors. Intrathecal (i.t.) administration of endomorphin-1 and endomorphin-2 at doses from 0.039 to 5 nmol dose-dependently produced antinociception with the paw-withdrawal test. The paw-withdrawal inhibition rapidly reached its peak at 1 min, rapidly declined and returned to the pre-injection levels in 20 min. The inhibition of the paw-withdrawal responses to endomorphin-1 and endomorphin-2 at a dose of 5 nmol observed at 1 and 5 min after injection was blocked by pretreatment with a non-selective opioid receptor antagonist naloxone (1 mg/kg, s.c.). The antinociceptive effect of endomorphin-2 was more sensitive to the mu (1)-opioid receptor antagonist, naloxonazine than that of endomorphin-1. The endomorphin-2-induced paw-withdrawal inhibition at both 1 and 5 min after injection was blocked by pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine (10 mg/kg, s.c.) or the delta(2)-opioid receptor antagonist naltriben (0.6 mg/kg, s.c.) but not the delta(1)-opioid receptor antagonist 7-benzylidine naltrexone (BNTX) (0.6 mg/kg s.c.). In contrast, the paw-withdrawal inhibition induced by endomorphin-1 observed at both 1 and 5 min after injection was not blocked by naloxonazine (35 mg/kg, s.c.), nor-binaltorphimine (10 mg/kg, s.c.), naltriben (0.6 mg/kg, s.c.) or BNTX (0.6 mg/kg s.c.). The endomorphin-2-induced paw-withdrawal inhibition was blocked by the pretreatment with an antiserum against dynorphin A-(1-17) or [Met(5)]enkephalin, but not by antiserum against dynorphin B-(1-13). Pretreatment with these antisera did not affect the endomorphin-1-induced paw-withdrawal inhibition. Our results indicate that endomorphin-2 given i.t. produces its antinociceptive effects via the stimulation of mu (1)-opioid receptors (naloxonazine-sensitive site) in the spinal cord. The antinociception induced by endomophin-2 contains additional components, which are mediated by the release of dynorphin A-(1-17) and [Met(5)]enkephalin which subsequently act on kappa-opioid receptors and delta(2)-opioid receptors to produce antinociception.

Topics: Analgesics; Animals; Benzylidene Compounds; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Methionine; Immune Sera; Injections, Spinal; Injections, Subcutaneous; Male; Mice; Naloxone; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain; Pain Measurement; Pain Threshold; Peptide Fragments; Time Factors

Dynorphin A-(1-17) has been found to produce spinal antianalgesia and allodynia. Thus, we studied whether dynorphin A-(1-17) modulates substance P release evoked by the C-fiber-selective stimulant capsaicin (1 microM) from trigeminal nucleus caudalis slices. Very low concentrations of dynorphin A-(1-17) (0.01-0.1 nM) strongly facilitated capsaicin-evoked substance P release. This dynorphin A-(1-17) effect was not blocked by the opioid receptor antagonists naloxone (100 nM), beta-funaltrexamine (20 nM), naloxonazine (1 nM), nor-binaltorphimine (3 nM) and ICI 174,864 (N,N-dialyl-Tyr-Aib-Phe-Leu; 0.3 microM). Yet, the effect of dynorphin A-(1-17) was blocked by the NMDA receptor antagonist MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5-10-imine maleate; 0.3 microM). Neonatal treatment with capsaicin (50 mg/kg s.c.), which destroys substance P-containing primary afferents, abolished the excitatory effect of dynorphin A-(1-17) on K+-evoked substance P release. In conclusion, dynorphin A-(1-17) increases substance P release from C-fibers by the activation of NMDA receptors which supports the involvement of presynaptic mechanisms in dynorphin-induced antianalgesia and allodynia.

Topics: Animals; Animals, Newborn; Capsaicin; Dizocilpine Maleate; Dynorphins; Enkephalin, Leucine; Excitatory Amino Acid Antagonists; In Vitro Techniques; Male; Naloxone; Naltrexone; Narcotic Antagonists; Nerve Fibers; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Substance P; Trigeminal Nuclei

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.

Topics: Animals; Dose-Response Relationship, Drug; Dynorphins; Eating; Electroshock; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Morphine; Naloxone; Narcotics; Rats; Rats, Inbred Strains

In the present study, we examined the binding of [3H][D-Ala2,D-Leu5]enkephalin ([ 3H]DADL) to bovine thalamic membranes. Scatchard plots were linear with a KD of 0.7 nM. However, competition experiments suggested binding heterogeneity. Approximately 20% of [3H]DADL binding was easily inhibited by [D-Pen2,D-Pen5]enkephalin (DPDPE) and was insensitive to morphine, implying labeling of delta receptors. The remaining 80% of binding was quite sensitive to both morphine and [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) and insensitive to DPDPE, consistent with a mu receptor. However, this binding did not correspond to classical morphine-selective mu receptors. Unlike morphine-selective receptors, this binding had similar affinities for morphine, DAGO, DADL and [D-Ser2,Leu5]enkephalin-Thr6 (DSLET). In addition, it was far more sensitive to naloxonazine's wash-resistant inhibition and magnesium-induced enhancement of binding than either the morphine-selective (mu 2) or delta sites. [3H]DSLET binding yielded results very similar to those using [3H]DADL. In conclusion, approximately 80% of [3H]DADL binding in thalamus corresponds to a mu receptor distinct from the classical morphine-selective site. Based upon the results of our studies, we feel that this binding represents mu 1 receptors. DPDPE (10 nM) can effectively inhibit the binding of [3H]DADL to delta receptors, leaving a relatively homogeneous labeling of mu 1 sites. The availability of this selective binding assay should facilitate additional studies of mu 1 receptors.

Topics: Animals; Cattle; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Hydrogen-Ion Concentration; In Vitro Techniques; Naloxone; Oligopeptides; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Thalamus