Analgesic and convulsant effects of guanidinoethylmercaptosuccinic acid (GEMSA)--a potent enkephalin convertase inhibitor

We studied behavioral effects of the intraventricularly and intrathecally administered guanidinoethylmercaptosuccinic acid (GEMSA) - a potent inhibitor of enkephalin convertase. When given intraventricularly in doses of 3 and 6 micrograms, GEMSA elicited analgesia; after doses of 12.5 and 25 micrograms the explosive motor behavior and convulsions occurred. Following the intrathecal administration of GEMSA (12.5, 25 and 50 micrograms), an increase in the tail-flick latency was observed; moreover that drug potentiated analgesic effects of the intrathecally applied Met5-enkephalin-Arg6-Phe7 and Met5-enkephalin-Arg6-Gly7-Leu8. All the above effects of GEMSA were significantly attenuated by naloxone. The rats subjected to chronic pain showed a weaker analgesic response to the intrathecally injected GEMSA. The 3H-GEMSA binding to enkephalin convertase in the spinal cord of these rats produced only a slight increase in KD; besides, no changes in the enzyme activity were observed. The study shows that GEMSA has a potent pharmacological action in the central nervous system. Furthermore, this effect is partly due to the influence of GEMSA on endogenous opioid peptide systems, possibly on proenkephalin A.     

Characterization of zeta (ζ): a new opioid receptor involved in growth

Endogenous opioid systems (i.e., opioids and opioid receptors) are known to play a role in neural cancer. Using [³H]-[Met⁵]enkephalin, a potent ligand involved in growth, specific and saturable binding was detected in homogenates of S20Y neuroblastoma transplanted into A/Jax mice; the data fit a single binding site. Scatchard analysis yielded aK_d of 0.49 nM and a binding capacity of 5.32 fmol/mg protein. Binding was dependent on protein concentration, time, temperature, and pH, and was sensitive to Na⁺ and guanine nucleotides. Optimal binding required protease inhibitors, and pretreatment of the tumor homogenates with trypsin markedly reduced [³H]-[Met⁵]enkephalin binding, suggesting that the binding site was proteinaceous in character. Displacement experiments indicated that [Met⁵]enkephalin was the most potent displacer of [³H]-[Met⁵]enkephalin; other ligands selective for μ, δ, κ, , and σ were not highly competitive. Given the functional significance of [Met⁵]enkephalin as a potent regulator of normal and abnormal growth, and that the receptor recognized by [Met⁵]enkephalin does not resemble any previously described, the present study has demonstrated the presence of a new opioid receptor termed zeta (ζ) (from the Greek ‘Zoe’, life) related to the proliferation of cells and tissues.     

The opioid octapeptide Met-enkephalin-Arg-Gly-Leu: Characterization and distribution in rat spinal cord

The regional quantitation, immunohistochemical localization and molecular heterogeneity of Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ were examined in rat spinal cord with a specific radioimmunoassay. A rostrocaudal gradient in Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ content was observed; the highest levels occurred in sacral cord. Dorsal cord content was higher than that of ventral cord at all spinal segments. Immunohistochemical staining supported and refined the latter observation: a dense network of perikarya and fibers was found in Laminae I and II of the dorsal horn. Cell bodies were frequently observed in lamina IV. Additional terminals were seen around the central canal and in the ventral gray matter, often outlining perikarya of motor neurons. Total Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ immunoreactivity could be fractionated into two main components using gel filtration chromatography. Nearly half of the total immunoreactivity eluted as a high molecular weight peptide; the other half which co-eluted with Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ was further identified to be authentic Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ on reverse phase high pressure liquid chromatography. The present data, in conjunction with our previous study of Met⁵-enkephaline and Met⁵-enkephalin-Arg⁶-Phe⁷ indicates that all opioid peptides derived from preproenkephalin A are present in spinal cord and most likely are stored in the same neurons. Immunohistochemical localization of Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ in dorsal and ventral cord suggest a role for this peptide in both sensory and motor integration.     

Streptozotocin-induced diabetes selectively alters the potency of analgesia produced by μ-opioid agonists, but not by δ- and κ-opioid agonists

To investigate the possible mechanisms of the alterations in morphine-induced analgesia observed in diabetic mice, we examined the influence of streptozotocin-induced (STZ-induced) diabetes on analgesia mediated by the different opioid receptors. The antinociceptive potency of morphine (10 mg/kg), administered s.c., as determined by both the tail-pinch and the tail-flick test, was significantly reduced in diabetic mice as compared to that in controls. Mice with STZ-induced diabetes had significantly decreased sensitivity to intracerebroventricularly (i.c.v.) administered μ-opioid agonists, such as morphine (10 μg) and [d-Ala², N-Me Phe⁴,Gly-ol⁵]enkephalin (DAMGO, 0.5 μg). However, i.c.v. administration of [d-Pen^2,5]enkephalin (DPDPE, 5 μg), a δ-opioid agonist, and U-50,488H (50 μg), a κ-opioid agonist, produced pronounced antinociception in both control and diabetic mice. Furthermore, there were no significant differences in antinociceptive potency between diabetic and control mice when morphine (1 μg), DAMGO (10 μg), DPDPE (0.5 μg) or U-50,488H (50 μg) was administered intrathecally. In conclusion, mice with STZ-induced diabetes are selectively hyporesponsive to supraspinal μ-opioid receptor-mediated antinociception, but they are normally responsive to activation of δ- and κ-opioid receptors.     

Role of μ1-opiate receptors in supraspinal opiate analgesia: a microinjection study

Microinjection of opiates into either the periaqueductual gray, locus coeruleus, nucleus raphe magnus, or nucleus reticularis gigantocellularis elicits a profound naloxone-sensitive analgesia. μ-Opioid receptors have been implicated in supraspinal analgesia and studies from our laboratory have demonstrated the importance of the μ₁-receptor subtype. In an effort to examine the receptor subtypes responsible for opioid analgesia in specific brain regions, we examined dose-response relationships and naloxonazine sensitivity of morphine and two enkephalin derivatives in the above 4 brain regions. Both morphine and [ -Ser², Leu⁵]enkephalin-Thr⁶ (DSLET) were effective analgesics in all regions examined. The poor affinity of DSLET for μ₂-receptors and of morphine for δ-receptors, combined with their similar, high affinity for μ₁-receptors, implied a μ₁-mechanism of action. The μ₁-selective antagonist naloxonazine effectively blocked the analgesic responses of both compounds in all regions. [ -Pen², -Pen⁵]enkephalin (DPDPE), a potent δ-ligand which does not interact with μ₁-receptors, did not elicit analgesia in either the periaqueductal gray or locus coeruleus at any dose tested. These results suggest that opiates and opioid peptides produce analgesia in these 4 brain regions through μ₁-receptors. The inactivity of DPDPE argues against a role for δ-receptors and the similar analgesic potencies of morphine and DSLET makes a significant role for μ₂-receptors unlikely.     

Demonstration and characterization of zeta (ζ), a growth-related opioid receptor, in a neuroblastoma cell line

Endogenous opioids and opioid receptors (i.e. endogenous opioid systems) are involved in carcinogenesis. Using homogenates of S20Y neuroblastoma (NB) cells grown in culture, the binding of a growth-selective ligand, [Me⁵]enkephalin, was examined to ascertain the zeta (ζ) opioid receptor. Specific and saturable binding of [³H]-[Met⁵]enkephalin was detected in NB cells; the data were consistent with a single binding site. Scatchard analysis yielded a K_d of 1.6 nM and a binding capacity B_max off 48.1 fmol/mg protein; 14,000 receptors per cell were estimated. Binding was dependent on protein concentration, time, temperature, and pH, and was sensitive to 100 nM, but not 5 nM, Na⁺, Ca²⁺, and Mg²⁺; GppNHp at concentrations of 100–500 mM had little effect on binding. Optimal binding required protease inhibitors, and pretreatment of the tumor cell homogenates with trypsin markedly reduced [³H]-[Met⁵]enkphalin winding, suggesting that the binding site was proteinaceous in character. Displacement experiments indicated that [Met⁵]enkephalin was the most potent displacer of [³H]-[Met⁵]enkephalin. Cell density (log, confluence, postconfluence) did not alter the K_d or B_max. This study serves as the first demonstration and characterization of the zeta (ζ) opioid receptor in tissue culture cells. The homogenous nature of NB cell cultures, along with the enrichment in receptor number, provides an excellent model system to isolate and purify the ζ receptor.     

Depression by morphine-6-glucuronide of nociceptive activity in rat thalamus neurons: comparison with morphine

To assess the contribution of the active metabolite of morphine, morphine-6-glucuronide (M6G), to the analgesic effect of systemically administered morphine, experiments were carried out on rats under urethane anesthesia in which nociceptive activity was evoked by electrical stimulation of afferent C fibers in the sural nerve and recorded from single neurons in the ventrobasal complex of the thalamus. Intravenous (i.v.) injections of morphine completely blocked the activity at doses of 500 and 1000 μg/kg, the ED,, being 44 μg/kg. M6G administered by i.v. injection reduced the evoked nociceptive activity only by about 40% at 80 and 160 μg/kg, the ED₅₀ being 6 μg/kg. After intrathecal (i.t.) injection, morphine produced maximum depression of 55% of the control activity at 20 μg the ED₅₀ is 18 μg. M6G injected i.t. produced maximum depression of 40% at doses ranging from 0.2 to 10 μg. The ED₅₀ of M6G i.t. is below 0.2 μg. The effects of morphine and M6G were reversed by naloxone (200 μg/kg i.v.). The results show that M6G is more potent than morphine, regardless of the route of administration, while morphine is more effective when injected i.v. Due to the low efficacy of M6G, it seems unlikely that this glucuronide contributes substantially to the analgesic effect of morphine when renal function is normal. The results also make evident that the maximum effect of morphine results from an action at spinal and supraspinal sites.     

Opiate mechanisms in the central amygdala and gastric stress pathology in rats

Bilateral microinjections of the opiate antagonist substance (0.1, 1.0 and 10.0 μg) into the central nucleus of the amygdala (CEA) produced a significant potentiation of cold restraint-induced gastric pathology in rats. The opiate agonist, β-endorphin (0.1, 1.0 and 10.0 μg), on the other hand, inhibited stress ulcer formation in a dose-related manner. Stress ulcer-attenuating effects were also seen with intra-CEA injections of the enkephalin analogs [ -Ala², -Leu⁵]enkephalin (10.0 μg) and [ -Ala²]Met-enkephalinamide (10.0 μg). Pretreatment of rats with naloxone (1.0 μg) completely antagonized and even reversed the gastric cytoprotective effects of β-endorphin (1.0 and 10.0 μg). The results indicate that the CEA is important in the gastric cytomodulatory effects of endogenous opiates during stressful experiences.     

Opioid supraspinal analgesic synergy between the amygdala and periaqueductal gray in rats

Analgesia can be elicited following microinjections of morphine, μ-selective agonists and β-endorphin into the amygdala. These analgesic responses are mediated by opioid synapses in the periaqueductal gray (PAG) since general (naltrexone), μ (β-funaltrexamine) and δ₂ (naltrindole isothiocyanate) opioid antagonists administered into the PAG significantly reduce both morphine and β-endorphin analgesia elicited from the amygdala. Supraspinal multiplicative opiate analgesic interactions have been observed between the PAG and rostroventromedial medulla (RVM), the PAG and locus coeruleus (LC), and the RVM and LC. The present study further examined the relationship between the amygdala and PAG in analgesic responsiveness by determining whether multiplicative analgesic interactions occur following paired administration of subthreshold doses of morphine into both structures, β-endorphin into both structures, morphine into one structure and β-endorphin into the other structure, or morphine and β-endorphin into one structure. Co-administration of subthreshold doses of morphine into both the amygdala and PAG results in a profound synergistic interaction on the jump test, but not the tail-flick test. Co-administration of subthreshold doses of β-endorphin into both structures also results in a profound test-specific synergistic interaction. In both cases, the magnitude of the interaction was similar regardless of the site receiving the fixed dose of the opioid, and the site receiving the variable dose of the opioid. Co-administration of β-endorphin (1 μg) into the amygdala and morphine (1 μg) into the PAG produced a potent interaction, but co-administration of morphine (1 μg) into the amygdala and β-endorphin (1 μg) into the PAG failed to produce interactive effects. Finally, co-administration of morphine (1 μg) and β-endorphin (1 μg) into either the amygdala alone or the PAG alone failed to produce an interaction, indicating the importance of regional opioid activation. These data are discussed in terms of the test-specificity of nociceptive processing in the amygdala, in terms of the multiple modulatory mechanisms mediating β-endorphin analgesia in the PAG, and in terms of whether the interactions are either mediated by anatomical connections between the amygdala and PAG or by mechanisms initiated by these two sites converging at another site or sites.     

GABA inhibition of central angiotensin II and hypertonic CSF pressor responses

Angiotensin II (AII), hypertonic cerebrospinal fluid (CSF) and serotonin produced an increase in arterial pressure when administered intraventricularly (IVT) in conscious rats. Injection of 25 and 100 μg (IVT) of the inhibitory neurotransmitter γ-aminobutyric acid (GABA), while producing slight hypotension, reduced the pressor effect of centrally administered AII and hypertonic CSF, but not serotonin. IVT-administered muscimol, a potent GABA agonist, also attenuated the pressor effect of IVT AII and hypertonic CSF. Thus, in addition to the profound depressor effect of large doses of centrally administered GABA, very low doses (25–100 μg, IVT) of this amino acid can alter the pressor responses caused by IVT injection of AII and hypertonic CSF.     

Slow-wave sleep induced in cats by extremely small amounts of synthetic and pineal vasotocin injected into the third ventricle of the brain

A single injection of 10⁻⁶ pg synthetic arginine vasotocin (AVT), corresponding to about 600 molecules AVT, into the third ventricle of unanesthetized cats, induced slow-wave sleep 5 min after the injection. An equivalent amount, of a partially purified pineal AVT injected into the third ventricle, produced the same effects. After incubation with trypsin, pineal AVT completely lost its ability to induce slow-wave sleep. The slow-wave sleep induced by 10⁻⁶ pg synthetic AVT injected intraventricularly could be matched by lμg synthetic AVT injected intraperitoneally. Neither synthetic arginine vasopressin, nor synthetic oxytocin, injected intraventricularly in the amount of 10⁻⁶ pg, was able to induce slow-wave sleep. Whereas in the control animals injected with pineal AVT after incubation with trypsin, or in the control animals injected with vasopressin or oxytocin, the paradoxical sleep averaged 21.9–22.8% of the sleep time, during a total recording time of 5 hr, in the cats injected with synthetic or pineal AVT, the paradoxical sleep was completely suppressed.     

Hip-Arg-Phe-, Hip-Phe-Arg- and Hip-His-Leu-cleaving dipeptidyl carboxypeptidases in human adrenal tumors

Hip-Arg-Phe-, Hip-Phe-Arg-and Hip-His-Leu-cleaving dipeptidyl carboxypeptidase activities were measured in the supernatant (S₂) and pellet (P₂) fractions obtained by ultracentrifugation of human adrenal tumor preparations. Negligible enzyme activity was found in cortical tumor whereas highly significant activities were present in the P₂ fractions of the two pheochromocytoma specimens. The hydrolysis rates, expressed in terms of the percent of added substrate were 58–66%/60 min for Hip-Phe-Arg, 55–58%/60 min for Hip-Arg-Phe and 19–30%/60 min for HipHis-Leu. The angiotensin-converting enzyme inhibitor, captopril, differentially inhibited the enzyme splitting Hip-His-Leu versus the one cleaving Hip-Arg-Phe; Hip-Phe-Arg is probably the substrate of both. It is concluded that the Hip-ArgPhe-cleaving enzyme in adrenomedullary tumor is probably identical to the purportedly novel dipeptidyl carboxypeptidase that we detected earlier in rabbit ear artery wall, which converts (Met⁵)-enkephalin-Arg⁶,Phe⁷ to (Met⁵)-enkephalin.     

Evidence that naloxonazine produces prolonged antagonism of central delta opioid receptor activity in vivo

Intracerebroventricular (i.c.v.) administrations of the postulated μ₁ opioid receptor antagonist naloxonazine produced an increase in the frequency of urinary bladder contractions recorded isometrically in the anesthetized rat. This substance also antagonized the inhibition of spontaneous bladder contractions produced by submaximal i.c.v. doses of the highly selective μ opioid agonist [D-Ala²-MePhe⁴,Gly-(ol)⁵]enkephalin (DAGO) and the δ opioid agonist [D-Pen²,D-Pen⁵]enkephalin (DPDPE). The antagonism of DAGO was reversible but that of DPDPE lasted up to 30 h. These data suggest that endogenous opioids are involved in the central control of bladder motility and that naloxonazine is a long-lasting δ opioid receptor antagonist.     

Distribution of Met-enkephalin-Arg,Phe in rat spinal cord

The distribution of Met⁵-enkephalin-Arg⁶,Phe⁷ (YGGFMRF) in rat spinal cord was determined by a specific RIA and compared with that of Met⁵-enkephalin. The concentration of YGGFMRF on a per mg protein basis was highest in sacral cord and successively decreased in more rostral segments. A similar rostro-caudal distribution was observed for Met⁵-enkephalin. Regional microdissection revealed the dorsal grey matter to be highest in YGGFMRF content followed by ventral gray, ventral white and dorsal white matter; a similar pattern was observed for Met⁵-enkephalin. The ratio of Met⁵-enkephalin. The ratio of Met⁵-enkephalin: YGGFMRF concentration was5.4 ± 0.15 (S.E.M.) on average in all regions measured, indicating a very close quantitative relationshp between the two molecules. Our data suggest that YGGFMRF may act as a precursor of or cotransmitter with Met⁵-enkephalin in spinal cord tissue.     

Opioid and cannabinoid receptor-mediated regulation of the increase in adrenocorticotropin hormone and corticosterone plasma concentrations induced by central administration of Δ-tetrahydrocannabinol in rats

The purpose of this study was to investigate the cannabinoid and opioid mediated regulation on the effects of central Δ⁹-tetrahydrocannabinol (Δ⁹-THC) administration on hypothalamus–pituitary–adrenal (HPA) axis activity in the male rat. Intracerebroventricular (i.c.v.) administration of Δ⁹-THC (25, 50, 100 μg/rat) markedly increased plasma adrenocorticotropin hormone (ACTH) and corticosterone concentrations. Time course effect studies revealed that both hormones secretion peaked at 60 min after Δ⁹-THC i.c.v. administration (50 μg/rat), decreased gradually and returned to baseline levels by 480 min. The i.c.v. administration of the specific cannabinoid receptor antagonist SR-141716A (3 μg/rat) significantly attenuated the increase of both hormones secretion induced by Δ⁹-THC (50 μg/rat). Nevertheless, higher doses (12.5 and 50 μg/rat) of this compound increased both ACTH and corticosterone plasma concentrations. Subcutaneous (s.c.) administration with the opiate receptor antagonist naloxone (0.3 mg/kg) was without effect but significantly diminished the increase of both hormones secretion induced by Δ⁹-THC (50 μg/rat). Taken together, these results indicate that opiate and cannabinoid receptors are involved in the activation of the HPA axis induced by Δ⁹-THC. Furthermore, the increase of ACTH and corticosterone secretion after the administration of higher doses of SR-141716A than those required to block such activation, suggests that endogenous cannabinoids are tonically inhibiting the release of both hormones or that this agonist-like activity may be part of an uncharacterized action of this compound not mediated by cannabinoid receptors.     

Modulation of endomorphin-2-induced analgesia by dipeptidyl peptidase IV

The tetrapeptide, endomorphin-2 (Tyr-Pro-Phe-PheNH₂) possesses high affinity for μ opioid receptors, and produces potent analgesia in mice. Its structure appears to satisfy the substrate requirements of the proteinase, dipeptidyl peptidase IV which removes dipeptides from the amino terminus of peptides containing proline as the penultimate amino acid. A potent, stable and specific inhibitor of this enzyme, Ala-Pyrrolidonyl-2-nitrile, has been described which should potentiate endomorphin-2-induced analgesia. Further, since dipeptidyl peptidase IV has an absolute requirement for -Pro, a more metabolically-stable -Pro²-endomorphin-2 analog should produce longer analgesic actions at lower doses. The present study found that endomorphin-2 was degraded approximately twice as fast than the chromogenic substrate, Ala-Pro-2naphthylamide, by dipeptidyl peptidase IV, whereas -Pro²-endomorphin-2 was totally resistant to this enzyme's action. -Pro²-endomorphin-2 (ED₅₀=0.05 μg) was more potent than endomorphin-2 (ED₅₀=30 μg) in significantly increasing tail-flick latencies with longer durations of action. Both the peptide and analogue were equipotent (ED₅₀=0.5 μg) in significantly increasing jump thresholds. Ala-Pyrrolidonyl-2-nitrile (10–75 nmol) elicited a dose-dependent analgesia, and potentiated the analgesic actions of endomorphin-2, particularly on the tail-flick test. Whereas systemic naltrexone (2.5, 10 mg/kg) dose-dependently eliminated each of the three forms of analgesia on the jump test as well as the peak (15 min) effect on the tail-flick test, analgesia elicited by either endomorphin-2, -Pro²-endomorphin-2 or Ala-Pyrrolidonyl-2-nitrile returned after 30–60 min in naltrexone-treated rats on the tail-flick test. These data strongly suggest that dipeptidyl peptidase IV plays a role in the inactivation of endomorphin-2 in vivo, and thereby modulates its central analgesic actions.     

Differential ontogeny of rat brain peptidases: prenatal expression of enkephalin convertase and postnatal development of angiotensin-converting enzyme

We quantitated the levels of two peptidases in the developing rat brain as a means to determine their function. Enkephalin convertase (EC 3.4.17.10), a carboxypeptidase B-like enzyme detected by [3H]guanidinoethylmercaptosuccinic acid (GEMSA) autoradiography, is present in high concentration throughout the brains of rat fetuses 3 days prior to birth. During the first 3 postnatal weeks, the density of [3H]GEMSA-labeled enkephalin convertase drops to adult levels. The expression of enkephalin convertase prior to that of most neuropeptides supports a role for this enzyme in propeptide processing. The regional distribution of [3H]GEMSA binding is similar in fetal and adult rats except that the thalamus exhibits the highest levels of [3H]GEMSA binding prenatally, and among the lowest levels in adult rats. Thus, peptide(s) formed in high concentration in the prenatal thalamus may be substrates for enkephalin convertase. Angiotensin-converting enzyme (ACE, EC 3.14.15.1) was visualized in the perinatal period by [3H]captopril autoradiography. Striatonigral ACE is undetectable at birth and increases to adult levels by two weeks of age. The expression of ACE after the initial presence of known peptides in the basal ganglia implies that the enzyme is not essential for peptide synthesis, suggesting instead a degradative role. In contrast to the striatonigral system, the choroid plexus contains high concentrations of ACE prior to birth, consistent with previous proposals of different substrates for ACE in the choroid plexus and the basal ganglia.     

Differential effects of 3-isobutyl-1-methylxanthine injected intrathecally or intracerebroventricularly on antinociception induced by opioids administered intracerebroventricularly in the mouse

Various doses of 3-isobutyl-1-methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor, injected intrathecally (i.t.) or intracerebroventricularly (i.c.v.) alone did not show any antinociceptive effect. IBMX (0.01 to 1 ng) pretreatment i.t. for 10 min dose-dependently attenuated the inhibition of the tail-flick response induced by i.c.v. administered morphine (2 μg), β-endorphin (1 μg), and U50, 488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzeocetamide), 60 μg. However, pretreatment with IBMX i.c.v. did not affect the inhibition of the tail-flick response induced by morphine, β-endorphin, and U50, 488H administered i.c.v. Neither i.c.v. nor i.t. pretreatment with IBMX attenuated the inhibition of the tail-flick response induced by D-Pen²-D-Pen⁵-enkephalin (DPDPE; 10 μg) administered i.c.v. Our results suggest that spinal but not supraspinal cAMP phosphodiesterases are involved in mediating antinociception induced by morphine, β-endorphin and U50, 488H administered supraspinally. However, neither spinal nor supraspinal cAMP phosphodiesterase is involved in mediating antinociception induced by DPDPE administered supraspinally.     

Antinociceptive effects of isoleucine derivatives of deltorphin I and deltorphin II in rat spinal cord: a search for selectivity of delta receptor subtypes

Deltorphins show a high affinity and selectivity for δ opioid receptors. Analogs of deltorphins with substitution of Val^5,6 residues with more hydrophobic lle^5,6 appear to have a higher in vitro activity and selectivity than parent deltorphins. In our study, changes in the nociceptive threshold after intrathecally injected deltorphin I (DELT I), deltorphin II (DELT II) and their lle^5,6-derivatives (ILE-DELT I and ILE-DELT II, respectively) were investigated in a tail-flick (TF) and a paw pressure (PP) tests. Male Wistars rats (260–350 g) with a chronically implanted catheter in the lumbar enlargement of the spinal cord were used. DELT I and DELT II, injected i.th. in doses of 0.15, 1.5 and 15 μg, increased the TF latency in a dose-dependent manner. The effect of their derivatives was similar, but the action of ILEDELT II was shorter than that of the parent peptide. In the PP test, the antinociceptive effects of DELT I and their derivative ILE-DELT I were similar, but the effect of a higher dose of ILE-DELT I lasted longer in comparison with the parent peptide. Both DELT II and ILE-DELT II exhibited a low and short-lasting antinociceptive potency in the PP test. The effect of DELT 1 (1.5 μg) was antagonized by pretreatment with NTI (30 μg), a non-selective δ opioid receptor antagonist, as well as by the δ2 receptor antagonist NTB (3 μg) and the δ1 antagonist BNTX (1 μg) in both those tests used. The antinociceptive effect of DELT II(1.5 μg) was antagonized by pretreatment with NTI (30 μg) and NTB (3 μg) in the TF test, but not in the PP test. In the latter test, the antinociceptive effect of DELT II was potentiated by pretreatment with BNTX (1 μg). The effects of both the derivatives ILE-DELT I and ILE-DELT II were antagonized by NTl (30 μg) in the TF test, and by NTI (30 μg) and NTB (3 μg) in the PP test. Like in the case of the parent peptide, the effect of ILE-DELT II was potentiated by pretreatment with the δ1 antagonist BNTX (1 μg). Summing up, modification of the DELT I and II by substituting lle^5,6 for Val^5,6 residues appears to influence the δ1 selectivity rather then the potency of the peptides at spinal δ receptors.     

Activation of central μ-opioid receptors is involved in clonidine analgesia in rats

The analgesic effect of clonidine in spontaneously hypertensive rats (SHR) and in normotensive Sprague-Dawley (SD) rats was assessed by using the formalin pain test. The analgesic response of SD rats to low doses (15–60 μg/kg i.p.) but not to a high dose (150 μg/kg i.p.) of clonidine was inhibited by naloxone, 2 mg/kg i.p., and a similar interaction was noted in SHR. In both rat strains, the analgesic response to low i.p. doses of clonidine was also inhibited by injection of 5 μg of naloxone or 7 μg of β-funaltrexamine, a μ-receptor antagonist, into the lateral cerebral ventricle. I.c.v. injection of 5 μg of ICI 174864, a δ-receptor antagonist, potentiated or did not influence clonidine analgesia in SD rats and SHR, respectively. It is concluded that the analgesic response to clonidine involves activation of central μ-opioid receptors in both SHR and SD rats, possibly by an endogenous opioid released by clonidine.