N-terminal tetrapeptide of dermorphin and D-Arg-substituted tetrapeptides: inactivation process of the antinociceptive activity by peptidase

Degradation products of the N-terminal tetrapeptide of dermorphin, H-Tyr-D-Ala-Phe-Gly-OH (ALPG) and D-Arg2-substituted tetrapeptide analogs of dermorphin, H-Tyr-D-Arg-Phe-Gly-OH (ARPG), H-Tyr-D-Arg-Phe-Gly-NH2 (TDAPG-NH2) and H-Tyr-D-Arg-Phe-beta-Ala-OH (TDAPA) by enkephalin degrading enzymes were studied by using reversed-phase high-performance liquid chromatography. After 5 and 25 hr incubations of the peptides with solubilized enzymes of mouse brain or spinal cord, liberation of the appreciable Tyr1 residue was observed in ALPG but not in ARPG, TDAPG-NH2 and TDAPA. When ARPG and TDAPG-NH2 were incubated with enzymes for 25 hr, a main degradation product was the N-terminal tripeptide produced from the hydrolysis of Phe3-Gly4 bond. Conversely, TDAPA did not produce the N-terminal tripeptide after 25 hr incubation with enzymes. In the enzyme assay, Tyr1-D-Arg2 bond of ARPG, TDAPG-NH2 and TDAPA was more stable than that of ALPG to the cleavage by aminopeptidase M (AP-M). Phe3-Gly4 bond of ALPG, ARPG and TDAPG-NH2 were easily hydrolyzed by carboxypeptidase Y (CP-Y) within 3 hr incubation, whereas the hydrolysis of Phe3-beta-Ala4 bond of TDAPA by CP-Y was not observed after 3 hr incubation. The present results and previous behavioural data suggest that a potent and prolonged antinociceptive activity of the D-Arg-substituted tetrapeptides is mainly attributed to the stability of Tyr1-D-Arg2 bond against aminopeptidase of peptidases.     

D-Arg2-dermorphin tetrapeptide analogs: a potent and long-lasting analgesic activity after subcutaneous administration

To examine pharmacological properties of D-Arg2-dermorphin tetrapeptides, six tetrapeptide analogs based on the following formulas, H-Tyr-D-Arg-Phe-Gly-OX (X = H, ethyl, n-propyl), H-Tyr-D-Arg-Phe-Sar-OX (Sar = sarcosine; X = H, methyl, ethyl), were prepared. All these analogs exhibited highly potent and long-lasting analgesia as compared with that of morphine after subcutaneous administration in mice. Among analogs tested, H-Tyr-D-Arg-Phe-Sar-OH showed the highest activities, which were 21, 30 and 58 times more active than morphine in the tail pressure, tail flick and phenylbenzoquinone writhing tests, respectively, on a molar basis.     

Environmental and intraperitoneal ethanol influences morphine antinociceptive effect in mice

The ability of acute environmental or intraperitoneal (i.p.) ethanol to influence morphine antinociceptive effect was studied in mice. In order to induce tolerance to morphine analgesia, mice received daily injections of 10 mg/Kg morphine over a period of 10 days. Mice were divided into three groups: i.p. ethanol (E), environmental ethanol (E*), and control saline (M). During the induction of tolerance these groups were treated identically except on days 1 and 11. On these days, 10 minutes prior to morphine injection, mice received either i.p. ethanol (1g/Kg), environmental ethanol (a bottle of 10% ethanol placed next to the animals cage during the experiments), or an equivalent volume of saline. Analgesia was assessed using a standard hot plate protocol and dose-response cumulative curves for morphine analgesia were obtained on days 1 and 11. On day 1, both the i.p. and environmental administration of ethanol showed similar morphine-potentiation effects [Mean Effective Dose: ED50 (M1)=4.5 mg/kg; ED50 (E1)=2.4 mg/kg; ED50 (E*1)=2.1 mg/kg]. On day 11, control group mice showed a reduction of morphine analgesia at test [ED50 (M11)=14.1 mg/kg]. Mice receiving i.p. and environmental ethanol again showed a leftward shift in dose-response cumulative curves for morphine antinociception with respect to controls [ED50 (E11)=9.1 mg/kg; ED50 (E*11)=4.7 mg/kg]. I.p. ethanol administration at non-antinociceptive doses enhances the morphine antinociception effect similarly in tolerant and non-tolerant (naive) mice. The presence of environmental ethanol can also induce a similar pattern of increase in morphine antinociception effect.     

Modulation of acute morphine tolerance by corticotropin-releasing factor and dynorphin A in the mouse spinal cord.

Previously, we have demonstrated that intrathecally (i.t.) administered corticotropin-releasing factor (CRF) in mice produces stimulus-specific antinociception and modulation of morphine-induced antinociception by mechanisms involving spinal kappa opioid receptors. Recently, we also have found that CRF releases immunoreactive dynorphin A, a putative endogenous kappa opioid receptor agonist, from superfused mice spinal cords in vitro. Dynorphin A administered intracerebroventricularlly (i.c.v.) to mice has been shown to modulate the expression of morphine tolerance. In the present study, the possible modulatory effects of i.t. administered CRF as well as dynorphin A on morphine tolerance were studied in an acute tolerance model. Subcutaneous administration of 100 mg/kg of morphine sulfate (MS) to mice caused an acute tolerance to morphine-induced antinociception. The antinociceptive ED50 of MS was increased from 4.4 mg/kg (naive mice) to 17.9 mg/kg (4 hours after the injection of 100 mg/kg MS). To study the modulatory effects of spinally administered CRF and dynorphin A on the expression of morphine tolerance, CRF and dynorphin A were injected i.t. at 15 min and 5 min, respectively, before testing the tolerant mice by the tail-flick assay. The antinociceptive ED50 of MS in tolerant mice was decreased to 8.8 mg/kg and 7.1 mg/kg, respectively, after i.t. administration of CRF (0.1 nmol) and dynorphin A (0.2 nmol). In contrast, 0.5 nmol of alpha-helical CRF (9-41), a CRF antagonist and 0.4 nmol of norbinaltorphimine, a highly selective kappa opioid receptor antagonist, when administered i.t. at 15 min before the tail-flick test in tolerant mice, increased the antinociceptive ED50 of MS to 56.6 mg/kg and 88.8 mg/kg, respectively. These data confirmed the modulatory effect of dynorphin A on morphine tolerance and suggested that CRF, which releases dynorphin A in several central nervous system regions, also plays a modulatory role in the expression of morphine tolerance.     

Antinociceptive effects of [D-Ala2]deltorphin II, a highly selective delta agonist in vivo

The present study has characterized the antinociceptive actions of [D-Ala2]deltorphin II following intracerebroventricular (i.c.v.) administration in the mouse tail-flick test. [D-Ala2]deltorphin II produced dose- and time-related antinociception, with maximal effects at +10 min and significant antinociception which lasted for 40-60 min. [D-Ala2]deltorphin II was 13-fold more potent than i.c.v. [D-Pen2, D-Pen5]enkephalin (DPDPE), a second highly selective delta agonist, and approximately equipotent with i.c.v. morphine in producing antinociception. The antinociceptive effects of i.c.v. [D-Ala2]deltorphin II and DPDPE, but not those of morphine, were antagonized by the selective delta antagonist, ICI 174,864. In contrast, pretreatment with the non-equilibrium mu antagonist, beta-funaltrexamine blocked morphine antinociception, but failed to antagonize [D-Ala2]deltorphin II and DPDPE antinociception. These data indicate that [D-Ala2]deltorphin II produced its antinociceptive effects at a supraspinal delta receptor. [D-Ala2]deltorphin II appears to be the most appropriate delta opioid agonist currently available for studies in vivo and support the involvement of delta receptors in supraspinal antinociception.     

Effects of neomycin on the development of tolerance to morphine antinociception.

The effects of neomycin on the development of tolerance to morphine antinociception were examined in mice. Because neomycin did not readly cross blood brain barrier, we examined the effects of neomycin following systemic, intracerebroventricular (i.c.v.) and intrathecal (i.t.) injections on the morphine tolerance. Daily subcutaneous (s.c.), i.c.v. and i.t. injections of morphine produced tolerance regardless of route of administration. Both i.c.v. and i.t. neomycin, which alone produced no changes in the basal tail flick latencies, significantly attenuated the development of tolerance to antinociception produced by repeated systemic morphine, while intraperitoneal (i.p.) administration of neomycin did not affect morphine tolerance. Further, i.c.v. and i.t. neomycin attenuated the development of tolerance to antinociception produced by repeated i.c.v. and i.t. morphine, respectively, which were not attenuated by systemic neomycin. This results indicate a potential role for neomycin-sensitive Ca2+ channels on the development of tolerance to the morphine antinoception.     

Characterization of intrathecal vasopressin-induced antinociception, scratching behavior, and motor suppression.

Intrathecal (IT) administration of vasopressin produces antinociception, scratching behavior, and motor suppression. The present experiments characterized these effects with regards to the following: 1) VP receptor specificity, 2) possible involvement of endogenous opiates, 3) possible involvement of seizure activity, and 4) whether the antinociception is due to direct actions of VP at the spinal cord. These studies showed that IT administration of a V1-specific vasopressin antagonist completely blocked the antinociception, scratching behavior, and motor suppression produced by 25 ng IT vasopressin. Furthermore, IT administration of the vasopressin metabolite, [pGlu4,Cyt6]AVP(4-9), produced none of the effects produced by vasopressin. Systemic administration of the opiate antagonists naloxone (1 mg/kg IP) and naltrexone (10 mg/kg IP) had no significant effect on the antinociception produced by IT vasopressin, whereas naltrexone potentiated the scratching behavior. Neither the IT vasopressin-induced antinociception nor scratching behavior was affected by pretreatment with the anticonvulsant sodium valproate. In addition, IT vasopressin inhibited the tail flick reflex in rats with transected spinal cords, demonstrating direct spinal effects of vasopressin. In conclusion, IT administration of vasopressin produces antinociception, scratching behavior, and motor suppression via activation of VP-specific receptors in the spinal cord.     

Involvement of kappa/dynorphin system in the development of tolerance to nicotine-induced antinociception

The aim of the present study was to explore the possible role of kappa/dynorphin system in the development of tolerance to nicotine antinociception in mice. First, we observed that kappa-opioid receptor (KOP-r) participates in the acute spinal antinociception produced by nicotine (3 and 5 mg/kg, s.c.) since the pre-treatment with the selective kappa antagonist nor-binaltorphimine (3 mg/kg, i.p.) attenuated this response in the tail-immersion test but not in the hot-plate test nor in locomotor responses. Possible changes in the expression of KOP-r were investigated in tolerant mice to nicotine antinociception by using autoradiography of [³H]CI-977 binding. The density of KOP-r decreased in the spinal cord of tolerant mice. In addition, bi-directional cross-tolerance between nicotine (3 and 5 mg/kg, s.c.) and the selective kappa agonist U50,488H (10 mg/kg, s.c.) was found in the tail-immersion test. Recent evidences indicate that an up-regulation of dynorphin levels in the spinal cord and subsequent activation of NMDA receptors participate in the development of tolerance to opioid and cannabinoid antinociception. In this study, dynorphin content in the lumbar spinal cord was similar in control and nicotine tolerant mice. Furthermore, the administration of the NMDA antagonist MK-801 (0.03 and 0.01 mg/kg, i.p.) before each daily nicotine injection did not modify the development of nicotine tolerance. In summary, these data indicate that KOP-r is directly involved in the development of tolerance to nicotine antinociception by a mechanism independent from dynorphin and NMDA receptors.     

Chimeric peptide of Met-enkephalin and FMRFa induces antinociception and attenuates development of tolerance to morphine antinociception.

A synthetic chimeric peptide of Met-enkephalin and FMRFamide (YGGFMKKKFMRFa), based on MERF was synthesized. This peptide was tested for possible antinociceptive effects using the tail flick test in mice. The effect of the chimeric peptide on morphine antinociception and development of tolerance to the antinociceptive action of morphine was also investigated. The chimeric peptide produced significant, dose-dependent antinociception (40, 60 and 90 mg/kg) in the tail flick test. Pretreatment with naloxone (5 mg/kg, IP) significantly attenuated the antinociceptive effect induced by the chimeric peptide (90 mg/kg, IP), indicating involvement of an opioidergic mechanism. In combination experiments with morphine, the antinociceptive dose of the chimeric peptide (60 mg/kg, IP) potentiated morphine (7 mg/kg, IP) antinociception. A low dose of the chimeric peptide (10 mg/kg, IP), that did not produce significant antinociception on its own, also potentiated morphine antinociception. In the tolerance studies, male albino mice received twice daily injections of morphine (20 mg/kg, IP) followed by either saline (0.1 ml) or chimeric peptide (80 mg/kg, IP) for a period of 4 days. A control group received twice daily injections of saline (0.1 ml) for the same period. When tested on Day 5, tolerance to antinociceptive action of morphine (15 mg/kg, IP) was evidenced by decreased response in chronic morphine plus saline treated mice compared to control group. Concurrent administration of chimeric peptide (80 mg/kg, IP) with morphine significantly attenuated the development of tolerance to the antinociceptive action of morphine. The preliminary results of this study demonstrate that peripherally administered chimeric peptide can produce dose dependent, naloxone reversible, antinociception; potentiate morphine antinociception and attenuate morphine tolerance, indicating a possible role of these type of amphiactive sequences in antinociception and its modulation. These chimeric peptides may also prove to be useful tools for further ascertaining the role of FMRFa family of peptides in mechanisms leading to opiate tolerance and dependence.     

Substance P antagonist-induced spinal cord vasoconstriction: Effects of thyrotropin-releasing hormone and substance P agonists

Local spinal cord vasomotor effects of 3 substance P (SP) antagonists were studied in the rat following intrathecal (IT) administration. Each SP antagonist (3.3 nmol) increased spinal cord vascular resistance and reduced blood flow. A LH-RH antagonist analog (10 nmol) of similar molecular weight and which also contained multiple D-Trp residues did not cause spinal cord vasoconstriction. The vasoconstrictor action of the SP antagonist, [D-Arg¹, D-Pro², D-Trp^7,9, Leu¹¹]-SP ([D-Arg]-SP) was unaffected by pretreatment with a stable SP receptor agonist (5 nmol IT). Given evidence for a cerebral vasodilator action of TRH agonists, the effects of TRH (IV) and a stable TRH analog (MK-771, IT) on [D-Arg]-SP-induced vasoconstriction were also assessed. Neither TRH nor MK-771 prevented the [D-Arg]-SP-induced vasoconstriction. However, TRH (IV) but not MK-771 (IT) partially opposed [D-Arg]-SP-induced reduction in thoracic spinal cord blood flow. Thus, SP antagonists cause spinal cord vasoconstriction by a non-SP receptor mediated phenomenon. In addition, the attenuation of SP-antagonist-induced neuropathological changes previously reported with IV. TRH administration is likely due to less severe consequences of vasoconstriction in the presence of a higher initial baseline blood flow rather than direct prevention of the vasoconstriction.     

Effects of acute and chronic morphine and narcotic antagonists on brain energy metabolism

Morphine sulphate (4 mg/kg to 32 mg/kg) produced a dose-dependent decrease in brain malate as antinociception increased. Decreased brain malate persisted 72 hours after implantation of morphine pellets by which time mice had become tolerant to antinociception. This finding suggests that malate decrease, unlike changes of other metabolites in other studies, might not be simply a result of general metabolic changes. Malate change as well as antinociception was prevented by prior injection of naloxone (3.0 mg/kg) or naltrexone (0.6 mg/kg) in acute experiments. Malate decrease in pelleted mice was no longer present if withdrawal was produced by naloxone or naltrexone in mice implanted with morphine pellets for 72 hours. Brain P-creatine was elevated in all mice implanted with morphine pellets even after withdrawal, thus, apparently, representing a more generalized effect than malate change.     

Effect of methamphetamine on the development of acute morphine tolerance and dependence in mice

The effect of methamphetamine on morphine analgesia (tail-flick assay) was studied in non-tolerant mice and in mice made acutely tolerant to morphine following a single injection of 100 mg/kg morphine. The analgesic potency of morphine was increased in non-tolerant and tolerant mice to the same extent by 3.2 mg/kg methamphetamine (3.3 and 4.4 fold increases, respectively). In contrast, the ED50's for morphine analgesia and naloxone-precipitated jumping in mice pretreated with either 100 mg/kg morphine or both morphine and 3.2 mg/kg methamphetamine were not significantly different, indicating that methamphetamine had no effect on the development of acute morphine tolerance and dependence. Although methamphetamine had no effect on the development of acute tolerance to morphine, 4-day pretreatment with methamphetamine produced cross-tolerance to morphine analgesia. However, cross-tolerance to morphine was not accompanied by enchanced sensitivity to naloxone.     

Morphine tolerance in mice changes response of heroin from mu to delta opioid receptors

Heroin produced antinociception in the tail flick test through mu receptors in the brain of ICR and CD-1 mice, a response inhibited by 3-O-methylnaltrexone. Tolerance to morphine was produced by subcutaneous morphine pellet implantation. By the third day, the heroin response was produced through delta opioid receptors. The response was inhibited by simultaneous intracerebroventricular (i.c. v.) administration of naltrindole, a delta opioid receptor antagonist. More specifically, delta1 rather than delta2 receptors were involved because 7-benzylidenenaltrexone, a delta1 receptor antagonist, inhibited but naltriben, a delta2 antagonist, did not. Also, antinociception produced by i.c.v. heroin was inhibited by intrathecal administration of bicuculline and picrotoxin consistent with the concept that delta1 receptors in the brain mediated the antinociceptive response through descending neuronal pathways to the spinal cord to activate GABAA and GABAB receptors rather than spinal alpha2-adrenergic and serotonergic receptors activated originally by the mu agonist action in naive mice. The mu response of 6-monoacetylmorphine, a metabolite of heroin, was changed by morphine pellet implantation to a delta2 response (inhibited by naltriben but not 7-benzylidenenaltrexone). The agonist action of morphine in these morphine-tolerant mice remained mu. Thus, the opioid receptor selectivity of heroin and 6-monoacetylmorphine in the brain is changed by production of tolerance to morphine. Such a change explains how morphine tolerant mice are not cross-tolerant to heroin.     

Heterologous mu-opioid receptor adaptation by repeated stimulation of kappa-opioid receptor: up-regulation of G-protein activation and antinociception

The present study was designed to investigate the effect of repeated administration of a selective kappa-opioid receptor agonist (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride [(-)U-50,488H] on antinociception and G-protein activation induced by mu-opioid receptor agonists in mice. A single s.c. injection of (-)U-50,488H produced a dose-dependent antinociception, and this effect was reversed by a selective kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Furthermore, a single s.c. pre-treatment with (-)U-50,488H had no effect on the mu-opioid receptor agonist-induced antinociception. In contrast, repeated s.c. administration of (-)U-50,488H resulted in the development of tolerance to (-)U-50,488H-induced antinociception. Under these conditions, we demonstrated here that repeated s.c. injection of (-)U-50,488H significantly enhanced the antinociceptive effect of selective mu-opioid receptor agonists endomorphin-1, endomorphin-2 and [d-Ala2,N-MePhe4,Gly-ol5] enkephalin (DAMGO). Using the guanosine-5'-o-(3-[35S]thio) triphosphate ([35S]GTP gamma S) binding assay, we found that (-)U-50,488H was able to produce a nor-BNI-reversible increase in [35S]GTP gamma S binding to membranes of the mouse thalamus, which has a high level of kappa-opioid receptors. Repeated administration of (-)U-50,488H caused a significant reduction in the (-)U-50,488H-stimulated [35S]GTP gamma S binding in this region, whereas chronic treatment with (-)U-50,488H exhibited the increase in the endomorphin-1-, endomorphin-2- and DAMGO-stimulated [35S]GTP gamma S bindings in membranes of the thalamus and periaqueductal gray. These results suggest that repeated stimulation of kappa-opioid receptors leads to the heterologous up-regulation of mu-opioid receptor functions in the thalamus and periaqueductal gray regions, which may be associated with the supersensitivity of mu-opioid receptor-mediated antinociception.     

Pharmacological manipulation of gamma-aminobutyric acid (GABA) in morphine analgesia,tolerance and physical dependence

The findings from our laboratory indicated that pharmacological manipulations of GABA system modified morphine analgesia, tolerance and physical dependence. Elevating brain levels of GABA by slowing its destruction with aminooxyacetic acid not only antagonized the analgesic action of morphine in both non-tolerant and tolerant mice, but also enhanced the development of tolerance and physical dependence. On the other hand, blockade of postsynaptic sites of GABA receptors by bicuculline resulted in an inhibition of tolerance and dependence development. Administration of 2,4-diaminobutyric acid, an inhibitor of GABA uptake in the neurons, antagonized morphine analgesia in both non-tolerant and tolerant mice. However, it did not modify naloxone precipitated withdrawal jumping. On the contrary, β-alanine, an inhibitor of the GABA uptake process in glial cells, potentiated naloxone precipitated withdrawal jumping in morphine dependent mice, but it had no effect on morphine antinociception in both non-tolerant and tolerant mice.     

Neurotensin-induced antinociception and hypothermia in mice: antagonism by TRH and structural analogs of TRH

Intracisternal (IC) administration of neurotensin (NT) in a dose of 10 micrograms produced a significant hypothermia and antinociception in the hot-plate test in mice. Both of these effects of IC NT were completely antagonized by concomitant administration of equimolar doses of thyrotropin-releasing hormone (TRH) and several TRH congeners including 3-methyl-His-TRH (pGlu-3-methyl-His-Pro-NH2), MK-771 (pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide), beta-ala-TRH (pGlu-His-Pro-beta-ala-NH2), and RX-77368 (pGlu-His-dimethyl-Pro-NH2). The antagonism by TRH and TRH analogs on NT-induced hypothermia and antinociception was dose-dependent. Of particular interest was the finding that RX-77368 not only blocked the effects of NT but also produced hyperalgesia. It appears that TRH analogs that are more resistant to biologic degradation are, like TRH, capable of blocking NT-induced behaviors.     

Synthesis, biological activity and conformation of enkephalin analogs structurally related to kyotorphin

[D-Arg2,Leu5]Enkephalin and two series of its N-terminal short-chain analogues with a free and modified C-terminal carboxylic group, viz. amides and ethyl esters of tri- and tetrapeptides, were synthesized in solution and by solid-phase method. Their analgesic activity, assayed by the "tail pinch" method following intracisternal and intravenous administration to mice, was compared with activity of enkephalins and morphine. To study the space structure of the synthesized compounds, conformational calculations and fluorescence spectroscopy were applied to measure distance between aromatic nuclei of tyrosine and phenylalanine residues in the two tetrapeptides. Ethyl esters of the tri- and tetrapeptides exceed in analgesic activity the corresponding carboxylic acids and amides. In contrast to the pentapeptide, the tetrapeptide analogues were active upon intravenous administration. Conformational aspects of this series of analogues are discussed in detail; the abrupt increase in activity upon transition from tri- to tetrapeptides does not appear to be related to conformational changes.     

Further Characterization of a Novel Tetrapeptide with an Analgesic Action in the Central and Peripheral Nervous System in Rats

The novel tetrapeptide FLPS has been previously shown to induce antinociception in a model of post-incisional pain in the rat. It has been demonstrated in membrane preparations of rat brain that 10 μM tetrapeptide did not compete with [³H]naloxone for its binding site on the opioid receptors which differentiates it from typical opioids. In the first set of experiments, the stimulation of [³⁵S]GTPγS binding by the tetrapeptide to membrane preparations of recombinant cell lines expressing human μ, δ, and κ receptors has been investigated. The specific [³⁵S]GTPγS binding did not change in the presence of 100 μM tetrapeptide and DAMGO, DPDPE, or U-69593 at submaximal and maximal concentrations, indicating that the tetrapeptide was not an agonist or antagonist of the opioid receptors and it did not stimulate or blocked the stimulation of G-proteins coupled to these receptors. Studies with naloxone and naloxone methiodide pretreatment suggested that the tetrapeptide was either directly or indirectly affected by a naloxone-binding site located primarily within the central nervous system and to a lesser extent in the periphery. Naloxone pretreatment had a dual effect on morphine antinociception based on the concentration used. At 2 mg/kg, naloxone competed reversibly with 25 mg/kg morphine on its binding site on the opioid receptors, while at 5.4 mg/kg it blocked antinociception induced by 10 mg/kg morphine, 2.7 mg/kg aspirin, or 75 mg/kg tetrapeptide. These data suggest that a new naloxone-binding site is involved in alleviation of pain by at least three classes of analsegics (opioids, cycloogenase 2 inhibitors, and the tetrapeptide). The biological activity of the peptide was discovered first and the mechanism of action is now being studied. In an attempt to identify the therapeutic target of the tetrapeptide, a total of 23 radioligand binding assays to targets within the CNS were conducted. The results of these assays were negative and they reinforce the notion that the tetrapeptide activates an unknown mechanism involved in pain perception after surgery.     

The role of T-type calcium channels in morphine analgesia,development of antinociceptive tolerance and dependence to morphine,and morphine abstinence syndrome

Involvement of T-type voltage dependent Ca2+ channels (VDCCs) on morphine antinociception, in the development of tolerance and dependence to morphine, and naloxone-precipitated abstinence syndrome in morphine dependent mice was examined by using mibefradil, a T-type VDCCs blocker. Mice were rendered tolerant and dependent on morphine by subcutaneous (s.c.) implantation of a morphine pellet containing 75 mg of morphine base for 72 hr. The tail-flick test was used to assess the nociceptive threshold. Coadministration of acute mibefradil (10 mg/kg, i.p.) with morphine enhanced the antinociceptive effects of acute morphine. Repeated mibefradil administration (10 mg/kg, i.p., just before, 24 and 48 hr after morphine pellet implantation) completely blocked the development of tolerance to the antinociceptive effect of morphine and even by this effect reached supersensitivity to morphine. However, repeated mibefradil treatment did not alter the development of dependence to morphine assessed by the A(50) values of naloxone (s.c.) required to precipitate withdrawal jumping 72 hr after morphine pellet. But, acute mibefradil (10, 30, and 50 mg/kg, i.p.) dose dependently decreased the expression of morphine abstinence syndrome when given directly 30 min prior to naloxone (0,05 mg/kg, s.c.) 72 hr after morphine pellet. These results indicate a critical role of T-type VDCCs in morphine antinociception, the development of tolerance to the antinociceptive effects of morphine and in morphine abstinence syndrome.     

Evidence that ginsenosides prevent the development of opioid tolerance at the central nervous system

The analgesic effect of ginsenosides or morphine was first determined following intrathecal (i.t.) administration in rat tail-flick test. The effect of chronic i.t. co-administration of ginsenosides with morphine on the development of opioid tolerance were also examined using rat tail-flick test. Administration of ginsenosides (i.t.) produced a weak antinociception in a dose-dependent manner. Administration of morphine (i.t.) also produced antinociception in a dose-dependent manner. The ED50 was 1.20 microg (1.14-1.29 microg). However, acute i.t. co-administration of ginsenosides with morphine was not additive in antinociception. Repeated i.t. co-administration of 200 microg ginsenosides with 10 microg morphine inhibited the development of tolerance induced by 10 microg morphine in rat tail-flick test, although i.t. co-administration of 50 or 100 microg ginsenosides with morphine was without effect. In conclusion, these results indicate that i.t. administered ginsenosides produce an antinociception in rat tail-flick test and also prevent opioid tolerance caused by chronic treatment with morphine at the spinal sites.