Endogenous Opioid Signaling in the Mouse Retina Modulates Pupillary Light Reflex.

Opioid peptides and their receptors are expressed in the mammalian retina; however, little is known about how they might affect visual processing. The melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), which mediate important non-image-forming visual processes such as the pupillary light reflex (PLR), express ß-endorphin-preferring, µ-opioid receptors (MORs). The objective of the present study was to elucidate if opioids, endogenous or exogenous, modulate pupillary light reflex (PLR) via MORs expressed by ipRGCs. MOR-selective agonist [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAMGO) or antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) was administered via intravitreal injection. PLR was recorded in response to light stimuli of various intensities. DAMGO eliminated PLR evoked by light with intensities below melanopsin activation threshold but not that evoked by bright blue irradiance that activated melanopsin signaling, although in the latter case, DAMGO markedly slowed pupil constriction. CTAP or genetic ablation of MORs in ipRGCs slightly enhanced dim-light-evoked PLR but not that evoked by a bright blue stimulus. Our results suggest that endogenous opioid signaling in the retina contributes to the regulation of PLR. The slowing of bright light-evoked PLR by DAMGO is consistent with the observation that systemically applied opioids accumulate in the vitreous and that patients receiving chronic opioid treatment have slow PLR.

Inhibiting the breakdown of endogenous opioids and cannabinoids to alleviate pain.

Chronic pain remains unsatisfactorily treated, and few novel painkillers have reached the market in the past century. Increasing the levels of the main endogenous opioid peptides - enkephalins - by inhibiting their two inactivating ectopeptidases, neprilysin and aminopeptidase N, has analgesic effects in various models of inflammatory and neuropathic pain. Stemming from the same pharmacological concept, fatty acid amide hydrolase (FAAH) inhibitors have also been found to have analgesic effects in pain models by preventing the breakdown of endogenous cannabinoids. Dual enkephalinase inhibitors and FAAH inhibitors are now in early-stage clinical trials. In this Review, we compare the effects of these two potential classes of novel analgesics and describe the progress in their rational design. We also consider the challenges in their clinical development and opportunities for combination therapies.

A2A receptor knockout worsens survival and motor behaviour in a transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative genetic disorder that leads to motor, cognitive, and psychiatric disturbances. The primary neuropathological hallmark is atrophy of the striatum. HD preferentially affects efferent striato-pallidal neurons that express enkephalin as well as dopamine D2 and A(2A) adenosine receptors (A(2A)Rs). Expression and function of A(2A)Rs are altered in HD but, despite being an important modulator of the striato-pallidal function, the subsequent pathophysiological consequence of such changes remains unclear. Whether blockade of A(2A)Rs is of therapeutic interest in HD remains ill-defined. In the present work, we aimed to determine the pathophysiological consequences of genetic deletion of A(2A)Rs in HD by crossing A(2A)R knockout mice with the N171-82Q HD transgenic model. Our data demonstrate that knockout of A(2A)Rs moderately but significantly worsens motor performances and survival of N171-82Q mice and leads to a decrease in striatal enkephalin expression. These results support that early and chronic blockade of A(2A)Rs might not be beneficial in HD.

The effect of botulinum neurotoxin A on sciatic nerve injury-induced neuroimmunological changes in rat dorsal root ganglia and spinal cord.

Botulinum neurotoxin serotype A (BoNT/A) acts by cleaving synaptosome-associated-protein-25 (SNAP-25) in nerve terminals to inhibit neuronal release and shows long-lasting antinociceptive action in neuropathic pain. However, its precise mechanism of action remains unclear. Our study aimed to characterize BoNT/A-induced neuroimmunological changes after chronic constriction injury (CCI) of the sciatic nerve. In the ipsilateral lumbar spinal cords of CCI-exposed rats, the mRNA of microglial marker (complement component 1q, C1q), astroglial marker (glial fibrillary acidic protein, GFAP), and prodynorphin were upregulated, as measured by reverse transcription-polymerase chain reaction (RT-PCR). No changes appeared in mRNA for proenkephalin, pronociceptin, or neuronal and inducible nitric oxide synthase (NOS1 and NOS2, respectively). In the dorsal root ganglia (DRG), an ipsilateral upregulation of prodynorphin, pronociceptin, C1q, GFAP, NOS1 and NOS2 mRNA and a downregulation of proenkephalin mRNA were observed. A single intraplantar BoNT/A (75 pg/paw) injection induced long-lasting antinociception in this model. BoNT/A diminished the injury-induced ipsilateral spinal upregulation of C1q mRNA. In the ipsilateral DRG a significant decrease of C1q-positive cell activation and of the upregulation of prodynorphin, pronociceptin and NOS1 mRNA was also observed following BoNT/A admistration. BoNT/A also diminished the injury-induced upregulation of SNAP-25 expression in both structures. We provide evidence that BoNT/A impedes injury-activated neuronal function in structures distant from the injection site, which is demonstrated by its influence on NOS1, prodynorphin and pronociceptin mRNA levels in the DRG. Moreover, the silence of microglia/macrophages after BoNT/A administration could be secondary to the inhibition of neuronal activity, but this decrease in neuroimmune interactions could be the key to the long-lasting BoNT/A effect on neuropathic pain.

Morphine-like opiates selectively antagonize receptor-arrestin interactions.

The addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of mu and delta receptors with G protein or beta-arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affected by receptor signaling. To assess receptor-G protein coupling without competition from arrestins, we employed a cell-free BRET assay using membranes isolated from cells expressing luminescent receptors and fluorescent Gbeta(1). In this system, the agonist-induced enhancement of BRET (indicating shortening of distance between the two proteins) was G alpha-mediated (as shown by sensitivity to pertussis toxin and guanine nucleotides) and yielded data consistent with the known pharmacology of the ligands. We found marked differences of efficacy for G protein and arrestin, with a pattern suggesting more restrictive structural requirements for arrestin efficacy. The analysis of such differences identified a subset of structures showing a marked discrepancy between efficacies for G protein and arrestin. Addictive opiates like morphine and oxymorphone exhibited large differences both at delta and mu receptors. Thus, they were effective agonists for G protein coupling but acted as competitive enkephalins antagonists (delta) or partial agonists (mu) for arrestin. This arrestin-selective antagonism resulted in inhibition of short and long term events mediated by arrestin, such as rapid receptor internalization and down-regulation.

Delta opioid receptor-mediated immunoregulatory role of methionine-enkephalin in freshwater teleost Channa punctatus (Bloch.).

The immunoregulatory role of methionine-enkephalin (Met-enk) is well studied in mammals, but has not been explored in ectotherms despite the fact that this peptide is highly conserved in vertebrates. The present study demonstrates the diverse effects of Met-enk depending on its concentration and specific function of splenic phagocytes in the freshwater fish Channa punctatus. Although Met-enk increased both phagocytic as well as respiratory burst activity, the concentration-related response was opposite to each other. It had the maximum stimulatory effect on phagocytosis at 10(-9)M, while the same concentration was least effective in increasing superoxide production. Similarly, Met-enk at concentrations lower or higher than 10(-9)M was either ineffective or less effective in case of phagocytosis, while highly effective in stimulating superoxide production. On the other hand, concentration-independent inhibitory effect of Met-enk was observed in case of nitrite production. Nonetheless, Met-enk regulated all the functions of phagocyte through opioid receptors since non-specific opioid receptor antagonist naltrexone completely blocked the effect of Met-enk on phagocytosis, superoxide and nitrite production by splenic phagocytes of C. punctatus. Among selective opioid receptor antagonists, delta-opioid receptor antagonist naltrindole completely antagonized the effect of Met-enk on phagocytosis, superoxide and nitrite production, while mu- and kappa-opioid receptor antagonist, CTAP and norbinaltorphimine, respectively, were ineffective in influencing any of the functions. This suggests that Met-enk modulates splenic phagocyte functions in the fish C. punctatus via delta-opioid receptor. This is further substantiated by using highly selective delta-opioid receptor agonist, SNC80.

Protection of endogenous enkephalin catabolism as natural approach to novel analgesic and antidepressant drugs.

The most efficient drugs to alleviate severe pain are opioid compounds. However, their chronic use could be associated with serious drawbacks, such as tolerance, respiratory depression and constipation. Therefore, there is a need for compounds able to efficiently alleviate inflammatory and neurogenic pain following chronic treatment. The discovery that the endogenous opioid peptides, enkephalins, are inactivated by two metallopeptidases, neutral endopeptidase and aminopeptidase N, which can be blocked by synthetic dual inhibitors, represents a promising way to develop 'physiological' analgesics devoid of morphine side effects. These dual inhibitors also have antidepressant-like properties through enkephalin-related activation of delta-opioid receptors. This is expected to reduce the emotional component of pain in humans. This article reviews the promising data obtained for future development of a new class of analgesic that could be of major interest in a number of severe and chronic pain syndromes.

Species-specific effects on hemolymph glucose control by serotonin, dopamine, and L-enkephalin and their inhibitors in Squilla mantis and Astacus leptodactylus (crustacea).

Hemolymph glucose level is controlled by crustacean Hyperglycemic Hormone (cHH) released from the eyestalk neuroendocrine centers under conditions of both physiological and environmental stress. Biogenic amines and enkephalin have been found to mediate the release of several neurohormones from crustacean neuroendocrine tissue. We investigated the effect of serotonin, dopamine, and Leucine-enkephalin in vivo--injected into the stomatopod Squilla mantis and the decapod Astacus leptodactylus--whether increasing or depressing glycemia. Serotonin had a marked effect in elevating glucose level compared with initial values in both species. 5-HT1-like receptors are more involved in mediating serotonin action as co-injected cyproheptadine was a more effective antagonist than ketanserin (5-HT2-like receptor inhibitor). Dopamine injection in intact animals produced a decrease below initial levels of hemolymph glucose. This effect was significantly antagonized by domperidone. No significant effect of both amines occurred in eyestalkless animals. L-enkephalin shows a differential effect: in S. mantis it induced hypoglycemia while in A. leptodactylus it caused an increase of glucose level. Co-injected antagonist naloxone affected the direction of the response. Serotonin appears to provide a major control on glucose mobilization, whereas dopamine and L-enkephalin act as modulators whose plasticity in use or action varies among species.

Reversal of ongoing thermal hyperalgesia in mice by a recombinant herpesvirus that encodes human preproenkephalin.

Herpesvirus-mediated transfer of the human preproenkephalin gene to primary afferent nociceptors prevents phasic thermal allodynia/hyperalgesia in mice. It is not known, however, whether similar viral treatments would reverse ongoing or chronic pain and allodynia/hyperalgesia. To this end, mice were given intrathecal injections of pertussis toxin (PTX), which produces a weeks-long thermal hyperalgesia apparently by uncoupling certain G proteins from inhibitory neurotransmitter receptors. This treatment produced profound thermal hyperalgesia in both Adelta and C-fiber thermonociceptive tests lasting at least 6 weeks. However, treatment of skin surfaces with an enkephalin-encoding herpesvirus, but not control virus or vehicle, completely reversed this hyperalgesia. This profound anti-hyperalgesia was observed for both Adelta- and C-fiber-mediated responses. Interestingly, however, while the anti-hyperalgesic effect of the enkephalin-encoding virus on C-fiber-mediated responses was reversed by intrathecal application of micro or delta opioid antagonists, only delta antagonists reversed the effect of this virus on Adelta hyperalgesia. Thus, virus-mediated delivery of the proenkephalin cDNA reverses thermal hyperalgesia produced by PTX-induced ribosylation of inhibitory G proteins by an opioid-mediated mechanism. These results suggest that herpesvirus vectors encoding analgesic peptides may be useful in attenuating centrally mediated, ongoing neuropathic pain and/or hyperalgesia.

Further evidence for the interaction of mu- and delta-opioid receptors in the antinociceptive effects of the dual inhibitor of enkephalin catabolism, RB101(S). A spinal c-Fos protein study in the rat under carrageenin inflammation.

We have previously shown that RB101, a dual inhibitor of enkephalin-degrading enzymes, decreased carrageenin-evoked c-Fos protein expression at the spinal cord level in awake rats. Moreover, we have also shown that c-Fos expression is a useful marker of the possible direct or indirect interactions between neural pathways, such as opioid and cholecystokinin systems. We now investigated the respective roles of the three main types of opioid receptors (mu, delta, or kappa) and their possible interactions, in the depressive effects of RB101 in inflammatory nociceptive conditions induced by intraplantar carrageenin (6 mg/150 microl of saline). We used beta-funaltrexamine (beta-FNA), naltrindole (NTI), and nor-binaltorphimine (BNI) as specific antagonists for mu, delta- and kappa-opioid receptors, respectively. c-Fos protein-immunoreactivity (c-Fos-IR) was evaluated as the number of c-Fos-IR nuclei in the lumbar spinal cord 90 min after carrageenin. c-Fos-IR nuclei were preferentially located in the superficial (I-II) and deep (V-VI) laminae of segments L4-L5 (areas containing numerous neurons responding exclusively, or not, to nociceptive stimuli). RB101(S) (30 mg/kg, i.v.) significantly reduced the total number of carrageenin-evoked c-Fos-IR nuclei (30% reduction, P<0.01). This effect was completely blocked by beta-FNA (10 mg/kg, i.v.), or NTI (1 mg/kg, i.v.). In contrast, BNI (2.5 mg/kg, i.v.) did not reverse the reducing effects of RB101(S) on carrageenin-evoked c-Fos protein expression. These results suggest that functional interactions occur between mu- and delta-opioid receptors in enkephalin-induced antinociceptive effects.

Enkephalinergic involvement in substantia nigra in the modulation of hypothalamically-induced predatory attack behavior.

The present study was carried out in five cats which did not attack the rats spontaneously. Predatory attack on an anaesthetized rat was elicited by electrical stimulation of lateral hypothalamus at a mean current strength of 650 microA. The attack was accompanied by minimal affective display and culminated in neck biting. Microinfusions of DAME (delta-alanine methionine enkephaline) in 500 ng dose in substantia nigra facilitated the predatory attack and there was a significant reduction in the threshold current strength for affective display as well as somatomotor components. Microinfusions of naloxone, an opioid antagonist in 1.0 microg dose when DAME effect was at its peak reversed the facilitatory effects and the threshold returned to the control levels within 10 minutes of naloxone infusion at the same locus. Microinfusions of naloxone alone in similar dosage completely blocked the predatory attack response as indicated by an increase in the threshold current strength for somatomotor as well as affective display components. The somatomotor were completely inhibited and could not be elicited even when the current strength was increased to 1000 microA. Control injections of saline in similar volumes (0.5 microl) failed to produce any response Microinfusions of naloxone in lower dose (250 ng) failed to produce any blocking effect. These findings indicate that hypothalamically elicited predatory attack is facilitated by enkephalinergic mechanisms operating at the midbrain level.

Synthesis and characterization of the first potent inhibitor of yapsin 1. Implications for the study of yapsin-like enzymes.

The potent peptidic inhibitor, Y1, of the basic residue-specific yeast aspartyl protease, yapsin 1, was synthesized and characterized. The inhibitor was based on the peptide sequence of a cholecystokinin(13-33) analog that yapsin 1 cleaved with an efficiency of 5.2 x 10(5) m(-1) s(-1) (Olsen, V., Guruprasad, K., Cawley, N. X., Chen, H. C., Blundell, T. L., and Loh, Y. P. (1998) Biochemistry 37, 2768-2777). The apparent K(i) of Y1 for the inhibition of yapsin 1 was determined to be 64.5 nm, and the mechanism is competitive. Y2 was also developed as an analog of Y1 for coupling to agarose beads. The resulting inhibitor-coupled agarose beads were successfully used to purify yapsin 1 to apparent homogeneity from conditioned medium of a yeast expression system. Utilization of this new reagent greatly facilitates the purification of yapsin 1 and should also enable the identification of new yapsin-like enzymes from mammalian and nonmammalian sources. In this regard, Y1 also efficiently inhibited Sap9p, a secreted aspartyl protease from the human pathogen, Candida albicans, which has specificity for basic residues similar to yapsin 1 and might provide the basis for the prevention or control of its virulence. A single-step purification of Sap9p from conditioned medium was also accomplished with the inhibitor column. N-terminal amino acid sequence analysis yielded two sequences indicating that Sap9p is composed of two subunits, designated here as alpha and beta, similar to yapsin 1.

Roles of endogenous opioid peptides in modulation of nocifensive response to formalin.

Roles of endogenous opioid peptides and their receptors in modulation of the nocifensive responses to formalin in mice were studied. Mice were pretreated i.c.v. or intrathecally (i.t.) with selective opioid receptor antagonists or intrathecally with antisera against endogenous opioid peptides and the nocifensive licking responses to intraplantar injection of formalin (0.5%, 25 microl) were then observed. Pretreatment with the epsilon-opioid receptor antagonist beta-endorphin(1-27) or the selective mu-opioid receptor antagonist D-Phe-Cys-Tyr-Orn-Thr-Pen-Thr-NH(2) (CTOP) given i.c.v. dose dependently enhanced the second, but not the first phase of the nocifensive response. However, i.c.v. pretreatment with the selective delta-receptor antagonist naltrindole or kappa-receptor antagonist nor-binaltrophimine did not affect the nocifensive responses. Intrathecal pretreatment with selective delta(1)-opioid antagonist 7-benzylidene naltrexamine significantly enhanced both the first and second phases of nocifension. Intrathecal pretreatment with CTOP also increased the second but not the first phase of the nocifension. However, i.t. pretreatment with the selective delta(2)-receptor antagonist naltriben or nor-binaltrophimine did not affect the second phase of the nocifension. Intrathecal pretreatment with antiserum against Leu-enkephalin, Met-enkephalin, or dynorphin A(1-17), but not beta-endorphin, enhanced only the second phase of nocifensive response to formalin. It is concluded that the blockade of epsilon- and mu-receptors, but not delta- or kappa-receptors, at the supraspinal sites enhanced the second phase of formalin-induced nocifension. In the spinal cord, Leu-enkephalin, and to a lesser extent, Met-enkephalin and dynorphin A(1-17) and mu- and delta(1)-opioid receptors, but not delta(2)- or kappa-opioid receptors, are involved in modulating the feedback inhibition of the second phase of formalin-induced nocifension.

The role of the 7B2 CT peptide in the inhibition of prohormone convertase 2 in endocrine cell lines.

Prohormone convertase (PC) 2 plays an important role in the processing of neuropeptide precursors via the regulated secretory pathway in neuronal and endocrine tissues. PC2 interacts with 7B2, a neuroendocrine protein that is cleaved to a 21-kDa domain involved in proPC2 maturation and a carboxyl-terminal peptide (CT peptide) that represents a potent inhibitor of PC2 in vitro. A role for the CT peptide as an inhibitor in vivo has not yet been established. To study the involvement of the CT peptide in PC2-mediated cleavages in neuroendocrine cells, we constructed a mutant proenkephalin (PE) expression vector containing PE with its carboxyl-terminal peptide (peptide B) replaced with the 7B2 inhibitory CT peptide. This PECT chimera was stably transfected into two PC2-expressing cell lines, AtT-20/PC2 and Rin cells. Although recombinant PECT proved to be a potent (nM) inhibitor of PC2 in vitro, cellular PC2-mediated cleavages of PE were not inhibited by the PECT chimera, nor was proopiomelanocortin cleavage (as assessed by adrenocorticotropin cleavage to alpha-melanocyte-stimulating hormone) inhibited further than in control cells expressing only the competitive substrate PE. Tests of stimulated secretion showed that both the CT peptide and the PE portion of the chimera were stored in regulated secretory granules of transfected clones. In both AtT-20/PC2 and Rin cells expressing the chimera, the CT peptide was substantially internally hydrolyzed, potentially accounting for the observed lack of inhibition. Taken together, our data suggest that overexpressed CT peptide derived from PECT is unable to inhibit PC2 in mature secretory granules, most likely due to its inactivation by PC2 or by other enzyme(s).

Opioid modulation of calcium current in cultured sensory neurons: mu-modulation of baroreceptor input.

We used the whole cell open-patch or perforated-patch technique to characterize mu-opioid modulation of Ca(2+) current (I(Ca)) in nodose sensory neurons and in a specific subpopulation of nodose cells, aortic baroreceptor neurons. The mu-opiate receptor agonist Tyr-D-Ala-Gly-MePhe-Gly-ol enkephalin (DAGO) inhibited I(Ca) in 95% of neonatal [postnatal day (P)1-P3] nodose neurons. To the contrary, only 64% of juvenile cells (P20-P35) and 61% of adult cells (P60-P110) responded to DAGO. DAGO-mediated inhibition of I(Ca) was naloxone sensitive, irreversible in the presence of guanosine 5'-O-(3-thiotriphosphate), absent with guanosine 5'-O-(2-thiodiphosphate), and eliminated with pertussis toxin; DAGO's inhibition of I(Ca) was G protein mediated. Incubation of neurons with omega-conotoxin GVIA eliminated the effect of DAGO in neonatal but not in juvenile cells. In the latter, DAGO reduced 37% of the current remaining in the presence of omega-conotoxin. In the subset of nodose neurons, aortic baroafferents, the effect of DAGO was concentration dependent, with an IC(50) of 1.82 x 10(-8) M. DAGO slowed activation of I(Ca), but activation curves constructed from tail currents were the same with and without DAGO (100 nM). In summary, mu-opiate modulation of I(Ca) in nodose neurons was demonstrated in three age groups, including specifically labeled baroafferents. The demonstration of a mechanism of action of mu-opioids on baroreceptor afferents provides a basis for the attenuation of the baroreflex that occurs at the level of the nucleus tractus solitarii.

Enkephalin inhibits vagal control of heart rate, contractile force and coronary blood flow in the canine heart in vivo.

The following studies were conducted to determine if the ability of the intrinsic cardiac opioid, met-enkephalin-arg-phe to interrupt vagal bradycardia can be generalized to include the disruption of vagal effects on atrial contraction and coronary blood flow. Anesthetized dogs were instrumented to measure heart rate and left atrial contractile force or heart rate and coronary blood flow. The response of each variable was recorded at rest and during vagal stimulation. During the evaluation of vagal effects on contractile activity and coronary blood flow, heart rate was maintained constant by electrically pacing the hearts above their resting heart rate. In the first protocol, vagal stimulation reduced both heart rate and atrial contractile force in a frequency dependent fashion. When met-enkephalin-arg-phe (MEAP) was infused systemically for three min at 3 nmol min(-1) kg(-1), there were no observed changes in resting heart rate or atrial contraction. However, when the vagal stimuli were reapplied during the peptide infusion, the previously observed vagal effects on rate and contractile force were reduced in magnitude by one-half to two-thirds. The ability of MEAP to interrupt the vagal control of heart rate and contractile activity involves opiate receptors since the effect was eliminated in both cases by prior opiate receptor blockade with the high affinity antagonist, diprenorphine. In the second protocol, vagal stimulation produced a transient increase in coronary blood flow and an accompanying increase in myocardial oxygen consumption. These effects were reduced by approximately 80% during the systemic infusion of MEAP. A similar increase in coronary blood flow mediated by the direct acting muscarinic agonist, methacholine, was unaltered by the infusion of peptide. In summary, these data suggest that the intrinsic cardiac enkephalin, MEAP, is capable of inhibiting the vagal control of heart rate, contractile force and coronary blood flow and probably does so through a common opiate receptor located prejunctionally on vagal nerve terminals or within nearby parasympathetic ganglia.

[Enkephalin catabolism inhibitors and antalgics of the future: from preclinical research to clinical trials]. / Les inhibiteurs du catabolisme des enképhalines comme antalgiques du futur: de la recherche préclinique aux ouvertures cliniques.

It is now well accepted that the pain suppression effect of morphine is related to the interaction of this alkaloid with binding sites located in the central nervous system. The wide distribution of opioid receptors probably accounts for the multiplicity of pharmacological responses elicited by morphine administration, as in addition to its strong analgesic potency morphine induces side effects. Therefore, there is a critical need for new analgesics able to fulfil the gap existing between opioid analgesics and antalgics. These new analgesics could be of major interest in a number of severe pain syndromes. The discovery that the endogenous morphine-like peptides enkephalins are degraded by well-defined metabolic pathways represents a promising outlook for the development of new analgesics. The complete inhibitors of enkephalin catabolism produce their physiological effects by increasing the extracellular levels of endogenous opioid peptides released either tonically or following stimuli-evoked depolarization. Under these conditions, their effects will depend upon the magnitude and duration of the enkephalin release evoked by a particular stimulus, which probably varies in the different enkephalinergic pathways. It is expected that increasing the levels of endogenous opioid peptides would avoid serious drawbacks inasmuch as they appear related to a ubiquitous overstimulation of brain opioid receptors. Some mixed inhibitors of enkephalin degrading enzymes are now undergoing preclinical trials.

Differences in opioidergic inhibition of spinal reflexes and Fos expression evoked by mechanical and chemical noxious stimuli in the decerebrated rabbit.

Noxious mechanical and chemical stimuli were applied to the toes of the left hind limb of decerebrated, spinalized rabbits and their effects on a hind limb spinal withdrawal reflex and expression of Fos-like immunoreactivity in the spinal cord were measured. The animals were prepared so as to minimize nociceptive inputs arising from surgery. A single crush stimulus applied with a pair of haemostatic forceps caused long-lasting (c. 20 min) inhibition of reflexes evoked in medial gastrocnemius motoneurons by electrical stimulation of the skin at the heel. Naloxone (0.25 mg/kg i.v.) increased reflexes to more than 1000% of pre-drug controls and reversed crush-evoked inhibition. Mustard oil applied to the toes had no consistent effects on the heel-gastrocnemius reflex before or after naloxone. Both crush and mustard oil stimuli gave rise to unilateral increases in the number of Fos-immunopositive profiles in the superficial dorsal horn of spinal segments L7 and S1. There were significantly more Fos-immunoreactive elements in the central and lateral parts of lamina I of both segments in animals receiving the crush stimulus than there were in animals receiving the mustard oil stimulus. Immunochemical localization of enkephalins in rabbit spinal cord showed a dense network of fibres and terminals in laminae I and II, accompanied by infrequent but distinctly stained neuronal cell bodies. The same pattern, with increased numbers of visible cell bodies, was seen after treatment with colchicine. The present data show that tonic and stimulus-evoked opioidergic inhibition of the heel-gastrocnemius reflex of the rabbit are not epiphenomena of surgical preparation of the hindlimb. Opioid-mediated inhibition of the heel-gastrocnemius withdrawal reflex of the rabbit was evoked by noxious mechanical but not by chemical stimulation of the toes. Of these stimuli, the former gave rise to greater activation of neurons in central and lateral lamina I of segments L7 and S1, the region of termination of afferent fibres from the heel and the location of some enkephalin-positive neuronal cell bodies. Thus, noxious mechanical stimulation of the toes elicits inhibition of the heel-gastrocnemius withdrawal reflex, probably via activation of enkephalinergic neurons in the lateral half of lamina I in the L7 and S1 segments.