A randomized phase I trial evaluating the effects of inhaled 50-50% N2 O-O2 on remifentanil-induced hyperalgesia and allodynia in human volunteers.

BACKGROUND: Opioids are known to relieve pain, and also aggravate pre-existing hyperalgesia. In animal studies, the N-methyl-d-aspartate-receptor antagonist nitrous oxide (N2 O) was able to prevent hyperalgesia. The present study evaluated the effect of N2 O on hyperalgesia after remifentanil infusion in healthy volunteers. METHODS: Twenty-one healthy volunteers were enrolled in this placebo-controlled cross-over study. Transcutaneous electrical stimulation at high current densities induced spontaneous acute pain and stable areas of hyperalgesia. Each volunteer underwent the following four sessions: (1) 50-50% N2 -O2 and i.v. saline; (2) 50-50% N2 -O2 and i.v. remifentanil 0.1 µg/kg/min; (3) 50-50% N2 O-O2 and i.v. saline; (4) 50-50% N2 O-O2 and i.v. remifentanil 0.1 µg/kg/min. Inhaled gas mixtures lasted for 60 min, i.v. drug administration for 30 min. Visual analogue scale pain intensity, areas of pinprick hyperalgesia and touch-evoked allodynia were assessed repeatedly for 160 min. RESULTS: Data of 19 volunteers were analysed. There were significant time and treatment effects regarding areas of hyperalgesia and allodynia (p < 0.02). The area of hyperalgesia was significantly reduced in the N2 O + remifentanil session compared to the remifentanil session (35.88 ± 22.37 vs. 43.55 ± 18.48 cm(2) , p = 0.004). The area of allodynia was significantly reduced in the N2 O + remifentanil session compared to the remifentanil session (29.95 ± 16.15 vs. 34.80 ± 15.35 cm(2) , p = 0.008). The pain intensity was significantly reduced in the N2 O + remifentanil session compared to the remifentanil session (37.96 ± 12.78 vs. 42.15 ± 13.34 mm, p < 0.0001). CONCLUSIONS: Nitrous oxide significantly reduced hyperalgesia, allodynia and pain intensity aggravated after remifentanil administration in a human volunteer model. WHAT DOES THIS STUDY ADD?: This study brings the evidence that N2 O reduces the remifentanil aggravated secondary hyperalgesia in human volunteers exposed to a well-known model of electrical pain. N2 O was able to oppose the hyperalgesia, the allodynia and the pain intensity consecutive to remifentanil use in this specific pain model.

Progressive enhancement of delayed hyperalgesia induced by repeated heroin administration: a sensitization process.

It is difficult to conceive that tolerance and sensitization processes, two apparently opposite phenomena, can concomitantly modify one given biological process, i.e., the processing of pain. We have shown recently that opiates produce not only analgesia but also long-lasting hyperalgesia in rats. This suggests that tolerance to the analgesic effect of an opiate, especially heroin, could be in part the result of an actual sensitization of pronociceptive systems. Here, we show that both magnitude and duration of heroin-induced delayed hyperalgesia increase with intermittent heroin administrations, leading to an apparent decrease in the analgesic effectiveness of a given heroin dose. Our observation that a small dose of heroin which is ineffective for triggering a delayed hyperalgesia in non-heroin-treated rats induced an enhancement in pain sensitivity for several days after a series of heroin administrations is in agreement with the sensitization hypothesis. The effectiveness of the opioid receptor antagonist naloxone to precipitate hyperalgesia in rats that had recovered their pre-drug nociceptive value after single or repeated heroin administrations indicates that heroin-deprived rats were in a new biological state associated with a high level balance between opioid-dependent analgesic systems and pronociceptive systems. Because the NMDA receptor antagonist dizocilpine maleate (MK-801) prevented both heroin-induced long-lasting enhancement in pain sensitivity and naloxone-precipitated hyperalgesia, these findings further suggest that tolerance, sensitization, and one withdrawal symptom, hyperalgesia, are issued from a neuroadaptive process in which NMDA systems play a critical role.

[Low doses ketamine: antihyperalgesic drug, non-analgesic]. / Kétamine à faibles doses: antihyperalgésique, non analgésique.

Recent data in animal experiments as in clinical trials have clearly reported that pain modulation is related to an equilibrium between antinociceptive and pronociceptive systems. Therefore, the apparent pain level could not only be a consequence of a nociceptive input increase but could also result from a pain sensitization process. Glutamate, via NMDA receptors, plays a major role in the development of such a neuronal plasticity in the central nervous system, leading to a pain hypersensitivity that could facilitate chronic pain development. By an action on NMDA receptors opioids also induce, in a dose dependent manner, an enhancement of this postoperative hypersensitivity. "Antihyperalgesic" doses of ketamine, an NMDA receptor antagonist, were able to decrease this central sensitization not only in painful animal but also in human volunteers exposed to different pain models, or in the postoperative period. Many studies have reported that ketamine effects are elicited when this drug is administered the following manner: peroperative bolus (0.1 to 0.5 mg/kg), followed by a constant infusion rate (1 to 2 microg/kg per min) during the peroperative period and for 48 to 72 hours after anaesthesia. Those ketamine doses improved postoperative pain management by reducing hyperalgesia due to both surgical trauma and high peroperative opioid doses. This antihyperalgesic action of ketamine also limited the postoperative morphine tolerance leading to a decrease in analgesic consumption and an increase in the analgesia quality.

[Complexity and physiology of the analgesic effects of opioids]. / Comptexité et logique physiologiques du mode d'action des substances opioïdes.

Nowadays, it is considered that clinical pain is not only a reflexion of the nociceptive stimulus intensity, but is to a large extent the expression of neural plasticity including both peripheral and central sensitization. N-Methyl-D-aspartate receptors (NMDA-Rs) play a critical role in the central pain sensitization process leading to exaggerated pain and chronic pain. Although opioids are unsurpassed analgesics, we observed that opioids induced delayed and sustained NMDA-related hyperalgesia after an initial analgesia suggesting a pain sensibilisation process. It is shown that NMDA receptor antagonists and specific diets able to negatively modulate NR2B subunit containing NMDA receptors prevented abnormal pain hypersensitivity, partially reversed chronic pain and restored the opioid effectiveness on opioid-resistant pain models.

Characterization of a morphine-modulating peptide, FLFQPQRFamide, in the rat hypophysis: biochemical and immunocytochemical studies.

The octapeptide FLFQPQRFamide (F8Fa) is a FMRFamide-like peptide with a certain number of antiopiate properties. Previous studies have shown that both F8Fa specific receptors and F8Fa-like material are present in the rat central nervous system. In this study, RIA revealed that the rat neurohypophysis also contains F8Fa immunoreactive (IR) material (230 +/- 49 pg/neural lobe). HPLC profiles revealed several forms of F8Fa IR. Neurohypophysis extracts can also inhibit the binding of F8Fa to rat spinal cord preparations, which suggests that this F8Fa-like material has a biological activity. Immunocytochemical observations, at the light and electron microscopic levels, confirmed the presence throughout the neural lobe of F8Fa IR, in axonal fibers and terminals similar to those containing the more classical neurohypophysial hormones. Immunogold staining showed that F8Fa IR was restricted to neurosecretory granules in certain axonal and terminal profiles. Double staining of the same ultrathin sections, using our anti-F8Fa antiserum and vasopressin or its neurophysin specific antibodies, revealed that F8Fa IR was colocalized with vasopressin. F8Fa IR was not visible in ocytocinergic fibers or terminals. A striking depletion of F8Fa IR (80%) was observed in rats which were given 2% saline to drink for 6 days. Similarly, an ip injection of an hypertonic saline solution was shortly followed by a 20% drop of F8Fa IR. In vitro F8Fa IR release from isolated neurohypophysis was evoked under a 56 mM KCl depolarization. These results suggest that F8Fa IR may act as a paracrine/endocrine mediator released from the rat neurohypophysis.

Characterization of a potent agonist for NPFF receptors: binding study on rat spinal cord membranes.

Specific receptors for the octapeptide FLFQPQRFamide (NPFF), a mammalian FMRFamide-like neuropeptide with anti-opiate properties have been identified in rat central nervous system. However, exploration of the biological role of this peptide requires a peptidase-resistant agonist. In this study, the stability and binding characteristics of [125I]DYLMeFQPQRFamide, a radioiodinated analogue of NPFF, on rat spinal cord tissue were determined and compared with those of [125I]YLFQPQRFamide, the reference ligand which previously permitted to characterize NPFF binding sites. In a binding assay, [125I]DYLMeFQPQRFamide remained intact in the presence of membranes without peptidase inhibitors, whereas [125I]YLFQPQRFamide was completely hydrolysed. The specific binding was time-dependent, dose-dependent, saturable and reversible. [125I]DYLMeFQPQRFamide shared the same binding characteristics as [125I]YLFQPQRFamide (Kd = 0.07 nM; Bmax = 14.7 fmol/mg protein). Binding was not affected by various spinal cord opioids or peptides. Autoradiographic studies indicated that binding sites were mainly located in the most external layers of dorsal horn where high densities of NPFF binding sites have previously been described. [125I]YLFQPQRFamide and [125I]DYLMeFQPQRFamide binding sites were both GTP-regulated. These findings indicate that DYLMeFQPQRFamide should be of value in studies on NPFF-mediated actions in vivo.

Lessons from an unpleasant surprise: a biochemical strategy for the diagnosis of pheochromocytoma.

OBJECTIVE: To audit the performances of the analytes used in the diagnosis of pheochromocytoma and to present a graphical guideline to help the diagnosis. DESIGN: A 5 year retrospective study. SETTINGS: Laboratory and departments of a university hospital. PARTICIPANTS: In-patients, suspected of bearing a pheochromocytoma, were investigated for urinary metanephrines and catecholamines (photometric method) and vanillylmandelic acid, fractionated catecholamines and metanephrines [high pressure liquid chromatography (HPLC) coupled to electrochemical detection (ED)] urinary excretion. MAIN OUTCOME: Patients with a pheochromocytoma (24 out of 2003 patients) were diagnosed by the combination of normetanephrine and metanephrine determination. RESULTS: All analytes but dopamine were significantly elevated in patients with a pheochromocytoma. The area under the receiver operating characteristics (ROC) curves were the highest for total metanephrines, normetanephrine and metanephrine determinations. Because of analytical interferences in the metanephrines determination, the normetanephrine and metanephrine performed better. It is noteworthy that all pheochromocytomas had either normetanephrine or metanephrine levels above their respective optimal threshold (sensitivity 100%). The best optimal threshold performance was reached by the mean of three daily samples. Total or fractionated catecholamines or vanillylmandelic acid were less accurate tools. CONCLUSION: Amongst urinary tests, the combined use of HPLC/ED determination of normetanephrine and metanephrine seems the most effective screening strategy for the diagnosis of pheochromocytoma. The older total metanephrine photometric assay is grieved by analytical interferences.

Structure-activity study of neuropeptide FF: contribution of N-terminal regions to affinity and activity.

Twenty neuropeptide FF (NPFF) analogs having various lengths were synthesized by solid-phase peptide synthesis to gain more information on the role of N-terminal residues for the NPFF receptor affinity. The affinities were evaluated in the rat spinal cord membrane preparations, and the biological activities were measured on morphine analgesia in the mouse tail-flick test. Shortening of the NPFF sequence from the N-terminal produced only a moderate decrease in affinity until NPFF (4-8) was reached. In the same way, NPFF(3-8) significantly decreased morphine analgesia, while NPFF(4-8) had no significant effect at a dose of 22 nmol. The introduction in the N-terminal part of NPFF of a D-enantiomer at positions 2 and 1 or the presence of an N-methyl group on position 3 did not modify affinity and activity. Substitution of proline5 by the D-isomer decreased the affinity of NPFF analogs whatever their length, and [Tyr1,D-Pro5]NPFF(1-8) was 2.5-fold less potent than [Tyr1]NPFF(1-8) in reversing morphine-induced analgesia. In contrast, the presence of a glycine residue in position 5 did not influence the affinity toward NPFF receptors. Data provide evidence that the N-terminal segment of neuropeptide FF is responsible for high-affinity binding.

Modulation of human lymphocyte proliferation by FLFQPQRFamide, a FMRFamide-like peptide with anti-opiate properties.

The octapeptide Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F8Fa), originally detected in mammalian brain by antisera raised against the invertebrate peptide Phe-Met-Arg-Phe-NH2 (FMRFamide) is a neuropeptide able to antagonize the actions of both endogenous and exogenous opiates. Since it is well accepted that lymphocytes are targets for opiates, we have tested the effect of F8Fa on T cell proliferation from normal human peripheral blood lymphocytes. Our study shows that F8Fa exerts a concentration-dependent diphasic modulation of human T lymphocyte proliferation. Thus, despite a great variability between individuals, 10(-13) M F8Fa was found to enhance the proliferation of T cells induced by phytohemagglutinin or anti-CD2 monoclonal antibodies, while 10(-7) M F8Fa inhibited T cell proliferation, without affecting cell viability. When F8Fa was tested on monocyte-depleted cell preparations, only the inhibitory effect was observed. These results indicate that F8Fa may stimulate T cells via monocytes, but may also directly inhibit T lymphocyte proliferation. Given the presence of F8Fa-like peptide in human plasma, we suggest that F8Fa may act as a neurohormone in the control of the immune system.

Autoradiographic distribution of receptors to FLFQPQRFamide, a morphine-modulating peptide, in rat central nervous system.

The neuropeptide FLFQPQRFamide is a structure related to FMRFamide which is able to inhibit the effects of both endogenous and exogenous opiates. This morphine-modulating activity is mediated via the stimulation of specific FLFQPQRFamide receptors, different from opiate receptors. In vitro quantitative receptor autoradiography was performed on frozen sections of rat central nervous system to characterize binding properties and visualize FLFQPQRFamide receptors using the specific ligand [125I]YLFQPQRFamide, a radio-iodinated analogue of FLFQPQRFamide. [125I]YLFQPQRFamide appeared to interact reversibly with a single class of binding sites (KD = 0.2 nM). The specific binding represented 80% of the total binding at 0.05 nM, the FLFQPQRFamide concentration used in this mapping study. Sites labelled with [125I]YLFQPQRFamide were distributed heterogeneously within the brain and spinal cord. A high density of FLFQPQRFamide binding sites was detected in the most external layers of the dorsal horn of spinal cord and various nuclei of pons and medulla including trigeminal, dorsal tegmental and reticular nuclei. Nucleus of solitary tract, parabrachial, ambiguous and facial nuclei are also intensively labelled. Some structures of mesencephalon and diencephalon exhibited a high density of FLFQPQRFamide binding sites: central gray, raphe nuclei and thalamic nuclei such as parafascicular, laterodorsal, central median, paratenial and paraventricular nuclei. Suprachiasmatic and mammillary nuclei, lateral, posterior and anterior areas of hypothalamus and medial preoptic area exhibited high labelling. FLFQPQRFamide binding sites were also seen in some structures of the dopaminergic meso-cortico-limbic system including ventral tegmental area, cingulate cortex, lateral septum and the head of the caudate-putamen. Dense labelling appeared in the presubiculum of hippocampus. The dissimilar mapping of FLFQPQRFamide and opiate brain receptors confirms our previous pharmacological findings in FLFQPQRFamide binding studies on rat spinal cord membranes, showing that FLFQPQRFamide receptors are different from opiate receptors. There was a good correspondence between localization of binding sites and that of the putative endogenous peptide. Both occur in brain areas previously associated with analgesic action of opiates. However, the mapping of FLFQPQRFamide receptors in the central nervous system suggests that the FLFQPQRFamide system could be implicated in other physiological functions.

Interaction between neuropeptide FF and opioids in the ventral tegmental area in the behavioral response to novelty.

Considerable evidence has focused on the interaction between endogenous opioid peptides and the dopaminergic mesocorticolimbic system in behavioral responses to stress. Recently, it has been proposed that the CNS synthesizes and secretes neuropeptides that act as part of a homeostatic system to attenuate the effects of morphine or endogenous opioid peptides. Among these antiopioids, neuropeptide FF (NPFF) is particularly interesting since both NPFF immunoreactive-like terminals and NPFF binding sites are located in the vicinity of the dopaminergic cell bodies within the ventral tegmental area (VTA) suggesting an interaction at this level. The purpose of the present study was to evaluate the respective implication of opioid and antiopioid peptides at the level of the VTA in the locomotor response to novelty in rats. The results indicate that s.c. naloxone pretreatment, an opiate receptor antagonist, reduced locomotor activity in rats placed in a novel environment without having any effect in a familiar environment. This effect takes place in the VTA since intra-VTA administration of naloxone methobromide diminished similarly and dose-dependently the motor response to novelty. This effect is mainly dependent on opioid peptides released at VTA level since local injections of thiorphan, an inhibitor of enkephalin degradation, strongly increased locomotor response to novelty and this effect is completely prevented by the co-administration of naloxone methobromide. When injected in the VTA, NPFF is acting as an antiopioid compound, i.e. it reduces the locomotor activity triggered by exposure to novelty to the level recorded in a familiar environment. Moreover, NPFF decreased dose-dependently the potentiation of novelty-induced locomotor response produced by VTA injection of thiorphan. Taken together, these results suggest that NPFF neurons may participate at the level of the VTA to a homeostatic regulating process counteracting opioid effects induced by a mild stress such as novelty.

Characteristics and specific localization of receptors for atrial natriuretic peptides at non-neuronal cells in cultured mouse spinal cord cells.

Characteristics of atrial natriuretic peptide receptors were determined in cultured mouse spinal cord cells. Saturation and competition experiments demonstrated the presence of a single class of atrial natriuretic peptide binding sites with high affinity (KD = 0.054 nM) and a density of 1.92 fmoles/10(6) cells. A similar affinity (KD = 0.070 nM) was observed in rat spinal cord membrane preparations. These atrial natriuretic peptide binding sites were functional receptors since the treatment of cells with atrial natriuretic peptide increased cyclic guanosine monophosphate levels within these cells in a classical time-dependent manner. When atrial natriuretic peptide was applied onto the cell body of intracellularly recorded spinal cord neurons, this peptide did not evoke a change of the input resistance or of the resting membrane potential value. Light-microscopic autoradiography studies showed that no atrial natriuretic peptide binding could be detected on typical birefringent neurons but it could be located on astroglial and epithelial cells as identified by immunocytochemical markers. These results show that functional atrial natriuretic peptide receptors with high affinity exist in cultured mouse spinal cord cells and are not located on neurons. The presence of atrial natriuretic peptide receptors on astrocytes suggests that this neuropeptide might be a good candidate for neuron-glial communication. As the atrial natriuretic peptide binding sites previously shown in epithelia responsible for maintaining fluid and electrolyte gradients, the atrial natriuretic peptide receptors on epithelial cells in these spinal cord cultures may be involved in vivo in the control of water balance in the central nervous system.

Neuropeptide FLFQRFamide receptors within the ventral mesenchephalon and dopaminergic terminal areas: localization and functional antiopioid involvement.

The neuropeptide FLFQPQRF amide is a FMRFamide-like peptide with some anti-opiate properties. Neuropeptide FLFQPQRFamide receptors are present in the mammalian central nervous system and have been clearly identified as different from opiate receptors. Autoradiography has revealed neuropeptide FLFQPQRFamide receptor localization within the ventral mesencephalon, where opiate receptors are also located. In order to delineate anatomical localization of neuropeptide FLFQPQRFamide receptors, we used selective chemical lesions of dopaminergic cells and intrinsic perikarya of the ventral mesencephalon, coupled with in vitro autoradiographic techniques. We show that: (i) unilateral lesions of dopaminergic perikarya produced by 6-hydroxydopamine did not affect either ipsi or contralateral neuropeptide FLFQPQRFamide receptor density within the mesencephalon; (ii) unilateral lesions of intrinsic perikarya by ibotenic acid injected into the ventral tegmental area produced a significant reduction of neuropeptide FLFQPQRFamide receptors (40%) in this region; (iii) in the substantia nigra pars compacta, ibotenic acid unilateral lesions did not affect the density of neuropeptide FLFQPQRF-amide receptors; (iv) unilateral 6-hydroxydopamine or ibotenic acid lesions failed to affect neuropeptide FLFQPQRFamide binding in the dopaminergic projection areas. These results suggest that, like opiate receptors, the neuropeptide FLFQPQRFamide binding sites are not localized on dopaminergic neurons but are distributed on both soma of non dopaminergic cells in the ventral tegmental area and on fibers afferent to the ventral tegmental area and substantia nigra pars compacta. To evaluate the possibility that the stimulation of neuropeptide FLFQPQRFamide receptors may affect the opioid modulation of mesocorticolimbic dopaminergic neuron activity, we tested the effects of neuropeptide FLFQPQRFamide ventral tegmental area infusion (0.25-10 micrograms) on the behavioral activation induced by intra-ventral tegmental area morphine infusion. We observed that in the ventral tegmental area, the stimulation of neuropeptide FLFQPQRFamide receptors inhibits morphine-induced locomotor hyperactivity. These results suggest that in the ventral tegmental area, neuropeptide FLFQPQRFamide may participate, via an indirect mechanism, to the control of the mesocorticolimbic dopaminergic system activity by counteracting the effect of opioids.

Characterization and localization of a putative morphine-modulating peptide, FLFQPQRFamide, in the rat spinal cord: biochemical and immunocytochemical studies.

The bovine octapeptide Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (FLFQPQRFamide), originally detected by antisera raised against the invertebrate peptide, Phe-Met-Arg-Phe-NH2, is a neuropeptide which antagonizes the actions of endogenous and exogenous opiates. Using a sensitive radioreceptor assay, we show that rat spinal cord extracts were able to inhibit binding of FLFQPQRFamide, suggesting that a biologically active FLFQPQRFamide-like material exists in the rat spinal cord. We also raised antibodies against the peptide and used them, together with radioimmunological and immunohistochemical methods, to characterize this material further and analyse its cellular and subcellular localization in this area of the central nervous system. Radioimmunoassay showed that extracts from cervical and thoracolumbar levels contained measurable amounts of FLFQPQRFamide-immunoreactive material (about 3 ng/g tissue), present essentially in the dorsal horn. Analytical reverse-phase chromatography revealed that this material existed in several molecular forms. One of these fractions (about 20% of the total immunoreactivity) had the elution characteristics of synthetic FLFQPQRFamide. Light microscopic immunohistochemistry showed FLFQPQRFamide immunoreactivity at all spinal levels, localized mainly in a dense plexus of fibers in the superficial layers of the dorsal horn. Immunoreactive profiles were also seen in the lateral funiculi and around the central canal at all levels and in the intermediolateral columns; some rare immunoreactive fibers were also noted in the ventral horn at cervical and thoracic levels. FLFQPQRFamide-positive cell bodies were never detected in any of our sections. Electron microscopy of ultrathin sections of the dorsal horn and central gray treated with our antisera and a post-embedding immunogold procedure revealed that the immunoreactivity, at least within these areas, was restricted to dense-cored vesicles (90-120 nm in diameter) in axonal and terminal profiles. As seen by radioimmunoassay and immunohistochemistry, unilateral rhizotomy of all dorsal roots between segments C4 and T2 did not change the levels of FLFQPQRFamide immunoreactivity in the ipsilateral C6-C8 segments. Taken together with our recent data showing the existence of specific FLFQPQRFamide receptors at the spinal cord level, our present observations suggest that the dorsal horn of the rat spinal cord may be a site where vertebrate Phe-Met-Arg-Phe-like peptides, and in particular, FLFQPQRFamide, may exert opiate modulating activities.

Acute tolerance associated with a single opiate administration: involvement of N-methyl-D-aspartate-dependent pain facilitatory systems.

Mechanisms underlying the development of acute tolerance to the analgesic effect of opiates were investigated. In the rat tail-flick test, administration of naloxone (1 mg/kg, s.c.) 40 min after heroin (1 mg/kg, s.c.) was shown to induce hyperalgesia, indicative of a short-onset, opiate-activated pain facilitatory systems masking the opiate analgesia. Pretreatment with the N-methyl-D-aspartate receptor antagonist dizocilpine maleate blocked, in a dose-dependent manner, the naloxone-induced hyperalgesia and potentiated the heroin-induced analgesia. Using a schedule of two successive injections of 1 mg/kg heroin, acute tolerance was indicated by a marked reduction (-52%) in analgesia induced by the second dose. After pretreatment with dizocilpine maleate, the acute tolerance was abolished and the analgesic effects of both injections of heroin were strongly potentiated. These observations indicate that acute tolerance appears after the first exposure to opiates and stems from opiate activation of N-methyl-D-aspartate-dependent pain facilitatory systems.

Distribution of neuropeptide FF (FLFQPQRFamide) receptors in the adult rat spinal cord: effects of dorsal rhizotomy and neonatal capsaicin.

By using quantitative autoradiography and highly selective iodinated ligands, we quantified modifications in neuropeptide FF binding sites in the superficial layers (laminae I and II) of the cervical (C6-C8 segments) and lumbar (L3-L5 segments) enlargements in two models: (i) rats neonatally treated with capsaicin; (ii) rat submitted 15 days before to unilateral dorsal rhizotomies. We comparatively analysed the distribution of mu-opioid binding sites in the same animals. We have shown that the [125I]YLFQPQRFamide (neuropeptide FF sites) labelling is not significantly modified following selective damage of fine afferent fibres by neonatal capsaicin treatment. In the cervical and lumbar enlargements, capsaicin-treated/control binding ratios for [125I]YLFQPQRFamide were 0.90 and 0.86, respectively. While unilateral dorsal rhizotomy induced a drastic decrease in [125I]FK-33-824 labelling in the side ipsilateral to the lesion as compared to the intact side of (yielding ratios of 0.29 and 0.31 for cervical and lumbar levels, respectively), [125I]YLFQPQRFamide labelling was not significantly modified, yielding ratios of 0.98 and 0.91 for cervical and lumbar levels, respectively. These data suggest that, in contrast with a majority of mu-opioid receptors, neuropeptide FF receptors are not located on fine primary afferent fibers carrying nociceptive information from the fore- or hindlimb in the rat. This preferential postsynaptic localization, together with the reported "morphine modulating" action of this peptide, support the proposal of a role for neuropeptide FF in intraspinal modulation of nociceptive input.

Opiate tolerance to daily heroin administration: an apparent phenomenon associated with enhanced pain sensitivity.

From a classical viewpoint, tolerance to analgesic effects of opiates refers to the decreased effectiveness of a given opiate following its repeated use. Despite much research, it has not been conclusively demonstrated in vivo that functional changes observed at the opioid receptor level in the responsiveness to opiates account for development of tolerance. An alternative hypothesis is that opioid receptors remain operative following repeated opiate administration but that opioid receptor activation rapidly induces a prolonged increase in pain sensitivity which opposes the predominant opiate analgesic effect following repeated opiate administration. We recently showed that a single heroin administration induces an enhanced pain sensitivity for several days, a phenomenon which is prevented by the non-competitive N-methyl-D aspartate receptor antagonist MK-801. Herein we report that repeated once-daily heroin injections induced a gradual lowering of the nociceptive threshold which progressively masked a sustained heroin analgesic functional effect. MK-801 prevented such opiate-induced allodynia and thereby prevented development of an apparent decrease in the effectiveness of heroin. These results indicate that intermittent heroin administration induced a persistent increase in the basal pain sensitivity which, if not taken into account gives the impression of less analgesia, i.e. apparent tolerance.

Aldosterone secretion and adrenal angiotensin II receptors in the Brattleboro rat.

The basal secretion of aldosterone, measured in adrenal venous blood, was three- to fourfold lower in Brattleboro than in Long-Evans rats used as controls. Infusion of a low dose of angiotensin II (1 ng/min per 100 g body/wt) to Long-Evans rats caused a fourfold increase in aldosterone release but neither the low dose nor a tenfold higher dose changed the rate of release in Brattleboro rats. Only a very high dose (300 ng/min per 100 g body wt) succeeded in increasing the secretion of aldosterone in Brattleboro rats but throughout the time-course of the infusion, secretion remained about fivefold lower than in Long-Evans rats and the incremental response was reduced by 74.9%. Adrenal zona glomerulosa angiotensin II receptor sites had similar affinity and maximum binding capacity in the two groups of rats. It is suggested that the reduced corticosteroidogenic capacity of the adrenal cortex of Brattleboro rats results from an impairment of the post-receptor mechanisms involved in the biosynthesis of aldosterone.

Neuropeptide FF in the rat adrenal gland: presence, distribution and pharmacological effects.

Neuropeptide FF (NPFF, FLFQPQRFamide) is an FMRFamide-like octapeptide exhibiting antiopiate activity. The presence of both NPFF-immunoreactivity (NPFF-IR) and NPFF-specific receptors has been described in the mammalian central nervous system (CNS). The peripheral effects of NPFF indicate that NPFF-IR material is present outside the CNS. Biochemical and immunohistochemical methods enabled us to determine the presence and distribution of NPFF-IR in the rat adrenal gland. The amount of NPFF-IR material in whole gland was estimated by radioimmunoassay to be 19.00 +/- 4.00 fmol/gland. High performance liquid chromatography analysis of adrenal extracts revealed a single molecular form which coeluted with authentic NPFF. Demedullation decreased adrenal NPFF-IR content, indicating that NPFF-IR was present in both cortex and medulla. Light microscopy revealed NPFF-IR in beaded fibers confined in the outer part of the cortex and in medullary cells. Double-labeling with antityrosine-hydroxylase and anti-NPFF antibodies showed NPFF-IR in cortical catecholaminergic postganglionic fibers restricted to the subcapsular and glomerulosa zonae. NPFF-IR was also located in medullary chromaffin cells and in rays and islets of chromaffin cells dispersed throughout the cortex. Insulin-induced hypoglycemia did not alter NPFF-IR content. Denervation lowered adrenal NPFF-IR content. These data indicate that this peptide is present in nerve fibers of extrinsic origin. In vitro approaches using adrenal slices have shown that NPFF inhibited aldosterone release in a dose-dependent manner. Taken together, these data suggest that NPFF may participate in the control of aldosterone production and adrenal blood supply.

Evidence for multiple opioid receptors in the human posterior pituitary.

The distribution and quantification of opioid receptor types in post-mortem human pituitary cryostat sections was determined by quantitative in vitro receptor autoradiography. Highly specific radioligands were used for each opioid receptor type i.e. [125l]-FK-33-824 for mu-opioid sites, [125l][D.Ala2]-Deltorphin-l for delta-opioid sites and 3H-U69,593 for kappa-opioid sites. None of the five specimens tested exhibited any labeling in the anterior lobe of the pituitary for the three radioligands. As for the posterior pituitary, all 5 specimens contained both mu and kappa-opioid binding sites whereas none of them showed delta-binding sites. The presence of both mu- and kappa-opioid binding sites in the human posterior pituitary contrasts with previous findings in the rat where only kappa-sites have been found. The present study could contribute to understanding of the functional action of opioids in the human pituitary.