Exposure to GSM 900-MHz mobile radiation impaired inhibitory avoidance memory consolidation in rat: Involvements of opioidergic and nitrergic systems.

The use of mobile phones is increasing, and the main health concern is the possible deleterious effects of radiation on brain functioning. The present study aimed to examine the effects of exposure to a global system for mobile communication (GSM) with mobile phones on inhibitory avoidance (IA) memory performance as well as the involvement of endogenous opioids and nitric oxide (NO) in this task. Male Wistar rats, 10-12 weeks old, were used. The results showed that four weeks of mobile phone exposure impaired IA memory performance in rats. The results also revealed that post-training, but not pre-training, as well as pre-test intracerebroventricular (i.c.v.) injections of naloxone (0.4, 4 and 40 ng/rat), dose-dependently recovered the impairment of IA memory performance induced by GSM radiation. Additionally, the impairment of IA memory performance was completely recovered in the exposed animals with post-training treatment of naloxone (40 ng/rat) plus pre-test i.c.v. injections of L-arginine (100 and 200 nmol/rat). However, pre-test i.c.v. injections of L-NAME (10 and 20 nmol/rat), impaired IA memory performance in the animals receiving post-training naloxone (40 ng/rat). In the animals receiving post-training naloxone treatment, the impairment of IA memory performance due to pre-test i.c.v. injections of L-NAME was recovered by the pre-test co-administration of L-arginine. It was concluded that the recovery from impairment of IA memory in GSM-exposed animals with post-training naloxone treatment was the result of blockade of the opioidergic system in early memory consolidation as well as activation of the nitrergic system in the retrieval phase of memory.

The anti-inflammatory effects of low- and high-frequency electroacupuncture are mediated by peripheral opioids in a mouse air pouch inflammation model.

BACKGROUND: Although acupuncture has been widely used for complementary therapeutic approaches to treat inflammatory diseases and inflammation-induced pain, the potential anti-inflammatory effects of acupuncture treatment remain controversial in clinical trials, and the underlying mechanisms are still unclear. OBJECTIVE: The objective was to determine whether electroacupuncture (EA) is able to suppress the peripheral inflammatory response (e.g., zymosan-induced leukocyte migration into air pouch). As part of a mechanistic approach, it was further evaluated whether endogenous opioid systems are involved in the "EA-induced anti-inflammatory effect" (EA-AI). METHODS: EA (1 or 120 Hz) was performed bilaterally in the Zusanli acupoint (ST36) or in a nonacupoint (gluteal muscle) for 30 min in ICR mice under anesthetic condition. The number of leukocytes that migrated into the air pouch was counted 4 hours after zymosan injection. EA was performed at 0, 0.5, 1, or 2 hours prior to zymosan injection, respectively. To evaluate opioid involvement in EA-AI, intrathecal naloxone (36 microg/mouse) and intraperitoneal naloxone methiodide (30 mg/kg) were administered 10 min before EA stimulation. RESULTS: Both the 1 and 120 Hz frequencies of EA into Zusanli acupoint at the same time with zymosan injection significantly reduced leukocyte migration into the air pouch as compared with those of control groups (i.e., anesthetic control and needling control into Zusanli acupoint without electrical stimulation). The EA stimulation into nonacupoint did not produce any significant anti-inflammatory effect. EA treatment at 0.5 hours prior to zymosan injection also produced an anti-inflammatory effect but 1 and 2 hours prior to zymosan injection did not elicit any effect. Peripheral opioid blockage significantly reversed EA-AI, whereas spinal opioid blockage did not alter EA-AI. CONCLUSION: EA can suppress peripheral inflammation through a peripheral opioid mechanism. To achieve the full effectiveness of EA, repeated application is recommended for the treatment of a variety of inflammatory diseases.

Pulsed magnetic field induced "analgesia" in the land snail, Cepaea nemoralis, and the effects of mu, delta, and kappa opioid receptor agonists/antagonists.

A brief exposure to a pulsed magnetic field (Cnp: patent pending) had significant antinociceptive or "analgesic" effects in the land snail, Cepaea nemoralis, as evidenced by an increase in the latency of response to a warmed (40 degrees C) surface. This analgesia was in part opioid mediated being significantly reduced, but not eliminated: by the prototypic opiate antagonist, naloxone; the mu (mu) opioid receptor directed antagonists, naloxazine or beta-funaltrexamine, and the delta (delta) opioid receptor directed antagonists, naltrindole-5'-isothiocyanate or ICI 174,864. However the Cnp induced analgesia was unaffected by the kappa (kappa) opioid receptor directed antagonist, nor-binaltorphimine. The delta 1 and delta 2 opioid receptor directed agonists, (DPDPE, [D-Pen2,D-Pen5]enkephalin), (deltorphin, [D-Ala2,Glu4]), respectively, also had significant differential analgesic effects, supporting a functional delta opioid receptor mediated enkephalinergic mechanism in Cepaea. These results suggest that this specific pulsed magnetic field (Cnp) elicits significant analgesic effects through mechanisms that, in part, involve delta and, to a lesser extent mu opioid receptors.

Cranial irradiation with Gaalas laser leads to naloxone reversible analgesia in rats.

Laser irradiation of the rat cranium can produce analgesia. The present experiment investigated the mechanism of such action. 27 rats received all possible combinations of laser (0, 6.4, and 12J/cm2) and naloxone (0, 5, and 10 mg/kg) prior to a hot plate test. Laser (820 nm, KHz pulsing, Omega Laser Systems, London) was applied to the rats' skulls and hind paw lick latencies (in seconds) were recorded immediately, 30 min., and 24 hr. after the administration of treatment. When animals were tested immediately following laser irradiation at 12J/cm2 significant analgesia resulted. Treatment with naloxone at either dose antagonised this effect, but naloxone produced no significant hyperalgesia when given alone. This suggests that opioid peptide mechanisms mediate the analgesic action of low-intensity laser irradiation of the cranium.

[The role of the endogenous opioid system in the mechanisms of the development of early postradiation autonomic and behavioral disorders]. / K voprosu o roli éndogennoi opioidnoi sistemy v mekhanizmakh razvitiia rannikh postluchevykh vegetativnykh i povedencheskikh narushenii.

In the experiments with rats and dogs it was shown that irradiation within the dose range of 50-100 Gy is accompanied by the increasing of enkephaline content in various brain structures and beta-endorphine level in blood plasma. The blocking of the opiate receptors naloxone promotes the decrease of post-radiation vomiting in dogs, gastrostasis and hypokinesia in rats. That supports the participation of endogenous opioid system in the primary clinical reaction response to irradiation.

Effects of chronic postnatal opioid receptor blockade by naltrexone upon proliferation capacity in the prenatally X-irradiated brain of the rat.

We recently reported that in rats prenatally X-irradiated on gestation day 14 with 1 Gy, postnatal chronic application of the opioid antagonist naltrexone (NX) led to a remarkable growth spurt of the microencephalic brain. In the present study we present histological and autoradiographic results found in the subependymal layer (SEL) of the forebrain lateral ventricles. NX led to an intermittent augmentation of the mitotic index of the X-irradiated brains within a postnatal observation period of 24 weeks. The most conspicuous finding was transient hyperplasia of the SEL at 4-6 weeks of age which occurred in close proximity to an intact ependymal lining. Districts of the lateral ventricles which were denuded from ependyme and where the rest of the ependymal layer (EL) was dislocated peripherally showed upon NX treatment a long-lasting SEL hyperplasia with a tendency towards dysplasia. These results revealed that repair proliferation of embryotoxic X-irradiation is normally under strong control by the opioid system. If that system, which exerts a suppressing effect upon glial growth, is blocked by NX, prominent hyperplastic reactions occur which may be useful for repairing the lesion pattern.

Enkephalin photoaffinity probes: synthesis and binding properties.

The diazomethyl ketone derivative of D-Ala2-Leu-enkephalin and Leu-enkephalin were synthesized. Replacement of the C-terminal carboxyl group with CO-CHN2 resulted in a potency decrease, the new compounds display micromolar affinities to 3H-naloxone and 3H-DALE binding sites. Photolysis of the ligands bound to rat brain membranes resulted in an approximately 30% irreversible loss of the receptors. Photoinactivation was prevented by the opiate antagonist, naloxone, thus providing that the ligands are specific photoaffinity probes of the opioid receptors.

Sodium modulates opioid receptors through a membrane component different from G-proteins. Demonstration by target size analysis.

The target size for opioid receptor binding was studied after manipulations known to affect the interactions between receptor and GTP-binding regulatory proteins (G-proteins). Addition of GTP or its analogs to the binding reaction, exposure of intact cells to pertussis toxin prior to irradiation, or treatment of irradiated membranes with N-ethylmaleimide did not change the target size (approximately equal to 100 kDa) for opioid receptors in NG 108-15 cells and rat brain. These data suggest that the 100-kDa species does not include an active subunit of a G-protein or alternatively that GTP does not promote the dissociation of the receptor-G-protein complex. The presence of Na+ (100 mM) in the radioligand binding assay induced a biphasic decay curve for agonist binding and a flattening of the monoexponential decay curve for a partial agonist. In both cases the effect was explained by an irradiation-induced loss of the low affinity state of the opioid receptor produced by the addition of Na+. This suggests that an allosteric inhibitor that mediates the effect of sodium on the receptor is destroyed at low doses of irradiation, leaving receptors which are no longer regulated by sodium. The effect of Na+ on target size was slightly increased by the simultaneous addition of GTP but was not altered by pertussis toxin treatment. Thus, the sodium unit is distinct from G-proteins and may represent a new component of the opioid receptor complex. Assuming a simple bimolecular model of one Na+ unit/receptor, the size of this inhibitor can be measured as 168 kDa.

Mu-, but not delta-, opioid receptor-mediated antinociception in mice is attenuated by gamma-irradiation.

Mice were exposed to whole-body irradiation (500 rads) from a 137Cs gamma-source and tested 2 h later for antinociception (tail-flick test) produced by intracerebroventricular administration of morphine or the more delta-selective opioid peptide, [D-Pen2,L-Pen5]enkephalin (DPLPE). Irradiation significantly attenuated the antinociception produced by morphine, but not by DPLPE. These results demonstrate a differential sensitivity of mu- and delta-opioid receptors to gamma-irradiation and, in addition, may be of clinical relevance for cancer patients receiving concurrent radiation therapy and opioid analgesics.

Laser photobiostimulation-induced hypoalgesia in rats is not naloxone reversible.

Laser photobiostimulation (LPBS) at the pulsing frequency of 4 Hz applied to the low resistance point located at the base of the tail of the rat, (Governing Vessel Meridian 1), produced a hypoalgesic effect, measured by tail-flick and hot-plate techniques. Pre-treatment with low dose naloxone (2 mg/kg) did not reverse the hypoalgesic effect of LPBS. High dose naloxone (20 mg/kg) reversed only partially, but significantly, the hypoalgesic effect of LPBS measured by hot-plate, but not that measured by the tail-flick technique. These data suggest that mechanisms other than endogenous opioids may be involved in LPBS-induced hypoalgesia.

Exposure to time varying magnetic fields associated with magnetic resonance imaging reduces fentanyl-induced analgesia in mice.

The effects of exposure to clinical magnetic resonance imaging (MRI) on analgesia induced by the mu opiate agonist, fentanyl, was examined in mice. During the dark period, adult male mice were exposed for 23.2 min to the time-varying (0.6 T/sec) magnetic field (TVMF) component of the MRI procedure. Following this exposure, the analgesic potency of fentanyl citrate (0.1 mg/kg) was determined at 5, 10, 15, and 30 min post-injection, using a thermal test stimulus (hot-plate 50 degrees C). Exposure to the magnetic-field gradients attenuated the fentanyl-induced analgesia in a manner comparable to that previously observed with morphine. These results indicate that the time-varying magnetic fields associated with MRI have significant inhibitory effects on the analgesic effects of specific mu-opiate-directed ligands.

Physical characterization of native opiate receptors. Additional information from detailed binding analysis of a radiation-inactivated receptor.

Target size analyses of the etorphine receptor were performed on frozen rat brain P2 homogenates using the radiation inactivation technique. Multi-point saturation curves at each radiation dose revealed that the apparent dissociation constant for the binding of this ligand to its receptor is a function of the dose. Analysis of the results shows clearly that the ligand-binding macromolecule is functionally coupled to at least one other macromolecule. When the coupling is destroyed the ligand dissociation constant becomes larger by over an order of magnitude. Thus, the variation of the dissociation constant with dose yields interesting new information on the nature of the native receptor which has implications with respect to the conformation of the binding site and to solubilization and cloning methods directed towards sequencing the ligand-binding component of opiate receptors.

Photoinactivation of the mu opioid receptor using a novel synthetic morphiceptin analog.

The benzophenone chromophore has been incorporated into a synthetic amino acid (p-benzoyl-L-phenylalanine; L-Bpa) to produce a chemically stable photoaffinity probe. L-Bpa was found to retain the photochemical reactivity of benzophenone. To test the utility of this synthetic amino acid as a photo-reactive probe for receptors, a tetrapeptide analog of morphiceptin was made as a model peptide in which the C-terminal prolinamide was replaced by L-Bpa amide. The affinity of the mu opioid receptor for this peptide is comparable to that for the parent compound, morphiceptin. Irradiation of the peptide-receptor complex reduced the subsequent binding of [3H]naloxone and virtually eliminated that of [3H]Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAGO). Binding studies with [3H]naloxone indicated that both the affinity and the capacity were reduced. Competition studies with [3H]D-Ala2-D-Leu5-enkephalin (DADLE) and naloxone indicated selective inactivation of a mu type opioid receptor.

Photoaffinity crosslinking of etorphine with opioid binding sites in the bovine adrenal medulla.

The covalent crosslinking of [3H]etorphine with opioid binding sites in the bovine adrenal medulla is reported. Of all the radiolabeled opiates tested (ethylketocyclazocine, etorphine, [D-Ala2, D-Leu5]enkephalin, [D-Ala2, Me-Phe4, Gly5-ol]enkephalin only etorphine could be crosslinked under uv irradiation. In our conditions (black uv lamp, 160 W, peak mean 360 nm, from a distance of 10 cm) maximum covalent binding was observed after a 10-min irradiation. Protein concentration was a crucial factor for the irreversible/total binding ratio. A good ratio (50%) was obtained at protein concentrations of about 1.0 mg/ml. Covalent binding of nonmodified opiates could be of interest for the biochemical characterization of their binding sites.

Determination of the sizes of the opioid receptors in their membrane environment by radiation inactivation.

Opioids, like other drugs, are thought to initiate their effects by association with their specific receptors. However, very little is known about the opioid receptor as a molecular entity. The binding components have been solubilized in detergent and purified by different approaches, but the molecular size of soluble opioid receptor complexes reported by different groups varied from 23,000 to 750,000. In this study, the technique of radiation inactivation by gamma rays was used to investigate the apparent size of the opioid receptor in rat brain membranes under different conditions. The molecular sizes of opioid receptor complexes were estimated as 313,000 +/- 13,500 in the presence of [D-Ala2, D-Leu5] enkephalin, NaCl and Gpp (NH)p; as 165,000 +/- 8,500 in the presence of NaCl only, or of both NaCl and Gpp (NH)p; as 217,000 +/- 6,600 in the presence of Gpp (NH)p only; and as 286,000 +/- 60,900 in the presence of MgCl2 only. A simple model has been proposed to explain these different apparent target sizes of opioid receptors obtained under different conditions.

Target size analysis of opioid receptors. No difference between receptor types, but discrimination between two receptor states.

Target size analysis of opioid receptor is complicated by the presence of multi-exponential inactivation curves. Irradiation of intact frozen tissue proved essential to eliminate such artifacts, due to indirect irradiation effects. Upon irradiation condition, opioid binding activity was inactivated in a single mono-exponential manner. Identical inactivation curves were obtained for mu, delta and kappa binding activities in brain membranes from rat, guinea-pig and frog and in NG 108-15 cells: the molecular mass obtained was 98 +/- 2 kDa. However, when opioid binding was assayed in the presence of Na+, Mg2+ and GTP, the molecular mass was found to be only 56 +/- 4.4 kDa. We suggest that the opioid recognition site comprises a unit of 56 kDa and that in the absence of Na+, Mg2+ and GTP an additional membrane component of 40-44 kDa is necessary for high-affinity opioid binding.

Photoaffinity labeling of opiate receptors with 3H-etorphine: possible species differences in glycosylation.

Opiate receptors from whole rat brain (minus cerebellum) and cow striatum were labeled irreversibly using the intrinsic photolability of 3H-etorphine. After incubation with 2 nM 3H-etorphine and centrifugal washing, membranes were irradiated with light of 254 nm. Non-specific binding was determined by carrying out incubations in presence and absence of 10 microM levallorphan. Specific binding in photolabeled membranes was 75-80%, with a photo-incorporation yield of approximately 50%. Photolabeled membranes were extracted with CHAPS/Lubrol and unbound 3H-etorphine was removed by dialysis and passage over Sephadex G-25. Solubilized proteins were then subjected to chromatography on wheat germ agglutinin, and retained proteins were eluted with N-acetyl D-glucosamine (NAG). Protein profiles from rat brain and cow striatum were identical, with 89% of the total protein flowing through unretained and 11% eluted by NAG. However, the profile of radioactivity was markedly different in the two species. With rat, the specific activity (cpm/A280) was the same for flow-through and NAG-eluate. With cow, the specific activity of the NAG-eluate was 17 times greater than the flow-through. These results indicate that cow striatum and rat whole brain contain populations of opiate receptors which are glycosylated differently.

Differential sensitivity of major types of opioid binding sites to U.V. irradiation.

The three major types of opioid binding sites exhibited differential sensitivity to inactivation by ultraviolet irradiation. The order of sensitivity was delta greater than mu greater than kappa. Two separate rates of inactivation by U.V. light were observed for each type, an initial fast rate and a secondary slow rate. Addition of EDTA or EGTA to the buffer medium decreased only the initial fast rate of inactivation. The photosensitive tryptophan and tyrosine moieties have been shown to be photolyzed in a two step manner in a number of protein systems. It is suggested that the differential sensitivity of the multiple opioid binding sites may reflect either differing amino acid composition or differences in tertiary structure.

Sizes of opioid receptor types in rat brain membranes.

The sizes of the receptors binding opiates and enkephalins in rat brain membranes were investigated by the radiation inactivation technique. By comparison with enzymes of known size added as internal standards, the mu and delta binding sites both gave a molecular weight of about 110000. Other opiate-binding components, which may include the kappa site, showed a much lower rate of inactivation when in reducing conditions, implying a subunit molecular weight of the order of 30000 for such a site.