RESUMO
AIM: The objective of this study is to develop a serovar-specific loop-mediated isothermal amplification (LAMP) method for sensitive, rapid, and inexpensive detection of Salmonella serovar Enteritidis under field conditions. METHODS: A set of six specific primers was designed with Salmonella Enteritidis DNA as the target. LAMP conditions were optimized by incubating the target DNA with the Bst DNA polymerase large fragment in a simple water bath. The sensitivity and specificity of LAMP was then compared with those of fluorescent quantitative real-time polymerase chain reaction (FQ-PCR). RESULTS: The results were as follows. (1) Serovar-specific Salmonella Enteritidis DNA was amplified at 65°C in as early as 20min in a water bath. (2) A colour change visible to the naked eye indicated a positive amplification reaction. (3) The detection limit of the LAMP assay was 4 copies µl(-1) ; thus, the sensitivity and specificity of this assay is similar to those of the FQ-PCR. CONCLUSIONS: LAMP is a high-throughput detection technique with high sensitivity, specificity, and simplicity; these factors make it suitable for specifically detecting Salmonella Enteritidis under field conditions and in laboratory settings. Thus, LAMP eliminates the need for complicated equipment and technical training in the detection of this specific serovar. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study involving the use of LAMP to detect Salmonella serovar-specific DNA sequences. It is also the first to report an ideal method of distinguishing between Salmonella Enteritidis and other Salmonella under field conditions.
Assuntos
Microbiologia de Alimentos/métodos , Técnicas de Amplificação de Ácido Nucleico , Salmonella enteritidis/fisiologia , Salmonella enteritidis/genética , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Studies of direct excitatory effects elicited by opioid agonists on various types of neurone have been confirmed and expanded in numerous laboratories following the initial findings reviewed previously by Stanley Crain and Ke-Fei Shen. However, the critical role of the endogenous glycolipid GM1 ganglioside in regulating Gs-coupled, excitatory opioid receptor functions has not been addressed in any of the recent reviews of opioid stimulatory mechanisms. This article by Stanley Crain and Ke-Fei Shen focuses on crucial evidence that the concentration of GM1 in neurones might, indeed, play a significant role in the modulation of opioid receptor-mediated analgesia, tolerance and dependence.
Assuntos
Analgesia , Analgésicos Opioides/farmacologia , Gangliosídeo G(M1)/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Receptores Opioides/efeitos dos fármacos , Animais , Células CHO , Cricetinae , Tolerância a Medicamentos , Gangliosídeo G(M1)/fisiologia , Humanos , Antagonistas de Entorpecentes/farmacologia , Transtornos Relacionados ao Uso de Opioides/etiologia , Receptores Opioides/agonistasRESUMO
Activation of opioid receptors has generally been considered to produce inhibitory effects on neuronal activity. However, recent studies indicate that specific mu-, delta- and kappa-opioid receptor agonists can elicit excitatory, as well as inhibitory, modulation of the action potentials of sensory neurons isolated in culture. Stanley Crain and Ke-Fei Shen review the evidence for mediation of these direct excitatory effects by naloxone-reversible opioid receptors. They propose that this dual modulatory mechanism may help to account for previously unexplained enhancement by opioids of transmitter release, paradoxical hyperalgesic and aversive effects of opioids, and some aspects of opioid tolerance and addiction.
Assuntos
Entorpecentes/farmacologia , Neurônios Aferentes/fisiologia , Receptores Opioides/efeitos dos fármacos , Humanos , Neurônios Aferentes/efeitos dos fármacos , Receptores Opioides/fisiologiaRESUMO
Recent preclinical and clinical studies have demonstrated that cotreatments with extremely low doses of opioid receptor antagonists can markedly enhance the efficacy and specificity of morphine and related opioid analgesics. Our correlative studies of the cotreatment of nociceptive types of dorsal-root ganglion neurons in vitro and mice in vivo with morphine plus specific opioid receptor antagonists have shown that antagonism of Gs-coupled excitatory opioid receptor functions by cotreatment with ultra-low doses of clinically available opioid antagonists, e.g. naloxone and naltrexone, markedly enhances morphine's antinociceptive potency and simultaneously attenuates opioid tolerance and dependence. These preclinical studies in vitro and in vivo provide cellular mechanisms that can readily account for the unexpected enhancement of morphine's analgesic potency in recent clinical studies of post-surgical pain patients cotreated with morphine plus low doses of naloxone or nalmefene. The striking consistency of these multidisciplinary studies on nociceptive neurons in culture, behavioral assays on mice and clinical trials on post-surgical pain patients indicates that clinical treatment of pain can, indeed, be significantly improved by administering morphine or other conventional opioid analgesics together with appropriately low doses of an excitatory opioid receptor antagonist.
Assuntos
Analgésicos Opioides/farmacologia , Morfina/farmacologia , Antagonistas de Entorpecentes/farmacologia , Entorpecentes/farmacologia , Animais , Sinergismo Farmacológico , Tolerância a Medicamentos , Transtornos Relacionados ao Uso de Substâncias/prevenção & controleRESUMO
Previous studies have reported that large (microM) concentrations of kappa opioids, e.g. dynorphin and 3,4 dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)benzene-acetamide (U-50,488H), shorten the duration of the calcium component of the action potential of dorsal root ganglion neurons by decreasing a voltage-sensitive Ca2+ conductance. The present study showed that, in addition to these inhibitory modulatory effects, small (nM) concentrations of dynorphin, as well as U-50,488H, prolonged the action potential in about 75% of the neurons of dorsal root ganglia in ganglion spinal cord explants of mouse (tested in 5 mM Ba2+). Both the excitatory and inhibitory effects of these kappa opioids were prevented by perfusion together with the opioid antagonist, diprenorphine (10 nM). However, when responsivity tests with opioids were carried out in the presence of multiple K+ channel blockers [Ba2+, Cs+ and tetraethylammonium (TEA)], 1 nM dynorphin prolonged the action potential in only 7% of the neurons (n = 28), whereas 1 nM U-50,488H still elicited the prolongation of the action potential in 60% of the cells (n = 39). These data suggest that dynorphin prolongs the action potential of neurons of dorsal root ganglion by activating a kappa subtype of receptor that decreases a voltage-sensitive K+ conductance, whereas U-50,488H produces similar excitatory modulation of the action potential by activating another kappa subtype of receptor that increases a voltage-sensitive Ca2+ conductance. Thus, U-50,488H-induced prolongation of the action potential appears to be mediated by a kappa subtype of receptor that produces the opposite effect on Ca2+ channels to that which occurs during kappa opioid-induced shortening of the action potential.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cálcio/fisiologia , Dinorfinas/farmacologia , Gânglios Espinais/efeitos dos fármacos , Entorpecentes/farmacologia , Neurônios/efeitos dos fármacos , Potássio/fisiologia , Receptores Opioides/fisiologia , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida , Potenciais de Ação/efeitos dos fármacos , Animais , Encefalina Leucina/análogos & derivados , Encefalina Leucina/farmacologia , Leucina Encefalina-2-Alanina , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Técnicas In Vitro , Camundongos , Condução Nervosa/efeitos dos fármacos , Neurônios/fisiologia , Gravidez , Pirrolidinas/farmacologia , Receptores Opioides kappaRESUMO
Electrophysiologic studies of dorsal-root ganglion (DRG) neurons in culture have demonstrated both excitatory (Gs-coupled) as well as inhibitory (Gi/Go-coupled) opioid receptor-mediated actions. Brief treatment of DRG neurons with cholera toxin-beta which binds specifically to GM1 sites on neuronal membranes, selectively blocks opioid excitatory but not inhibitory effects. Conversely, after brief treatment of DRG neurons with GM1, but not with GM2, GM3, or other related gangliosides, the threshold concentration of opioid agonists for eliciting excitatory effects is markedly decreased from nM to pM-fM levels and opioid antagonists, for example, naloxone (NLX), at low concentrations paradoxically elicit excitatory effects. These studies suggest that the excitatory opioid supersensitivity of GM1-treated DRG neurons is due primarily to increased efficacy of excitatory opioid-receptor activation of Gs. Recent studies of cloned delta opioid receptors transfected into CHO cells suggest that this supersensitivity of GM1-treated DRG neurons may be further augmented by rapid conversion of many opioid receptors from a Gi/Go-coupled inhibitory mode to a Gs-coupled excitatory mode. The opioid excitatory supersensitivity elicited in DRG neurons by acute elevation of exogenous GM1 provides novel insights into mechanisms underlying opioid tolerance and dependence, since remarkably similar supersensitivity occurs in DRG and other neurons after chronic treatment with morphine or other opioid agonists that upregulate endogenous GM1.
Assuntos
Gangliosídeo G(M1)/farmacologia , Neurônios/fisiologia , Receptores Opioides/fisiologia , Animais , Células Cultivadas , Toxina da Cólera/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Gânglios Espinais/fisiologia , Humanos , Modelos Neurológicos , Naloxona/farmacologia , Neurônios/efeitos dos fármacos , Receptores Opioides/efeitos dos fármacos , Receptores Opioides delta/efeitos dos fármacos , Receptores Opioides delta/fisiologiaRESUMO
Multiple modulatory effects of opioids on the duration of the calcium component of the action potential (APD) of dorsal-root ganglion (DRG) neurons of mouse spinal cord-ganglion explants were studied. The APD of DRG neuron perikarya has been previously shown to be shortened by exposure to high concentrations of opioids (ca. 0.1-1 microM) in about 1/2 of the cells tested. The present study demonstrates that in addition to these inhibitory modulatory effects of opioids, lower concentrations (1-10 nM) of present study demonstrates that in addition to these inhibitory modulatory effects of opioids, lower concentration (1-10 nM) of delta- mu, and kappa-opioid agonists elicit excitatory modulatory effects, i.e. prolongation of the APD, in about 2/3 of the sensory neurons tested. APD prolongation as well as shortening elicited by delta, mu, and kappa agonists were prevented by coperfusion with the opioid antagonists, naloxone or diprenorphine (10 nM). APD prolongation induced by the delta-agonist [D-Ala2-D-Leu5]enkephalin (DADLE) was prevented in the presence of multiple K+ channel blockers, whereas excitatory modulation by the specific kappa-agonist, U-50,488H was not attenuated under these conditions. After treatment of DRG neurons with pertussis toxin (1 micrograms/ml for several days) or forskolin (50 muM for less than 15 min), a much smaller fraction of cells showed opioid-induced APD shortening; moreover, a much larger fraction of cells showed opioid-induced APD prolongation, even when tested with high concentrations of DADLE (1-10 muM). These data indicate that opioid-induced APD prolongation is not mediated by pertussis toxin-sensitive G proteins (which have been shown to regulate opioid inhibitory effects) and suggest that elevation of cyclic AMP levels may enhance opioid excitatory responsiveness. Furthermore, our analyses indicate that mu-, delta- and kappa-subtypes of excitatory as well as inhibitory opioid receptors may be expressed on the same DRG neuron perikaryon under in vitro conditions. If dual opioid modulation of the APD of DRG perikarya also occurs in central DRG terminals this may play a significant role both in nociceptive signal transmission as well as tolerance to opioid analgesia.
Assuntos
Endorfinas/farmacologia , Encefalina Leucina/análogos & derivados , Gânglios Espinais/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Células Cultivadas , Encefalina Leucina/farmacologia , Leucina Encefalina-2-Alanina , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Camundongos , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Receptores Opioides/efeitos dos fármacos , Receptores Opioides/fisiologiaRESUMO
We previously showed that mouse sensory dorsal root ganglion (DRG) neurons chronically exposed to 1 microM D-ala2-D-leu5-enkephalin (DADLE) or morphine for > 2-3 days in culture become tolerant to the usual opioid inhibitory receptor-mediated effects, i.e. shortening of the duration of the calcium-dependent component of the action potential (APD), and supersensitive to opioid excitatory APD-prolonging effects elicited by low opioid concentrations. Whereas nanomolar concentrations of dynorphin(1-13) or morphine are generally required to prolong the APD of naive DRG neurons (by activating excitatory opioid receptors), femtomolar levels become effective after chronic opioid treatment. Whereas 1-30 nM naloxone or diprenorphine prevent both excitatory and inhibitory opioid effects but do not alter the APD of native DRG neurons, both opioid antagonists unexpectedly prolong the APD of most of the chronic opioid-treated cells. In the present study, chronic exposure of DRG neurons to 1 microM DADLE together with cholera toxin-B subunit (which selectively blocks GM1 ganglioside-regulated opioid excitatory, but not inhibitory, receptor functions) prevented the development of opioid excitatory supersensitivity and markedly attenuated tolerance to opioid inhibitory effects. Conversely, sustained exposure of DRG neurons to 1 nM DADLE, which selectively activates excitatory opioid receptor functions, resulted in characteristic opioid excitatory supersensitivity but no tolerance. These results suggest that 'dependence'-like properties can be induced in chronic opioid-treated sensory neurons in the absence of tolerance. On the other hand, development of some components of tolerance in these cells may require sustained activation of both excitatory, as well as inhibitory, opioid receptor functions.
Assuntos
Leucina Encefalina-2-Alanina/farmacologia , Gânglios Espinais/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Receptores Opioides/efeitos dos fármacos , Transtornos Relacionados ao Uso de Substâncias/fisiopatologia , Animais , Toxina da Cólera/farmacologia , Tolerância a Medicamentos/fisiologia , Gânglios Espinais/citologia , Camundongos , Fragmentos de Peptídeos/farmacologia , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Effects of temperature on population spike and cellular activities have been assessed in the CA1 region of hippocampal slices from mature and immature rabbit. In field potential recordings, population spike amplitude was maximal at near 30 degrees C for both mature and immature tissue, and fell off as temperature was either raised (to a maximum of 44 degrees C) or lowered (to a minimum of 20 degrees C). With cooling below 30 degrees C, population spikes decreased in amplitude and became broader; stimuli always elicited some response, and changes due to cooling were reversible. With increases in temperature, however, irreversible decrease and/or loss of population spikes occurred when tissue was warmed beyond 43 degrees C. Input-output curves established for mature and immature slices indicated that, at all temperatures, population spike amplitude grew more rapidly with small increases in stimulus intensity in immature slices as compared to mature slices. Intracellular recordings were made from CA1 pyramidal cells in mature and immature hippocampal slices. For both mature and immature tissues, moderate warming (to 40 degrees C) produced membrane hyperpolarizations in many cells, especially in the mature hippocampus. Increasing temperature beyond 40 degrees C led to marked depolarizations in a number of cells, a depolarization that was irreversible, particularly in mature neurons. Cooling generally produced a depolarizing shift in membrane potential and an accompanying increase in input resistance; these effects, however, were reversible. Temperature changes in both warming and cooling directions had effects on repetitive firing patterns in both mature and immature neurons. In particular, spike trains elicited by a constant current pulse at a given membrane potential became shorter. The effects of cooling on this cell parameter were reversible, but warming-induced changes were usually permanent. Irreversibility of the warming effects was more pronounced in cells from mature than from immature hippocampus. As reported previously, cooling produced marked spike broadening and changes in synaptic potentials in both mature and immature neurons. These studies confirm previously reported temperature sensitivities of neuronal properties in hippocampal slices. On the basis of these data, and reports from other laboratories, it is clear that relatively small changes in temperature can have rather dramatic effects on properties of single cells and cell populations. Such temperature sensitivity is critical in evaluating data obtained from in vitro slice preparations.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Envelhecimento/fisiologia , Hipocampo/fisiologia , Temperatura , Potenciais de Ação/efeitos dos fármacos , Animais , Estimulação Elétrica , Hipocampo/crescimento & desenvolvimento , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , CoelhosRESUMO
In a previous study, we demonstrated that cholera toxin-A subunit, as well as the whole toxin, selectively blocks opioid-induced prolongation of the Ca2+ component of the action potential duration (APD) in dorsal root ganglion (DRG) neurons, indicating mediation of this excitatory effect by Gs-linked opioid receptors. The present study shows that pretreatment of DRG neurons with the B subunit of cholera toxin (1-10 ng/ml; greater than 15 min) can also block mu/delta and kappa opioid-induced APD prolongation, but not shortening. Since the B subunit binds selectively to GM1 ganglioside located on the cell surface, these results suggest that this ganglioside may regulate Gs-linked excitatory opioid receptor functions in DRG neurons. Possible contamination of purified B subunit preparations of cholera toxin with traces of the more potent A subunit was eliminated by heating the stock solution to 56 degrees C for 20 min. Exposure of DRG neurons to an affinity-purified anti-GM1 antiserum also blocked opioid-induced APD prolongation, providing further evidence that GM1 ganglioside may play an essential role in excitatory opioid modulation of the action potential of these cells. The blockade by cholera toxin-B subunit and anti-GM1 antibodies of opioid-induced APD prolongation is best accounted for by the following hypothesis: CTX-B interferes with an endogenous GM1 ganglioside component of the excitatory, but not inhibitory, opioid receptor complex on DRG neurons that may allosterically regulate coupling of the receptors via Gs to adenylate cyclase/cyclic adenosine monophosphate-dependent ionic conductances.
Assuntos
Toxina da Cólera/farmacologia , Dinorfinas/farmacologia , Gangliosídeo G(M1)/fisiologia , Gânglios Espinais/fisiologia , Neurônios Aferentes/fisiologia , Receptores Opioides/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Feto , Gangliosídeo G(M1)/imunologia , Gânglios Espinais/efeitos dos fármacos , Soros Imunes , Camundongos , Neurônios Aferentes/efeitos dos fármacos , Técnicas de Cultura de ÓrgãosRESUMO
Our previous studies indicated that opioid-induced prolongation of the Ca2+ component of the action potential duration (APD) in dorsal root ganglion (DRG) neurons is mediated by excitatory opioid receptors that are coupled to cyclic AMP-dependent voltage-sensitive ionic conductances. In the present study, DRG neurons were treated with cholera toxin (CTX), or with the A subunit of CTX, in order to determine if these excitatory opioid receptors are positively coupled via the GTP-binding protein Gs to the adenylate cyclase/cyclic AMP system. In contrast, inhibitory opioid receptors have been shown to be linked to pertussis toxin-sensitive Gi/Go regulatory proteins that mediate APD shortening responses. After pretreatment of DRG-spinal cord explants with remarkably low concentrations of CTX-A (1 pg/ml-1 ng/ml; greater than 15 min) or whole toxin (1 pg/ml-1 microgram/ml) the APD prolongation elicited in DRG neurons by 1-10 nM delta/mu (DADLE) or kappa (U-50,488H) opioids was blocked (29 out of 30 cells), whereas APD shortening by microM opioid concentrations was unaffected. Opioid-induced APD prolongation was blocked even when the initial treatment with CTX or CTX-A alone did not prolong the APD. The blocking effects of CTX and CTX-A were reversed in tests made 2 h after return to control medium. The mechanisms underlying the unusually potent blocking effects of CTX and CTX-A on opioid excitatory modulation of the APD of DRG neurons require correlative biochemical analyses.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Toxina da Cólera/farmacologia , Leucina Encefalina-2-Alanina/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Neurônios Aferentes/fisiologia , Receptores Opioides/fisiologia , Medula Espinal/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Feto , Gânglios Espinais/fisiologia , Substâncias Macromoleculares , Camundongos , Neurônios Aferentes/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Receptores Opioides/efeitos dos fármacosRESUMO
We recently showed that the opioid alkaloids, etorphine, dihydroetorphine and diprenorphine, have remarkably potent antagonist actions on excitatory opioid receptor functions in mouse sensory dorsal root ganglion (DRG) neurons. Pretreatment of naive nociceptive types of neurons with pM concentrations of these antagonists blocks excitatory prolongation of the calcium-dependent component of the action potential duration (APD) elicited by pM-nM morphine or other bimodally acting mu, delta and kappa opioid agonists and unmasks inhibitory APD shortening which usually requires much higher (ca. microM) concentrations. The present study demonstrates that pM concentrations of [des-Tyr1] fragments of dynorphin and beta-endorphin, as well as beta-endorphin-(1-27), can also selectively block excitatory opioid receptor functions in DRG neurons and unmask potent inhibitory effects of low concentrations of bimodally acting mu, delta and kappa opioid peptides and alkaloid agonists. These N- or C-terminus modified dynorphin or beta-endorphin peptides can be readily formed in neurons by specific peptidase activities. Since sustained activation of excitatory opioid receptor functions is essential for the development of tolerance/dependence in chronic morphine-treated DRG neurons in culture, the present in vitro study may help to account for the unexplained efficacy of [des-Tyr1] dynorphin fragments, as well as the endogenous opioids dynorphin A and beta-endorphin, in suppressing development and expression of naloxone-precipitated withdrawal and morphine tolerance in vivo.
Assuntos
Dinorfinas/farmacologia , Gânglios Espinais/efeitos dos fármacos , Receptores Opioides delta/efeitos dos fármacos , beta-Endorfina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Dinorfinas/química , Ala(2)-MePhe(4)-Gly(5)-Encefalina , D-Penicilina (2,5)-Encefalina , Encefalinas/farmacologia , Gânglios Espinais/citologia , Camundongos , Morfina/farmacologia , Neurônios/efeitos dos fármacos , Receptores Opioides delta/agonistas , beta-Endorfina/químicaRESUMO
Mouse sensory dorsal-root ganglion (DRG) neurons chronically exposed to 1 microM D-Ala2-D-Leu5-enkephalin (DADLE) for greater than 1 week in culture become tolerant to opioid inhibitory effects, i.e. shortening of the duration of the calcium-dependent component of the action potential (APD). Acute application of higher concentrations of DADLE (ca. 10 microM) to these treated neurons not only fails to shorten the APD but, instead, generally elicits excitatory effects, i.e. prolongation of the APD. The present study shows that chronic DADLE- or morphine-treated DRG neurons also become supersensitive to the excitatory effects of opioids. Whereas nM concentrations of dynorphin(1-13) are generally required to prolong the APD of naive DRG neurons, fM levels become effective after chronic opioid treatment. Whereas 1-30 nM naloxone or diprenorphine do not alter the APD of naive DRG neurons, both opioid antagonists unexpectedly prolong the APD of most of the treated cells. Similar supersensitivity to the excitatory effects of opioid agonists and antagonists was previously observed after acute treatment of naive DRG neurons with GM1 ganglioside. Our results suggest that both chronic opioid and acute GM1 treatments of DRG neurons greatly enhance the efficacy of opioid excitatory receptor functions so that even the extremely weak agonist properties of naloxone and diprenorphine become effective in prolonging the APD of these treated cells when tested at low concentrations, whereas their antagonist properties at inhibitory opioid receptors do not appear to be altered. Furthermore, whereas cholera toxin-B subunit (CTX-B; 1-10 nM) blocks opioid-induced APD prolongation in naive DRG neurons (presumably by interfering with endogenous GM1 modulation of excitatory opioid receptors functions), even much higher concentrations of CTX-B were ineffective in chronic opioid-treated as well as acute GM1-elevated neurons. These and related data suggest that opioid excitatory supersensitivity in chronic opioid-treated DRG neurons may be due to a cyclic AMP-dependent increase in GM1 ganglioside levels. Our results may clarify mechanisms of opioid dependence and the paradoxical supersensitivity to naloxone which triggers withdrawal symptoms after opiate addiction.
Assuntos
Leucina Encefalina-2-Alanina/administração & dosagem , Gangliosídeo G(M1)/farmacologia , Gânglios Espinais/efeitos dos fármacos , Morfina/administração & dosagem , Neurônios Aferentes/efeitos dos fármacos , Receptores Opioides/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Células Cultivadas , Toxina da Cólera/administração & dosagem , Diprenorfina/administração & dosagem , Gangliosídeo G(M1)/metabolismo , Gânglios Espinais/citologia , Camundongos , Naloxona/administração & dosagem , Neurônios Aferentes/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Fatores de TempoRESUMO
Our previous electrophysiologic studies on nociceptive types of dorsal root ganglion (DRG) neurons in culture demonstrated that extremely low fM-nM concentrations of morphine and many other bimodally-acting mu, delta and kappa opioid agonists can elicit direct excitatory opioid receptor-mediated effects, whereas higher (microM) opioid concentrations evoked inhibitory effects. Cotreatment with pM naloxone or naltrexone (NTX) plus fM-nM morphine blocked the excitatory effects and unmasked potent inhibitory effects of these low opioid concentrations. In the present study, hot-water-immersion tail-flick antinociception assays at 52 degrees C on mice showed that extremely low doses of morphine (ca. 0.1 microg/kg) can, in fact, elicit acute hyperalgesic effects, manifested by rapid onset of decreases in tail-flick latency for periods >3 h after drug administration. Cotreatment with ultra-low-dose NTX (ca. 1-100 pg/kg) blocks this opioid-induced hyperalgesia and unmasks potent opioid analgesia. The consonance of our in vitro and in vivo evidence indicates that doses of morphine far below those currently required for clinical treatment of pain may become effective when opioid hyperalgesic effects are blocked by coadministration of appropriately low doses of opioid antagonists. This low-dose-morphine cotreatment procedure should markedly attenuate morphine tolerance, dependence and other aversive side-effects.
Assuntos
Analgésicos Opioides/farmacologia , Hiperalgesia/tratamento farmacológico , Hiperalgesia/fisiopatologia , Morfina/farmacologia , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Doença Aguda , Animais , Relação Dose-Resposta a Droga , Tolerância a Medicamentos/fisiologia , Etorfina/farmacologia , Temperatura Alta , Masculino , Camundongos , Dependência de Morfina/prevenção & controle , Limiar da Dor/efeitos dos fármacos , Receptores Opioides/fisiologiaRESUMO
In a previous study we demonstrated that injection (i.p.) of low doses of GM1 ganglioside in mice rapidly attenuates morphine's analgesic effects. This result is consonant with our electrophysiologic studies in nociceptive types of dorsal root ganglion (DRG) neurons in culture, which showed that exogenous GM1 rapidly increased the efficacy of excitatory (Gs-coupled) opioid receptor functions. By contrast, treatment of DRG neurons with the non-toxic B-subunit of cholera toxin (CTX-B) which binds selectively to GM1, blocked the excitatory, but not inhibitory, effects of morphine and other bimodally-acting opioid agonists, thereby resulting in a net increase in inhibitory opioid potency. The present study provides more direct evidence that endogenous GM1 plays a physiologic role in regulating excitatory opioid receptor functions in vivo by demonstrating that cotreatment with remarkably low doses of CTX-B (10 ng/kg, s.c.) selectively blocks hyperalgesic effects elicited by morphine or by a kappa opioid agonist, thereby unmasking potent opioid analgesia. These results are comparable to the effects of cotreatment of mice with morphine plus an ultra-low dose of the opioid antagonist, naltrexone (NTX) which blocks opioid-induced hyperalgesic effects, unmasking potent opioid analgesia. Low-dose NTX selectively blocks excitatory opioid receptors at their recognition site, whereas CTX-B binds to, and interferes with, a putative allosteric GM1 regulatory site on excitatory opioid receptors. Furthermore, chronic cotreatment of mice with morphine plus CTX-B attenuates development of opioid tolerance and physical dependence, as previously shown to occur during cotreatment with low-dose NTX.
Assuntos
Toxina da Cólera/farmacologia , Tolerância a Medicamentos , Hiperalgesia , Antagonistas de Entorpecentes , Transtornos Relacionados ao Uso de Opioides , Medição da Dor , Receptores Opioides/fisiologia , Adjuvantes Imunológicos/farmacologia , Administração Oral , Analgésicos Opioides/farmacologia , Animais , Células Cultivadas , Toxina da Cólera/administração & dosagem , Esquema de Medicação , Tolerância a Medicamentos/imunologia , Temperatura Alta , Hiperalgesia/induzido quimicamente , Hiperalgesia/fisiopatologia , Injeções Subcutâneas , Masculino , Camundongos , Morfina/farmacologia , Naltrexona/administração & dosagem , Transtornos Relacionados ao Uso de Opioides/fisiopatologia , Medição da Dor/efeitos dos fármacos , Síndrome de Abstinência a Substâncias/etiologia , Síndrome de Abstinência a Substâncias/prevenção & controleRESUMO
The mechanism of action of the dimeric enkephalin peptide, biphalin (Tyr-D-Ala-Gly-Phe-NH2)2, which was previously shown to have remarkable high antinociceptive potency and low dependence liability in vivo, has now been studied by electrophysiologic analyses of its effects on the action potential duration (APD) of nociceptive types of sensory dorsal root ganglion (DRG) neurons in culture. Acute application of biphalin (pM-microM) elicited only dose-dependent, naloxone-reversible inhibitory (APD-shortening) effects on DRG neurons. Furthermore, at pM concentrations that evoked little or no alteration of the APD of DRG neurons biphalin selectively antagonized excitatory (APD-prolonging) effects of low (fM-nM) concentrations of bimodally-acting mu and delta opioid agonists and unmasked potent inhibitory effects of these opioids. This dual opioid inhibitory-agonist/excitatory-antagonist property of biphalin is remarkably similar to that previously observed in studies of the ultra-potent opioid analgesic, etorphine on DRG neurons and in sharp contrast to the excitatory agonist action of most mu, delta and kappa opioid alkaloids and peptides when tested at low (pM-nM) concentrations. Chronic treatment of DRG neurons with high (microM) concentrations of biphalin did not result in supersensitivity to the excitatory effects of naloxone nor in tolerance to opioid inhibition effects, in contrast to the excitatory opioid supersensitivity and tolerance that develop in chronic morphine- or DADLE-treated, but not chronic etorphine-treated, neurons. These studies on DRG neurons in vitro may help to account for the unexpectedly high antinociceptive potency and low dependence liability of biphalin as well as etorphine in vivo.
Assuntos
Analgésicos/farmacologia , Encefalinas/farmacologia , Antagonistas de Entorpecentes , Neurônios Aferentes/efeitos dos fármacos , Sequência de Aminoácidos , Analgésicos Opioides/farmacologia , Animais , Células Cultivadas , Técnicas de Cultura , Tolerância a Medicamentos , Eletrofisiologia , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Morfina/farmacologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologiaRESUMO
Chronic morphine-treated dorsal-root ganglion (DRG) neurons in DRG/spinal cord explant cultures were previously shown to become supersensitive to the excitatory effects of remarkably low concentrations of the opioid agonists, morphine and dynorphin, and the opioid antagonist, naloxone. The present study demonstrates that this opioid excitatory supersensitivity of chronic morphine-treated DRG neurons (1 microM for > 1 week) is retained for periods > 3 months after return to control culture medium. Acute application of femtomolar dynorphin, as well as nanomolar naloxone, to the treated neurons after months in control medium evoked characteristic prolongation of the action potential duration (APD), as occurs in cells tested during or shortly after chronic opioid exposure. The threshold concentrations for eliciting these excitatory effects in naive DRG neurons are > 1000-fold higher. Furthermore, treatment of micromolar morphine-sensitized neurons with 1 nM etorphine (which is a potent excitatory opioid receptor antagonist) for I week prior to return to control medium blocked further expression of opioid excitatory supersensitivity when tested after an additional 1-7 weeks in culture. These results provide a unique in vitro model system for analyses of some of the cellular mechanisms underlying protracted opioid dependence in vivo.
Assuntos
Gânglios Espinais/efeitos dos fármacos , Morfina/farmacologia , Naloxona/farmacologia , Entorpecentes , Neurônios/efeitos dos fármacos , Transtornos Relacionados ao Uso de Substâncias , Animais , Meios de Cultura , Etorfina/farmacologia , Gânglios Espinais/citologia , Camundongos , Camundongos Endogâmicos , Antagonistas de Entorpecentes/farmacologia , Entorpecentes/farmacologia , Valores de Referência , Fatores de TempoRESUMO
At low (< nM) concentrations, mu, delta or kappa opioid peptides as well as morphine and other opioid alkaloids elicit dose-dependent excitatory prolongation of the calcium-dependent component of the action potential duration (APD) of many mouse sensory dorsal root ganglion (DRG) neurons, whereas application of the same opioids at higher (uM) concentrations results in inhibitory shortening of the APD. These bimodal opioid excitatory/inhibitory effects on DRG neurons are blocked by naloxone. In contrast to bimodally acting opioids, the opioid alkaloids, etorphine and dihydroetorphine (thebaine-oripavine derivatives) uniquely elicited only dose-dependent, naloxone-reversible inhibitory effects on sensory neurons in DRG-spinal cord explants, even at concentrations as low as 1 pM, and showed no excitatory effects at lower concentrations. These remarkably potent inhibitory opioid receptor agonists also act as antagonists at excitatory opioid receptors since pretreatment of DRG neurons with subthreshold concentrations (< pM) blocked excitatory APD prolongation by nM morphine (or other opioids) and unmasked inhibitory APD shortening which generally requires much higher concentrations. Furthermore, acute application of pM-nM etorphine to chronic microM morphine- or D-Ala2-D-Leu5 enkephalin (DADLE)-treated DRG neurons blocked the nM naloxone-precipitated APD prolongation that generally occurs in DRG cells sensitized by bimodally acting opioids. In the presence of pM etorphine, chronic treatment of DRG neurons with microM morphine or DADLE no longer resulted in development of tolerance/dependence effects, as previously observed after similar chronic opioid treatment in the presence of cholera toxin-B subunit. These in vitro studies may clarify the mechanisms underlying the potent analgesic effects of etorphine and dihydroetorphine in vivo and to guide the use of these and other excitatory opioid receptor antagonists in attenuating development of opiate dependence/addiction.