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Publications and source records attributed to S M Crain.
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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.
Application of low concentrations (pM-nM) of NGF to mouse dorsal root ganglion (DRG)-spinal cord explants in long-term organotypic cultures rapidly prolongs the duration of the Ca(2+)-dependent component of the action potential (APD) in a major subset of DRG neurons that were previously shown to have characteristic responsiveness to exogenous opioids. These NGF-elicited excitatory modulating effects are blocked by pretreatment of the DRG neurons with monoclonal antibodies to rodent NGF receptors. NGF-induced APD prolongation is also prevented by the opioid receptor antagonist naloxone and the specific kappa-opioid antagonist nor-binaltorphimine (but not by specific mu- and delta-opioid antagonists). The results suggest that NGF stimulates the release of endogenous opioids (e.g., dynorphin) from DRG neurons and that prolongation of the APD occurs secondarily by activation of excitatory kappa-opioid receptor functions on these same or nearby cells. NGF-induced release of small quantities of opioids by DRG neurons would be expected to prolong the APD in view of the remarkable sensitivity of these neurons to the excitatory effects of extremely low (fM-nM) concentrations of exogenous opioid agonists. NGF-induced APD prolongation is blocked by the same cholera toxin A or B subunit treatments previously shown to block Gs coupling and GM1 ganglioside regulation of excitatory opioid receptors, respectively. These in vitro studies suggest that excitatory opioid receptor-mediated functions may play a role in mediating some types of rapid NGF-induced hyperalgesic and other physiologic effects on the nervous system.
Evidence is presented for linkage of opioid receptors directly to the stimulatory G protein (guanine nucleotide-binding protein), Gs, in addition to the generally accepted linkage to the inhibitory and "other" G proteins, gi and Go, in F-11 (neuroblastoma-dorsal root ganglion neuron) hybrid cells. Treatment of intact F-11 cells with cholera toxin decreased specific binding of the opioid agonist [D-Ala2,D-Leu5]enkephalin to F-11 cell membranes by 35%, with the remaining binding retaining high affinity for agonist. Under these conditions cholera toxin influenced the alpha subunit of Gs (Gs alpha) but had no effect on the alpha subunit of Gi/o (Gi/o alpha), based on ADP-ribosylation studies. Pertussis toxin treatment decreased high-affinity opioid agonist binding by about 50%; remaining binding was also of high affinity, even though pertussis toxin had inactivated Gi/o alpha selectively and essentially completely. Simultaneous treatment with both toxins had an additive effect, reducing specific binding by about 80%. While opioid agonists inhibited forskolin-stimulated adenylate cyclase activity of F-11 cells as expected, opioids also stimulated basal adenylate cyclase activity, indicative of interaction with Gs as well as Gi. Cholera toxin treatment attenuated opioid-stimulation of basal adenylate cyclase, whereas pertussis toxin treatment enhanced stimulation. In contrast, inhibition by opioid of forskolin-stimulated activity was attenuated by pertussis toxin but not by cholera toxin. It is concluded that a subset of opioid receptors may be linked directly to Gs and thereby mediate stimulation of adenylate cyclase. This Gs-adenylate cyclase interaction is postulated to be responsible for the novel excitatory electrophysiologic responses to opioids found in our previous studies of sensory neurons and F-11 cells.
In a previous study, we showed that microM concentrations of mu or delta opioid agonists increase voltage-dependent outward K+ currents in neuroblastoma x DRG neuron hybrid F11 cells via pertussis toxin-sensitive receptors. The present study demonstrates that much lower concentrations (fM to nM) of these opioids (DAGO and DPDPE) decreased voltage-dependent outward K+ currents during step depolarization. The opioid antagonist, naloxone (3 nM) prevented these decreases in K+ current as did the cholera toxin subunits A or B (ca. 1 nM). Furthermore, the specific mu opioid receptor antagonist, beta-funaltrexamine (5 nM) blocked the decrease by DAGO and the specific delta antagonist, naltrindole (1 nM) blocked that by DPDPE. Acute GM1 ganglioside (1 microM) treatment markedly enhanced the efficacy of opioid-induced decrease in K+ current. After treating the cells with pertussis toxin (1 microgram/ml) for 2 days or more, these opioids decreased the K+ current even when tested at concentrations as high as 1 microM. These results indicate that the decrease in K+ current elicited in F11 cells by low concentrations of mu and delta opioid agonists resembles the opioid-induced prolongation of the action potential duration and decrease in voltage-dependent K+ conductance that occur in DRG neurons in primary cultures. The F11 cell line provides therefore a valuable model system for correlative pharmacologic, electrophysiologic and biochemical analyses of Gs-coupled, GM1 ganglioside-regulated excitatory opioid receptor functions, in addition to G(i)/G(o)-coupled inhibitory receptor functions, in sensory neurons.
Dynorphin A-(1-17) (Dyn A) administered intrathecally (IT) or released spinally in the mouse produces an antianalgesic action. The present experiments indicate that IT administration of cholera toxin inhibited the antianalgesic action of Dyn A. When clonidine was administered intracerebroventricularly (ICV) to release spinal Dyn A, IT cholera toxin inhibited the antianalgesic action of Dyn A so that the analgesic component of action of clonidine became evident. Dyn A given IT inhibited the analgesic action of morphine given ICV. Cholera toxin given IT eliminated the antagonistic action of Dyn A. These results, in addition to others, indicate that IT cholera toxin antagonized the action of Dyn A. When the antianalgesic action of Dyn A was attenuated by pretreatment with dynorphin antiserum or by pretreatment that produced desensitization to Dyn A, cholera toxin had no effect. These results suggested that the antianalgesic action of Dyn A is mediated by activation of opioid receptors that are positively coupled to adenylate cyclase through a Gs regulatory protein.
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.
The F11 cell line is a fusion product of cells of mouse neuroblastoma cell line N18TG-2 with embryonic rat dorsal-root ganglion (DRG) neurons. Previous biochemical results suggest that they express mu- and delta-opioid receptors that are negatively coupled to adenylate cyclase. The present study provides direct agonist-binding and electrophysiologic evidence of mu and delta, but not kappa, receptor expression in F11 cells. Radioligand binding assays show that F11 cell membranes bind the mu- and delta-opioid receptor agonists, DAGO and DPDPE with Kd = 4.5 and 4.9 nM and Bmax = 111 and 195 fmol/mg, respectively. Tight-seal patch-clamp recordings of F11 cells after several days in a differentiating culture medium (low serum, cyclic AMP and nerve growth factor) showed that: (i) the outward K+ current during pulsed depolarization in most of these cells was increased by either DAGO or DPDPE, but none were responsive to both opioids or to the kappa-opioid receptor agonist, U-50,488H. The response was blocked by relevant receptor antagonists, naloxone, beta-funaltrexamine or naltrindole; (ii) cells without processes responded neither to DAGO nor to DPDPE; (iii) treatment with pertussis toxin blocked all opioid-induced increases in outward K+ current. The opioid-induced increase in voltage-dependent membrane K+ current in F11 cells resembles the inhibitory effect elicited by mu- and delta-opioid agonists in primary cultures of mouse DRG neurons.
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.
Low (nanomolar) concentrations of opioid agonists prolong the calcium-dependent component of the action potential duration (APD) of many dorsal root ganglion (DRG) neurons, whereas higher (micromolar) levels shorten the APD. Both effects are blocked by naloxone (1-10 nM). Opioid-induced APD prolongation appears to be mediated by excitatory opioid receptors that are positively coupled via a cholera toxin-A-sensitive Gs protein to adenylate cyclase/cyclic AMP-dependent ion conductances, whereas opioid-induced APD shortening is mediated by inhibitory receptors linked via pertussis toxin-sensitive Gi/Go proteins. Cholera toxin-B subunit, which binds to GM1 ganglioside, also selectively blocks opioid-induced APD prolongation. After brief treatment with GM1 ganglioside, the opioid agonists, dynorphin (1-13) or morphine, prolong the APD at femtomolar vs. the usual nanomolar concentrations, whereas no significant alterations were observed in the sensitivity of these GM1-treated cells to opioid inhibitory effects elicited by higher opioid concentrations. The present study shows that the opioid antagonists, naloxone or diprenorphine (1-30 nM), did not alter the APD of naive DRG neurons. In contrast, after GM1 treatment (1 microM, greater than 10 min), both opioid antagonists (but not (+)naloxone) unexpectedly prolonged the APD of most of the GM1-treated cells, but still continued to antagonize opioid-induced APD shortening. These results suggest that the supersensitivity of GM1-treated DRG neurons to the excitatory effects of opioid agonists and antagonists is due primarily to a remarkably increased efficacy of excitatory Gs-coupled opioid receptor functions, similar to the opioid excitatory supersensitivity that we have recently observed in chronic opioid-treated DRG neurons.
In previous studies, we showed that low (nM) concentrations of opioid prolong the action potential duration (APD) of many mouse dorsal root ganglion (DRG) neurons via Gs-linked excitatory opioid receptors, whereas micromolar opioid levels shorten the APD via Gi/Go-linked inhibitory receptors. In addition, cholera toxin-B subunit (CTX-B) selectively blocks opioid- but not forskolin-induced prolongation of the APD in DRG neurons. Since CTX-B binds with selective high affinity to GM1 ganglioside located on the cell surface, the results suggest that GM1 plays an essential role in regulating excitatory opioid receptor functions. This hypothesis was tested by treating DRG neurons in mouse DRG-cord explants with exogenous gangliosides and determining whether the efficacy of opioid agonists in prolonging the APD is enhanced. The threshold concentration of the opioids, dynorphin(1-13) and morphine required to prolong the APD in many DRG neurons was markedly decreased from nM to fM levels after bath exposure to 10 nM to 1 microM GM1 ganglioside for less than 5 min. In contrast, GM2 and GM3 gangliosides and asialo-GM1 ganglioside were ineffective, even when DRG neurons were exposed to high concentrations (1-10 microM) for periods greater than 1 h. Although GD1a, GD1b and GQ1b gangliosides appeared to be as effective as GM1 when tested at microM concentrations for 15 min, tests at lower concentrations, shorter periods, and/or at lower temperature (24 degrees vs 34 degrees C), showed that they were significantly less effective than GM1.(ABSTRACT TRUNCATED AT 250 WORDS)
Previous studies showed that low concentrations of opioids prolong the calcium-dependent component of the action potential duration (APD) of dorsal root ganglion (DRG) neurons, whereas higher concentrations shorten the APD. In the present study whole-cell voltage-clamp, as well as cell-attached membrane-patch voltage-clamp, recordings demonstrate that application of picomolar to nanomolar concentrations of mu, delta or kappa opioid agonists (DAGO, DPDPE or dynorphin) to DRG neurons in dissociated cell cultures reversibly decreased the activities of voltage-sensitive K+ channels. Pretreatment of DRG neurons with the opioid receptor antagonists, naloxone (30 nM) or diprenorphine (1 nM) prevented mu/delta or kappa opioid-induced decreases in K+ channel activities, respectively. Since opioids added to the bath solution decreased the activities of K+ channels in the membrane patch sealed off by the pipette tip, our results provide strong evidence that some modes of excitatory modulation of the action potential of DRG neurons are mediated by diffusible second messengers. The data are consonant with our previous studies indicating that opioids can elicit excitatory effects on sensory neurons via cholera toxin-sensitive Gs-linked excitatory opioid receptors coupled to cyclic AMP-dependent ionic channels.
Previous studies have shown that chronic morphine increases levels of the G-protein subunits Gia and Goa, adenylate cyclase, cyclic AMP-dependent protein kinase, and certain phosphoproteins in the rat locus coeruleus, but not in several other brain regions studied, and that chronic morphine decreases levels of Gia and increases levels of adenylate cyclase in dorsal root ganglion/spinal cord (DRG-SC) co-cultures. These findings led us to survey the effects of chronic morphine on the G-protein/cyclic AMP system in a large number of brain regions to determine how widespread such regulation might be. We found that while most regions showed no regulation in response to chronic morphine, nucleus accumbens (NAc) and amygdala did show increases in adenylate cyclase and cyclic AMP-dependent protein kinase activity, and thalamus showed an increase in cyclic AMP-dependent protein kinase activity only. An increase in cyclic AMP-dependent protein kinase activity was also observed in DRG-SC co-cultures. Morphine regulation of G-proteins was variable, with decreased levels of Gia seen in the NAc, increased levels of Gia and Goa in amygdala, and no change in thalamus or the other brain regions studied. Interestingly, chronic treatment of rats with cocaine, but not with several non-abused drugs, produced similar changes compared to morphine in G-proteins, adenylate cyclase, and cyclic AMP-dependent protein kinase in the NAc, but not in the other brain regions studied. These results indicate that regulation of the G-protein/cyclic AMP system represents a mechanism by which a number of opiate-sensitive neurons adapt to chronic morphine and thereby develop aspects of opiate tolerance and/or dependence. The findings that chronic morphine and cocaine produce similar adaptations in the NAc, a brain region important for the reinforcing actions of many types of abused substances, suggest further that common mechanisms may underlie psychological aspects of drug addiction mediated by this brain region.
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.
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)
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.
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