Magnetic fields inhibit opioid-mediated ‘analgesic’ behaviours of the terrestrial snail,Cepaea nemoralis

1. The terrestrial snail, Cepaea nemoralis, when placed on a warmed surface (40 degrees C) displays a thermal avoidance behaviour that entails an elevation of the anterior portion of the fully extended foot. The latency of this nociceptive response was increased by the prototypical mu and specific kappa opiate agonists, morphine and U-50, 488H, respectively, in a manner indicative of anti-nociception and the induction of 'analgesia'. Pretreatment with the prototypical opiate antagonist, naloxone, blocked the morphine- and reduced the U-50, 488H-induced analgesia. Naloxone had no effects on the thermal response latencies of saline treated animals. 2. Exposure to either cold (7 degrees C) or warm (38 degrees C) temperature stress increased the nociceptive thresholds of Cepaea in a manner indicative of the induction of 'stress-induced analgesia'. The warm stress-induced analgesia was opioid mediated, being blocked by naloxone, whereas, the cold stress-induced analgesia was insensitive to naloxone. 3. Exposure for 15-30 min to 0.5 Hz weak rotating magnetic fields (1.5-8.0 G) significantly reduced the analgesic effects of the mu and kappa opiate agonists in a manner similar to that observed with naloxone. The magnetic stimuli also inhibited the endogenous opioid mediated warm stress-induced analgesia and significantly reduced the cold stress-induced analgesia. The magnetic stimuli had no evident effects on the nociceptive responses of saline-treated animals. The dihydropyridine (DHP) and non-DHP calcium channel antagonists diltiazem, verapamil. and nifedipine differentially and significantly reduced, while the DHP calcium channel agonist, BAY K8644, significantly enhanced the inhibitory effects of the magnetic fields on morphine-induced analgesia.     

Phorbol ester suppression of opioid analgesia in rats

Protein kinase C (PKC) has been shown to be an important substrate in intracellular signal transduction. Very little is known concerning its possible role in mediating opiate-induced analgesia. In the present study, 12-O-tetradecanoylphorbol 13-acetate (TPA), a selective activator of PKC, was injected intrathecally (ith) to assess its influence on the analgesia induced by intrathecal injection of the mu opioid agonist PL017, the delta agonist DPDPE and the kappa agonist 66A-078. Radiant heat-induced tail flick latency (TFL) was taken as an index of nociception. TPA in the dose of 25-50 ng, which did not affect the baseline TFL, produced a marked suppression of opioid antinociception, with a higher potency in blocking mu and delta than the kappa effect. In addition, mu and delta agonists induced remarkable decreases in spinal cyclic AMP (cAMP) content whereas the kappa effect was weak. The results suggest a cross-talk between the PKC system and the signal transduction pathway subserving opioid analgesia.     

Evidence for transduction of mu but not kappa opioid modulation of extracellular signal-regulated kinase activity by G(z) and G(12) proteins

Chronic treatment with micro or kappa opioid agonists (>/=2 h) inhibits EGF-induced ERK activation in opioid receptor overexpressing COS-7 cells. Although acute mu and kappa opioids activate ERK via a pertussis toxin-sensitive G protein, pertussis toxin insensitivity of the chronic mu (but not kappa) action was observed. Here, we tested several pertussis toxin-insensitive G proteins as candidates to transduce acute and/or chronic opioid modulation of ERK. Overexpressed Galpha(z) (but not Galpha(12)) transduced acute mu (but not kappa) ERK activation in pertussis toxin-treated COS-7 cells. Chronic mu (but not kappa) inhibited EGF stimulation of ERK in pertussis toxin-treated cells overexpressing Galpha(z) or Galpha(12). Transfection of Galpha(13) or Galpha(q) blocked inhibition under the same conditions. Overexpressed interfering and non-interfering Galpha(z) mutants differentially affected mu inhibition of ERK consistent with G(z) transduction. In this and prior studies, Galpha(z) and Galpha(12) immunoreactivity were detected in untransfected COS-7 cells, suggesting that these G proteins may be endogenous mediators of chronic mu inhibitory actions on ERK.     

MIF-1 and Tyr-MIF-1 antagonize morphine and opioid but not non-opioid stress-induced analgesia in the snail, Cepaea nemoralis

The effects of prolyl-leucyl-glycinamide (MIF-1, PLG), tyrosine-prolyl-leucyl-glycinamide (Tyr-MIF-1, YPLG) and naloxone on morphine and warm and cold stress-induced increases in the latency of the thermal (40 degrees C hot plate) avoidance behaviors of the terrestrial snail, Cepaea nemoralis, were examined. All three substances blocked the morphine- and warm stress-induced opioid analgesia, while having no effects on non-opioid cold stress-induced analgesia. Tyr-MIF-1 had a significantly greater inhibitory effect than MIF-1. These results indicate that MIF-1 and Tyr-MIF-1 antagonize the antinociceptive effects of exogenous opiates and opioid-mediated analgesia in snails in a manner analogous to that described for mammals. This raises the possibility of an evolutionary conservation of functional opioid antagonists.     

G regulatory proteins and muscarinic receptor signal transduction in mucous acini of rat submandibular gland

The involvement of G regulatory proteins in muscarinic receptor signal transduction was examined in electrically permeabilized rat submandibular acinar cells. The guanine nucleotide analog, GTP gamma S, caused the dose dependent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to release IP3. This response was insensitive to pertussis toxin treatment and was duplicated by NaF but not by GDP beta S. Enhanced IP3 synthesis was observed with a combination of GTP gamma S and carbachol. Exogenous IP3, as well as carbachol and GTP gamma S, provoked the release of sequestered 45Ca2+ from non-mitochondrial stores. In intact cells, carbachol significantly reduced the level of cyclic AMP induced by the beta-adrenergic agonist, isoproterenol, to 69% of its normal value. Pertussis toxin abolished this inhibitory action of carbachol on cyclic nucleotide levels. These results suggest that muscarinic receptors are coupled to two separate G regulatory proteins in submandibular mucous acini-the pertussis toxin-insensitive Gp of the phosphoinositide transduction pathway associated with elevated cytosolic calcium levels, and the pertussis toxin-sensitive Gi inhibitory protein of the adenylate cyclase complex.     

The effect of CNS opioid on autonomic nervous and cardiovascular responses in diet-induced obese rats

The intracerebroventricular (i.c.v.) infusion of beta-endorphin can cause either a decrease in blood pressure in normal rats or an increase in obese rats. Diet-induced obesity is associated with an increase of hypothalamic mu opioid receptors. Since beta-endorphins act by opioid receptors, we investigated the effect of CNS mu as well as kappa opioid receptor agonist and antagonist on mean blood pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) in male Wistar rats fed either a high fat (HF) (40% fat by weight) or a regular low fat (control) (4% fat by weight) diet. After a 12-week-feeding period the animals were implanted with i.c.v. cannulas and 3-5 days later they were anesthetized and instrumented to record MAP, HR and RSNA. HF rats have higher MAP and the i.c.v. injection of a mu opioid agonist (DAMGO) initially decreased the MAP and then increased MAP, HR and RSNA in the normal animals. The increase was greater in HF animals. The i.c.v. injection of the mu antagonist (beta-FNA) resulted in a significantly greater decrease in MAP in HF animals. beta-FNA increased the RSNA in the HF rats but decreased it in the normal rats. The kappa agonist (dynorphin) decreased MAP in normal rats followed by a return to baseline, but not in HF rats. The kappa antagonist, nor-binaltorphimine (N-BP), increased MAP and RSNA in normal rats and to a lesser extent in HF rats. These findings suggest that rats given a high fat diet have higher blood pressures and a greater mu opioid-mediated responsiveness with a greater mu opioid-mediated autonomic tone. Additionally there is a decreased kappa responsiveness and tone in the HF rats. Both these changes, increased mu and decreased kappa responsiveness could strongly contribute to the increased blood pressure in obese animals.     

The presence of a heterotrimeric G protein and its role in signal transduction of extracellular calmodulin in pollen germination and tube growth

The role of heterotrimeric G proteins in pollen germination, tube growth, and signal transduction of extracellular calmodulin (CaM) was examined in lily pollen. Two kinds of antibodies raised against animal Gzalpha, one against an internal sequence and the other against its N terminus, cross-reacted with the same 41-kD protein from lily pollen plasma membrane. This 41-kD protein was also specifically ADP ribosylated by pertussis toxin. Microinjection of the membrane-impermeable G protein agonist GTP-gamma-S into a pollen tube increased its growth rate, whereas microinjection of the membrane-impermeable G protein antagonist GDP-beta-S and the anti-Galpha antibody decreased pollen tube growth. The membrane-permeable G protein agonist cholera toxin stimulated pollen germination and tube growth. Anti-CaM antiserum inhibited pollen germination and tube growth, and this inhibitory effect was completely reversed by cholera toxin. The membrane-permeable heterotrimeric G protein antagonist pertussis toxin completely stopped pollen germination and tube growth. Purified CaM, when added directly to the medium of plasma membrane vesicles, significantly activated GTPase activity in plasma membrane vesicles, and this increase in GTPase activity was completely inhibited by pertussis toxin and the nonhydrolyzable GTP analogs GTP-gamma-S and guanylyl-5'-imidodiphosphate. The GTPase activity in plasma membrane vesicles was also stimulated by cholera toxin. These data suggest that heterotrimeric G proteins may be present in the pollen system where they may be involved in the signal transduction of extracellular CaM and in pollen germination and tube growth.     

Anti-Analgesic Effect of the Mu/Delta Opioid Receptor Heteromer Revealed by Ligand-Biased Antagonism

Delta (DOR) and mu opioid receptors (MOR) can complex as heteromers, conferring functional properties in agonist binding, signaling and trafficking that can differ markedly from their homomeric counterparts. Because of these differences, DOR/MOR heteromers may be a novel therapeutic target in the treatment of pain. However, there are currently no ligands selective for DOR/MOR heteromers, and, consequently, their role in nociception remains unknown. In this study, we used a pharmacological opioid cocktail that selectively activates and stabilizes the DOR/MOR heteromer at the cell surface by blocking its endocytosis to assess its role in antinociception. We found that mice treated chronically with this drug cocktail showed a significant right shift in the ED₅₀ for opioid-mediated analgesia, while mice treated with a drug that promotes degradation of the heteromer did not. Furthermore, promoting degradation of the DOR/MOR heteromer after the right shift in the ED₅₀ had occurred, or blocking signal transduction from the stabilized DOR/MOR heteromer, shifted the ED₅₀ for analgesia back to the left. Taken together, these data suggest an anti-analgesic role for the DOR/MOR heteromer in pain. In conclusion, antagonists selective for DOR/MOR heteromer could provide an avenue for alleviating reduced analgesic response during chronic pain treatment.     

Effects of G protein and cGMP on phytochrome-mediated amaranthin synthesis inAmaranthus caudatus seedlings

The effects of G protein and cGMP on phytochrome-mediated amaranthin biosynthesis inAmaranthus caudatus seedlings were studied. It was shown that G protein agonist cholera toxin induced amarathin synthesis in darkness, whereas G protein antagonist pertussis toxin inhibited red light-induced amaranthin synthesis. Amaranthin synthesis was also induced by exogenous cGMP, while the amaranthin biosynthesis induced by cholera toxin, red light and exogenous cGMP was inhibited by genistein. L Y-83583, an inhibitor of guanylyl cyclase, inhibited the amarenthin synthesis induced both by red light and cholera toxin, while it was not able to inhibit the amaranthin synthesis induced by exogenous cGMP. These results suggest that G protein, guanylyl cyclase and cGMP were the candidates in phytochrone signal transduction chain for red light-induced amaranthin biosynthesis and the red light signal transduction chain might be as follows: red light → phytochrome → G protein → guanylyl cyclase → cGMP.     

Effects of G protein and cGMP on phytochrome-mediated amaranthin synthesis in Amaranthus caudatus seedlings

The effects of G protein and cGMP on phytochrome-mediated amaranthin biosynthesis inAmaranthus caudatus seedlings were studied. It was shown that G protein agonist cholera toxin induced amarathin synthesis in darkness, whereas G protein antagonist pertussis toxin inhibited red light-induced amaranthin synthesis. Amaranthin synthesis was also induced by exogenous cGMP, while the amaranthin biosynthesis induced by cholera toxin, red light and exogenous cGMP was inhibited by genistein. L Y-83583, an inhibitor of guanylyl cyclase, inhibited the amarenthin synthesis induced both by red light and cholera toxin, while it was not able to inhibit the amaranthin synthesis induced by exogenous cGMP. These results suggest that G protein, guanylyl cyclase and cGMP were the candidates in phytochrone signal transduction chain for red light-induced amaranthin biosynthesis and the red light signal transduction chain might be as follows: red light → phytochrome → G protein → guanylyl cyclase → cGMP.     

The effect of opiates and opiate antagonists on heat latency response in the parasitic nematode Ascaris suum

The effects of the opiates morphine and morphine-6-glucuronide (M6G), the mu opioid receptor specific antagonist D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NH(2) (CTOP), and the general opiate antagonist naloxone on the latency of response to thermal stimulation were determined in the parasitic nematode Ascaris suum. Thermal detection and avoidance behaviors of the worms were evaluated with a tail flick analgesia meter using a modification of a technique employed for nociception experiments in rodents. Morphine and M6G were shown to have a dose dependent analgesic effect on A. suum's latency of response to heat with morphine being the most potent. The analgesic effect of morphine was reversed by naloxone but not CTOP. Neither naloxone nor CTOP was able to block the analgesia of M6G. CTOP but not naloxone had significant analgesic effects on its own. These findings are generally consistent with previous results on the effects of opiates and nitric oxide release from A. suum tissue. Apparently these nematodes possess opioid receptors that effect nociception.     

Kappa opiate agonists inhibit Ca2+ influx in rat spinal cord-dorsal root ganglion cocultures. Involvement of a GTP-binding protein

The aim of the present study has been to characterize the regulation by opiates of 45Ca2+ influx in rat spinal cord-dorsal root ganglion cocultures. We have demonstrated that K+-induced depolarization, in the presence of the Ca2+ channel agonist Bay K8644, stimulated Ca2+ influx (3-4-fold) via the dihydropyridine class of voltage-dependent Ca2+ channels. While mu and delta opiates had no effect, kappa opiate agonists (e.g. U50488, dynorphin) profoundly depressed the stimulated Ca2+ influx (86% inhibition at 100 microM U50488). The kappa agonist action was stereospecific and could be reversed by the opiate antagonist naloxone. The inhibition produced by kappa agonists was greatly diminished following pertussis toxin treatment, and this effect was accompanied by toxin-induced ADP-ribosylation of a 40-41-kDa protein. This suggests that kappa opiate receptors are negatively coupled to voltage-dependent Ca2+ channels, via a pertussis toxin-sensitive GTP-binding protein. Basal 45Ca2+ uptake, stimulated by adenylate cyclase activators (forskolin and cholera toxin), was potently inhibited by kappa opiates suggesting that, under conditions of neurohormonal stimulation of adenylate cyclase, kappa receptors are coupled to Ca2+ channels indirectly via the adenylate cyclase complex. In addition, cAMP-independent coupling pathways may also be involved.     

Dual coupling of the cloned 5-HT1A receptor to both adenylyl cyclase and phospholipase C is mediated via the same Gi protein.

The coloned 5-HT_1A receptor, stably expressed in HeLa cells, has been shown to mediate the effects of 5-hydroxytryptamine (5-HT) to inhibit cAMP formation and to stimulate the hydrolysis of phosphatidylinositol. Both responses were found to be pertussis toxin sensitive. We have examined these two responses in membranes derived from these cells and show that the 5-HT_1A receptor can directly regulate the activity of adenylyl cyclase and phospholipase C in response to agonist. In order to examine whether the same or distinct guanine nucleotide-binding regulatory protein(s) (G protein) are involved in these two signal transduction pathways, we used anti-peptide antibodies recognizing the -subunits of G_i1, G_i2, G_i3 as specific tools, since these pertussis toxin substrates are expressed in HeLa cells. These antibodies have previously been shown to prevent receptor-G protein coupling by binding to the regions of G proteins which are putatively involved in interaction with receptors. Our results indicate that the G_i proteins, but preferentially G₃, mediate the effects of 5-HT both to inhibit adenylyl cyclase and to stimulate phospholipase C. These findings demonstrate that the same receptor interacting with the same C protein can regulate several distinct effector molecules.     

Evolutionary Aspects of the Neuromodulation of Nociceptive Behaviors

There is accumulating evidence for a phylogentic continuityin the expression and regulation of fundamental behaviors ofessential survival value. The ability to detect and respondto aversive environmental stimuli is a basic feature of allanimals that is expressed in the term "nociception." Nociceptiveresponses provide an index of the sensitivity of individualsto actual or potential aversive physical stimuli. Measurementsof alterations in nociceptive responses (antinociception oranalgesia, hyperanalgesia) are commonly used to monitor thebehavioral and physiological status of animals following exposureto either noxious or potentially damaging stimuli. In this paperthe neuromodulation of the nociceptive and analgesic behaviorsof molluscs (the land snail, Cepaea nemoralis) and mammals (rodents)is considered. Behavioral and pharmacological evidence is presentedto suggest that opioid neuropeptides are similarly involvedin the modulation of the nociceptive responses of rodents andsnails. The FMRFamide-related family of neuropeptides are alsoshown to be involved in the modulation of nociceptive behaviors,though with apparently different roles in molluscs and mammals.It is proposed that comparative investigations of the mediationof basic phylogenetically conserved functions, such as nociception,are a useful means to determine and analyse, general featuresof behavioral neuromodulation by neuropeptides.     

Pertussis toxin abolishes angiotensin II-induced phosphoinositide hydrolysis and prostaglandin synthesis in rat renal mesangial cells.

Incubation of rat renal mesangial cells with angiotensin II (0.1 microM) resulted in transient breakdown of phosphatidylinositol 4,5-bisphosphate, rapid generation of diacylglycerol and phosphatidic acid, increased 45Ca2+ influx, increased intracellular [Ca2+] as measured by quin 2, and increased prostaglandin E2 synthesis. All of these processes were markedly inhibited time- and dose-dependently by prior exposure of cells to pertussis toxin. In contrast, the effects of the ionophore A23187 on 45Ca2+ influx and prostaglandin E2 synthesis were not altered by the exposure of the cells to pertussis toxin. The action of the toxin was not associated with alterations in cellular concentrations of cyclic AMP. Incubation of membrane fraction of mesangial cells with pertussis toxin resulted in ADP-ribosylation of Mr-42,000 protein. From all these results, it is likely that a G protein is involved in receptor-mediated signal transduction in renal mesangial cells.     

Cardiovascular responses to central administration of mu and kappa opioid receptor agonist and antagonist in normal rats

The response to centrally administered beta-endorphin has been characterized by decreasing sympathetic nervous activity and decreased cardiovascular tone. We investigated the effect of the central administration of both mu and kappa opioid receptor agonist and antagonists on cardiovascular responses. The administration of the mu agonist, DAMGO (0.2nmol) increased the mean arterial pressure (MAP) and stimulated iliac vasoconstriction while higher doses (2 and 20nmol) decreased MAP and stimulated iliac vasodilation. The administration of the kappa receptor agonist, Dynorphin decreased the MAP and stimulated superior mesenteric vasodilation. beta-Funaltrexamine reduced MAP and superior mesenteric vasodilation while nor-binaltorphimine increased MAP and iliac and superior mesenteric vasoconstriction. We conclude that mu receptor activation decrease or increase MAP depending on the mu agonist concentration. However, kappa receptor activation is consistently associated with a decrease in MAP.     

Differential sensitivity of progesterone- and zona pellucida-induced acrosome reactions to pertussis toxin

Pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins) have previously been shown to mediate the zona pellucida-induced acrosome reaction in mammalian sperm. In this study we compared the inhibitory effect of pertussis toxin on the zona-induced acrosome reaction in human spermatozoa with that on the reaction induced by progesterone, another physiological acrosome reaction-promoting stimulus associated with the ovulated oocyte. Up to the concentration of 1 μg/ml, pertussis toxin did not produce any direct effects on the acrosome reaction frequency nor did it influence sperm movement and viability. However, preincubation of spermatozoa with the toxin at a concentration of 100 ng/ml completely abolished the increase in the acrosome reaction frequency upon subsequent exposure to solubilized zona pellucida material. In contrast, the same treatment did not impair the ability of spermatozoa to initiate the acrosome reaction in response to progesterone. Moreover, the preincubation with pertussis toxin did not modify the changes in the intracellular concentration of calcium ions occurring after progesterone addition. These data suggest that different physiological stimuli may utilize different signal transduction pathways to induce the human sperm acrosome reaction. © 1993 Wiley-Liss, Inc.     

Agonists activate Gi1 alpha or Gi2 alpha fused to the human mu opioid receptor differently

As preferential coupling of opioid receptor to various inhibitory Galpha subunits is still under debate, we have investigated the selectivity of the human mu opioid receptor fused to a pertussis toxin insensitive C351I Gi1 alpha or C352I Gi2 alpha in stably transfected HEK 293 cells. Overall agonist binding affinities were increased for both fusion constructs when compared to the wild type receptor. [35 S]GTPgammaS binding was performed on pertussis toxin treated cells to monitor coupling efficiency of the fusion constructs. Upon agonist addition hMOR-C351I Gi1 a exhibited an activation profile similar to the non-fused receptor while hMOR-C352I Gi2 alpha was poorly activated. Interestingly no correlation could be drawn between agonist binding affinity and efficacy. Upon agonist addition, forskolin-stimulated cAMP production, as measured using a reporter gene assay, was inhibited by signals transduced via the fused Gi1 alpha and Gi2 alpha mainly. In contrast both fusion constructs were able to initiate ERK-MAPK phosphorylation via coupling to endogenous G proteins only. In conclusion our data indicate that hMOR couples more efficiently to Gi1 alpha than Gi2 alpha and that the coupling efficacy is clearly agonist-dependent.     

Effect of Pertussis Toxin on Radioligand Binding to Rat Brain Adenosine A1 Receptors

In a previous study we showed that in vivo treatment with pertussis toxin could inhibit some, but not all, effects of adenosine in the rat hippocampus. In this study we investigated the effect of pertussis toxin on the binding of adenosine analogues to A1 receptors in rat brain. Intraventricular injection of pertussis toxin (10 micrograms into the lateral ventricle) did not affect A1 receptor binding in any brain region studied, as evaluated by autoradiography. In vitro treatment of brain sections (10 microns) with pertussis toxin for 5 h, under conditions when greater than 80% of the G proteins were ADP ribosylated, did not alter radioligand binding to adenosine A1 receptors. GTP (10 microM) virtually abolished the high-affinity agonist binding to the A1 receptor. On the other hand, in solubilized cortical membrane preparations, pertussis toxin pretreatment induced a complete shift of the A1 receptors to the low-affinity state. This suggests that the ability of pertussis toxin to affect G proteins coupled to A1 receptors in brain depends not only on the distribution of the toxin but also on the configuration of receptors and G proteins.     

An alpha 40 subunit of a GTP-binding protein immunologically related to Go mediates a dopamine-induced decrease of Ca2+ current in snail neurons

Dopamine induces a decrease in voltage-dependent Ca2+ current in identified neurons of the snail H. aspersa. This effect is blocked by intracellular injection of activated B. pertussis toxin and of an affinity-purified antibody against the alpha subunit of bovine Go protein. The dopamine effect is mimicked by intracellular injection of mammalian alpha o. In snail nervous tissue, pertussis toxin ADP-ribosylates a single protein band on SDS gels, and this band is recognized in immunoblots by the anti-alpha o antibody. We propose that this is a 40 kd alpha subunit of a molluscan G protein immunologically related to alpha o and that it mediates the effect of dopamine on Ca2+ currents in identified snail neurons.