Electrophysiological studies on the spinal effects of dermorphin, an endogenous μ-opioid agonist

The intrathecal administration of dermorphin, an endogenous heptopeptide first discovered in amphibia, produces dose-dependent selective inhibitions of C fibre-evoked responses in rat dorsal horn nociceptive neurones (ED₅₀ 0.11 μg). Naloxone (10 μg) but not ICI 174,864 (125 μg) antagonised the effects of the peptide. Aβ-fibre-evoked activity was relatively unaffected. Thus dermorphin can profoundly inhibit nociceptive afferent input in the spinal cord, and in this preparation is more potent (approximately 40X) than morphine.     

Enkephalins modulate excitatory synaptic transmission in the superficial dorsal horn by acting at μ-opioid receptor sites

The effect of opioids on synaptic potentials of dorsal horn (DH) neurons has been investigated in a rat spinal cord DH slice-dorsal root ganglion (DRG) in vitro preparation. Conventional intracellular recording from DH and DRG neurons using 3 M potassium acetate-filled electrodes was employed. Dorsal roots were electrically isolated from the spinal cord slice and stimulated with pulses of different intensity and duration to evoke afferent action potentials monitored intracellularly from DRG neurons. Low-intensity single-shock stimulation of the dorsal roots (8–20 V pulses of 0.02–0.05 ms duration) activated large primary afferents and elicited excitatory postsynaptic potentials (EPSP) in all of the neurons tested. High-intensity stimulation of the dorsal roots (over 35 V pulses of 0.5 ms duration), sufficient to excite small myelinated and unmyelinated primary afferents resulted in a large and prolonged depolarization of DH neurons associated with firing of action potentials. Bath application (d-Ala², N-Me-Phe⁴,Gly⁵-ol)-enkephalin (DAGO), (d-Ala², d-Leu⁵)-enkephalinamide (DADLEA), or (d-Ala², d-Met⁵)-enkephalinamide (DADMEA) produced dose-dependent, reversible hyperpolarization in about 75% of the neurons tested. The hyperpolarization was associated with a fall in neuronal input resistance. In addition, opioids depressed the synaptic transmission in all of the neurons examined. This depressant effect of opioids was independent from their effects on resting membrane potential. Delta specific receptor opioid agonists (d-Pen^2.5)-enkephalin (DPDPE) and (d-Pen², l-Pen⁵)-enkephalin (DPLPE), were completely ineffective in producing an effect on neuronal membrane or synaptic transmission. All opioid effects were antagonized by naloxone.     

Mechanical activation of dorsal root ganglion cells in vitro: comparison with capsaicin and modulation by κ-opioids

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca²⁺]_i) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by κ-opioids. [Ca²⁺]_i responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca²⁺]_i increases which were abolished in Ca²⁺-free solution, but unaffected by lanthanum (25 μM) or tetrodotoxin (10⁻⁶ M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd³⁺; 250 μM) and amiloride (100 μM) abolished the [Ca²⁺]_i transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca²⁺]_i transients. The κ-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca²⁺]_i transients but had little effect on capsaicin-induced [Ca²⁺]_i transients. The inhibitory effect of U50,488 was abolished by the κ-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30–100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca²⁺]_i transients in small diameter DRG somas are mediated by influx of Ca²⁺ through a Gd³⁺- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca²⁺ transients are sensitive to κ-opioid agonists.     

Comparison of G-protein activation in the brain by μ-, δ-, and κ-opioid receptor agonists in μ-opioid receptor knockout mice

Mice lacking the μ-opioid receptor gene have been developed by a gene knockout procedure. In this study, the activity of opioid receptor coupled G-proteins was examined to investigate whether there is a change in the extent of coupling for μ-, δ-, and κ-opioid receptors in μ-opioid receptor knockout mice. Selective agonists of μ- (DAMGO), δ- (DPDPE), and κ- (U-69,593) opioid receptors stimulated [³⁵S]GTPγS binding in the caudate putamen and cortex of wild-type mice. In contrast, only U-69,593 stimulated [³⁵S]GTPγS binding in these regions of μ-opioid receptor knockout mice. These results confirmed the absence of G-protein activation by a μ-opioid receptor agonist in μ-opioid receptor knockout mice, and demonstrated that coupling of the κ-opioid receptor to G-proteins is preserved in these mice. However, G-protein activation by the δ-opioid receptor agonist, DPDPE, was reduced in the μ-opioid receptor knockout mice, at least in the brain regions studied using autoradiography.     

Opioid receptor subtypes in the rat spinal cord: electrophysiological studies with μ- and δ-opioid receptor agonists in the control of nociception

We have compared the ability of selective mu- and delta-opiate agonists to modulate nociceptive transmission at the level of the rat dorsal horn using electrophysiological approaches. Single-unit extracellular recordings were made from neurones in the lumbar dorsal horn of the intact rat under halothane anaesthesia. Neurones could be activated by both A- and C-fibre electrical stimulation (and by natural innocuous and noxious stimuli). Agonists were applied directly onto the cord in a volume of 50 microliters. The intrathecal administration of 3 agonists, Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO) (mu-selective) (2 X 10(-3)-10 nmol) Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET) (mu/delta) (7 X 10(-4)-70 nmol), and cyclic Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) (delta) (2 X 10(-2)-100 nmol) produced dose-dependent inhibitions of C-fibre-evoked neuronal activity whilst A-fibre activity was relatively unchanged. DAGO produced near-maximal inhibitions which could be completely reversed by naloxone (1.5 nmol) whilst DPDPE causes less marked inhibitions which could only be partially reversed by naloxone (1.5-13.5 nmol). DTLET produced effects intermediate to those of DAGO and DPDPE. The results suggest that both mu- and delta-opioid receptors can modulate the transmission of nociceptive information in the rat spinal cord.     

Streptozotocin-induced diabetes selectively alters the potency of analgesia produced by μ-opioid agonists, but not by δ- and κ-opioid agonists

To investigate the possible mechanisms of the alterations in morphine-induced analgesia observed in diabetic mice, we examined the influence of streptozotocin-induced (STZ-induced) diabetes on analgesia mediated by the different opioid receptors. The antinociceptive potency of morphine (10 mg/kg), administered s.c., as determined by both the tail-pinch and the tail-flick test, was significantly reduced in diabetic mice as compared to that in controls. Mice with STZ-induced diabetes had significantly decreased sensitivity to intracerebroventricularly (i.c.v.) administered μ-opioid agonists, such as morphine (10 μg) and [d-Ala², N-Me Phe⁴,Gly-ol⁵]enkephalin (DAMGO, 0.5 μg). However, i.c.v. administration of [d-Pen^2,5]enkephalin (DPDPE, 5 μg), a δ-opioid agonist, and U-50,488H (50 μg), a κ-opioid agonist, produced pronounced antinociception in both control and diabetic mice. Furthermore, there were no significant differences in antinociceptive potency between diabetic and control mice when morphine (1 μg), DAMGO (10 μg), DPDPE (0.5 μg) or U-50,488H (50 μg) was administered intrathecally. In conclusion, mice with STZ-induced diabetes are selectively hyporesponsive to supraspinal μ-opioid receptor-mediated antinociception, but they are normally responsive to activation of δ- and κ-opioid receptors.     

Effects of polyclonal and monoclonal antibodies to substance P on slow excitatory transmission in rat spinal dorsal horn

The effects of bath perfusion of polyclonal and monoclonal antibodies to substance P (SP) on slow excitatory transmission in rat dorsal horn have been investigated by intracellular recording in the immature rat spinal cord slice preparation. Both polyclonal and monoclonal antibodies to SP produced a significant decrease in the amplitude and the duration of the slow depolarization generated in dorsal horn neurons by high intensity, repetitive dorsal root stimulation or exogenous SP application. The effect of endogenous SP, or SP-related peptide, released during dorsal root stimulation appears likely since bath perfusion of a slice with a normal rabbit serum, or affinity chromatography preadsorbed SP antiserum, or non-specific IgG, or 5-hydroxytryptamine antiserum had no similar depressant effect. These results, if taken together with other experimental evidence, suggest that SP, or SP-like peptide, is in some way involved in a generation of the dorsal root-evoked slow depolarization. In addition, a novel approach is presented for using polyclonal and monoclonal antibodies to SP as pharmacological antagonists. Use of a specific characterized monoclonal antibody for the detection of physiological and pharmacological effects of putative peptide transmitters in vitro opens new avenues for further investigations.     

Analgesic synergy and improved motor function produced by combinations of μ-δ- and μ-κ-opioids

This study evaluated the effects of intrathecal administration of a low-analgesic dose of the selective μ-agonist DAMGO co-administered with sequentially increasing doses of either the selective δ-agonist DPDPE or the selective κ-agonist, U50,488H on mechanical nociceptive thresholds in the rat. Potent analgesic synergy was observed with both combinations. Since an elevation in nociceptive threshold can result from motor deficits, as well as true analgesia, we also evaluated the effects of the combination regimens on motor coordination using a rotarod apparatus. The combination regimens produced significantly less motor deficits than those observed when DPDPE and U50,488H were administered as single agents. These findings of enhanced analgesia with decreased motor side-effects associated with administration of fixed μ/δ or μ/κ combinations suggest that co-administration of opiates that act at different receptors may constitute a superior approach to the treatment of pain.     

Receptor trafficking induced by μ-opioid-receptor phosphorylation

Opiates, including morphine, are widely used drugs for antinociception in clinics. Prolonged treatments of opioids induce both tolerance and dependence, which are the major side effects of opioid therapy. One of the mechanisms for the development of tolerance and dependence is implicated to be opioid-receptor trafficking. Here we review the current understandings of opioid-receptor phosphorylation, endocytosis and desensitization after repeated agonist treatments. Also, the role of G-protein coupled receptor kinases in opioid-receptor phosphorylation is discussed. How to associate these observations to physiological and behavioral changes in animal models and clinics is still under investigation.     

Activation of central μ-opioid receptors is involved in clonidine analgesia in rats

The analgesic effect of clonidine in spontaneously hypertensive rats (SHR) and in normotensive Sprague-Dawley (SD) rats was assessed by using the formalin pain test. The analgesic response of SD rats to low doses (15–60 μg/kg i.p.) but not to a high dose (150 μg/kg i.p.) of clonidine was inhibited by naloxone, 2 mg/kg i.p., and a similar interaction was noted in SHR. In both rat strains, the analgesic response to low i.p. doses of clonidine was also inhibited by injection of 5 μg of naloxone or 7 μg of β-funaltrexamine, a μ-receptor antagonist, into the lateral cerebral ventricle. I.c.v. injection of 5 μg of ICI 174864, a δ-receptor antagonist, potentiated or did not influence clonidine analgesia in SD rats and SHR, respectively. It is concluded that the analgesic response to clonidine involves activation of central μ-opioid receptors in both SHR and SD rats, possibly by an endogenous opioid released by clonidine.     

Involvement of μ- and δ-opioid receptors in the ethanol-associated place preference in rats exposed to foot shock stress

The purpose of this study was to establish the ethanol-induced place preference in rats exposed to foot shock stress using the conditioned place preference paradigm. We also investigated the role of the endogenous opioid system in the development of the ethanol-induced place preference. The administration of ethanol (300 mg/kg, i.p.) with foot shock stress, but not without such stress, induced a marked and significant place preference. Naloxone (1 and 3 mg/kg, s.c.), a non-selective opioid receptor antagonist, significantly attenuated the ethanol-induced place preference. Moreover, the selective μ-opioid receptor antagonist β-funaltrexamine (3 and 10 mg/kg, i.p.) and selective δ-opioid receptor antagonist naltrindole (1 and 3 mg/kg, s.c.), but not the selective κ-opioid receptor antagonist nor-binaltorphimine (1 and 3 mg/kg, i.p.), significantly attenuated the ethanol-induced place preference. Furthermore, 150 mg/kg ethanol (which tended to produce a place preference, although not significantly) combined with each dose (that did not produce a place preference) of the μ-opioid receptor agonist morphine (0.1 mg/kg, s.c.) or selective δ-opioid receptor agonist 2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahydroquinolino [2,3,3-g] isoquinoline (TAN-67; 20 mg/kg, s.c.), but not the selective κ-opioid receptor agonist trans-3,4-dichloro-N-(2-(1-pyrrolidinyl)cyclohexyl)benzenacetamide methanesulfonate (U50,488H; 1 mg/kg, s.c.), produced a significant place preference. These data indicate that stress may be important for development of the rewarding effect of ethanol, and that μ- and δ-opioid receptors may be involved in the rewarding mechanism of ethanol under stressful conditions.     

μ-Opiate receptor binding in the medial preoptic area is cyclical and sexually dimorphic

The density and distribution of μ- and κ-opiate receptors in the medial preoptic area (MPOA) of male and female rats across the estrous cycle was examined using quantitative in vitro autoradiography of [³H]D-Ala²,MePhe⁴,Gly-ol⁵-enkephalin (DAGO), [³H]naloxone and [³H]bremazocine binding. While no difference in κ-receptor labeling was observed across sex or estrous stage, selective μ-receptor labeling with [³H]DAGO revealed a significant variation of density and distribution in the MPOA across the estrous cycle and between sexes. A dense concentration of μ-receptors located in the central, sexually dimorphic portion of the MPOA was observed during metestrus and diestrus in females, but not during proestrus nor in males. This region appeared to be the same as that labeled similarly using [³H]naloxone. These results suggest that a regional substrate for functional activation by endogenous opiod peptides (e.g. β-endorphin) is cyclically regulated in females, which may explain the gonadal streoid-dependent effects of MPOA β-endorphin on lordosis and luteinizing hormone secretion in females.     

The effects of RB101, a mixed inhibitor of enkephalin-catabolizing enzymes, on carrageenin-induced spinal c-Fos expression are completely blocked by β-funaltrexamine, a selective μ-opioid receptor antagonist

We have demonstrated that pre-administered RB101 (40 mg/kg, i.v.), a mixed inhibitor of enkephalin-catabolizing enzymes, decreased spinal c-Fos expression induced 1 h and 30 min after intraplantar (i.pl.) carrageenin (41% reduction, p<0.01). These effects were completely blocked by pre-administered β-funaltrexamine (10 mg/kg, i.v., 24 h prior to stimulation), a selective long-lasting μ-opioid receptor antagonist. In conclusion, these results clearly demonstrate that the effects of endogenous enkephalins on noxiously evoked spinal c-Fos expression are essentially mediated via μ-opioid receptors.     

Up-regulation of spinal μ-opioid receptor function to activate G-protein by chronic naloxone treatment

The effects of repeated s.c. administrations of an μ-opioid receptor antagonist naloxone on the G-protein activation induced by μ-opioid receptor agonists [ -Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin (DAMGO), endomorphin-1 and endomorphin-2 in the mouse spinal cord was studied, monitoring guanosine-5′-o-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding. All μ-opioid receptor agonists concentration-dependently increased the [³⁵S]GTPγS binding. The increases of [³⁵S]GTPγS binding induced by agonists were significantly enhanced in mice pretreated with naloxone. Under the present condition, chronic treatment with naloxone significantly increased the density of [³H]DAMGO binding sites with an increase in K_d values in spinal cord membranes, indicating an increase in μ-opioid receptors on the membrane surface. These findings suggest that chronic treatment with an μ-opioid receptor antagonist naloxone leads to the supersensitivity to activate G-protein by μ-opioid receptor agonists with an increase in μ-opioid receptors in membranes of the mouse spinal cord.     

The effect of increased NaCl intake on rat brain endogenous μ‐opioid receptor signalling

Numerous studies demonstrate the significant role of central β‐endorphin and its receptor, the μ‐opioid receptor (MOR), in sodium intake regulation. The present study aimed to investigate the possible relationship between chronic high‐NaCl intake and brain endogenous MOR functioning. We examined whether short‐term (4 days) obligatory salt intake (2% NaCl solution) in rats induces changes in MOR mRNA expression, G‐protein activity and MOR binding capacity in brain regions involved in salt intake regulation. Plasma osmolality and electrolyte concentrations after sodium overload and the initial and final body weight of the animals were also examined. After 4 days of obligatory hypertonic sodium chloride intake, there was clearly no difference in MOR mRNA expression and G‐protein activity in the median preoptic nucleus (MnPO). In the brainstem, MOR binding capacity also remained unaltered, although the maximal efficacy of MOR G‐protein significantly increased. Finally, no significant alterations were observed in plasma osmolality and electrolyte concentrations. Interestingly, animals that received sodium gained significantly less weight than control animals. In conclusion, we found no significant alterations in the MnPO and brainstem in the number of available cell surface MORs or de novo syntheses of MOR after hypertonic sodium intake. The increased MOR G‐protein activity following acute sodium overconsumption may participate in the maintenance of normal blood pressure levels and/or in enhancing sodium taste aversion and sodium overload‐induced anorexia.     

Modulation of rat brain α- and β-adrenergic receptor populations by lesions of the dorsal noradrenergic bundle

Bilateral lesion of the ascending noradrenergic fibers in the dorsal bundle of adult Wistar rats with 4 μg 6-hydroxydopamine caused extensive depletion of norepinephrine in all forebrain areas, but led to a 54% increase in norepinephrine levels in the cerebellum. β-Adrenergic receptor binding of [³Hdihydroalprenolol was significantly increased in all forebrain areas depleted of norepinephrine except hypothalamus. The increase in [³Hdihydroalprenolol binding was due to 62% and 34% increases in the number of β-receptor sites in the frontal cerebral cortex and hippocampus respectively. Binding of [³HWB-4101 toα₁-adrenergic receptors after dorsal bundle lesion was augmented generally to a lesser extent than β-receptor binding, with significantly increased numbers of sites only in the frontal cortex (74%), thalamus (20%) and septum. Bothα₁-andβ-receptor binding sites were reduced in number by 25–28% in the cerebellum of dorsal bundle-lesioned rats, whereas intraventricular administration of 6-hydroxydopamine to adult rats, which depletes norepinephrine in the cerebellum by 96%, increased cerebellarα₁-andβ-receptor binding by 33–40%. Binding of [³Hclonidine to forebrainα₂-adrenergic receptors was significantly elevated in the frontal cortex, but reduced in the amygdala and septum, after dorsal bundle lesion.     

Modulation of humoral immune response by central administration of leucine-enkephalin: Effects of μ, δ and κ opioid receptor antagonists

The effect of leucine-enkephalin (Leu-Enk) on primary humoral immune response was investigated following intracerebroventricular (i.c.v.) administration of the peptide in the rat. Leu-Enk stimulated plaque-forming cell (PFC) response in rats i.c.v. injected with 0.1 and 1 μg/kg, whereas doses of 20 and 50 μg/kg exerted immunosuppressive effects. I.c.v. treatment of rats with δ opioid receptor antagonist ICI 174864 and κ opioid receptor antagonist nor-binaltorphimine (nor-BNI) blocked stimulation and suppression of PFC response induced by Leu-Enk, respectively. The μ opioid receptor antagonist β-funaltrexamine (β-FNA) reversed both immunomodulatory effects produced by Leu-Enk. Since β-FNA alone had no effect on PFC response (unlike ICI 174 864 and nor-BNI), these data showed that central effects of Leu-Enk on PFC response were mediated by brain μ opioid receptors, and suggested a possible involvement of δ and κ opioid receptors.     

Opioid peptide receptor studies. 7. The methylfentanyl congener RTI-4614-4 and its four enantiomers bind to different domains of the rat μ opioid receptor

Mutational analysis of opioid receptors supports the hypothesis that dissimilar receptor domains contribute to the binding affinity of different ligands. To determine whether enantiomeric ligands can serve to distinguish between different binding pockets (which focuses the analysis on asymmetric structural factors while avoiding confounding changes in physiochemical characteristics), we analyzed the binding of the 3-methylfentanyl congeners RTI-4614-4 [(±)-cis-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N- phenylpropanamide HCl)], its four stereoisomers [(2S,3R,4S)-1 a, (2R,3R,4S)-1 b, (2R,3S,4R)-1 c, and (2S,3S,4R)-1 d ], and other μ agonists with cloned rat μ opioid receptors stably expressed in HEK-293 cells and μ/κ receptor chimeras. Chimera III (κ_{aminoacids 1–141}/μ_{aminoacids 151–398}), chimera IV (μ_{aminoacids 1–150}/κ_{aminoacids 142–380}), and chimera XII (κ_{aminoacids 1–262}/μ_{aminoacids 269–398}) bound [¹²⁵I]IOXY (6β-iodo-3,14-dihydoxy-17-cyclopropylmethyl-4,5α-epoxymorphinan) with high affinities. The Ki values of 1 a, 1 b, 1 c, and 1 d at the wild-type μ receptor were 0.55 nM, 0.66 nM, 124 nM, and 59.2 nM, respectively. When the region from the N terminal to the start of the transmembrane helix 3 (TMH3) of the μ receptor was substituted by that of the κ receptor (chimera III), the Ki value of 1 b was increased (relative to the μ receptor) 590-fold compared to a 73-fold increase for 1 a. When this portion of the κ receptor was replaced by that of the μ receptor (chimera IV), the loss of affinity was not as great: 11.7-fold for 1 a and 58.5-fold for 1 b. Replacement of the middle of the third intracellular loop and third extracellular loop (e3) of the κ receptor with that of the μ receptor (chimera XII) lowered (relative to their Ki values at the κ receptor) the Ki values of [D-Ala²,D-Leu⁵]enkephalin and [D-Ala²-MePhe⁴,Gly-ol⁵]enkephalin to a much greater extent than the Ki values of the isomers. The κ/chimera XII shift was greater for isomers 1 c and 1 d than for 1 b and 1 a. Viewed collectively, these data suggest that the region from the N terminal to the start of the TMH3 of the μ opioid receptor determines the binding affinity of RTI-4614-4 and its isomers and that the e3 loop also plays a major role in determining the binding affinity of μ agonist peptides. These data also show that the stereoisomers of RTI-4614-4 probably bind to different domains of the μ receptor and suggest that manipulation of stereochemistry may be a useful tool for designing domain-specific ligands. Synapse 28:117–124, 1998. © 1998 Wiley-Liss, Inc. This article is a US government work and, as such, is in the public domain of the United States of America.