Opposite effects of the δ- and μ-opioid receptor agonists on ventilation in conscious adult dogs

We studied ventilation and ventilatory pattern in adult unanesthetized dogs after intracisternal adminstration of morphiceptin analogue (MA) (Tyr-Pro-NMePhe-d-Pro-NH₂) and morphine sulfate (MS) which are μ-receptor opioid agonists and afterd-Ala-d-Leuenkephalin (DADLE), a preferential δ-receptor opioid agonist. DADLE induced a prolongation in expiratory time, T_e, and a reduction in instantaneous minute ventilation, V_t/T_tot, which lasted for about 2 h and was dose dependent. In contrast, MA and MS induced a striking decrease in T_e and tidal volume with a net increase in V_t/T_tot. Both MA and DADLE increased the number of sighs per unit time while morphine did not. Naloxone increased V_t/T_tot when used after both DADLE or MA but larger doses were required for an observable effect after DADLE than after MA. These data suggest that in the unanesthetized dog: (1) the effect of intracisternal opioids on ventilation and ventilatory pattern is not uniform and (2) the opioid μ-receptor subsystem may involve different neuronal pathways from those of the opioid δ-receptor subsystem to modulate breathing.     

Kappa-Selective Opioid Receptor Agonists Leumorphin and Dynorphin Inhibit Supraoptic Neurons in Rat Hypothalamic Slice Preparations

To clarify the effects of opioid peptides, and in particular the effects of kappa-receptor agonists on the activity of supraoptic neurons, extracellular recordings were made from 71 spontaneously firing neurons in the rat hypothalamic slice preparation. Of 71 neurons, 28 showed a phasic firing pattern (phasic neurons: putative vasopressin neurons). The mean firing rate of phasic neurons was 2.6 spikes/s (intraburst firing rate 5.4 ± 2.2 spikes/s). The mean firing rate of neurons classified as non-phasic neurons (putative oxytocin neurons) was 4.5 spikes/s. Following bath application of leumorphin (LM) at 10^?7 M, which has potent opioid activity at kappa-receptors, 17 (61%) of 28 phasic neurons were inhibited and 22 (51%) of 43 non-phasic neurons were inhibited. Excitation was observed in only one non-phasic neuron. The dose-dependence of the response to LM was tested in five supraoptic neurons. There was an inverse relationship between LM concentration and percent change in firing rate. The threshold concentration of LM was approximately 10^?8 M. The relatively selective kappa-receptor antagonist, MR-2266, completely blocked the LM-induced responses. Its effects were long-lasting and only partial recovery was observed 2 h after the application of MR-2266. Dynorphin had similar inhibitory effects on supraoptic neurons to those obtained with LM when tested on the same neurons. In another series of experiments the mu-receptor agonist morphine and the delta-receptor agonist [D-Ala, D-leu]-enkephalin (DADLE) were applied to 28 supraoptic neurons (12 phasic and 16 non-phasic neurons) at 10^?7 M and their actions compared directly with that of LM. Only two of 12 phasic neurons tested were inhibited by DADLE and none of five phasic neurons tested was inhibited by morphine, while eight of the 12 neurons tested were inhibited by LM. By contrast the non-phasic neurons tested were inhibited by application of each of the peptides; seven of 16 neurons tested were not only inhibited by LM, but also five of 11 neurons by DADLE and seven of 15 by morphine. The magnitude of the responses varied from cell to cell. These results suggest that LM acts at the same receptors as dynorphin, and that opioids acting preferentially at kappa-receptors inhibit both vasopressin and oxytocin neurons while delta- and mu-receptor agonists inhibit primarily oxytocin neurons.     

Hyperpolarizing action of enkephalin on neurons in the dorsal motor nucleus of the vagus, in vitro

Intracellular recordings were made from neurons of the dorsomotor vagal nucleus (DMV) in slices of rat medulla oblongata. [D-Ala2, D-Leu5]-enkephalin (DADLE), applied by perfusion (0.01-3 microM) or droplets, dose-dependently hyperpolarized 85% of the DMV neurons tested. The hyperpolarization, associated with a decrease in membrane resistance, persisted after elimination of synaptic activity by perfusion with Ca2(+)-free/high-Mg2+ solution or with 1 microM TTX solution. The opioid antagonist, naloxone, reversibly inhibited DADLE-induced hyperpolarization. The hyperpolarization depended on extracellular K+ concentration and reversed at about -90 mV. DADLE also decreased Ca2(+)-dependent spike duration and after-hyperpolarization (AHP). DAGO (a selective mu-receptor agonist), but not DPLPE (a selective delta-receptor agonist), mimicked DADLE's effects on membrane potential, Ca2(+)-dependent spike duration, and AHP. It is concluded that DADLE, through postsynaptic mu-type opioid receptors, hyperpolarized DMV neurons by increasing K+ conductance, which may have an inhibitory effect on DMV output. DADLE-induced decrease of spike duration and AHP was also mediated by mu-receptors and could have additional effects on functions of the DMV neuron by virtue of reduction in Ca2+ entry.     

Ventilatory muscle endurance training in quadriplegia: effects on breathing pattern

We examined the effects of ventilatory muscle endurance training on resting breathing pattern in 12 C6-C7 traumatic quadriplegics at least 1 year post-injury. All subjects had complete motor loss below the lesion level. Subjects were randomly assigned to a training (N = 6), or a control group (N = 6). Baseline tests included measurement of resting ventilation and breathing pattern using mercury in rubber strain gauges for 20 minutes in a seated position; maximum inspiratory mouth pressure (MIP) at FRC, and sustainable inspiratory mouth pressure for 10 minutes (SIP); lung volumes, and arterial blood gases (ABG's). The training protocol consisted of breathing through an inspiratory resistor equivalent to 85% SIP for 15 minutes twice daily, 5 days a week for 8 weeks. Both trainers and controls attended the lab every 2 weeks for reassessment of MIP and SIP and the inspiratory resistance was increased in the training group as SIP increased. At the end of 8 weeks, baseline tests were repeated. All subjects had normal ABG's. There was a significant increase in mean MIP and SIP in both the control group (30% +/- 19% and 31% +/- 18% respectively), and in the training group (42% +/- 24% and 78% +/- 49% respectively). Although the absolute values for both MIP and SIP were greater in the training group than in the control group, the differences were not significant. The alterations in resting breathing pattern were also the same in both groups. Mean frequency decreased significantly in the control group (20.2/minute to 16.9/minute) and, while insignificant, the change in frequency in the training group was the same, 19.4/minute to 16.4/minute. Mean tidal volume (Vt) increased 18.2% of baseline Vt in the control group and 17.0% baseline in the trainers, resulting in no change in minute ventilation. As MIP and SIP increased similarly in both groups, the data from the control and trainers was pooled and timing changes re-evaluated pre- and post-study. A significant decrease in mean Ti/Ttot was observed, while no change in Vt/Ti was found. We concluded that the testing procedure itself provided the stimulus resulting in a significant increase in MIP and SIP. The addition of training did not increase MIP and SIP further. The increased MIP and SIP resulted in a slower and deeper breathing pattern and a significantly shorter Ti/Ttot in both trainers and control subjects.     

mu- but not delta- and kappa-opioid receptors in the ventrolateral orbital cortex mediate opioid-induced antiallodynia in a rat neuropathic pain model

Previous studies have indicated that the ventrolateral orbital cortex (VLO) is involved in opioid-mediated antinociception in the tail flick test and formalin test. The aim of the current study was to examine the effect of opioids microinjected into the VLO on allodynia in the rat L5/L6 spinal nerve ligation (SNL) model of neuropathic pain and determine the roles of different subtypes of opioid receptors in this effect. The allodynia was assessed by both mechanical (von Frey filaments) and cold plate (4 degrees C) stimuli. Morphine (1.0, 2.5, and 5.0 microg) microinjected into the VLO contralateral to the nerve ligation dose-dependently depressed the mechanical and cold allodynia and these effects were reversed by nonselective opioid receptor antagonist naloxone (1.0 microg) administrated into the same site. Microinjection of endomorphin-1 (5.0 microg), a highly selective mu-opioid receptor agonist, and [D-Ala2, D-Leu5]-enkephalin (DADLE, 10 microg), a delta-/mu-opioid receptor agonist, also depressed the allodynia, and the effects of both drugs were blocked by selective mu-receptor antagonist beta-funaltrexamine (beta-FNA, 3.75 microg), but the effects of DADLE were not influenced by the selective delta-receptor antagonist naltrindole (5.0 microg). Microinjection of U-62066 (100 microg), a kappa-opioid receptor agonist, into the VLO had no effect on the allodynia. These results suggest that the VLO is involved in opioid-induced antiallodynia and mu- but not delta- and kappa-opioid receptor mediates these effects in the rat with neuropathic pain.     

Opioid receptor subtypes involved in the regulation of prolactin secretion during pregnancy and lactation

Afferent endogenous opioid neuronal systems facilitate prolactin secretion in a number of physiological conditions including pregnancy and lactation, by decreasing tuberoinfundibular dopamine (TIDA) inhibitory tone. The aim of this study was to investigate the opioid receptor subtypes involved in regulating TIDA neuronal activity and therefore facilitating prolactin secretion during early pregnancy, late pregnancy and lactation in rats. Selective opioid receptor antagonists nor-binaltorphimine (kappa-receptor antagonist, 15 micro g/5 micro l), beta funaltrexamine (mu-receptor antagonist, 5 microg/5 microl) and naltrindole (delta-receptor antagonist, 5 microg/5 microl) or saline were administered intracerebroventricularly (i.c.v.) on day 8 of pregnancy during a nocturnal prolactin surge, on day 21 of pregnancy during the ante partum prolactin surge or on day 7 of lactation before the onset of a suckling stimulus. Serial blood samples were collected at regular time intervals, via chronic indwelling jugular cannulae, before and after drug administration and plasma prolactin was determined by radioimmunoassay. TIDA neuronal activity was measured using the 3,4-dihydroxyphenylacetic acid (DOPAC) : dopamine ratio in the median eminence 2 h 30 min after i.c.v. drug injection. In each experimental condition, plasma prolactin was significantly inhibited by both kappa- and mu-receptor antagonists, whereas the delta-receptor antagonist had no effect compared to saline-injected controls. Similarly, nor-binaltorphimine and beta funaltrexamine significantly increased the median eminence DOPAC : dopamine ratio during early and late pregnancy, and lactation whereas naltrindole had no effect compared to saline-injected controls. These data suggest that TIDA neuronal activity, and subsequent prolactin secretion, is regulated by endogenous opioid peptides acting at both kappa- and mu-opioid receptors during prolactin surges of early pregnancy, late pregnancy and lactation.     

Mu-, delta-, kappa- and epsilon-opioid receptor modulation of the hypothalamic-pituitary-adrenocortical (HPA) axis: subchronic tolerance studies of endogenous opioid peptides

In opiate-naive rats, the endogenous opioid peptides, beta-endorphin, dynorphin(1-13) and Met-Enk-Arg-Phe (MEAP) and the synthetic enkephalin analogue D-Ala2-D-Leu5-Enk (DADLE) potently stimulated plasma corticosterone in a dose-dependent, naloxone reversible manner. To characterize their in vivo affinities, the effects of these peptides on plasma corticosterone release were tested in rats made tolerant to morphine, U50488H, DADLE/morphine or beta-endorphin. These cross-tolerance studies showed that dynorphin and MEAP exerted their action on plasma corticosterone release at kappa-opioid receptors. The action of DADLE occurred at delta-opioid receptors, while the action of beta-endorphin occurred principally at another receptor site. These results indicate that there is independent modulation of the hypothalamic-pituitary-adrenal axis by endogenous opioid peptides at mu-, delta- and kappa-opioid receptors. In addition there may be modulation by beta-endorphin at a separate site that we suggest could be a central epsilon-receptor site. This cross-tolerance paradigm, using a neuroendocrine model, provides in vivo evidence for the action of centrally active endogenous opioid peptides at multiple and independent opioid receptors.     

Opioid receptors in the human cerebellum: evidence from [11C]diprenorphine PET, mRNA expression and autoradiography

Little is known regarding opioid receptors in the human cerebellum. The present [11C]diprenorphine PET study investigated opioid receptor binding in the human cerebellum in vivo, and showed a differential binding level in cerebellar cortex, vermis and dentate nuclei. The additional study in vitro of opioid receptors in human cerebellar cortex and rat brain corroborated the presence of opioidergic mechanisms in the human cerebellum in contrast to the rat. A differential cellular distribution pattern was detected for the three major opioid receptors investigated. For the mu-receptor, and at a lower level for the kappa-receptor, mRNA expression was mainly observed over granule cells. Binding sites were most prominent in the molecular layer. For the delta-receptor no signal was detected. The consideration of cerebellar opioidergic mechanisms and the distribution patterns of the various opioid receptors may promote the understanding of cerebellar function and of opioidergic pharmacology in the human.     

Differential ontogeny of divalent cation effects on rat brain delta-, mu-, and kappa-opioid receptor binding

The effect of the divalent cations, Mn2+, Mg2+ and Ca2+ on rat forebrain delta-, mu- and kappa-receptor binding was examined during postnatal development. It was found that delta-receptor binding, assessed with [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) (+ 10 nM D-Ala2- MePhe4-Gly-ol5-enkephalin (DAMGE)), was stimulated by the 3 cations in a dose- and developmental time-dependent manner. delta-Binding was most sensitive to the cations during the first week postnatal, prior to the appearance of high-affinity delta-binding. In contrast, inhibition of mu-receptor binding ([3H]DAMGE) by divalent cations appeared early in development and remained constant throughout the postnatal period. Divalent cation inhibition of kappa-binding ([3H]ethylketocyclazocine ([3H]EKC) + 100 nM DAMGE and 100 nM DADLE) appeared after the second week postnatal. These results demonstrate that the characteristics and postnatal development of divalent cation modulation of mu-, delta- and kappa-binding is distinctly different. Thus, the neonate may be a good model system to examine the binding properties and functions of delta- and kappa-receptor subtypes.     

Chronic selective activation of excitatory opioid receptor functions in sensory neurons results in opioid 'dependence' without tolerance.

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.     

Opioid-receptor-mediated excitation of rat mesenteric afferent fibres supplying the rat jejunum

The aim of the present study was to examine the sensitivity to opioid-receptor agonists of mesenteric afferents supplying the small intestine and to characterize the subpopulations of any responsive fibres. Mesenteric afferent discharge was recorded electrophysiologically in response to cumulative doses (1-400 microgram kg-1) of the mu-receptor agonist [D-ala,2 N- me-Phe4, Gly5-ol]-enkephalin (DAMGO), the delta-receptor agonist [D-ala,2 D-leu5]-enkephalin (DADLE) and the kappa-receptor agonist U-50488. DAMGO and DADLE, but not U-50488, markedly stimulated whole nerve mesenteric afferent discharge (P < 0.05) that was unrelated to intestinal motor events. Subpopulations of afferent fibres responding to DAMGO were examined using waveform analysis to identify single units from within the whole mesenteric nerve bundles. One population was CCK-sensitive (15/15 fibres) and the other was a subpopulation of mechanosensitive afferents that responded to distension (17/28). 5-HT-sensitive afferents did not respond to DAMGO (0/11). We conclude that specific subpopulations of mesenteric afferents respond to mu- and possibly delta- but not kappa-receptor agonists. This sensitivity to opioids may contribute to the antinociceptive property of vagal afferents.     

Regulation of proopiomelanocortin messenger RNA concentrations by opioid peptides in primary cell cultures of rat hypothalamus.

The effects of opioid peptides on a 1.1-kb long proopiomelanocortin messenger RNA (POMC mRNA) have been investigated in rat hypothalamic cells maintained in culture. Most opioid peptides exerted an inhibitory control on POMC mRNA steady-state concentrations. beta-Endorphin caused a 65% maximal inhibitory effect (IC50 = 6.1 x 10(-9) M) while slightly less inhibition was caused by Met- and Leu-enkephalin, dynorphin A and DADLE ([D-Ala2,D-Leu5] enkephalin). The effects of beta-endorphin and of Met-enkephalin were completely reversed by the delta opioid antagonist ICI 174,864 while the kappa-receptor specific antagonist binaltorphimine or the sigma-receptor specific antagonist DTG (1,3-di(2-tolyl) guanidine) respectively blocked the inhibitory actions of dynorphin A and of DADLE. The mu-receptor specific agonist DAGO ([D-Ala2,N-Me-Phe4,Gly5-OL]enkephalin) did not affect POMC mRNA levels. The failure of the dopaminergic D2 antagonist haloperidol to modify the inhibitory effects of opioid peptides argues for a direct inhibitory opioid peptide modulation of hypothalamic POMC mRNA levels mediated by the delta-, kappa- and sigma- (but not mu-) receptors in vivo.     

Differential effects of excitatory amino acid receptor antagonism in the ventral respiratory group.

This study tested the hypothesis that selective antagonism of excitatory amino acid (EAA) receptors within the ventral respiratory group (VRG) would induce changes in both respiratory rhythm and pattern. In the paralyzed, decerebrate, vagotomized and ventilated cat, baseline values for respiratory (Ttot), inspiratory (Ti), and expiratory (Te) durations and peak integrated phrenic nerve (integralPN) amplitude were established. Microinjection of the non-NMDA (N-methyl-d-aspartate) receptor antagonist NBQX (2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) into rostral/inspiratory-modulated (iVRG) or caudal/expiratory-modulated VRG elicited an immediate apnea. When PN activity resumed, Ttot was significantly decreased, and integralPN amplitude was attenuated. NMDA receptor antagonism with microinjections of AP5 (2-amino-5-phosphonopentanoic acid) into iVRG decreased Te for more than 30 min. NMDA receptor antagonism in inspiratory/expiratory-modulated VRG (level of obex, transitional VRG) yielded either apnea or a significant reduction in Ttot, Ti and integralPN amplitude. Our data suggest that endogenous EAA receptor-mediated neurotransmission throughout the VRG is active in the determination of both respiratory timing and pattern. Our data further suggest that tVRG serves a unique function within the respiratory network.     

Facilitation of ACTH secretion by morphine is mediated by activation of CRF releasing neurons and sympathetic neuronal pathways

Exogenously applied opioid agonists have a stimulatory effect on adrenocorticotropic hormone (ACTH) secretion. The present experiments were designed to examine the mechanisms involved in the stimulatory effect of the mu-receptor agonist morphine on ACTH release in chronically cannulated, freely moving, non-stressed rats. Morphine (7.5 mg/kg, i.v.) treatment was followed by a significant increase in plasma levels of ACTH. Pretreatment with the peripheral ganglionic blocker chlorisondamine (3 mg/kg, i.p.) attenuated the response to morphine. The morphine stimulatory effect was also partially inhibited if the rats were pretreated with a specific antiserum to corticotropin-releasing factor (CRF). In rats given both CRF antiserum and chlorisondamine, the plasma ACTH levels remained unchanged after morphine application. These findings indicate that morphine stimulates the release of ACTH by activating both CRF-secretion and peripheral sympathetic neuronal pathways.     

Stress-induced changes in the analgesic and thermic effects of opioid peptides in the rat

Stress (e.g. restraint) potentiates analgesia and alters changes in body temperature induced by morphine administered either systemically or intracerebroventricularly (i.c.v.) in rats. In order to extend the generality of this phenomenon to opioid peptides, we determined whether the analgesic and thermic effects of i.c.v. D-Ala2-D-Leu5-enkephalin (DADLE) or D-Ala2-N-MePhe4-Gly5(ol)-enkephalin (DAGO), agonists selective for delta- and mu-opioid receptors, respectively, were affected by restraint stress. Analgesia was measured in the tail-flick test and core body temperature by rectal probe. The unstressed rats exhibited a dose-dependent increase in tail-flick latencies after administration of either DAGO or DADLE. Restrained rats treated with DAGO or DADLE had a greater analgesic response to each dose of peptide than did unstressed rats; both the magnitude and duration of the drug effect were increased. The unstressed group of rats responded to all doses of DAGO and DADLE with an increase of core temperature. In contrast, restrained rats showed a decrease of core temperature following injection with either DAGO or DADLE. Thus, restraint stress can significantly modify the effects of DAGO and DADLE on analgesia and body temperature in a manner that is qualitatively and quantitatively similar to that observed previously for morphine administered by the i.c.v. route.     

μ-, δ-, κ- and ε-Opioid receptor modulation of the hypothalamic-pituitary-adrenocortical (HPA) axis: subchronic tolerance studies of endogenous opioid peptides

In opiate-naive rats, the endogenous opioid peptides, β-endorphin, dynorphin(1–13) and Met---Enk---Arg---Phe (MEAP) and the synthetic enkephalin analogue -Ala²- -Leu⁵-Enk (DADLE) potently stimulated plasma corticosterone in a dose-dependent, naloxone-reversible manner. To characterize their in vivo affinities, the effects of these peptides on plasma corticosterone release were tested in rats made tolerant to morphine, U50488H, DADLE/morphine or β-endorphin. These cross-tolerance studies showed that dynorphin and MEAP exerted their action on plasma corticosterone release at κ-opioid receptors. The action of DADLE occurred at δ-opioid receptors, while the action of β-endorphin occurred principally at another receptor site. These results indicate that there is independent modulation of the hypothalamic-pituitary-adrenal axis by endogenous opioid peptides at μ-, δ- and κ-opioid receptors. In addition, there may be modulation by β-endorphin at a separate site that we suggest could be a central ε-receptor site. This cross-tolerance paradigm, using a neuroendocrine model, provides in vivo evidence for the action of centrally active endogenous opioid peptides at multiple and independent opioid receptors.     

Effect of dermorphin and morphine on the sympathetic and cardiovascular system of the pithed rat

Dermorphin is a recently discovered opioid peptide which is unique in having a D-amino acid in its sequence. Dermorphin binding sites have been shown in central and peripheral organs and central administered dermorphin produces profound autonomic responses. The purpose of this study was to examine the effect of intravenous dermorphin on heart rate and blood pressure of the pithed rat in basal condition and in response to controlled sympathetic stimulation. Also, since dermorphin is a selective mu-receptor agonist, its effects were compared to morphine, an opiate selective for mu receptors. Dermorphin (0.0001-10 mumol/kg, i.v) or morphine (1-10 mg/kg) had no effect on basal heart rate or blood pressure and failed to modify sympatho-adreno-medullary evoked pressor and tachycardic responses. Furthermore, dermorphin or morphine did not affect the increase in plasma norepinephrine and epinephrine in response to spinal cord stimulation. It is concluded that the dermorphin and morphine have no direct peripheral effects on heart rate or blood vessel tone nor do these mu-receptor agonists have any effect on norepinephrine and epinephrine release from the sympathetic nerves and the adrenal medulla in the rat.     

Blockade of D-2 dopamine receptors strongly enhances the potency of enkephalins to inhibit dopamine-sensitive adenylate cyclase in rat neostriatum: involvement of delta- and mu-opioid receptors

The interactions between dopamine receptors and opioid receptors coupled to adenylate cyclase in rat neostriatum were investigated. cAMP efflux from neostriatal slices induced by simultaneous activation of (stimulatory) D-1 and (inhibitory) D-2 dopamine receptors with 30 microM dopamine was inhibited by the preferential delta-opioid receptor agonist [D-Ala2-D-Leu5] enkephalin (DADLE) and the mu-opioid receptor agonist morphine with an EC50 of 100 and 800 nM, respectively. On selective D-1 receptor activation (i.e., with D-2 receptors blocked by 10 microM (-)sulpiride), the EC50 of DADLE was strongly reduced to 3 nM, whereas that of morphine was unaffected. When D-1 and D-2 receptors were activated simultaneously, the inhibitory effects of DADLE (0.3 microM) and morphine (3 microM) on cAMP efflux were antagonized equally well by naloxone, a mu-opioid receptor antagonist. In contrast, on selective D-1 receptor activation, naloxone was about 20 times more potent in antagonizing the inhibitory effect of morphine than DADLE. Moreover, the delta-opioid receptor antagonist ICI 174864 (0.75 microM) did not affect the inhibitory effect of morphine but antagonized that of DADLE, provided that D-2 receptors were blocked. The highly selective delta-opioid receptor agonist [D-Pen2-D-Pen5] enkephalin (DPDPE) inhibited dopamine-stimulated cAMP efflux only when D-2 receptors were blocked. Similar results were obtained when the agonists SKF 38393 and LY 141865 were used to activate D-1 and D-2 receptors, respectively. These data indicate that blockade of D-2 receptors in the neostriatum elicits the coupling of delta-opioid receptors to dopamine-sensitive adenylate cyclase, thereby making it considerably more sensitive to inhibition by the enkephalins.     

Kappa opioid receptors in human lumbo-sacral spinal cord

[3H]Etorphine and [3H]ethylketocyclazocine bind with high affinity (Kd between 0.25-2.0 nM) to a single class of sites in human lumbo-sacral spinal cord. Other ligands such as [3H]morphine, [3H]dihydromorphine and [3H]D-Ala2, D-Leu5-enkephalin (DADLE) did not bind to significant number of sites under our incubation conditions. Ligand selectivity pattern strongly suggests that [3H]etorphine labels kappa opioid binding sites in the human lumbo-sacral spinal cord since benzomorphans and oripavines are much more potent than mu and delta agonists. Furthermore, [3H]etorphine and [3H]ethylketocyclazocine binding is sensitive to high concentrations of DADLE suggesting that these sites are of the kappa 2 sub-type. Finally, the visualization of these sites by receptor autoradiography demonstrates that they are mainly concentrated in lamina II and III of the dorsal horn. Moderate densities of sites are present around the central canal. Thus, it is possible that kappa opioid binding sites could be involved in the control of sensory and autonomic functions in the human lumbo-sacral spinal cord.     

Postnatal development of opioid regulation of micturition in the kitten.

Endogenous opioids tonically regulate micturition in adult mammals. The present study sought to determine if opioids regulate micturition in neonatal kittens. Naloxone (up to 2 mg/kg given i.p. or i.v. to unanesthetized/ketamine-anesthetized or chloralose-anesthetized kittens, respectively), an opioid receptor antagonist, produced no effects in unanesthetized, ketamine-anesthetized, or chloralose-anesthetized kittens that had been prepared for bladder pressure recording, until 3 weeks of age. This indicates that endogenous opioids are not tonically regulating micturition in neonatal kittens. From 3 weeks up to at least 6 weeks of age, naloxone (100 micrograms/kg i.p. or i.v.) weakly facilitated bladder activity by transiently increasing the amplitude and/or duration of bladder contractions, but no effects on frequency of contractions was recorded. Morphine (up to 2 mg/kg given i.p. or i.v. to unanesthetized/ketamine-anesthetized or chloralose-anesthetized kittens, respectively), an opioid agonist, did not inhibit bladder contractions in unanesthetized or ketamine-anesthetized neonatal kittens, but it did inhibit (at a threshold dose of 100 micrograms/kg) and completely abolished (at a dose of 300 micrograms/kg) bladder activity in chloralose-anesthetized kittens in a dose-dependent, naloxone-reversible manner. Surprisingly, following morphine administration to unanesthetized or ketamine-anesthetized neonatal kittens, naloxone now produced an adult-like enhancement of bladder activity. These latter results indicate that opioid receptors, whose inhibitory effects are anesthetic-dependent, are present along the micturition reflex pathway in neonates. Immunohistochemical studies of the sacral spinal cord revealed that opioid peptides are distributed similarly in neonatal and adult cats.