Delta Opiate Receptors Research Articles

Opiate drugs and delta-receptor-mediated myocardial protection.

Hypothermic myocardial arrest is necessary to complete most cardiac surgery, which limits the success of such operations. Similarly, cold, inhospitable environments limit the survival of warm-blooded animals. Animals have successfully adapted to this challenge through hibernation. Hibernation is an energy-conserving state, now known to be governed by cyclical variation in endogenous opiate compounds. It may also be induced in nonhibernators via hibernating animal serum factors or delta-opiate peptides. Furthermore, hibernation-induction triggers extend organ preservation in many models. This study examined whether opiate drugs with an affinity for the delta-opiate receptor confer similar protection. Isolated hearts harvested from New Zealand White rabbits were treated with either cardioplegia alone or delta-opiate drugs (fentanyl, morphine, buprenorphine, pentazocine) followed by 2 hours of 34 degrees C ischemia. Hearts were then reperfused, and functional and metabolic indices of treated groups were compared with untreated controls. Isovolumic developed pressure, coronary flow, and oxygen consumption were compared as a percent of preischemia versus 45 minutes after reflow. Developed pressure and oxygen consumption were better preserved in the morphine, buprenorphine, and pentazocine groups when compared with cardioplegia alone. Drugs with delta-opiate activity confer myocardial protection, which is additive to cardioplegia. Use of delta-opiate drugs in this context may have important clinical implications.

Dynorphin selectively augments the M-current in hippocampal CA1 neurons by an opiate receptor mechanism.

Most electrophysiological studies of opioids on hippocampal principal neurons have found indirect actions, usually through interneurons. However, our laboratory recently found reciprocal alteration of the voltage-dependent K(+) current, known as the M-current (I(M)), by kappa and delta opioid agonists in CA3 pyramidal neurons. Recent ultrastructural studies have revealed postsynaptic delta opiate receptors on dendrites and cell bodies of CA1 and CA3 hippocampal pyramidal neurons (HPNs). Reasoning that previous electrophysiological studies may have overlooked voltage-dependent postsynaptic effects of the opioids in CA1, we reevaluated their role in CA1 HPNs using the rat hippocampal slice preparation for intracellular current- and voltage-clamp recording. None of the delta and mu; receptor-selective opioids tested, including [D-Pen(2,5)]-enkephalin (DPDPE), [D-Ala(2)]-deltorphin II (deltorphin), [D-Ala(2), NMe-Phe(4), Gly-ol]-enkephalin (DAMGO), and [D-Ala(2), D-Leu(5)] enkephalin (DADLE), altered membrane properties such as I(M) or Ca(2+)-dependent spikes in CA1 HPNs. The nonopioid, Des-Tyr-dynorphin (D-T-dyn), also had no effect. By contrast, dynorphin A (1-17) markedly increased I(M) at low concentrations and caused an outward current at depolarized membrane potentials. The opioid antagonist naloxone and the kappa receptor antagonist nor-binaltorphimine (nBNI) blocked the I(M) effect. However, the kappa-selective agonists U69,593 and U50,488h did not significantly alter I(M) amplitudes when averaged over all cells tested, although occasional cells showed an I(M) increase with U50,488h. Our results suggest that dynorphin A postsynaptically modulates the excitability of CA1 HPNs through opiate receptors linked to voltage-dependent K(+) channels. These findings also provide pharmacological evidence for a functional kappa opiate receptor subtype in rat CA1 HPNs but leave unanswered questions on the role of delta receptors in CA1 HPNs.

Delta Opiate Receptors Account for the Castration-Induced Unmasking of Gonadotropin-Releasing Hormone Binding Sites in the Rat Pituitary

Under control incubation conditions, gonadotropin-releasing hormone (GnRH) binds only a fraction of its receptors in rat-cultivated pituitary cells. Unmasking of the remaining receptors, which have been termed ‘cryptic’, requires drug- or peptide-induced protein kinase activation. Spontaneous masking however is not observed on pituitary cells sampled from castrated male rats, suggesting the presence of an intrinsic unmasking factor. Many endogenous factors could theoretically account for the effect. Here we attempted to identify the factor involved by taking advantage of their differential dependency upon second messengers and transduction cascades. Spontaneous unmasking of GnRH binding was found reversed by pertussis toxin (PTX), an inhibitor of α_i and α_o subunits of heterotrimeric G proteins, and by U73122, a phospholipase C (PLC) inhibitor. In contrast, desensitization of protein kinase C (PKC) or inhibition of tyrosine kinase by herbimycin were ineffective. Among endogenous pituitary factors able to unmask GnRH receptors in pituitary cells from normal male rats, as EGF, NPY or opiate peptides, only the latter were found to correspond to this transduction profile. In an attempt to characterize the pharmacology of opiate effects, naloxone (10 µM), a poorly selective opiate antagonist, restored masking of GnRH binding in cells from castrates. Only the δ antagonist naltrindole (1 µM) was able to mimick the action of naloxone. Conversely, when tested on cells from intact animals, morphine (10 µM), as well as dslet (1 µM) and met-ENK (10 nM), preferential δ agonists, but not dago and β-endorphin or U50488 H and dynorphin, respectively µ and ĸ agonists, were able to suppress masking. Among opioid peptides endogenous to the pituitary, only met-ENK was able to unmask cryptic receptors, an effect antagonized by naltrindole. We conclude that an opiate δ receptor subtype is endogenously activated in the pituitary of castrated male rats to prevent masking of GnRH binding.

Opioid inhibition of rat medial vestibular nucleus neurones in vitro and its dependence on age

Extracellular and whole-cell patch clamp intracellular recordings were made from rat medial vestibular nucleus (MVN) neurones in vitro, and their responses to selective mu-, kappa- and delta-opioid receptor agonists and antagonists were examined. Of 127 neurones tested, the large majority were inhibited in a dose-dependent manner by the delta-opioid receptor agonists [D-Ala2, D-Leu5]-enkephalin (DADLE) and [D-Pen2, Pen5]-enkephalin (DPLPE). The mu-opioid receptor agonist morphine and the kappa-receptor agonist U50,488 did not affect the tonic discharge rate of any of the 63 MVN cells tested. The delta-receptor antagonist naltrindole effectively antagonised the inhibitory effects of DADLE and DPLPE. Weak excitatory responses to high doses of DADLE were seen in only two MVN cells. These results demonstrate the presence of delta- but not mu- or kappa-opioid receptors on tonically active MVN neurones. Whole-cell intracellular recordings from MVN cells in a current clamp showed that the DADLE-induced inhibition was accompanied by membrane hyperpolarisation and decrease in input resistance, while voltage clamp experiments showed that DADLE induced an outward membrane current that was reduced but not abolished by 20 mM tetraethylammonium bromide. Thus the mechanisms of action of DADLE in inhibiting MVN cells involve the potentiation of outward K currents, in a similar way to the effects of opioids in other areas of brain. The inhibitory effects of DADLE increased linearly with age, so that the responses to DADLE in the youngest animals used here (60-80 g, approx. 3 weeks of age) were relatively small, increasing significantly over the following 2-3 weeks. This age-dependence may be due to post-natal changes in the density of delta-opiate receptors or the efficacy of the signalling pathways activated by them in the MVN cells over this time.

Intracerebroventricular injection of mu- and delta-opiate receptor antagonists block 60 Hz magnetic field-induced decreases in cholinergic activity in the frontal cortex and hippocampus of the rat

In previous research, we have found that acute exposure to a 60 Hz magnetic field decreased cholinergic activity in the frontal cortex and hippocampus of the rat as measured by sodium-dependent high-affinity choline uptake activity. We concluded that the effect was mediated by endogenous opioids inside the brain because it could be blocked by pretreatment of rats before magnetic field exposure with the opiate antagonist naltrexone, but not by the peripheral antagonist naloxone methiodide. In the present study, the involvement of opiate receptor subtypes was investigated. Rats were pretreated by intracerebroventricular injection of the mu-opiate receptor antagonist, beta-funaltrexamine, or the delta-opiate receptor antagonist, naltrindole, before exposure to a 60 Hz magnetic field (2 mT, 1 hour). It was found that the effects of magnetic field on high-affinity choline uptake in the frontal cortex and hippocampus were blocked by the drug treatments. These data indicate that both mu- and delta-opiate receptors in the brain are involved in the magnetic field-induced decreases in cholinergic activity in the frontal cortex and hippocampus of the rat.

Intracerebroventricular injection of mu‐ and delta‐opiate receptor antagonists block 60 Hz magnetic field‐induced decreases in cholinergic activity in the frontal cortex and hippocampus of the rat

In previous research, we have found that acute exposure to a 60 Hz magnetic field decreased cholinergic activity in the frontal cortex and hippocampus of the rat as measured by sodium-dependent high-affinity choline uptake activity. We concluded that the effect was mediated by endogenous opioids inside the brain because it could be blocked by pretreatment of rats before magnetic field exposure with the opiate antagonist naltrexone, but not by the peripheral antagonist naloxone methiodide. In the present study, the involvement of opiate receptor subtypes was investigated. Rats were pretreated by intracerebroventricular injection of the mu-opiate receptor antagonist, β-funaltrexamine, or the delta-opiate receptor antagonist, naltrindole, before exposure to a 60 Hz magnetic field (2 mT, 1 hour). It was found that the effects of magnetic field on high-affinity choline uptake in the frontal cortex and hippocampus were blocked by the drug treatments. These data indicate that both mu- and delta-opiate receptors in the brain are involved in the magnetic field-induced decreases in cholinergic activity in the frontal cortex and hippocampus of the rat. Bioelectromagnetics 19:432–437, 1998. © 1998 Wiley-Liss, Inc.

Delta-opiate DPDPE in magnetically oriented phospholipid micelles: binding and arrangement of aromatic pharmacophores

D-Penicillamine(2,5)-enkephalin (DPDPE) is a potent opioid peptide that exhibits a high selectivity for the delta-opiate receptors. This zwitterionic peptide has been shown, by pulsed-field gradient 1H NMR diffusion studies, to have significant affinity for a zwitterionic phospholipid bilayer. The bilayer lipid is in the form of micelles composed of dihexanoylphosphatidylcholine (DHPC) and dimyristoylphosphatidylcholine (DMPC) mixtures, where the DMPC forms the bilayer structure. At high lipid concentration (25% w/w) these micelles orient in the magnetic field of an NMR spectrometer. The resulting 1H-13C dipolar couplings and chemical shift changes in the natural abundance 13C resonances for the Tyr and Phe aromatic rings were used to characterize the orientations in the bilayer micelles of these two key pharmacophores.

Dual label in situ hybridization studies provide evidence that luteinizing hormone-releasing hormone neurons do not synthesize messenger ribonucleic acid for mu, kappa, or delta opiate receptors.

Abundant evidence suggests that opiatergic neurons play an important intermediary role in the regulation of LHRH release by ovarian steroids; however, it is unclear whether opiates communicate directly or indirectly with LHRH neurons. To investigate this issue, we used dual label in situ hybridization histochemistry to determine whether LHRH neurons synthesize messenger RNA (mRNA) for mu, kappa, and/or delta opiate receptors. For these studies, we examined both intact (n = 3) and ovariectomized, steroid-treated rats. Ten of the ovariectomized rats were implanted 1 week later (day 0) with SILASTIC brand (Dow Corning) capsules of estradiol. On the morning of day 2, half of the estradiol-treated rats were injected with 5 mg progesterone. All animals were killed at approximately 1530 h on day 2. We found that cells expressing mu, kappa, and delta opiate receptor mRNAs were in all sections that also contained LHRH neurons. In every case, LHRH neurons were seen to be surrounded by or in close proximity to cells containing mu, kappa, or delta mRNAs. However, regardless of steroid treatment, we found no neurons containing both LHRH mRNA and mRNAs encoding any of the three receptor subtypes. These results support the hypothesis that LHRH neurons are regulated indirectly by opiatergic neurons.

Beta-endorphin enhances Concanavalin-A-stimulated calcium mobilization by murine splenic T cells.

Intracellular calcium mobilization is an important early event involved in T cell activation. The endogenous opioid peptide beta-endorphin is known to modulate immune functions that depend on T cell activation, therefore its effect on intracellular calcium mobilization was investigated. The intracellular calcium concentration ([Ca2+]i) of T cell-enriched splenocytes was measured by flow cytofluorometric analysis using the calcium-sensitive dye, Fluo-3. By gating on the T cell marker, Thy-1, a 95%-pure population of T cells was identified for study. Cells preincubated with beta-endorphin showed significantly enhanced [Ca2+]i responses to the mitogen, Concanavalin A (Con A). This was detectable with concentrations of beta-endorphin as low as 10(-13) M; maximal enhancement required 10(-10) to 10(-9) M doses. The efficacy of beta-endorphin was dependent on the duration of pretreatment. beta-Endorphin amplified the Con A-induced increase in [Ca2+]i by reducing the lag time for the response to Con A and by increasing the mean [Ca2+]i of the cells. N-Ac-beta-endorphin, which shows minimal potency at neuronal opiate receptors, was unable to substitute for beta-endorphin. Naltrindole, a highly selective delta opiate receptor antagonist, inhibited the action of beta-endorphin, whereas a selective mu opiate receptor antagonist was ineffective. Although less potent than beta-endorphin, the delta opiate receptor agonist D-Ala2-D-Leu5-enkephalin also significantly enhanced [Ca2+]i responses. In summary, concentrations of beta-endorphin, within the physiological range found in the systemic circulation, modulate the increase in T cell [Ca2+]i induced by Con A. Both the efficacy of D-Ala2-D-Leu5-enkephalin alone and the antagonism of beta-endorphin by naltrindole suggest that a delta-type opiate receptor may mediate these effects.

Thr353, Located within the COOH-terminal Tail of the ´ Opiate Receptor, Is Involved in Receptor Down-regulation

Prolonged exposure to abused drugs such as opiates causes decreased response to the drug; this reduced sensitivity is thought to be due to the loss of receptors, or down-regulation. The molecular mechanism of the opiate receptor down-regulation is not known. In order to address this, we generated a number of mutants of the delta opiate receptor COOH-terminal tail. When expressed in the Chinese hamster ovary cells, both the wild type and the receptor with a deletion of 37 COOH-terminal residues bind diprenorphine with comparable affinities and show similar decreases in cAMP levels in response to D-Ala2, D-Leu5, enkephalin (DADLE). However, the truncated receptor does not show down-regulation from the cell surface upon prolonged exposure (2-48 h) to DADLE. In contrast, both the wild type receptor and the receptor with the deletion of only 15 COOH-terminal residues show substantial down-regulation upon long term DADLE treatment. These results suggest that the region located between 15 and 37 residues from the COOH terminus is involved in the receptor down-regulation. In order to identify residues that play a key role in down-regulation, point mutations of residues within this region were examined for their ability to modulate receptor down-regulation. The receptor with a mutation of Thr353 to Ala does not down-regulate, whereas the receptor with a mutation of Ser344 to Gly down-regulates with a time course similar to that of the wild type receptor. Taken together, these results suggest that the COOH-terminal tail is not essential for functional coupling but is necessary for down-regulation and that Thr353 is critical for the agonist-mediated down-regulation of the delta opiate receptor.

The peristaltic reflex in the rat ileum: evidence for functional mu- and delta-opiate receptors.

The potency order of opiate agonists at decreasing the rate of peristalsis in the rat isolated ileum was: difenoxin > loperamide > DADLE (D-Ala2-D-Leu5-enkephalin) > morphine > DSLET (D-Ser2,Leu5-Thr6-enkephalin). U-50488 (trans 3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl) cyclohexyl) benzeneacetamide methane sulphonate) was inactive at 300 nM. Naloxone (400 nM) caused a significant 1.52-fold increase in the rate of peristaltic contractions and inhibited the effects of the active opiate agonists. The apparent pA2 values of naloxone were similar using difenoxin, loperamide and morphine as agonists, but the value was slightly, though significantly lower when DADLE was the agonist. It is suggested that the previously identified delta-opiate receptors of the rat small intestine have a functional role in suppressing the peristaltic reflex. The same response is subserved by mu-opiate receptors and either of these opiate-receptor subtypes could be activated by endogenous enkephalins.

The site of inhibitory action of endogenous opioids in the superior cervical ganglion of the cat

A low-frequency stimulus train to the preganglionic input inhibits synaptic transmission in the superior cervical ganglion (SCG) of the cat. The inhibition is blocked by naloxone as well as by selective antagonists at mu and delta opiate receptors, which suggests that the mediator is an endogenous opioid [27, 29]. Exogenous opioid peptides, including methionine-enkephalin (Met-Enk), which is present in preganglionic axons of the SCG, inhibit ganglionic transmission by a naloxone-sensitive mechanism. In the present study we test, in the anesthetized cat, whether the naloxone-sensitive synaptic inhibition is mediated by a pre- and/or post-synaptic mechanism. As a test of presynaptic inhibition, we measured the acetylcholine (ACh) released by preganglionic stimulation into the venous effluent of the perfused SCG. As a test of post-synaptic inhibition, we measured the effect of a preganglionic conditioning train on the ganglion cell firing evoked by ganglion-stimulant drugs injected into the arterial supply of the ganglion. In presence of naloxone (3 μM), which blocked the synaptic inhibition, the amount of ACh released by stimulated preganglionic axons did not change. Thus, the endogenous opioid which mediates the naloxone-sensitive inhibition does not act by depressing ACh release. In contrast, the ganglion cell firing evoked by ganglion-stimulant drugs was markedly depressed by a conditioning train, and naloxone blocked the depression, which suggests that the endogenous mediator of the naloxone-sensitive inhibition acts postsynaptically to decrease the excitability of ganglion cells. Exogenous Met-Enk depressed both ACh release by preganglionic stimulation and the firing of ganglion cells evoked by ganglion-stimulant drugs. These findings suggest that although opiate receptors in the cat SCG are present at both pre- and post-synaptic sites, under the conditions of the present experiment the naloxone-sensitive, synaptically mediated inhibition is mediated exclusively by activation of post-synaptic opiate receptors.

Comparison of the distribution of mu and delta opiate receptor mRNAs in rat and mouse brain by in situ hybridization

Comparison of the distribution of mu and delta opiate receptor mRNAs in rat and mouse brain by in situ hybridization

Participation of opiate delta-receptors in immunomodulation.

The activation of the opiate delta-receptors by the highly specific agonist DSLET evokes an immunoinhibitory effect in CBA mice which disappears with preliminary blockade of S2 serotonin receptors by cyproheptadine. The blockade of delta-receptors by the antagonist ICI 174,864 leads to stimulation of the immune response, which is tested on the basis of the number of rosette-forming cells. Neither stimulation nor suppression of immunogenesis are manifested in animals with a transsected hypophyseal stalk; this suggests the central action of the opioids. The increase in the immune response obtained with the administration of ICI 174,864 is associated with the dopaminergic system, since it is prevented by haloperidol. It was demonstrated that the thymus participates in the immune-stimulating influence of ICI 174,864.

Comparison of delta opiate receptor agonist induced reward and motor effects between the ventral pallidum and dorsal striatum

The role of the ventral pallidum and the dorsal striatum in mediating the rewarding effects of the delta receptor specific agonist [2-D-penicillamine, 5-D-penicillamine]enkephalin (DPDPE) were evaluated in the rat using the intracranial self-stimulation paradigm. Reward shifts were indicated by the change in frequency required to maintain half-maximal responding while motor/performance changes were identified by increases or decreases in the maximum responding. Each hour-long test session consisted of three identical, consecutive 20 min rate-frequency curves. In an effort to ascertain possible heterogeneity of function along the rostrocaudal axis, DPDPE (0.0 nmol = saline dose, 0.3 nmol = low dose, 1.0 nmol = medium dose, 3.0 nmol = high dose) was microinjected into either the rostral or caudal region of the two structures. Microinjections into the caudate were positioned directly above the ventral pallidum placements resulting in centromedial or caudomedial caudate placements. DPDPE microinjections into the rostral ventral pallidum resulted in a significant reward increase (28% increase or -0.14 log Hg shift) only at the high dose. In contrast, caudal ventral pallidal DPDPE microinjections showed a dose-response effect with reward increases of 19, 22 and 31% (-0.09, -0.11 and -0.16 log Hz) for the low, medium and high dose, respectively. DPDPE microinjections into the centromedial caudate resulted in a large reward increase (29% or -0.15 log Hz) at the high dose, while caudomedial caudate DPDPE microinjections had no effect on reward. Motor/performance effects tended to follow the pattern of reward effects, with most regions showing motor increases ranging from 25 to 75% over baseline activity. The only exception was found in the caudomedial caudate, where microinjections of the high dose of DPDPE resulted in an approximate 20% suppression of motor/performance activity. These results demonstrate that the ventral pallidum and the mediocentral caudate play a role in modulating opiate rewards, and adds to the growing body of literature regarding the regional heterogeneity within the caudate and ventral pallidum.

Evidence for functional delta-opiate receptors in the rat intestine.

The selective delta-opiate agonists D-Ser2, Leu5, Thr6-enkephalin (DSLET), D-Ala2, D-Leu5-enkephalin and D-Pen2, D-Pen5-enkephalin caused inhibition of the cholinergic contraction produced by transmural stimulation of the rat isolated jejunum. Dynorphin A, which is an agonist at both kappa- and delta-opioid receptors also inhibited the cholinergic contraction, as did leu- and met-enkephalin. The selective mu-receptor agonist D-Ala2-NMe-Phe4, Gly-ol5-enkephalin was the least potent of all peptides tested. In general, the order of potency of the peptides was similar to that reported for the delta-receptor-rich mouse vas deferens with potency values similar to those recorded previously for the hamster vas deferens. The selective delta-opioid antagonist naltrindole caused parallel shifts to the concentration-effect curve to DSLET giving a pA2 value of 10.15. The results indicate that the previously identified delta-binding sites in the rat jejunum may correspond to functional delta-opiate receptors involved in attenuating acetylcholine release.

Direct excitatory opiate effects mediated by non-synaptic actions on rat medial vestibular neurons

Opiates increase firing of rat medial vestibular nucleus neurons. We have attemped to dermined the mechanism of these excitatory opiate actions by extracellular recording of neronal activity with ionophoretic application of opiate agonists and bath application of antagonists. The spontaneous activity of approximately 30% of medial vestibular neurons, scattered throughout nucleus, was increased by ionophoretic application of either morphine or [D-Ala 2]leucine enkephalin, implicating the presence of both mu and delta opiate receptors. The responses to both were blocked by the opiate receptors antagonist, naloxine. In only a few neuron opiates decrease firing. Most previous reports of direct opiate excitation have proven to be due to disinhibition. This is not the case here, as indicated by three observations: 1) the excitatory opiate response was sustained when γ-aminobutyric acid (GABA) receptors were blocked by Bicuculline; 2) perfusion of a solution containing 0.1 nM Ca 2+ and mM Mg 2+ blocks synaptic transmission, but does noe block the excitatory responses to both opiates and 3) the opiate-induced depolarization and action potential generation was evident in neurons whose spontaneous firing was almost totally depressed by adenosine. These results indicate that the excitation is neither due to disinhibition nor to a presynaptic opiate action. We conclude that medial vestibular neuron have postsynaptic opiate receptors that mediate direct neuronal excitation.

Mesoaccumbens dopamine-opiate interactions in the control over behaviour by a conditioned reinforcer

These experiments examined the role of dopamine-opiate interactions in the ventral tegmental area (VTA) and nucleus accumbens in the mediation of reinforcement-related behaviour. It has been shown previously that opiates induce a dopamine-dependent increase in locomotor activity in rats when infused into the VTA, and a dopamine-independent hyperactivity when infused into the nucleus accumbens. The present study investigated the generality and significance of these two findings, by examining dopamine-opiate interactions in the control over behaviour exerted by a conditioned reinforcer (CR), an arbitrary stimulus which gains control by association with primary reinforcement. Rats were trained to associate a light/noise stimulus with sucrose reinforcement, and the efficacy of the CR in controlling behaviour was assessed by measuring its ability to support a new lever pressing response. Responding on one lever (CR lever) produced the CR, responding on the other lever had no programmed consequences. In experiment 1, intra-accumbens infusions of d-amphetamine (10 micrograms), the D1 dopamine receptor agonist SKF-38393 (0.1 microgram), the D2 dopamine receptor agonist LY-171555 (quinpirole; 0.1 microgram) or the opiate receptor agonist [D-Ala2]-methionine enkephalinamide (DALA; 1 microgram) selectively increased responding on the CR lever. Infusion with DALA intra-VTA had no effect. However, pretreatment with DALA intra-VTA (10 x 1 microgram/day) subsequently reduced the selectivity of the response to infusions intra-accumbens with d-amphetamine or SKF-38393, and blocked the response to LY-171555 or DALA. Pretreatment also shifted to the right the dose-response function for DALA intra-accumbens. In experiment 2, intra-accumbens infusions of d-amphetamine, SKF-38393, LY-171555 or DALA again increased responding on the CR lever only. Pretreatment with intra-accumbens d-amphetamine (5 x 1 microgram/day) reduced the selectivity of the response subsequently to d-amphetamine, and blocked the response to SKF-38393, LY-171555 or DALA. In experiment 3, intra-accumbens infusions of the mu-opiate receptor agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (0.003-0.1 microgram), or the delta-opiate receptor agonist [D-Pen2,5]-enkephalin (0.03-1 microgram) enhanced selectively responding on the CR lever. Thus, the dopamine-dependent locomotor-stimulant properties of intra-VTA infusions of opiates are associated with impaired conditioned reinforcer efficacy. Finally, repeated stimulation of the mesoaccumbens dopamine pathway may compromise the dopamine-independence of the opiate system within the nucleus accumbens.

Novel peptides and mechanisms of opiate tolerance

We have used two diverse approaches for studying cellular and tissue effects of chronic exposure to opiates. First, receptor regulation by agonists and antagonists was characterized in a human neuroblastoma cell line, SH-SY5Y. Morphine down-regulated mu and delta opiate receptors. Mu receptor down-regulation was selectively blocked by the mu antagonist CTOP and delta receptor changes by the delta antagonist ICI 174864. Naloxone up-regulated opiate receptors in these cells, as it does in brain. Thus, SH-SY5Y cells provided a neuronal model for studying selective mu and delta receptor regulation in response to agonists and antagonists. In a second approach, we have shown that peptides recently isolated from brain in our laboratory (Tyr-MIF-1 and Tyr-W-MIF-1) can act as opiate agonists in several test paradigms, but as antagonists under specific circumstances in these test systems. These include chronic exposure to morphine or reduction of receptor reserve by alkylating agents. These peptides may serve as prototypes for endogenous compounds that act as agonists in the naive state and as antagonists in the tolerant state.

Opiate and opiate antidiarrhoeal drug action on rat isolated intestine

1. Stimulation of segments of rat jejunum (2.5, 5, 10, 20 and 40 Hz for 8 s) and ileum (10 Hz) resulted in a fast atropine-sensitive contraction during stimulation and a non-cholinergic after-contraction. Stimulation of segments with single pulses at 0.1 Hz had no effect. The colon (10 Hz) usually responded only with single large atropine-sensitive contractions. 2. The responses of the jejunum (2.5-40 Hz, 8 s) were unaffected by the mu-receptor ligands morphine (0.3 microM) and RX 783006 (0.3 microM) or the kappa-receptor ligand, ethylketocyclazocine (0.3 microM). The cholinergic contraction of the colon was unaffected by ethylketocyclazocine (0.3 microM), but was reduced slightly by morphine at high concentrations only. 3. The prototype delta-opiate receptor ligand D-Ala2-D-Leu5-enkephalin (DADLE; 30 nM) reduced the contraction during stimulation (2.5, 5, 10 and 20 Hz for 8 s) of the rat jejunum. The IC50 value for inhibition of the contraction elicited by stimulation at 10 Hz was 3.2 in the jejunum and was 12 nM in the ileum. Contractions of the colon were only slightly inhibited at a high concentration of DADLE (1 microM). 4. DADLE had no effect on contractions elicited by acetylcholine, at a concentration of up to 3 microM in segments of jejunum. Naloxone (1 microM) abolished the inhibitory effect of DADLE on the cholinergic contraction of the electrically stimulated jejunum. In addition, naloxone caused a large enhancement of control responses to stimulation. 5. Only high concentrations of the antidiarrhoeals loperamide and diphenoxylate inhibited the contraction elicited at 10 Hz in the jejunum (IC50, 1.3 and 7.1 microM, respectively) and ileum (loperamide IC50, 0.5 microM). The same concentrations of these drugs also inhibited the effect of exogenously added acetylcholine. A similar pattern of findings was made in the colon. 6. The results show marked differences in responses to transmural stimulation and to opiate drugs compared to those previously obtained from guinea-pig ileum.(ABSTRACT TRUNCATED AT 400 WORDS)