Current status of inverse agonism at serotonin2A (5-HT2A) and 5-HT2C receptors.

Contemporary receptor theory was developed to account for the existence of constitutive activity, as defined by the presence of receptor signaling in the absence of any ligand. Thus, ligands acting at a constitutively active receptor, can act as agonists, antagonists, and inverse agonists. In vitro studies have also revealed the complexity of ligand/receptor interactions including agonist-directed stimulus trafficking, a finding that has led to multi-active state models of receptor function. Studies with a variety of cell types have established that the serotonin 5-HT(2A) and 5-HT(2C) receptors also demonstrate constitutive activity and inverse agonism. However, until recently, there has been no evidence to suggest that these receptors also demonstrate constitutive activity and hence reveal inverse agonist properties of ligands in vivo. This paper describes our current knowledge of constitutive activity in vitro and then examines the evidence for constitutive activity in vivo. Both the serotonin 5-HT(2A) and 5-HT(2C) receptors are involved in a number of physiological and behavioral functions and are the targets for treatment of schizophrenia, anxiety, weight control, Parkinsonism, and other disorders. The existence of constitutive activity at these receptors in vivo, along with the possibility of inverse agonism, provides new avenues for drug development.

Effect of serotonin depletion on 5-HT2A-mediated learning in the rabbit: evidence for constitutive activity of the 5-HT2A receptor in vivo.

RATIONALE: Associative learning during Pavlovian eyeblink conditioning has been shown to be regulated by 5-HT2A receptors. The existence of inverse agonists that retard learning through an action at the 5-HT2A receptor suggests the existence of constitutive activity at that receptor and that depletion of serotonin should have minimal effects on learning. OBJECTIVES: We examined whether depletion of serotonin would impair trace eyeblink conditioning or the enhancement of conditioning produced by the agonist lysergic acid diethylamide (LSD) and the retardation of conditioning produced by the inverse agonist MDL11,939. METHODS: Animals received bilateral intraventricular injections of 5,7-dihydroxytryptamine (5,7-DHT) at doses of 760 or 1,140 microg/side (1.88 or 2.82 micromol/side) and were later exposed to eight daily conditioning sessions. RESULTS: Serotonin depletion produced by the lower dose of 5,7-DHT was 71 and 72% in cortex and hippocampus, respectively, with no change in 5-HT2A receptor density, no effect on learning, and no effect on the ability of LSD to enhance and MDL11,939 to retard learning. The higher dose of 5,7-DHT produced serotonin decreases of 85 and 90% in cortex and hippocampus, respectively, accompanied by a 96% decrease in the density of the serotonin transporter, but no significant effect on learning. CONCLUSIONS: Pavlovian trace eyeblink conditioning is regulated predominantly by the constitutive activity of the 5-HT2A receptor rather than by serotonin release onto the receptor during learning. It was suggested that the 5-HT2A receptor regulates learning by modulating the release of dopamine, acetylcholine, and glutamate, transmitters known to affect eyeblink conditioning.

Selective and divergent regulation of cortical 5-HT(2A) receptors in rabbit.

It is well established that repeated administration of both 5-hydroxytryptamine(2) (5-HT(2)) receptor agonists and antagonists decreases the density of 5-HT(2A) and 5-HT(2C) receptors. However, the regulation of these two receptors has not been studied in the same tissue. Therefore, we examined the effects of repeated daily injections of the 5-HT(2) receptor agonists (+/-)-2,5-dimethoxy-4-iodoamphetamine (DOI) and D-lysergic acid diethylamide (LSD) and the antagonists d-2-bromolysergic acid diethylamide hydrogen tartrate (BOL) and alpha-phenyl-2-(2-phenylethyl)-4-piperidinemethanol (MDL 11,939) on rabbit cortical 5-HT(2A) and 5-HT(2C) receptors. Repeated administration of DOI, LSD, or BOL decreased cortical 5-HT(2A) receptor density but had no effect on the density of cortical 5-HT(2C) receptors. Repeated administration of the selective 5-HT(2A) receptor antagonist MDL 11,939 significantly increased 5-HT(2A) receptor density. This unexpected outcome also occurred without any change in cortical 5-HT(2C) receptor density. The down-regulation of 5-HT(2A) receptors produced by chronic administration of BOL was associated with a decrease in DOI-elicited head bobs, whereas 5-HT(2A) receptor up-regulation produced by MDL 11,939 was associated with an increase in DOI-elicited head bobs compared with controls. These studies demonstrate that 5-HT(2A) receptor antagonists can both down- and up-regulate the density of cortical 5-HT(2A) receptors and these changes in receptor density have functional consequences for 5-HT(2A) receptor-mediated behaviors. Furthermore, because DOI, LSD, and BOL have approximately equal affinities for the 5-HT(2A) and 5-HT(2C) receptors, these results suggest that different mechanisms regulate 5-HT(2A) and 5-HT(2C) receptor density, in that chronic occupation of 5-HT(2C) receptors does not modulate their density in rabbit frontal cortex.

Effects of prenatal exposure to cocaine on the developing brain: anatomical, chemical, physiological and behavioral consequences.

Earlier studies of human infants and studies employing animal models had indicated that prenatal exposure to cocaine produced developmental changes in the behavior of the offspring. The present paper reports on the results obtained in a rabbit model of in utero exposure to cocaine using intravenous injections (4 mg/kg, twice daily) that mimic the pharmacokinetics of crack cocaine in humans. At this dose, cocaine had no effect on the body weight gain of dams, time to delivery, litter size and body weight or other physical characteristics of the offspring. In spite of an otherwise normal appearance, cocaine-exposed neonates displayed a permanent impairment in signal transduction via the D1 dopamine receptor in caudate nucleus, frontal cortex and cingulate cortex due to an uncoupling of the receptor from its associated Gs protein. This uncoupling in the caudate nucleus was shown to have behavioral consequences in that young or adult rabbits, exposed to cocaine in utero, failed to demonstrate amphetamine-elicited motor responses normally seen after activation of D1 receptors in the caudate. The cocaine progeny also demonstrated permanent morphological abnormalities in the anterior cingulate cortex due to uncoupling of the D1 receptor and the consequent inability of dopamine to regulate neurite outgrowth during neuronal development. Consistent with the known functions of the anterior cingulate cortex, adult cocaine progeny demonstrated deficits in attentional processes. This was reflected by impairment in discrimination learning during classical conditioning that was due to an inability to ignore salient stimuli even when these were not relevant to the task. The impairment in discrimination learning also occurred in an instrumental avoidance task and could be shown to be due to an impairment of cingulothalamic learning-related neuronal coding. It was proposed that the selective loss of D1-related neurotransmission in the anterior cingulate cortex prevented an appropriate activation of GABA neurons and thus a loss of inhibitory regulation that is necessary for processes involved in associative attention. Taken together, these findings suggest that the uncoupling of the D1 receptor from its G protein may be the fundamental source of the anatomic, cognitive and motor disturbances seen in rabbits exposed to cocaine in utero. Moreover, the long-term cognitive and motor deficits observed in the rabbit model are in agreement with the recent reports indicating that persistent attentional and other behavioral deficits may be evident in cocaine-exposed children as they grow older and are challenged to master more complex cognitive tasks.

Antagonist binding at 5-HT(2A) and 5-HT(2C) receptors in the rabbit: high correlation with the profile for the human receptors.

This study examined the binding of serotonin receptor antagonists at the 5-HT(2A) and 5-HT(2C) receptors of the rabbit's cerebral cortex. The 5-HT(2A) receptor was characterized by the binding of [3H]MDL 100,907 (R(+)-alpha-(2, 3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methan ol) to cortical membranes and the 5-HT(2C) receptor by the binding of [3H]mesulergine in the presence of the selective 5-HT(2A) receptor ligand spiperone. Both [3H]MDL 100,907 and [3H]mesulergine demonstrated high affinity binding to single sites in rabbit membranes. Based on Scatchard plots of [3H]MDL 100,907 binding, the mean B(max) was 8.5+/-0.7 fmol/mg tissue and the mean K(d) was 33. 1+/-3.5 pM. For [3H]mesulergine binding the mean B(max) was 3.70+/-0. 58 fmol/mg tissue and the mean K(d) was 0.35+/-0.05 nM. Binding of [3H]MDL 100,907 to the 5-HT(2A) receptor and of [3H]mesulergine to the 5-HT(2C) receptor was confirmed by displacement studies with highly selective 5-HT(2A) and 5-HT(2C) receptor ligands. The pharmacological profile of these ligands in rabbits correlated highly with published values for 5-HT(2A) (r=0.91, P<0.001) and 5-HT(2C) (r=0.94, P<0.001) receptors in humans. There was also a high correlation between the profiles for human and rat 5-HT(2C) receptor (r=0.92, P<0.001), but not for 5-HT(2A) receptors (r=0.53, P>0.10). It was concluded that the rabbit provides an appropriate animal model for studies attempting to predict the pharmacology of human 5-HT(2A) and 5-HT(2C) receptors.

Selective attenuation of psychostimulant-induced behavioral responses in mice lacking A(2A) adenosine receptors.

A(2A) adenosine receptors are highly expressed in the striatum where they modulate dopaminergic activity. The role of A(2A) receptors in psychostimulant action is less well understood because of the lack of A(2A)-selective compounds with access to the central nervous system. To investigate the A(2A) adenosinergic regulation of psychostimulant responses, we examined the consequences of genetic deletion of A(2A) receptors on psychostimulant-induced behavioral responses. The extent of dopaminergic innervation and expression of dopamine receptors in the striatum were indistinguishable between A(2A) receptor knockout and wild-type mice. However, locomotor responses to amphetamine and cocaine were attenuated in A(2A) knockout mice. In contrast, D(1)-like receptor agonists SKF81297 and SKF38393 produced identical locomotor stimulation and grooming, respectively, in wild-type and A(2A) knockout mice. Similarly, the D(2)-like agonist quinpirole produced motor-depression and stereotypy that were indistinguishable between A(2A) knockout and wild-type mice. Furthermore, attenuated amphetamine- (but not SKF81297-) induced locomotion was observed in pure 129-Steel as well as hybrid 129-SteelxC57BL/6 mice, confirming A(2A) receptor deficiency (and not genetic background) as the cause of the blunted psychostimulant responses in A(2A) knockout mice. These results demonstrate that A(2A) receptor deficiency selectively attenuates psychostimulant-induced behavioral responses and support an important role for the A(2A) receptor in modulating psychostimulant effects.

Effects of prenatal cocaine exposure on amphetamine-induced dopamine release in the caudate nucleus of the adult rabbit.

Acute amphetamine (AMPH) challenge has been used to probe the neurochemical and behavioral integrity of dopaminergic neurons under various conditions including prenatal cocaine exposure. In this study, we employed in vivo microdialysis to examine the effects of prenatal cocaine exposure on AMPH-induced dopamine (DA) release in the caudate nucleus of the awake adult rabbit. Pregnant rabbits were given intravenous injections of either saline or cocaine (4 mg/kg) twice a day from gestational day 8 (G8) through G29. Microdialysis was performed in adult saline and cocaine progeny at approximately postnatal day 70 (P70). There were no significant differences between cocaine and saline progeny in their basal concentrations of DA or its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). AMPH (5 mg/kg, i.v.) significantly increased extracellular DA in the caudate of both groups. However, AMPH-induced DA release was 2 to 3-fold greater in cocaine progeny than in the saline controls. Although, DOPAC decreased in both groups following AMPH injection, there was no significant group effect. In addition, there were no significant changes in concentrations of HVA. AMPH is known to release DA by a mechanism of exchange diffusion via the presynaptic DA transporter (DAT). Therefore, we examined the binding of [(3)H]WIN 35,428 to membrane fractions prepared from fresh caudate tissue to determine whether prenatal exposure to cocaine had altered the density (B(max)) or affinity (K(d)) of the DAT. While the B(max) for [(3)H]WIN 35,428 binding increased 3-fold between P3 and P120, there were no significant differences between saline and cocaine progeny at any age examined. The K(d) for [(3)H]WIN 35,428 binding did not change with postnatal age and did not differ between cocaine and saline progeny. These findings suggest that prenatal exposure to cocaine produces a long-term increase in the size of the presynaptic, AMPH releasable, cytoplasmic pool of DA.

Influence of age and dietary restriction on norepinephrine uptake into cardiac synaptosomes.

The purpose of this study was to determine whether aging alters neuronal uptake of norepinephrine (NE) in the rat heart and if dietary restriction influenced the effect of age on this system. Cardiac synaptosomes were prepared from 6-, 12- and 24-month-old male F344 rats fed ad libitum (AL) or a diet restricted (DR) to 60% of AL intake. Cardiac synaptosomes were incubated with 50, 100, 200, or 400 nM [3H]NE for 10 min at 37 degrees C with and without desmethylimipramine (DMI), a selective neuronal-uptake blocker. DMI-sensitive [3H]NE uptake was calculated as the difference between samples with and without DMI. NE uptake was adjusted for the number of cardiac synaptosomes in each sample by dividing by the endogenous NE content in each sample. The Vmax for uptake ([3H]NE/min/ng NE) declined significantly between 6 and 12 months in AL rats and between 12 and 24 months in DR rats. Km was not significantly different between age or diet groups. The change in Vmax with age suggests that the number of NE transporters per synaptosome may decline with age and that DR delays this effect of age. There were no differences in the sensitivity to DMI between age or diet groups.

Infusion of the serotonin1B (5-HT1B) agonist CP-93,129 into the parabrachial nucleus potently and selectively reduces food intake in rats.

Unilateral infusion of the selective 5-HT1B agonist, CP-93,129 (3-(1,2,5,6-tetrahydropyrid-4-yl) pyrrolo[3,2-b]pyrid-5-one) into the parabrachial nucleus (PBN) of the pons reduced food consumption by rats. The hypophagia was dose-related (ED50 approximately 1 nmol) and associated with fewer observations of feeding and more periods of inactivity. Water intake, grooming and exploratory activity were unaffected. CP-93,129 also decreased food intake when injected into the hypothalamic paraventricular nucleus, but this action was 50-fold less potent than administration into the PBN. Autoradiography demonstrated 5-HT1B sites in the PBN; this binding was displaced by CP-93,129. The results implicate parabrachial 5-HT1B receptors in mediating serotonergic enhancement of satiation.

Harmaline competitively inhibits [3H]MK-801 binding to the NMDA receptor in rabbit brain.

Harmaline, a beta-carboline derivative, is known to produce tremor through a direct activation of cells in the inferior olive. However, the receptor(s) through which harmaline acts remains unknown. It was recently reported that the tremorogenic actions of harmaline could be blocked by the noncompetitive NMDA channel blocker, MK-801. This study examined whether the blockade of harmaline's action, in the rabbit, by MK-801 was due to a pharmacological antagonism at the MK-801 binding site. This was accomplished by measurement of [3H]MK-801 binding in membrane fractions derived from tissue containing the inferior olivary nucleus and from cerebral cortex. Harmaline completely displaced saturable [3H]MK-801 binding in both the inferior olive and cortex with apparent IC50 values of 60 and 170 microM, respectively. These IC50 values are consistent with the high doses of harmaline required to produce tremor, e.g., 10-30 mg/kg. Non-linear curve fitting analysis of [3H]MK-801 saturation experiments indicated that [3H]MK-801 bound to a single site and that harmaline's displacement of [3H]MK-801 binding to the NMDA receptor was competitive as indicated by a shift in Kd but not in Bmax. In addition, a Schild plot gave a slope that was not significantly different from 1 indicating that harmaline was producing a displacement of [3H]MK-801 from its binding site within the NMDA cation channel and not through an action at the glutamate or other allosteric sites on the NMDA receptor. These findings provide in vitro evidence that the competitive blockade of harmaline-induced tremor by MK-801 occurs within the calcium channel coupled to the NMDA receptor. Our hypothesis is that harmaline produces tremor by acting as an inverse agonist at the MK-801 binding site and thus opening the cation channel.

Age-related changes in cardiac norepinephrine release: role of calcium movement.

The purpose of this study was to determine if the age-related decrease in norepinephrine (NE) release from cardiac adrenergic nerve terminals is due to a defect in Ca2+ movement into the nerve terminal or to an alteration in Ca2+ activation of intracellular events leading to NE release. NE release was assessed in cardiac synaptosomes prepared from 6- and 24-month-old male F344 rats. K(+)-induced NE release was significantly greater in young vs old rats. Raising extracellular [Ca2+] increased NE release, but NE release always remained higher in the younger animals. Ionomycin, a Ca2+ ionophore, induced NE release from cardiac synaptosomes, and there was no age difference in the response. The age-related reduction in NE release induced by K+ and the capacity of ionomycin to induce similar NE release in young and old cardiac synaptosomes points to a reduction in Ca2+ movement during depolarization.

Hypotensive stress retards associative learning in rabbits.

This study examined the effects of hypotensive stress on classical conditioning of the rabbit's nictitating membrane response. Hypotension, consisting of an approximately 45% decrease in blood pressure, was maintained for 30 min by the i.v. infusion of sodium nitroprusside. Twenty minutes later animals were exposed to a conditioning session consisting of 60 pairings of a 200 ms tone conditioned stimulus with a 100 ms airpuff unconditioned stimulus directed at the cornea. This procedure was repeated for four consecutive days. Animals exposed to the hypotensive stress demonstrated a significantly retarded acquisition of conditioned responses as measured by their frequency and onset latency as well as by an increase in the number of trials required to reach acquisition criteria of five and 10 consecutive conditioned responses as compared with controls. A separate group of animals received a nitroprusside infusion one day after the acquisition of conditioned responses to the tone conditioned stimulus. These animals demonstrated a normal retention of conditioned responses and a normal response to varying intensities of the conditioned stimulus. Hypotensive stress also had no effect on the frequency and topography of the unconditioned response. It was concluded that a decrease in blood pressure can serve as a physiological stressor. One of the reactions to this stress consists of a retardation in the formation of associations during a learning task, without any decrease in the ability to retrieve previously learned material.

Behavioral sensitization, behavioral tolerance, and increased [3H]WIN 35,428 binding in rabbit caudate nucleus after repeated injections of cocaine.

This study examined whether changes in the behavioral response to repeated intravenous injections of cocaine hydrochloride (4 mg/kg, twice daily for 22 days) might be related to alterations in the dopamine (DA) transporter as measured by the binding of the potent cocaine analog [3H]WIN 35,428 to membranes derived from fresh caudate tissue. Rabbits demonstrated both tolerance and sensitization. Tolerance occurred for cocaine elicited convulsions, whereas sensitization occurred to the ability of cocaine to elicit motor activity, facial twitches, and head bobbing. Cocaine-exposed animals demonstrated a significant 17% increase in the Bmax of specific [3H]WIN 35,428 binding to caudate membranes with no change in Kd. The increase in Bmax was observed at 42 but not 96 h after the last chronic cocaine administration. There was no change in [3H]WIN 35,428 binding at 42 h after a single injection of cocaine. We suggest that the upregulation of the dopamine transporter in the caudate nucleus reflected the mechanisms involved in tolerance rather than sensitization.

[3H]WIN 35,428 binding in the caudate nucleus of the rabbit: evidence for a single site on the dopamine transporter.

The binding of the potent cocaine analog, WIN 35,428 ((-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane 1,5-napthalenedisulfonate), was investigated in adult Dutch Belted rabbits by using membrane fractions prepared from fresh caudate nucleus. The consistent finding of this study was that [3H]WIN 35,428 binds to a single class of sites. For example: 1) kinetic analysis revealed that the rate of association and dissociation of [3H]WIN 35,428 was linear; 2) analyses of saturation experiments or homotopic displacement with cold WIN 35,428 by three separate methods statistically favored a one-site model; and 3) heterotropic displacement with drugs that bind to the dopamine (DA) transporter consistently yielded only a single class of binding sites as reflected by a complete displacement of [3H]WIN 35,428 and Hill slopes of approximately 1 (range, 0.89-1.06). The rank order of potencies (Ki) obtained in the competition experiments was: mazindol > nomifensine > (-)-cocaine > bupropion > (-)-norcocaine >> desipramine > DA > (+)-cocaine >> norepinephrine > citalopram > 5-hydroxytryptamine. The affinities of these drugs at the [3H]WIN 35,428 binding site was significantly correlated (r = 0.96, P < .001) with their potencies for inhibiting the uptake of DA, but not the uptake of norepinephrine or 5-hydroxytryptamine. Because [3H]WIN 35,428 binding was fully displaced by cocaine and the displacement was stereoselective, with (-)-cocaine being 200-fold more potent than (+)-cocaine, we conclude that [3H]WIN 35,428 was binding to a single cocaine site. Taken together, these findings indicate that [3H]WIN 35,428 binds to a single cocaine site on the DA transporter of the rabbit with a Kd of 3.2 +/- 0.4 nM and a maximum binding of 0.39 +/- 0.04 pmol/mg of caudate tissue.

Evidence that beta-endorphin is an agonist at bovine pineal delta-opioid receptors.

Since beta-endorphin is the putative endogenous ligand for epsilon-opioid receptors, the previous demonstration of saturable, high affinity beta-endorphin binding sites on bovine pineal membranes suggests the possible presence of epsilon-opioid receptors. To determine the identity of pineal beta-endorphin binding sites, the inhibition of [125I]beta-endorphin binding by ligands with varying affinities for epsilon-, mu-, delta- or kappa-opioid receptors was investigated. A high positive correlation was observed between the Ki values for these drugs to inhibit [125I]beta-endorphin binding to pineal membranes and for these drugs to bind to delta-opioid receptors but not to mu-, kappa- or epsilon-opioid receptors, demonstrating that in the pineal beta-endorphin binds to delta-opioid receptors. Both NaCl and a GTP analogue were potent inhibitors of [125I]beta-endorphin binding, providing evidence that beta-endorphin is an agonist at pineal delta-opioid receptors. These results suggest that endogenous bovine beta-endorphin may modulate pineal function.

Modulation of mu opioid binding by protein kinase inhibitors.

Several isoquinolinesulfonamide protein kinase inhibitors, including 1-(5-isoquinolinesulfonyl)-methylpiperazine dihydrochloride (H7), inhibited [3H]-[D-Ala2, MePhe4, Gly5-ol]- enkephalin (DAMGO) binding to rabbit cerebellar mu opioid receptors with Ki values similar to those reported for kinase inhibition by these compounds, suggesting that their mechanism of action may involve inhibition of protein kinase activity. However, since the binding assays were performed in the absence of exogenous ATP, it is unlikely that protein phosphorylation is taking place during the binding assay, making it improbable that H7 and its congeners inhibit DAMGO binding by inhibition of protein kinase activity. In support of this hypothesis, K252a, a structurally unrelated, broad spectrum protein kinase inhibitor, was inactive in modulating DAMGO binding, even at a concentration 5-fold greater than its Ki for inhibiting protein kinase activities. Inhibition of DAMGO binding through inhibition of kinase activity implies a noncompetitive or allosteric mechanism. Scatchard analysis of [3H]DAMGO binding combined with Schild analysis demonstrated that the inhibition of DAMGO binding by the isoquinolinesulfonamides was competitive. These results show that the isoquinolinesulfonamide protein kinase inhibitors directly interact with the mu opioid receptor. Thus, these compounds are unsuitable for the investigation of the potential role of protein phosphorylation in the modulation of mu opioid receptor binding.

Irreversible blockade of D2 dopamine receptors by fluphenazine-N-mustard increases D2 dopamine receptor mRNA and proenkephalin mRNA and decreases D1 dopamine receptor mRNA and mu and delta opioid receptors in rat striatum.

The consequences of irreversibly-inhibiting D2 dopaminergic receptors on the expression of D1 and D2 dopamine receptor mRNAs and proenkephalin mRNA and on the levels of mu- and delta-opioid receptors in rat striatum were studied following single or repeated administration of the irreversibly-acting D2 dopamine receptor antagonist, fluphenazine-N-mustard (FNM). The density of dopamine and opioid receptors was determined by receptor autoradiography and the levels of the mRNA for the D1 and D2 dopamine receptors and proenkephalin were measured by in situ hybridization histochemistry. Repeated treatment of rats with FNM for 6 days produced more than 80% inhibition of D2 dopamine receptors but less than 25% inhibition of D1 dopamine receptors. Repeated treatment with FNM also resulted in statistically significant increases in D2 dopamine receptor mRNA but decreases in D1 dopamine receptor mRNA. In contrast, acute treatment with FNM for 3 h had no significant effects on D1 or D2 dopamine receptor mRNAs in striatum. An examination of the effects of FNM on the opioid system showed that repeated treatment with FNM for 6 days produced more than a 2-fold increase in the expression of proenkephalin mRNA in striatum. This was accompanied by significant decreases in mu- and delta-opioid receptors in striatum, mainly by reducing the size of the patch compartment of striatum. Acute treatment with FNM for 3 h produced small increases in proenkephalin mRNA and mu-opioid receptors in striatum but had no significant effects on delta-opioid receptors. These results suggest that persistent inhibition of D2 dopamine receptors differentially regulates the expression of D1 and D2 dopamine receptor mRNA in striatum, and that the magnitude, duration and interval of inhibiting dopaminergic transmission may be important factors in regulating dopamine receptor mRNA expression. These results also suggest that D2 dopamine antagonists indirectly down-regulate opioid receptors by increasing the expression of proenkephalin mRNA, thereby increasing enkephalin which, in turn, decreases opioid receptors in striatum.

Characterization and localization of delta opioid binding sites in the bovine pineal gland.

Opioid binding sites in the bovine pineal were characterized using the highly selective delta opioid agonist 3H-[D-Pen2,pCl-Phe4,D-Pen5] enkephalin (DPDP(C1)E). Pineal membranes possess a single class of high affinity binding sites for this delta ligand (Kd = 0.26 nM; Bmax = 250 fmol/mg protein). The specific opioid antagonist naloxone dose dependently inhibited 3H-DPDP(C1)E binding, confirming that this ligand is indeed binding to opioid receptors. The delta selective ligands deltorphin and [D-Pen2,5]enkephalin (DPDPE) were much more potent than the mu selective compounds dermorphin and [D-Ala2,MePhe4,Gly5- ol]enkephalin (DAMGO) in inhibiting 3H-DPDP(C1)E binding. These results demonstrate that in bovine pineal membranes, DPDP(C1)E binds to delta opioid sites. Autoradiographic studies showed a uniform distribution of 3H-DPDP(C1)E binding over the bovine pineal in the sections we analyzed. This distribution suggests that delta opioid binding sites are associated with pinealocytes which account for the majority of cell types in the pineal. However, it is not possible to rule out that these receptors may also be associated with other cell types which are present in the bovine pineal. The density and widespread distribution of delta opioid receptors supports the hypothesis that endogenous opioid peptides directly modulate pineal function.

Evidence for an involvement of the mu-type of opioid receptor in the modulation of learning.

Pavlovian conditioning of the rabbit's nictitating membrane response was used to determine whether the retardation of learning produced by enkephalin derivatives was mediated through an action on mu- or delta-opioid receptors. Intraventricular injection of the highly selective mu-agonist, [D-Ala2, MePhe4, Gly-ol5] enkephalin (DAMGO) significantly retarded (2 nmol) or completely blocked (20 nmol) the acquisition of conditioned responses. The retarded or blocked acquisition of conditioned responses produced by DAMGO could still be detected when the rabbits were tested two days after cessation of drug injections, suggesting that DAMGO had affected learning and not simply performance of conditioned responses. Intraventricular injection of a comparable dose (20 nmol) of the highly selective delta-agonist, [D-Pen2,5] enkephalin (DPDPE) did not retard learning. A higher dose of DPDPE (200 nmol), which would be expected to act at both mu- and delta-receptors, did produce a retardation of acquisition that was comparable to the effects of a 2-nmol dose of DAMGO. However, unlike DAMGO, the retardation of learning produced by this dose of DPDPE appeared to be due, at least in part, to a reduction in the aversive properties of the unconditioned stimulus. The retardation of learning produced by 2 nmol of DAMGO and 200 nmol of DPDPE was due to an action on an opioid receptor because this effect was blocked by a subcutaneous injection of naloxone (2.5 mumol/kg), a specific opioid antagonist. The stereoselective effects of the mu-opioid agonist levorphanol further confirmed that the retarded learning was mediated through actions at the mu-opioid receptor. Autoradiographic studies indicated that a 2-nmol, intraventricular dose of DAMGO achieved contents greater than 1 pmol/mg in tissue lying within 2 mm of the lateral ventricle, third ventricle and cerebral aqueduct. These data suggest that enkephalins produce a retardation of learning through an effect on the mu-type of opioid receptor and that at least one locus of their actions is produced at periventricular sites lying within 2 mm of the ventricles.(ABSTRACT TRUNCATED AT 250 WORDS)