Intrathecal delivery of a mutant micro-opioid receptor activated by naloxone as a possible antinociceptive paradigm.

Direct injection of double-stranded adeno-associated virus type 2 (dsAAV2) with a mu-opioid receptor (MOR) mutant [S4.45(196)A], and a reporter protein (enhanced green fluorescent protein) into the spinal cord (S2/S3) dorsal horn region of ICR mice resulted in antinociceptive responses to systemic injection of opioid antagonist naloxone without altering the acute agonist morphine responses and no measurable tolerance or dependence development during subchronic naloxone treatment. To develop further such mutant MORs into a therapeutic agent in pain management, a less invasive method for virus delivery is needed. Thus, in current studies, the dsAAV2 was locally injected into the subarachnoid space of the spinal cord by intrathecal administration. Instead of using the MORS196A mutant, we constructed the dsAAV2 vector with the MORS196ACSTA mutant, a receptor mutant in which naloxone has been shown to exhibit full agonistic properties in vitro. After 2 weeks of virus injection, naloxone (10 mg/kg s.c.) elicited antinociceptive effect (determined by tail-flick test) without tolerance (10 mg/kg s.c., b.i.d. for 6 days) and significant withdrawal symptoms. On the other hand, subchronic treatment with morphine (10 mg/kg s.c., b.i.d.) for 6 days induced significant tolerance (4.8-fold) and withdrawal symptoms. Furthermore, we found that morphine, but not naloxone, induced the rewarding effects (determined by conditioned place preference test). These data suggest that local expression of MORS196ACSTA in spinal cord and systemic administration of naloxone has the potential to be developed into a new strategy in the management of pain without addiction liability.

dsAAV type 2-mediated gene transfer of MORS196A-EGFP into spinal cord as a pain management paradigm.

We previously reported that mutations in the mu-opioid receptor (MOR), S196L or S196A, rendered MOR responsive to the opioid antagonist naloxone without altering the agonist phenotype. Subsequently, a mouse strain carrying the S196A mutation exhibited in vivo naloxone antinociceptive activity without the development of tolerance. In this study we investigated the possibility of combining the in vivo site-directed delivery of MORS196A and systemic naloxone administration as a paradigm for pain management. Double-stranded adenoassociated virus type 2 (dsAAV2) was used to deliver MORS196A-EGFP by injecting the virus into the spinal cord (S2/S3) dorsal horn region of ICR mice. MORS196A-EGFP fluorescence colocalized with some calcitonin gene-related peptide and neuron-specific protein immunoreactivity in the superficial layers of the dorsal horn 1 week after injection and lasted for at least 6 months. In mice injected with the mutant receptor, morphine induced similar antinociceptive responses and tolerance development or precipitated withdrawal symptoms and reward effects, similar to those in the control mice (saline injected into the spinal cord). Conversely, in the dsAAV2-injected mice, naloxone produced antinociceptive effects at the spinal level but not at the supraspinal level, whereas naloxone had no measurable effect on the control mice. Furthermore, the chronic administration of naloxone to mice injected with dsAAV2-MORS196A-EGFP did not induce tolerance, dependence, or reward responses. Thus, our current approach to activate a mutant receptor, but not the endogenous receptor, with an opioid antagonist represents an alternative to the use of traditional opioid agonists for pain management.

Prenatal morphine alters the synaptic complex of postsynaptic density 95 with N-methyl-D-aspartate receptor subunit in hippocampal CA1 subregion of rat offspring leading to long-term cognitive deficits.

Infants who are passively exposed to morphine or heroin through their addicted mothers usually develop neurobiological changes. The postsynaptic density 95 (PSD-95) protein, a submembranous cytoskeletal specialization, is dynamically linked with N-methyl-d-aspartate receptors (NMDARs) to form a synaptic complex in postsynaptic neurons. This complex serves important neurobiological functions, including mammalian learning and memory. However, the effects of prenatal morphine exposure on this synaptic complex are not well understood. In this study, we determined whether prenatal morphine exposure altered the synaptic complex association between PSD-95 and three major NMDAR subunits (NR1, NR2A, and NR2B), at the mRNA and protein levels, within the hippocampal CA1 subregion (an important integration area for mammalian learning and memory) of rat offspring along with the performance of long-term cognitive functions. Sprague-Dawley rat offspring from morphine-addicted mothers were studied at a younger age (postnatal day 14; P14) and at an older age (P45). Subsequently, an eight-arm radial maze task was applied to analyze the working and cued reference memory in such offspring (P45). The real-time polymerase chain reaction results showed that prenatal morphine exposure caused significant decreases in mRNA levels of the PSD-95 and three NMDAR subunits (NR1, NR2A, and NR2B) in offspring (P14 and P45). Similarly, at the protein level, immunoblotting showed that decreased whole levels of PSD-95 and NMDAR subunits were seen in offspring subjected with prenatal morphine. Furthermore, the protein interaction of the synaptic complex between the PSD-95 and NMDAR subunit, as indicated by coimmunoprecipitation, was less in prenatal morphine samples than in vehicle controls (P14 and P45). The prenatal morphine group also showed poorer performance for an eight-arm radial maze task than the vehicle-control group. These results are particularly important for a better understanding of certain opioid-mediated neurobehavioral cognitive changes in offspring associated with altered protein interaction between PSD-95 and NMDAR subunits within the developing brain.

Studies on quinazolines. 5. 2,3-dihydroimidazo[1,2-c]quinazoline derivatives: a novel class of potent and selective alpha 1-adrenoceptor antagonists and antihypertensive agents.

A series of 2-[(substituted phenylpiperazin-1-yl)methyl]- and 2-[(substituted phenylpiperidin-1-yl)methyl]-2,3-dihydroimidazo[1,2- c]quinazolin-5(6H)-ones or -5(6H)-thiones, and 3-[(substituted phenylpiperazin-1-yl)methyl]-2,3-dihydroimidazo[1,2-c]quinaz oline derivatives were synthesized, as conformationally restricted analogues of SGB-1534 and ketanserin, for evaluation as alpha-antagonists and antihypertensive agents. Most compounds containing a (substituted phenylipiperazinyl)methyl side chain displayed high binding affinity for alpha 1-adrenoceptor with no significant activity at alpha 2-sites. Compounds having a (substituted phenylpiperazinyl)methyl at the 3-position of 2,3-dihydroimidazo[1,2-c]quinazolin-5(6H)-one ring system had a better activity than those with the same substituent at the 2-position. Structure-activity relationships for alpha 1-adrenoceptor affinity are presented and indicate that compounds with substitution at the ortho position on the benzene ring of the phenylpiperazine side chain moiety are more potent than those without substitution and/or substitutions at the 3- and 4-positions. Computer-assisted superimposition of SGB-1534 and 20b showed little structural correspondence between the quinazolinone and 2,3-dihydroimidazo[1,2-c]quinazoline nucleus, and specific interactions of these molecular fragments with the receptor protein appear unlikely. Antihypertensive activity was evaluated via intravenous administration of each compound to spontaneously hypertensive rats, and compounds (16a, 16b, 20b, and 28b) illustrated similar efficacy to SGB-1534 when assessed after 6 h. The pA2 value for 16a against phenylephedrine in rat aorta was much higher than that of prazosin. On the basis of alpha 1-adrenoceptor affinity/selectivity in vitro and duration of antihypertensive action in vivo, compounds 20b and 28b warrant further evaluation.

Studies on quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides. 8.1, 2 synthesis and pharmacological evaluation of tricyclic fused quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides as potential alpha1-adrenoceptor antagonists.

A series of 2-substituted methyl 2,3-dihydroimidazo[1, 2-c]quinazolin-5(6H)-ones (4), 3-substituted methyl 2, 3-dihydroimidazo[1,2-c]quinazolin-5(6H)-ones (5), 3-substituted methyl 2,3-dihydro-5H-thiazolo[2,3-b]quinazolin-5-ones (15a,b), 3-substituted methyl 2,3-dihydroimidazo[2,1-b]quinazolin-5(1H)-ones (16a,b), 3-substituted methyl 2,3-dihydro-1H-imidazo[1,2-b][1,2, 4]benzothiadiazine 5,5-dioxides (33a,b), 2-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (42-45a,b), 3-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (50-53a,b), 3-substituted methyl 5H-thiazolo[2,3-b]quinazolin-5-ones (55-56a,b), and 3-substituted methyl 5-(methylthio)-2,3-dihydroimidazo[1,2-c]quinazoline (57) were synthesized as compound 1conformational rigid congeners for pharmacological evaluation as potential alpha1-adrenoceptor antagonists. Compounds 4, 5, 33a,b, 44a,b, 45a,b, 52a,b, 53a,b, and 57 were found to possess high affinity for the alpha1-adrenoceptor. Compounds 5 and 57 were the most highly selective and potent alpha1 antagonists with Ki = 0.21 +/- 0.02 and 0.90 +/- 0.08 nM, respectively. The S-enantiomers of these two compounds (Ki = 0.13 +/- 0.01 nM for (S)-(-)-5; Ki = 1.0 +/- 0.2 nM for (S)-(+)-57) were 144-200-fold more potent than the R-enantiomers (Ki = 26 +/- 8 nM for (R)-(+)-5; Ki = 144 +/- 23 nM for (R)-(-)-57). Compound 4 showed 8-fold higher affinity to alpha1A-AR better than alpha1B-AR. These compounds possessed weak to no activity against the 5-HT1A receptor.

Blockade of the development of morphine tolerance by U-50,488, an AVP antagonist or MK-801 in the rat hippocampal slice.

1. In this study, we investigated the effects of different drugs (a kappa-opioid receptor agonist U-50,488, a vasopressin receptor antagonist dPTyr(Me)AVP or an N-methyl-D-aspartate (NMDA) receptor antagonist MK-801) on the development of morphine tolerance in rat hippocampal slices. 2. Hippocampal slices (450 microm) of Sprague-Dawley rats (250-300 g) were used. Slices were continuously superfused with artificial CSF or drugs at 1 ml min(-1). Nichrome wire electrodes were placed in the Schaffer-collateral pathway and used to deliver biphasic 0.2 ms pulses of 5-30 V (0.033 Hz). A glass microelectrode was placed in the CA1 area to record population spikes. 3. When the slices were superfused with 10 microM morphine, the amplitude of population spikes increased 2-3 fold in 30-40 min. However, this effect of morphine decreased, i.e. tolerance developed after continuous superfusion of morphine for 2-6 h. 4. When either U-50,488 (200 nM) or dPTyr(Me) AVP (500 pM) or MK-801 (500 pM) was co-superfused with morphine (10 microM), it significantly blocked the development of morphine tolerance. Nor-BNI (a kappa-opioid receptor antagonist, 200 nM) significantly reversed the inhibitory effect of U-50,488 but not those of dPTyr(Me)AVP or MK-801 on the development of morphine tolerance. 5. These data indicate that kappa-opioid receptors, AVP receptors and NMDA receptors are all involved in the development of morphine tolerance. The suppression of kappa-opioid receptor activity after chronic morphine may occur before the activation of AVP receptors or NMDA receptors during the development of morphine tolerance.

Endomorphin-1 and -2 induce naloxone-precipitated withdrawal syndromes in rats.

In 1997, endomorphin-1 (EM-1) and -2 (EM-2) were identified as the most specific endogenous mu-opioid ligands. These two peptides have shown analgesic effects and many other opioid functions. In the present study, we attempt to investigate the possible ability of endomorphins to induce naloxone-precipitated withdrawal in comparison with that induced by morphine. Using the previously established scoring system in rats, 12 withdrawal signs (chewing, sniffing, grooming, wet-dog shakes, stretching, yawning, rearing, jumping, teeth grinding, ptosis, diarrhea, and penile erection) were observed and scored following naloxone (4 mg/kg, i.p.) challenge. Compared with the sham control, EM-1 and EM-2 (20 microg, i.c.v., b.i.d. for 5 days) both produced significant naloxone-induced withdrawal syndromes with similar severity to that induced by the same dose of morphine. There was no significant difference between EM-1, EM-2, and morphine-treated group for naloxone-induced withdrawal signs, except for grooming. EM-1 and EM-2 induced more grooming than that caused by morphine. Although EM-1 and EM-2 both led to the withdrawal, they displayed different potency for certain signs and suggest their distinct regulations. The present results indicate EM-1 and EM-2 could initiate certain mechanism involved opiate dependence.

Decrease in delta-opioid receptor density in rat brain after chronic [D-Ala2,D-Leu5]enkephalin treatment.

Chronic treatment of Sprague-Dawley rats with [D-Ala2,D-Leu5]enkephalin (DADLE) resulted in the development of tolerance to the antinociceptive effect of this opioid peptide. When opioid receptor binding was measured, time-dependent decreases in [3H]diprenorphine binding to the P2 membranes prepared from the cortex, midbrain and striatum were observed. Scatchard analysis of the saturation binding data revealed a decrease in Bmax values and no change in the Kd values of [3H]diprenorphine binding to these brain regions, indicative of down-regulation of the receptor. This reduction in the opioid receptor binding activities could be demonstrated to be due to the DADLE effect on the delta-opioid receptors in these brain regions. When [3H]DADLE binding was carried out in the presence of morphiceptin, a significant reduction in the delta-opioid receptor binding was observed in all brain areas tested. mu-Opioid receptor binding decrease was observed only in the striatum after 5 days of DADLE treatment. Additionally, the onset of delta-opioid receptor decrease in the midbrain area was rapid, within 6 h of the initiation of the chronic DADLE treatment. Thus, analogous to previous observations in which chronic etorphine treatment preferentially reduced mu-opioid receptor binding, chronic DADLE treatment preferentially reduced delta-opioid receptor binding activity.

Decrease in mu-opioid receptor binding capacity in rat brain after chronic PL017 treatment.

In previous studies, we have demonstrated that chronic treatment of rats with either etorphine or D-Ala2, D-Leu5-enkephalin (DADLE) resulted in the reduction of opioid receptor binding activities during the course of tolerance development. In both cases, mu-opioid receptor binding capacity was attenuated together with the delta-opioid receptor binding capacity. Because both etorphine and DADLE are relatively non-specific opioid ligands, interacting with both mu and delta receptors, these studies could not determine whether down-regulation of a specific receptor type is possible. Therefore, in the current studies, animals were rendered tolerant to the mu-opioid receptor-selective ligand PL017 and the receptor binding capacity was measured afterwards. Treating Sprague-Dawley rats with increasing doses of PL017 (2.5-20 micrograms/kg) i.c.v. for 5 days resulted in a 30- to 40-fold increase in the AD50 of the peptide to elicit the antinociceptive response and about 14-fold increase in the ED50 of the peptide to elicit the catatonic effect. When mu- and delta-binding was determined using [3H]diprenorphine in the presence of morphiceptin or DPDPE respectively, a significant decrease (20-30%) in the mu-opioid receptor binding but not in delta-opioid receptor binding was observed in all the brain areas tested after 5 days of PL017 treatment. Scatchard analysis of the [3H]DAMGO saturation binding data revealed a decrease in Bmax values and no change in the Kd values. Hence, mu-opioid receptors can be specifically regulated by ligand in the brain as delta-receptors are in neuroblastoma x glioma NG 108-15 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic morphine treatment causes down-regulation of spinal adenosine A1 receptors in rats.

Recent studies suggest that the release of adenosine in the spinal cord may be a significant component of the morphine antinociceptive action. We wanted to know whether the spinal adenosine system is involved in morphine tolerance. Animals were rendered tolerant to morphine, and A1 adenosine receptor binding activity was measured. Treating Sprague-Dawley rats with multiple, increasing doses of morphine i.p. for 6 days resulted in an about 10-fold increase in the median antinociceptive dose (AD50) of morphine to elicit an antinociceptive response. On the other hand, this treatment also caused a 4 to 5-fold increase in the AD50 of cyclopentyladenosine (CPA). When A1 adenosine receptor binding was determined by using [3H]cyclohexyladenosine ([3H]CHA) a significant decrease in binding (P less than 0.05) in the spinal cord but not in the cortex was observed. Scatchard analysis of the [3H]CHA saturation binding data revealed a decrease in Bmax values (from 185.5 fmol/mg to 110.2 fmol/mg) and no significant change in Kd values.

Effects of diuretics on thyroid function of guinea pigs.

Uptake of radioiodide by the isolated thyroid and serum concentrations of thyroxine (T4) and tri-iodothyronine (T3) were determined in guinea pigs pretreated with ethacrynic acid (20 mg/kg), furosemide (40 mg/kg) or hydrochlorothiazide (40 mg/kg). It was found that both ethacrynic acid and furosemide suppressed the 131I uptake by the isolated thyroid tissues. In addition, thyroid weight and serum T3 concentration were lower in ethacrynic acid-treated animals. It seems that some diuretics, particularly ethacrynic acid, depressed the function of thyroidal follicular cells.

U-50,488 blocks the development of morphine tolerance and dependence at a very low dose in guinea pigs.

U-50,488, (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide hydrochloride), is a selective kappa-opioid receptor agonist. In this study, we found that U-50,488 antagonized morphine-induced antinociception in morphine-naive guinea pigs at doses which did not have any antinociceptive effect by themselves (0.01-3 mg/kg). On the other hand, U-50,488 (3 mg/kg) partially restored morphine-induced antinociception in morphine-tolerant guinea pigs (8 mg/kg/day i.p. morphine HCl for 6 days). Furthermore, the development of tolerance to morphine antinociception was completely blocked by coadministration of U-50,488 at a very low dose (0.003 mg/kg i.p.) which neither exerted an antinociceptive effect by itself nor affected the antinociception induced by 8 mg/kg of morphine HCl. The withdrawal signs induced by 8 mg/kg (i.p.) naloxone HCl on the 7th day were also depressed by coadministration of 0.003 mg/kg U-50,488 with morphine HCl (8 mg/kg i.p.) every day for 7 days. These effects of U-50,488 could be applied to humans to prevent morphine tolerance and dependence.

Autoradiographic evidence for decrease in binding of mu- and delta-opioid receptors after subchronic [D-Ala2,D-Leu5]enkephalin treatment in rats.

Tolerance to the antinociceptive effect of [D-Ala2,D-Leu5]enkephalin (DADLE) developed in Sprague-Dawley rats given the peptide chronically. delta-Opioid receptor binding is significantly reduced in P2 membranes from various brain areas after 1-3 days' treatment. mu-Opioid receptor binding, however, is reduced only in striatum, and only after 5 days. To study this finding further, receptor autoradiography was used to quantify mu- and delta-opioid binding sites in rat brain sections after subchronic DADLE treatment. Autoradiograms were made following equilibrium binding of the highly selective opioid radioligands, [3H]Tyr-D-Ala-Gly-MePhe-Gly-ol ([3H]DAMGO) and [3H][D-Pen2,5]enkephalin ([3H]DPDPE) to brain sections. Computerized grain counting was applied to discrete regions of the autoradiograms corresponding to caudate and interpeduncular nuclei. We found that [3H]DAMGO binding decreased in caudate after 3 days of DADLE treatment and [3H]DPDPE binding decreased in the interpeduncular nucleus, rostral portion, after 1-day DADLE treatment. These autoradiographic changes are consistent with our earlier results for biochemical binding, although we now detected an earlier change in mu-opioid receptor binding, by using autoradiography.

Chronic intracerebroventricular administration of morphine down-regulates spinal adenosine A1 receptors in rats.

Previous studies from our laboratory have shown that systemic chronic morphine treatment causes down-regulation of spinal adenosine A1 receptors in rats. In this study, we further investigated whether supraspinal morphine treatment causes this effect. Adult male Sprague-Dawley rats were rendered tolerant to morphine by multiple intracerebroventricular (i.c.v.) injections for 2 or 4 days. Adenosine A1 receptor binding activities were measured with [3H]cyclohexyladenosine in the spinal cord and midbrain. A significant decrease in [3H]cyclohexyladenosine binding was found in the spinal cord but not in the midbrain region after 2 or 4 days of chronic i.c.v. morphine treatment. A decrease in the number of binding sites (Bmax) with no change in the affinity (Kd) of the ligand for the adenosine A1 receptor was observed. These results suggest that supraspinal morphine administration could cause the down-regulation of spinal adenosine A1 receptors and this may play a role in the mechanism of morphine tolerance.

Doridosine derivatives: binding at adenosine receptors and in vivo effects.

Doridosine, an adenosine analogue, causes, in vivo, hypotension, reduction of heart rates, muscle relaxation and anti-inflammatory effects through adenosine A1 and A2 receptors. A series of doridosine derivatives was synthesized in a search for compounds with more selective adenosine A1 receptor activity. These derivatives were characterized for binding to the respective adenosine receptors and for their cardiovascular effects. We used competition binding studies with highly selective radioligands: [3H]cyclohexyladenosine for adenosine A1 and [3H]CGS 21680 for adenosine A2 binding assays. The results for eight doridosine derivatives revealed that 1-cyclopropylisoguanosine (BN-063) and 1-allylisoguanosine (AZ-108-1) were more selective for the adenosine A1 receptor. In vivo, both BN-063 and AZ-108-1 caused significant bradycardia but no obvious effect on blood pressure. The bradycardia was almost completely blocked by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, a specific adenosine A1 receptor antagonist).

Chronic opioid treatment may uncouple opioid receptors and G-proteins: evidence from radiation inactivation analysis.

Radiation inactivation (target size analysis) was used in this study to determine whether uncoupling of opioid receptor and G-protein is a contributing mechanism to opioid tolerance. Male Sprague-Dawley rats (160-260 g) were rendered tolerant to morphine or [D-Ala2,D-Leu5]enkephalin (DADLE) by multiple i.p. or i.c.v. injections twice a day for 6 or 5 days. Control rats were injected with saline instead of opioids. The animals were killed, the midbrains excised and pooled together for each group. The washed P2 membranes were suspended in buffer and irradiated with 1-10 Mrad doses of 60Co irradiation, following which mu- or delta-opioid receptor binding activity of each sample was assayed. The molecular weight of the receptor was calculated from a standard irradiation curve constructed using several enzyme markers of known molecular weight. We found that the functional molecular size of mu-opioid receptor significantly decreased from 349 kDa to 228 kDa after 6 days of chronic morphine treatment, while, the molecular size of delta-opioid receptor decreased from 303 kDa to 223 kDa after 5 days of chronic DADLE treatment. These results are consistent with the uncoupling of opioid receptor from G-protein during chronic opioid treatment.

The role of nitric oxide in the development of morphine tolerance in rat hippocampal slices.

In the present study, we investigated the effects of a nitric oxide (NO) precursor, L-arginine, on the effect of different drugs, [trans-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid e hydrochloride] (U-50,488, a kappa-opioid receptor agonist); dPTyr(Me)AVP (a vasopressin receptor antagonist); dizocilpine (MK-801, a N-methyl-D-aspartate (NMDA) receptor antagonist), to block the development of morphine tolerance or NO release in Sprague-Dawley rat hippocampal slices (450 microm). Slices were continuously superfused with artificial cerebrospinal fluid (ACSF) or drugs at 1 ml/min. Nichrome wire electrodes were placed in the Schaffer-collateral pathway and used to deliver biphasic 0.2-ms pulses of 5-30 V (0.033 Hz). A glass microelectrode was placed in the CA1 area to record population spikes. The amount of NO released in the superfusate was measured as nitrite formation. When the slices were superfused with 10 microM morphine, the amplitude of population spikes increased 200%-300% in 30-40 min. However, this effect of morphine decreased, i.e., tolerance developed, after continuous superfusion of morphine for 2-6 h. On the other hand, the nitrite level was increased about 250% of the control level through 6 h of morphine superfusion. Co-superfusion of L-arginine with morphine could further increase the nitrite level and also facilitate the development of morphine tolerance. On the other hand, 3-Br-7-nitroindazole (a neuronal NO synthase inhibitor) decreased the nitrite level significantly and blocked the development of morphine tolerance. When either U-50,488 (200 nM) or dPTyr(Me)AVP (500 pM) or MK-801 (500 pM) was co-superfused with morphine (10 microM), the development of morphine tolerance was blocked significantly and the nitrite level decreased to 100%-150% of the control level. L-arginine (500 nM) significantly reversed the effect of these drugs to block the development of morphine tolerance or to decrease the nitrite level through 6 h of superfusion. These data suggest that NO may play a key role in the development of morphine tolerance. Drugs which suppress the synthesis or release of NO would be expected to block the development of morphine tolerance.

Chronic effect of [D-Pen2,D-Pen5]enkephalin on rat brain opioid receptors.

In previous studies, we have demonstrated that chronic etorphine or [D-Ala2,D-Leu5]enkephalin (DADLE) treatment of rats results in the reduction of mu- and delta-opioid receptor binding activities as tolerance develops. As both etorphine and DADLE are relatively non-specific opioid ligands, interacting with both mu- and delta-receptors, these studies could not determine whether down-regulation of a specific receptor type occurs. Therefore, in the present studies, animals were rendered tolerant to the delta-opioid receptor-selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE), and receptor binding activities were measured. Treating Sprague-Dawley rats with increasing doses of DPDPE (80-160-240-320 micrograms/kg) i.c.v. for 1 to 4 days resulted in a time-dependent increase in the AD50 of DPDPE to elicit an antinociceptive response. When delta-receptor binding was determined by using [3H]DPDPE, a 40-50% decrease in binding in the midbrain and cortex, and 25-35% decrease in binding in the striatum were observed after 3 or 4 days of DPDPE treatment. Scatchard analysis of the [3H]DPDPE saturation binding data revealed a decrease in Bmax values and no significant change in Kd values. To our surprise, when mu-receptor binding was determined by using [3H]Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO), a 10-15% decrease in binding was also observed in the midbrain and cortex after 4 days of DPDPE treatment. Our conclusion is that chronic DPDPE treatment preferentially reduces delta-opioid receptor binding activity. Its minor effect on the mu-opioid receptor maybe due to an interaction between delta cx and mu cx binding sites.

Immunohistochemical evidence of down-regulation of mu-opioid receptor after chronic PL-017 in rats.

In a previous study, mu-opioid receptor binding was decreased by chronic treatment of rats with a mu-opioid receptor-selective agonist [CH3Phe3, D-Pro4]morphiceptin (PL-017) [Tao, P.L., Lee, H.Y., Chang, L.R., Loh, H.H., 1990. Decrease in mu-opioid receptor binding capacity in rat brain after chronic PL-017 treatment. Brain Res. 526, 270-275]. However, there was a lack of correlation between the time course of receptor down-regulation and the loss of pharmacological effects of the drug. In the current study, we used immunohistochemistry to reinvestigate this issue. Male Sprague-Dawley rats were chronically treated with PL-017 i.c.v. for 1, 3 or 5 days, using an escalating dosage paradigm (0.75-6.0 microg), which resulted in a 1.4 to 32-fold increase in the AD50. Rat brains were removed, frozen, coronally sectioned (14 microm) and processed for mu-, delta- or kappa-opioid receptor immunohistochemistry by the avidin-biotin complex (ABC) method. Significant decreases in OP3 immunodensity were found in many brain regions which are enriched with OP3 after chronic treatment of PL-017. Time-dependent decreases in OP3 were detected and reached a plateau around 3 days of PL-017 treatment. No significant change in OP1 or OP2 immunodensity after chronic treatment with PL-017 was found. Our conclusion is that chronic treatment with PL-017 of rats selectively down-regulates mu-opioid receptors in the brain. This may be an important mechanism for PL-017 tolerance.

1-Methyl-4-phenyl-pyridinium (MPP+) uptake does not explain the differential toxicity of MPP+ in the nigrostriatal and mesolimbic dopaminergic pathways.

The present study examined the possible differential toxicity of 1-methyl-4-phenyl-pyridinium (MPP+) in the nigrostriatal and mesolimbic dopaminergic pathways in BALB/c mice, and investigated whether MPP+ uptake may account for this differential toxicity. Results indicated that chronic MPP+ infusions to the striatum (ST) and nucleus accumbens (NAc) both produced a toxicity on dopamine (DA) neurons. However, MPP+ produced a more severe DA depletion in the ST than in the NAc. Kinetic analyses from MPP+ uptake studies revealed a similar Km value in both the ST and NAc, suggesting that the affinity for MPP+ uptake is not different. The Vmax was approximately 1.6-fold higher in the ST than in the NAc. These results together suggest that chronic MPP+ infusions produce a differential toxicity on DA neurons in the nigrostriatal and mesolimbic dopaminergic pathways and that the ability of MPP+ uptake in DA terminals probably does not account for this differential toxicity.