Autoradiographic evidence that prolonged withdrawal from intermittent cocaine reduces mu-opioid receptor expression in limbic regions of the rat brain.

Numerous reports support evidence that dopaminergic mesolimbic pathways interact with opioid systems to influence the reinforcing properties of cocaine. Withdrawal from chronic administration of cocaine in rats causes an upregulation of mesocorticolimbic mu-opioid receptors during early stages, but information about prolonged cocaine abstinence is lacking. We addressed this issue by treating rats with cocaine or saline (control) intermittently (1 mg/kg, i.v., every 12 min for 2 h daily) for 10 days followed by a 10- or 20-day withdrawal period. The animals were then decapitated and the brains removed for quantitative in vitro autoradiographic analysis of 14 brain regions with (125)I-DAMGO. A separate group of animals received two consecutive cycles of the 10-day cocaine/10-day withdrawal regimen. Only the group that participated in the two consecutive cycles showed a significant effect of treatment: downregulation of mu-opiate receptors in limbic cortical layer 3 (17% lower than saline-treated controls, P = 0.03), the core of the nucleus accumbens (16% decrease, P = 0.05), and the nucleus of the diagonal band (18% decrease, P = 0.05). The mu-receptor may manifest, as do other neural markers (e.g., dopamine transporter, dopamine efflux), a biphasic temporal pattern with upregulation during early phases of cocaine withdrawal but a downregulation at later times.

Chronic exposure to antibodies directed against anti-opiate peptides alter delta-opioid receptor levels.

The development of addictive states in response to chronic opioid use may be regulated partially by the release of endogenous peptides. These anti-opiate peptides (AOP) are secreted or released into the CNS and produce diverse actions that counterbalance the effects of prolonged opiate exposure. Though the mechanism(s) by which these peptides exert their physiological properties remain largely unknown, there is some indication that AOP's modulate opioid receptor levels. In this study, we investigated the effects of chronically infused alpha-melanocyte stimulating hormone (alpha-MSH), dynorphin(1-8) (DYN(1-8)), dynorphin A (DYNA), and NPFF antibodies on delta-opioid receptor expression in rat brains. Quantitative autoradiographic experiments revealed that antibodies directed against alpha-MSH and DYNA produced significant increases in delta receptor levels in the caudate, claustrum, and cingulate cortex of the rat brain. Conversely, NPFF monoclonal antibodies caused significant decreases in the caudate, nucleus accumbens, olfactory tubercle, and cingulate cortex. These results suggest that the density of delta-opioid receptors is affected by changes in the levels of the anti-opioid peptides in the extracelluar fluid in the rat brain.

The analgesic effects of tryptophan and its metabolites in the rat.

Male Sprague-Dawley rats weighing 150-200 g were given doses of tryptophan methyl ester or its metabolites; kynurenine sulphate, kynurenic acid, xanthurenic acid, quinolinic acid, anthranilic acid methyl ester or picolinic acid methyl ester. Doses administered intraperitoneally were 50, 100, 200, 300, 400 and 600 mg kg-1. Pain sensitivity was assessed using the hotplate and tailflick methods at 30 min before and at 30-min interval after the injection of test compounds. The administrations of tryptophan, kynurenic acid, quinolinic acid, anthranilic acid, xanthurenic acid, picolinic acid, and kynurenine were associated with analgesia. Animals given 300 or 600 mg kg-1 of tryptophan exhibited a significant decrease (P<0.05; P<0.01, respectively) in pain sensitivity with the hotplate test. l-Kynurenic acid (300 mg kg-1) produced analgesia (P<0.01) 30 min after drug administration. Quinolinic and anthranilic acids both produced prolonged decrease in pain sensitivity (P<0.05) using the tailflick test. These results indicate that tryptophan and some of its metabolites possess analgesic properties.

Regulation of mu binding sites after chronic administration of antibodies directed against specific anti-opiate peptides.

There is some indication that anti-opiate peptides (AOP) modulate opioid receptor systems by altering mu-receptor density. To further characterize this phenomenon, we investigated the effects of continuous infusion of anti-AOP IgG on mu binding sites in the brains of rats. Specifically, male Sprague-Dawley rats received intracerebroventricular (i.c.v.) infusions for 13 days of either control (rabbit) IgG or test IgGs: anti-dynorphin A IgG, anti-dynorphin A1-8 IgG, anti-alpha-MSH IgG, or the monoclonal anti-NPFF IgG. Administration of anti-NPFF IgG or the anti-dynorphin1-8 IgG significantly increased mu labeling by 40-70% in several brain regions at the caudate level. Contrary to these findings, anti-alpha-MSH IgG decreased (19-32%) [125I]-DAMGO labeling in several thalamic nuclei. The results suggest that the density of mu-opioid receptors is regulated in part by anti-opiate peptides in the extracellular fluid of the brain.

Downregulation of mu-opioid binding sites following chronic administration of neuropeptide FF (NPFF) and morphine.

The effect of continuous ICV infusion of NPFF (10 micrograms/microliter) and morphine (40 micrograms/microliter) on mu-opioid binding sites was examined in rats using the in vitro radiolabeled techniques of whole-brain homogenate receptor binding and quantitative autoradiography. Mu receptors were labeled with [3H][D-Ala2-MePhe4,Glyol5] enkephalin in the homogenate binding experiments and with [125I][D-Ala2-MePhe4,Gly-ol5]enkephalin in autoradiographic studies. In homogenate binding studies, chronic administration of NPFF or morphine significantly downregulated mu receptors by 20% and 44%, respectively. Quantitative autoradiographic experiments demonstrated downregulation of mu opioid receptors in specific brain nuclei for both NPFF- and morphine-treated animals. Within the striatum and several nuclei of the thalamus, the mu receptors of the NPFF- and morphine-treated animals were decreased by 20-30% and 38-73%, respectively. These results suggest that NPFF may modulate opioid-mediated responses in part by altering the density of mu-opioid receptors.

Studies of the biogenic amine transporters. VI. Characterization of a novel cocaine binding site, identified with [125I]RTI-55, in membranes prepared from whole rat brain minus caudate.

Previous studies showed that the cocaine analog [125I]RTI-55 labels dopamine and serotonergic (5-HT) biogenic amine transporters (BATs) with high affinity. Here we characterized [125I]RTI-55 binding to membranes prepared from whole rat brain minus the caudate nuclei. Paroxetine (50 nM) was used to block [125I]RTI-55 binding to 5-HT transporter sites. Initial experiments identified drugs that displaced [125I]RTI-55 binding with moderately low slope factors. Binding surface analysis of the interaction of 3 beta-(4-chlorophenyl)tropan-2 beta-carboxylic acid phenyl ester hydrochloride (RTI-113) and 3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid phenyl ester hydrochloride (RTI-122) with [125I]RTI-55 binding sites readily resolved two binding sites for [125I]RTI-55 with Kd values of 0.44 nM and 17 nM and Bmax values of 31 and 245 fmol/mg protein. Potent 5-HT and noradrenergic uptake inhibitors had low affinity for both sites. Whereas cocaine, CFT and WIN35,065-2 were 6.0-, 25- and 14-fold selective for the first site, benztropine, PCP and the novel pyrrole, (+-)-(2RS,3aSR,8bRS)-1,2,3,3a,4,8b-hexahydro- 2-benzyl-1-methylindeno-[1,2-b]pyrrole resorcylate [(+-)-HBMP, formerly called (+-)-RTI-4793-14], were moderately selective for the second site. A single binding site with the characteristics of site 1 was resolved using COS cells transiently expressing the cloned rat dopamine transporter. Lesion studies with 6-hydroxydopamine and 5,7-dihydroxytryptamine were conducted to test the hypothesis that site 1 and site 2 are physically distinct. The data showed that these neurotoxins differentially decreased [125I]RTI-55 binding to sites 1 and 2. The differential distribution of sites 1 and 2 in rat brain provides further support for this hypothesis. Viewed collectively, these data show that [125I]RTI-55 labels a novel binding site in rat brain membranes, termed DATsite2, which is not associated with the classic dopamine, serotonin or norepinephrine transporters.

Use of the neutrophil:lymphocyte ratio in the diagnosis of appendicitis.

In patients with right lower quadrant pain, the total white cell count is an unreliable predictor of appendicitis. It has been reported that the lymphocyte count can fall in acute appendicitis. This study was undertaken to investigate whether the neutrophil:lymphocyte ratio is a more sensitive indicator than the total leucocyte count. A retrospective study was performed of patients undergoing appendectomy for suspected appendicitis over a 2-year period. A total of 402 patients were identified; histopathology confirmed appendicitis in 367 (91%). Other significant pathology was found in 13 (3.2%). Twenty-two (5.5%) had a histologically normal appendix and recovered uneventfully with no other diagnosis being made. A total of 298 (79%) patients with appendicitis had an elevated preoperative total white cell count. The neutrophil:lymphocyte ratio was calculated for each patient. Using an upper limit of 3.5:1, it was found that 324 (88%) of patients with appendicitis had a ratio equal to or greater than this value. This was significantly different from the proportion with a raised total leucocyte count (P = 0.001). We suggest that the simple calculation of the neutrophil:lymphocyte ratio may provide a parameter that is more sensitive than the total leucocyte count in the prediction of appendicitis.

6 beta-[125iodo]-3, 14-dihydroxy-17-methyl-4, 5 alpha-epoxymorphinan ([125I]IOXY-AGO): a potent and selective radioligand for opioid mu receptors.

The recent cloning and expression of an opioid mu receptor has opened up new opportunities for research in opioid pharmacology. The relatively low level of transient receptor expression in COS cells emphasizes the need for radioligands with high specific activity and low nonspecific binding with which to label receptors. In addition, recent data indicating that agonists and antagonists bind to different domains on the same receptor protein indicate the utility of having both agonist and antagonist radioligands available for the study of opioid receptor mechanisms. Previous studies characterized the binding of the opioid antagonist 6 beta-[125iodo]-3,14-dihydroxy-17-cyclopropylmethyl-4,5 alpha-epoxymorphinan ([125I]IOXY) and showed that this naltrexone analog labels mu and kappa 2 receptors in rat and guinea pig brain with high affinity and low nonspecific binding. In the present study, we synthesized the agonist congener of IOXY, 6 beta-iodo-3,14-dihydroxy-17-methyl-4,5 alpha-epoxymorphinan. We named this novel agent IOXY-AGO for IOXY-agonist. Competition binding studies showed that IOXY-AGO has high affinity for mu receptors (Ki = 0.28 nM) and lower affinity for delta (Ki = 18.7 nM) and kappa 1 (Ki = 33.9 nM), kappa 2a (Ki = 38.4 nM) and kappa 2b (Ki = 58.2 nM) binding sites. IOXY-AGO was radioiodinated to a specific activity of 2,200 Ci/mmol. [125I]IOXY-AGO binding was rapid, readily reversible, and characterized by low nonspecific binding.(ABSTRACT TRUNCATED AT 250 WORDS)

RTI-4793-14, a new ligand with high affinity and selectivity for the (+)-MK801-insensitive [3H]1-]1-(2-thienyl)cyclohexyl]piperidine binding site (PCP site 2) of guinea pig brain.

[3H]TCP, an analog of the dissociative anesthetic phencyclidine (PCP), binds with high affinity to two sites in guinea pig brain membranes, one that is MK-801 sensitive and one that is not. The MK-801-sensitive site (PCP site 1) is associated with NMDA receptors, whereas the MK-801-insensitive site (PCP site 2) may be associated with biogenic amine transporters (BAT). Although several "BAT ligands" are known that bind selectively to PCP site 2 and not to PCP site 1 (such as indatraline), these compounds have low affinity for site 2 (Ki values > 1 microM). Here we demonstrate that the novel pyrrole RTI-4793-14 is a selective, high affinity ligand for PCP site 2. We determined the IC50 values of RTI-4793-14 and several reference compounds [PCP, (+)-MK801 and indatraline] for PCP site 1 (assayed with [3H](+)-MK801), PCP site 2 (assayed with [3H]TCP in the presence of 500 nM (+)-MK801) and a variety of BAT-related measures ([3H]CFT binding to the DA transporter, [3H]nisoxetine binding to the norepinephrine transporter, [3H]dopamine uptake, [3H]serotonin uptake). In addition, we determined the ability of RTI-4793-14 to block NMDA responses in cultured hippocampal neurons under voltage clamp. (+)-MK801 had high affinity for PCP site 1 (4.6 nM) and potently inhibited NMDA-induced responses, but was much less potent in the BAT-related measures (IC50 s > 10 microM). PCP had high affinity at PCP site 1 (IC50 = 92 nM) and PCP site 2 (IC50 = 117 nM), and was moderately potent in all BAT-related measures except [3H]nisoxetine binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of solubilized bradykinin B2 receptors from smooth muscle and mucosa of guinea pig ileum.

Bradykinin (BK) B2 receptors in guinea pig ileum were characterized in both membrane and soluble form. [3H]BK bound to a single class of sites with almost identical affinities in membranes prepared from the longitudinal muscle, circular muscle and mucosal layers of the ileum. The pharmacology of the binding in the distinct layers was indistinguishable. The detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) maximally solubilized nearly 80% of membrane binding activity in a very stable conformation. In soluble preparations, [3H]BK labeled a single class of sites but with about 10-fold lower affinity. The affinities of BK analogs in competition studies were similarly reduced. There was no difference in the pharmacology of the binding in soluble receptors prepared from the different layers of the ileum. The results show that the ileum is a good source of solubilized B2 receptors and that the receptors in the smooth muscle and the mucosa are very similar.

Bradykinin stimulation of phosphoinositide hydrolysis in guinea-pig ileum longitudinal muscle.

1. Bradykinin (BK)-induced contraction of ileal smooth muscle is assumed to be due to phosphoinositide hydrolysis but this has never been reported. We have investigated whether BK receptors are linked to this transduction mechanism in guinea-pig ileum longitudinal muscle and determined whether these receptors are equivalent to those labelled in [3H]-BK binding assays. 2. In membranes prepared from longitudinal muscle, [3H]-BK bound to a single class of sites with high affinity. Characterization of the binding with BK analogues indicated that the radioligand selectivity labelled a B2 type receptor. 3. BK significantly elevated tissue levels of [3H]-inositol phosphates in longitudinal muscle slices preincubated with [3H]-myo-inositol. The agonists potencies of BK, Lys-BK, Met-Lys-BK, Tyr5-BK and Tyr8-BK were in agreement with their relative potencies in the binding assay. The B1 receptor agonist des-Arg9-BK, did not stimulate inositol phosphate production. The response to BK was blocked by known B2 receptor antagonists but not by the B1 antagonist des-Arg9, Leu8-BK. 4. BK-induced phosphoinositide hydrolysis was unaffected by exposure of muscle slices to either atropine or indomethacin. 5. The results indicate that the B2 receptors linked to phosphoinositide turnover in ileal longitudinal muscle exhibit properties similar to those involved in contractile responses. Also, the receptor mediating the phosphoinositide response is likely to be that labelled in the [3H]-BK binding studies.

Altered brain cholinergic enzymes activity in the genetically obese rat.

Genetically obese male Zucker rats (fa/fa) and their lean littermates (Fa/-) were used in this experiment. Fourteen-week-old obese and lean littermates were sacrificed and choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) enzymes were assayed in specific brain regions. The assays of these enzymes indicate that obese animals had a significantly lower ChAT activity in the cerebellum, pons, and cerebral cortex and a significant increase in ChAT activity in the thalamus and hypothalamus. Meanwhile, the cerebral cortex, cerebellum, midbrain, thalamus and hypothalamus of the obese animals showed significantly higher AChE activity than their lean littermates. It was concluded from this study that obesity may be associated with changes in the enzymes of the brain cholinergic system.