Dexamethasone inhibition of acute opioid physical dependence in vitro is reverted by anti-lipocortin-1 and mimicked by anti-type II extracellular PLA2 antibodies

Dexamethasone has been shown to inhibit opiate withdrawal, in an in vitro model. In this respect, we suggested that dexamethasone could reduce opiate withdrawal by blocking the release of prostaglandins' precursor, arachidonic acid through protein synthesis dependent-mechanisms (1). Since arachidonic acid is released by the enzyme phospholipase A₂ (PLA₂) in the present paper we evaluate whether dexamethasone effect may by related to inhibition of PLA₂ activity. Therefore, the effect of a neutralizing anti-lipocortin-1 antibody and a polyclonal anti-type II extracellular phospholipase A₂ antibody on the opiate withdrawal in vitro was considered. Following a 4 min in vitro exposure to morphine a strong contracture of guinea-pig isolated ileum was observed after the addition of naloxone. Dexamethasone at concentration of 5x10⁻⁵ M reduces of 50% morphine withdrawal and the polyclonal anti-type II extracellular PLA₂ antibody (in a dilution 1:1000) mimicked dexamethasone inhibitory effect. Incubation of the ileum preparation with a neutralizing anti-lipocortin-1 antibody (at a dilution of 1:10.000) 30 min before dexamethasone reverted the steroid effects. These results suggest that dexamethasone inhibition of opiate withdrawal is due to extracellular type II PLA₂ inhibition through lipocortin-1.     

Morphine withdrawal causes subsensitivity of adrenergic receptor response

To determine whether the hyperactivity of noradrenergic neurons that occurs during opiate withdrawal might be associated with a reciprocal alteration in noradrenergic receptor response, isoproterenol-stimulated adenylate cyclase activity was examined in the cerebella of morphine-addicted monkeys at two time periods: a) prior to withdrawal, and b) during late withdrawal. Compared to controls, the chronically-addicted group showed a significant increase in maximal enzyme velocity, a finding consistent with the observed hypoactivity of noradrenergic neurons which occurs during opiate administration. In contrast, the morphine withdrawal group demonstrated a significant decrease in enzyme activity. Piperoxan, known to mimic the effects of withdrawal in causing noradrenergic hyperactivity, also decreased enzyme activity. In no group was there a change in K_a for isoproterenol. These findings, indicating that opiate withdrawal is associated with a subsensitivity of noradrenergic receptor response, suggest a possible etiology for certain of the physiological changes of noradrenergic hypoactivity seen during the secondary (or protracted) abstinence syndrome.     

Spontaneous Withdrawal from Long-Term Treatment with Morphine Accelerates the Turnover of α2-Adrenoceptors in the Rat Brain: Up-Regulation of Receptors Associated with Increased Receptor Appearance

Abstract: The aim of this study was to quantify and compare the turnover of brain α₂-adrenoceptors during chronic morphine treatment and after spontaneous morphine withdrawal in rats. The oral administration of increasing doses of morphine (10–90 mg/kg) for 20 days did not alter the specific binding of the agonist [³H]clonidine in the cerebral cortex. However, spontaneous opiate withdrawal (24 h) significantly increased the density of cortical α₂-adrenoceptors (B_max for [³H]clonidine was 21% greater). The recovery of [³H]clonidine binding after irreversible inactivation by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (1.6 mg/kg) was assessed in naive, morphine-dependent, and morphine-withdrawn rats to study the process of α₂-adrenoceptor repopulation and to calculate receptor turnover parameters. The simultaneous analysis of receptor recovery curves revealed that the turnover of brain α₂-adrenoceptors in morphine-withdrawn rats was accelerated [appearance rate constant (r) = 21 fmol/mg of protein/day; disappearance rate constant (k) = 0.25 day^?1] compared with those in morphine-dependent (r = 13 fmol/mg of protein/day; k = 0.14 day^?1) and naive (r = 15 fmol/mg of protein/day; k = 0.16 day^?1) rats. Moreover, this analysis also indicated that the increased density of cortical α₂-adrenoceptors observed during morphine withdrawal was due to a significantly higher receptor appearance (Δr = 37–57%) and not to a decreased receptor disappearance, which in fact showed also an increase (Δk = 56–79%). It is proposed that the increased rate of α₂-adrenoceptor production in the brain of morphine-dependent rats during spontaneous withdrawal is most probably mediated by the overactivity of the adenylyl cyclase/cyclic AMP system induced by opiate addiction.     

Opiate Modulation of Striatal Dopamine and Hippocampal Norepinephrine Release Following Morphine Withdrawal

When opiates are abruptly withdrawn after chronic treatment, increases in hippocampal noradre-nergic function are observed which are accompanied by decreases in striatal dopamine release. The latter effects have to shown to persist for several weeks following the onset of opiate withdrawal. We examined the long-term effects of opiate withdrawal on 4-aminopyridine and potassium stimulated release of striatal dopamine and hippocampal norepinephrine. Tissue samples were obtained either from rats that had been exposed to opiate withdrawal following a seven day morphine infusion or sham treated control subjects. At 48 hours after the onset of withdrawal (cessation of morphine infusions), slices were loaded with [³H] neurotransmitter, washed extensively, and exposed to different drug treatments. 4-aminopyridine induced concentration related increases in striatal dopamine release, which was 36% calcium independent. Similar values for fractional release of striatal dopamine were obtained in morphine withdrawn and control subjects, for both potassium and 4-aminopyridine induced release. In addition, thresholds for 4-aminopyridine or potassium induced release of striatal dopamine did not differ between control and morphine withdrawn subjects. Treatment with 1.0 M morphine sulfate potentiated potassium evoked release of norepinephrine to an equal extent in both morphine withdrawn and sham treated hippocampal tissue. Exposure to a threshold concentration of potassium (8.0 mM), stimulated increased release of hippocampal norepinephrine in a significantly greater fraction of tissue samples obtained from morphine withdrawn animals. Although these results do not support changes in striatal dopamine release following opiate withdrawal, opiate mechanisms appear to be important determinants of in vitro hippocampal norepinephrine release.     

A novel analogue of clonidine with opiate-receptor agonist activity

A new analogue of the α₂-adrenergic receptor ligand clonidine, N-(4-hydroxphenacetyl)-4-aminoclonidine, was synthesized. The analogue possesses opiate-receptor agonist activity in addition to α-adrenergic partial agonist activity. The analogue elicits inhibition of adenylate cyclase of NG108-15 neuroblastoma × glioma hybrid cells; most of the inhibition is reversed by the opiate-receptor antagonist naloxone. The analogue also inhibits the binding of [³H]D-Ala²-Met⁵-enkephalinamide and [³H]dihydromorphine to rat brain opiate receptors. The structure of the analogue suggests common elements in the ligand binding sites of α- and opiate receptors and may lead to a new class of opiate analgesics.     

Aspects of opiate dependence in the myenteric plexus of the guinea-pig.

Myenteric plexus-longitudinal muscle strips prepared from tolerant/dependent guinea-pigs and continuously exposed to normorphine, display a contracture upon naloxone challenge. This phenomenon represents a sign of abstinence. Removal of the opiate by extensive washing resulted in the failure of naloxone to induce the abstinence sign, while the plexus still displayed considerable, although reduced, tolerance to morphine. Reexposure of withdrawn preparations to normorphine reinduced the ability to display the abstinence sign. Highly tolerant preparations exhibited a 30 fold increase in sensitivity to serotonin and prostaglandin E₁ when tested a few minutes after naloxone-precipitated withdrawal. Supersensitivity rapidly declined when normorphine was washed off the preparation, while reincubation of withdrawn tissues with the opiate resulted in reinduction of supersensitivity. The data confirms a close relationship between a state of tolerance and dependence (including display of the abstinence sign) and supersensitivity to putative neurotransmitters or neuromodulators, becoming evident following administration of naloxone.     

Behavioral profile of ψ-MSH: Relationship with acta and β-endorphin action

In view of the close structural similarity of the pro-opiocortin fragment γ-MSH and of ACTH/MSH type peptides, the behavioral profile of γ-MSH was explored. Attention was first focussed on behavioral procedures in which ACTH/MSH related neuropeptides have been found effective. Using different procedures to test avoidance behavior, it was found that γ-MSH and ACTH-like neuropeptides had opposite effects. In this respect the activity of γ-MSH resembles that of opiate antagonists rather than that of β-endorphin. Accordingly, ACTH_1–24-induced excessive grooming which is blocked by opiate antagonists, is attenuated by γ-MSH. In addition, γ-MSH injected into the periaqueductal gray matter of the brainstem of opiate naive rats elicited symptoms reminiscent of those seen after opiate withdrawal. γ-MSH attenuated more or less several effects of intracerebroventricularly administered β-endorphin (e.g. antinociception, hypothermia, α-MSH release) and decreased acquisition of heroin self-administration. Although γ-MSH at rather high doses displaced naloxone from its specific binding sites in brain homogenates, it did not interfere with β-endorphin-induced effects on in vitro muscle preparations (guinea pig ileum, rat rectum). Interestingly, γ-MSH induced relaxation of the rat rectum in vitro. It is postulated that γ-MSH may attenuate β-endorphin-induced effects by acting via γ-MSH receptor sites (functional antagonism), although a pharmacological antagonism cannot be excluded as yet.     

Interference with withdrawal signs of naloxone-induced opiate withdrawal under anesthesia is anesthetic-specific in opiate-dependent rats.

We hypothesized that interference of opiate antagonist-precipitated withdrawal signs under anesthesia is anesthetic-specific. Three groups of morphine-dependent rats were compared in different experimental conditions using a protocol of rapid withdrawal induction by an antagonist under anesthesia. We observed that ketamine and midazolam have different effects on the expression of withdrawal. This brings specific insights into the pharmacological basis of therapy with induction of opiate antagonist.     

Enlarged Cavum Septum Pellucidum as a Neurodevelopmental Marker in Adolescent-Onset Opiate Dependence

Objective

Adolescent-onset exposure to highly addictive substances such as opiates may induce far-reaching deleterious effects on later mental and physical health. However, little is known about the neurodevelopmental basis for adolescent-onset opiate dependence. Here we examined whether having an abnormally large cavum septum pellucidum (CSP), a putative marker of limbic structural maldevelopment, is associated with opiate dependence particularly beginning in adolescence.

Method

The overall length of the CSP and the prevalence of abnormal enlargement of the CSP were assessed and compared in 65 opiate-dependent subjects (41 adolescent-onset opiate users and 24 adult-onset opiate users) and 67 healthy subjects.

Results

Opiate-dependent subjects showed a greater prevalence of abnormal CSP enlargement relative to healthy subjects (odds ratio [OR]=3.64, p=0.034). The overall CSP length of adolescent-onset opiate-dependent subjects was greater, as compared not only with healthy subjects (F_1,104=11.03, p=0.001) but also with those who began opiate use during adulthood (F_1,61=4.43, p=0.039).

Conclusions

The current findings provide the first evidence that abnormal CSP enlargement, which reflects limbic system dysgenesis of neurodevelopmental origin, may be linked to later development of opiate dependence. In addition, a greater CSP length, which indicates more severe limbic abnormalities, appears to confer higher risk for earlier onset of opiate use.     

Effects of anaesthetic agents in interference of naloxone-induced opiate-withdrawal are dose-dependent in opiate-dependent rats

In opiate-dependent rats previous studies showed that anaesthetic agents, such as chloral hydrate, midazolam and ketamine interfere with naloxone-precipitated opiate withdrawal. Each anaesthetic induces a specific pattern of interference, indicating that the interference is agent-dependent. In order to further investigate these effects and highlight a potential pharmacological basis of opiate withdrawal interference through anaesthetic agents, we hypothesized that anaesthetic-mediated interference of opiate withdrawal is also dose-dependent. Three groups of rats were compared in an experimental procedure of rapid withdrawal induction by an antagonist under anaesthesia using sub-anaesthetic dosage of midazolam, ketamine or saline. We observed that sub-anaesthetic dosage of ketamine, or midazolam, interferes significantly with opiate withdrawal expression. This brings arguments in favour of a pharmacological basis underlying rapid antagonists induction in opiate dependent rats.     

Enhancement of opiate binding by various molecular forms of phosphatidylserine and inhibition by other unsaturated lipids

A study was undertaken on the possible involvement of phospholipids on stereospecific opiate binding to a rat brain membrane fraction comprised mainly of synaptic membranes. The addition of acidic phospholipids such as phosphatidylserine, phosphoinositides, and phosphatidic acid significantly enhanced opiate binding. With the exception of phosphatidylserine, when the acidic phospholipids contained a polyunsaturated acyl group, they were actually inhibitory, along with neutral phospholipids derived from brain. Both the C_18:0, C_18:1 form (derived from myelin) and the C_18:0, C_22:6 form of phosphatidylserine (derived from synaptic membranes) produced as much as a 45% enhancement in opiate binding. Unsaturated fatty acids were highly inhibitory, the degree of inhibition being related to the degree of unsaturation. Bot phospholipase A and C were inhibitory; and the inhibitory effect of A could not be prevented by albumin or overcome with the addition of phosphatidylserine. With the use of the cross-linking agent, dinitrodifluorobenzene, it could be demonstrated that the phosphatidylserine of synaptic membranes appeared to be preferentially associated with membrane protein. The enhancement of opiate binding by phosphatidylserine diminished with increasing degree of cross-linking.     

Naltrexone precipitated opiate withdrawal in methadone addicted human subjects: Evidence for noradrenergic hyperactivity

The noradrenergic alpha-2 receptor agonist, clonidine, suppresses many of the effects of opiate withdrawal in both humans and other animals and this is consistent with the data indicating important interactions of opiate and noradrenergic systems in brain. This evidence supports the hypothesis that central noradrenergic hyperactivity is involved in the expression of major signs and symptoms of the opiate withdrawal syndrome, but to date clinical studies have not provided biochemical data consistent with this idea. In order to assess whether naltrexone precipitated opiate withdrawal in methadone addicted human subjects is associated with changes in noradrenergic function, a double-blind study was completed in which 15 methadone-dependent subjects received naltrexone and 8 subjects received placebo. Signs and symptoms of the opiate abstinence syndrome increased significantly in the 15 subjects who received naltrexone compared with the 8 who received placebo. Plasma concentrations of free MHPG also increased significantly in those subjects who took naltrexone compared to the placebo treated subjects. In addition, withdrawal signs and symptoms were significantly correlated with plasma MHPG concentration. Since several compounds which suppress central noradrenergic activity also reduce the severity of the withdrawal syndrome, the noradrenergic hyperactivity suggested by the present clinical study may be a functional mechanism for at least part of the opiate withdrawal syndrome.     

Suppression of opiate withdrawal by cyclosporin A and dietary modification

It has been demonstrated in a murine model that a defined diet (Purina Basal Diet 5755) has immunosuppressive effects similar to cyclosporin A (CsA). It was also shown that CsA treatment in opiate dependent rats can attenuate the severity of opiate withdrawal. In this study, an opiate dependence model was established in Balb/c mice to assess the effects of the 5755 diet and CsA on morphine withdrawal - a CNS mediated phenomenon. Three groups of mice were used; a chow-fed control group (Purina 5008), a chow fed CsA treated group, and a group maintained on the 5755 diet. Morphine dependence was established by subcutaneous implantation of a 100 mg morphine base pellet under ether anesthesia. Seventy-two hours after pellet implantation, withdrawal was precipitated by a single injection of the opiate antagonist naloxone (2 mg/kg ip). Two indicators of withdrawal were assessed; jumping and diarrhea. The data demonstrated that both CsA and the 5755 diet resulted in significant attenuation of withdrawal symptoms with the 5755 diet being the most effective of the two. These findings suggest that immune modulation elicited by the 5755 diet and CsA treatment has a direct impact on the CNS opioid function.     

Neonatal animal models of opiate withdrawal

The symptoms of opiate withdrawal in infants are defined as neonatal abstinence syndrome (NAS). NAS is a significant cause of morbidity in term and preterm infants. Factors, such as polysubstance abuse, inadequate prenatal care, nutritional deprivation, and the biology of the developing central nervous system contribute to the challenge of evaluating and treating opiate-induced alterations in the newborn. Although research on the effects of opiates in neonatal animal models is limited, the data from adult animal models have greatly contributed to understanding and treating opiate tolerance, addiction, and withdrawal in adult humans. Yet the limited neonatal data that are available indicate that the mechanisms involved in these processes in the newborn differ from those in adult animals, and that neonatal models of opiate withdrawal are needed to understand and develop effective treatment regimens for NAS. In this review, the behavioral and neurochemical evidence from the literature is presented and suggests that mechanisms responsible for opiate tolerance, dependence, and withdrawal differ between adult and neonatal models. Also reviewed are studies that have used neonatal rodent models, the authors' preliminary data based on the use of neonatal rat and mouse models of opiate withdrawal, and other neonatal models that have been proposed for the study of neonatal opiate withdrawal.     

Failure of opiates to increase the hydrolysis of GTP in neuroblastoma-glioma 108-15 cells

It has been repeatedly demonstrated that the neuroblastoma-glioma (NG 108-15) cell line has opiate receptors that inhibit adenylate cyclase and it has been proposed that this inhibition is mediated by a naloxone reversible stimulation of a low K_m GTPase (Koski and Klee, Proc. Natl. Acad. Sci. 78:4185, 1981). The guanine nucleotides of NG cells were labeled with [³H]guanine followed by incubation with 10^?6M guanine. Etorphine (10^?6M] or vehicle were added and the incubations continued for 1–4 min. The reaction was stopped with 5 percent TCA containing nucleotides as carriers and markers for the HPLC. Marker nucleotides were detected at 254 nm and the labeled nucleotides by liquid scintillation spectrometry. In several experiments, etorphine failed to produce any measurable change in the labeled nucleotides or in the GTP/GDP ratios. To verify that the opiate receptors were functional we measured its capacity to inhibit the formation of cAMP induced by PGE₁. We also studied the effects of naloxone and PGE₁ on the formation of cAMP in opiate tolerant cells. Tolerant cells responded to naloxone with a 50 percent increase in cAMP, indicating again that the opiate receptors were functional. Our results are consistent with the idea that in intact NG108-15 cells the opiate-mediated hydrolysis of GTP observed in cell membrane preparations is of very small magnitude.     

beta-Endorphin: structure-activity relationships in the guinea pig ileum and opiate receptor binding assays.

The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of β_h-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of β_h-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.     

beta-adrenergic supersensitivity in rat brainstem during clonidine withdrawal

The clonidine withdrawal syndrome was studied in the rat by measuring β-adrenergic responses as isoproterenol stimulated cyclic 3′, 5′-Adenosine monophosphate accumulation in brain slices and β-adrenergic membrane receptors as [³H] dihydroalprenolol binding. Supersensitivity of cyclic AMP accumulation was evident in brain-stems of clonidine-treated animals 18 and 24 hours after the last dose, but not in cerebral cortex. In addition there was no indication of changes in either number or affinity of beta-receptors in brainstem. The similarity of these findings to changes in adenylate cyclase activity seen during opiate withdrawal is intriguing.     

Genetic association of a GABAA receptor γ2 subunit variant with severity of acute physiological dependence on alcohol

The ultimate goal of quantitative trait locus (QTL) mapping is to identify the genes affecting complex traits. Using animal models, we recently identified QTLs on mouse Chromosomes (Chrs) 1, 4, and 11 affecting genetic predisposition to acute alcohol withdrawal. Among mice derived from the C57BL/6J (B6) and DBA/2J (D2) inbred strains, the locus identified on Chr 11 (∼20 cM) accounted for 12% of the genetic variability in withdrawal liability. Candidate genes in proximity to this QTL encode the γ₂, α₁, and α₆ subunits of GABA_A receptors. The present studies identify a polymorphism between the B6 and D2 strains in the γ₂ subunit gene, Gabrg2, and expand genotypic analysis to their BXD recombinant inbred strains. This polymorphism predicts a difference in amino acid sequence (Ala-11 vs. Thr-11) within the extracellular amino-terminal region of the γ₂ subunit. Analysis using BXD strain means for acute alcohol withdrawal severity suggests that the γ₂ subunit polymorphism is genetically correlated with alcohol withdrawal severity. This is the first report that QTL mapping for an alcohol-related trait has successfully led to the identification of a polymorphic candidate gene product that is genetically associated with the trait. Received: 15 September 1998 / Accepted: 8 October 1998     

Growth factor withdrawal in combination with sodium butyrate addition extends culture longevity and enhances antibody production in CHO cells

The effect of growth factor (GF) and sodium butyrate (NaBu) on Chinese hamster ovary (CHO) cell growth, cell viability and antibody production was investigated using shaking flasks in GF-containing and GF-deficient medium containing 0, 1 and 3 mM NaBu. The withdrawal of GF and the addition of NaBu suppressed cell growth, but they significantly increased specific antibody productivity, q_Ab. Interestingly, the withdrawal of GF in combination with the addition of NaBu markedly retarded cell death, leading to extended culture longevity. For instance, at 3 mM NaBu, cell viability fell below 80% after day 4 in GF-containing medium, but it remained over 80% until day 18 in GF-deficient medium. Due to the enhanced q_Ab and the extended culture longevity, approximately 2-fold increase in total antibody production was achieved in pseudo-perfusion culture with 1 mM NaBu in GF-deficient medium, compared to the culture in GF-containing medium. The effect of GF and NaBu on the change in the expression and activity of cellular proteins, c-Myc, Bcl-2 and pyruvate dehydrogenase (PDH), was also investigated. Both the withdrawal of GF and the addition of NaBu decreased the expression of c-Myc. The expression of Bcl-2 was enhanced by the addition of NaBu in a dose-dependent manner while it was not affected by the withdrawal of GF. In addition, both the withdrawal of GF and the addition of NaBu reduced metabolic rates, q_Glc, q_Lac and Y_Lac/Glc, and increased PDH activity while not affecting PDH expression, suggesting that they may reduce the glycolytic rates, but enhance the conversion rates of pyruvate to TCA intermediates. Taken together, the withdrawal of GF in combination with the addition of NaBu can be considered as a relevant strategy for alleviating NaBu-induced cell apoptosis and enhancing antibody production since it can be easily implemented as well as enhance q_Ab and extend culture longevity.     

Effects of morphine on adenylate cyclase activity in lymphocytes of healthy subjects and patients with alcoholism and opiate addiction]

The morphine dose 10(-7) M had practically no effect on adenylate cyclase (AC) activity in lymphocytes of healthy controls (n = 20). The same dose of morphine had a pronounced stimulating effect on the AC activity in lymphocytes of alcoholics in withdrawal (n = 16). In the group of opiate addicts in withdrawal (n = 9) morphine had also a stimulating effect, which differed significantly from controls (33.7 +/- 13.8; P. 0.02). The range of fluctuation of morphine influence on AC activity during the first week of hospitalization was 162.9 +/- 33.0% in alcoholics and 30.4 +/- 4.6% in opiate addicts (P 0.01).