Search

Although dopamine (DA) has been extensively implicated in the morphine-induced conditioned place preference (CPP; a measure of reward), noradrenaline (NA) and other systems may play a larger role than previously suspected. The mesolimbic DA system, comprised of projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), receives noradrenergic innervations from the nucleus tractus solitaries (NTS)-A2 cell group and is modulated by NA. The purpose of the present study was to evaluate the turnover of DA and NA in the NAc and the site-specific phosphorylation of TH in the NAc, VTA, and NTS on the CPP mice conditioned by morphine. A dose-effect curve for morphine-induced CPP (0.5-8 mg/kg, s.c.) was obtained using 6-day conditioning sessions followed by a CPP test. TH phosphorylation was determined by quantitative blot immunolabeling and immunohistochemistry using phosphorylation state-specific antibodies; NA and DA turnover was evaluated by high-performance liquid chromatography. Morphine-induced CPP phosphorylates TH at serine (Ser)40 but not Ser31 in NAc, which is associated with an enhanced of DA and NA turnover. We also found that morphine-induced CPP increased levels of TH phosphorylated at Ser31 and Ser40 in the NTS. The present study demonstrates that morphine-induced CPP might stimulate TH activity and accelerate DA and NA turnover in the NAc via a mechanism involving phosphorylation of TH.

Opioids are used in humans in the treatment of chronic osteoarticular pain, but the development of tolerance to the analgesic effects after continuous administration is still not well understood. The aim of the present study was to evaluate the expression of phospho-ERK 1/2 and phospho-p38 in mice with monoarthritis chronically exposed to morphine as a possible explanation for the development of tolerance. Inflammation was induced by intraplantar injection of complete Freund's adjuvant (CFA) and the tolerance by implantation of 75 mg morphine pellets. The results of the present study show that ERKs phosphorylation is unaltered by inflammation or morphine tolerance, each one individually, in the plantar tissue. In contrast, phospho-p38 is similarly decreased by inflammation or morphine tolerance. In naïve but not in tolerant animals, acute injection of morphine induces significant increase in phospho-p38 without any changes in phospho-ERK 1/2 expression. During inflammation, the acute injection of morphine induces a significant increase in the expression of ERK 1/2, but not in phospho-p38, in naïve animals. Phospho-ERK 1/2 expression was significantly decreased in the presence of inflammation plus tolerance. In contrast, no significant differences in phospho-p38 expression were observed between naïve and tolerant animals acutely injected with saline or morphine in presence of CFA inflammation. These results suggest that ERK but not p38 could be implicated in the development of morphine tolerance during peripheral inflammation. These experiments could contribute to establish the mechanisms implicated in the development of morphine tolerance in presence of inflammatory pain.

Our previous studies have shown that morphine withdrawal increases the hypothalamic-pituitary-adrenocortical axis activity, which is dependent on a hyperactivity of noradrenergic pathways (nucleus tractus solitarius-A(2)) innervating the hypothalamic paraventricular nucleus. The extracellular signal-regulated kinase has been implicated in drug addiction, but its role in activation of paraventricular nucleus and nucleus tractus solitarius during morphine dependence remain poorly understood. We have determined the activation of extracellular signal-regulated kinase during morphine dependence and withdrawal as well as its involvement in morphine withdrawal-induced gene expression. We show that naloxone-induced morphine withdrawal activates extracellular signal-regulated kinases(1/2) and increases c-Fos expression in rat paraventricular nucleus and nucleus tractus solitarius-A(2) neurons. Activated extracellular signal-regulated kinases(1/2) was colocalized with c-Fos in both nuclei, and this response was blocked by SL327, a drug that prevents extracellular signal-regulated kinase activation. In the paraventricular nucleus from morphine-withdrawn rats, the number of neurons expressing CRF was increased. Immunohistochemical study showed a dramatic increase in c-Fos immunoreactivity within CRF-positive cells. These results suggest that extracellular signal-regulated kinases1/2 signaling pathway is necessary for morphine withdrawal-induced activation of brain areas associated with the stress system.

We previously demonstrated that morphine withdrawal induced hyperactivity of the heart by the activation of noradrenergic pathways innervating the left and right ventricle, as evaluated by noradrenaline (NA) turnover and Fos expression. We investigated whether cAMP-dependent protein kinase (PKA) plays a role in this process by estimating changes in PKA immunoreactivity and the influence of inhibitor of PKA on Fos protein expression, tyrosine hydroxylase (TH) immunoreactivity levels and NA turnover in the left and right ventricle. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (5 mg/kg). When opioid withdrawal was precipitated, an increase in PKA immunoreactivity and phospho-CREB (cyclic AMP response element protein) levels were observed in the heart. Moreover, morphine withdrawal induces Fos expression, an enhancement of NA turnover and an increase in the total TH levels. When the selective PKA inhibitor HA-1004 was infused, concomitantly with morphine pellets, it diminished the increase in NA turnover and the total TH levels observed in morphine-withdrawn rats. However, this inhibitor neither modifies the morphine withdrawal induced Fos expression nor the increase of nonphosphorylated TH levels. The present findings indicate that an up-regulated PKA-dependent transduction pathway might contribute to the activation of the cardiac catecholaminergic neurons in response to morphine withdrawal and suggest that Fos is not a target of PKA at heart levels.

We previously demonstrated that morphine withdrawal induced hyperactivity of the heart by activation of noradrenergic pathways innervating the left and right ventricle, as evaluated by noradrenaline (NA) turnover and Fos expression. The present study was designed to investigate the role of protein kinase C (PKC) in this process, by estimating whether pharmacological inhibition of PKC would attenuate morphine withdrawal induced Fos expression and changes in tyrosine hydroxylase (TH) immunoreactivity levels and NA turnover in the left and right ventricle. Dependence on morphine was induced on day 8 by an injection of naloxone. Morphine withdrawal induced Fos expression and increased TH levels and NA turnover in the right and left ventricle. Infusion of calphostin C, a selective PKC inhibitor, did not modify the morphine withdrawal-induced increase in NA turnover and TH levels. However, this inhibitor produced a reduction in the morphine withdrawal-induced Fos expression. The results of the present study provide new information on the mechanisms that underlie morphine withdrawal-induced up-regulation of Fos expression in the heart and suggest that TH is not a target of PKC during morphine withdrawal at heart levels.

We previously demonstrated an increase in Fos expression in the heart during morphine withdrawal. In the present study we examined the role of beta- and alpha-adrenoceptors in naloxone-precipitated increases in Fos expression in the heart. Dependence on morphine was induced by 7-day chronic subcutaneous implantation of six morphine pellets (75 mg). Morphine withdrawal was precipitated by administration of naloxone (5 mg/kg subcutaneously) on day 8. Using immunohistochemical staining of Fos, the present results indicate that morphine withdrawal induced marked Fos immunoreactivity (Fos-IR) within the cardiomyocyte nuclei. Moreover, Western blot analysis revealed a peak expression of c-fos in the right and left ventricles after naloxone-precipitated withdrawal in parallel with an increase in noradrenaline (NA) turnover. In the second study, the effects of the administration of adrenoceptor antagonists on withdrawal-induced Fos expression in the heart were studied. Pretreatment with the beta antagonist, propranolol (3 mg/kg intraperitoneally) or alpha1-adrenoceptor antagonist, prazosin (1 mg/kg intraperitoneally) did not block the marked Fos-IR or the hyperactivity of catecholaminergic neurons observed in the heart during withdrawal. However, pre-treatment with alpha2-adrenoceptor antagonist, yohimbine (1 mg/kg intraperitoneally), 20 min before naloxone administration to morphine-dependent rats antagonized Fos expression and the enhancement of NA turnover in the heart. Collectively, these results suggest that noradrenergic neurons in the heart are active during morphine withdrawal, and that activation of transcriptional responses mediated by Fos are dependent upon cardiac alpha2-adrenoceptor.

Studies involving heart catecholaminergic systems in morphine-dependent rats have not established whether the adaptive changes observed in the heart during morphine withdrawal are mediated peripherally or centrally. In this study, naloxone (Nx), naloxone methiodide (NxM) and N-methyl levallorphan (NML), quaternary derivatives of Nx and levallorphan, respectively, that do not cross the blood-brain barrier, were administered to morphine-dependent rats and catecholamines and their metabolites determined in the right ventricle. Rats were made dependent on morphine by implantation of morphine pellets for 7 days. On day 8 animals received s.c. injections of saline, Nx (1 mg/kg), NxM (5 mg/kg) or NML (5 mg/kg) and were decapitated 30 min later. Noradrenaline (NA) and its metabolites normetanephrine (NMN) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) and dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were determined by high-performance liquid chromatography with electrochemical detection. After NxM or NML administration to morphine-dependent rats there was a pronounced increase in NMN and DOPAC levels, as well as in NA and DA turnovers (as estimated by NMN/NA and DOPAC/DA ratios, respectively) in the right ventricle. Similarly, giving Nx to morphine-dependent rats increased NMN and DOPAC levels and NA and DA turnovers. In addition, in the paraventricular nucleus of the hypothalamus (PVN) NA and DA turnover, measured as the MHPG/NA or DOPAC/DA ratios, increased after Nx administration but not after NxM or NML These results suggest that the changes in cardiac sympathetic activity observed during morphine withdrawal are due to intrinsic mechanisms outside the central nervous system. These data may be important for understanding the adaptive changes induced in the heart in subjects dependent on opioids.

This study evaluated the adaptive changes in noradrenergic neurons and the concomitant production of cAMP during morphine dependence and withdrawal in the right ventricle of the rat. Rats were made dependent on morphine by morphine pellet implantation for 7 days. On the day of sacrifice animals received an acute injection of saline or naloxone (1 mg/kg s.c.) and were decapitated 30 min later. Pretreatment with propranolol 15 min prior to naloxone was conducted to evaluate the possible implication of beta-adrenoceptors. The contents of noradrenaline and dopamine and their metabolites were examined. After naloxone administration to morphine-dependent rats (withdrawal) there was a pronounced increase in the content of normetanephrine and 3,4-dihydroxyphenylacetic acid and increased noradrenaline and dopamine turnover. In addition cAMP levels were increased after naloxone administration to morphine-treated rats. Propranolol did not block the hyperactivity of catecholaminergic neurons or the enhancement of cAMP observed in the heart during withdrawal. The present results indicate that heart catecholaminergic neurons play a significant role in the alterations in heart functions during morphine abstinence syndrome and suggest that those alterations are mediated through cAMP.

The changes in the content of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in discrete brain nuclei during chronic opioids administration have not been well established. We evaluated the effects of acute and chronic morphine administration on the content of CRF and AVP in different hypothalamic and extrahypothalamic (bed nucleus of the stria terminalis, BNST) nuclei in rats. Concomitantly, changes in hypothalamic noradrenaline (NA) turnover [estimated by the 3-methoxy-4-hydroxyphenylethyleneglycol MHPG/NA ratio] and in plasma corticosterone release (as a marker of the activity of the hypothalamus-pituitary-adrenal axis) were determined. Male rats were implanted with placebo (naïve) or morphine (tolerant) pellets for 7 days. On day 8, groups of rats received an acute injection of either saline i.p. or morphine (30 mg/kg i.p.) and were sacrificed 30 min later. Acute morphine injection to naïve rats increased both the release of corticosterone and the hypothalamic NA turnover. CRF and AVP showed no modifications in the paraventricular nucleus (PVN) or in the median eminence (ME). CRF content decreased in the ventromedian nucleus (VMN) and increased in the BNST, but did not change in the arcuate nucleus (AN). AVP was elevated in the supraoptic nucleus (SON) but not changed in the suprachiasmatic nucleus (SCN). In chronic morphine-treated rats, there was a pronounced decrease in the NA turnover and in the release of corticosterone, which indicates that tolerance develops to the acute effects of morphine. Correspondingly, CRF and AVP were enhanced in the PVN and decreased in the ME, when compared with naïve rats injected with morphine. CRF content was decreased in the AN and in the BNST, but increased in the VMN. The AVP content was decreased in the SON, and no modifications were seen in the SCN. The present study shows that, in addition to the modifications in corticosterone secretion and in hypothalamic NA turnover, chronic morphine administration produces a complex response in the CRF and AVP systems. These modifications might contribute to the behavioral, emotional and neuroendocrine alterations produced during opioid tolerance.

The influence of the L-type calcium channel antagonist nimodipine on both the activity of noradrenergic neurons projecting to the hypothalamus and the pituitary-adrenal response during morphine tolerance and withdrawal was analysed. Tissue concentration of hypothalamic noradrenaline (NA) and its metabolite 3-methoxy-4-hydroxy-phenylethylen-glycol (MHPG) were determined by high-pressure liquid chromatography. Plasma corticosterone concentration (a marker of pituitary-adrenal activity) was measured by radioimmunoassay. Rats rendered tolerant to morphine decreased hypothalamic MHPG concentration, and reduced hypothalamic NA turnover. Chronic infusion of nimodipine concurrently with morphine prevented the decrease in NA turnover during tolerance. After naloxone administration to tolerant rats we found a striking parallelism between an increased activity of the hypothalamic-pituitary-adrenal axis and an enhanced activity of noradrenergic neurons projecting to the hypothalamus. However, hypothalamic NA turnover and MHPG concentration, both elevated during withdrawal, returned to control levels in rats infused chronically with nimodipine, concomitantly with a reduction of the secretion of corticosterone. Taken together, these data indicate that increased noradrenergic neuronal activity in the hypothalamic nerve terminals is associated with the neuroendocrine morphine withdrawal syndrome and suggest that an up-regulated calcium system might contribute to the activation of these neurons.

The influence of the L-type Ca2+ channel modulators nimodipine (a Ca2+ blocker) and BAY K 8644 (a Ca2+ activator) on the expression of tolerance to the inhibitory effects of kappa- and mu-opioid agonists in the guinea-pig ileum from guinea-pigs rendered tolerant to the kappa-opioid receptor agonist U-50,488H was investigated. Tolerance to U-50,488H was induced by its administration (15 mg/kg twice a day) for 4 days. Control groups received saline at the same time schedule. Chronic infusion of guinea-pigs with nimodipine (2 micrograms/microliters/h for 7 days) or BAY K 8644 (0.5 microgram/microliters/h for 7 days), did not cause any modification of the height of contractions induced by electrical stimulation of the myenteric plexus-longitudinal muscle (MPLM) preparation from naïve guinea-pigs. The Ca2+ antagonist nimodipine increases the potency of U-50,488H (selective kappa agonist) to reduce the amplitude of neurogenic contractions of the MPLM strip in naïve animals, whereas the Ca2+ activator BAY K 8644 induced the opposite effect. However, the effect of DAMGO (selective mu agonist) was not modified in guinea-pigs infused with nimodipine or BAY K 8644. Tolerance to the inhibitory effects of both U-50,488H and DAMGO was observed following administration of U-50,488H for 4 days and was revealed as a rightward shift of the concentration-response curves for the two agonists. Chronic infusion of guinea-pigs with nimodipine concurrently with chronic U-50,488H, markedly attenuated the expression of selective tolerance to U-50,488H as well as the cross-tolerance between U-50,488H and DAMGO. By the contrary, the magnitude of tolerance to U-50,488H and to DAMGO was enhanced by concomitant infusion of BAY K 8644. The results suggest that, in the GPI, kappa-opioid receptor may be functionally linked to the dihydropyridine sensitive Ca2+ channel: The blockade of the channel increased whereas its activation reduced the potency of U-50,488H. In chronic experiments, nimodipine prevented the expression of tolerance to U-50,488H and the cross-tolerance between U-50,488H and DAMGO, whereas BAY K 8644 produced the opposite effect. These results suggest that, in the GPI, selective tolerance to kappa-agonist as well as cross-tolerance between kappa- and mu-opioid agonists would involve activation of L-type Ca2+ channels, which could indicate that intracellular Ca2+ may be the final common pathway through which myenteric neurons adapt to the chronic opioid exposure.