Extracellular signal-regulated kinase (ERK) and nitric oxide synthase mediate intrathecal morphine-induced nociceptive behavior

Intrathecal (i.t.) administration of morphine at a high dose of 60nmol into the spinal lumbar space in mice produces a severe hindlimb scratching followed by biting and licking. Nitric oxide (NO) is thought to play an important role in signal transduction pathways that enhance nociceptive transmission in the spinal cord. The present study was designed to determine whether high-dose i.t. morphine could influence the activation of the extracellular signal-regulated kinase (ERK), a mitogen-activated protein (MAP) kinase in neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) activation. Both 7-NI and TRIM, selective inhibitors of nNOS, resulted in a dose-dependent inhibition of high-dose i.t. morphine-induced behavior. The selective iNOS inhibitor W1400 in relatively large doses inhibited in a non dose-dependent manner. The i.t. injection of morphine evoked a definite activation of ERK in the lumbar dorsal spinal cord. Behavioral experiments showed that U0126 (0.5-2.5nmol), a MAP kinase-ERK inhibitor, dose-dependently attenuated the behavioral response to i.t. morphine. In mice treated with high-dose morphine, 7-NI was very effective in blocking ERK activation, whereas W1400 had no effect. Taken together, these results suggest that the behavioral response to high-dose i.t. morphine may be triggered by the nNOS-ERK pathway in the dorsal spinal cord.     

Footshock- and psychological-stress prevent the development of tolerance to spinal but not supraspinal morphine.

The site of action involved in the suppression by exposure to footshock (FS)- and psychological (PSY)-stress of the development of antinociceptive tolerance to morphine has been investigated. Daily treatment with 10 mg/kg, s.c.; 3 micrograms, i.t.; and 5 micrograms, i.c.v. of morphine, regardless of the administration route, resulted in the development of tolerance. Daily exposure to FS- or PSY-stress suppressed the development of tolerance to s.c. and i.t. administered morphine but not that to i.c.v. administered morphine. Pretreatment with 2 mg/kg, i.p. of nor-binaltorphimine (nor-BNI) abolished the suppressive effect of PSY-stress on the development of tolerance to morphine given s.c. The suppression by PSY-stress was also antagonized by 2 micrograms, i.t. of nor-BNI and not by 2 micrograms, i.c.v. of nor-BNI. Thus, the development of tolerance in the spinal cord due to interaction of morphine at mu-opioid receptors can be suppressed by exposure to these stresses, probably through the descending signals from the supraspinal area, and activation of kappa-opioid receptors in the spinal cord could also participate in the suppression by PSY-stress.     

Chronic inhibition of intracellular Ca release or protein kinase C activation significantly reduces the development of morphine dependence

We have previously shown that chronic antagonism of metabotropic glutamate receptors in the brain attenuates naloxone-precipitated withdrawal symptoms in rats treated chronically with subcutaneous (s.c.) morphine. Several subtypes of metabotropic glutamate receptors are directly linked, through a guanine nucleotide regulatory protein, to the phosphatidylinositol (PI) second messenger system. In the present investigation, we assessed the effect of inhibiting the products of PI hydrolysis on the development of opioid dependence. Thus, concurrently with subcutaneous morphine, we infused intracerebroventricularly (i.c.v.) in rats, various doses of chelerythrine, which selectively inhibits the activation of protein kinase C, and thapsigargin, which inhibits the release of intracellular Ca²⁺ when given chronically. Both chelerythrine and thapsigargin reduced the severity of naloxone-precipitated abstinence symptoms when infused i.c.v. at a dose of 10 nmol/day. A single injection of either chelerythrine or thapsigargin immediately prior to the precipitation of withdrawal failed to decrease the severity of abstinence symptoms. Our results suggest that by chronically inhibiting activity of the phosphatidyl-inositol system, the development of morphine dependence can be attenuated.     

Chronic inflammatory pain does not attenuate the development of tolerance to chronic morphine in adult male rats

The overall impact of chronic pain on the response to opioids is ambiguous in the literature, and comparisons between human and animal studies are complicated by vast differences between the manner and dosage of opioids given to humans treated for pain in comparison to rodents as well as a lack of healthy participant studies examining the impact of chronic opioids. The purpose of this study was to evaluate the impact of chronic pain on the development of tolerance to morphine and to assess how the concentration of drug affects this process. Twenty-four hours after the injection of CFA or normal saline in the left hind paw, the level of mechanical hypersensitivity was assessed and animals were randomly assigned to a morphine dose (1, 3 or 8 mg/kg or saline). Morphine was administered by subcutaneous injection twice a day for 5 days. On Day 6, animals were challenged with a single dose of 3 mg/kg morphine prior to formalin testing. Evidence of tolerance was mixed, and the results varied widely among the conditions. Analysis of mean paw withdrawal thresholds indicated that the analgesic efficacy of subcutaneous morphine diminished following repeated dosing. The presence of the chronic inflammatory pain condition during the morphine dosing period produced an increase in formalin pain behaviors compared to saline controls, such that animals given any dose of morphine during the 5-day dosing period showed higher responding to formalin following the 3 mg/kg dose than animals that had received saline injections. These results indicate that chronic pain does influence the development of opioid tolerance, but it does not prevent this phenomenon from occurring as suggested by some researchers.     

The effects of FK506 on the development and expression of morphine tolerance and dependence in mice

FK506 is an immunophilin-binding ligand that inhibits calcineurin and decreases nitric oxide (NO) production in the nervous tissues. We examined the effects in mice of systemic treatment with FK506 on the induction and expression of morphine (s.c.) tolerance and dependence and compared them with the effects of the non-specific NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), and specific inducible NO synthase inhibitor, aminoguanidine. FK506 (0.5-10 mg/kg, s.c.) exerted inhibitory effects on both development and expression of tolerance to morphine-induced antinociception. FK506 also significantly decreased the expression of morphine dependence, as assessed by naloxone-precipitated (2 mg/kg, i.p.) withdrawal syndrome, but a similar effect was not found for the development of morphine dependence. A similar pattern of effects was observed with L-NAME (3-20 mg/kg, i.p.), while aminoguanidine (50-100 mg/kg, i.p.) did not alter tolerance or dependence. Examining the possible interaction between their inhibitory effects on tolerance and dependence, we combined the subeffective doses of FK506 (0.5 or 1 mg/kg) with L-NAME (3 mg/kg) or aminoguanidine (100 mg/kg). The combination of FK506 with L-NAME, but not with aminoguanidine, significantly decreased the development and expression of tolerance and expression of dependence. These data show the effectiveness of FK506 on morphine tolerance and dependence and suggest an additive effect between FK506 and the inhibition of constitutive NO synthesis in this regard.     

The angiotensin II-receptor antagonist losartan does not prevent hemodynamic or vascular tolerance to nitroglycerin.

Tolerance may involve increased production of angiotensin II. We tested the hypothesis that losartan would prevent the development of tolerance to continuous transdermal nitroglycerin (GTN). Twenty volunteers received losartan, 75 mg/day, or placebo in a randomized, double-blind, parallel fashion. After 1 week, continuous transdermal GTN, 0.6 mg/h, was given, in addition to losartan or placebo, to all volunteers for 1 week. Standing systolic blood pressure (SBP) and heart rate were measured, and forearm venous volume responses to sublingual GTN were evaluated. Measurements were made at baseline, after 1 week of losartan versus placebo, 3 h after initial therapy with transdermal GTN, and after 1 week of continuous transdermal GTN given in combination with losartan versus placebo. After sustained GTN therapy, SBP was unchanged from baseline in both groups, indicating that losartan did not prevent the development of tolerance. Tolerance also developed to the forearm venous volume responses and was not prevented by losartan. Therapy with an angiotensin II-receptor antagonist does not prevent the development of tolerance to continuous transdermal GTN.     

β-内酰胺类抗生素对吗啡耐受及依赖作用

为了研究头孢曲松及其类似物是否具有对抗吗啡耐受及依赖作用和头孢曲松对抗吗啡耐受的量效关系,本文采用小鼠热板舔足模型及纳洛酮戒断模型对头孢曲松、头孢拉定和头孢替安等6种β-内酰胺类抗生素进行观察,并与生理盐水对照组比较。100 mg.mL-1头孢曲松和头孢噻肟在吗啡形成耐受模型后第10天可能最大镇痛百分率(PMAP)分别为74.95%和65.94%,与生理盐水组(22.99%)比较具有显著性。100和50 mg.kg-1头孢曲松对戒断实验小鼠30 m in内平均跳跃次数为3.8和3.6,与对照组(20.9)比较具有显著性。结果表明,100 mg.kg-1头孢曲松和头孢噻肟对吗啡诱导的耐受和躯体依赖具有显著的抑制作用。头孢曲松具有剂量依赖性的抗吗啡耐受作用。     

Nitroglycerin inhibits the development of morphine tolerance and dependence in rats

The development of tolerance to and physical dependence on opioids remains a significant barrier to their clinical use. N-Methyl-D-aspartate (NMDA) receptor antagonists inhibit tolerance and dependence. However, many NMDA antagonists have undesirable side effects. It has been shown that nitroglycerin (NTG) can antagonize NMDA receptor activity. This study was designed to determine whether NTG could inhibit the development of morphine tolerance and dependence. Rats were anesthetized and implanted with either morphine or placebo pellets, and pumps infusing vehicle or NTG (doses from 0.1 microg/kg/day to 10 mg/kg/day). Tolerance development was assessed by tail-flick latency (TFL). After 6 days, withdrawal was precipitated by subcutaneous injection of 2 mg/kg naloxone. Withdrawal signs were observed for 15 min. Placebo-pelleted rats showed no changes in TFL over the course of the study and no withdrawal signs. Morphine-pelleted rats developed tolerance. The 0.1 mg/kg/day NTG dose significantly attenuated tolerance development, while the other doses had no significant effect. The 0.1 mg/kg/day dose also attenuated some withdrawal signs. Higher or lower doses were not effective, possibly because of competing biochemical effects.     

The importance of the number of NMDA receptors in the development of supersensitivity or tolerance to and dependence on morphine.

Opiate or NMDA receptor antagonists given during and/or after the development of tolerance and dependence have been reported to prevent these developments. In the present study, MK801 (dizolcipine) and naltrexone (NX), two antagonists of NMDA and opiate receptors, respectively were used in rats to find any correlations between changes in NMDA receptor kinetics, and the intensity of tolerance and dependence. Thus, six different groups of rats were formed. The rats in the groups were given saline (S)+S, S+morphine (M), NX+S, NX+M, MK801+S and MK801+M, respectively, once per day for 8 days. On day 9, the rats from each group were divided into four subgroups. The rats of the first subgroup were subjected to the determination of tail-flick latency. The rats of the second subgroup were administered 1 mg kg-1 naloxone (NL) 2 h after administration of 3 mg kg-1M. The rats of the third subgroup were implanted with two M pellets and after 72 h they were challenged with NL. The remaining rats received drugs also on day 9 according to the previous administration paradigm. Two hours after the administrations, their brains were utilised for the determination of NMDA receptor kinetics, employing [3H]glutamate. The measurement of tail-flick latency showed the prevention by NX or MK801 of the development of tolerance to M. The rats, which were administered 3 mg kg-1M 2 h before 1 mg kg-1 NL injection, on day 9 showed that only NX given previously along with M attenuated the intensity of the development of M dependence. NX administered alone intensified the development of dependence on a single dose of M. The development of M dependence upon the M pellet implantation was intensified by the previous administration of NX or MK801 concomitantly with M. The administration of M or MK801 alone, or NX together with M, caused significant upregulation of NMDA receptors. NX alone, and MK801 given concurrently with M led to a significant downregulation. So, in light of the previous findings and the present experimental data it can be said that: (1) supersensitivity to opioids may be a downregulation of NMDA as well as an upregulation of the opioid receptor; (2) either upregulation or downregulation of NMDA receptors may facilitate subsequent development of opioid dependence; (3) tolerance to opioid may necessitate both upregulation of NMDA receptors and downregulation of opioid receptors; and (4) beneficial effects of opioid antagonists in the treatment of opiate dependence and CNS injuries may be strongly related to the down regulation of NMDA receptors.     

Effects of nor-binaltorphimine on the development of analgesic tolerance to and physical dependence on morphine.

The effects of a highly selective kappa antagonist, nor-binaltorphimine (nor-BNI), on the development of tolerance to morphine analgesia and physical dependence on morphine were examined. Pretreatment with nor-BNI (5 mg/kg s.c.) 2 h prior to injection of morphine or a selective kappa agonist, U-50,488H, significantly antagonized the analgesic effect of U-50,488H, but not morphine analgesia in mice. The development of tolerance to morphine analgesia was significantly potentiated by pretreatment of mice with nor-BNI 2 h prior to morphine treatment during chronic morphine treatment for 5 days. Additionally, the pretreatment with nor-BNI during chronic treatment with the high dose of morphine for 5 days significantly potentiated the naloxone-induced body weight loss in morphine-dependent mice and rats. These findings suggest that inactivation of the kappa opioid system may potentiate the development of tolerance to morphine analgesia in mice and may aggravate the naloxone-precipitated body weight loss in morphine-dependent mice and rats.     

Oxytocin and a C-terminal derivative (Z-prolyl-D-leucine) attenuate tolerance to and dependence on morphine and interact with dopaminergic neurotransmission in the mouse brain

The effects of oxytocin (OXT) and of dipeptides derived from the C-terminal portion of oxytocin (Z-prolyl-leucine and Z-prolyl-D-leucine) on the development of acute and chronic tolerance to, and dependence on morphine were tested in the mouse. Oxytocin and the dipeptides attenuated the development of acute and chronic tolerance to the antinociceptive effect of morphine and delayed the onset of the naloxone-precipitated withdrawal syndrome. Both oxytocin and Z-prolyl-D-leucine affected drug-induced behavioural responses related to dopamine (DA) in the brain. Thus, oxytocin potentiated the hypermotility induced by a large dose of apomorphine and decreased the supersensitivity of the DA receptors. Small doses of Z-prolyl-D-leucine inhibited the hypomotility elicited by a small dose of apomorphine and potentiated the hyperactivity induced by amphetamine. The data indicate that both oxytocin and Z-prolyl-D-leucine affect tolerance to and dependence on morphine. While oxytocin interacts mainly with postsynaptic DA-ergic neuronal elements, the dipeptide primarily affects DA-ergic neurotransmission at the presynaptic level.     

Acrylonitrile-induced extracellular signal-regulated kinase (ERK) activation via protein kinase C (PKC) in SK-N-SH neuroblastoma cells

Acrylonitrile (ACN) is classified by IARC as a probable carcinogen. Chronic exposure to ACN increases the incidence of tumors in various organs of test animals, including the brain and lung. ERK1/2 activation plays crucial roles in cell proliferation and is involved in many steps of tumor progression. Therefore, this study examined whether ACN altered the activation state of ERK1/2 in human neuroblastoma SK-N-SH cells. Treatment of these cells with ACN greatly increased phosphorylation of ERK1/2 in dose- and time-dependent manners. This effect was inhibited by PD 98059 and U 0126, specific inhibitors of MEK, indicating that MEK, an upstream activator of ERK1/2, was directly involved in ACN-induced ERK1/2 activation. Furthermore, the activation of ERK1/2 by ACN was attenuated by inhibition of PKC with GF 109203X, rottlerin and prolonged incubation with PMA (phorbol 12-myristate 13-acetate). This demonstrated the participation of PKC in the ACN-stimulated activation of ERK1/2. Taken together, our results indicate that ACN-induced ERK1/2 activation involves PKC through a MEK-dependent pathway.     

Quercetin,a bioflavonoid,reverses development of tolerance and dependence to morphine

Quercetin, a bioflavonoid (25 and 50 mg/kg), when chronically administered for 9 days, failed to produce any significant change in tail flick latency as compared to control mice. However, repeated administration of quercetin (25 and 50 mg/kg) for 9 days attenuated the development of tolerance to the analgesic effect of morphine (10 mg/kg). Quercetin (25 and 50 mg/kg) also suppressed naloxone (2 mg/kg)‐precipitated withdrawal jumps on day 10 in morphine‐tolerant mice. Pretreatment of mice with quercetin (10–50, ip) significantly reversed the morphine (5 mg/kg)‐induced delay in gastric transit, whereas a higher dose of quercetin (100 mg/kg) did not have any significant effect on morphine‐induced delay in gastric transit. Quercetin per se had no effect on gastric transit. In conclusion, the results of the present study suggest potential use of quercetin in alleviating the adverse effects of opioid treatment. Drug Dev. Res. 57:167–172, 2002. © 2003 Wiley‐Liss, Inc.