Maaja Sutak

Antinociceptive effects of intrathecally administered F8Famide and FMRFamide in the rat

The effects of intrathecal injections of F8Famide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2, 0.05-17.5 nmol) and FMRF-amide (Phe-Met-Arg-Phe-NH2, 0.002-25 nmol), known as anti-opioid agents, were investigated by using noxious thermal (tail flick) and mechanical (paw pressure) tests in the rat. Both peptides produced significant long-lasting (24-48 h) analgesia in both tests without causing detectable motor dysfunction. Pretreatment with systemic naloxone (5.5 mumol/kg i.p.) attenuated the initial antinociceptive effects (first hour) induced by both peptides (8.8 nmol) in the tail flick test and only by FMRFamide in the paw pressure test. A subeffective dose of F8Famide (0.05 nmol) enhanced both the intensity and the duration of spinal morphine (6.6 nmol) analgesia in both tests. In contrast, a subanalgesic dose of FMRFamide (0.002 nmol) decreased the intensity and enhanced the duration of the effect of morphine. These results show that, besides acting as antinociceptive agents in the spinal cord, F8Famide and FMRFamide could differentially modulate spinal opioid functions.

Blockade and reversal of spinal morphine tolerance by peptide and non‐peptide calcitonin gene‐related peptide receptor antagonists

This study examined the effects of the peptide CGRP receptor antagonist CGRP8‐37 and the newly‐developed non‐peptide CGRP receptor antagonist BIBN4096BS for their potential to both inhibit the development and reverse tolerance to the antinociceptive action of morphine. Repeated administration of intrathecal morphine (15 μg), once daily, produced a progressive decline of antinociceptive effect and an increase in the ED50 value in the tailflick and paw pressure tests. Co‐administration of CGRP8‐37 (4 μg) or BIBN4096BS (0.05, 0.1 μg) with morphine (15 μg) prevented the decline of antinociceptive effect and increase in ED50 value in the tailflick test. Intrathecal administration of the CGRP receptor antagonists did not alter the baseline responses in either tests. Acute CGRP8‐37 also did not potentiate the acute actions of spinal morphine. In animals rendered tolerant to intrathecal morphine, subsequent administration of CGRP8‐37 (4 μg) with morphine (15 μg) partially restored the antinociceptive effect and ED50 value of acute morphine, reflecting the reversal of tolerance. Animals tolerant to intrathecal morphine expressed increased CGRP and substance P‐like immunostaining in the dorsal horn of the spinal cord. The increase in CGRP, but not substance P‐like immunostaining, was blocked by a co‐treatment with CGRP8‐37 (4 μg). In animals already tolerant to morphine, the increase in CGRP but not substance P‐like immunostaining was partially reversed by CGRP8‐37 (4 μg). These data suggest that activation of spinal CGRP receptors contributes to both the development and expression of spinal opioid tolerance. CGRP receptor antagonists may represent a useful therapeutic approach for preventing as well as reversing opioid tolerance.

Comparative effects of cyclo-oxygenase and nitric oxide synthase inhibition on the development and reversal of spinal opioid tolerance.

This study examined the effects of the COX inhibitors, ketorolac and ibuprofen, and the NOS inhibitor L‐NAME for their potential to both inhibit the development and reverse tolerance to the antinociceptive action of morphine. Repeated administration of intrathecal morphine (15 μg), once daily, resulted in a progressive decline of antinociceptive effect and an increase in the ED50 value in the tailflick and paw pressure tests. Co‐administration of ketorolac (30 and 45 μg) or S(+) ibuprofen (10 μg) with morphine (15 μg) prevented the decline of antinociceptive effect and increase in ED50 value. Similar treatment with L‐NAME (100 μg) exerted weaker effects. Administration of S(+) but not R(−) ibuprofen (10 mg kg−1) had similar effects on systemic administration of morphine (15 mg kg−1). Intrathecal or systemic administration of the COX or NOS inhibitors did not alter the baseline responses in either tests. Acute keterolac or S(+) ibuprofen also did not potentiate the acute actions of spinal or systemic morphine, but chronic intrathecal administration of these agents increased the potency of acute morphine. In animals already tolerant to intrathecal morphine, subsequent administration of ketorolac (30 μg) with morphine (15 μg) partially restored the antinociceptive effect and ED50 value of acute morphine, reflecting the reversal of tolerance. Intrathecal L‐NAME (100 μg) exerted a weaker effect. These data suggest that spinal COX activity, and to a lesser extent NOS activity, contributes to the development and expression of opioid tolerance. Inhibition of COX may represent a useful approach for the prevention as well as reversal of opioid tolerance.

Role of opioid receptors in the spinal antinociceptive effects of neuropeptide FF analogues

1 Neuropeptide FF (NPFF) has been shown to produce antinociceptive effects and enhance morphine‐induced antinociception after intrathecal (i.t.) injection. In this study, the spinal effects of two NPFF analogues, [D‐Tyr1, (NMe)Phe3]NPFF (1DMe) and [D‐Tyr1, D‐Leu2, D‐Phe3]NPFF (3D), which are resistant to degradation and exhibit a high affinity for NPFF binding sites, were examined in tests of thermal and mechanical nociception. 2 1DMe and 3D produced potent dose‐dependent spinal antinociception in the tail‐flick test. On a molar basis, 1DMe was 20 and 50 times more potent than 3D and morphine, respectively, and high doses of 1DMe and 3D produced a sustained antinociceptive effect without visible signs of motor impairment. 3 Spinal antinociceptive effects produced by 1DMe (0.86 nmol) or 3D (8.6 nmol) were significantly reduced by i.t. co‐administration of naloxone (11 nmol) or i.t. pre‐administration of D‐Phe‐Cys‐Tyr‐D‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP, 9.25 nmol) or β‐funaltrexamine (β‐FNA, 2 nmol) or naltrindole (2.2 nmol). The doses of the μ‐antagonists (CTOP and β‐FNA) or the δ‐antagonist (naltrindole) used in 1DMe and 3D experiments blocked the antinociceptive effects of μ‐or δ‐receptor‐selective agonists. 4 When administered in combination with antinociceptive doses of the μ‐receptor agonist, morphine (13.2 nmol) or the δ‐receptor agonist, [D‐Ala2]deltorphin I (20 nmol), sub‐effective dose of 1DMe or 3D (0.009 nmol) enhanced and prolonged the spinal effects of these opioid agonists. 5 The results of this study show that spinal μ‐and δ‐opioid receptors play a role in antinociception produced by NPFF analogues. These results also suggest a role for NPFF in modulation of nociceptive signals at the spinal level.

Augmentation of spinal morphine analgesia and inhibition of tolerance by low doses of μ- and δ-opioid receptor antagonists

Ultralow doses of naltrexone, a non‐selective opioid antagonist, have previously been found to augment acute morphine analgesia and block the development of tolerance to this effect. Since morphine tolerance is dependent on the activity of μ and δ receptors, the present study investigated the effects of ultralow doses of antagonists selective for these receptor types on morphine analgesia and tolerance in tests of thermal and mechanical nociception.

MODIFICATION OF BRAIN ACETYLCHOLINE RELEASE BY MORPHINE AND ITS ANTAGONISTS IN NORMAL AND MORPHINE‐DEPENDENT RATS

1 The spontaneous release of acetylcholine (ACh) from the cerebral cortex of control and morphine‐dependent rats was investigated. The rate of resting output of ACh in morphine‐dependent animals was lower than that in the control animals. 2 Administration of naloxone and nalorphine to morphine‐dependent rats was followed by a significant rise in the release of cortical ACh. In control rats no such increase in the release of ACh occurred after similar injections of narcotic antagonists. 3 Injections of morphine produced a consistent decrease in the rate of spontaneous release of cortical ACh in the control rats, but similar injections in the dependent rats did not produce a decrease in the rate of cortical ACh release. 4 The relevance of these results with regard to development of the narcotic abstinence syndrome is discussed.

The role of spinal neuropeptides and prostaglandins in opioid physical dependence

This study examined the role of spinal calcitonin gene‐related peptide (CGRP), substance P, and prostaglandins in the development and expression of opioid physical dependence. Administration of escalating doses (5 – 100 mg kg−1, i.p.) of morphine for 7 days markedly elevated CGRP and substance P− immunoreactivity in the dorsal horn of the rat spinal cord. Naloxone (2 mg kg−1, i.p.) challenge decreased both CGRP and substance P immunoreactivity and precipitated a robust withdrawal syndrome. Acute intrathecal pre‐treatment with a CGRP receptor antagonist, CGRP8 – 37 (4, 8 μg), a substance P receptor antagonist, SR 140333 (1.4, 2.8 μg), a cyclo‐oxygenase (COX) inhibitor, ketorolac (30, 45 μg), and COX‐2 selective inhibitors, DuP 697 (10, 30 μg) and nimesulide (30 μg), 30 min before naloxone challenge, partially attenuated the symptoms of morphine withdrawal. CGRP8 – 37 (8 μg), but no other agents, inhibited the decrease in CGRP immunoreactivity. Chronic intrathecal treatment with CGRP8 – 37 (4, 8 μg), SR 140333 (1.4 μg), ketorolac (15, 30 μg), DuP 697 (10, 30 μg), and nimesulide (30 μg), delivered with daily morphine injection significantly attenuated both the symptoms of withdrawal and the decrease in CGRP but not substance P immunoreactivity. The results of this study suggest that activation of CGRP and substance P receptors, at the spinal level, contributes to the induction and expression of opioid physical dependence and that this activity may be partially expressed through the intermediary actions of prostaglandins.

Intrathecal Clonidine: Analgesia and Effect on Opiate Withdrawal in the Rat

Clonidine, an α2, adrenergic agonist, has analgesic properties and recently has been used to suppress opiate withdrawal. These two properties theoretically make it a suitable analgesic substitute in patients tolerant to opioids. The objectives of this study were to see if intrathecal clonidine is analgesic and whether it can modify morphine withdrawal at the spinal level. Rats chronically implanted with catheters in the lumbar subarachnoid space were utilized. In analgesia experiments, intrathecal clonidine produced analgesia with the peak effect in the paw-lick test occurring at 200 nM, and in the tail-flick test analgesia was apparent at 100 nM and peaked at 400 nM (in 10 μL Ringers lactate). In dependency experiments, animals dependent on morphine (300 mg · kg−1) received intrathecal clonidine 25, 50, 200 nM in 10 μ1 Ringers lactate 72 h after morphine. Following this, a naloxone challenge, 3 mg · kg−1 was administered and withdrawal assessed. Clonidine-treated animals showed significant weight loss and decrease in temperature, and those treated with high doses showed marked hypothermia and hind-limb flaccidity. Intrathecal clonidine prevented the hyperalgesia associated with opiate withdrawal but did not affect the occurrence of the majority of behavioral signs (e.g., piloerection, irritability) associated with morphine withdrawal. Intrathecal clonidine prevented the naloxone-induced increase in blood pressure during withdrawal and in animals not treated with morphine-produced hypotension. Thus, intrathecal clonidine is analgesic, and part of the antiwithdrawal action of clonidine may be exerted at the spinal level.

Analgesia and tolerance to intrathecal morphine and norepinephrine infusion via implanted mini-osmotic pumps in the rat☆

&NA; The objectives of this study were to investigate the duration of analgesia and the development of tolerance following continuous intrathecal administration of morphine and norepinephrine alone, and morphine followed by norepinephrine via mini‐osmotic pumps in the rat. Analgesia was assessed by the tail‐flick test. In single pump experiments morphine 1 &mgr;l (10 &mgr;g)/h (7 days) and 0.5 &mgr;l (10 &mgr;g)/h (14 days) produced analgesia with tolerance by days 5–7. Norepinephrine 1 &mgr;l (15 &mgr;g)/h (7 days) produced analgesia equivalent to that of morphine with tolerance developing by day 3. Following continuous intrathecal morphine 1 &mgr;l (10 &mgr;g)/h for 5 days, norepinephrine 1 &mgr;l (15 &mgr;g)/h for 7 days failed to produce a significant increase in analgesia. This was in contrast to the increase in analgesia seen when the norepinephrine infusion followed a saline infusion. Determination of the norepinephrine concentration in the solution from the osmotic pumps verified that the norepinephrine is stable for the treatment period.

ACTION OF ENKEPHALIN ANALOGUES AND MORPHINE ON BRAIN ACETYLCHOLINE RELEASE: DIFFERENTIAL REVERSAL BY NALOXONE AND AN OPIATE PENTAPEPTIDE

1 Methionine (Met)‐enkephalin, leucine (Leu)‐enkephalin and their synthetic analogues were tested for effects on the spontaneous release of cortical acetylcholine (ACh) in vivo. The ability of naloxone to reverse the action of enkephalins on ACh release was compared with its action against morphine. An enkephalin analogue, structurally related to Met‐enkephalin, was tested for opiate antagonistic activity in ACh release experiments. 2 Intraventricular administration of Met‐enkephalin, Leu‐enkephalin, d‐Ala2‐Met5‐enkephalinamide (DALA) and d‐Ala2‐d‐Leu5‐enkephalin (DALEU) produced a dose‐related inhibition of cortical ACh release. Met‐ and Leu‐enkephalin were very similar both in their potency and the time course of their action on ACh release. Both DALA and DALEU were more potent and had a longer duration of action than Leu‐enkephalin. Systemic injections of two pentapeptides, d‐Met2‐Pro5‐enkephalinamide and d‐Ala2‐MePhe4‐Met5(O)‐ol‐enkephalin (33,824), produced a sustained inhibition of cortical ACh release. 3 Naloxone, administered systemically following the depression of ACh release induced by either intraventricular injections of enkephalins (DALA or DALEU), or systemic injections of enkephalins (d‐Met2‐Pro5‐enkephalinamide or 33,824), reversed this depression and restored the release to baseline levels. The effect of d‐Met2‐Pro5‐enkephalinamide on the release of ACh was reversed by naloxone with difficulty. Naloxone also reversed the inhibitory effect of systemic morphine and this reversal was associated with a large overshoot of ACh release. The latter was never observed in the enkephalin experiments. 4 Intraventricular injection of the pentapeptide, d‐Ala2‐d‐Ala3‐Met5‐enkephalinamide (TAAPM), at doses that did not influence the basal ACh release, blocked or reversed the inhibitory effect of morphine on this release. This peptide did not block the effect of the non‐opiate, chlorpromazine, under similar conditions. In two experiments TAAPM failed to reverse the inhibition of ACh release produced by systemically injected enkephalin, D‐Met2‐Pro5‐ enkephalinamide. 5 Effects of morphine and enkephalin on ACh release are discussed in terms of their action on different opiate receptors.