Supraspinal and spinal cord opioid receptors are responsible for antinociception following intrathecal morphine injections.

BACKGROUND AND OBJECTIVE: The clinical practice of spinal morphine administration for pain relief is based on observations in animals that opioid receptors exist in the spinal cord and intrathecal injections of opioids in those species (mostly rats) lead to antinociceptive effects. Clinicians are well aware that administration of spinal opioids is associated with side-effects, such as nausea and respiratory depression, that indicate supraspinal spread of the drug administered. Those observations call into question how much of the observed pain relief is due to action of the drug in the brain. This study investigated the spinal cord actions of morphine given intrathecally to rats in a model that allows investigation of drug-receptor interaction at the spinal cord level. Experiments were performed on male Wistar rats with chronically implanted lumbar subarachnoid catheters. METHODS: Nociceptive thresholds were measured in rats given morphine intrathecally alone and in combination with intrathecal injections of selective opioid receptor antagonists: beta-funaltrexamine (mu), naltrindole (delta) and nor-binaltorphimine (kappa). RESULTS: Intrathecal morphine caused dose-related antinociceptive effects that were reversed totally by naloxone. Intrathecal beta-funaltrexamine and naltrindole did not reverse the effects of intrathecal morphine. However, intrathecal nor-binaltorphimine did reverse the electrical current threshold effects of morphine but not tail flick latency. CONCLUSIONS: Antinociception following intrathecal morphine involves spinal and supraspinal opioid receptors. The tail flick effect described in rat experiments involves actions at opioid receptors in the brain that override any action that may be caused by combination of morphine with mu-opioid receptors in the spinal cord.

Antinociceptive properties of neurosteroids: a comparison of alphadolone and alphaxalone in potentiation of opioid antinociception.

In this study, we investigated the antinociceptive and sedative effects of the opioids fentanyl, morphine, and oxycodone given alone and in combination with two neurosteroids: alphadolone and alphaxalone. An open-field activity monitor and rotarod apparatus were used to define the sedative effects caused by opioid and neurosteroid compounds given alone intraperitoneally to male Wistar rats. Dose-response curves for antinociception were constructed using only nonsedative doses of these drugs. At nonsedating doses, fentanyl, morphine, and oxycodone all caused dose-dependent tail flick latency (TFL) antinociceptive effects. Because neither neurosteroid altered TFL, electrical current was used as the test to determine doses of neurosteroid that caused antinociceptive effects at nonsedative doses. Alphadolone 10 mg/kg intraperitoneally caused significant antinociceptive effects in the electrical test but alphaxalone did not. All three opioid dose-response curves for TFL antinociception were shifted to the left by coadministration of alphadolone even though alphadolone alone had no effect on TFL. Alphaxalone given alone had no antinociceptive effects at nonsedative doses and it had no effect on opioid antinociception. Neither neurosteroid caused sedative effects when combined with opioids. We conclude that coadministration of alphadolone, but not alphaxalone, with morphine, fentanyl, or oxycodone potentiates antinociception and that this effect is not caused by an increase in sedation.

Antinociceptive role of 5-HT1A receptors in rat spinal cord.

BACKGROUND: Intrathecal administration of 5-hydroxytryptamine (5-HT) is antinociceptive to noxious heat and electrical stimuli. The contributions of different receptor subtypes to the antinociceptive effects of 5-HT are controversial. The main reasons for this are the poor receptor subtype selectivity of some agonist drugs and the difficulty of restricting drug action to the spinal cord in some experimental paradigms. This study investigated the roles of different 5-HT receptor subtypes involved in the spinal cord control of the nociception produced by these two nociceptive testing paradigms. METHODS: Tail-flick latency and electric current threshold for nociception were measured in an acute pain model that allowed the study of the antinociceptive effects of intrathecally administered drugs that were due to actions of these drugs at spinal cord receptors. Experiments were performed in male Wistar rats with chronically implanted lumbar subarachnoid catheters. Dose-response curves for spinally mediated antinociceptive effects of agonists selective for 5-HT receptor subtypes were constructed. RESULTS: The 5-HT1 agonist 1-(3-chlorophenyl)-piperazine dihydrochloride caused a dose-dependent antinociceptive effect, measured by both nociceptive tests. However, 8-hydroxy-DPAT (selective 5-HT1A agonist) produced antinociception assessed by electric current but not tail flick. A 5-HT1A-selective antagonist, 4-[3-(benzotriazol-1-yl)propyl]-1-(2-methoxyphenyl)-piperazine, reversed the antinociception in the electrical test produced by both of these agonists but the tail-flick latency effects after intrathecal 1-(3-chlorophenyl)-piperazine were not suppressed by this antagonist. CONCLUSIONS: We conclude that 5-HT1A receptors in the spinal cord are involved in the nociceptive mechanisms assessed by noxious electrical stimuli. Other 5-HT1 receptors (non 5-HT1A receptors) are involved in the spinally mediated antinociception assessed by thermal noxious stimuli.

Potentiation by ketamine of fentanyl antinociception. I. An experimental study in rats showing that ketamine administered by non-spinal routes targets spinal cord antinociceptive systems.

BACKGROUND: Ketamine has been found to exert antinociceptive effects in animals and to be analgesic at subanaesthetic doses in humans. This study was designed to investigate the involvement of spinal cord mechanisms in the potentiation of opioid analgesia by parenteral non-spinal administration of ketamine. METHODS: Thresholds for nociception were measured in an acute pain model in rats that allowed identification of antinociceptive effects due to drug action in the spinal cord. Dose-response curves for the antinociceptive effects of ketamine alone and ketamine in conjunction with the mu opioid fentanyl were constructed. RESULTS: Intraperitoneal ketamine up to 3.75 mg kg(-1) caused no sedative or antinociceptive effects and intrathecal ketamine caused dose-dependent, spinally mediated antinociceptive effects. Injections of ketamine doses that caused no antinociceptive effects when given alone (intrathecal 25 microg and intraperitoneal 3.75 mg/kg) significantly increased spinally mediated antinociception produced by intrathecal fentanyl injections when assessed using noxious heat (tail-flick test) but not when assessed by noxious electrical current (electrical current threshold test). CONCLUSIONS: We conclude that ketamine can potentiate the effects of fentanyl by an interaction at the level of the spinal cord even when ketamine is given via a non-spinal route of administration.

Antinociceptive properties of neurosteroids IV: pilot study demonstrating the analgesic effects of alphadolone administered orally to humans.

Fourteen patients scheduled for orthopaedic knee reconstruction surgery were enrolled in a prospective, double-blind, randomized study in which they received alphadolone (25-500 mg, n = 9) or placebo (lactose, n = 5) given orally 1 h after operation. All the subjects received a standardized general anaesthetic and the same type of surgery followed by physiotherapy using a continuous passive movement machine. Morphine was administered intravenously after operation by patient-controlled analgesia. Verbal rating and visual analogue scores assessed pain experiences for 6 h. Orally administered alphadolone up to 500 mg caused no increase in sedation, respiratory depression, nausea or vomiting. The experiences of these side-effects were all rated as none, mild or moderate. Orally administered alphadolone caused statistically significant reductions in morphine use and simultaneous highly significant reductions in pain scores. We conclude that alphadolone is a useful analgesic in humans when given by the oral route.

Antinociceptive properties of neurosteroids III: experiments with alphadolone given intravenously, intraperitoneally, and intragastrically.

The veterinary neurosteroid anaesthetic Saffan has the same formulation as Althesin now withdrawn from human use and is a mixture of two neurosteroids, alphadolone, and alphaxalone. The molecular structures of these two pregnanes and their properties as i.v. anaesthetics were reported to be similar. Preliminary experiments showed that alphadolone caused powerful antinociceptive effects without sedation when given i.p. In this study, alphadolone was given to rats (weight 100-200 g) i.v., i.p., and intragastrically. I.v. injections of alphadolone (25 mg kg(-1)) caused anaesthesia and sedation, whereas i.p. (0.1-100 mg kg(-1)) and intragastric administration (750 mg kg(-1)) produced no such effects. Intragastric alphadolone caused antinociceptive effects assessed with the electrical current threshold test (response 2.2 x pre-drug control values) without sedation. These effects were reversed at the level of the spinal cord by intrathecally-administered bicuculline (10 pmol). We conclude that a metabolite of alphadolone acetate produced in the liver leads to antinociceptive effects after i.p. and intragastric administration of the parent compound. This antinociception involves spinal cord GABA(A) receptors, even though the drug was administered via a non-spinal route.

Spinal and supraspinal components of opioid antinociception in streptozotocin induced diabetic neuropathy in rats.

This study investigated the antinociceptive effect of opioids given via intraperitoneal and intrathecal routes in a diabetes-induced neuropathic pain model in rats. Streptozotocin induced diabetes in 91% of juvenile male Wistar rats at the dose of 150 mg/kg (75 mg/kg intraperitoneal on 2 successive days). When compared with younger weight-matched saline treated rats, the diabetic rats developed hyperalgesia assessed by the paw pressure nociceptive test. Nociceptive thresholds and responses to fentanyl in all nociceptive tests in these younger normal rats were the same as those described previously for older normal rats. Fentanyl (10-100 microg/kg, i.p.) produced a dose-related antinociceptive effect in both neuropathic (n=6-8) and non-neuropathic (n=6-8) rats in electrical current, paw pressure and tail flick nociceptive tests. Higher doses of fentanyl were needed in neuropathic animals to achieve similar antinociceptive effects to those in non-neuropathic animals. Intrathecal injections of fentanyl (0.05-0.5 microg) in non-neuropathic rats, produced a spinally-mediated, dose-related antinociceptive effect assessed by all tests. In contrast, intrathecal administration of fentanyl that confined the drug action to the spinal cord produced little antinociceptive effect in neuropathic rats in all three tests. These experiments suggest that supraspinal mu opioid receptors are responsible for the antinociceptive effect of opioids in this model of neuropathic pain and that spinal cord opioid systems are in some way rendered ineffective for antinociception assessed with noxious heat, electrical and pressure stimuli.

Antinociceptive properties of neurosteroids I. Spinally-mediated antinociceptive effects of water-soluble aminosteroids.

Four water-soluble aminosteroid intravenous anaesthetic agents (ORG 20380, 20549, 21047 and 20599) were investigated for antinociceptive properties following intrathecal injection in rats. Two compounds, ORG 20380 and 20549, produced spinally-mediated antinociception assessed by tail flick and electrical current nociceptive tests. These effects were dose-related and suppressed by concurrent administration of the GABA(A) receptor antagonist, bicuculline. ORG 21047 and 20599 caused no antinociceptive effects when given intrathecally. Experiments in which nociceptive thresholds were measured after intravenous injections of ORG 20549 showed that subanaesthetic doses of this compound caused antinociceptive effects revealed by both nociceptive tests. This was equal in magnitude to that obtained with intrathecal administration of the same drug. We conclude that ORG 20380 and 20549 produce spinally-mediated antinociception by combination with spinal cord GABA(A) receptors. These spinal receptors are different from the GABA(A) receptors responsible for the anaesthetic effects of these drugs.

Antinociceptive properties of neurosteroids II. Experiments with Saffan and its components alphaxalone and alphadolone to reveal separation of anaesthetic and antinociceptive effects and the involvement of spinal cord GABA(A) receptors.

Studies have shown that the steroid anaesthetic alphaxalone positively modulates gamma-aminobutyric acid (GABA) receptors in vitro. It has also been reported that positive modulation of GABA(A) receptors in the rat spinal cord can produce antinociception in vivo. This present study looks at the interaction of an intraperitoneal injection (i.p.) of the steroid anaesthetic combination Saffan (alphaxalone 9 mg/ml, alphadolone acetate 3 mg/ml) with GABA(A) receptors in the spinal cord. Full recovery from anaesthesia induced by Saffan 2 ml/kg i.p., as assessed by the rotarod test, occurred after 28.78 +/- 0.86 min. Residual antinociceptive effects were assessed by application of electrical current at two skin sites (neck and tail) and also tail withdrawal from noxious heat. Residual antinociception was observed at both skin sites assessed by the electrical test but not when assessed by noxious heat. The antinociceptive effects in the tail but not the neck were suppressed by intrathecal administration of GABA(A) antagonists (bicuculline and SR-95531). In a separate group of experiments alphaxalone and alphadolone were given i.p. individually at the same doses that were given when formulated in Saffan. Alphaxalone produced sedative and anaesthetic effects with no antinociception. Alphadolone caused no sedation but it did cause antinociceptive effects equal in magnitude to those produced by Saffan. We conclude that Saffan produces antinociception in rats when given i.p. by an interaction with spinal GABA(A) receptors. Furthermore, this antinociception is due to the alphadolone content of the neurosteroid anaesthetic and not the alphaxalone.

Antinociceptive properties of propofol: involvement of spinal cord gamma-aminobutyric acid(A) receptors.

In this study, we investigated the interaction of propofol (a compound used widely as an intravenous anesthetic) with gamma-aminobutyric acid(A) (GABA(A)) and delta opioid receptors at the level of the spinal cord. Nociceptive thresholds were measured in rats through the use of electrical current testing (ECT) and tail-flick latency. Full recovery from sedation occurred 36.3 +/- 1.7 min (mean +/- S.E.M.; n = 20) after 40 mg/kg propofol i.p. Forty minutes after administration, there was residual antinociception when assessed by ECT but not when assessed by noxious heat. The ECT antinociceptive effects of propofol at tail but not neck sites were suppressed by intrathecal injection of the GABA(A) antagonists bicuculline and SR-95531 and the delta opioid antagonist naltrindole. These results suggest that there is an interaction between propofol and antagonists at receptors in the caudal segments of the spinal cord responsible for tail innervation. Antagonist dose-response curves were compared with those for suppression of intrathecal midazolam-induced antinociception. All intrathecal antagonists reversed the antinociceptive effect of propofol with the same dose-response curves as those previously obtained for suppression of the effect of intrathecal midazolam. We conclude that propofol, when given intraperitoneally, produces antinociception in rats through an interaction with spinal GABA(A) receptors. This combination leads to activation of a spinal cord system involving a delta opioid receptor; the same mechanisms involved with midazolam-induced spinal antinociception.

gamma-Aminobutyric acidA receptors and spinally mediated antinociception in rats.

Experiments were performed on rats with lumbar subarachnoid catheters, using four agonist drugs [gamma-aminobutyric acid (GABA), muscimol, midazolam and 5-hydroxytryptamine (5-HT)] and two GABA(A) antagonists (bicuculline and SR-95531) given intrathecally. All four agonists caused dose-related antinociception assessed by the electrical current threshold test. These effects were spinally mediated because the agonists caused increases in nociceptive thresholds in the skin of the tail and not the neck. In the same experiments, 5-HT and GABA caused simultaneous increases in tail-flick latency and electrical current thresholds in the tail. Both GABA(A) antagonists caused dose-related suppression of the antinociceptive effects of equieffective doses of all four agonists. Tail-flick latency increases caused by 5-HT were not suppressed by bicuculline in the same experiments in which bicuculline had suppressed the electrical current threshold effects of intrathecal 5-HT. The log dose-response curves for both antagonists for suppression of GABA effects were coincident, having a very shallow slope and covering the whole range of doses effective against the other agonists. The two GABA(A) antagonists were very different in relative potency for suppression of the spinally mediated antinociceptive effects of the other three agonists. The rank order of potency for bicuculline suppression of the effects of equieffective doses of the other agonists was muscimol > 5-HT > midazolam, whereas the rank order for SR-95531 was muscimol >> midazolam > 5-HT. We conclude that there exist in the spinal cord at least three different GABA(A) receptors responsible for spinally mediated antinociception caused by intrathecal injections of midazolam, muscimol and 5-HT. These are all targets for endogenous GABA.

The marine natural product 3,5-dibromo-2-(2,4-dibromo-phenoxy)phenol, inhibits contractile activity in the guinea-pig ileum.

1. The tetrabrominated diphenyl ether 3,5-dibromo-2-(2,4-dibromophenoxy)phenol (BPE), a natural marine product isolated from a sponge, was tested for pharmacological activity in guinea-pig ileum. 2. BPE (2 mumol/L) decreased basal force and the frequency of spontaneous contractions of the ileum. It also significantly decreased contractions of the ileum induced by 5 mmol/L barium and to electrical stimulation at parameters which stimulated intrinsic nerves. 3. The slopes of concentration-response curves to acetylcholine (ACh), histamine and 5-hydroxytryptamine (5-HT) were significantly reduced by BPE at concentrations of 2 mumol/L or greater. 4. BPE (2 mumol/L) did not affect calcium-induced contractions of longitudinal muscle fibres from guinea-pig ileum which were stripped of their cellular membrane. It (6 mumol/L) also had no effect on ATP levels in longitudinal muscle fibres. 5. BPE (2 mumol/L) reduced both phasic and tonic components of contractions induced by raising the extracellular concentration of K+ to 15, 30, 45 or 60 mmol/L (in the presence of atropine, propranolol, phentolamine and desensitization to 5-HT to inhibit the effects of nerve transmitter release). 6. BPE (2 mumol/L) reduced carbachol-induced contractions of ileum pre-incubated in 1 mumol/L felodipine, a blocker of L-type voltage-operated calcium channels (VOCC). 7. BPE dose dependently (0.6-6 mumol/L) reduced contractions induced by Ca2+ in both K+ depolarized ileum and in tissue exposed to carbachol (10 mumol/L) in the presence of felodipine (0.1 mumol/L). 8. These results suggest that BPE affects intracellular messenger systems controlling cytosolic calcium and/or blocks entry of calcium into the cell through both VOCC and receptor-operated channels (ROC).