Clonidine-induced Antinociception Research Articles

Analgesia, enhancement of spinal morphine antinociception, and inhibition of tolerance by ultra-low dose of the α2A-adrenoceptor selective antagonist BRL44408

Ultra-low doses of non-selective α2-adrenoceptor antagonists augment acute spinal morphine antinociception and block morphine tolerance; however, the receptor involved in mediating these effects is currently unknown. Here, we used tail flick and paw pressure tests on the rat to investigate the acute analgesic and tolerance-inducing effects of spinal morphine and norepinephrine alone or in combination with an ultra-low dose of the α2A-adrenoceptor antagonist, BRL44408. We also assessed the potential antinociceptive effects of BRL44408 alone following spinal administration. A spinal dose of BRL44408, over 1000-fold lower than that required to inhibit clonidine-induced antinociception (1.65ng/10µL), significantly prolonged morphine and norepinephrine action in both nociception tests. Following repeated morphine or norepinephrine injections, 1.65ng BRL44408 attenuated both the decline of antinociceptive effect and increase in morphine ED50 values, responses indicative of acute morphine tolerance. BRL44408 administered alone produced a delayed antinociceptive effect unrelated to repeated nociceptive testing. This response was partially reduced by the α2-adrenoceptor antagonist atipamezole (10µg). Ultra-low dose BRL44408 was able to inhibit the loss of morphine- and norepinephrine-induced antinociceptive response, and prevent the loss of drug potency due to repeated agonist exposure. This implicates the spinal α2A-adrenoceptor subtype in the action of ultra-low dose α2-adrenoceptor antagonists on morphine and norepinephrine tolerance. The BRL44408-induced analgesia is partially dependent on its interaction with the α2-adrenoceptors. Thus, this agent class may be useful in pain therapy.

Stereo-selective inhibition of spinal morphine tolerance and hyperalgesia by an ultra-low dose of the alpha-2-adrenoceptor antagonist efaroxan

Ultra-low doses of alpha-2 (α2)-adrenoceptor antagonists augment spinal morphine antinociception and inhibit tolerance, but the role of receptor specificity in these actions is unknown. We used the stereo-isomers of the α2 adrenoceptor antagonist, efaroxan to evaluate the effect of receptor specificity on the induction of spinal morphine tolerance and hyperalgesia. Tail flick and paw pressure tests were first used to evaluate high dose efaroxan (12.6μg) and its stereo-isomers on clonidine analgesia in intrathecally catheterized rats. Ultra-low doses of individual isomers (1.3ng) were then co-administered with morphine (15μg) to determine their effects on acute antinociceptive tolerance and hyperalgesia induced by low dose spinal morphine (0.05ng). Results demonstrate that high dose (+) efaroxan antagonized clonidine-induced antinociception, while (−) efaroxan had minimal effect. In addition, an ultra-low dose of (+) efaroxan (1.3ng), substantially lower than required for receptor blockade, inhibited the development of acute morphine tolerance, while (−) efaroxan was less effective. Racemic (±) efaroxan effects were similar to those of (+) efaroxan. Furthermore, low dose morphine (0.05ng) produced sustained hyperalgesia in the tail flick test and this was blocked by co-injection of (+) but not (−) efaroxan (1.3ng). Given the isomer-specific efaroxan effects and their different receptor potencies, we suggest that inhibition of opioid tolerance by ultra-low dose efaroxan involves a specific interaction with spinal α2-adrenoceptors in this model. Likewise, inhibitory effects of adrenoceptor antagonists on morphine tolerance may be due to blockade of opioid-induced hyperalgesia.

Low Dose Alpha-2 Antagonist Paradoxically Enhances Rat Norepinephrine and Clonidine Analgesia

Ultralow-dose opioid antagonists prolong opioid antinociception and block tolerance. In this study we determined whether low doses of the α-2 adrenergic receptor (A2-R) antagonist, atipamezole, similarly influenced A2-R-induced antinociception and tolerance. In rats, intrathecal norepinephrine (NE) or clonidine in combination with atipamezole was tested using tail-flick and paw pressure tests. Acute tolerance to NE was induced by serial injections. Low-dose atipamezole significantly prolonged NE and clonidine-induced antinociception. Coadministration of atipamezole with A2-R agonists also prevented loss of agonist potency in the acute tolerance model. This study demonstrates paradoxical effects of low-dose A2-R antagonists augmenting A2-R agonist-induced analgesia.

Clonidine-induced antinociception and locomotor hypoactivity are reduced by dexamethasone in mice.

The effects of dexamethasone pretreatment on clonidine-induced antinociception and locomotor hypoactivity were investigated in mice. In the hot-plate and the tail-flick tests, dexamethasone administered intraperitoneally at a dose of 1 mg kg(-1), 30 or 60 min before clonidine, reduced clonidine antinociception in both tests and reduced clonidine-induced locomotor hypoactivity in the activity cage. When administered 15 min before clonidine, dexamethasone had no effect on clonidine antinociception. A higher dexamethasone dose (10 mg kg(-1)) induced the same effects observed at a dose of 1 mg kg(-1) in the hot-plate and the tail-flick tests, but the former dose had a stronger effect on locomotor hypoactivity. Dexamethasone (10 ng/mouse) administered intracerebroventricularly 30 min before clonidine was also able to reduce both clonidine-induced antinociception and locomotor hypoactivity. The protein synthesis inhibitor, cycloheximide, administered intraperitoneally at the dose of 10 mg kg(-1), 2 h before clonidine, was able to prevent dexamethasone effects on clonidine-induced antinociception. The glucocorticoid receptor antagonist RU-38486, administered intracerebroventricularly at the dose of 1 ng/mouse, was also able to block dexamethasone effects on clonidine-induced antinociception and locomotor hypoactivity, whereas both cycloheximide and RU-38486 per se did not influence pain sensitivity or locomotor activity. These results suggest that the dexamethasone effects on clonidine-induced antinociception and locomotor hypoactivity depend on the stimulating effects that dexamethasone exert, on the protein synthesis via the glucocorticoid receptor in the brain.

Comparative study of α 2-adrenoceptors in Fischer 344 and Lewis rats. Evidence for clonidine-induced place aversion

Fischer 344 (F344) and Lewis rat strains have been shown to exhibit different vulnerability to development or maintenance of opioid seeking behaviours probably due to differences in the endogenous opioid system. Since opioid and α 2-adrenergic mechanisms closely interact in nociception and substance abuse, strain differences may be expected to affect α 2-adrenoceptor-mediated events. The sensitivity of these two strains to α 2-adrenoceptor-mediated antinociception has been reported to be markedly different. In this work we have further studied the function of α 2-adrenoceptors in F344 and Lewis rats by means of several in vivo and in vitro procedures. Comparative studies of [ 3H]RX821002 and [ 35S]GTPγS binding revealed that α 2-adrenoceptors could be slightly more responsive to agonist stimulation in the brain cortex of F344 rats, which is in agreement with previous antinociception studies. However, these differences were modest, not observed in the spinal cord and did not translate into functional differences concerning the effects of clonidine on vas deferens contractility and body temperature. Conditioning experiments showed that a moderate dose of clonidine, which is relevant in antinociceptive and opioid antiwithdrawal studies, induces a robust place aversion which is also equivalent in F344 and Lewis rats. This finding underlies the consistency of the effect and its independency of genetic differences between both rat strains. It seems therefore that the pharmacological properties of α 2-adrenoceptors are similar in F344 and Lewis rats, and thus the previously reported differences in clonidine-induced antinociception could be attributed to other factors such as dissimilar endogenous function of specific noradrenergic pathways.

Persistent exercise attenuates nicotine- but not clonidine-induced antinociception in female rats

Exercise decreases the antinociceptive effects of opiate drugs. It has been hypothesized that the exercise-induced attenuation of opiate drug action is the result of the development of cross-tolerance between endogenous opioids released during exercise and exogenous opiates. The present study was designed to evaluate the role of exercise on non-opiate antinociception. Female Long–Evans rats were allowed ad lib access to running wheels. After 3 weeks, antinociceptive responses of animals were measured using the tail flick test following the administration of clonidine or nicotine. Nicotine and clonidine both produced dose-dependent increases in antinociceptive responses. Active animals were significantly less sensitive to nicotine-induced antinociception than inactive animals. There was no difference between the two groups in clonidine-induced antinociception. The results of these experiments suggest that exercise does not attenuate non-opioid, clonidine-induced antinociception. However, exercise does attenuate nicotine-induced antinociception. Therefore, the effect of persistent exercise on analgesic drugs is not specific to opiates.

Inhibition of the I(h) current in isolated peripheral nerve: a novel mode of peripheral antinociception?

Although the alpha2-adrenergic agonist clonidine has been shown to promote peripheral antinociception, its mechanism of action has not yet been clearly elucidated. By the use of the sucrose-gap method, we have shown that in C fibers of the rabbit vagus nerve, clonidine at micromolar concentrations enhances activity-dependent hyperpolarizations generated by the Na+-K+ pump during and after repetitive stimulation. Similar results were obtained with 10 microM of ZD 7288, a specific blocker of the hyperpolarization-activated cation current (I(h)) and with 2 mM of Ba2+ that blocks the inwardly rectifying potassium current (I(KIR)). Furthermore, clonidine had no added effect on the ZD 7288-induced response, whereas it produced a marked enhancement of Ba2+induced response. From these results, it can be concluded that clonidine enhances activity-dependent hyperpolarization by inhibiting the current I(h). We propose that clonidine, by increasing the threshold for initiating the action potential, induces a slowing or block of conduction and that this mechanism is the origin of the clonidine-induced antinociception. Finally, this study suggests a novel role for inwardly rectifying hyperpolarization-activated conductances in peripherally mediated antinociception.

Inhibition of the Ih current in isolated peripheral nerve: A novel mode of peripheral antinociception?

Although the alpha2-adrenergic agonist clonidine has been shown to promote peripheral antinociception, its mechanism of action has not yet been clearly elucidated. By the use of the sucrose-gap method, we have shown that in C fibers of the rabbit vagus nerve, clonidine at micromolar concentrations enhances activity-dependent hyperpolarizations generated by the Na+-K+ pump during and after repetitive stimulation. Similar results were obtained with 10 microM of ZD 7288, a specific blocker of the hyperpolarization-activated cation current (I(h)) and with 2 mM of Ba2+ that blocks the inwardly rectifying potassium current (I(KIR)). Furthermore, clonidine had no added effect on the ZD 7288-induced response, whereas it produced a marked enhancement of Ba2+induced response. From these results, it can be concluded that clonidine enhances activity-dependent hyperpolarization by inhibiting the current I(h). We propose that clonidine, by increasing the threshold for initiating the action potential, induces a slowing or block of conduction and that this mechanism is the origin of the clonidine-induced antinociception. Finally, this study suggests a novel role for inwardly rectifying hyperpolarization-activated conductances in peripherally mediated antinociception.

Involvement of supraspinal GABA-ergic systems in clonidine-induced antinociception in the tail-pinch test in mice

We investigated the involvement of supraspinal GABAergic systems in the antinociceptive effect of clonidine using the tail-pinch test in mice. Muscimol (31.2–250 ng/mouse, i.c.v.) and R(+)-baclofen (10–100 ng/mouse, i.c.v.), selective agonists for the GABA a and GABA b receptors, respectively, significantly attenuated the antinociceptive effect of subcutaneously (s.c.) administered clonidine (1 mg/kg) in a dose-dependent manner. The attenuating effect of muscimol (62.5 ng/mouse, i.c.v.) on the clonidine-induced antinociception was significantly blocked by the GABA a antagonists bicuculline (100–400 ng/mouse, i.c.v.) and picrotoxin (250 ng/mouse, i.c.v.) but not by the GABA B antagonist 2-hydroxysaclofen (10 μg/mouse, i.c.v.). On the other hand, the attenuating effect of R(+)-baclofen (50 ng/mouse, i.c.v.) was blocked by the coadministration with 2-hydroxysaclofen (20 μg/mouse), but was not affected by the coadministration with bicuculline (400 ng/mouse). These results indicate that both supraspinal GABA a and GABA b receptors play inhibitory roles in the antinociception caused by systemically administered clonidine.

Involvement of supraspinal GABA receptors in majonoside-R2 suppression of clonidine-induced antinociception in mice

Majonoside-R2 (MR2) is a major constituent of Vietnamese ginseng ( Panax vietnamensis, Ha et Grushv. Araliaceae) that is known to exhibit antagonistic activity against opioid-induced antinociception. In this study, we investigated the effect of MR2 on the antinociception caused in mice by the α2-adrenoceptor agonist clonidine, and elucidated the role of supraspinal GABAergic systems in this effect of MR2. The systemic administration of clonidine (0.5–2.5 mg kg , s.c.) dose-dependently suppressed the nociceptive response of mice in the tail-pinch and hot-plate tests. The intraperitoneal (i.p.), intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration of idazoxan (a selective α2-adrenoceptor antagonist) significantly antagonized the antinociceptive effect of clonidine in both tests. MR2 administered systemically (1.5–6.2 mg kg , i.p.) or centrally (5–10 μg/mouse, i.c.v. or i.t) dose-dependently antagonized the clonidine (1 mg kg , s.c.)-induced antinociception in the tail-pinch test but not in the hot-plate test. The antagonistic effect of i.c.v. MR2 on the systemic clonidine-induced antinociception in the tail-pinch test was significantly reversed by i.c.v. administrations of the selective benzodiazepine receptor antagonist flumazenil (5 μg/mouse) and the GABA A antagonist picrotoxin (0.25 μg/mouse). These results suggest that the supraspinal GABA A/benzodiazepine receptors are involved in the antagonistic effect of MR2 on the clonidine-induced antinociception in the tail-pinch test in mice.

P-87 - Involvement of supraspinal GABAA receptors and spinal α2-adrenoceptors in the suppression of clonidine-induced antinociception by majonoside-R2 in mice

P-87 - Involvement of supraspinal GABAA receptors and spinal α2-adrenoceptors in the suppression of clonidine-induced antinociception by majonoside-R2 in mice

ω-Agatoxin IVA blocks spinal morphine/clonidine antinociceptive synergism

Involvement of P-type voltage-dependent Ca 2+ channels in spinal morphine- or clonidine-induced antinociception and in the synergistic interaction between morphine and clonidine was examined in the present studies. Coadministration of the selective P-type antagonist, ω-agatoxin IVA (25 ng) intrathecally (i.t.) to mice along with morphine or clonidine enhanced the tail flick antinociception of each agonist 5–6-fold. The greater-than-additive (synergistic) interaction that occurred when morphine and clonidine were coadministered i.t. decreased to an additive interaction in the presence of ω-agatoxin IVA. In mice pretreated with pertussis toxin (10 ng) to inactivate G proteins, ω-agatoxin IVA did not alter the morphine/clonidine synergism. Surprisingly, ω-agatoxin IVA reversed the additive morphine/clonidine interaction that occurs in morphine-tolerant mice back to synergism. These results suggest that functional P-type Ca 2+ channels play an essential role in the antinociceptive synergism between spinal morphine and clonidine.

α-Subunit G-protein antisense oligodeoxynucleotide effects on supraspinal ( i.c.v.) α 2-adrenoceptor antinociception in mice

An in vivo antisense strategy was used to examine the involvement of G-protein subunits in supraspinal (intracerebroventricular; i.c.v.) α 2-adrenoceptor-mediated antinociception. Mice that were injected with 33-mer antisense oligodeoxyribonucleotides (6 nmol) or vehicle were tested (tailflick) with an agonist (clonidine, guanfacine or BH-T 920) administered i.c.v. 18–24 h later. G i3α antisense treatment attenuated BH-T 920 and clonidine-induced antinociception. G i2α antisense produced differential effects on the three agonists. G i1α and G s α antisense treatment had no significant effect. Together with the previous demonstration that i.c.v. μ-opioid antinociception is mediated via G i2α, the present results suggest that different receptors may mediate antinociception via different G-protein subunits and, hence, that specific subunits might offer novel targets for drug discovery.

Antibodies raised against the α subunits of G 1 and Gx/x transducer proteins diminished clonidine-induced antinociception in mice

Antibodies raised against the α subunits of G 1 and Gx/x transducer proteins diminished clonidine-induced antinociception in mice

Antagonism of clonidine antinociception by buspirone and 1-(2-pyrimidinyl)-piperazine

The effects of pretreatment with buspirone and its major metabolite 1-(2-pyrimidinyl)-piperazine (1-PP) on antinociception produced by clonidine were investigated in mice. Buspirone and 1-PP dose dependently attenuated the antinociceptive action of s.c. administered clonidine in the writhing and tail-flick assays. In both assays, 1-PP was more potent than buspirone in antagonizing clonidine-induced antinociception. After s.c. pretreatment with buspirone (8 mg/kg) and 1-PP (4 mg/kg), the antinociceptive ED 50 values of s.c. clonidine were significantly increased. The antagonistic effects of buspirone and 1-PP on clonidine-induced antinociception may be due to their α 2-adrenoceptor antagonist activity.

Differential effects of K+ channel blockers on antinociception induced by alpha 2-adrenoceptor, GABAB and kappa-opioid receptor agonists.

1. The effects of several K+ channel blockers (sulphonylureas, 4-aminopyridine and tetraethylammonium) on the antinociception induced by clonidine, baclofen and U50,488H were evaluated by use of a tail flick test in mice. 2. Clonidine (0.125-2 mg kg-1, s.c.) induced a dose-dependent antinociceptive effect. The ATP-dependent K+ (KATP) channel blocker gliquidone (4-8 micrograms/mouse, i.c.v.) produced a dose-dependent displacement to the right of the clonidine dose-response line, but neither 4-aminopyridine (4-AP) (25-250 ng/mouse, i.c.v.) nor tetraethylammonium (TEA) (10-20 micrograms/mouse, i.c.v.) significantly modified clonidine-induced antinociception. 3. The order of potency of sulphonylureas in antagonizing clonidine-induced antinociception was gliquidone > glipizide > glibenclamide > tolbutamide, which is the same order of potency as these drugs block KATP channels in neurones of the CNS. 4. Baclofen (2-16 mg kg-1, s.c.) also induced a dose-dependent antinociceptive effect. Both 4-AP (2.5-25 ng/mouse, i.c.v.) and TEA (10-20 micrograms/mouse, i.c.v.) dose-dependently antagonized baclofen antinociception, producing a displacement to the right of the baclofen dose-response line. However, gliquidone (8-16 micrograms/mouse, i.c.v.) did not significantly modify the baclofen effect. 5. None of the K+ channel blockers tested (gliquidone, 8-16 micrograms/mouse; 4-AP, 25-250 ng/mouse and TEA, 10-20 micrograms/mouse, i.c.v.), significantly modified the antinociception induced by U50,488H (8 mg kg-1, s.c.). 6. These results suggest that the opening of K+ channels is involved in the antinociceptive effect of alpha 2 and GABAB, but not kappa-opioid, receptor agonists. The K+ channels opened by alpha2-adrenoceptor agonists seem to be ATP-dependent channels, whereas those opened by GABAB receptor agonists are not.

Dissociation of hypertension and enhanced clonidine-induced antinociception in spontaneously hypertensive rats

In the present study, we evaluated the relationship between hypertension and the antinociceptive effect of clonidine in genetically hypertensive rats. Intraperitoneal (i.p.) administration of clonidine (0.69 mg/kg) produced hypoalgesia without lowering blood pressure in conscious spontaneously hypertensive (SH), spontaneously hypertensive-stroke prone (SH-SP) and Wistar-Kyoto (WKY) rats. The antinociceptive effect of clonidine, measured by the hot plate method, was greater in the hypertensive rats (SH and SH-SP) than in their normotensive controls (WKY). The enhancement of clonidine-induced antinociception in these genetically hypertensive rats was not altered after acute lowering of blood pressure with guanethidine. Furthermore, the development of this hyper-antinociceptive effect of clonidine was earlier than that of hypertension in SH rats. In conclusion, our data suggest that the enhanced antinociceptive effect of clonidine in hypertensive animals is not directly related to blood pressure.

Spinal 5-HT pathways and the antinociception induced by intramedullary clonidine in rats

The possible involvement of spinal 5-hydroxytryptamine (5-HT) pathways in antinociception induced by microinjection of clonidine into the ventrolateral surface of the medulla oblongata was investigated in rats. Microinjection of clonidine (10-20 micrograms), but not yohimbine (1 microgram) or 0.9% saline, into the lateral medulla prolonged the hot plate latency in rats. This clonidine-induced antinociception was abolished by intramedullary injection of the alpha 2-adrenoceptor antagonist, yohimbine. Selective destruction of spinal 5-HT neurons produced by intraspinal injection of 5,7-dihydroxytryptamine (5,7-DHT; 10 micrograms) or postsynaptic blockade of spinal 5-HT receptors produced by intrathecal injection of cyproheptadine (1 microgram; a mixed 5-HT1/5-HT2 antagonist) also abolished clonidine-induced antinociception. Rats given 5,7-DHT intraspinally or cyproheptadine intrathecally showed a decrease in hot plate latency as compared with the controls. In anesthetized rats, the 5-HT release from the thoracic spinal cord was enhanced by microinjection of clonidine into the lateral medulla. This enhanced spinal 5-HT release evoked by intramedullary injection of clonidine was abolished by pretreatment of rats with intraspinal injection of 5,7-DHT. These results indicate that 5-HT pathways to the spinal cord mediate the antinociceptive effect induced by microinjection of clonidine into the ventrolateral surface of the medulla oblongata in rats.

Mode of antinociceptive action of flupirtine in the rat

1. Flupirtine is a novel, centrally acting, non-opioid analgesic agent. The present investigation was undertaken to ascertain which neuronal systems might be responsible for its antinociceptive effect in rodents. The antinociceptive responses to the test compounds were examined in the tail-flick test. 2. The selective destruction of noradrenergic pathways by 6-hydroxydopamine considerably reduced the flupirtine-induced inhibition of nociceptive responses but not the clonidine-induced antinociception which was significantly enhanced. Depletion of spinal 5-hydroxytryptaminergic pathways by pretreatment with 5,7-dihydroxytryptamine failed to affect the action of flupirtine and clonidine. 3. The depletion of neurotransmitters by reserpine totally abolished the antinociceptive action of flupirtine. By contrast, clonidine-induced inhibition of nociceptive responses remained unchanged. 4. Inhibition of the synthesis of noradrenaline by alpha-methyl-L-p-tyrosine attenuated the antinociception induced by flupirtine. In contrast, inhibition of the synthesis of 5-hydroxytryptamine by (+/-)-6-fluorotryptophan did not influence the antinociceptive activity of flupirtine. 5. Inhibition of noradrenaline uptake by imipramine led to a significant augmentation of flupirtine-induced antinociception. 6. Selective antagonists at alpha-adrenoceptors significantly decreased the antinociceptive action of flupirtine. Antinociception induced by clonidine was significantly diminished by idazoxan but not by prazosin. 7. The 5-hydroxytryptamine (5-HT) antagonist, ketanserin diminished the antinociceptive activity of flupirtine, probably due to its additional alpha 1-adrenoceptor antagonist activity. The antinociceptive effect of clonidine was not influenced by ketanserin. 8. Cholinoceptor antagonists such as mecamylamine and pirenzepine did not alter the antinociceptive action of flupirtine. Flupirtine-induced antinociception also remained unchanged after pretreatment with haloperidol. 9. Flupirtine has no pharmacologically relevant affinity for alpha 1-, alpha 2-adrenoceptors, 5-HT1- and 5-HT2-receptors as shown in direct binding studies. 10. The present results indicate that the antinociceptive action induced by flupirtine depends on the descending noradrenergic pain-modulating system.

Differential effects of clonidine on pain, arterial blood pressure, and heart rate in the cat: Lack of interactions with naloxone

In cats anesthetized with α-chloralose and urethane, intravertebral administration of clonidine (4 and 10 μg/kg) dose-dependently suppressed the jaw-opening reflex, arterial blood pressure, and heart rate. For a given dose, there was a differential degree of inhibition in the order of analgesia ⪢ hypotension > bradycardia. Naloxone injections (0.4 and 1.0 mg/kg, i.vert.) essentially failed to antagonize these effects, suggesting the lack of involvement of the opiate receptors or endogenous opioids in these processes. Furthermore, pain suppression by clonidine appeared to be independent of the vasodepression and cardioinhibition it promoted. It is possible that neural mechanisms responsible for clonidine-induced antinociception, hypotension, and bradycardia are likely to have differential sensitivities to the imidazoline compound, regardless of whether they exist in separate central sites or in subpopulations of neurons within common neural substrates.