Igor Dimitri Gama Duarte

Peripheral analgesia and activation of the nitric oxide-cyclic GMP pathway

We have previously described the analgesic effect of dibutyryl cyclic GMP or acetylcholine (ACh) injected into rat paws. Since ACh induces nitric oxide (NO) release from endothelial cells, we investigated the possible involvement of the NO-cyclic GMP pathway in ACh-induced analgesia, using a modification of the Randall-Selitto rat paw test. We found that sodium nitroprusside, which releases NO non-enzymatically, caused antinociception in the rat paw made hyperalgesic with prostaglandin E2. The analgesic effect of sodium nitroprusside and ACh was enhanced by intraplantar injection of an inhibitor of cyclic GMP phosphodiesterase (MY 5445) and was blocked by a guanylate cyclase inhibitor, methylene blue (MB). The analgesia induced by ACh, but not by sodium nitroprusside, was blocked by NG-monomethyl-L-arginine (L-NMMA), an inhibitor of the formation of NO from L-arginine. L-arginine itself had little or no effect upon prostaglandin-induced hyperalgesia but caused significant analgesia in paws inflamed with carrageenin. This analgesia was blocked by MB, as well as by L-NMMA, and was potentiated by MY 5445. These results suggest that ACh-induced analgesia was mediated via the release of NO. The results also indicate that the guanylate cyclase system is stimulated in the inflammatory reaction. The analgesia resulting from activation of this system is possibly overshadowed by substances that concomitantly stimulate nociceptor hyperalgesic mechanisms.

The molecular mechanism of action of peripheral morphine analgesia: stimulation of the cGMP system via nitric oxide release

In addition to the central and spinal sites of action of morphine both our laboratory and others have demonstrated that opiates can also cause analgesia through a peripheral mechanism (Ferreira, 1983). However, the molecular basis of the mechanism of the analgesic actions of opiates remains unknown. In vitro studies show that morphine is able to inhibit activation of adenylate cyclase (Collier and Roy, I974) as well as to stimulate formation of cGMP (Minneman and Iversen, I976). Recently, using the rat paw hyperalgesia test, we showed that in the periphery, acetylcholine-induced analgesia was mediated via the activation of the nitric oxide/cGMP pathwny (Duarte et al., I990). This conclusion was based upon the fact that intraplantar injection of sodium nitroprusside, a substance which nonenzymatically releases nitric oxide (NO), caused analgesia. Furthermore, the analgesic effects of acetylcholine (ACh) and sodium nitroprusside were enhanced by intraplantar injection of an inhibitor of cyclic GMP phosphodiesterase, My5445. In addition, methylene bk:e (MB), an inhibitor of guanylate cyclase, blo1.:ked the analgesia induced by acetylcholine and sodium nitroprusside. On the other hand, the analgesia induced by acetylcholine, but not by sodium nitroprusside, was blocked by N°-monomethyl-L-arginine (LNMMA), an inhibitor of the formation of NO from L-arginine. Due to the similarity of the local action of ACh and opiates we investigated whether agents which affect the arginine/NO-cGMP pathway also interfere with morphine-induced peripheral analgesia as tested with our modification of the Randall-Selitto rat paw pressure test (see Duarte et al., I990). In this test a constant pressure of 20 mmHg is applied to the hind paw of rats

Analgesia by direct antagonism of nociceptor sensitization involves the arginine-nitric oxide-cGMP pathway

We tested the hypothesis that activation of the nitric oxide (NO)-cGMP pathway is involved in the mechanism of two directly acting non-opiate peripheral analgesics, myrcene and dipyrone, using our modification of the Randall-Selitto test. The NO inhibitor, NG-monomethyl-L-arginine (50 micrograms/paw) and methylene blue (500 micrograms/paw) abolished the analgesic effect of dipyrone and myrcene. Dibutyryl cyclic adenosine monophosphate (DbcAMP) caused a dose-dependent hyperalgesia (20, 50 and 100 micrograms/paw). Only responses to low doses of DbcAMP were inhibited by the two analgesics. Pretreatment with MY5445 (50 micrograms/paw) resulted in potentiation of the effects of both analgesics. These results support our hypothesis that the sensitivity of nociceptors may be controlled by the balance between the levels of cAMP and cGMP. Stimulation of the NO-cGMP pathway is probably the common denominator for the mode of action of peripheral analgesics which block hyperalgesia directly.

The peripheral antinociceptive effect induced by morphine is associated with ATP-sensitive K+ channels

The effect of several K+ channel blockers such as glibenclamide, tolbutamide, charybdotoxin (ChTX), apamin, tetraethylammonium (TEA), 4‐aminopyridine (4‐AP) and cesium on the peripheral antinociceptive effect of morphine was evaluated by the paw pressure test in Wistar rats. The intraplantar administration of a carrageenan suspension (250 μg) resulted in an acute inflammatory response and a decreased threshold to noxious pressure. Morphine administered locally into the paw (25, 50, 100 and 200 μg) elicited a dose‐dependent antinociceptive effect which was demonstrated to be mediated by a peripheral site up to the 100 μg dose. The selective blockers of ATP‐sensitive K+ channels glibenclamide (20, 40 and 80 μg paw−1) and tolbutamide (40, 80 and 160 μg paw−1) antagonized the peripheral antinociception induced by morphine (100 μg paw−1). This effect was unaffected by ChTX (0.5, 1.0 and 2.0 μg paw−1), a large conductance Ca2+‐activated K+ channel blocker, or by apamin (2.5, 5.0 and 10.0 μg paw−1), a selective blocker of a small conductance Ca2+‐activated K+ channel. Intraplantar administration of the non‐specific K+ channel blockers TEA (160, 320 and 640 μg), 4‐AP (10, 50 and 100 μg) and cesium (125, 250 and 500 μg) also did not modify the peripheral antinociceptive effect of morphine. These results suggest that the peripheral antinociceptive effect of morphine may result from activation of ATP‐sensitive K+ channels, which may cause a hyperpolarization of peripheral terminals of primary afferents, leading to a decrease in action potential generation. In contrast, large conductance Ca2+‐activated K+ channels, small conductance Ca2+‐activated K+ channels as well as voltage‐dependent K+ channels appear not to be involved in this transduction pathway.

Activation of ATP-sensitive K(+) channels: mechanism of peripheral antinociceptive action of the nitric oxide donor, sodium nitroprusside.

Using the rat paw pressure test, in which sensitivity is increased by intraplantar injection of prostaglandin E(2) (PGE(2)), we conducted a study using several K(+) channel blockers. The objective was to determine what types of K(+) channels could be involved in the peripheral antinociceptive action of the nitric oxide donor sodium nitroprusside (SNP). SNP elicited a dose-dependent (250 and 500 microgram/paw) peripheral antinociceptive effect, which was considered local, since only higher doses produced an effect in the contralateral paw. The effect of SNP (500 microgram/paw) was dose-dependently antagonized by intraplantar administration of the sulfonylureas tolbutamide (20, 40 and 160 microgram) and glibenclamide (40, 80 and 160 microgram), selective blockers of ATP-sensitive K(+) channels. Charybdotoxin (2 microgram/paw), a selective blocker of high conductance Ca(2+)-activated K(+) channels, and apamin (10 microgram/paw), a selective blocker of low conductance Ca(2+)-activated K(+) channels, did not modify the peripheral antinociception induced by SNP. Tetraethylammonium (2 mg/paw), 4-aminopyridine (200 microgram/paw) and cesium (800 microgram/paw) also had no effect. Based on this experimental evidence, we conclude that the activation of ATP-sensitive K(+) channels could be the mechanism by which nitric oxide, donated by SNP, induces peripheral antinociception, and that Ca(2+)-activated K(+) channels and voltage-dependent K(+) channels appear not to be involved in the process.

Involvement of ATP-sensitive K+ channels in the peripheral antinociceptive effect induced by dipyrone

We evaluated the effect of several K(+) channel blockers on the peripheral antinociception induced by dipyrone using the rat paw pressure test, in which sensitivity is increased by intraplantar injection (2 micro g) of prostaglandin E(2). Dipyrone administered locally into the right hindpaw (50, 100 and 200 micro g) elicited a dose-dependent antinociceptive effect which was demonstrated to be local, since only higher doses produced an effect when injected in the contralateral paw. The specific blockers of ATP-sensitive K(+) channels glibenclamide (40, 80 and 160 micro g/paw) and tolbutamide (80, 160 and 320 micro g/paw) antagonized the peripheral antinociception induced by dipyrone (200 micro g/paw). Charybdotoxin (2 micro g/ paw), a blocker of large conductance Ca(2+)-activated K(+) channels, and dequalinium (50 micro g/paw), a selective blocker of small conductance Ca(2+)-activated K(+) channels, did not modify the effect of dipyrone. This effect was also unaffected neither by intraplantar administration of non-specific voltage-dependent K(+) channel blockers tetraethylammonium (1700 micro g) and 4-aminopyridine (100 micro g) nor cesium (500 micro g), a non-specific K(+) channel blocker. These results suggest that the peripheral antinociceptive effect of dipyrone may result from activation of ATP-sensitive K(+) channels, while other K(+) channels appear not to be involved in the process.

The molecular mechanism of central analgesia induced by morphine or carbachol and the L-arginine-nitric oxide-cGMP pathway.

The role of the L-arginine-NO-cGMP pathway in morphine-induced central analgesia was investigated in two nociceptive tests: PGE2-induced hind paw hyperalgesia and tail-flick. The central analgesic effect of morphine was potentiated by MY5445, a specific cGMP phosphodiesterase inhibitor. I.c.v. injections of morphine or carbachol caused dose-dependent analgesia, which was prevented by methylene blue, an inhibitor of guanylate cyclase. The NO synthase inhibitor, N-iminoethyl-L-ornithine, prevented carbachol-induced analgesia, but did not affect morphine-induced analgesia. Our results suggest that activation of cGMP may underlies analgesia induced by morphine and carbachol. The activation of guanylate cyclase by carbachol seems to depend on the L-arginine-NO pathway, but that caused by morphine remains to be further characterized.

Dibutyryl-cyclic GMP induces peripheral antinociception via activation of ATP-sensitive K+ channels in the rat PGE2-induced hyperalgesic paw

Using the rat paw pressure test, in which increased sensitivity is induced by intraplantar injection of prostaglandin E2, we studied the action of several K+ channel blockers in order to determine what types of K+ channels could be involved in the peripheral antinociception induced by dibutyrylguanosine 3 : 5′‐cyclic monophosphate (DbcGMP), a membrane permeable analogue of cyclic GMP. DbcGMP elicited a dose‐dependent (50, 75, 100 and 200 μg paw−1) peripheral antinociceptive effect. The effect of the 100 μg dose of DbcGMP was considered to be local since only a higher dose (300 μg paw−1) produced antinociception in the contralateral paw. The antinociceptive effect of DbcGMP (100 μg paw−1) was dose‐dependently antagonized by intraplantar administration of the sulphonylureas tolbutamide (20, 40 and 160 μg) and glibenclamide (40, 80 and 160 μg), selective blockers of ATP‐sensitive K+ channels. Charybdotoxin (2 μg paw−1), a selective blocker of high conductance Ca2+‐activated K+ channels, and apamin (10 μg paw−1), a selective blocker of low conductance Ca2+‐activated K+ channels, did not modify the peripheral antinociception induced by DbcGMP. Tetraethylammonium (2 mg paw−1), 4‐aminopyridine (200 μg paw−1) and cesium (800 paw−1), non‐selective voltage‐gated potassium channel blockers, also had no effect. Based on this experimental evidence, we conclude that the activation of ATP‐sensitive K+ channels could be the mechanism by which DbcGMP induces peripheral antinociception, and that Ca2+‐activated K+ channels and voltage‐dependent K+ channels appear not to be involved in the process.

Midazolam-induced hyperalgesia in rats: modulation via GABAA receptors at supraspinal level

The effect of benzodiazepines on the nociceptive threshold was studied in rats using the tail-flick and the formalin tests. Systemic injection of midazolam (10 mg/kg, i.p.) induced a significant decrease of the tail-flick latency and produced a long-lasting nociceptive effect in the formalin test, thus characterising a hyperalgesic state. The hyperalgesia induced by midazolam in the tail-flick test was blocked by flumazenil, a specific antagonist for benzodiazepine sites associated with GABA(A) receptors. Picrotoxin, a Cl- channel blocker, inhibited midazolam-induced hyperalgesia in both tests. Midazolam caused hyperalgesia when administered intracerebroventricularly (i.c.v.; 25 microg) but not intrathecally (i.t.; 75 microg). I.c.v. but not i.t. (5 microg) injection of flumazenil suppressed the hyperalgesia induced by midazolam (10 mg/kg, i.p.). Combination of non-hyperalgesic doses of diazepam (10 mg/kg, i.p.) or ethanol (0.48 g/kg, oral) with midazolam (5 mg/kg, i.p.) also induced hyperalgesia. Our results demonstrate that midazolam and diazepam alone or in combination with ethanol can produce hyperalgesia by interacting with GABA(A) receptors at the supraspinal level in rats. The risk of hyperalgesia should be taken in account when these drugs are used in combination in humans.

The μ‐opioid receptor agonist morphine, but not agonists at δ‐ or κ‐opioid receptors, induces peripheral antinociception mediated by cannabinoid receptors

Although participation of opioids in antinociception induced by cannabinoids has been documented, there is little information regarding the participation of cannabinoids in the antinociceptive mechanisms of opioids. The aim of the present study was to determine whether endocannabinoids could be involved in peripheral antinociception induced by activation of μ‐, δ‐ and κ‐opioid receptors.