Involvement of GABAergic systems in manifestation of pharmacological activity of desipramine.

We have conducted this study to elucidate the influence of GABAergic systems on manifestation of pharmacological activity of desipramine using both pharmacological and electrophysiological methods. Desipramine (20 mg/kg, i.p.) significantly blocked the adjuvant-induced thermal hyperalgesia, which was facilitated by treatment with the GABA(A) antagonist picrotoxin (2 mg/kg, i.p.) or the GABA(B) antagonist saclofen (2 mg/kg, i.p.). This analgesic effect of desipramine was antagonized by post-treatment with picrotoxin or saclofen. However, none of these compounds showed any effect in normal animals without adjuvant-induced inflammation. In a slice preparation of the hippocampus, treatment with GABA (10(-5)-5 x 10(-4) M), baclofen (10(-5)-10(-4) M) or muscimol (10(-5)-10(-4) M) inhibited the field potential evoked in pyramidal neurons by Schaffer collateral stimulation. The inhibitory effect of GABA was facilitated by concurrent application of desipramine, carbamazepine or diazepam at a concentration of 5 x 10(-5)-2 x 10(-4) M. The rank of order of facilitation is: desipramine > carbamazepine > diazepam. Desipramine also enhanced the inhibitory effect of baclofen and muscimol. These results suggest that desipramine causes GABAergic systems to activate still more, and this phenomenon appears to be involved in manifestation of the pharmacological activity of desipramine such as antinociception.

Influence of serotonin receptor antagonists on substance P and serotonin release evoked by tooth pulp stimulation with electro-acupuncture in the trigeminal nucleus cudalis of the rabbit.

We studied the effect of NAN-190 (5-HT(1A) antagonist), ketanserin (5-HT(2) antagonist) and ICS 205-930 (5-HT(3) antagonist) on tooth pulp stimulation (TPS)-induced 5-HT release and substance P (SP) release in the superficial layers of the trigeminal nucleus caudalis (SpVc-I,II) in the presence or absence of electro-acupuncture (EAP). TPS slightly increased 5-HT release and significantly increased SP release. In combination with EAP, TPS-induced 5-HT release was remarkably enhanced, whereas SP release was significantly suppressed. Pretreatment with NAN-190 (3.5 mg/kg, i.v.) significantly enhanced the increase in TPS-induced 5-HT release in the presence of EAP. On the other hand, the increase of 5-HT release induced following TPS in the presence of EAP was inhibited by pretreatment with ketanserin (2.5 mg/kg, i.v.) and ICS 205-930 (1 mg/kg, i.v.). When NAN-190 was pre-treated in the animals combined TPS and EAP, the amount of SP release was significantly reduced compared with the absence of this drug. On the other hand, pretreatment with ketanserin and ICS 205-930 reversed the inhibitory effect of EAP on the TPS-generated SP release, especially ICS 205-930, which remarkably enhanced TPS-induced SP release compared with the absence of this drug. On the basis of the obtained results, we concluded that NAN-190 and ICS 205-930 act on EAP-induced analgesia positively and suppressively, respectively, by regulation of TPS-generated SP release through activation of their subtype receptors. On the other hand, ketanserin does not affect TPS-induced 5-HT release and SP release in the presence of EAP.

Interaction between nitric oxide and substance P on heat-induced inflammation in rat paw.

To elucidate the interaction between nitric oxide (NO) and substance P (SP) in neurogenic inflammatory responses, we measured the change in the degree of Evans blue leakage and NO levels in perfusate from the subcutaneous space in the rat instep following noxious heat stimulation (47 degrees C for 30 min). Furthermore, the effects of drugs affecting nitric oxide synthase were examined. Noxious heat stimulation caused on an increase in NOx, or NO2- and NO3- into the perfusate in parallel with plasma extravasation. Nw-nitro-L-arginine methylester (L-NAME: 100 mg/kg once daily.) intraperitoneally (i.p.) given five times (chronic treatment) significantly suppressed the increase in Evans blue extravasation induced by heat stimulation, whereas acute treatments with L- and D-NAME (100 mg/kg once, i.p.) did not show any significant effect. NO release induced by heating also was significantly suppressed by chronic pretreatment with L-NAME, but not by acute treatment. SP (10(-5) M) applied into the perfusate caused a remarkable increase in the NOx release into the perfusate. Intra-arterial injection of RP67580 (1 mg/kg) on the perfused side, but not SR48968 (1 mg/kg), significantly attenuated the increases in Evans blue leakage and NOx release during heat stimulation. These results suggest that heat-induced SP release from the peripheral endings of small-diameter afferent fibers causes NO generation through NK-1R, and that this gas act to elicit or enhance inflammatory responses.

Serotonin receptor subtypes involved in modulation of electrical acupuncture.

We examined the effects of intravenous injection of several serotonin (5-HT) antagonists on the inhibitory action of electro-acupuncture (EAP) against the nociceptive responses in the trigeminal nucleus caudalis in rabbits. The inhibitory effect of EAP was suppressed by pindolol, methysergide and ICS 205-930, whereas NAN-190 and ketanserin amplified the EAP effect. These results suggest that 5-HT1, except 5-HT1A; 5-HT2, except 5-HT2A; and 5-HT3 receptors are positively involved in EAP-induced analgesia, whereas the activation of 5-HT1A and 5-HT2A receptors suppressively act on EAP-induced analgesia.

Nitric oxide in the rat spinal cord in Freund's adjuvant-induced hyperalgesia.

To elucidate the involvement of nitric oxide in spinal nociceptive processing, the correlation of thermal withdrawal latency with nitric oxide synthase-stained neurons in the rat lumbar dorsal horn was analyzed after adjuvant-induced inflammation. From 4 hr through 5 days after subcutaneous injection of complete Freund's adjuvant into the hind paw, a marked thermal hyperalgesia was observed for heat stimulus applied to the affected region. NADPH-diaphorase- and nitric oxide synthase-positive neurons increased significantly in the superficial layers of the dorsal horn ipsilateral to the inflamed hind paw at day 3 of adjuvant-induced inflammation. No change in NADPH-diaphorase-positive neurons was observed at 1 hr and 1 day of adjuvant-induced inflammation. The intravenous administration of N omega-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg), an inhibitor of nitric oxide synthase, significantly blocked the adjuvant-induced thermal hyperalgesia at day 3 of inflammation, but not at day 1; and it had no effect in non-inflamed rats. This anti-hyperalgesic effect of L-NAME at day 3 of inflammation was reversed by the prior administration of L-arginine (600 mg/kg, i.p.), a substrate of nitric oxide synthase. These data suggest that nitric oxide producing neurons in the spinal dorsal horn are involved in maintaining and facilitating the hyperalgesia associated with chronic nociception.

A possible involvement of monoaminergic and opioidergic systems in the analgesia induced by electro-acupuncture in rabbits.

To elucidate mechanisms involved in analgesia induced by effects of electro-acupuncture (EAP), effects of EAP on evoked potentials and release of substance P (SP) following tooth pulp stimulation (ST) in the superficial layers of the trigeminal nucleus caudalis (Vc-I-II) were studied in the rabbit. The potentials evoked by ST were composed of two main components with conduction velocity of ca. 30 m/sec (fast component) and ca. 12 m/sec (late component). The late component was significantly inhibited by morphine (10 mg/kg, i.v.) or CP-96,345 (5 mg/kg, i.v.), an SP antagonist. This inhibitory effect of morphine was antagonized by naloxone (1 mg/kg, i.v.) or methysergide (5 mg/kg, i.v.). In addition, the late component was significantly inhibited by EAP, which was observed in ca. 70% of the rabbits examined. This EAP-induced inhibitory effect was antagonized by naloxone (1 mg/kg, i.v.) or methysergide (5 mg/kg, i.v.), but not by prazosin (5 mg/kg, i.v.) and yohimbine (1 mg/kg, i.v.). The stimulus-evoked SP release was inhibited by EAP, which was significantly antagonized by pretreatment with naloxone (1 mg/kg, i.v.) or methysergide (5 mg/kg, i.v.). These results suggest that one of the mechanisms of analgesia induced by EAP is due to inhibition of the stimulus-evoked SP release in the Vc-I-II through activation of the descending serotonergic systems linking up with opioidergic systems.

Influence of electro-acupuncture on the release of substance P and the potential evoked by tooth pulp stimulation in the trigeminal nucleus caudalis of the rabbit.

The effects of electro-acupuncture (EAP) on the release of substance P (SP) and the responses evoked by tooth pulp stimulation (ST) in superficial layers of the trigeminal nucleus caudalis (Vc-I,II) were studied in rabbits. ST evoked increase in release of immunoreactive SP (iSP). This increase was inhibited by EAP in 9 of 13 animals. The potentials evoked by ST were composed of two main components with latency times of ca 4.3 msec and ca. 9.4 msec. The latter component, reflecting the excitation of A delta fibers, was significantly inhibited by CP-96,345 (3 mg/kg, i.v.), an SP antagonist. EAP also inhibited the latter component in 8 of 11 animals. These results suggest that one of the mechanisms of analgesia induced by EAP is inhibition of stimulus-evoked SP release in the Vc-I,II.

Contribution of substance P to heat-induced edema in rat paw.

To investigate a physiological function of substance P (SP) present in the peripheral ending of sensory neurons, we determined immunoreactive SP (iSP) levels in the s.c. perfusate and the amount of edema evoked in rat paw by noxious heat stimulation. We found that immersion of rat paw into hot water (47 degrees C) for 30 min led to a significant increase of iSP in the perfusate and about 50% increase in paw volume. Neonatal pretreatment with capsaicin inhibited significantly the increase in both iSP and paw volume evoked by noxious heat stimulation. Acute and chronic denervation of the sciatic and saphenous nerves also inhibited the heat-evoked iSP release and edema remarkably. Intraplantar injection of SP evoked an increase in paw volume in dose-dependent manner. This increasing effect of SP on paw volume was more substantial than that produced by histamine. Simultaneous treatment with stem bromelain and emorfazone decreased significantly the heat-evoked iSP release and edema. These results suggest that 1) SP produced by noxious heat stimulation in the periphery may be released from the afferent fibers with small-diameter, 2) bradykinin may intervene in this SP release and 3) SP released in the periphery may be closely related to the edema formation of the thermal injury reaction.

Is a calmodulin-opiopeptide interaction related to the mechanism of opioid action?

The effect of several opioids: methadone, etorphine, beta-endorphin and D-ala2met enkephalin on Ca++/calmodulin stimulation of enzyme activities either in pure solution (cyclic nucleotide phosphodiesterase) or in striatal membranes (protein kinases in synaptic membranes) were compared to see if a direct opioid/calmodulin interaction could eliminate the stimulation of enzyme activity as part of the mechanism by which opioids alter ion flow and neurotransmitter release. In other experiments, in which endogenous phosphorylation of proteins in striatal synaptic membranes was altered by opioid treatments, the possibility of restoring protein kinase activity to normal levels in the membrane preparation by supplementation with calmodulin at optimal Ca++ concentration was examined. Some opioids (methadone and D-ala2met enkephalin) did not inhibit calmodulin-stimulated phosphodiesterase, which suggests that they were not able to bind to calmodulin. In addition, it was not possible to restore decreases in protein kinase activity to normal levels by adding calmodulin to the assay in the presence of optimal Ca++. We conclude that a direct binding of opioids to calmodulin is not a general mechanism of opioid action, although the binding may participate in the action of some neuropeptides, including beta-endorphin.