Ryuichiro Ando

Evidence that nitric oxide-glutamate cascade modulates spinal antinociceptive effect of morphine: a behavioural and microdialysis study in rats

We evaluated the ability of spinally administered nitric oxide (NO) synthase inhibitor to modulate antinociceptive action of intrathecal (i.t.) morphine in rats by measuring the early and late phases of flinching and licking/biting in the formalin test. To determine the contribution of spinal NO and glutamate, we measured the release of NO metabolites (nitrite/nitrate) and glutamate from the spinal cord in rats, using a microdialysis probe placed in the lumbar space. The i.t. administration of NG-nitro L-arginine methyl ester (L-NAME) produced a dose-dependent reduction in the number of flinches during the late phase, whereas there were no significant alterations in the late phase licking/biting, and early phase flinching and licking/biting. Spinal administration of morphine at low doses produced a significant antinociceptive activity in the early and late phases of the flinching behaviour, whereas higher doses of morphine were required to obtain a significant effect in the licking/biting behaviour during both phases. Combination of L-NAME with morphine resulted in an enhanced reduction in the early and late phase flinching. Enhanced antinociceptive activity was observed in the late phase licking/biting by i.t. combined administration of L-NAME (400 nmol) and morphine (1.25 nmol). In the present study, we have confirmed our prior results that injection of formalin (5.0%) into the plantar surface of the paw evoked a biphasic spinal release of nitrite/nitrate and a transient release of glutamate. Formalin-evoked release of nitrite/nitrate and glutamate was also reduced markedly by i.t. combined administration of L-NAME and morphine. These behavioural and biochemical results suggest that i.t. administered L-NAME may enhance morphine-induced antinociception through an increased inhibition of nitrite/nitrate and glutamate releases evoked by formalin injection at the spinal cord level.

The role of spinal nitric oxide and glutamate in nociceptive behaviour evoked by high-dose intrathecal morphine in rats.

&NA; Injection of high‐dose of morphine into the spinal lumbar intrathecal (i.t.) space of rats elicits a nociceptive behavioural syndrome characterized by periodic bouts of spontaneous agitation and severe vocalization. The induced behavioural response such as vocalization and agitation was observed dose‐dependently by i.t. administration of morphine (125–500 nmol). Pretreatment with naloxone (s.c. and i.t.), an opioid receptor antagonist, failed to reverse the morphine‐induced behavioural response. The excitatory effect of morphine was inhibited dose‐dependently by pretreatment with 3‐((+)2‐carboxy‐piperazin‐4‐yl)‐propyl‐1‐phosphonic acid (CPP), a competitive N‐methyl‐d‐aspartate (NMDA) receptor antagonist and MK‐801, a non‐competitive NMDA receptor antagonist. The non‐selective nitric oxide (NO) synthase inhibitor NG‐nitro l‐arginine methyl ester (l‐NAME) inhibited dose‐dependently the behavioural response to high‐dose i.t. morphine (500 nmol), whereas d‐NAME was without affecting the response to high‐dose i.t. morphine. In the present study, we measured NO metabolites (nitrite/nitrate) in the extracellular fluid of rat dorsal spinal cord using in vivo microdialysis. The i.t. injection of morphine (500 nmol) evoked significant increases in NO metabolites and glutamate from the spinal cord. Not only NO metabolites but also glutamate released by high‐dose morphine were reduced significantly by pretreatment with l‐NAME (400 nmol). Pretreatment with CPP and MK‐801 showed a significant reduction of the NO metabolites and glutamate levels elevated by high‐dose i.t. morphine. These results suggest that the excitatory action of high‐dose i.t. morphine may be mediated by an NMDA–NO cascade in the spinal cord.

Characterization of p-chloroamphetamine-induced penile erection and ejaculation in anesthetized rats.

Methodological shortcomings present in elicitation of male sexual reflexes in anesthetized animals. The present study has demonstrated, however, that intraperitoneal (i.p.) injection of p-chloroamphetamine (PCA), an indirect serotonin (5-HT) agonist, elicited simultaneously both penile erection and ejaculation in anesthetized rats. PCA (2.5-10.0 mg/kg, i.p.) caused an intermittent cluster of genital responses consisting of penile erection, glans erections, and penile cups, which closely resembles the response observed during the ex copula tests in unanesthetized rats. Measurements of intracavernous penile pressure showed that rhythmic changes in penile pressure were produced by PCA, together with glans erections and penile cups. PCA also caused a frequent ejaculations and the weighing of ejaculate accumulated over 0.5 hr was increased in a bell-shaped pattern, and the maximum effect was observed at 5.0 mg/kg. Pretreatment with p-chlorophenylalanine, a serotonin (5-HT)-synthesis inhibitor, significantly inhibited the expression of PCA-induced penile erection and ejaculation, while acute spinal transection at thoracic level did not affect the sexual responses. These results indicate that PCA-induced penile erection and ejaculation in anesthetized rats are mainly produced by the release of 5-HT, which is limited to the lower spinal cord and/or the peripheral sites. Furthermore, the sexual responses can be easily and reliably elicited by administration of PCA, which may be useful for the study of the mechanisms underlying male sexual functions.

Immunohistochemical determination of rat spinal cord substance P, and antinociceptive effect during development of thiamine deficiency

During 30 days of thiamine deficiency (TD) feeding, the rat antinociceptive effect (pain threshold) to noxious heat stimulation was significantly increased in proportion to the decrease substance P (SP) fluorescent intensity in the spinal cord. Only a single injection of thiamine HCl (0.5 mg/kg, s.c.) on the early treatment day during TD feeding effectively reversed the analgesic effect to the pair-fed control level. Whereas this reversal effect by thiamine treatment was not found if this treatment was done on the relatively late day. However, either treatment day, except muricide, complete disappearance of various animal behaviours induced by TD was found. These results indicate that, after certain degree of TD development, TD-induced behavioral effects might be reversible, but the afferent nerve fibers might be irreversibly damaged, probably by the similar mechanism as found for an excitotoxin(s) mediated injury in the certain brain region(s). The results also suggest a possibility that SP and an excitotoxin, glutamate, in the dorsal part of the spinal cord greatly contribute to the pain transmission induced by noxious heat stimulation.

α2-Adrenoceptor antagonists: Effects on ejaculation, penile erection and pelvic thrusting behavior in dogs

We previously reported that systemic administration of yohimbine, an alpha2-adrenoceptor antagonist, exerts a biphasic effect (stimulating and suppressing) on ejaculation in dogs, when this function is analyzed using the amount of ejaculated semen in response to genital stimulation. To clarify the effect of alpha2-adrenoceptor blockade on male sexual function, we investigated the effects of four selective alpha2-adrenoceptor antagonists, rauwolscine, idazoxan, RX821002 and mydaglizole, on sexual responses (ejaculation, penile erection and pelvic thrusting behavior) elicited by manual penile stimulation in dogs. Rauwolscine (intraperitoneal, 30 min before the testing) caused a biphasic effect on ejaculation; the amount of ejaculated semen produced by the stimulation was significantly increased by the lower doses (0.1 and 0.3 mg/kg), whereas it was decreased by the higher doses (1.0 and 2.0 mg/kg). The higher doses of rauwolscine also markedly inhibited both penile erection and pelvic thrusting behavior. Idazoxan and RX821002, at doses of 0.1 and 0.3 mg/kg, caused a significant increase in the amount of ejaculated semen without affecting other sexual functions. RX821002 (2.0 mg/kg), but not idazoxan (2.0 mg/kg), moderately inhibited both penile erection and pelvic thrusting behavior. Mydaglizole, a peripherally acting alpha2-adrenoceptor antagonist, did not affect the sexual responses at any doses (0.1-4.0 mg/kg). In the ejaculatory declining test, all alpha2-adrenoceptor antagonists (0.1 mg/kg), except for mydaglizole, completely prevented the decrease in ejaculatory capacity produced by antecedent ejaculation. These results indicate that, though the range of the effective dose is narrow, the alpha2-adrenoceptor antagonists that can block the central alpha2-adrenoceptors have the stimulatory effects on ejaculatory function. The difference of the sexual effects may be based on the action except for the alpha2-adrenoceptor blockade.

Biphasic effects of yohimbine on the ejaculatory response in the dog.

The effects of various doses (0.01-1.00 mg/kg) of yohimbine, an alpha-2 adrenoceptor antagonist, on the erectile and ejaculatory response elicited by manual penile stimulation were investigated in male dogs. Systemic administration of yohimbine caused a biphasic effect on ejaculatory response; the amount of ejaculate produced by the genital stimulation (for 5 min) was dose-dependently increased by low doses (0.01-0.10 mg/kg) of yohimbine, whereas it was decreased by the highest dose (1.00 mg/kg) of yohimbine. The erectile potency was attenuated only, by the highest dose of yohimbine. The most effective dose (0.10 mg/kg) of yohimbine on ejaculation did not affect the duration of penile erection after removing the genital stimulation. In a stereoisomers testing, the stimulatory effect on ejaculation was also observed by rauwolscine, an alpha-2 adrenoceptor antagonist (0.03 and 0.10 mg/kg), but not by corynanthine, an alpha-1 adrenoceptor antagonist (0.10 and 0.30 mg/kg). These results suggest that yohimbine at low doses specifically facilitate the ejaculatory response through the blockade of the alpha-2 adrenoceptors. This study also indicates that the effects of yohimbine on male genital responses vary with its dosage used.

Spinal actions of GR73632, a novel tachykinin NK1 receptor agonist.

Behavioral characterization of GR73632, a newly synthesized tachykinin NK1 receptor agonist, was examined in mice. Intrathecal (IT) injection of GR73632 in the spinal subarachnoid space of mice elicited a dose-dependent behavioral syndrome, consisting of scratching, biting and licking. The time course of the response to GR73632 was almost similar to that of substance P. GR73632 evoked much more licking and biting than did substance P, that in turn caused less scratching. GR73632 was approximately 200-fold more potent than substance P in inducing the characteristic behavioral response. The GR73632-induced behavioral response was inhibited by IT co-administration of CP-96,345, a non-peptide NK1 receptor antagonist, but not its inactive enantiomer CP-96,344. CP-96,345, co-injected IT with substance P, also inhibited the behavioral response to substance P. These results demonstrate that the scratching, biting and licking response induced by IT GR73632 may be mediated by the NK1 receptor in the spinal cord. These findings suggest that GR73632 may be useful as a tachykinin NK1 receptor agonist and also for evaluating spinal pharmacological activities of NK1 receptor antagonists.

[Effects of capsaicin on spontaneous unit discharges in medial thalamic single neurons of cats (author's transl)].

The effect of capsaicin was studied in gallamine triethiodide immobilized adult cats. Single neurons were recorded from the medial thalamus with a stainless steel microelectrode. Out of 21 neurons recorded in this experiment, 10 were responsive to both nociceptive (pinch) and non-nociceptive (hair bending and/or tapping) stimuli. Six neurons were responsive to only non-nociceptive stimuli and 5 were not responsive to these stimuli. Out of 10 neurons responding to both nociceptive and non-nociceptive stimuli, 9 were responsive to both bradykinin (3 microgram) and capsaicin (3 microgram). Out of 6 neurons responding to only non-nociceptive stimuli, 5 were not responsive to either bradykinin and capsaicin. All neurons responding to bradykinin were also responsive to capsaicin. The latency for bradykinin and capsaicin was 7.64 +/- 1.12 sec and 0.97 +/- 0.07 sec, respectively. The increased in firing frequency produced by capsaicin was depressed by morphine. However, these depressant effects of morphine on single unit activity were antagonized by naloxone.

Involvement of spinal NK2 and NMDA receptors in aversive behavior induced by intra-arterial injection of capsaicin

The spinal processing by which intra-arterial injection of capsaicin (CAP) induces vocalization response (VOR) was investigated in guinea pigs. Intrathecal pre-treatment with CP-96,345 (a selective NK(1) receptor antagonist, 50 nmol) did not affect the CAP-induced VOR. However, significant attenuation of the VOR was observed by intrathecal pre-treatment with a selective NK(2) receptor antagonist MEN-10,376 (40 nmol) accompanied with a significant change in the response modality. MK-801 [an N-methyl-D-aspartate (NMDA) receptor antagonist, 20 and 40 nmol] inhibited the CAP-induced VOR dose-dependently without affecting the response modalities. Furthermore, intrathecal co-treatment with 40-nmol MEN-10,376 and 40-nmol MK-801 resulted in a marked inhibitory effect on the VOR followed by a significant alteration of response modalities. Intrathecal pre-treatment with neurokinin A (NKA; a tachykinin NK(2) receptor agonist, 1 nmol) enhanced the CAP-induced VOR. These behavioral results suggested that spinal NK(2) and NMDA receptors might have priority over NK(1) receptors in the spinal processing of nociceptive information from the CAP-sensitive nociceptor.

Chronic clonidine treatment and its termination: Effects of penile erection and ejaculation in the dog

The effects of chronic administration (4 weeks) of the alpha-2 adrenoceptor agonist clonidine (CL) and its termination on penile erection and ejaculation were investigated in male dogs. Penile erection and ejaculation were elicited by manual penile stimulation (for 5 min). CL (10 micrograms/kg/hr, s.c.) was delivered via osmotic minipump (Alza, 2ML-4). 3 or 7 days after the minipump implantation, CL caused a significant decrease in the amount of ejaculate produced by the genital stimulation without affecting the erectile potency. Ejaculatory ability returned to pretreatment levels despite continued CL administration, becoming evident in tests 14 days after initiation of treatment. Further, chronic CL (23 days) antagonized the inhibitory effects of acute administration of CL (0.05 mg/kg, i.p.). These data indicate tolerance to continued delivery of low doses as well as to acute administration of a higher dose. In the acute drug experiments, the ejaculatory inhibition elicited by CL (0.05 mg/kg, i.p.) was completely antagonized by pretreatment with yohimbine (0.05 and 0.10 mg/kg, i.p.), an alpha-2 adrenoceptor antagonist, but not with naloxone (1.0 mg/kg, i.p.), an opioid receptor antagonist. Furthermore, DG-5128 (1.0 and 2.0 mg/kg, i.p.), a selective alpha-2 adrenoceptor antagonist that poorly penetrates the blood-brain barrier, failed to antagonize the CL-induced ejaculatory inhibition. This study suggests that functional alterations in the central alpha-2 adrenoceptor mechanism may be related to the changes in the ejaculatory capacity during chronic treatment with CL.