Behavioural and antinociceptive effects of intrathecally injected substance P analogues in mice

The antinociceptive effects and antagonism of substance P (SP)-induced behaviour were examined after intrathecal injections in mice. When coadministered with SP, the SP analogues attenuated the SP-induced behaviour to various extents with [D-Arg1,D-Pro2,4,D-PHe7,D-His9]SP as the most potent antagonist. Physalaemin and eledoisin brought about a SP-like behavioural response. [D-Arg1,D-Trp7,9,Leu11]SP inhibited the response to eledoisin but not that to physalaemin. The SP analogues substituted with D-Trp7,9 and with Leu or Val in position 11 produced the strongest antinociceptive effects. Pretreatment with naloxone (5 mg/kg, i.p.) abolished the antinociceptive effects of [D-Trp7,9,Leu11]SP, implying that opiate receptors in the spinal cord mediate at least in part the antinociceptive effects. Animals under pentobarbital anesthesia showed a potentiated antinociceptive effect in the tail-flick test. In conclusion, we found that the SP analogues tested exerted varying degrees of antinociception, and that this was probably at least in part mediated by activation of spinal opiate receptors, whereas the behavioural antagonism was probably due to the blockade of spinal SP receptors.     

Behavioural effects of intrathecally injected tachykinins in rats with peripheral nerve transection

Summary The effect of unilateral sciatic nerve transection on behavioural responses produced by intrathecal administration of substance P (SP), neurokinin A, eledoisin and physalaemin was investigated in the rat. The injection of SP (3 nmol/rat) into the subarachnoid space was followed by reciprocal scratching, biting and licking of the fore- and hind-limbs. There was no observable difference in the behavioural response to SP between rats with nerve transection and sham operated rats at 5 days after operation. Whereas at 10, 20, and 30 days after nerve transection the response to SP was significantly increased as compared with sham operated rats. This phenomenon was also observed with neurokinin A (1.5, 3.0 and 6.0 nmol/rat), eledoisin (0.05 and 0.10 nmol/rat) and physalaemin (0.05 and 0.10 nmol/ rat) at 10 days after operation. Ipsilateral depletion of SP from the lumbar (L4-L6) spinal cord was observed at 5, 10, 20, and 30 days after the unilateral transection of the sciatic nerve. These results suggest that sciatic nerve transection may produce an increased response to tachykinins through an enhanced sensitivity of tachykinin receptors in the lumbar cord. Send offprint requests to T. Sakurada     

Intrathecally injected Ile-Pro-Ile, an inhibitor of membrane ectoenzyme dipeptidyl peptidase IV, is antihyperalgesic in rats by switching the enzyme from hydrolase to synthase functional mode to generate endomorphin 2

We have found recently that membrane-bound dipeptidyl peptidase IV (DPP-IV) generated extracellularly immunoreactive endomorphin-2 from Tyr-Pro precursor in a depolarisation-sensitive manner in rat isolated L4,5 dorsal root ganglia when the enzyme was switched to synthase mode by the hydrolase inhibitor Ile-Pro-Ile. Presently, we induced hyperalgesia in rats by injecting carrageenan into the right hindpaw and measured the reduction in nociceptive threshold (hyperalgesia) to pressure (Randall–Selitto test). The hyperalgesia, peaking at 180 min after injection, was fully reversed by intrathecal administration of 30 nmol/rat Ile-Pro-Ile. The antihyperalgesic action was antagonized by s.c. naloxone (1 mg/kg) and intrathecally injected specific antiserum to endomorphin-2 indicating that the opioid receptor-mediated effect was produced by an endogenously generated endomorphin-2-like immunoreactive substance. Intrathecal Ile-Pro-Ile was ineffective as an analgesic in the acute nociceptive test such as the rat tail-flick, whereas endomorphin-2 (EC₅₀ = 13.3 nmol/rat), endomorphin-1 (6.8 nmol/rat), morphine (0.11 nmol/rat) and DAMGO (0.0059 nmol/rat) exerted opioid receptor-mediated analgesia given by the same route. We concluded that carrageenan-induced C-fiber barrage (wind-up) may create ideal conditions for the de novo synthesis of endomorphin-2 in rat spinal cord dorsal horns if the DPP-IV enzyme is switched to the synthase functional mode by Ile-Pro-Ile.     

Antinociceptive effects of intrathecally injected cholinomimetic drugs in the rat and cat

Rats chronically implanted with intrathecal catheters displayed a dose-dependent increase in the hot-plate, tail-flick response latencies, and decreased the magnitude of the writhing response following the injection of certain cholinomimetics into the subarachnoid space through the indwelling catheter. The structure-activity relationship for these agents is oxotremorine greater than carbachol greater than acetylcholine + physostigmine much much greater than acetylcholine = nicotine-HCl = 0. Atropine, but not naloxone, strychnine, picrotoxin, curare or methysergide and phentolamine, reversed the antinociceptive effect. This suggests the involvement of muscarinic cholinergic mechanisms. Experiments with intrathecal injection of carbachol into the spinal subarachnoid space of cats fitted with intrathecal catheters also revealed a potent antinociceptive effect which was completely antagonized by atropine. The effect was somatotopically limited with the skin surfaces innervated by cord segments nearest the catheter tip showing the most significant effect with the shortest latency of onset. This observation, together with the absence of changes in general reflex motor function or postural control further indicated a selective spinal effectiveness of muscarinic agonists after low dose intrathecal administration.     

Similar behavioural effects of 5-hydroxytryptamine and substance P injected intrathecally in mice

Intrathecal injection of 5-hydroxytryptamine (5-HT) or of substance P in mice elicited a behavioural syndrome consisting of reciprocal hindlimb scratching and biting or licking, directed towards the caudal parts of the body. 5-Hydroxtryptamine elicited more scratching than did substance P, which in turn caused a greater number of biting or licking responses. The 5-HT-induced responses were mimicked by 5,6-dihydroxytryptamine and inhibited by the 5-HT receptor blocker metergoline. The present findings indicate that 5-HT, injected intrathecally, may have similar effects as substance P in stimulating sensory pathways in the spinal cord.     

The dipeptidyl peptidase IV (CD26, EC 3.4.14.5) inhibitor vildagliptin is a potent antihyperalgesic in rats by promoting endomorphin-2 generation in the spinal cord

We have reported previously that the dipeptidyl peptidase IV inhibitor Ile-Pro-Ile had an antihyperalgesic action in rats when given intrathecally in the carrageenan-induced hyperalgesia, as detected by the Randall–Selitto test. Vildagliptin, a non-peptide inhibitor of the same enzyme, which is already on the market as an “euglycemic” agent in diabetics, has a slightly more potent and more sustained antihyperalgesic effect in the same test when given by the same route. The action of 3 nmol/rat vildagliptin could be antagonized by subcutaneous naltrexone (0.5 mg/kg) pretreatment, or by intrathecally co-administered specific antiserum to endomorphin-2. Thus, the antihyperalgesia by vildagliptin, similarly to Ile-Pro-Ile, was opioid receptor-mediated and could be attributed to the promotion of endomorphin-2 generation in rat spinal cord dorsal horn. Furthermore, vildagliptin (1 mg/kg) is a potent antihyperalgesic also when given subcutaneously.     

Enhancement of the hypotensive effects of intrathecally injected endocannabinoids by the entourage compound palmitoylethanolamide

The intrathecal (i.t.) injection of 50 and 100 nmol anandamide to urethane anesthetized rats induced a dose-dependent decrease in the mean blood pressure (− 10.6 ± 1.6 mmHg and − 15.0 ± 1.7 mmHg, respectively; n = 6) whereas a lower dose of this endocannabinoid (25 nmol) was devoid of effect. Similar responses were obtained both with the non-metabolizable analog methanandamide and with the endocannabinoid N-arachidonoyldopamine. When the sub-effective dose (25 nmol) of each compound was co-injected with palmitoylethanolamide (100 nmol), significant decreases in the blood pressure were observed (− 12.3 + 1.3 mmHg for anandamide; − 12.1 ± 0.8 mmHg for methanandamide; − 12.1 ± 0.8 mmHg for N-arachidonoyldopamine; n = 4–6). Palmitoylethanolamide also enhanced the hypotensive responses to the 50 nmol-dose of both anandamide and methanandamide. The hypotensive response induced by co-administration of palmitoylethanolamide and 25 nmol anandamide was prevented both by the cannabinoid CB₁ receptor antagonist SR 144716A (20 nmol; i.t.) and by the vanilloid TRPV1 receptor antagonist capsazepine (20 nmol; i.t.) and enhanced by pretreatment with URB602 (3.5 nmol; i.t.), a putative inhibitor of palmitoylethanolamide degradation. These results suggest that in the spinal cord palmitoylethanolamide acts as an entourage compound for the hypotensive effects of i.t. administered endocannabinoids. The facilitative action of palmitoylethanolamide affects the vanilloid TRPV1 as well as the cannabinoid CB₁ receptor-mediated effects of endocannabinoids on the blood pressure control.     

Antinociceptive effects of ginsenosides injected intracerebroventricularly or intrathecally in substance P-induced pain model

We have examined the effects of several ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg3) administered intracerebroventricularly (i.c.v.) or intrathecally (i.t.) on the nociceptive behavior induced by substance P (0.7 microg) injected i.t. Among the several ginsenosides studied, Rb2, Rc, Rd, and Re, but not Rb1, Rf, Rg1 and Rg3, treated i.c.v. (50 microg) attenuated the nociceptive behavior induced by substance P injected i.t. On the other hand, we found that i.t. treatment with 50 microg of Rb1, Rb2, Rd, or Rf effectively attenuated the nociceptive behavior induced by i.t. injected substance P. However, the i.t. treatment with the same doses of Rc, Re, Rg1 or Rg3 was not effective for antagonizing i.t. injected substance P-induced nociceptive behavior. Our results show that ginsenosides Rb2, Rc, Rd, or R2 injected supraspinally exert a antinociceptive effect in the substance P-induced pain model. Furthermore, Rb1, Rb2, Rd, or Rf treated spinally produce antinociception in the substance P-induced pain model.     

硫辛酰维格列汀对2型糖尿病（T2DM）大鼠降糖作用的研究

目的观察硫辛酰维格列汀对2型糖尿病大鼠血糖的影响。方法选用雄性Wistar大鼠,高脂高糖饲料喂养4周后小剂量注射链脲佐菌素建立2型糖尿病模型,随机将大鼠分为正常对照组、糖尿病模型组、胰岛素糖尿病组、硫辛酰维格列汀糖尿病组。分别测定给药后0、0．5、1、2、3、4、5、6、7、8、9、10、12、24h的血糖变化。结果注射链脲佐菌素72h后造模大鼠血糖均明显高于注射前（P％0．05）和正常对照组（P〈0．05）。与正常对照组和糖尿病模型组相比,胰岛素糖尿病组在注射胰岛素后0．5～4h,血糖变化差异有统计学意义（P〈0．05）;硫辛酰维格列汀糖尿病组在灌胃给药3～10h,血糖变化差异有统计学意义（P〈0．05）。结论硫辛酰维格列汀和胰岛素有不同程度的降血糖作用,相比胰岛素,硫辛酰维格列汀降糖作用更加持久。     

Effects of muscimol, amphetamine, and DAMGO injected into the nucleus accumbens shell on food-reinforced lever pressing by undeprived rats

Previous studies have shown that large increases in food intake in nondeprived animals can be induced by injections of both the GABA_A agonist muscimol and the μ-opioid agonist DAMGO into the nucleus accumbens shell (AcbSh), while injections of the catecholamine agonist amphetamine have little effect. In the current study we examined whether injections of these drugs are able to increase food-reinforced lever pressing in nondeprived rats. Twelve subjects were trained to lever press on a continuous reinforcement schedule while food deprived and were then tested after being placed back on ad libitum feeding. Under these conditions, responding was markedly increased by injections of either muscimol or DAMGO, although the onset of the effects of the latter drug was delayed by 30-40 min. In contrast, amphetamine injections failed to increase reinforced lever pressing, although they did enhance responding on a non-reinforced lever, presumably reflecting alterations in behavioral activation. These results demonstrate that stimulation of GABA_A and μ-opioid receptors within the AcbSh is able to promote not only food intake, but also food-directed operant behavior. In contrast, stimulation of AcbSh dopamine receptors may enhance behavioral arousal, but does not appear to specifically potentiate behaviors directed toward food procurement.     

Renal effects of intrathecally injected tachykinins in the conscious saline-loaded rat: receptor and mechanism of action

1. The effects of intrathecally (i.t.) injected substance P (SP), neurokinin A (NKA), [β-Ala⁸]NKA (4–10) and [MePhe⁷]neurokinin B (NKB) at T₁₃ thoracic spinal cord level were investigated on renal excretion of water, sodium and potassium in the conscious saline-loaded rat. Antagonists selective for NK₁ (RP 67580), NK₂ (SR 48968) and NK₃ (R 820; 3-indolylcarbonyl-Hyp-Phg-N(Me)-Bzl) receptors were used to characterize the spinal effect of SP on renal function.
2. Saline gavage (4.5% of the body weight) enhanced renal excretion of water, sodium and potassium over the subsequent hour of measurement. Whereas these renal responses were not affected by 0.65 nmol SP, the dose of 6.5 nmol SP blocked the natriuretic response (aCSF value 3.9±0.8; SP value 0.7±0.3 μmol min⁻¹, P<0.01) as well as the renal excretion of water (aCSF value 48.9±5.8; SP value 14.5±4.0 μl min⁻¹, P<0.01) and potassium (aCSF value 4.8±0.6; SP value 1.5±0.6 μmol min⁻¹, P<0.01) at 30 min post-injection. SP had no significant effect on urinary osmolality. The SP-induced renal inhibitory effects during the first 30 min were abolished in rats subjected to bilateral renal denervation 1 week earlier or in rats injected i.t. 5 min earlier with 6.5 nmol RP 67580. In contrast, the co-injection of SR 48968 and R 820 (6.5 nmol each) did not affect the inhibitory responses to SP. On their own, these antagonists had no direct effect on renal excretion function. Since SP induced only transient changes in mean arterial blood pressure (−18.8±3.8 mmHg at 1 min and +6.3±2.4 mmHg at 5 min post-injection), it is unlikely that the renal effects of SP are due to systemic haemodynamic changes.
3. NKA (6.5 nmol but not 0.65 nmol) produced a transient drop in renal excretion of water (aCSF value 31.2±5.1; NKA value 11.3±4.2 μl min⁻¹, P<0.05), sodium (aCSF value 1.7±0.8; NKA value 0.4±0.2 μmol min⁻¹, P<0.05) and potassium (aCSF value 4.1±0.7; NKA value 1.5±0.4 μmol min⁻¹, P<0.05) at 15 min post-injection. However, the same doses (6.5 nmol) of selective agonists for tachykinin NK₂ ([β-Ala⁸]NKA(4-10)) and NK₃ ([MePhe⁷]NKB) receptors were devoid of renal effects.
4. This study provided functional evidence that tachykinins may be involved in the renal control of water and electrolyte excretion at the level of the rat spinal cord through the activation of NK₁ receptors and the sympathetic renal nerve.

     

GABA_A受体对鞘内注射氧化槐定碱镇痛作用的影响

观察氧化槐定碱(oxysophoridine,OSR)鞘内注射(intrathecal injection,ith)的镇痛作用并探讨与GABAA受体相关的作用机制。采用小鼠温浴法测定痛阈,观察OSR(ith)的镇痛作用及GABAA受体激动剂和拮抗剂对OSR镇痛作用的影响;采用免疫组化法检测OSR对小鼠脊髓背角GABAARα1蛋白表达的影响。结果显示,OSR(12.5和6.25 mg·kg-1,ith)能明显延长热甩尾潜伏期(P<0.05,P<0.01),痛阈提高率达68.45%。GABA和蝇蕈醇(MUS)与OSR有协同镇痛作用,印防己毒素(PTX)和荷包牡丹碱(BIC)能拮抗OSR的镇痛作用。OSR(12.5 mg·kg-1,ith)可使脊髓背角GABAARα1表达明显增多(P<0.01)。结果提示,OSR(ith)具有明显的镇痛作用,脊髓GABAA受体参与了OSR的镇痛机制。     

The antihyperalgesic and selective analgesic effects of neurotropin]

In experiments of albino rats neurotropin injection was shown to increase the latency of the tail-flick test in response to the nociceptive thermal stimulus and didn't change the threshold of the test in response to the nociceptive electroskin stimulus. The administration of the antiserum to beta-endorphin lowered the threshold and the latency of the tail-flick test in response to both stimuli. The preliminary administration of neurotropin reduced the hyperalgesic effect of the antiserum on both nociceptive stimuli.     

The antihyperalgesic effects of the T-type calcium channel blockers ethosuximide, trimethadione, and mibefradil

The purpose of the present study was to explore the analgesic effects of the low voltage-activated T-type Ca2+ channel blockers ethosuximide, trimethadione, and mibefradil in persistent and acute nociceptive tests. The anticonvulsant effects of the compounds were also determined in the intravenous pentylenetetrazol seizure model. Following intraperitoneal administration, ethosuximide and trimethadione dose-dependently reversed capsaicin-induced mechanical hyperalgesia. Similarly, the highest dose of mibefradil tested (30 microg, intracisternal) reversed capsaicin-induced mechanical hyperalgesia. Ethosuximide and mibefradil produced statistically significant analgesic effects in both early and late phase formalin-induced behaviors and trimethadione reduced late phase behaviors. Additionally, ethosuximide and trimethadione produced antinociceptive effects in the rat-tail flick reflex test. In contrast, following intracisternal administration, mibefradil had no effect in the tail flick reflex test. In addition, the anticonvulsants ethosuximide and trimethadione increased the doses of pentylenetetrazol required to produce both first twitch and clonic seizures. In contrast however, mibefradil had no anticonvulsant effect. The present results demonstrate that the clinically used anticonvulsants ethosuximide and trimethadione provide analgesic effects at doses, which are anticonvulsant. Furthermore, the data further supports the idea that T-type Ca2+ channels may be important targets for treating persistent pain syndromes.     

The effects of locally injected antibiotic on carrageenan-induced granuloma in rats

Indomethacin (0.5 mg/100 g b.w./day) and chloramphenicol (0.5 mg or 15 mg/100 g b.w./day) were tested for their anti-inflammatory effects on 7th day carrageenan-induced granuloma formation. Neither of the drugs modified granuloma or pouch wall weight but they decreased the exudate and the cluster of dead cells. Indomethacin and chloramphenicol decreased glucosamine in the dead cell granuloma fraction and increased the level of collagen in the pouch wall. The drugs differed in their inhibitory effect on lysozyme and prostaglandin E₂ accumulation in the exudate. The increase in collagen was related to a drop in the level of prostaglandin E₂ which seems to regulate collagen deposition in the granuloma. However, the prostaglandin E₂-lysozyme correlation — which was only significant with chloramphenicol — suggests a mode of action for chloramphenicol different from that of indomethacin. Chloramphenicol could act by a myelodepressive and/or chemotactic effect. The effects of chloramphenicol on the macrophages are discussed.     

The antinociceptive effects of endomorphin-1 and endomorphin-2 in diabetic mice

The antinociceptive effects of endomorphin-1 and endomorphin-2, endogenous mu-opioid receptor agonists, were examined using the tail-flick test in non-diabetic and diabetic mice. Endomorphin-1, at doses of 1 to 10 microg, i.c.v., and endomorphin-2, at doses of 3 to 30 microg, i.c.v., each dose dependently inhibited the tail-flick response in both non-diabetic and diabetic mice. There was no significant difference between the antinociceptive effects of endomorphin-1 in non-diabetic mice and diabetic mice. The antinociceptive effect of endomorphin-2 was greater in non-diabetic mice than in diabetic mice. In non-diabetic mice, the antinociceptive effects of endomorphin-1 and endomorphin-2 were significantly reduced by beta-funaltrexamine, a mu-opioid receptor antagonist, and naloxonazine, a selective mu(1)-opioid receptor antagonist, but not by naltrindole, a delta-opioid receptor antagonist, or nor-binaltorphimine, a kappa-opioid receptor antagonist. In diabetic mice, the antinociceptive effect of endomorphin-2 was significantly reduced by beta-funaltrexamine and naloxonazine. However, these micro-opioid receptor antagonists had no significant effect on the antinociceptive effect of endomorphin-1 in diabetic mice. The antinociception induced by endomorphin-1 in diabetic mice was significantly reduced by naltrindole and 7-benzylidenenaltrexon, a selective delta(1)-opioid receptor antagonist, administered i.c.v. However, nor-binaltorphimine had no significant effect on the antinociceptive effects of endomorphin-1 and endomorphin-2 in diabetic mice. These results indicate that the antinociceptive effects of endomorphin-1 and endomorphin-2 in non-diabetic mice are mediated through the activation of mu(1)-opioid receptors, whereas in diabetic mice, endomorphin-1 and endomorphin-2 may produce antinociception through different actions at delta(1)- and mu(1)-opioid receptors, respectively.     

The antinociceptive effects of alpha7 nicotinic agonists in an acute pain model

Nicotinic receptors have been found to play a role in modulating pain transmission in the CNS. Activation of cholinergic pathways by nicotine and nicotinic agonists has been shown to elicit antinociceptive effects in a variety of species and pain tests. The involvement of alpha(7) nicotinic receptors in nicotinic analgesia was assessed after spinal (i.t.) and intraventricular (i.c.v.) administration in mice. Dose-dependent antinociceptive effects were seen with the alpha(7) agonist choline after spinal and supraspinal injection using the tail-flick test. Furthermore, alpha(7) antagonists MLA and alpha-BGTX significantly blocked the effects of choline. Dihydro-beta-erythroidine and mecamylamine failed to block choline-induced antinociception. These results strongly support the involvement of alpha(7) subunits in choline's antinociceptive effects. DMXB and 4-OH-DMXB, partial alpha(7) agonists, failed to elicit a significant antinociceptive effect. However, they blocked choline-induced antinociception in a dose-dependent manner following i.t. injection. This antagonism is probably related to their partial agonistic properties of the alpha(7) receptors. These studies suggest that activation of alpha(7) receptors in the CNS elicits antinociceptive effects in an acute thermal pain model.     

Evidence for adenosine- and serotonin-mediated antihyperalgesic effects of cizolirtine in rats suffering from diabetic neuropathy

Cizolirtine is a novel non-opioid drug which demonstrated antinociceptive activity in numerous pain models in rodents. Yet, its mechanism of action remains unknown. Several lines of evidence support the idea that adenosine (ADO) and serotonin (5-HT) modulate nociceptive signaling. Our study aimed at investigating whether these neuroactive molecules could be implicated in the mechanism of action of cizolirtine. Cizolirtine-induced antihyperalgesia was compared before and after pretreatment with ADO A(1)-A(2A) and 5-HT(1B/1D) receptor ligands in rats rendered diabetic by streptozotocin pretreatment and suffering from neuropathic pain. Cizolirtine alone (30-80 mg/kg, i.p.) significantly increased mechanical nociceptive thresholds. Acute pretreatment with the A(1)-A(2A) receptor antagonist caffeine (5 mg/kg, i.p.) or the 5-HT(1B/1D) receptor antagonist GR-127,935 (3 mg/kg, i.p.) significantly reduced the antihyperalgesic effects of cizolirtine. Conversely, cizolirtine-induced antihyperalgesia was promoted by pretreatment with either the selective A(1) receptor agonist CPA (0.3 mg/kg, i.p.) or the selective 5-HT(1B) receptor agonist CP-94,253 (3mg/kg, i.p.), and this potentiation was totally prevented by acute pretreatment with respective antagonists. Interestingly, A(1) receptor blockade by DPCPX inhibited the promoting effect of CP-94,253 on cizolirtine-induced antihyperalgesia, suggesting that the adenosine A(1)-mediated step takes place downstream the serotonin 5-HT(1B)-mediated step in the neurobiological mechanisms underlying cizolirtine action.