Spinal Metabotropic Glutamate Receptors (mGluRs) are Involved in the Melittin-induced Nociception in Rats

Intraplantar injection of melittin has been known to induce sustained decrease of mechanical threshold and increase of spontaneous flinchings. The present study was undertaken to investigate how the melittin-induced nociceptive responses were modulated by changes of metabotropic glutamate receptor (mGluR) activity. Changes in paw withdrawal threshold (PWT), number of flinchings and paw thickness were measured at a given time point after injection of melittin (10microgram/paw) into the mid-plantar area of rat hindpaw. To observe the effects of mGluRs on the melittin-induced nociceptions, group I mGluR (AIDA, 100microgram and 200microgram), mGluR1 (LY367385, 50microgram and 100microgram) and mGluR5 (MPEP, 200microgram and 300microgram) antagonists, group II (APDC, 100microgram and 200microgram) and III (L-SOP, 100microgram and 200microgram) agonists were intrathecally administered 20 min before melittin injection. Intraplantar injection of melittin induced a sustained decrease of mechanical threshold, spontaneous flinchings and edema. The effects of melittin to reduce mechanical threshold and to induce spontaneous flinchings were significantly suppressed following intrathecal pre-administration of group I mGluR, mGluR1 and mGluR5 antagonists, group II and III mGluR agonists. Group I mGluR antagonists and group II and III mGluR agonists had no significant effect on melittin-induced edema. These experimental findings indicate that multiple spinal mGluRs are involved in the modulation of melittin-induced nociceptive responses.

Mechanical Hyperalgesia Induced by Blocking Calcium-activated Potassium Channels on Capsaicin-sensitive Afferent Fiber

Small and large conductance Ca2+-activated K+ (SKCa and BKCa) channels are implicated in the modulation of neuronal excitability. We investigated how changes in peripheral KCa channel activity affect mechanical sensitivity as well as the afferent fiber type responsible for KCa channel-induced mechanical sensitivity. Blockade of SKCa and BKCa channels induced a sustained decrease of mechanical threshold which was significantly attenuated by topical application of capsaicin onto afferent fiber and intraplantar injection of 1-ethyl-2-benzimidazolinone. NS1619 selectively attenuated the decrease of mechanical threshold induced by charybdotoxin, but not by apamin. Spontaneous flinching and paw thickness were not significantly different after KCa channel blockade. These results suggest that mechanical sensitivity can be modulated by KCa channels on capsaicin-sensitive afferent fibers.

Multiple 5-Hydroxytryptamine (5-HT) Receptors Are Involved in the Melittin-induced Nociceptive Responses in Rat I. Role of Peripheral 5-HT Receptor

Melittin-induced tonic pain model is characterized by local inflammation, edema, spontaneous flinchings, and sustained mechanical hypersensitivity. These nociceptive responses are mediated through selective activation of capsaicin-sensitive primary afferent fibers by melittin. The present study was undertaken to elucidate the role of peripheral 5-hydroxytryptamine (5-HT) receptors in the melittin-induced nociceptive responses. Changes in mechanical threshold, flinching behaviors and paw thickness were measured in rat intraplantarly injected with melittin (40microgram/paw) alone or treated together with melittin and 5-HT receptor antagonists. WAY-100635 (100microgram & 200microgram/paw), isamoltane hemifumarate (100microgram & 200microgram/paw), methysergide maleate (60microgram, 120microgram & 200microgram/paw) and ICS-205,930 (100microgram & 200microgram/paw) were intraplantarly injected 20 min before melittin injection. All 5-HT receptor antagonists tested in this experiment significantly attenuated the ability of melittin to reduce mechanical threshold and to induce flinching behaviors. 5-HT receptor antagonists, except ICS-205,930, had mild inhibitory effect on melittin-induced edema. These experimental findings suggest that multiple peripheral 5-HT receptors are involved in the melittin-induced nociceptive responses.

Calcium Ions are Involved in Modulation of Melittin-induced Nociception in Rat: I. Effect of Voltage-gated Calcium Channel Antagonist

Melittin-induced nociceptive responses are mediated by selective activation of capsaicin-sensitive primary afferent fibers and are modulated by excitatory amino acid receptor, cyclooxygenase, protein kinase C and serotonin receptor. The present study was undertaken to investigate the peripheral and spinal actions of voltage-gated calcium channel antagonists on melittin-induced nociceptive responses. Changes in mechanical threshold and number of flinchings were measured after intraplantar (i.pl.) injection of melittin (30microg/paw) into mid-plantar area of hindpaw. L-type calcium channel antagonists, verapamil [intrathecal (i.t.), 6 or 12microg; i.pl.,100 & 200microg; i.p., 10 or 30 mg], N-type calcium channel blocker, omega-conotoxin GVIA (i.t., 0.1 or 0.5microg; i.pl., 5microg) and P-type calcium channel antagonist, omega-agatoxin IVA (i.t., 0.5microg; i.pl., 5microg) were administered 20 min before or 60 min after i.pl. injection of melittin. Intraplantar pre-treatment and i.t. pre- or post-treatment of verapamil and omega-conotoxin GVIA dose-dependently attenuated the reduction of mechanical threshold, and melittin-induced flinchings were inhibited by i.pl. or i.t. pre-treatment of both antagonists. P-type calcium channel blocker, omega-agatoxin IVA, had significant inhibitory action on flinching behaviors, but had a limited effect on melittin-induced decrease in mechanical threshold. These experimental findings suggest that verapamil and omega-conotoxin GVIA can inhibit the development and maintenance of melittin-induced nociceptive responses.

Calcium Ions are Involved in Modulation of Melittin-induced Nociception in Rat: II. Effect of Calcium Chelator

Melittin, a major component of bee venom, produces a sustained decrease in mechanical threshold, and an increase in spontaneous flinchings and paw thickness, which are characteristics similar to those induced by whole bee venom. Melittin-induced nociception has been known to be modulated by the changes in the activity of excitatory amino acid receptors, voltage-dependent calcium channels, cyclooxygenase and serotonin receptors. The present study was undertaken to investigate the role of calcium chelators (TMB-8 & Quin 2) in melittin-induced nociceptive responses. Changes of mechanical threshold and spontaneous flinching behaviors were measured at a given time point following intraplantar injection of melittin (30microgram/paw). Intrathecal or intraplantar pre-administration and intrathecal post-treatment of TMB-8 and Quin 2 significantly prevented the melittin-induced reduction of mechanical threshold, and intraplantar or intrathecal pre-treatment of TMB-8 and Quin 2 suppressed melittin-induced flinching behaviors. These results indicate that calcium ion in the spinal dorsal horn neurons and peripheral nerves plays an important role in the production and maintenance of mechanical allodynia and spontaneous pain by melittin.

Melittin-induced Nociceptive Responses are Alleviated by Cyclooxygenase-1 Inhibitor

Melittin-induced pain model has been known to be very useful for the study of pain mechanism. Melittin-induced nociceptive responses are reported to be modulated by the changes in the activity of excitatory amino acid receptor, calcium channel, spinal serotonin receptor and extracellular signaling-regulated kinase. The present study was undertaken to investigate the role of cyclooxygenase (COX) in the melittin-induced nociception. Changes in mechanical threshold, flinchings and paw thickness were measured before and after intraplantar injection of melittin in the rat hind paw. Also studied were the effects of intraperitonealy administered diclofenac (25 mg & 50 mg/kg), piroxicam (10 mg & 20 mg/kg) and meloxicam (10 mg & 20 mg/kg) on the melittin-induced nociceptions. Intraplantar injection of melittin caused marked reduction of mechanical threshold that was dose-dependently attenuated by non-selective COX inhibitor (diclofenac) and selective COX-1 inhibitor (piroxicam), but not by COX-2 inhibitor (meloxicam). Melittin-induced flinchings were strongly suppressed by non-selective COX and COX-1 inhibitor, but not by COX-2 inhibitor. None of the COX inhibitors had inhibitory effects on melittin-induced increase of paw thickness (edema). These experimental findings suggest that COX-1 plays an important role in the melittin-induced nociceptive responses.

N-methyl-D-aspartate (NMDA) and Non-NMDA Receptors are Involved in the Production and Maintenance of Nociceptive Responses by Intraplantar Injection of Bee Venom and Melittin in the Rat

Whole bee venom (WBV) and its major component, melittin, have been reported to induce long-lasting spontaneous flinchings and hyperalgesia. The current study was designed to elucidate the peripheral and spinal mechanisms of N-methyl-D-aspartate (NMDA) and non-NMDA receptors by which intraplantar (i.pl.) injection of WBV and melittin induced nociceptive responses. Changes in mechanical threshold and flinching behaviors were measured after the injection of WBV (0.04 mg or 0.1 mg/paw) and melittin (0.02 mg or 0.05 mg/paw) into the mid-plantar area of a rat hindpaw. MK-801 and CNQX (6-cyano-7-nitroquinoxaline-2, 3-dione disodium) were administered intrathecally (i.t. 10microgram) or i.pl. (15microgram) 15 min before or i.t. 60 min after i.pl. WBV and melittin injection. Intrathecal pre- and post- administration of MK-801 and CNQX significantly attenuated the ability of high dose WBV and melittin to reduce paw withdrawal threshold (PWT). In the rat injected with low dose, but not high dose, of WBV and melittin, i.pl. injection of MK-801 effectively suppressed the decrease of PWTs only at the later time-points, but the inhibitory effect of CNQX (i.pl.) was significant at all time-point after the injection of low dose melittin. High dose WBV- and melittin-induced spontaneous flinchings were significantly suppressed by i.t. administration of MK-801 and CNQX, and low dose WBV- and melittin-induced flinchings were significantly reduced only by intraplantarly administered CNQX, but not by MK-801. These experimental flinchings suggest that spinal, and partial peripheral mechanisms of NMDA and non-NMDA receptors are involved in the development and maintenance of WBV- and melittin-induced nociceptive responses.

Comparative Study on the Nociceptive Responses Induced by Whole Bee Venom and Melittin

The present study was undertaken to confirm whether melittin, a major constituent of whole bee venom (WBV), had the ability to produce the same nociceptive responses as those induced by WBV. In the behavioral experiment, changes in mechanical threshold, flinching behaviors and paw thickness (edema) were measured after intraplantar (i.pl.) injection of WBV (0.1 mg & 0.3 mg/paw) and melittin (0.05 mg & 0.15 mg/paw), and intrathecal (i.t.) injection of melittin (6microgram). Also studied were the effects of i.p. (2 mg & 4 mg/kg), i.t. (0.2microgram & 0.4microgram) or i.pl. (0.3 mg) administration of morphine on melittin- induced pain responses. I.pl. injection of melittin at half the dosage of WBV strongly reduced mechanical threshold, and increased flinchings and paw thickness to a similar extent as those induced by WBV. Melittin- and WBV-induced flinchings and changes in mechanical threshold were dose- dependent and had a rapid onset. Paw thickness increased maximally about 1 hr after melittin and WBV treatment. Time-courses of nociceptive responses induced by melittin and WBV were very similar. Melittin-induced decreases in mechanical threshold and flinchings were suppressed by i.p., i.t. or i.pl. injection of morphine. I.t. administration of melittin (6microgram) reduced mechanical threshold of peripheral receptive field and induced flinching behaviors, but did not cause any increase in paw thickness. In the electrophysiological study, i.pl. injection of melittin increased discharge rates of dorsal horn neurons only with C fiber inputs from the peripheral receptive field, which were almost completely blocked by topical application of lidocaine to the sciatic nerve. These findings suggest that pain behaviors induced by WBV are mediated by melittin-induced activation of C afferent fiber, that the melittin- induced pain model is a very useful model for the study of pain, and that melittin-induced nociceptive responses are sensitive to the widely used analgesics, morphine.

Carrageenan-Induced Hyperalgesia Is Partially Alleviated by Endomorphin-1 Locally Delivered into Inflamed Paws In Rat

This study was performed to test whether endomorphin-1 has analgesic effect, when locally administrated into inflamed peripheral tissue. Carrageenan suspension (0.5%) was injected intraplantarly into the right paw of Sprague-Dawley male rats, and the rats were subjected to a series of mechanical stimuli with von Frei filaments before and after the injection. Carrageenan-injected rats showed typical inflammatory hyperalgesic signs and decrease of withdrawal threshold, peaked at 3 to 6 hours after the injection and lasted more than 3 days. Endomorphin-1 was intraplantarly injected with carrageenan, simultaneously or 3~4 hours after carrageenan. Simultaneous injection of endomorphin-1 with carrageenan significantly reduced hyperalgesia and thd analgesic effect was prolonged up to 8 hours. The delivery of endomorphin-1 (50 microgram) into the inflamed area after 3 to 4 hours of carrageenan injection significantly increased the threshold of hyperalgesic mechanical withdrawal response, but only partially. Intrathecal treatment of endomorphin-1 completely reversed carrageenan-induced hyperalgesia. This report is the first to show that peripherally delivered endomorphin-1 relieved inflammatory hyperalgesia. But a control through peripheral mu-opioid receptors appears to be not sufficient for complete pain treatment.

Antagonists of NMDA Receptor, Calcium Channel and Protein Kinase C Potentiate Inhibitory Action of Morphine on Responses of Rat Dorsal Horn Neuron

he present study was designed to examine whether the co-application of morphine with Ca2+ channel antagonist (Mn2+, verapamil), N-methyl-D-aspartate (NMDA) receptor antagonist (2-amino-5-phosphonopentanoic acid[AP5], Mg2+) or protein kinase C inhibitor (H-7) causes the potentiation of morphine- induced antinociceptive action by using an in vivo electrophysiological technique. A single iontophoretic application of morphine or an antagonist alone induced weak inhibition of wide dynamic range (WDR) cell responses to iontophoretically applied NMDA and C-fiber stimulation. Although there was a little difference in the potentiating effects, the antinociceptive action of morphine was potentiated when morphine was iontophoretically applied together with Mn2+, verapamil, AP5, Mg2+ or H-7. However, the potentiating action between morphine and each antagonist was not apparent, when the antinociceptive action evoked by morphine or the antagonist alone was too strong. These results suggest that the potentiating effect can be caused by the interaction between morphine and each antagonist in the spinal dorsal horn.

Involvement of NMDA Receptor and L-type Calcium Channel in the Excitatory Action of Morphine

We studied the excitatory action of morphine on the responses of dorsal horn neuron to iontophoretic application of excitatory amino acid and C-fiber stimulation by using the in vivo electrophysiological technique in the rat. In 137 of the 232 wide dynamic range (WDR) neurons tested, iontophoretic application of morphine enhanced the WDR neuron responses to N-methyl-D-aspartate (NMDA), kainate, and graded electrical stimulation of C-fibers. Morphine did not have any excitatory effects on the responses of low threshold cells. Morphine-induced excitatory effect at low ejection current was naloxone-reversible and reversed to an inhibitory action at high ejection current. NMDA receptor, calcium channel and intracellular Ca2+ antagonists strongly antagonized the morphine-induced excitatory effect. These results suggest that changes in intracellular ionic concentration, especially Ca2+, play an important role in the induction of excitatory effect of morphine in the rat dorsal horn neurons.

Responsiveness of dendrites to the glutamate applied focally with pressure ejector and iontophoresis into hippocampal slices

Glutamate is the most common excitatory amino acid in the brain. Responsiveness of dendrites to the glutamate greatly varies depending on the application sites. Especially, a point of the maximal response to the glutamate of the dendrite is called as 'hot spot'. In our experiment, the responsiveness of the hot spot to the glutamate was investigated in the CA1 pyramidal neuron of the rat hippocampal slice. CNQX, the antagonist of AMPA receptor, blocked 95% of membrane current to the glutamate focal application (Igl). Train ejection of glutamate on one point of the dendrite increased or decreased the amplitude of Igl with the pattern of train, and the changes were maintained at least for 30 min. In some cases, glutamate train ejection also induced calcium dependent action potentials. To evoke long-term change of synaptic plasticity, we adopted theta-burst in the glutamate train ejection. The theta-burst decreased the amplitude of glutamate response by 60%. However, after theta-burst glutamate train ejection, the calcium dependent action potential appeared. These results indicated that the focal application of glutamate on the neuronal dendrite induced response similar to the synaptic transmission and the trains of glutamate ejection modulated the change of AMPA receptor.

Zn2+ modulates the responses of rat dorsal horn neuron to C-fiber stimulation and excitatory amino acids

Zinc contained in the neurons of central nervous system is activity-dependently released and then attenuates NMDA (N-methyl-D-aspartate)-induced neurotoxicity while augmenting non-NMDA-induced neurodegeneration. Zinc also has been reported to produce antinociceptive action on the inflammation- and nerve injury-induced hyperalgesia in the behavioral test. In this study, we investigated the effects of zinc on the responses of dorsal horn cells to NMDA, kainate and graded electrical stimulation of C-fibers. In the majority of WDR cells (70.6%), zinc current-dependently inhibited WDR cell responses to NMDA and in the remaining cells, produced biphasic responses; excitation followed by inhibition. Zinc augmented the responses of WDR cells to iontophoretical application of kainate. The dominant effect of Zn2+ on the responses of WDR cells to C-fiber stimulation was excitatory, but inhibition, excitation-inhibition and no change of the responses to C-fiber stimulation were induced. Ca2+-EDTA antagonized the excitatory or inhibitory effects of Zn2+ on the WDR cell responses. These experimental findings suggest that Zn2+ modulates the transmission of sensory information in the rat spinal cord.

Magnesium suppresses the responses of dorsal horn cell to noxious stimuli in the rat

Magnesium ion is known to selectively block the N-methyl-D-aspartate (NMDA)-induced responses and to have anticonvulsive action, neuroprotective effect and antinociceptive action in the behavioral test. In this study, we investigated the effect of Mg2+ on the responses of dorsal horn neurons to cutaneous thermal stimulation and graded electrical stimulation of afferent nerves as well as to excitatory amino acids and also elucidated whether the actions of Ca2+ and Mg2+ are additive or antagonistic. Mg2+ suppressed the thermal and C-fiber responses of wide dynamic range (WDR) cell without any effect on the A-fiber responses. When Mg2+ was directly applied onto the spinal cord, its inhibitory effect was dependent on the concentration of Mg2+ and duration of application. The NMDA- and kainate-induced responses of WDR cell were suppressed by Mg2+, the NMDA-induced responses being inhibited more strongly. Ca2+ also inhibited the NMDA-induced responses current-dependently. Both inhibitory actions of Mg2+ and Ca2+ were additive, while Mg2+ suppressed the EGTA-induced augmentation of WDR cell responses to NMDA and C-fiber stimulation. Magnesium had dual effects on the spontaneous activities of WDR cell. These experimental findings suggest that Mg2+ is implicated in the modulation of pain in the rat spinal cord by inhibiting the responses of WDR cell to noxious stimuli more strongly than innocuous stimuli.

Calcium modulates excitatory amino acid (EAA)- and substance P-induced rat dorsal horn cell responses

Excitatory amino acid (EAA) and substance P (SP) have been known to be primary candidates for nociceptive neurotransmitter in the spinal cord, and calcium ions are implicated in processing of the sensory informations mediated by EAA and SP in the spinal cord. In this study, we examined how Ca2+ modified the responses of dorsal horn neurons to single or combined iontophoretical application of EAA and SP in the rat. All the LT cells tested responded to kainate, whereas about 55% of low threshold (LT) cells responded to iontophoretically applied NMDA. NMDA and kainate excited almost all wide dynamic range (WDR) cells. These NMDA- and kainate-induced WDR cell responses were augmented by iontophoretically applied EGTA, but suppressed by Ca2+, Mn2+ verapamil and omega-conotoxin GVTA, effect of verapamil being more prominent and well sustained. Ca2+ and Mn2+ antagonized the augmenting effect of EGTA. On the other hand, prolonged spinal application of EGTA suppressed the response of WDR cell to NMDA. SP had triple effects on the spontaneous activity as well as NMDA-induced responses of WDR cells: excitation, inhibition and no change. EGTA augmented, but Ca2+, Mn2+ and verapamil suppressed the increase in the NMDA-induced responses and spontaneous activities of WDR cells following iontophoretical application of SP. These results suggest that in the spinal cord, sensory informations mediated by single or combined action of EAA and SP can be modified by the change in calcium ion concentration.

Dopaminergic inhibition of dorsal horn cell activity in the cat

Dopamine has been generally known to exert antinociceptive action in behavioral pain test, such as tail flick and hot plate test, but there appears to be a great variance in the reports on the antinociceptive effect of dopamine depending on the dosage and route of drug administration and type of animal preparation. In the present study, the effects of dopamine on the responses of wide dynamic range (WDR) cells to mechanical, thermal and graded electrical stimuli were investigated, and the dopamine-induced changes in WDR cell responses were compared between animals with an intact spinal cord and the spinal animals. Spinal application of dopamine (1.3 & 2.6 mM) produced a dose-dependent inhibiton of WDR cell responses to afferent inputs, the pinch-induced or the C-fiber evoked responses being more strongly depressed than the brush-induced or the A-fiber evoked responses. The dopamine-induced inhibition was more pronounced in the spinal cat than in the cat with intact spinal cord. The responses of WDR cell to thermal stimulation were also strongly inhibited. Dopamine D2 receptor antagonist, sulpiride, but not D1 receptor antagonist, significantly blocked the inhibitory action of dopamine on the C-fiber and thermal responses of dorsal horn cells. These findings suggest that dopamine strongly suppresses the responses of WDR cells to afferent signals mainly through spinal dopamine D2 receptors and that spinal dopaminergic processes are under the tonic inhibitory action of the descending supraspinal pathways.

Calcium channel blockers suppress the responses of rat dorsal horn cell to nociceptive input

Calcium ions are implicated in a variety of physiological functions, including enzyme activity, membrane excitability, neurotransmitter release, and synaptic transmission, etc. Calcium antagonists have been known to be effective for the treatment of exertional angina and essential hypertension. Selective and nonselective voltage-dependent calcium channel blockers also have inhibitory action on the acute and tonic pain behaviors resulting from thermal stimulation, subcutaneous formalin injection and nerve injury. This study was undertaken to investigate the effects of iontophoretically applied Ca++ and its antagonists on the responses of WDR (wide dynamic range) cells to sensory inputs. The responses of WDR cells to graded electrical stimulation of the afferent nerve and also to thermal stimulation of the receptive field were recorded before and after iontophoretical application of Ca++, EGTA, Mn++, verapamil, omega-conotoxin GVIA, omega-conotoxin MVIIC and omega-agatoxin IVA. Also studied were the effects of a few calcium antagonists on the C-fiber responses of WDR cells sensitized by subcutaneous injection of mustard oil (10%). Calcium ions and calcium channel antagonists (Mn++, verapamil, omega-conotoxin GVIA & omega-agatoxin IVA) current-dependently suppressed the C-fiber responses of WDR cells without any significant effects on the A-fiber responses. But omega-conotoxin MVIIC did not have any inhibitory actions on the responses of WDR cell to A-fiber, C-fiber and thermal stimulation. Iontophoretically applied EGTA augmented the WDR cell responses to C-fiber and thermal stimulations while spinal application of EGTA for about 20 ~ 30 min strongly inhibited the C-fiber responses. The augmenting and the inhibitory actions of EGTA were blocked by calcium ions. The WDR cell responses to thermal stimulation of the receptive field were reduced by imtophoretical application of Ca++, verapamil, omega -agatoxin IVA, and omega-conotoxin GVIA but not by omega-conotoxin MVIIC. The responses of WDR cells to C-fiber stimulation were augmented after subcutaneous injection of mustard oil (10%, 0.15 ml) into the receptive field and these sensitized C-fiber responses were strongly suppressed by iontophoretically applied Ca++, verapamil, omega-conotoxin GVIA and omega-agatoxin IVA. These experimental findings suggest that in the rat spinal cord, L-, N-, and P-type, but not Q-type, voltage-sensitive calcium channels are implicated in the calcium antagonist-induced inhibition of the normal and the sensitized responses of WDR cells to C-fiber and thermal stimulation, and that the suppressive effect of calcium and augmenting action of EGTA on WDR cell responses are due to changes in excitability of the cell.