Norifumi Yonehara

Release of Calcitonin Gene-Related Peptide-Like Immunoreactive Substance from Neuromuscular Junction by Nerve Excitation and Its Action on Striated Muscle

Abstract: In a rat phrenic nerve‐hemidiaphragm preparation, calcitonin gene‐related peptide (CGRP) increased the twitch contraction induced by nerve or transmural stimulation dose dependently. Either electrical or high K+ stimulation of the phrenic nerve caused release of a CGRP‐like immunoreactive substance (CGRP‐LIS) in a Ca2+‐dependent manner. Electrical stimulation of the phrenic nerve also increased the cyclic AMP content in diaphragm. This increase was not observed in Ca2+‐free medium and was blocked by antiserum against CGRP. These results indicate that excitation of the motor nerve causes release of CGRP‐LIS at nerve terminals and that the released CGRP‐LIS increases the cyclic AMP content of skeletal muscles and potentiates twitch contraction.

Influence of opioids on substance P release evoked by antidromic stimulation of primary afferent fibers in the hind instep of rats

The effect of opioids on the release of immunoreactive substance P (iSP) following simultaneous electrical stimulation of the sectioned sciatic and saphenous nerves was examined by perfusion of the subcutaneous space in the rat instep. Antidromic stimulation of both the nerves caused an increase in iSP release, which was dependent on the intensity of stimulation, and an approx. 200% increase in Evans blue extravasation. Stimulation-induced iSP release and extravasation were suppressed by pretreatment with capsaicin (50 mg/kg s.c.) and spantide (10 mumol/kg i.p.), respectively. Intra-arterial infusion of morphine (30 mumol/kg) or ethylketocyclazocine (30 mumol/kg) or [D-Ala2,D-Leu5]enkephalin (30 mumol/kg) inhibited the increase in iSP release evoked by antidromic stimulation at 10 V. This inhibitory effect of morphine was antagonized by pretreatment with naloxone (2 mg/kg, i.p.). These results suggest existence of multiple types of opioid receptor on the peripheral endings of primary afferent fibers, that regulate SP release from the peripheral nerve endings into the extravascular space.

Effects of opioids and opioid peptide on the release of substance P-like material induced by tooth pulp stimulation in the trigeminal nucleus caudalis of the rabbit

The superficial layer in subnucleus caudalis of the brain-stem trigeminal sensory nuclear complex (SpVc) in the rabbit was perfused with artificial cerebrospinal fluid using a push-pull perfusion cannula system. Immunoreactive substance P (iSP) and [Met5]enkephalin (iME) released into the perfusates following electrical stimulation of the lower incisor pulp were measured. An increase in the release of iSP and iME lasting for 1 h or more was observed following electrical stimulation with 40 V. The increase in iSP release depended on the intensity of stimulation. Systemic morphine (10 mg/kg i.v.) completely inhibited the stimulus-evoked iSP release and this inhibition was antagonized by pretreatment with naloxone (5 mg/kg i.v.). The stimulus-evoked iSP release was also inhibited by local application of morphine (10(-6) M) or the opioid peptide [D-Ala2,Met5]enkephalinamide (10(-4) M). However, the local application of naloxone (5 X 10(-7) M) only partially antagonized the inhibitory effects of locally applied morphine and the opioid peptide. These results suggest that there is a functional interaction between SP and enkephalin systems in the superficial layer of SpVc for the regulation of dental pain transmission.

Alteration in sensitivity of ionotropic glutamate receptors and tachykinin receptors in spinal cord contribute to development and maintenance of nerve injury-evoked neuropathic pain

Allodynia or hyperalgesia induced by peripheral nerve injury may be involved in changes in the sensitivity of neurotransmitters at the spinal cord level. In order to clarify the functional role of neurotransmitters in peripheral nerve injury, we used rats with nerve injury induced by chronic constriction of the sciatic nerve (CCI rat model) and estimated the effects of the intrathecal injection of drugs known to affect glutamate and tachykinin receptors. In sham-operated rats, the NMDA receptor agonist NMDA and AMPA-kinate receptor agonist RS-(5)-bromowillardin reduced withdrawal latency. The non-competitive NMDA receptor antagonist MK-801, competitive NMDA receptor antagonist AP-5 and AMPA-kinate receptor antagonist NBQX increased withdrawal latency. Substance P (SP) increased the withdrawal latency but only transitorily. The NK1 receptor antagonist RP67580 increased withdrawal latency, but the NK2 receptor antagonist SR48968 did not show an effect. In CCI rats, RS-(5)-bromowillardin reduced withdrawal latency, but NMDA did not show an effect. NBQX increased withdrawal latency, while MK-801 and AP-5 showed little or no effect. SP reduced withdrawal latency, and both RP67580 and SR48968 increased it. These results indicate that the alteration in sensitivity of ionotropic glutamate receptors and tachykinin receptors in the spinal cord contribute to development and maintenance of nerve injury-evoked neuropathic pain.

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.

Effect of morphine on the release of excitatory amino acids in the rat hind instep: Pain is modulated by the interaction between the peripheral opioid and glutamate systems

Behavioral evidence supports a role for peripheral glutamate receptors in normal nociceptive transmission. In this study, we examined the release of the excitatory amino acids, glutamate and aspartate, in the s.c. perfusate of the rat hind instep by in vivo microdialysis. Antidromic stimulation of the sciatic nerve and noxious stimuli in the form of heat stimulation and local application of capsaicin cream (1%) to the instep caused an increase in excitatory amino acid release. This capsaicin-induced excitatory amino acid release was suppressed by pretreatment with capsaicin. Both systemic (10 mg/kg, i.v.) and local injections (10(-5) M in the perfusate) of morphine inhibited the increase in excitatory amino acid release evoked by local application of capsaicin cream to the instep. This inhibitory effect of morphine was antagonized by naloxone either given systemically (5 mg/kg, i.v.) or locally (10(-5) M). These results suggest that excitatory amino acids are released from small diameter afferent fibers by heat stimulation in the periphery or local application of capsaicin cream, and that activation of opioid receptors, present on the peripheral endings of small-diameter afferent fibers, can regulate noxious stimulus-induced excitatory amino acid release.

Influence of painful chronic neuropathy on neurogenic inflammation

&NA; The effect of topical application of capsaicin cream on neurogenic inflammation was investigated in a neuropathic pain model in rat. The neuropathic state was induced by loose ligation of the sciatic nerve with a chromic gut suture. A marked thermal hyperalgesia was observed in response to heat stimulation applied to the operated side from 3 days through 2 weeks, followed by a gradual return to the control level 35 days after surgery. In sham‐operated animals, topical application of capsaicin cream to both sides of the hind paw, the instep and sole, as well as antidromic stimulation of the sciatic nerve led to a significant increase in the amounts of Evans blue and substance P (SP) released into the perfusates. This stimulus‐induced extravasation was significantly suppressed by pretreatment with RP67580, an NK1 antagonist. On day 7 after ligation, capsaicin‐ and antidromic stimulation‐induced extravasation were significantly reduced. At this time, both amount of SP released immediately after application of capsaicin and during antidromic stimulation were almost similar to that in sham‐operated rats, whereas the basal amount of SP release significantly increased in ligated animals. In particular a major release of SP was detected immediately after the start of the perfusion compared with that in sham‐operated rats. Plasma extravasation evoked by SP (10−4 M) applied to the subcutaneous perfusate was significantly less in ligated than in sham‐operated rats. These results suggest that nerve injury with chronic pain may produce increase in basal SP release into the peripheral tissues, and then such enhanced SP release cause reduction of SP‐induced extravasation.

Effects of opioids on the heat stimulus-evoked substance P release and thermal edema in the rat hind paw

We examined the effect of opioids on the heat stimulus-evoked release of substance P (SP) into the subcutaneous space and the formation of edema in the rat hind paw. Immersion of the rat hind paw for 30 min into hot water adjusted to 47 degrees C led to a marked increase in the release of SP into the subcutaneous perfusate with the formation of thermal edema. Intra-arterial infusion of morphine (10-100 mumol/kg) or ethylketocyclazocine (30-100 mumol/kg) inhibited dose dependently the heat stimulus-evoked increase in SP release and the thermal edema and the inhibitory effects were antagonized by pretreatment with N-methyl levallorphan (10 mg/kg i.p.) and Win 44,441-3 (10 mg/kg i.p.). The heat stimulus-evoked release of SP was reduced significantly during the intra-arterial infusion of [D-Ala2,Met5] enkephalinamide (100 mumol/kg). These results suggest that the opioid-induced inhibition of heat-induced edema could result from inhibition of the release of SP from peripheral sensory nerve endings.

Involvement of NMDA-nitric oxide pathways in the development of tactile hypersensitivity evoked by the loose-ligation of inferior alveolar nerves in rats.

To investigate whether or not NMDA/nitric oxide (NO) pathways in the trigeminal system are involved in the development and/or maintenance of such pathological pain states as the hyperalgesia and allodynia observed after dental surgery, loose-ligation on the left inferior alveolar nerves of rats were performed. The responses to mechanical stimulation were then measured using von Frey filaments. Hypersensitivity to tactile stimulation developed on the ipsilateral side in ligated animals 5 days after surgery and lasted for at least 30 days. In addition, the effects of drugs on these pain states during the period 2-3 weeks following surgery were investigated. As a result, it was observed that tactile hypersensitivity was inhibited by the intraperitoneal (i.p.) administration of both MK-801 hydrogen maleate (0.05-0.1 mg/kg) and N(G)-monomethyl-L-arginine acetate (L-NMMA: 10-100 mg/kg). Still further, NO production and the number of neuronal NO synthase (nNOS)-positive neurons in the trigeminal nucleus caudalis (SpVc) was evaluated. As a result of these experiments, it was found that the changes in NO levels evoked by the intravenous infusions of N-methyl-D-aspartate (NMDA; 10 mg/kg) and MK-801 (0.5 mg/kg) were significantly larger in the loose-ligated rats compared to the sham-operated rats. Moreover, the number of nNOS-positive neurons was found to have increased on the ipsilateral side in layers I/II of the SpVc. These results would suggest that tactile hypersensitivity develops after inferior alveolar nerve injury and that NMDA receptor/NOS/NO production pathways in the SpVc may be involved in the development of such pathophysiological states.

Involvement of substance P present in primary afferent neurones in modulation of cutaneous blood flow in the instep of rat hind paw

1 The participation of small‐diameter afferent fibres in the microcirculatory haemodynamics of cutaneous tissue was examined by studies on the effects of antidromic stimulation of primary afferent neurones on cutaneous blood flow (CBF) and tachykinin release into the subcutaneous space in the instep of the hind paw of rats. 2 Antidromic stimulation of the sectioned sciatic nerve induced a biphasic flow response, an initial transient decrease followed by an increase, with no alteration in the blood pressure. 3 Neither phase was affected by pretreatment with phentolamine (0.1 mg kg−1, i.a.), propranolol (0.5 mg kg−1, i.a.), atropine (0.5 mg kg−1, i.a.), methysergide (0.5 mg kg−1, i.a.) or mepyramine (10 mg kg−1, i.a.) plus cimetidine (10 mg kg−1, i.a.), but both were significantly inhibited by pretreatment with capsaicin (50 mg kg−1, s.c). 4 Spantide (1–2 μmol kg−1, i.a.), a substance P (SP) antagonist, reduced the basal CBF, and also inhibited both phases of the biphasic flow response evoked by antidromic stimulation of the sectioned sciatic nerve. 5 Intra‐arterial infusion of SP (0.5 μmol kg−1, i.a.) induced a biphasic flow response similar to that elicited by antidromic stimulation of the sectioned sciatic nerve. 6 Antidromic stimulation of the sectioned sciatic nerve caused a marked increase in SP release into the subcutaneous perfusate of the instep of the rat hind paw, but no detectable increase in neurokinin A release. 7 We suggest that SP and its receptors are mainly responsible for the vascular response induced by stimulation of the sectioned sciatic nerve, and that small‐diameter afferent fibres containing SP tonically regulate vascular tone in cutaneous microvessels.