Reizo Inoki

Modified formalin test: characteristic biphasic pain response

&NA; A modified formalin test in mice was investigated. The pain response curve induced by 0.5% formalin was biphasic, having 2 peaks, from 0 to 5 min (first phase) and from 15 to 20 min (second phase). A low concentration of formalin was used, allowing the effects of weak analgesics to be detected. Centrally acting drugs such as narcotics inhibited both phases equally. Peripherally acting drugs such as aspirin, oxyphenbutazone, hydrocortisone and dexamethasone only inhibited the second phase. Aminopyrine and mefenamic acid which acted on both central and peripheral sites inhibited both phases, but the second phase was inhibited by lower doses. Thus, this method enables one to easily distinguish the site of action of analgesics. Furthermore, pain response in the first phase was inhibited by capsaicin‐treated desensitization and Des‐Arg9‐(Leu8)‐bradykinin (bradykinin inhibitor). The second phase was inhibited by compound 48/80 pretreatment, indomethacin and bradykinin inhibitor. Therefore, it is suggested that substance P and bradykinin participate in the manifestation of the first phase response, and histamine, serotonin, prostaglandin and bradykinin are involved in the second phase. These results indicate that the first and second phase responses induced by formalin have distinct characteristic properties, and it is a very useful method for examining pain, nociception and its modulation by pharmacological or other means.

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.

Elaboration of a bradykinin-like substance in dog's canine pulp during electrical stimulation and its inhibition by narcotic and nonnarcotic analgesics.

SummaryA substance capable of contracting rat uterine smooth muscle was detected in the saline superfusate bathing the exposed pulp of the dogs canine tooth. Mechanical, thermal or electrical stimulation applied directly to the pulp or electrical stimulation of the cervical sympathetic nerve triggered the release of the substance into the superfusate. Its peptide nature and similarity to synthetic bradykinin was demonstrated by a number of enzymatic tests. The substance was considered to be elaborated from blood and its elaboration was principally preceded by a vasomotor response.Morphine, aspirin, pentazocine, pethidine and aminopyrine prevented the appearance of the bradykinin-like substance in the superfusate collected after stimulation of the pulp or the cervical sympathetic nerve. The inhibitory action of morphine was antagonized by levallorphan, naloxone and nalorphine.

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.

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.

Intracerebroventricular treatment of mice with pertussis toxin induces hyperalgesia and enhances 3H-nitrendipine binding to synaptic membranes: Similarity with morphine tolerance

SummaryThe effect of intracerebroventricular treatment of mice with pertussis toxin (PTX) on pain perception and 3H-nitrendipine binding was examined to study a possible change in the GTP-binding proteins in morphine tolerant rodents. It was observed that both PTX treatment and chronic administration of morphine cause hyperalgesia in the acetic acid-induced writhing test. Analgesic effects brought by the acute administration of morphine or nifedipine, a calcium antagonist, were not affected by PTX treatment. In synaptic membrane fractions prepared from mice treated with PTX or morphine chronically, specific binding of 3H-nitrendipine was enhanced approximately 41.8% and 35.7%, respectively, without alteration in its affinity. Chronic administration of morphine followed by PTX treatment did not display further increases in 3H-nitrendipine binding.These results suggest that the PTX-sensitive GTP-binding proteins may not be involved in the manifestation of the analgesic effect of morphine in mice.

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.

Effect of ethanol on hippocampal GABA receptors in the rat brain

The effect of ethanol on GABA receptors was studied in hippocampal slices and membrane preparations of the rat brain. In slice preparation, ethanol enhanced the GABA inhibition of the population spikes evoked in CA 1 pyramidal cells by Schaffer collateral stimulation. The effect of ethanol was dose-dependent being observed with minimal concentration of 70 mM. In 3H-GABA binding experiments, ethanol enhanced the binding to fresh membranes with no change in the affinity. Maximal stimulation was observed at ethanol concentration of 70 mM. In hippocampal slices or membrane preparations obtained from ethanol- or barbital-administered rats chronically, ethanol failed to enhance GABA function. Thus, the present study demonstrated the involvement of GABA in central effect of ethanol. Also suggested in GABA was the involvement in the development of cross-tolerance between ethanol and barbiturates.

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.