Local inhibitory effects of dynorphin A-(1-17) on capsaicin-induced thermal allodynia in rhesus monkeys

Although dynorphin A-(1-17) has been characterized in vitro as a high efficacy kappa-opioid receptor agonist, functional studies of dynorphin A-(1-17) following central or systemic administration indicate the involvement of both opioid and non-opioid components. The aim of this study was to investigate whether local administration of dynorphin-related analogs can attenuate capsaicin (8-methyl-N-vanillyl-6-nonenamide)-induced nociception and what type of opioid receptor mediates the local action of dynorphin A-(1-17) in monkeys. Capsaicin (100 microg) was used to evoke a nociceptive response, thermal allodynia, which was manifested as a reduced tail-withdrawal latency in normally innocuous 46 degrees C warm water. Co-administration of dynorphin A-(1-17) (0.3-10 microg) with capsaicin in the tail dose-dependently inhibited thermal allodynia; however, both non-opioid fragments dynorphin A-(2-17) (10-300 microg) and dynorphin A-(2-13) (10-300 microg) were ineffective. Local antiallodynia of dynorphin A-(1-17) was antagonized by a small dose (100 microg) of an opioid receptor antagonist, quadazocine, applied s.c. in the tail. Pretreatment with a selective kappa-opioid receptor antagonist, nor-binaltorphimine (nor-BNI), s.c. 320 microg in the tail also reversed local antiallodynia of dynorphin A-(1-17). Both locally effective doses of antagonists, when applied s.c. in the back, did not antagonize local dynorphin A-(1-17), indicating that peripheral kappa-opioid receptors selectively mediated the local action of dynorphin A-(1-17) in the tail. In addition, a much larger dose of dynorphin A-(1-17) (1000 microg), when administered s. c. in the back or i.m. in the thigh, did not cause sedative or diuretic effects. These results suggest that in vivo opioid actions of dynorphin-related peptides can be differentiated locally in this procedure. They also indicate that local application of peptidic ligands may be a useful medication for localized pain.     

Tachykinin NK(2) receptors facilitate acetylcholine release from guinea-pig isolated trachea

The release of newly synthesised [3H]acetylcholine was evoked by electrical field stimulation (5 Hz, 600 pulses) of epithelium-deprived guinea-pig trachea strips after sensory neuropeptides depletion with 3 microM capsaicin. The selective tachykinin NK(2) receptor agonist [betaAla(8)]neurokinin A-(4-10) increased in a concentration-dependent manner the electrically-induced release of [3H]acetylcholine. The facilitatory effect was antagonised by the selective non-peptide tachykinin NK(2) receptor antagonist, SR 48968 (apparent pK(B) 8.9). The tachykinin NK(1) and NK(3) receptor agonists substance P methyl ester and senktide (both 10 and 100 nM), respectively, did not affect the evoked release of [3H]acetylcholine. It is concluded that the cholinergic nerves of guinea-pig trachea are endowed with prejunctional facilitatory tachykinin receptors of the NK(2) subtype.     

Role of kappa opioid receptors in modulating cholinergic twitches in the circular muscle of guinea-pig colon.

1. Single pulse electrical field stimulation (EFS, 0.5 ms pulse width, 60 V at a frequency of 0.05 Hz) induced twitch contractions of mucosa-free circular muscle strips from the guinea-pig proximal colon which were abolished by atropine (0.3 microM), tetrodotoxin (0.3 microM) or omega-conotoxin GVIA (0.1 microM). 2. Various opioid receptor agonist concentration-dependently inhibited twitches with the following rank order of potency (EC50 values in brackets): U 50488 (0.31 nM) > dermorphin (4.3 nM) = dynorphin A (1-13) (6.2 nM) > [D-Ala2, N-MePhe4, Gly5-ol]-enkephalin (DAMGO, 33.5 nM) = [D-Ala2, D-Leu5]-enkephalin (DADLE, 60 nM) > [D-Pen2, D-Pen2, D-Pen5]-enkepahlin (DPDPE, 1144 nM). 3. Peptidase inhibitors (captopril, thiorphan and bestatin, 1 microM each) did not modify the amplitude of twitches. In the presence of peptidase inhibitors the concentration-response curve to dynorphin A (1-13) was displaced to the left to yield an EC50 of 0.35 nM, comparable to that of the selective kappa receptor agonist, U50488. The curves to the other opioid receptor agonist were unaffected by peptidase inhibitors. 4. DPDPE, DADLE, dermorphin and DAMGO consistently induced a concentration-unrelated transient increase in basal tone and a small and transient facilitation of twitches before development of their inhibitory effect. These transient excitatory effects were not observed upon application of dynorphin A (1-13) or U 50488. The contraction produced by DPDPE (30 nM) was largely inhibited (> 80%) by 1 microM atropine. 5. Twitches suppression induced by dynorphin A (1-13) (30 nM) was partly reversed (46 +/- 8%, n = 6) by naloxone (0.3 microM). The potent and selective kappa opioid receptor antagonist nor-binaltorphimine (Nor-BNI, 3-100 nM)) did not affect the amplitude of twitches and potently antagonized (pKB 9.83 +/- 0.09, n = 10) the inhibitory effect of dynorphin. 6. Naloxone (1-300 nM) concentration-dependently depressed the cholinergic twitches: this depressant effect was largely counteracted in the presence of apamin (0.1 microM) and NG-nitro-L-arginine (30 microM) which potentiated cholinergic twitches on their own. 7. Dynorphin A (1-13) (10 nM, n = 6) did not affect the contractile response to exogenous acetylcholine (1 microM), indicating that depression of evoked twitches occurs prejunctionally. 8. We conclude that multiple opioid receptors modulate cholinergic twitches in the circular muscle of guinea-pig proximal colon. While mu and delta opioid receptor agonists produced mixed excitatory and inhibitory effects, kappa opioid receptors, activated by sub-nanomolar concentrations of dynorphin A (1-13), mediate a powerful and pure prejunctional inhibition of acetylcholine release.     

Release of dynorphin, somatostatin and substance P from the vascularly perfused small intestine of the guinea-pig during peristalsis.

The release of dynorphin-(1-17), somatostatin and substance P into the venous effluate of the isolated and vascularly perfused guinea-pig small intestine was measured during rest and peristaltic activity. The peptides were determined by specific radioimmunoassays. Increasing the intraluminal pressure by 5 mbar increased the release of dynorphin-(1-17), somatostatin and substance P. A substantial increase in the release of substance P was only seen in the presence of naloxone (1.5 microM) indicating an inhibitory influence of opioid peptide-containing neurones on the release of substance P. The pressure-induced release of substance P and dynorphin-(1-17) was completely prevented by tetrodotoxin (1.3 microM), which suggests a neural origin of these two peptides. The pressure-induced release of somatostatin was only partially inhibited by tetrodotoxin (1.3 microM) suggesting that somatostatin may also be released from non-neuronal sources, i.e. endocrine mucosal cells. Dimethylphenylpiperazinium (32 microM) increased the release of somatostatin and substance P and this effect was inhibited by tetrodotoxin (1.3 microM). Cholecystokinin-octapeptide (38 nM) induced a large increase in the release of somatostatin but only a minute increase in the release of substance P; these effects of cholecystokinin-octapeptide were not blocked by tetrodotoxin (1.3 microM). Noradrenaline (59 microM) inhibited the pressure-induced release of substance P but not that induced by dimethylphenylpiperazinium (32 microM). Neither the pressure-induced nor the dimethylphenylpiperazinium-evoked release of somatostatin was significantly diminished by noradrenaline. These results indicate that dynorphin-(1-17), somatostatin and substance P may be transmitters involved in the coordination of the peristaltic reflex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gastric acid secretion by central injection of dynorphin A-(1-17), an endogenous ligand of kappa-opioid receptor, in urethane-anesthetized rats.

Gastric acid secretion has been proposed to be regulated by opioid receptors in the central nervous system (CNS). Previously, we reported that central injection of synthetic agonists of kappa-opioid receptors stimulated gastric acid secretion in rats, and the secretion by the agonists was inhibited by norbinaltorphimine (an antagonist of kappa-opioid receptor). In the present study, we investigated the effect of dynorphin A-(1-17), an endogenous ligand of kappa-opioid receptor on the gastric acid secretion in the perfused stomach of urethane-anesthetized rats. Injection of dynorphin A-(1-17) (0.1-1 microg per rat) into the lateral cerebroventricle (LV) stimulated the secretion in a dose-dependent manner. The effect of dynorphin A-(1-17) was almost completely inhibited by the LV injection of norbinaltorphimine (10 microg) and in vagotomized rats. Although some studies of dynorphin A-(1-17) after central injection showed non-opioid effects such as the involvement of N-methyl-D-aspartate (NMDA) receptor, the effect of dynorphin A-(1-17) was not inhibited by a selective antagonist of the NMDA receptor ((+/-)-3-(2-carboxypiperazin-4-yl)-1-propylphosphonic acid, 10 microg). The LV injection of naloxone benzoylhydrazone (a kappa3-opioid receptor agonist, 100 microg) also stimulated the secretion in norbinaltorphimine-sensitive manner. These findings showed that both an endogenous ligand dynorphin A-(1-17) and a synthetic kappa3-opioid receptor agonist stimulated gastric acid secretion via kappa-opioid receptors in the CNS of rats in vivo.     

Delta-opioid-receptor activation by [D-Pen2,D-Pen5]enkephalin and morphine inhibits substance P release from trigeminal nucleus slices.

The release of substance P (SP) from spinal dorsal horn slices is partially inhibited by micromolar concentrations of selective delta-opioid receptor agonists. In the present study, we have examined the effect of nanomolar concentrations of [D-Pen2,D-Pen5]enkephalin (DPDPE, delta-opioid receptor agonist) and low micromolar of concentrations morphine on K(+)-evoked SP release from rat trigeminal nucleus caudalis (TNC) slices. DPDPE and morphine inhibited SP release with an apparent maximal effect at 3 nM and at 3 microM, respectively. DPDPE and morphine produced U-shaped concentration-response curves that were completely autoinhibited at 100 nM DPDPE and 1 microM morphine. The inhibition of SP release produced by 3 nM DPDPE and 3 microM morphine was blocked by the opioid receptor antagonists naloxone (30 nM; non-selective) and ICI 174,864 (0.3 microM; delta-selective) but not by nor-binaltorphimine (3 nM n-BNI; kappa-selective), naloxonazine (1 nM; micro 1-selective) or beta-funaltrexamine (20 nM beta-FNA; mu-selective). These findings indicate that delta-opioid receptor-mediated inhibition of SP release from TNC can be achieved by nanomolar concentrations of selective delta-opioid receptor agonists. Activation of delta-opioid receptors by morphine might be involved in the residual analgesia observed after mu 1-opioid receptor blockade and in the analgesia produced by high doses of morphine.     

Bronchodilatation by tachykinins and capsaicin in the mouse main bronchus.

1. The effect of sensory neuropeptides and capsaicin on basal and stimulated tone of mouse bronchial smooth muscle has been evaluated. 2. In basal conditions neither sensory neuropeptides (substance P, neurokinin A or calcitonin gene-related peptide (CGRP) nor capsaicin exerted any contractile effects. However, when a tonic contraction was induced with carbachol (1 microM) a prompt relaxation was induced by substance P (1- 100 nM) and by neurokinin A (1- 100 nM), with substance P being more potent. A second application of substance P was without effect. CGRP (10 nM) produced only a very small and erratic relaxation. Relaxation was also induced by capsaicin (1 microM), and this response could be evoked only once in each preparation. In 4 out of 6 preparations a cross-desensitization between substance P and capsaicin was observed. 3. The selective NK1 tachykinin agonist, [Pro9]-SP sulphone (1 microM), exerted potent bronchodilator actions on carbachol-contracted mouse bronchial preparations. In contrast, neither [beta Ala8]-NKA (4-10) nor [MePhe7]-NKB (both at a concentration of 1 microM), selective synthetic agonists for NK2 and NK3 receptors, exerted significant relaxant effects. Furthermore, the selective NK1 tachykinin antagonist, (+/-)-CP 96,345 (1 microM), abolished substance P (1 nM)- but not isoprenaline (0.1 microM)-induced relaxations. 4. Application of electrical field stimulation (EFS) (20 Hz, supramaximal voltage, 0.5 ms for 10 s) to carbachol-contracted preparations evoked a transient contraction followed by a relaxation. The tetrodotoxin-sensitive slow component of this relaxation was reduced following capsaicin desensitization. 5. In the presence of indomethacin (5 microM) the relaxation induced by substance P, capsaicin or EFS was suppressed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of effect of sumatriptan and UK-14,304 on capsaicin-induced relaxation of guinea-pig isolated basilar artery.

1. The objectives of this study were to assess the effects of sensory neuropeptide antagonists and presynaptically acting receptor agonists on capsaicin-induced relaxations of guinea-pig isolated basilar artery (GPBA). 2. Capsaicin, human alpha-calcitonin gene-related peptide (CGRP) and substance P (SP) caused concentration-related relaxations of GPBA which had been pre-contracted with prostaglandin F2 alpha (PGF2 alpha). Responses to capsaicin were not modified by the peptidase inhibitors, phosphoramidon (1 microM) and bestatin (100 microM). 3. The relaxant responses to capsaicin were blocked in a selective manner by ruthenium red (3 microM) and by the CGRP antagonist, CGRP8-37 (1 microM). CGRP8-37 also selectively inhibited the relaxant effects of CGRP. 4. The selective NK1 receptor antagonist, GR82334 (10 microM), inhibited SP-induced relaxations but had little effect on capsaicin-induced relaxations. 5. The 5-HT1 receptor agonist, sumatriptan, produced small contractions of GPBA under conditions of resting tone. In the presence of PGF2 alpha, sumatriptan had no further contractile effect. Sumatriptan (0.3 and 3 microM) did not modify capsaicin-induced relaxations of GPBA. 6. The alpha 2-adrenoceptor agonist, UK-14,304 (0.1 microM), had no effect on basal or PGF2 alpha-induced tone. UK-14,304 did not modify capsaicin-induced relaxations. 7. These results suggest that capsaicin causes relaxation of GPBA via a release of CGRP. This process is amenable to blockade by CGRP8-37 and ruthenium red, but not to modulation by either sumatriptan or UK-14,304.     

Phoneutria nigriventer spider venom activates 5-HT4 receptors in rat-isolated vagus nerve

1. The venom of Phoneutria nigriventer spider (PNV) causes intense pain and inflammation following an attack. We have investigated the involvement of capsaicin-sensitive nerve fibres by utilizing an in vitro nerve preparation. Extracellular DC potential recordings were made from the rat-isolated vagus nerve, a preparation that is rich in capsaicin-sensitive, that is, nociceptive, C-fibres. 2. PNV (1-10 microg ml(-1)), capsaicin (0.03-0.3 microM) or 5-hydroxytriptamine (5-HT) (0.3-3 microM) induced dose-dependent depolarizations of vagus nerve fibres. Depolarizing responses to capsaicin were blocked by ruthenium red (RR, 10 microM), but responses to PNV were not. Depolarizing responses to PNV or veratridine (50 microM) were inhibited by tetrodotoxin (TTX, 10 microM), but those to capsaicin were not. This suggests that capsaicin and PNV depolarize the nerve fibres by distinct mechanisms. 3. Depolarization in response to 5-HT (3 microM) was reduced by the 5-HT(3) receptor antagonists Y25130 (0.5 micro M) and tropisetron (10 nM) or, to a lesser extent, by the 5-HT(4) receptor antagonist RS39604 (1 or 10 microM). Depolarizing responses to PNV were not affected significantly by Y25130 or tropisetron, but were blocked by RS39604. 4. These data show that 5-HT(4) receptors play a significant role in the activation of nociceptive sensory nerve fibres by PNV and suggest that this is of importance in the development of the pain and inflammation associated with bites from the P. nigriventer spider.     

Morphine, but not sodium cromoglycate, modulates the release of substance P from capsaicin-sensitive neurones in the rat trachea in vitro.

1. Opioids have been shown to inhibit substance P (SP) release from primary afferent neurones (PAN). In addition, opioid receptors have been identified on PAN of the vagus nerves. Sodium cromoglycate (SCG) decreases the excitability of C-fibres in the lung of the dog in vivo. We have utilised a multi-superfusion system to investigate the effect of opioids and SCG on the release of SP from the rat trachea in vitro. 2. Pretreatment of newborn rats with capsaicin (50 mg kg-1 s.c. at day 1 and 2 of life) resulted in a 93.2 +/- 6.3% reduction in tracheal substance P-like immunoreactivity (SP-LI) content when determined by radioimmunoassay in the adult. 3. Exposure to isotonically elevated potassium concentrations (37-90 mM), capsaicin (100 nM-10 microM), and bradykinin (BK; 10nm-1 microM) but not des-Arg9-BK (1 microM) stimulated SP-LI release by a calcium-dependent mechanism. 4. SCG (1 microM and 100 microM) did not affect spontaneous, potassium (60 mM)- or BK (1 microM)-stimulated SP-LI release. 5. Morphine (0.1-100 microM) caused dose-related inhibition of potassium (60 mM)-stimulated SP-LI release with the greatest inhibition of 60.4 +/- 13.7% at 100 microM. The effect of morphine was not mimicked by the kappa-opioid receptor agonist, U50,488H (10 microM) or the delta-opioid receptor agonist, Tyr-(D-Pen)-Gly-Phe-(D-Pen) (DPDPE). 6. The effect of morphine was totally abolished by prior and concomitant exposure to naloxone (100 nM) which had no effect on control release values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased release of immunoreactive dynorphin A1-17 from the spinal cord after intrathecal treatment with endomorphin-2 in anesthetized rats

We previously demonstrated pretreatment with antiserum against dynorphin A1-17 attenuates endomorphin-2-induced analgesia and antianalgesia, suggesting that these endomorphin-2 effects are mediated by the release of dynorphin A1-17. Lumbar-cisternal spinal perfusion was used to measure the release of immunoreactive dynorphin A1-17 into spinal perfusates from urethane-anesthetized rats following endomorphin-2 or endomorphin-1 treatment within the perfusion solution. Treatment with endomorphin-2 (5-50 nmol) for 3 min caused a dose-dependent increase of immunoreactive dynorphin A1-17 in spinal perfusates, with a maximal increase detected between 24 and 48 min after endomorphin-2 treatment, while levels returned to baseline within 60 min. Endomorphin-2-induced release of immunoreactive dynorphin A1-17 was attenuated by pretreatment with mu-opioid receptor antagonist naloxone or 3-methoxynaltrexone. Endomorphin-1 induced a slight increase in immunoreactive dynorphin1-17 as well, but only at the highest dose used (50 nmol). Our results suggest that endomorphin-2 stimulated a specific subtype of mu-opioid receptor to induce the release of immunoreactive dynorphin A1-17 in spinal cords of rats.     

Antagonists of the kappa-opioid receptor enhance allodynia in rats and mice after sciatic nerve ligation

The administration of kappa-opioid receptor antagonists, nor-binaltorphimine (norBNI) and 5'-guanidinonaltrindole (GNTI) enhanced allodynia in rats and mice after sciatic nerve ligation. In order to understand the mechanism underlying this effect, we examined the possible involvement of the endogenous ligand of kappa-opioid receptor dynorphin. The experiments were carried out on male Wistar rats and on Albino-Swiss mice. The rats had been implanted with a catheter 7 days earlier in the subarachnoid space of the spinal cord. Intrathecal (i.t.) administrations in mice were made by lumbar puncture. The animals were i.t. injected with norBNI, GNTI (kappa-opioid receptor antagonists), dynorphin A1-17 antiserum (DYN A/S), ketamine (NMDA receptor antagonist) and their combinations. The nociceptive sensitivity was assessed using the mechanical (von Frey) and thermal allodynia tests on days 2-4 and 8-10 after the sciatic nerve ligation. Both antagonists, norBNI and GNTI, significantly enhanced mechanical and thermal allodynia in rats and mice with neuropathic pain. The potentiation of allodynia after the administration of norBNI or GNTI was inhibited by earlier administration of DYN A/S or by ketamine. Our results suggest that allodynia is mediated through nonopioid effect of the endogenous opioid peptide, dynorphin. The nonopioid action is potentiated by the blockade of kappa-opioid receptors, and corresponding to the elevation of prodynorphin mRNA level in neuropathic pain. Furthermore, the potentiation of allodynia after the administration of the above drugs appears to be mediated through the activation of NMDA receptors directly by dynorphin.     

Sensory nerve-mediated relaxation of guinea-pig isolated pulmonary artery: prejunctional modulation by alpha 2-adrenoceptor agonists but not sumatriptan.

1. Effects of the alpha 2-adrenoceptor agonists, UK14304 and clonidine, the 5-HT1 receptor agonist, sumatriptan and the kappa-opioid receptor agonist, GR103545, on sensory neurotransmission in histamine-contracted guinea-pig isolated pulmonary artery (GPPA) have been studied. 2. Electrical field stimulation (EFS) induced frequency-dependent relaxations of histamine-contracted GPPA, which were attenuated by tetrodotoxin and capsaicin pretreatment but not by the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME). 3. Substance P (0.3 microM) induced relaxations which were subject to rapid tachyphylaxis. Neither the NK1 receptor antagonist, (+/-)-CP 96,345, nor desensitization to substance P had any effect of EFS-induced relaxations of histamine-contracted GPPA. 4. Calcitonin gene-related peptide (CGRP; 3 and 30 nM) induced concentration-dependent relaxations of histamine-contracted GPPA. The putative CGRP receptor antagonist, CGRP8-37 (1 microM), markedly attenuated EFS-induced relaxations as well as relaxations induced by a low concentration of CGRP. 5. Sumatriptan (0.1 and 1 microM) and the selective kappa-opioid receptor agonist, GR103545 (10 and 100 nM) had no effect on EFS-induced relaxations of histamine-contracted GPPA. In contrast, the alpha 2-adrenoceptor agonists UK14304 (1-100 nM) and clonidine (300 nM) attenuated responses to EFS, the attenuation of UK14304 (100 nM) being reversed by yohimbine (300 nM). 6. It is concluded that in GPPA, where a presynaptic inhibition of sensory neurotransmission by alpha 2-adrenoceptor activation could be shown, there was no evidence for such modulation by either sumatriptan-sensitive 5-HT1 receptors or kappa-opioid receptors.     

Modulatory effect of bradykinin on the release of noradrenaline from rat isolated atria.

1. We investigated the modulation by bradykinin (BK) of electrically induced noradrenaline release in rat isolated atria preincubated with [3H]-noradrenaline. 2. BK (1-100 nM) enhanced significantly the stimulation-induced outflow of radioactivity in a concentration-dependent manner with a calculated EC50 of 0.58 nM. 3. Des-Arg9-BK (0.1-100 nM), a selective B1 receptor agonist, did not modify the stimulation-induced outflow of radioactivity. Hoe 140 (10 nM), a selective B2 receptor antagonist, but not [Leu8]-des-Arg9-BK (100 nM), a selective B1 receptor antagonist, blocked the facilitatory effect of BK. 4. The effect of BK was not affected by diclofenac (1 microM), a cyclo-oxygenase inhibitor. Bisindolylmaleimide (1 microM), a protein kinase C inhibitor, significantly reduced the facilitatory effect of BK (10 nM), angiotensin II (0.3 microM) and phorbol dibutyrate (0.1 and 1 microM) but not of fenoterol (1 microM). 5. The results suggest that BK enhances noradrenaline release via a prejunctional B2 kinin receptor in the rat atrium. The effect appears to involve protein kinase C as a second messenger.     

Blockade by ONO-NT-012, a unique prostanoid analogue, of prostaglandin E2-induced allodynia in conscious mice.

1. In the presence of atropine (0.2 microM) and indomethacin (2 microM), the effects of 5-hydroxytryptamine (5-HT) have been studied on electrically-evoked, neurogenic contractions of the guinea-pig proximal colon in vitro. 2. 5-HT, at higher concentrations than 1 nM, caused an increase in electrically (1 Hz, 0.3 ms, 160 mA)-evoked, atropine-resistant contractions in a concentration-dependent manner and at 30 nM produced a maximal effect (pEC50 value of 8.20 +/- 0.11, n = 6). The enhancing effects of 5-HT on the electrically evoked contractions were mimicked by alpha-methyl-5-HT (pEC50 value of 6.59 +/- 0.05, n = 6). 3. Both hexamethonium (100 microM) and spantide (10 microM), selective antagonists for nicotinic and tachykinin receptors respectively, significantly reduced the enhancement of the electrically evoked contractions by 5-HT (30 nM). 4. DAU 6285 (3 microM), a 5-HT4 receptor antagonist, abolished the enhancing action of 5-HT (30 nM), but metitepine (0.03 microM), a 5-HT1/5-HT2 receptor antagonist, ketanserin (0.01 microM), a 5-HT2 receptor antagonist, and ondansetron (1 microM), a 5-HT3 receptor antagonist, had no effect on the enhancement. The enhancing effects of alpha-methyl-5-HT (1 microM) were also abolished by DAU 6285 (3 microM). 5. Both 5-HT (30 nM) and alpha-methyl-5-HT (1 microM) had no effect on contractions to exogenous substance P (0.15-0.3 nM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide is involved in the relaxant effect of capsaicin in the human sigmoid colon circular muscle

The relaxant effect of capsaicin (300 nM) has been studied on mucosa-free circular strips of the human sigmoid colon in vitro. The response of precontracted preparations to capsaicin (sub-maximal relaxation) was reduced by over 50% by the nitric oxide synthase inhibitor N(G)-nitro- L-arginine (L-NOARG; 20 microM or 100 microM) or by the guanylate cyclase inhibitor 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), but not by tetrodotoxin (1 microM) or the P(2) purinoceptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS; 50 microM). L-NOARG or ODQ caused moderate contraction of the circular muscle, indicating a tonic "nitrergic" control. Anandamide (1-100 microM), an endogenous cannabinoid and capsaicin VR(1) receptor stimulant, failed to either mimic or modify the response to capsaicin (300 nM).It is proposed that capsaicin causes the release of smooth muscle relaxant substance(s) from afferent nerve endings in the gut wall, in a tetrodotoxin-resistant manner. Nitric oxide (possibly released from capsaicin-sensitive afferents) plays an important role in the capsaicin-evoked response. No evidence has been found for an involvement of PPADS-sensitive P(2) purinoceptors in the response to capsaicin or for a stimulation or inhibition of capsaicin-sensitive receptors by anandamide in the human sigmoid colon.     

ATP- and adenosine-mediated signaling in the central nervous system: the role of extracellular ATP in hippocampal long-term potentiation

Application of 10 microM ATP for 10 min transiently depressed, then slowly augmented, synaptic transmission in CA1 neurons, leading to long-term potentiation (LTP) (ATP-induced LTP). This ATP-induced LTP was blocked by addition of an N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, D,L-2-amino-5-phosphonovalerate (5 microM). For ATP-induced LTP, delivery of test synaptic inputs once every 20 s to CA1 neurons could be substituted by application of 100 nM NMDA during ATP perfusion. In addition, ATP-induced LTP was blocked by co-application of an ecto-protein kinase inhibitor, K-252b (40 nM), whereas a P2X purinoceptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (50 microM), or a P2Y purinoceptor antagonist, basilen blue (10 microM), had no effect. These results, therefore, indicate that the mechanisms of ATP-induced LTP involve the modulation of NMDA receptors / Ca(2+) channels and the phosphorylation of extracellular domains of synaptic membrane proteins, one of which could be the NMDA receptor / Ca(2+) channel.     

Modulatory effect of imetit, a histamine H3 receptor agonist, on C-fibers, cholinergic fibers and mast cells in rabbit lungs in vitro.

The pharmacological mechanisms involved in the interactions between C-fibers, cholinergic fibers and mast cells were investigated in tracheally perfused rabbit lungs by measuring the simultaneous release of substance P and histamine in lung effluents. The amounts of substance P and histamine released in lung superfusates were measured by radioimmunoassay (RIA) after administration of capsaicin and carbachol. Capsaicin (10(-4) M) induced a simultaneous increase in substance P (273 +/- 56% of baseline) and histamine (460 +/- 138%) release. Similarly, carbachol (10(-4) M) caused an increase in the release of both substance P (367 +/- 111%) and histamine (1379 +/- 351%). The effect of capsaicin was prevented by pretreating the lungs with the tachykinin NK1 receptor antagonist SR 140333 (10(-7) M), and atropine (10(-6) M). SR 140333 prevented the carbachol-induced release of substance P but not of histamine. Exogenous substance P induced an increase in histamine release (136 +/- 7%) which was significantly greater in lungs perfused with the neutral endopeptidase inhibitor, thiorphan (10(-5) M) (272 +/- 35%). This effect was prevented by atropine (10(-6) M). Pretreatment of lungs with imetit (5 x 10(-8) M), a selective H3 receptor agonist, prevented the capsaicin-induced release of both mediators. Imetit also blocked the carbachol-induced release of substance P but not of histamine. Exogenous substance P-evoked histamine release was inhibited by imetit. Therefore, it can be concluded that substance P released through the action of capsaicin can activate cholinergic fibers, leading to cholinoceptor stimulation with subsequent activation of C-fibers and mast cells. While the presence of presynaptic H3 receptors modulating substance P-induced acetylcholine release was only surmised, the existence of modulating histamine H3 receptors on C-fibers was confirmed.     

Dynorphin A-(1-13)-Tyr14-Leu15-Phe16-Asn17-Gly18-Pro19 : a potent and selective kappa opioid peptide.

Dynorphin A-(1-13)-Tyr14-Leu15-Phe16-Asn17-Gly18-Pro19+ ++ (dynorphin Ia: a peptide derived from the structure of adrenal dynorphin I) was synthesized by the solid-phase procedure. The product was purified and compared with dynorphin A-(1-13) and [D-Pro10]dynorphin A-(1-11) for its ability to inhibit the electrically evoked contractions of the guinea pig ileum (GPI) and mouse vas deferens (MVD) and to compete with the binding of [3H]ethylketocyclazocine (kappa ligand), [3H][D-Ala2,MePhe4,Glyol5]enkephalin (mu ligand) and [3H][D-Ser2,Thr6]Leu-enkephalin (delta ligand) to membrane preparations of the guinea pig cerebellum or rat brain. Additionally, the antinociceptive effects of the synthetic peptide were assessed in rat paw-pressure and tail-flick tests. In the GPI, dynorphin Ia possessed a relative potency (IC50 0.5 nM) that was comparable to that of [D-Pro10]dynorphin A-(1-11) (IC50 0.5 nM) or dynorphin A-(1-13) (IC50 0.7 nM). In the delta specific MVD assay, dynorphin Ia displayed a reduced potency (IC50 235 nM) as compared with that of dynorphin A-(1-13) (IC50 20 nM) or [D-Pro10]dynorphin A-(1-11) (IC50 46 nM). The affinity of dynorphin Ia for the kappa site in the guinea pig cerebellum (Ki 0.25 nM) was comparable to those of dynorphin A-(1-13) (Ki 0.11 nM) and [D-Pro10]dynorphin A-(1-11) (Ki 0.10 nM). However, the peptide possessed reduced affinities for the mu (Ki 6.7 nM) and delta (Ki 71 nM) opioid receptors as compared with [D-Pro10]dynorphin A-(1-11) (Ki 1.7 and 1.5 nM) an dynorphin A-(1-13) (Ki 0.5 and 4.4 nM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Paradoxical hyperalgesia induced by mu-opioid receptor agonist endomorphin-2, but not endomorphin-1, microinjected into the centromedial amygdala of the rat

The effects of endomorphin-2 or endomorphin-1 microinjected into the centromedial amygdala on the thermally-induced tail-flick response were studied in male CD rats. Microinjection of endomorphin-2 (8.7-35.0 nmol) given into the centromedial amygdala time- and dose-dependently decreased the tail-flick latencies. On the other hand, endomorphin-1 (8-32.6 nmol) given into the same site did not cause any change of the tail-flick latency. However, endomorphin-1 (32.6 nmol) or endomorphin-2 (35.0 nmol) given into the basolateral site of amygdala did not affect the tail-flick latency. Pretreatment with the antiserum against dynorphin A(1-17) (200 microg) significantly reversed the decrease of the tail-flick latency induced by endomorphin-2. The decrease of the tail-flick latency induced by endomorphin-2 was also blocked by the endomorphin-2 selective micro-opioid receptor antagonist 3-methoxynaltrexone (6.4 pmol) and by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (30 nmol), but not by the kappa-opioid receptor antagonist nor-binaltorphimine (6.6 nmol). It is concluded that endomorphin-2, but not endomorphin-1, given into the centromedial amygdala stimulates a 3-methoxynaltrexone-sensitive mu-opioid receptor subtype to induce the release of dynorphin A(1-17), which then acts on the NMDA receptor, but not kappa-opioid receptor for producing hyperalgesia. This conclusion is further supported by the additional findings that dynorphin A(1-17) (2.3 nmol) given into the centromedial amygdala also caused the decrease of the tail-flick latency, which was similarly blocked by the NMDA receptor antagonist MK-801 (30 nmol), but not kappa-opioid receptor antagonist nor-binaltorphimine (6.6 nmol).