Crotalphine, a novel potent analgesic peptide from the venom of the South American rattlesnake Crotalus durissus terrificus

We have shown that the venom of the South American rattlesnake Crotalus durissus terrificus induces a long-lasting antinociceptive effect mediated by activation of kappa- and delta-opioid receptors. Despite being mediated by opioid receptors, prolonged treatment with the crotalid venom does not cause the development of peripheral tolerance or abstinence symptoms upon withdrawal. In the present study, we have isolated and chemically characterized a novel and potent antinociceptive peptide responsible for the oral opioid activity of this crotalid venom. The amino acid sequence of this peptide, designated crotalphine, was determined by mass spectrometry and corroborated by solid-phase synthesis to be 相似文献   

The neuronal NO synthase participation in the peripheral antinociception mechanism induced by several analgesic drugs

The production of nitric oxide (NO) from l-arginine is catalyzed by NO synthase (NOS), which exists as the following three isoforms: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). The participation of this pathway in peripheral antinociception has been extensively established by our group with the use of several types of drugs, including opioids, cannabinoids, cholinergic, and α(2C) adrenoceptor agonists and nonsteroidal anti-inflammatory drugs (NSAIDS), and even non-pharmacological procedures such as electroacupuncture. In this study, we aimed to refine the previous data to investigate which type of NOS isoform is involved in the peripheral antinociception mechanism induced by anandamide, morphine, SNC80, bremazocine, acetylcholine, xylazine, baclofen, dipyrone, and diclofenac. After hyperalgesia was induced by intraplantar injection of prostaglandin E(2) in male Wistar rats, we measured peripheral nociception with the paw pressure test. All drugs that were used induced a peripheral antinociception effect that was completely blocked by injection of the selective neuronal NO synthase inhibitor, L-NPA (24μg/paw). The exception was the GABA(B) agonist baclofen, which induced an effect that was not antagonized. We used the inhibitors L-NIO and -NIL (24μg/paw) to exclude the involvement of endothelial and inducible NO synthase, respectively. These drugs were ineffective against the antinociception effect induced by all analgesic drugs that we utilized. Based on the experimental evidence, we conclude that the local injection of analgesic drugs activates nNOS to release NO and induce peripheral antinociception.     

Inhibition of inducible nitric oxide synthase in persistent pain

The present study was undertaken to investigate the role of inducible nitric oxide synthase in a rat model of persistent pain. The effects of L-N6 (1-iminoethyl) lysine (L-NIL), a relatively potent and relatively selective inhibitor of inducible nitric oxide synthase, were investigated in carrageenan induced hyperalgesia L-NIL (0.1 microMole) injected intraplantar or intrathecal markedly enhanced carrageenan induced hyperalgesia. These effects were reversed during the third hour by co-administration of L-arginine (900 mg/kg i.p.) but not D-arginine. Methylene blue (MB), a soluble guanylate cyclase inhibitor, administered intrathecally (0.1 microg) had no effect on L-NIL potentiation of carrageenan hyperalgesia but abolished antinociception induced by L-arginine. Obtained results suggest that nitric oxide derived from inducible nitric oxide synthase play an inhibitory role in carrageenan produced hyperalgesia in rat.     

Antinociceptive action of FK506 in mice

Immunophilins are abundantly present in the brain as compared to the immune system. Immunophilin-binding agents like FK506 are known to inactivate neuronal nitric oxide synthase (nNOS) by inhibiting calcineurin and decrease the production of nitric oxide. Nitric oxide is involved in the mediation of nociception at the spinal level. In the present study, the effect of FK506 on the tail flick response in mice and the possible involvement of NO-L-arginine pathway in this paradigm was evaluated. FK506 (0.5, 1 and 3 mg/kg, ip) produced a significant antinociception in the tail flick test. Nitric oxide synthase (NOS) inhibitor L-NAME significantly and dose dependently (10-40 mg/kg, ip) potentiated the FK506 (0.5 mg/kg)-induced antinociception. On the other hand, NOS substrate L-arginine (100, 200 and 400 mg/kg) inhibited the FK506-induced antinociception in a dose-dependent manner. Concomitant administration of L-NAME (20 and 40 mg/kg) with L-arginine (200 mg/kg) blocked the inhibition exerted by L-arginine on the FK506-induced antinociception. Thus, it was concluded that NO- L-arginine pathway may be involved in the FK506-induced antinociception in tail flick test.     

L-NAME causes antinociception by stimulation of the arginine-NO-cGMP pathway

NG-nitro-L-arginine methyl ester (L-NAME) has been used extensively as a paradigmatic inhibitor of NO synthase and has been shown to cause antinociception in several experimental models. We describe here how L-NAME produced a dose-dependent antinociceptive effect when injected intraperitoneally in the mouse after acetic acid induced writhings, or intraplantarly in the rat paw pressure hyperalgesia induced by carrageenin or prostaglandin E2. In contrast another NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), had no significant effect per se but inhibited L-NAME systemic induced antinociception in mice and local induced antinociception in the rat paw hyperalgesia test. D-NAME had no antinociceptive effect upon carrageenin-induced hyperalgesia. Pretreatment of the paws with two inhibitors of guanylate cyclase, methylene blue (MB) and 1H-:[1,2,4]-oxadiazolo-:[4,3-a] quinoxalin-1-one (ODQ) abolished the antinociceptive effect of L-NAME. L-Arginine and the cGMP phosphodiesterase inhibitor, MY 5445 significantly enhanced the L-NAME antinociceptive effect. The central antinociceptive effect of L-NAME was blocked by co-administration of L-NMMA, ODQ and MB. The present series of experiments shows that L-NAME, but not L-NMMA, has an antinociceptive effect. It can be suggested that L-NAME causes the antinociceptive effect by stimulation of the arginine/ NO/ cGMP pathway, since the antinociceptive effect of L-NAME can be antagonized by L-NMMA and abolished by the guanylate cyclase inhibitors (MB and ODQ). In addition, the NO synthase substrate, L-arginine and the cGMP phosphodiesterase inhibitor, MY5445 were seen to potentiate the effects of L-NAME. Thus, L-NAME used alone, has limitations as a specific inhibitor of the arginine-NO-cGMP pathway and may therefore be a poor pharmacological tool for use in characterising participation in pathophysiological processes.     

Early effect of Crotalus durissus terrificus venom on kidney circulation

Local blood flow was measured in renal cortex (1 mm below cortical surface) by means of the hydrogen clearance method in urethanized rats. Recording of blood pressure from femoral artery was performed. Crotalus durissus terrificus venom injection (one mg/kg i.v.) significantly decreased cortical blood flow at 10 min, without a significant arterial pressure modification. Posterior injection of mannitol 200 mg induced a significant increase in cortical blood flow, although initial values were not reached. Electron microscopy showed thromboses in the glomerular capillaries 35 minutes after venom injection. It is suggested that the precocious effect of this venom on renal cortical blood flow may be instrumental in the development of the renal acute insufficiency induced by Crotalus durissus terrificus venom.     

Geographic and ontogenic variability in the venom of the neotropical rattlesnake Crotalus durissus: pathophysiological and therapeutic implications

A comparative study was performed on the venoms of adult specimens of the neotropical rattlesnake, Crotalus durissus, from Guatemala, Costa Rica, Venezuela and Brazil, together with the venom of newborn specimens of C. d. durissus from Costa Rica. Venoms from Brazil (C. d. terrificus) and from newborn specimens of C. d. durissus presented an electrophoretic pattern characterized by the predominance of bands with molecular mass of 36 and 15 kDa, whereas those of adult specimens of C. d. durissus from Guatemala and Costa Rica, and C. d. cumanensis from Venezuela, showed a conspicuous band of 62 kDa, and additional bands of 36, 29 and 15 kDa. Moreover, venoms from C. d. terrificus and C. d. cumanensis showed a prominent band of < 10 kDa that probably corresponds to crotamine, since a 'crotamine-like' activity was detected in these venoms upon intraperitoneal injection in mice. Venoms of C. d. terrificus, C. d cumanensis and newborn C. d. durissus induced higher lethal and myotoxic effects than those of adult C. d. durissus. In contrast, adult C. d. durissus and C. d. cumanensis venoms induced hemorrhage, whereas venoms of C. d. terrificus and newborn C. d. durissus lacked this effect. All venoms showed coagulant effect in plasma, the highest activity being observed in the venom of newborn C. d. durissus. An anti-crotalic antivenom produced by Instituto Butantan (Brazil), using C. d. terrificus venom as antigen, was effective in the neutralization of lethal, myotoxic and coagulant effects of all venoms studied, being ineffective in the neutralization of hemorrhagic activity of the venoms of C. d. cumanensis and C. d. durissus. On the other hand, a polyvalent antivenom produced by Instituto Clodomiro Picado (Costa Rica), using the venoms of C. d. durissus. Bothrops asper and Lachesis stenophrys as antigens, was able to neutralize lethal, myotoxic, coagulant and hemorrhagic effects of C. d. durissus venom, but was ineffective in the neutralization of lethality and myotoxicity of C. d. terrificus, C. d. cumanensis and newborn C. d. durissus venom. The high toxicity of South American and newborn C. d. durissus venoms is related to the presence of high concentrations of the neurotoxic phospholipase A2 complex 'crotoxin'. Accordingly, antivenom from Instituto Butantan has a much higher titer of anti-crotoxin antibodies than antivenom from Instituto Clodomiro Picado. Crotalus durissus represents an example of intraspecies variation in venom composition and pharmacology that has relevant pathophysiologic and therapeutic implications.     

The venom of South American rattlesnakes inhibits macrophage functions and is endowed with anti-inflammatory properties

The injection of Crotalus durissus terrificus venom into the foot pad of mice did not induce a significant inflammatory response as evaluated by oedema formation, increased vascular permeability and cell migration. The subcutaneous injection of the venom, or its addition to cell cultures, had an inhibitory effect on the spreading and phagocytosis of resident macrophages, without affecting the viability of the cells. This effect was not observed when the venom was added to cultures of thioglycollate elicited macrophages, but it was able to inhibit these macrophage functions when the cells were obtained from animals injected simultaneously with the venom and thioglycollate. These observations suggest that the venom interferes with the mechanisms of macrophage activation. Leukocyte migration induced by intraperitoneal injection of thioglycollate was also inhibited by previous venom injection. This down-regulatory activity of the venom on macrophage functions could account for the mild inflammatory response observed in the site of the snake bite in Crotalus durissus terrificus envenomation in man.     

Participation of the components of L-arginine/nitric oxide/cGMP cascade by chemically-induced abdominal constriction in the mouse

The purpose of this study was to investigate the role of the L-arginine/nitric oxide (NO)/cGMP pathway in p-benzoquinone-induced writhing model in mouse. L-arginine, a NO precursor, displayed antinociceptive effects at the doses of 0.125-1.0 mg/kg. When the doses of L-arginine were increased gradually to 10-100 mg/kg, a dose-dependent triphasic pattern of nociception-antinociception-nociception was obtained. The NO synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) (18.7515 mg/kg), possessed antinociceptive activity. Methylene blue (MB), a guanylyl cyclase and/or NOS inhibitor, (5-160 mg/kg) also produced a dose-dependent triphasic response. When L-arginine (50 mg/ kg) was combined with L-NAME (75 mg/kg). L-arginine-induced antinociception did not change significantly. Cotreatment of L-arginine with 5 mg/kg MB significantly decreased MB-induced antinociception and reversed the nociception induced by 40 mg/kg MB to antinociception. It is concluded that the components of L-arginine/nitric oxide/cGMP cascade may participate in nociceptive processes both peripherally and centrally by a direct effect on nociceptors or by the involvement of other related pathways of nociceptive processes induced by NO.     

Isolation and characterization of the alpha and beta subunits of the platelet-activating glycoprotein from the venom of Crotalus durissus cascavella

It was concluded in a previous paper that the high Mr platelet-activating glycoprotein isolated earlier from the venom of Crotalus durissus cascavella has an hexameric structure of the alpha 3 beta 3 type involving two distinct subunits. Data reported here demonstrate that these two subunits are separable from each other by ion exchange chromatography under denaturating conditions, have similar Mrs (alpha = 12,540 et beta = 13,770) and exist in a one to one ratio within the native molecule. Carbohydrate analysis indicated that they are both similarly glycosylated to a small extent. They have slightly different amino-acid compositions, a common N-terminal sequence up to the fifth residue and similar extinction coefficients at 280 nm. The native molecule has a calculated Mr of 78,930. Additional data demonstrated that convulxin from the venom of Crotalus durissus terrificus is the same platelet-activating agent as the presently described platelet-activating glycoprotein (PAG) from the venom of Crotalus durissus cascavella.     

Isolation of a crotoxin-like protein from the venom of a South American rattlesnake (Crotalus durissus collilineatus)

1. A crotoxin-like protein was isolated from the venom of a South American rattlesnake Crotalus durissus collilineatus. 2. Many of its properties are similar to those of crotoxin, including its non-covalent heterodimeric structure, electrophoretic mobility on SDS-PAGE, isoelectric focusing properties, toxicity in mice, immunological reactivity, multiple isoforms, phospholipase activity, peptide map, and instability on an anion-exchange column. 3. Results indicate that "collilineatus toxin" is strongly homologous with crotoxin, found in the venom of Crotalus durissus terrificus, and all other characterized rattlesnake neurotoxins.     

Circadian-Rhythm-dependent Effects of L-NG-Nitroarginine Methyl Ester (L-Name) on Morphine-Induced Analgesia

Circadian changes in the interactions between L-N^G-nitroarginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, and morphine-induced antinociception were investigated by the mouse hot-plate test. Born the basal pain sensitivity and morphine-induced analgesia undergo significant 24h variations. L-NAME (40 mg/kg, ip) alone did not show any antinociceptive activity, but potentiated morphine-induced analgesia when combined with morphine at all injection times. In terms of percentage absolute potentiation (%AP), L-NAME dramatically augmented the analgesic effect of morphine in the late dark period at 19 hours after lights on (HALO). It is concluded that nitric oxide (NO) is involved in the modulation of the analgesic effect of morphine; thus, the L-NAME and morphine combination might be beneficial in alleviating pain.     

一氧化氮合酶的遗传、生理研究进展

一氧化氮具有广泛的生理功能,哺乳动物体内的NO是由NO合酶(NOS)氧化L-精氨酸而合成的,合成后的NO迅速跨膜扩散释放,NO合成失调能介导多种疾病。催化NO生物合成的NOS有三种亚型:神经元型NOS(nNOS)、内皮型NOS(eNOS)和诱导型NOS(iNOS),目前,人的三型NOS已纯化并且已分子克隆成功,对一氧化氮合酶的遗传研究确认了NOS家族的基因结构和染色体定位。     

Regulation and function of the protein inhibitor of nitric oxide synthase (PIN)/dynein light chain 8 (LC8) in a human mast cell line

The protein inhibitor of nitric oxide synthase (PIN) was independently identified as an inhibitor of nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS), and as a member of the cellular dynein light chain family, dynein light chain 8 (LC8), responsible for intracellular protein trafficking. Mast cells (MC) are involved in several homeostatic and pathological processes and can be regulated by NO. This study describes the expression of PIN/LC8 in the human MC line HMC-1. We also studied if PIN/LC8 binds nNOS, and what role this might have in leukotriene (LT) production. We found that PIN/LC8 mRNA and protein was expressed in HMC-1. Using a GST-PIN construct, we showed PIN binds to nNOS, but not endothelial (e)NOS in HMC-1; in our studies HMC-1 did not express inducible (i)NOS. Intracellular delivery of anti-PIN/LC8 antibody enhanced ionophore (A23187)-induced LT production through an unknown mechanism. Thus we established for the first time expression of PIN/LC8 in human MC, its ability to bind nNOS, and the effect that blocking it has on LT production in a human MC lines.     

The protective effect of aminoguanidine on cerebral ischemic damage in the rat brain

The NADPH-diaphorase (NADPH-d) histochemical technique is commonly used to localize the nitric oxide (NO) produced by the enzyme nitric oxide synthase (NOS) in neural tissue. The expression of inducible nitric oxide synthase (iNOS) is induced in the late stage of cerebral ischemia, and NO produced by iNOS contributes to the delay in recovery from brain neuronal damage. The present study was performed to investigate whether the increase in nitric oxide production via inducible nitric oxide synthase was suppressed by the administration of aminoguanidine, a selective iNOS inhibitor, as it follows a decrease of NADPH-diaphorase activity (a marker for NOS) after four-vessel occlusion used as an ischemic model. The administration of aminoguanidine (100 mg/kg i.p., twice per day up to 3 days immediately after the ischemic insult) reduced the number of NADPH-diaphorase positive cells to control levels. Our results indicated that aminoguanidine suppressed NADPH-diaphorase activity, and also decreased the number of NADPH-diaphorase positive cells in the CA1 region of the hippocampus following ischemic brain injury.     

Effects of nitric oxide synthase inhibitors on the febrile response to lipopolysaccharide and muramyl dipeptide in guinea pigs

We investigated the effect of N-nitro-L-arginine methyl ester (L-NAME), an unspecific nitric oxide synthase (NOS) inhibitor, and aminoguanidine, a relatively selective inhibitor of the inducible NOS enzyme, on both gram-negative lipopolysaccharide (LPS) and gram-positive muramyl dipeptide (MDP) fever in guinea pigs. Intraperitoneal injection of either 10 mg/kg L-NAME or 25 mg/kg aminoguanidine inhibited the febrile response to an intramuscular injection of 50 microg/kg MDP. However, LPS fever (20 microg/kg) was inhibited only by L-NAME. The development of LPS fever may therefore occur independently of the synthesis of nitric oxide by the inducible NOS enzyme, while MDP fever may involve synthesis of nitric oxide by both the inducible and the constitutively expressed NOS enzymes.     

Role of nitric oxide/cyclic GMP/K(+) channel pathways in the antinociceptive effect caused by 2,3-bis(mesitylseleno)propenol

The present study examined the antinociceptive effects induced by 2,3-bis(mesitylseleno)propenol, a bis-selenide alkene derivate, given orally, in chemical models of pain in rats and mice. Selenide administered orally (p.o.) into the rats caused antinociception against the first and second phases of the formalin test, with mean ID(50) values of 28.17 and 39.68 mg/kg, respectively. The antinociceptive effect caused by selenide (50 mg/kg, p.o.) on the formalin test was reversed by pretreatment with N(G)-L-nitro-arginine methyl ester (L-NAME, a nitric oxide (NO) synthase inhibitor), methylene blue (a non-specific NO/guanylyl cyclase inhibitor) and glibenclamide (an ATP-sensitive K(+) channel inhibitor), but not by atropine (a muscarinic antagonist). Given orally selenide in mice produced an inhibition of glutamate-, histamine- and compound 48/80-induced nociception with mean ID(50) values of 27.58, 36.18 and 44.53 mg/kg, respectively. Moreover, oral treatment with selenide in mice decreased licking -- induced by serotonin (mean ID(50) value of >50 mg/kg). The data show that selenide exerts pronounced systemic antinociception in chemical (formalin, glutamate, histamine, compound 48/80 and serotonin-induced pain) models of nociception. Taken together, these results suggest that the antinociceptive effect of selenide on the formalin test involves the participation of nitric oxide/cyclic GMP/K(+) channel pathways in rats.     

The primary structure of crotamine (author's transl)]

The primary structure of crotamine, a basic toxin isolated from the venom of the South American rattle-snake Crotalus durissus terrificus has been determined. The polypeptide chain is composed of 42 residues of amino acids. Crotamine shows a molecular weight of 4900 and contains 6 half cystine, 9 lysine, 2 arginine, 2 histidine and 2 tryptophan residues.     

Roles of nitric oxide and prostaglandins in pathogenesis of delayed colonic transit after burn injury in rats

Burn injury has been shown to impair gut transit, but the exact mechanism remains unknown. The present study investigated whether nitric oxide synthase (NOS) and cyclooxygenase (COX) mediated changes in burn-induced colonic transit. After rats underwent 30% total body surface area burn injury, they were injected with S-methylisothiourea (SMT, selective inducible NOS inhibitor), 7-nitronidazole (7-NI, selective neuronal NOS inhibitor), and nimesulide (NIM, selective COX-2 inhibitor), respectively. The protein and mRNA of NOS and COX-2 were measured by Western blot analysis and real-time RT-RCR, and localization of NOS and COX-2 protein was determined by immunohistochemistry. Our results showed that colonic transit assessed by the geometric center was delayed from 3.47+/-0.28 in controls to 2.21+/-0.18 after burn (P<0.009). SMT and NIM significantly improved colonic transit in burned rats but had no effect in sham-operated rats. 7-NI failed to modify delayed transit in burned rats but significantly delayed colonic transit in sham-operated rats. Both protein and mRNA of inducible NOS and COX-2 increased significantly but not neuronal NOS in burned rats. Inducible NOS protein expression was noted not only in epithelial cells but also in neurons of the myenteric ganglia in burned rats. These findings suggest that nitric oxide (NO) produced by neuronal NOS plays an important role in mediating colonic transit under the physiological condition. NO produced by inducible NOS and prostaglandins synthesized by COX-2 are both involved in the pathogenesis of delayed colonic transit after burn injury. Inducible NOS expression in neurons of the myenteric ganglia may contribute to dysmotility with burn injury.