Gisele Picolo

Pain and analgesia: The dual effect of nitric oxide in the nociceptive system.

Nitric oxide (NO) is involved in many physiological processes and several lines of evidence have indicated that NO plays a complex and diverse role in the modulation of pain. Nitric oxide is an important neurotransmitter involved in the nociceptive process and, in the dorsal horn of the spinal cord, it contributes to the development of central sensitization. On the other hand, experimental data have also demonstrated that NO inhibits nociception in the peripheral and also in the central nervous system. In addition, it has been shown that nitric oxide mediates the analgesic effect of opioids and other analgesic substances. The information included in the present review aims to present and analyze data about the dual effect of NO on pain transmission and control, the molecular mechanisms involved in these effects and also the potential use of nitric oxide in pain therapy.

Inflammation induced by Bothrops asper venom.

Inflammation is a major characteristic of envenomation by snakes from viperine and crotaline species. Bothrops asper snake venom elicits, among other alterations, a pronounced inflammatory response at the site of injection both in humans and experimental animals. This review describes the current status of our understanding of the inflammatory reaction, including pain, triggered by Bothrops asper venom. The experimental studies on the action of this venom as well as the complex network of chemical mediators involved are summarized. Moreover, aspects of the molecular mechanisms orchestrating this important response to envenomation by Bothrops asper are presented. Considering that isolated toxins are relevant tools for understanding the actions of the whole venom, studies dealing with the mechanisms of inflammatory and nociceptive properties of phospholipases A(2), a metalloproteinase and serine-proteases isolated from Bothrops asper venom are also described.

Hyperalgesia induced by Asp49 and Lys49 phospholipases A2 from Bothrops asper snake venom: pharmacological mediation and molecular determinants.

The ability of Lys49 and Asp49 phospholipases A(2) (PLA(2)), from Bothrops asper snake venom, to cause hyperalgesia was investigated in rats, using the paw pressure test. Intraplantar injection of both toxins (5-20 micro g/paw) caused hyperalgesia, which peaked 1h after injections. Incubation of both proteins with heparin, prior to their injection, partially reduced this response. Chemical modification of Asp49 PLA(2) with p-bromophenacyl bromide (p-BPB), which abrogates its PLA(2) activity, also abolished hyperalgesia. Intraplantar injection of a synthetic peptide corresponding to the C-terminal sequence 115-129 of Lys49 PLA(2), caused hyperalgesia of similar time course, but varying magnitude, than that induced by the native protein. In contrast, a homologous peptide derived from the Asp49 PLA(2) did not show any nociceptive effect. Hyperalgesia induced by both PLA(2)s was blocked by the histamine and serotonin receptor antagonists promethazine and methysergide, respectively, by the bradykinin B(2) receptor antagonist HOE 140 and by antibodies to tumor necrosis factor alfa (TNFalpha) and interleukin 1 (IL-1). Pretreatment with guanethidine, atenolol, prazosin and yohimbine, inhibitors of sympathomimetic amines, or with indomethacin, inhibitor of the cyclo-oxygenase pathway, reduced Lys49 PLA(2)-induced hyperalgesia without interfering with the nociceptive activity of Asp49 PLA(2). The hyperalgesic response to both myotoxins was not modified by pretreatment with celecoxib, an inhibitor of the cyclo-oxygenase type II, by zileuton, an inhibitor of the lipoxygenase pathway or by N(g)-methyl-L-arginine (LNMMA), an inhibitor of nitric oxide synthase. These results suggest that Asp49 and Lys49 PLA(2)s are important hyperalgesic components of B. asper venom, and that Lys49 and Asp49 PLA(2)s exert their algogenic actions through different molecular mechanisms.

Pharmacological modulation of hyperalgesia induced by Bothrops asper (terciopelo) snake venom

The ability of Bothrops asper snake venom to cause hyperalgesia was investigated in rats, using the paw pressure test. Intraplantar injection of the venom (5-15 microg/paw) caused a dose and time-related hyperalgesia, which peaked 2h after venom injection. Bothrops asper venom-induced hyperalgesia was blocked by the bradykinin B(2) receptor antagonist HOE 140 and attenuated by dexamethasone, an inhibitor of phospholipase A(2). Inhibition of the lipoxygenase pathway by NDGA abrogated the algogenic phenomenon. The hyperalgesic response was not modified by pretreatment with indomethacin, an inhibitor of the cyclo-oxygenase pathway, by meloxicam, an inhibitor of the type 2 cyclo-oxygenase pathway, by the PAF receptor antagonist BN52021 or by anti-TNF-alpha or anti-interleukin 1 antibodies. Intraplantar injection of the venom also caused an oedematogenic response which was not modified by any of these pharmacological treatments. These results suggest that hyperalgesia induced by Bothrops asper venom is, at least partially, mediated by bradykinin, phospholipase A(2) activity and leukotrienes. Distinct mechanisms are involved in the development of hyperalgesia and oedema induced by the venom.

δ-Opioid receptors and nitric oxide mediate the analgesic effect of Crotalus durissus terrificus snake venom

The antinociceptive effect of Crotalus durissus terrificus venom was investigated in a model of inflammatory hyperalgesia induced by carrageenin. The rat paw pressure test was applied before and 3 h after the intraplantar (i.pl.) injection of carrageenin. The venom administered per os before and 1 or 2 h after carrageenin blocked hyperalgesia. When carrageenin was injected in both hind paws and naloxone into one hind paw, antinociception was abolished only in the paw injected with naloxone. D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP) and nor-binaltorphimine, antagonists of micro- and kappa-opioid receptors, respectively, did not alter the effect of the venom. N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (ICI 174,864), an antagonist of delta-opioid receptors, antagonised this effect. Prolonged administration of the venom did not induce tolerance to this antinociceptive effect. N(G)-methyl-L-arginine (L-NMMA) and methylene blue, inhibitors of nitric oxide synthase and soluble guanylate cyclase, respectively, injected i.pl., antagonised antinociception. These data indicate that both delta-opioid receptors and nitric oxide participate in the mediation of the peripheral antinociceptive effect of C. durissus terrificus venom.

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 <EFSPENCQGESQPC, where <E is pyroglutamic acid and the two cysteine residues forming a disulfide bond. This 14-amino-acid residue sequence is identical to the gamma-chain sequence of crotapotin, a non-toxic component of this snake venom. Crotalphine, when orally administered (0.008-25mug/kg), induces antinociceptive effect in the prostaglandin E(2)- and carrageenin-induced mechanical hyperalgesia models in rats and in the hot-plate test in mice. Crotalphine was also effective when administered by intravenous (0.0032-0.04mug/kg) or intraplantar (s.c., 0.00006-0.3mug/paw) routes. In the mechanical hyperalgesia models, crotalphine shows a long-lasting (5 days) antinociceptive effect. d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr amide (CTOP) and N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (ICI 174,864), antagonists of mu- and delta-opioid receptors, respectively, did not alter the antinociceptive effect of the peptide, whereas nor-binaltorphimine, an antagonist of kappa-opioid receptors, blocked this effect. These results indicate that crotalphine induces antinociception mediated by activation of kappa-opioid receptors and may contribute to the antinociceptive effect of the crotalid venom.

Activation of peripheral ATP-sensitive K+ channels mediates the antinociceptive effect of Crotalus durissus terrificus snake venom.

The role of peripheral potassium channels on the antinociceptive effect of Crotalus durissus terrificus venom, a mixed delta- and kappa-opioid receptor agonist, was investigated in hyperalgesia induced by carrageenin or prostaglandin E(2). Rat paw pressure test was applied before and 3 h after the intraplantar (i.pl.) injection of the nociceptive stimuli. Oral administration of venom 2 h after carrageenin or prostaglandin E(2) induces antinociception. Local pretreatment with 4-aminopyridine and tetraethylammonium (blockers of voltage-dependent K(+) channel) or charybdotoxin and apamin (inhibitors of large- and small-conductance Ca(2+)-activated K(+) channel, respectively) did not modify venom effect. On the other hand, glybenclamide, an inhibitor of ATP-sensitive K(+) channel abolished antinociception induced by the venom. Glybenclamide also inhibited the antinociceptive effect of [D-Pen(2.5)] enkephalin (DPDPE), a delta opioid receptor agonist, but did not modify the effect of (+)-trans-(1R,2R)-U-50488 (U50488), a kappa opioid receptor agonist. Diazoxide and pinacidil, two ATP-sensitive K(+) channel openers, injected by intraplantar route, induced a long-lasting increment of pain threshold of the animals and produced antinociception in both models of hyperalgesia. These results suggest that the antinociceptive effect of crotalid venom is mediated by activation of ATP-sensitive K(+) channels at peripheral afferent neurons.

Crotalphine induces potent antinociception in neuropathic pain by acting at peripheral opioid receptors

Neuropathic pain is an important clinical problem and it is usually resistant to the current therapy. We have recently characterized a novel analgesic peptide, crotalphine, from the venom of the South American rattlesnake Crotalus durissus terrificus. In the present work, the antinociceptive effect of crotalphine was evaluated in an experimental model of neuropathic pain induced in rats by chronic constriction of sciatic nerve. The effect of the peptide was compared to that induced by the crude venom, which confirmed that crotalphine is responsible for the antinociceptive effect of the crotalid venom on neuropathic pain. For characterization of neuropathic pain, the presence of hyperalgesia, allodynia and spontaneous pain was assessed at different times after nerve constriction. These phenomena were detected 24 h after surgery and persisted at least for 14 days. The pharmacological treatments were performed on day 14 after surgery. Crotalphine (0.2-5 microg/kg) and the crude venom (400-1600 microg/kg) administered p.o. inhibited hyperalgesia, allodynia and spontaneous pain induced by nerve constriction. The antinociceptive effect of the peptide and crude venom was long lasting, since it was detected up to 3 days after treatment. Intraplantar injection of naloxone (1 microg/paw) blocked the antinociceptive effect, indicating the involvement of opioid receptors in this phenomenon. Gabapentin (200 mg/kg, p.o.), and morphine (5 mg/kg, s.c.), used as positive controls, blocked hyperalgesia and partially inhibited allodynia induced by nerve constriction. These data indicate that crotalphine induces a potent and long lasting opioid antinociceptive effect in neuropathic pain that surpasses that observed with standard analgesic drugs.

Evaluation of antivenoms in the neutralization of hyperalgesia and edema induced by Bothrops jararaca and Bothrops asper snake venoms

Neutralization of hyperalgesia induced by Bothrops jararaca and B. asper venoms was studied in rats using bothropic antivenom produced at Instituto Butantan (AVIB, 1 ml neutralizes 5 mg B. jararaca venom) and polyvalent antivenom produced at Instituto Clodomiro Picado (AVCP, 1 ml neutralizes 2.5 mg B. aspar venom). The intraplantar injection of B. jararaca and B. asper venoms caused hyperalgesia, which peaked 1 and 2 h after injection, respectively. Both venoms also induced edema with a similar time course. When neutralization assays involving the independent injection of venom and antivenom were performed, the hyperalgesia induced by B. jararaca venom was neutralized only when bothropic antivenom was administered iv 15 min before venom injection, whereas edema was neutralized when antivenom was injected 15 min or immediately before venom injection. On the other hand, polyvalent antivenom did not interfere with hyperalgesia or edema induced by B. asper venom, even when administered prior to envenomation. The lack of neutralization of hyperalgesia and edema induced by B. asper venom is not attributable to the absence of neutralizing antibodies in the antivenom, since neutralization was achieved in assays involving preincubation of venom and antivenom. Cross-neutralization of AVCP or AVIB against B. jararaca and B. asper venoms, respectively, was also evaluated. Only bothropic antivenom partially neutralized hyperalgesia induced by B. asper venom in preincubation experiments. The present data suggest that hyperalgesia and edema induced by Bothrops venoms are poorly neutralized by commercial antivenoms even when antibodies are administered immediately after envenomation.

The peripheral L-arginine-nitric oxide-cyclic GMP pathway and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine on neuropathic pain in rats

Crotalphine, a 14 amino acid peptide first isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, induces a peripheral long-lasting and opioid receptor-mediated antinociceptive effect in a rat model of neuropathic pain induced by chronic constriction of the sciatic nerve. In the present study, we further characterized the molecular mechanisms involved in this effect, determining the type of opioid receptor responsible for this effect and the involvement of the nitric oxide–cyclic GMP pathway and of K+ channels. Crotalphine (0.2 or 5 &mgr;g/kg, orally; 0.0006 &mgr;g/paw), administered on day 14 after nerve constriction, inhibited mechanical hyperalgesia and low-threshold mechanical allodynia. The effect of the peptide was antagonized by intraplantar administration of naltrindole, an antagonist of &dgr;-opioid receptors, and partially reversed by norbinaltorphimine, an antagonist of &kgr;-opioid receptors. The effect of crotalphine was also blocked by 7-nitroindazole, an inhibitor of the neuronal nitric oxide synthase; by 1H-(1,2,4) oxadiazolo[4,3-a]quinoxaline-1-one, an inhibitor of guanylate cyclase activation; and by glibenclamide, an ATP-sensitive K+ channel blocker. The results suggest that peripheral &dgr;-opioid and &kgr;-opioid receptors, the nitric oxide–cyclic GMP pathway, and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine. The present data point to the therapeutic potential of this peptide for the treatment of chronic neuropathic pain.