Essential oil of Sicilian Prangos ferulacea (L.) Lindl. and its major component, ß-ocimen, affect contractility in rat small and large intestine.

ETHNOPHARMACOLOGICAL RELEVANCE: Prangos ferulacea (L.) Lindl is an Apiaceae plant, widely used in traditional medicine. Recently, chemical composition and biological activities of its essential oil (Prangroil) have been reported, but there are no studies on possible effects on intestinal contractility. AIMS OF THE STUDY: We investigated the effects of essential oil Sicilian Prangoil on the contractility of rat small (duodenum) and large (colon) intestine and the related action mechanism. MATERIALS AND METHODS: Responses to Prangoil and to its major component ß-ocimen in intestinal segments were assessed in vitro as changes in isometric tension. RESULTS: Prangoil, induced in duodenum, depending upon doses, contraction and/or muscular relaxation. Instead, in colon Prangoil only reduced the phasic contractions and induced muscular relaxation. ß-ocimen, in both segments, produced only reduction of the spontaneous contractions without affecting basal tone. Prangoil contractile effects were abolished by ω-conotoxin, neural N-type Ca2+ channels blocker, atropine, muscarinic receptor antagonist, neostigmine, acetylcholinesterase (AChE) inhibitor, suggesting that Prangoil-induced contraction would be the result of an increase in neuronal cholinergic activity. Prangoil and ß-ocimen inhibitory effects were unaffected by ω-conotoxin, L-NAME, blocker of the NO synthase, ODQ, soluble guanylate cyclase inhibitor, excluding involvement of neurotransmitter release or NO synthesis in the inhibitory effects. Potassium channel blocker did not affect Prangoil or ß-ocimen inhibitory responses. Prangoil or ß-ocimen inhibited the Ca2+ and high-KCl solution -induced contractions and the Carbachol-induced contractions in calcium free solution. CONCLUSION: Prangoil affects the contractility of small and large intestine in rat, with regional differences, via potentiation of neural cholinergic activity, blockade of L-type voltage-gated calcium channel and reduction of Ca2+ release from the intracellular store. The Prangroil main components, ß-ocimen, contributes to the inhibitory effects.

Self-Assembly Nanostructure of Myristoylated ω-Conotoxin MVIIA Increases the Duration of Efficacy and Reduces Side Effects.

Chronic pain is one of the most prevalent health problems worldwide. An alternative to suppress or alleviate chronic pain is the use of peptide drugs that block N-type Ca2+ channels (Cav2.2), such as ω-conotoxin MVIIA. Nevertheless, the narrow therapeutic window, severe neurological side effects and low stability associated with peptide MVIIA have restricted its widespread use. Fortunately, self-assembly endows the peptide with high stability and multiple functions, which can effectively control its release to prolong its duration of action. Inspired by this, MVIIA was modified with appropriate fatty acid chains to render it amphiphilic and easier to self-assemble. In this paper, an N-terminal myristoylated MVIIA (Myr-MVIIA, medium carbon chain length) was designed and prepared to undergo self-assembly. The present results indicated that Myr-MVIIA can self-assemble into micelles. Self-assembled micelles formed by Myr-MVIIA at higher concentrations than MVIIA can prolong the duration of the analgesic effect and significantly reduce or even eliminate the side effects of tremor and coordinated motor dysfunction in mice.

Microneedle-mediated delivery of Ziconotide-loaded liposomes fused with exosomes for analgesia.

Ziconotide (ZIC) is an N-type calcium channel antagonist for treating severe chronic pain that is intolerable, or responds poorly to the administration of other drugs, such as intrathecal morphine and systemic analgesics. As it can only work in the brain and cerebrospinal fluid, intrathecal injection is the only administration route for ZIC. In this study, borneol (BOR)-modified liposomes (LIPs) were fused with exosomes from mesenchymal stem cells (MSCs) and loaded with ZIC to prepare microneedles (MNs) to improve the efficiency of ZIC across the blood-brain barrier. To evaluate local analgesic effects of MNs, the sensitivity of behavioral pain to thermal and mechanical stimuli was tested in animal models of peripheral nerve injury, diabetes-induced neuropathy pain, chemotherapy-induced pain, and ultraviolet-B (UV-B) radiation-induced neurogenic inflammatory pain. BOR-modified LIPs loaded with ZIC were spherical or nearly spherical, with a particle size of about 95 nm and a Zeta potential of -7.8 mV. After fusion with MSC exosomes, the particle sizes of LIPs increased to 175 nm, and their Zeta potential increased to -3.8 mV. The nano-MNs constructed based on BOR-modified LIPs had good mechanical properties and could effectively penetrate the skin to release drugs. The results of analgesic experiments showed that ZIC had a significant analgesic effect in different pain models. In conclusion, the BOR-modified LIP membrane-fused exosome MNs constructed in this study for delivering ZIC provide a safe and effective administration for chronic pain treatment, as well as great potential for clinical application of ZIC.

Analgesic effect of recombinant GABAergic precursors releasing ω-conotoxin MVIIA in a model of peripheral nerve injury in rats.

Development of chronic pain has been attributed to dysfunctional GABA signaling in the spinal cord. Direct pharmacological interventions on GABA signaling are usually not very efficient and often accompanied by side effects due to the widespread distribution of GABA receptors in CNS. Transplantation of GABAergic neuronal cells may restore the inhibitory potential in the spinal cord. Grafted cells may also release additional analgesic peptides by means of genetic engineering to further enhance the benefits of this approach. Conopeptides are ideal candidates for recombinant expression using cell-based strategies. The omega-conopeptide MVIIA is in clinical use for severe pain marketed as FDA approved Prialt in the form of intrathecal injections. The goal of this study was to develop transplantable recombinant GABAergic cells releasing conopeptide MVIIA and to evaluate the analgesic effect of the grafts in a model of peripheral nerve injury-induced pain. We have engineered and characterized the GABAergic progenitors expressing MVIIA. Recombinant and nonrecombinant cells were intraspinally injected into animals after the nerve injury. Animals were tested weekly up to 12 weeks for the presence of hypersensitivity, followed by histochemical and biochemical analysis of the tissue. We observed beneficial effects of the grafted cells in reducing hypersensitivity in all grafted animals, especially potent in the recombinant group. The level of pain-related cytokines was reduced in the grafted animals and correlation between these pain markers and actual behavior was indicated. This study demonstrated the feasibility of recombinant cell transplantation in the management of chronic pain.

Subcutaneous ω-Conotoxins Alleviate Mechanical Pain in Rodent Models of Acute Peripheral Neuropathy.

The peripheral effects of ω-conotoxins, selective blockers of N-type voltage-gated calcium channels (CaV2.2), have not been characterised across different clinically relevant pain models. This study examines the effects of locally administered ω-conotoxin MVIIA, GVIA, and CVIF on mechanical and thermal paw withdrawal threshold (PWT) in postsurgical pain (PSP), cisplatin-induced neuropathy (CisIPN), and oxaliplatin-induced neuropathy (OIPN) rodent models. Intraplantar injection of 300, 100 and 30 nM MVIIA significantly (p < 0.0001, p < 0.0001, and p < 0.05, respectively) alleviated mechanical allodynia of mice in PSP model compared to vehicle control group. Similarly, intraplantar injection of 300, 100, and 30 nM MVIIA (p < 0.0001, p < 0.01, and p < 0.05, respectively), and 300 nM and 100 nM GVIA (p < 0.0001 and p < 0.05, respectively) significantly increased mechanical thresholds of mice in OIPN model. The ED50 of GVIA and MVIIA in OIPN was found to be 1.8 pmol/paw and 0.8 pmol/paw, respectively. However, none of the ω-conotoxins were effective in a mouse model of CisIPN. The intraplantar administration of 300 nM GVIA, MVIIA, and CVIF did not cause any locomotor side effects. The intraplantar administration of MVIIA can alleviate incision-induced mechanical allodynia, and GVIA and MVIIA effectively reduce OIPN associated mechanical pain, without locomotor side effects, in rodent models. In contrast, CVIF was inactive in these pain models, suggesting it is unable to block a subset of N-type voltage-gated calcium channels associated with nociceptors in the skin.

Antidepressant-like and memory-enhancing effects of the N-type calcium channel blocker ziconotide in rats.

The lack of oral or injectable formulations of ziconotide (ω-conotoxin peptide), a novel analgesic agent, limits research on potential neurobehavioral protective properties of this substance, including antidepressant-like effects. Here we expose rats to a stress paradigm that induces depression and memory impairment to assess the effects of ziconotide treatment. Ziconotide was administered intracerebroventricular (i.c.v.) to rats undergoing stereotaxic surgery at a single dose (1 µg/rat) or in repeated long-term applications (dosage groups: 0.1, 0.3, and 1 µg/rat). The antidepressant activity and memory-enhancing effects of ziconotide were examined via the forced swimming test, the Morris water maze test, and the passive avoidance learning test. Behavioral results showed that long-term i.c.v. ziconotide administration significantly decreased the immobility time and delayed the latency period to immobility in a dose-dependent manner compared to controls. In the passive avoidance learning test, the latency period increased, and in the Morris water maze test, the platform location latency time decreased. A single dose of ziconotide (1 µg/rat) did not show a significant effect on memory function or depression parameters during the same tests. Animals were sacrificed immediately after behavioral testing, and both hippocampi were removed and prepared for BDNF evaluation. Hippocampal BDNF levels were significantly increased in rats receiving long-term i.c.v. ziconotide compared to controls. Our results suggest that long-term consumption of ziconotide may attenuate the severity of depression-like behavior and could be useful for preventing memory impairments in various learning models by elevating BDNF levels.

Neuropeptide Y release in the rat spinal cord measured with Y1 receptor internalization is increased after nerve injury.

Neuropeptide Y (NPY) modulates nociception in the spinal cord, but little is known about its mechanisms of release. We measured NPY release in situ using the internalization of its Y1 receptor in dorsal horn neurons. Y1 receptor immunoreactivity was normally localized to the cell surface, but addition of NPY to spinal cord slices increased the number of neurons with Y1 internalization in a biphasic fashion (EC50s of 1 nM and 1 µM). Depolarization with KCl, capsaicin, or the protein kinase A activator 6-benzoyl-cAMP also induced Y1 receptor internalization, presumably by releasing NPY. NMDA receptor activation in the presence of BVT948, an inhibitor of protein tyrosine phosphatases, also released NPY. Electrical stimulation of the dorsal horn frequency-dependently induced NPY release; and this was decreased by the Y1 antagonist BIBO3304, the Nav channel blocker lidocaine, or the Cav2 channel blocker ω-conotoxin MVIIC. Dorsal root immersion in capsaicin, but not its electrical stimulation, also induced NPY release. This was blocked by CNQX, suggesting that part of the NPY released by capsaicin was from dorsal horn neurons receiving synapses from primary afferents and not from the afferent themselves. Mechanical stimulation in vivo, with rub or clamp of the hindpaw, elicited robust Y1 receptor internalization in rats with spared nerve injury but not sham surgery. In summary, NPY is released from dorsal horn interneurons or primary afferent terminals by electrical stimulation and by activation of TRPV1, PKA or NMDA receptors in. Furthermore, NPY release evoked by noxious and tactile stimuli increases after peripheral nerve injury.

Evaluation of DNA damage in spinal cord and mutagenic effect of a Phα1ß recombinant toxin with analgesic properties from the Phoneutria nigriventer spider.

Phα1ß peptide isolated from the venom of the Phoneutria nigriventer spider has shown higher analgesic action in pre-clinical studies than ω-conotoxin MVIIA peptide used to treat severe chronic pain. In view of the great potential for the development of a new Phα1ß-based drug, a Phα1ß recombinant form (CTK 01512-2) has been studied for efficacy and safety. The aim of this study was to evaluate cytotoxic, genotoxic and mutagenic effects of a Phα1ß recombinant form and compare it with native Phα1ß and ω-conotoxin MVIIA. Cytotoxicity was evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colourimetric assay in L929 mouse fibroblast cells (0.5-10.0 µmol/L). Genotoxic and mutagenic activities were analysed using the alkaline comet assay in peripheral blood and spinal cord, and the micronucleus test in bone marrow from Wistar rats treated by intrathecal injection of CTK 01512-2 (200, 500 and 1000 pmol/site), native Phα1ß (500 pmol/site) and ω-conotoxin MVIIA (200 pmol/site). CTK 01512-2 decreased the cell viability of the L929, showing IC50 of 3.3 ± 0.1 µmol/L, while the Phα1ß and ω-conotoxin MVIIA did not show cytotoxicity (IC50  > 5.0 µmol/L). Native and recombinant Phα1ß forms induced DNA damage in the spinal cord, but not in peripheral blood. CTK 01512-2 at 1000 pmol/site increased the micronucleus frequency suggesting mutagenic effects. In conclusion, the recombinant form has cytotoxic, genotoxic and mutagenic effects, evidenced in doses five times above the therapeutic dose.

Novel analgesic ω-conotoxins from the vermivorous cone snail Conus moncuri provide new insights into the evolution of conopeptides.

Cone snails are a diverse group of predatory marine invertebrates that deploy remarkably complex venoms to rapidly paralyse worm, mollusc or fish prey. ω-Conotoxins are neurotoxic peptides from cone snail venoms that inhibit Cav2.2 voltage-gated calcium channel, demonstrating potential for pain management via intrathecal (IT) administration. Here, we isolated and characterized two novel ω-conotoxins, MoVIA and MoVIB from Conus moncuri, the first to be identified in vermivorous (worm-hunting) cone snails. MoVIA and MoVIB potently inhibited human Cav2.2 in fluorimetric assays and rat Cav2.2 in patch clamp studies, and both potently displaced radiolabeled ω-conotoxin GVIA (125I-GVIA) from human SH-SY5Y cells and fish brain membranes (IC50 2-9 pM). Intriguingly, an arginine at position 13 in MoVIA and MoVIB replaced the functionally critical tyrosine found in piscivorous ω-conotoxins. To investigate its role, we synthesized MoVIB-[R13Y] and MVIIA-[Y13R]. Interestingly, MVIIA-[Y13R] completely lost Cav2.2 activity and MoVIB-[R13Y] had reduced activity, indicating that Arg at position 13 was preferred in these vermivorous ω-conotoxins whereas tyrosine 13 is preferred in piscivorous ω-conotoxins. MoVIB reversed pain behavior in a rat neuropathic pain model, confirming that vermivorous cone snails are a new source of analgesic ω-conotoxins. Given vermivorous cone snails are ancestral to piscivorous species, our findings support the repurposing of defensive venom peptides in the evolution of piscivorous Conidae.

Potential Uses of Isolated Toxin Peptides in Neuropathic Pain Relief: A Literature Review.

Neuropathic pain is a subset of chronic pain that is caused by neurons that are damaged or firing aberrantly in the peripheral or central nervous systems. The treatment guidelines for neuropathic pain include antidepressants, calcium channel α2 delta ligands, topical therapy, and opioids as a second-line option. Pharmacotherapy has not been effective in the treatment of neuropathic pain except in the treatment of trigeminal neuralgia with carbamazepine. The inability to properly treat neuropathic pain causes frustration in both the patients and their treating physicians. Venoms, which are classically believed to be causes of pain and death, have peptide components that have been implicated in pain relief. Although some venoms are efficacious and have shown benefits in patients, their side-effect profile precludes their more widespread use. This review identifies and explores the use of venoms in neuropathic pain relief. This treatment can open doors to potential therapeutic targets. We believe that further research into the mechanisms of action of these receptors as well as their functions in nature will provide alternative therapies as well as a window into how they affect neuropathic pain.

Mechanisms of µ-opioid receptor inhibition of NMDA receptor-induced substance P release in the rat spinal cord.

The interaction between NMDA receptors and µ-opioid receptors in primary afferent terminals was studied by using NMDA to induce substance P release, measured as neurokinin 1 receptor internalization. In rat spinal cord slices, the µ-opioid receptor agonists morphine, DAMGO and endomorphin-2 inhibited NMDA-induced substance P release, whereas the antagonist CTAP right-shifted the concentration response of DAMGO. In vivo, substance P release induced by intrathecal NMDA after priming with BDNF was inhibited by DAMGO. ω-Conotoxins MVIIC and GVIA inhibited about half of the NMDA-induced substance P release, showing that it was partially mediated by the opening of voltage-gated calcium (Cav) channels. In contrast, DAMGO or ω-conotoxins did not inhibit capsaicin-induced substance P release. In cultured DRG neurons, DAMGO but not ω-conotoxin inhibited NMDA-induced increases in intracellular calcium, indicating that µ-opioid receptors can inhibit NMDA receptor function by mechanisms other than inactivation of Cav channels. Moreover, DAMGO decreased the ω-conotoxin-insensitive component of the substance P release. Potent inhibition by ifenprodil showed that these NMDA receptors have the NR2B subunit. Activators of adenylyl cyclase and protein kinase A (PKA) induced substance P release and this was decreased by the NMDA receptor blocker MK-801 and by DAMGO. Conversely, inhibitors of adenylyl cyclase and PKA, but not of protein kinase C, decreased NMDA-induced substance P release. Hence, these NMDA receptors are positively modulated by the adenylyl cyclase-PKA pathway, which is inhibited by µ-opioid receptors. In conclusion, µ-opioid receptors inhibit NMDA receptor-induced substance P release through Cav channel inactivation and adenylyl cyclase inhibition.

Bioactive Mimetics of Conotoxins and other Venom Peptides.

Ziconotide (Prialt®), a synthetic version of the peptide ω-conotoxin MVIIA found in the venom of a fish-hunting marine cone snail Conus magnus, is one of very few drugs effective in the treatment of intractable chronic pain. However, its intrathecal mode of delivery and narrow therapeutic window cause complications for patients. This review will summarize progress in the development of small molecule, non-peptidic mimics of Conotoxins and a small number of other venom peptides. This will include a description of how some of the initially designed mimics have been modified to improve their drug-like properties.

Spider peptide Phα1ß induces analgesic effect in a model of cancer pain.

The marine snail peptide ziconotide (ω-conotoxin MVIIA) is used as an analgesic in cancer patients refractory to opioids, but may induce severe adverse effects. Animal venoms represent a rich source of novel drugs, so we investigated the analgesic effects and the side-effects of spider peptide Phα1ß in a model of cancer pain in mice with or without tolerance to morphine analgesia. Cancer pain was induced by the inoculation of melanoma B16-F10 cells into the hind paw of C57BL/6 mice. After 14 days, painful hypersensitivity was detected and Phα1ß or ω-conotoxin MVIIA (10-100 pmol/site) was intrathecally injected to evaluate the development of antinociception and side-effects in control and morphine-tolerant mice. The treatment with Phα1ß or ω-conotoxin MVIIA fully reversed cancer-related painful hypersensitivity, with long-lasting results, at effective doses 50% of 48 (32-72) or 33 (21-53) pmol/site, respectively. Phα1ß produced only mild adverse effects, whereas ω-conotoxin MVIIA induced dose-related side-effects in mice at analgesic doses (estimated toxic dose 50% of 30 pmol/site). In addition, we observed that Phα1ß was capable of controlling cancer-related pain even in mice tolerant to morphine antinociception (100% of inhibition) and was able to partially restore morphine analgesia in such animals (56 ± 5% of inhibition). In this study, Phα1ß was as efficacious as ω-conotoxin MVIIA in inducing analgesia in a model of cancer pain without producing severe adverse effects or losing efficacy in opioid-tolerant mice, indicating that Phα1ß has a good profile for the treatment of cancer pain in patients.

Spinal actions of ω-conotoxins, CVID, MVIIA and related peptides in a rat neuropathic pain model.

BACKGROUND AND PURPOSE: Antagonists of the N-type voltage gated calcium channel (VGCC), Cav 2.2, have a potentially important role in the treatment of chronic neuropathic pain. ω-conotoxins, such MVIIA and CVID are effective in neuropathic pain models. CVID is reported to have a greater therapeutic index than MVIIA in neuropathic pain models, and it has been suggested that this is due to faster reversibility of binding, but it is not known whether this can be improved further. EXPERIMENTAL APPROACH: We examined the potency of CVID, MVIIA and two intermediate hybrids ([K10R]CVID and [R10K]MVIIA) to reverse signs of neuropathic pain in a rat nerve ligation model in parallel with production of side effects. We also examined the potency and reversibility to inhibit primary afferent synaptic neurotransmission in rat spinal cord slices. KEY RESULTS: All ω-conotoxins produced dose-dependent reduction in mechanical allodynia. They also produced side effects on the rotarod test and in a visual side-effect score. CVID displayed a marginally better therapeutic index than MVIIA. The hybrids had a lesser effect in the rotarod test than either of their parent peptides. Finally, the conotoxins all presynaptically inhibited excitatory synaptic neurotransmission into the dorsal horn and displayed recovery that was largely dependent upon the magnitude of inhibition and not the conotoxin type. CONCLUSIONS AND IMPLICATIONS: These findings indicate that CVID provides only a marginal improvement over MVIIA in a preclinical model of neuropathic pain, which appears to be unrelated to reversibility from binding. Hybrids of these conotoxins might provide viable alternative treatments.

Omega-conotoxins as experimental tools and therapeutics in pain management.

Neuropathic pain afflicts a large percentage of the global population. This form of chronic, intractable pain arises when the peripheral or central nervous systems are damaged, either directly by lesion or indirectly through disease. The comorbidity of neuropathic pain with other diseases, including diabetes, cancer, and AIDS, contributes to a complex pathogenesis and symptom profile. Because most patients present with neuropathic pain refractory to current first-line therapeutics, pharmaceuticals with greater efficacy in pain management are highly desired. In this review we discuss the growing application of ω-conotoxins, small peptides isolated from Conus species, in the management of neuropathic pain. These toxins are synthesized by predatory cone snails as a component of paralytic venoms. The potency and selectivity with which ω-conotoxins inhibit their molecular targets, voltage-gated Ca2+ channels, is advantageous in the treatment of neuropathic pain states, in which Ca2+ channel activity is characteristically aberrant. Although ω-conotoxins demonstrate analgesic efficacy in animal models of neuropathic pain and in human clinical trials, there remains a critical need to improve the convenience of peptide drug delivery methods, and reduce the number and severity of adverse effects associated with ω-conotoxin-based therapies.

Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge.

Slow changes in [Ca(2+)](i) reflect increased neuronal activity. Our study demonstrates that single-trial fast [Ca(2+)](i) imaging (≥200 Hz sampling rate) revealed peaks each of which are associated with single spike discharge recorded by consecutive voltage-sensitive dye (VSD) imaging in enteric neurones and nerve fibres. Fast [Ca(2+)](i) imaging also revealed subthreshold fast excitatory postsynaptic potentials. Nicotine-evoked [Ca(2+)](i) peaks were reduced by -conotoxin and blocked by ruthenium red or tetrodotoxin. Fast [Ca(2+)](i) imaging can be used to directly record single action potentials in enteric neurones. [Ca(2+)](i) peaks required opening of voltage-gated sodium and calcium channels as well as Ca(2+) release from intracellular stores.

Protective effects of ω-conotoxin on amyloid-ß-induced damage in PC12 cells.

The present study was designed to investigate the potential neuroprotective effect of ω-conotoxin (ω-CTX) in a cell-based model of Alzheimer's disease (AD) using cultured PC12 cells incubated with ß-amyloid (Aß). Immunohistochemical staining, Western blot, MTT and TdT-mediated dUTP-biotin nick end labeling (TUNEL) analysis were employed to assess the cell viability and cell death. Aß in this model was clearly neurotoxic, inducing necrosis and apoptosis. ω-CTX antagonized the effects of Aß: there was an increase in cell viability and a suppression of inflammatory- and oxidative stress-related factors. These data suggest that ω-CTX may have neuroprotective actions against Aß-induced neurotoxicity. The significance of these new findings relative to the etiology and treatment of AD is discussed.

Calcium channel subtypes for cholinergic and nonadrenergic noncholinergic neurotransmission in isolated guinea pig trachea.

The Ca(2+) channel subtypes in the neurotransmission of isolated guinea pig trachea were elucidated by monitoring the effects of specific Ca(2+) channel blockers on cholinergic contractions and nonadrenergic noncholinergic (NANC) relaxation elicited by electrical field stimulation (EFS). In isolated guinea pig trachea, cholinergic contractile responses to low- and high-frequency EFS were inhibited by the selective N-type calcium channel blocker, ω-conotoxin MVIIA. ω-Agatoxin IVA (a selective P-type blocker), ω-conotoxin MVIIC (a nonselective N-, Q-, and P-type blocker), and nifedipine (a selective L-type blocker) were ineffective, whereas Ni(2+) (a T- and R-type blocker) facilitated cholinergic contractions and produced a late contracture when its concentration exceeded 30 µM. The more the concentration of Ni(2+) increased, the greater the number of incidences and the late contracture areas which occurred. Late contracture did not seem to be due to the effects of acetylcholine, tachykinins, or other polypeptides, but disappeared in the absence of indomethacin. The NANC relaxant responses elicited by the low- and high-frequency EFS were inhibited by ω-conotoxin MVIIA or Ni(2+), but unaffected by ω-Agatoxin IVA, ω-conotoxin MVIIC, and nifedipine. In the absence of indomethacin, Ni(2+) did not alter the ω-conotoxin MVIIA (100 nM)-resistant component of cholinergic contraction, but significantly further inhibited that of NANC relaxation. These results suggest that in isolated guinea pig trachea, cholinergic contraction is regulated by N-type calcium channels which may mask T- and R-type calcium channels and may be co-modulated by both, while NANC relaxation is mainly and independently controlled by N-, T-, and R-type calcium channels.

Divergent M- and O-superfamily peptides from venom of fish-hunting Conus parius.

Six novel peptides from the piscivorous cone snail, Conus parius were purified by reverse-phase HPLC fractionation of crude venom. With the use of matrix-assisted laser desorption ionization mass spectrometry and standard Edman sequencing methods, the peptides were characterized. Two peptides were identified as members of the m-2 and m-4 branches of the M-superfamily and were designated as pr3a and pr3b, while four peptides were identified as members of the O-superfamily and were designated as pr6a, pr6b, pr6c and pr6d. Peptide pr3a differs from the majority of the M-superfamily peptides in the presence of two prolines, which are not modified to 4-trans-hydroxyproline. In peptide pr3b, five amino acids out of the 16 non-cysteine residues are identical with those of mu-GIIIA and mu-PIIIA, suggesting that pr3b may be a divergent mu-conotoxin. Peptide pr6a is notable because of its extreme hydrophobicity. Peptide pr6c has three prolines that are unhydroxylated. Peptides pr6b and pr6d differ from the previously characterized O-superfamily peptides in the presence of an extended N-terminus consisting of six amino acids. Peptides pr3a, pr3b, pr6a and pr6b were demonstrated to be biologically active when injected intraperitoneally in fish. The identification and characterization of these peptides in venom of a fish-hunting species establish the divergence of gene products and their patterns of post-translational modification within superfamilies in a single Conus species.

Drugs from slugs--past, present and future perspectives of omega-conotoxin research.

Peptides from the venom of carnivorous cone shells have provided six decades of intense research, which has led to the discovery and development of novel analgesic peptide therapeutics. Our understanding of this unique natural marine resource is however somewhat limited. Given the past pharmacological record, future investigations into the toxinology of these highly venomous tropical marine snails will undoubtedly yield other highly selective ion channel inhibitors and modulators. With over a thousand conotoxin-derived sequences identified to date, those identified as ion channel inhibitors represent only a small fraction of the total. Here we discuss our present understanding of conotoxins, focusing on the omega-conotoxin peptide family, and illustrate how such a seemingly simple snail has yielded a highly effective clinical drug.