Cloning of CTx-FVIA
Specimens of Conus Fulmen were collected from an area of subtropical sea south of Jeju island, Republic of Korea. Frozen hepatic tissue (0.1 g) from Conus Fulmen was added to 1 ml of GES reagent (5 M guanidinium thiocyanate, 100 mM EDTA, 0.5% v/v Sarkosyl) and then ground with a glass homogenizer . After centrifugation, the supernatant was mixed with phenol and chloroform and centrifuged again at room temperature. The colorless upper aqueous phase was then transferred to a fresh tube and mixed with 1 ml of isopropanol to precipitate the genomic DNA. The resultant pellet was washed twice in 70% ethanol and dissolved in 50 μl of 10 mM Tris-HCl (pH 8.5). The collected genomic DNA was used as a template for PCR with a forward primer (5'-CTCTCTCTCTCTCTGCTGGAC-3') and reverse primer (5'-CAGAAAAGGATAGAGCACAGAAGG-3') corresponding respectively to the third intron and 3' UTR in the genomic structure of the O-superfamily. The PCR protocol entailed 35 cycles of 94°C for 30 s, 55°C for 30 s and 70°C for 45 s. The purified PCR products were ligated into pGEM-Teasy vector (Promega) and then transformed to competent DH5α cells. The nucleic acid sequences of the expressed clones were determined using an ABI Prism 3700 DNA analyzer (Figure 1B).
The sequences of CTx-FVIA and CTx-MVIIA are shown in Figure 1C. The linear precursors of each peptide were synthesized using solid-phase methodology with Fmoc chemistry, starting from Rink-amide resin. Briefly, synthetic peptides were deprotected and cleaved using a mixture containing 82.5% TFA, 2.5% 1,2-ethanedithiol, 5% H2O, 5% thioanisole and 5% phenol (v/v). Each linear peptide was diluted to a final concentration of 2.5 × 10-5 M and subjected to oxidative disulfide bond formation for 3 days at 4°C in 0.3 M ammonium acetate buffer (pH 7.8) containing 0.5 M guanidine hydrochloride and reduced/oxidized glutathione (peptide:GSH:GSSG molar ratio was 1:100:10). Preparative RP-HPLC was performed using a Shimadzu LC-6AD system with an ODS column (20 × 250 mm). The purity of the synthetic peptides was confirmed by analytical RP-HPLC and MALDI-TOF mass spectrometry.
C2D7 cells expressing the α1B-1, α2bδ, and β1b subunits of N-type Ca2+ channels were cultured in DMEM medium supplemented with 1% penicillin/streptomycin, 5% bovine calf serum, 0.5 g/L geneticin and 30 U/ml hygromycin . Total Ba2+ currents were measured using the whole-cell patch clamp technique. Coverslips with cells were mounted in a perfusion chamber and constantly perfused using a gravity feed system with a modified HEPES-balanced external solution (151 mM tetraethylammonium chloride, 10 mM HEPES, 5 mM BaCl2, 1 mM MgCl2, and 10 mM glucose; pH was adjusted to 7.4 and osmolarity to 310 mOsm) to isolate the Ba2+ currents. Electrodes were made of borosilicate glass and had a resistance of 3-4 MΩ when filled with pipette solution. The pipette solution contained 100 mM CsCl, 1 mM MgCl2, 10 mM HEPES, 10 mM BAPTA, 3.6 mM MgATP, 14 mM phosphocreatine (CrP), 0.1 mM LiGTP and 50 U/ml creatine phosphokinase (CrPK). The solution pH was adjusted to 7.2 using CsOH. Ba2+ currents were recorded using an EPC-9 amplifier, and the data were analyzed using Pulse/Pulsefit (HEKA, Germany) and GraphPad Prism (GraphPad Inc.) software.
Drugs and animals
Formalin, acetic acid, substance P and glutamate were purchased from Sigma Chemical Co. (St. Louis, MO). TNF-α, IFN-γ and IL-1β were from R and D Systems Inc. (Minneapolis, MN, USA). All drugs were prepared just before use in 0.9% NaCl (w/v). Adult male Sprague-Dawley rats weighing 150-200 g or ICR mice weighing 23-25 g were used. Animals were housed together in groups in a room maintained at 22 ± 0.5°C and had unrestricted access to food and water. The animals were allowed to adapt to the experimental conditions in the laboratory for at least 2 h before pain testing. All procedures were conducted in accordance with the Guide for Care and Use of Laboratory Animals published by the U.S. National Institutes of Health and the ethical guidelines of the International Association for the Study of Pain.
Intrathecal and intravenous injection of CTx-FVIA and CTx-MVIIA
Using the method of Hylden and Wilcox [63, 64], conscious mice or enflurane-anesthetized rats were intrathecally injected using a 30-gauge needle connected to a 25-μl Hamilton syringe via polyethylene tubing. The injected volume was 5 μl, and the injection site was verified by injecting a similar volume of 1% methylene blue solution and determining the distribution of the dye in the spinal cord. The injected dye was distributed both rostrally and caudally but diffused only a short distance (about 0.5 cm), and no dye was found in the brain. Before any experiments were done, the success rate for intrathecal injections was consistently >95%. Intravenous injections were made into the lateral tail vein of conscious rats using a 26-gauge needle connected to 1 ml syringe.
Formalin tests were carried out as previously described by Hunskaar et al. . Briefly, mice were intrathecally injected with 3.2, 10, 32, or 100 ng/5 μl of CTx-FVIA or CTx-MVIIA, or physiological saline (control) 5 min before formalin testing. Thereafter, 10 μl of 5% formalin in saline (0.9% NaCl) were subcutaneously injected into the plantar surface of the left hindpaw, and the elicited behaviors, including licking, biting, scratching or shaking, were recorded and counted. Throughout this experiment the mice were observed in a transparent observation chamber (acrylic-plastic, 20 × 20 cm in diameter × height), and the counts were divided into two phases. The first phase extended from 0-5 min after injection, while the second phase extended from 20-40 min after injection. The total observation period was 40 min.
Acute thermal pain tests
Mice were intrathecally injected with 5 μl of CTx-FVIA (0.01 μg/5 μl) or physiological saline (control) 5 min before testing. The tail-flick test was then performed using a commercially available apparatus (Model TF6, EMDIE Instrument Co.). The body of each mouse was held with a hand, and their tail was extended so that the distal part could be irradiated with light at an intensity of 3.8 mWatt/cm2 to impose a heat stimulus. The time it took the mouse to flick its tail was then recorded with a cut-off time of 10 sec. Plantar test of pain threshold was also performed using a commercially available apparatus (Plantar test 7371, Ugo Basile). In this case, each mouse was subjected to a heat stimulus by irradiating the plantar pad of one paw with light at an intensity of 90 mWatt/cm2, and the latency of paw withdrawal was measured with a cut-off time of 15 sec. For the hot-plate test, mice were individually placed on a hot plate (54°C) and the reaction time, from placement of the mouse on the hot plate to the time the mouse began licking a front-paw was measured with a cut-off time of 30 sec. The dimensions of the hot plate apparatus were 30 × 30 × 30 cm (Model 39 Hot Plate, Itic Life Science).
Mice were intrathecally injected with 0.01 μg/5 μl of CTx-FVIA, -MVIIA, or physiological saline (control) 5 min before testing. For the writhing test, mice were intraperitoneally injected with 0.5 ml of 1% acetic acid in saline. The number of writhes was counted during a 30 min period following the injection. A writhe was defined as a contraction of the abdominal muscles accompanied by an extension of the forelimbs and elongation of the body.
Nociceptive behavior induced by substance P, glutamate or inflammatory cytokines
Mice were intrathecally injected with 0.01 μg/5 μl of CTx-FVIA, -MVIIA, or physiological saline (control) 5 min before testing. Thereafter, mice were intrathecally injected with 0.7 μg/5 μl of substance P, 20 μg/5 μl of glutamate or 100 pg/5 μl of TNF-α, IFN-γ or IL-1β and then immediately placed in an observation chamber (described above). The durations of the pain-like responses, which included licking, biting and scratching the abdomen and hind regions of the body, were recorded for 30 min. The cumulative durations of the pain-like responses were measured using a stopwatch. These characteristic behaviors induced by the pharmacological effects of the injected agents were not observed in the vehicle-treated control group.
Tail nerve injury (TNI) rat models
Under enflurane anesthesia, the left superior and inferior caudal trunks were isolated and resected between the S1 and S2 spinal nerves [31, 65]. To prevent possible rejoining of the proximal and distal ends of the severed trunk, a piece of the trunk (about 1-2 mm) was removed from the proximal end. Two weeks after surgical procedure, the mechanical sensitivity of the tail was measured based on the frequency of tail flicks induced by application of different von Frey filaments. Each rat was placed in a transparent plastic tube, 5.5 × 15 cm or 6.5 × 18 cm (diameter × length), depending on their body size; the tube had to be small enough to prevent the rat from turning around. For testing, a series of eight von Frey filaments (0.41, 0.70, 1.20, 2.00, 3.63, 5.50, 8.50 and 15.14 g) were used after calibration. The von Frey filaments were applied to the tails, starting with a filament of 2.00 g; initially the most sensitive area was determined by rubbing various areas of the tail with the shank of the von Frey hair, and then, this area was challenged systematically by applying static pressure with the von Frey hair to locate the spot. An abrupt tail movement of about 0.5-20 cm in response to von Frey hair stimulation is considered to be an abnormal response, indicative of mechanical allodynia. During repeated trials, the test stimuli can be easily delivered to the same spot without difficulty, since the tail is usually stationary. Rats were intrathecally injected with 6.5, 20, 65, or 200 ng/kg of CTx-FVIA, or physiological saline (control). The 50% withdrawal threshold was determined using the up-down method described by Chaplan et al. .
Thermal sensitivity was assessed by immersing the tail in cold (4°C) or warm (40°C) water. Following tail immersion, the investigator observed the tail to see if it moved abruptly, as in the tail-flick test, and measured the latency of the tail response with a cut-off time of 15 s. The tail immersion test was repeated five times at 5 min intervals to obtain the average tail response latency for each animal on each experimental day.
Mean arterial pressure test
All surgical procedures were performed using aseptic technique. A polyethylene-10 catheter was inserted into the femoral artery of Sprague-Dawley rats to record invasive arterial blood pressure (IntelliVue MP30, Philips). Catheterization and all of the measurement for blood pressure were done under general anesthesia with isoflurane. After taking the basal level of mean arterial pressure, 100 μg/kg of CTx-FVIA or -MVIIA was injected intravenously, and mean arterial pressure was measured over time.
Longer-term monitoring of blood pressure using a tail-cuff
Twenty Sprague-Dawley rats were randomly divided into two groups receiving CTx-FVIA (100 μg/kg) or CTx-MVIIA (100 μg/kg). Each group included 10 rats, and drugs were administered by intravenous injection. The systolic and diastolic blood pressures were measured before and 5 min, 3 h, 5 h and 24 h after drug administration using the tail-cuff method .
Data are presented as means ± SEM, and ED50 values are reported as geometric means accompanied by their respective 95% confidence limits. The statistical significance of differences between groups was assessed with t-tests (and nonparametric tests) or with one-way or two-way analysis of variance (ANOVA) followed by Bonferroni's or Dunnett's post-hoc test. All tests were performed using GraphPad Prism version 4.0 for Windows. Values of P < 0.05 were considered significant.