Neuroprotective Effect And The Mechanism Of Anti-excitatory Action Of ANEPⅢ, A New Recombinant Neurotoxic Peptide Originated From Buthus Martensii Karsch

It was first reported in 1989 that a peptide from the scorpion venom of Buthus martensii Karsch had an antiepileptic activity with medicinal value. This peptide had remarkable inhibitory effect on coriaria lactone-induced epilepsy in rats but had little toxicity in mice.Three genes of scorpion anti-neuroexcitation peptide were screened out from the cDNA library of the scorpion B. martensii Karsch. According to the cDNA sequence of anti-neuroexcitation peptide of scorpion B. martensii Karsch, the putative mature anti-neuroexcitation peptide (ANEP) encoding DNA fragment was obtained by a PCR method. The recombinant ANEP could apparently delay the convulsion seizure of model animals by 18 % and showed anti-neuroexcitatory activity.Hippocampal and cortical neurons were prepared from 1-day old rats. Low concentration and prolonged time of trypsin digestion and mechanical dissociation were adopted to conduct monolayer primary culture and its expression of ion channels and electrophysiology characters were observed using the whole-cell patch clamp recording techniques and RT-PCR techniques.To investigate the neuroprotective effects and mechanism of action of the ANEPIII, a new recombinant neurotoxic peptide originated from B. martensii Karsch, on trauma of cells, the primary cultured rat cortical and hippocampal neurons were used. Pharmacological experiments showed that the ANEPIII protected hippocampal and cortical neurons against Mg²⁺ free and glutamate insults in neurons trauma and LDH level, and elevated the number of surviving neurons of Mg²⁺ free-, glutamate- and hypoxic/hypoglycemic-induced death of hippocampal and cortical neurons. Lag-time microphotography was employed to detect cell volume. It was shown that ANEPâ…¢and MK-801 suppressed the cellular swelling induced by glutamate (n=8, p＜0.05). The trauma and apoptosis were evaluated by AO/EB staining.The neurons were treated with glutamate 600μmol/L for 20min after the hippocampal and cortical neurons were pretreated with ANEPâ…¢1000 nmol/L or MK-801 for 30 min, the neurons were stained with AO/EB. Fluorescent microscopy showed ANEPâ…¢and MK-801 reduced numbers ofhippocampal and cortical neurons undergoing death and apoptosis.To investigate the effect of ANEPâ…¢on intracellular calcium concentrations ([Ca²⁺]_i), hippocampal and cortical neurons were loaded with Ca²⁺-sensitive fluorescent indicator Fluo-3/AM. [Ca²⁺]_i represented by fluorescent intensity (FI) was measured using the laser scanning confocal microscope. The results showed that: (1) at resting level, [Ca²⁺]_i was not affected by ANEPâ…¢(3-3000 nM); (2) [Ca²⁺]_i was elevated markedly by KCI 60raM and FI was increase to 188ï¼…and 164ï¼…basal value in cultured rat hippocampal and cortical neurons, respectively; (3) ANEPâ…¢significantly inhibited the increase in [Ca²⁺]_i by KCl 60 mM in a dose-dependent manner; (4) dual peaks of [Ca²⁺]_i rise were observed at 40 seconds and 10 minutes after glutamate (500μmol/L) exposure, followed by a persistent rise; (5) ANEPâ…¢significantly suppressed the increase in [Ca²⁺]_i in a dose-dependent manner; (6) dual peaks induced by glutamate were also inhibited by the N-methyl-D-aspartate receptor antagonist MK-801.ANEPâ…¢was examined for its action on voltage-gated ion channels currents in primary cultured rat hippocampal and cortical neurons using the patch-clamp technique in the whole-cell configuration. ANEPâ…¢decreased the sodium currents in a voltage-dependent manner, which appeared as a shift of the current-voltage relation to positive potentials. The effect was reversible after washing. The concentration-responsiveness measured in hippocampal and cortical neurons revealed an IC₅₀ value of 214.76 nM and 124.57 nM at a potential of-30 mV and -20mV, respectively. For the different types of neurons, the shift of the current-voltage relation was distinct and was 9.7 mV in hippocampal neurons, and 5.7 mV in cortical cells with 1000 nM ANEPâ…¢. Furthermore, the time constant for recovery from inactivation was also prolonged by 1000 nM ANEPâ…¢. The delayed rectifier K⁺ current (I_k) was inhibited by externally applied recombinant BmK ANEPâ…¢, while the transient A-current (I_A) remained virtually unaffected. BmK ANEPâ…¢3000 nM, reduced the delayed rectifier current by 28.2ï¼…and 23.6ï¼…in cultured rat hippocampal and cortical neurons, respectively. The concentration of half-maximal block was 155.1 nM for hippocampal neurons and 227.2 nM for cortical neurons, respectively.The effect of ANEPâ…¢on the voltage-gated ion channels protein transcript levels in primary cultured rat hippocampal and cortical neurons were study by using RT-PCR technique. The results showed that: (1) Navl.1 mRNA expression was decreased markedly in the cultured hippocampal neurons 3h and 12h after exposure to ANEPâ…¢1000 nM; (2) Navl.2 mRNA expression was decreased markedly in the cultured cortical neurons 24h after exposure to ANEPâ…¢1000 nM; (3) ANEPâ…¢reduced the Kv1.5 channel mRNA expression on 3-48h and 24h in cultured rat hippocampal and cortical neurons, respectively; (4) Kv2.1 mRNA expression was increased markedly in the cultured cortical neurons 12h after exposure to ANEPâ…¢1000 nM; (5) Caα-1C mRNA expression was redeced in the cultured hippocarnpal neurons 24h after exposure to ANEPâ…¢1000 nM; (6) ANEPâ…¢reduced the Caα-1G mRNA expression on 24-48h and 24h in cultured rat hippocampal and cortical neurons, respectively.Our results indicate that ANEPâ…¢exerts neuroprotective and antineuroexcitatory properties, and the mechanism of action is related to calcium ion entry in cultured hippocampal and cortical neurons. These study provided data showing that ANEPâ…¢exerts a moderate inhibition of the delayed rectifier potassium currents and sodium currents in cultured hippocampal and cortical neurons in vitro. These effect might endow ANEPâ…¢with a genuinely novel antiepileptic mechanism, which could contribute to the antiepileptic action of this drug in vivo.