| Agmatine was first identified and characterized as a candidate for CDS (clonidine displacing substance) in the bovine brain in 1994. The following researches demonstrated that agmatine was a widely distributed endogenous substance and performed a lot of biological functions in the central nervous system. The evidences revealed its targets were diverse and its mechanisms were complicated. Now it is well accepted that agmatine is a new neurotransmitter and /or neuromodulator and it was assumed to be an endogenous ligand of imidazoline receptor. However, agmatine also had high affinity to the α2-adrenoceptors and many of its biological effects were related to the receptor. Thus, the assumption still needs to be confirmed. On the other side, as an organic cation, agmatine could block NMDA receptor channel and other ligand-gated cationic channels, including nicotinic acetylcholine and 5-HT receptors. However, there is no evidence to show whether agmatine interacts with the voltage-gated channels. As the voltage-gated channels underlie many important biological functions, such as cell excitability, neurotransmitters and hormone secretion, activity of enzymes and gene expression, we hypothesized that agmatine might present its physiological and pharmacological functions partially by modulating some kinds of these channels.Therefore, on one side, the effects of agmatine on several kinds of voltage-gated channels were observed in the present experiment. On the other side, as we found that agmatine could block the voltage-gated calcium channel(VGCC) in the cultured rat hippocampal neurons, we tried further to figure out whether agmatine had selective effect on the subtypes of VGCC such as N-, L-, T-, P- and Q- type. Further more, as agmatine is accepted as a central neurotransmitter and has a higher affinity to imidazoline and α2-adrenoceptors, we also wanted to determine if these receptors were involved in its blocking effect on VGCC currents.The acid-sensing ion channels (ASICs) or H+-gated cation channels belong to the epithelialsodium channel (ENaC)/degenerin gene family and are activated by drops of the extracellular pH. So far, several ASIC cation channel subunits have been cloned and functionally expressed and identified. ASICs were involved in many functional such as participating in nociception and mechanosensation, relating to the process of study and memory, and transmitting the synaptic signal. It should be noticed that some biological functions of agmatine such as increasing the volume of urine, participating in the pain was much similar to amiloride, the special antagonist of ASICs. Therefore, we tried to explore the effect of agmatine on the ASICs.The present study was conducted by the whole-cell patch recording technique in the cultured neonatal rat hippocampal neurons by applying drugs directly to the single neuron through a pressure injector or the DAD-12 injector system. Furthermore, to investigate the mechanism of agmatine on the L-type calcium channels, we transfected the subunits of aic/PiB/0125 mt0 the HEK-293 cell lines and expressed functional calcium channels. In summary, there are mainly three parts in the experiments.1. Agmatine blocks the voltage-gated calcium channel in cultured rat hippocampal neurons and its mechanism of underlying blockade.Agmatine (500 uM) had no significant effect on the voltage-gated potassium and sodium channels. Agmatine reversibly blocked the voltage-gated calcium channel and the blockade was enhanced with the increased concentration of agmatine. The IC50 was 1.18 ± 0.37 uM. Two - way ANOVA revealed that change of membrane potential displayed a significant interaction with the blockade of agmatine. Verapamil (100 uM), a selective blocker of L- type calcium channel, significantly inhibited VGCC current by 80 ± 7 %. Agmatine (100 |aM) could further depress the remained currents by 25 ± 6 %. The 012-adrenoceptor antagonist yohimbine (10 |aM) and the I2 imidazoline receptor antagonist idazoxon (10, 40 uM) had no significant effect on VGCC currents when used respectively. When the mixture of yohimbine and agmatine was applied, VGCC currents were still depressed remarkably. However, the blocking effect of agmatine decreased by 29 ± 8 % in the presence of idazoxon (10 uM). The effect of idazoxon did not increase at a higher concentration (40 uM).2. Agmatine could inhibit the L-type calcium currents functionally expressed in the HEK-293 cell lines.It was found that there were endogenous potassium currents but no sodium currents and... |
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