Modulation And Mechanism Of L-Type Calcium Channels In Cultured Hippocampal Neurons By Nitric Oxide

Nitric oxide (NO) is an important signaling messenger and has been shown to be involved in many physiological and pathophysiological processes in the brain. Considerable evidence indicates that there is an excessive amount of NO generation in hippocampal neurons following the hypoxic or ischemic insult, which is responsible for the brain injuries. During hypoxia or ischemia, there is a redistribution of ions across the membrane and the increase in the Ca2+ influx could greatly affect the activities of NO synthases, the amount of NO production and the level of NO in neural tissue. However, until now, it is not clear how the endogenous NO is related to the changes in the ionic events causing the Ca2+ influx. In addition, by what pathways NO could play a role in the mediation of the Ca2+ influx is not well understood. L-type Ca2+ channels are prevalent in hippocampal pyramidal neurons, contributing -30-50% of total calcium current. Therefore, modulation of L-type Ca2+ channel activity may have dramatic effects on Ca2+ influx. In this study, we examined the mechanistic effects of NO involved in the modulation of L-type calcium channel in the rat hippocampal neurons.Firstly, we examined the endogenous NO in the modulation of L-type calcium channels of cultured hippocampal neurons. L-arginine (L-Arg, l-3mM), a nitric oxide synthase (NOS) substrate, induced an 61% increase in the currents evoked by depolarizing step to 0 mV (n=30, p<0.01) and also a left shift of the I-V curve between -20 mV to 10 mV, an indication of increase in the channel sensitivity todepolarizing voltages. Nifedipine (NFDP, 20uM), a specific L-type calcium channel blocker, suppressed the currents by 60% (n=3, p<0.05) and such pretreatment completely abolished the enhancing effect of L-Arg on the currents (n=3), indicating a dominant action of L-Arg on L-type calcium channel. In contrast to L-Arg, application of NOS inhibitor N~G-Nitro-L-Arginine Methyl Ester (L-NAME, l-3mM) alone significantly reduced the currents amplitude about 51% compared with control (n=8, p<0.01), suggesting that there is a basal modulation of NO on the channel in cultured neuron. Moreover, pretreatment with lmM L-NAME (n=16) and 0.2mM 7-Nitroindazole (7-NINA, specific nNOS inhibitor, n=10) both partly blocked L-Arg-induced increase in the channel currents (p<0.05). These results indicate that endogenous NO enables the hippocampal L-type calcium channels to increase its openings and sensitivity to voltage.To determine the involvement of S-nitrosylation in mediating endogenous NO-induced channel activation, we examined the effects of the N-ethylmaleimide (NEM), which covalently modifies protein thiol groups thereby prevents subsequent S-nitrosylation reactions, and ascorbic acid (VitC), which decomposes nitrosotiol but not disulphide by reduction, on the modulation of L-type calcium channels produced by L-Arg. Pretreatment with lmM NEM completely precluded the action of L-Arg on the currents. Moreover, in the presence of NEM, L-Arg produced a significant reduction of the currents (n=14, p<0.01), implying that beside S-nitrosylation pathway, another inhibition mechanism such as cGMP pathway may be also involved in the channel modulation. Application of VitC (0.5mM) alone induced a right shift of the I-V curve between 20 mV to 60 mV (n=5, p<0.01), which is an opposite shift as compared to the L-Arg's action. Furthermore, Subsequent addition of VitC (2 mM) almost reversed L-Arg-induced I-V shift (n=4). The above results indicate that S-nitrosylation is involved in the modulation of L-type calcium channels produced by endogenous NO. In consistent, pretreatment with soluble guanylyl cyclase inhibitor lH-[l,2,4]Oxadiazolo[4,3-a]quinoxalin-l-one (ODQ, lOuM) showed no obvious effect on L-Arg-induced channel activation (n=8, p>0.05). However, such pretreatment reduced the concentration of L-Arg required to enhance the currents. In the presence of ODQ, lmM L-Arg increased the currents in all tested cells (8/8), while, in the absence of ODQ, only 57% cells (17/30) showed enhanced currents and the remained cells required 3mM L-Arg to induce a comparable response, implying that the cGMP pathway may also mediates the modulation of L-type calcium channels by endogenous NO but in a down-regulating way against S-nitrosylation.Then, we further explore the modulation and mechanism of NO by the means of NO donors on the hippocampal L-type calcium channel. NO donor DETA (0.5mM) stimulated the L-type calcium currents at steps between -20mV~10mV of the I-V curve, which is analog to the effect of L-Arg. The currents was increased about 23% at a depolarized step of OmV (n=ll, p<0.01). EDTA also shares the same mechanism as L-Arg. We found that when the cells were pretreated with lmM NEM to preclude the action of S-nitrosylation, 0.5mM DETA could not enhance the current anymore (n=8), indicating that DETA stimulates the channel via S-nitrosylation. When pretreated with lOuM ODQ to block the cGMP pathway, 0.5mM DETA could not only stimulate the channel, but also result in more stimulation than the control (n=6, p<0.01), which shows that cGMP pathway is playing an inhibition role to against S-nitrosylation. The above results further demonstrate that both S-nitrosylation and cGMP pathway are involved in the modulation of the hippocampal L-type calcium channel. For L-Arg and DETA, the stimulation via S-nitrosylation on the channel is much stronger while the inhibition via cGMP is less potent.In our experiments, we also found that another two kinds of NO donors showed different results from L-Arg and DETA. One is DEA and the other is SNP. 3mM DEA attenuated the currents significantly. When the voltage was depolarized from -50mV to 10mV, the currents were inhibited about 29% (n=5, p<0.01). SNP also showed inhibition on the channel and such inhibition was dose-dependent. O.lmM SNP had no obvious effect on the currents (n=3, p>0.05). 0.2-0.6mM showed dual effects: 5/12 cases showed inhibition and the currents were decreased about 16% at a step of 10mV (n=5, p<0.01), while 7/12 had no obvious effect on the currents. lmM SNP showed multiple effects: 6/11 cases were inhibited about 16% when the currents were elicited by a step of lOmV (n=6, p<0.01), 3/11 showed stimulating effect and 2/11 showed no obvious effect on the currents. For 3-9mM SNP, 7/34 cases SNP increased the currents while the others inhibited the channel: 3mM SNP decreased the currents about 20% at a step of lOmV (n=ll, p<0.01); 6mM decreased about 33% (n=10, p<0.01); 9mM showed 31% inhibition (n=8, p<0.01). Then we took 3-9mM SNP to explore their inhibition mechanism. We found that the presence of NEM could not affect the inhibition of 3mM SNP, indicating that little involvement of S-nitrosylation in the modulation of the hippocampal L-type calcium channel by 3mM SNP. While pretreated with ODQ to block the cGMP pathway, 4mM SNP still could inhibit the channel, suggesting that there are another inhibitory mechanism mediating the inhibition of SNP. Our further experiments showed that reducing agent DTT could partly reversed the effects of SNP, which suggesting that such inhibition might be viaoxidative mechanism. So we observed the inhibition of oxidizing agent DTNB on the channel. Similarly DTNB attenuated the currents by dose-dependent: 0.5mM DTNB decreased the currents about 10% at a step of lOmV (n=3, p<0.05); lmM decreased about 14% (n=5, p<0.01); 3mM decreased about 35% (n=5, p<0.05) and 5mM DTNB blocked the inwards currents completely (n=3, p<0.01). On the other hand, the reducing agent DTT had the contrary effects against DTNB. 3mM DTT caused right shift of the I-V curve and increased the currents from 20mV to 60mV of the I-V curve. These results demonstrate that hippocampal L-type calcium channel are sensitive to the redox modulation. Therefore NO donors DEA and SNP might inhibit the channel via oxidative mechanism.