| T-type Ca2+channel is a low voltage-activated calcium channel. T-typeCa2+channel family has three subtypes—CaV3.1, CaV3.2and CaV3.3. In thecentral nervous system, T-type Ca2+channel can induce pacemaker activityand low-threshold nerve impulse. In peripheral nervous system, T-type Ca2+channels mainly distribute in the small or middle size DRG nerouns. Themajor subtype of T-type Ca2+channel in DRG nerouns is CaV3.2. Recently,there are studies indicating that malfunction of the T-type Ca2+channel arerelated to many kinds of diseases, such as epilepsy, hypertension, heartdiseases, diabetes, pain, cancer. T-type Ca2+channel play important roles inboth physiological and pathophysiological conditions, and the moleculermechanisms of the T-type Ca2+channel modulation is the study focus of manygroups.The progress on the understanding of T-type Ca2+channel modulation hasbeen slow due to the lack of specific opener and inhibitor. Although withrecent discoveries of the specific opener and inhibitor, more work has beendone on T-type Ca2+channel modulation, our understanding on the mechanismof the T-type Ca2+channel modulation remains relatively limited. Acomprehensive understanding of T-type Ca2+channel modulation will providethe basis for the prevention and treatment of diseases related to abnormalT-type Ca2+channel functions.Pain has been a big problem plaguing human physical, mental health andnormal life. Ion channels play important role in pain physiolpgy and have beenthe interests of studies. Recent studies show that abnormal T-type Ca2+channel function is linked to neuropathic and inflammatory pain. However, nostudies have been performed on the effect of T-type Ca2+channel modulatorson pain behavior. This study will study modulation of T-type Ca2+channel function by substance P and the role of T-type Ca2+channel in pain behaviourPart oneStudy on regulation of T-type Ca2+channel by SP and theunderlying molecule mechanismObjective: To study the effect of SP on T-type Ca2+channel and theunderlying molecule mechanism.(1) To study the effect of SP on T-typeCa2+channel overexpressed in HEK293cells.(2) To study the effect of SP onT-type Ca2+channel in DRG neurons.(3) To understand the signalingmechansim of SP action.Methods: Whole-cell patch clamp technique will be used.Results:(1) The currents through the CaV3.2channel heterologouselyexpressed in HEK293cells (also overexpressing NK1receptors) wereinhibited by SP (1μM) about30.2%±6.8%(P＜0.05, n=13). The inhibitionwas poorly reversible upon washout of SP, but the inhibition were completelyrecovered by the reducing agent DTT (1mM),(recovered to99.3%±5.7%ofthe control, n=12).(2) The T-type Ca2+currents in DRG neurons were inhibited by SP (1μM) about25.9%±5.3%(P＜0.05, n=11). The inhibition could be completelyrecovered by the reducing agent DTT（1mM）（recovered to105.7%±11%ofcontrol, n=8）. The high voltage activated Ca2+currents were not affected bySP.(3) H2O2(300μM) inhibited the currents of CaV3.2expressed inHEK293cells (also overexpressing NK1receptors) about9.4%±1.7%(P＜0.05, n=5). The inhibition were completely recovered by the reducing agentDTT (1mM)(recovered to102.1%±1.3%of control, n=5). The inhibitioninduced by H2O2is less than that induced by SP.(4) Bath application of Antimycin A (1μM) increased the currents ofCaV3.2expressed in HEK293cells about142.1±7.6%(P＜0.05, n=8). Theincrease could not be recovered by the reducing agent DTT (1mM).(5) SP did not inhibit the currents of the mutant CaV3.2(H191Q). n=8. Conclusion: The results suggest that Substance P selectively inhibitedthe T-type Ca2+currents but not the HVA Ca2+currents.The inhibition was possibly through ROS/oxidation pathway. Histamineat the position191of CaV3.2is the critical site for this modification.Part twoThe effect of T-type Ca2+channel opener ST101on CaV3.2currents and the pain behaviorObjective: To study the effect of ST101on currents of CaV3.2overexpressed in HEK293cells, and the pain behavior of rats induced bythermal and mechanical stimulations.Methods: Whole-cell patch clamp technique; Rats spontaneous, thermaland mechanical pain models.Results:(1) ST101increased the currents of CaV3.2expressed in HEK293cells with the EC50of8.73±1.47nM.100nM ST101reached the maximumeffect, which increased the currents of CaV3.2about42.4±4.2%, n=5.(2) The behavior experiments show that ST101did not inducespontansous pian behavior in rats. The total spontaneous nocifencive behaviortime is: saline3.8±1.1s, Bradykinin (BK)100.3±17.8s and ST1018±1.5s,respectively (P=0.09, n=13).(3) ST101did not induce hyperalgesia with the thermal or mechanicalpain stimulations. The threshold of mechanical pain is: ST101group: beforeinjection68.4±7.9g, after injection59.3±4.8g（P＞0.05，n=5）; control group（saline）before injection60±0g, after injection53.2±6.8g（P＞0.05, n=5）,respectively. The threshold of thermal pain: ST101group36.3±3.2s, afterinjection38.6±2.0s（P＞0.05, n=5）; control group（saline）before injection38.9±0.9s, after injection38.6±0.6s（P＞0.05, n=5）, respectively.Conclusion: ST101increased the currents of CaV3.2expressed inHEK293cells. ST101did not induce spontansous pain or hyperalgesia in ratpain models. |
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