Investigation Of Mechanical Sensitive Channels On Mast Cells

Objective:To investigate mechano-sensitive property of the human mast cell line HMC-1 and further prove this property on mast cells(MCs) in vivo.Methods:(1) Light image technique was used to observe the effects of osmotic stress induced by hypotonic solution on degranulation of HMC-1.(2) Fluorescent image technique was performed to measure the change of intracellular calcium concentration([Ca²⁺]_i) in HMC-1 in response to osmotic stress.Calcium Green-1 AM was used as fluorescent probe.(3) Patch-clamp technique was applied to measure and analyze single-channel properties of stretch-activated(SA) channels on HMC-1 in outside-out and cell-attached modes.(4) Connective tissue slice in rat 'housanli' acupoint(ST36) was prepared as slice model.MCs in slices were labeled by toluidin blue(TB) or neutral red(NR).SA currents were recorded in whole-cell configuration from MCs in vivo.Results:(1) Hypotonic solution with 230 mOsm/kg could induce resting HMC-1 cells to degranulate in 5-30 min.The degranulation ratio was 54.9±8.5%(n=4 independent experiments,P≤0.01)(mean±SEM).This effects could be inhibited by DIDS,a classic blocker of Cl^- channels,and by SKF96365 that can block TRPV2(transient receptor potential vanilloid) channel.The corresponding degranulation ratio was 54.9±58.5%(n=4 independent experiments,P≤0.05) and 22.2±7.9%(n=2 independent experiments) respectively.(2) Hypotonic solution with 250 mOsm/kg could raise[Ca²⁺]_i as measured the fluorescent intensity of Calcium Green-1 AM in HMC-1.The relative fluorescent intensity increased to 116.7±3.6%(n=52,in 5 independent experiments,P≤0.01) of control,which could be cancelled by TRPV1-4 blocker Ruthenium Red(RuR) or SKF96365 to 100.3±0.9%(n=41,in 3 independent experiments,P≤0.0.1) and 103.7±3.2%(n=30,in 3 independent experiments,P≤0.05) respectively.200μM DIDS diminished fluorescent intensity of HMC-1 to 67.8±5.2%(n=29,in 4 independent experiments,P≤0.01).(3) Pressure(+30—+60 or -60—-30 cm H₂O) could activate SA channels.SA single-channel currents were strong outwardly certificating.The cord conductance at +100 mV was 55.1±3.4 pS.Channel open probability(P_o) exhibited voltage dependence.P_o increased greatly at positive membrane potential.P_o had pressure dependence as well.It rose with pressure increasing.At +20 mV pressure at which channel open probability reached the half value of maximum P_o was 26.4 cm H₂O.At +60 mV the open-time distribution of SA channel was fitted with three components with time constants ofÏ„_1o=755.1 ms,Ï„_2o=166.4 ms,Ï„_3o=16.5 ms and the closed-time distribution also required three components with time constants ofÏ„_1c=661.6 ms,Ï„_2c=253.2 ms,Ï„_3c=5.6 ms.(4) SA channel events showed extracellular Cl^- concentration([Cl^-]_o) dependence (n=9).The conductance at +100 mV was declined from 44.2±4.4 pS to 15.1±1.6 pS(P≤0.01) with[Cl^-]_o drop from 169 mM to 19 mM.At +60 mV P_o decreased to 62.2±13.6%of control(P≤0.01) and V_1/2 shifted from 5.5±2.7mV to 5.6±6.9 mV.(5) SA channel events could be reversibally inhibited by DIDS(n=9).The conductance at +100 mV declined from 58.5±4.1 pS to 18.9±7.0 pS(P≤0.01) and P_o at +60 mV decreased to 20.6±1.4%of control(P≤0.01).(6) The pressure need to active SA channel decrease when cytoskeleton of HMC-1 was disrupted by 10μM cytochalasin B(CB)(7) Amount of MCs are located in connective tissue under rat skin at 'housanli' acupoint(ST36).As HMC-1,SA channel on MCs in vivo could also be activated by pressure(-90—-30 cm H₂O).The whole-cell current of SA channel illustrated strong outwardly rectifying and increased with pressure raised.The conductance was 83.2±25.5 pS at +100 mV under -60 cm H₂O and reversal potential was about -35.8 mV.Conclusions:Degranulation of HMC-1 could be induced by mechanical stimuli.DIDS-sensitive Cl^-channel and TRPV may contribute to the mechanism.And intracellular calcium may be involved in signal pathway.Rat MCs in vivo also own mechano-sensitivity as HMC-1.