Effects Of Propofol And Ketamine On Intracellular Calcium Of Rabbits Tracheal Smooth Muscle Cells

Objective: Propofol and Ketamine are generally used for clinical anesthesia. Studies show that Propofol and Ketamine have different degree relaxation on tracheal smooth muscles, through which way to protect the airway. Propofol reacts mainly through inhibiting the cranial nerve tension; while in high concentrations, it has direct relaxing action on tracheal smooth muscles. Ketamine relaxes tracheal smooth muscles possibly concerned with inhibition of ROC, delivery of intracellular calcium PKC signal system, or dose dependent patency of kalium ion channel. There are less reports about whether the two have effects on intracellular calcium and react through endoplasmic reticulum. Our experiments approach the effects of different concentrations of Propofol and Ketamine on intracellular calcium and releasing of endoplasmic reticulum calcium through evaluating the variation of intracytoplasm calcium; and further identify their mechanisms of action on relaxing tracheal smooth muscles.Methods: New Zealand rabbit are chosen as experimental animals, no limits to their sex, and 1.5±0.3kg weight. The single rabbit tracheal smooth muscle cells are got by enzymolysis methods. Choose cells with complete cell membrane, three-dimensional, fusiform and well refraction function to begin fluorescent labeling. Shock the centrifuged cells into cell suspension, and put 5μM concentration FLuo-3/AM into the cell suspension. Incubate cell suspension in thermostatic waterbath box under 37℃for 30 min. Observe the labeling conditions of the trachea smooth muscle cells under the laser confocal microscopy.Groups 1.propofol Fractionate the cell suspension treated by the methods above into Propofol group, Pro+2-APB group and Pro+ryanodine group(Pro+RyD); and each group is fractionated into three subsets according to the density of propofol, that is 0, 30 and 300μM group. 2.ketamine Fractionate the cell suspension treated by the methods above into ketamine group ,ket+ 2-APB group and ket+ RyD group, and each group is fractionated into three subsets according to the density of ketamine, that is 0, 100 and 1000μM group.Experiment flow-sheetPropofol group(Cgroup) add 1μM ACH to the cell suspension signatured by FLuo-3, and record the calcium fluorescence peak intensity. Wash cell suspension with HBSS for 3 times. Then add different density propofol into each subset groups to incubate for 15 min; add 1μM ACH again . Record the calcium fluorescence peak intensity in each group.Pro+2-APB group add 1μM ACH to the cell suspension signatured by FLuo-3. Record the calcium fluorescence peak intensity. Then add 40μM 2-APB; incubate for 15 min; then add different density propofol respectively , and incubate with 2-APB together for 15 min; wash with HBSS for 3 times, add 1μM ACH again .Record the calcium fluorescence peak intensity in each group.Pro+RyD group add 1μM ACH to the cell suspension signatured by FLuo-3. Record the calcium fluorescence peak intensity. Wash cell suspension with HBSS for 3 times. Then add 10μM RyD incubate for 15 min; then add different density propofol; incubate with RyD together for 15 min; wash with HBSS for 3 times, add 1μM ACH again . Record the calcium fluorescence peak intensity in each group.In ketamine group(C group), ket+2-APB group and ket+RyD group, the density of ketamine is 0,100and 1000μM. The methods are the same to propofol.Results: 1 Cells aquired through enzyme separation method are pure, easy to discern, large quantity, and high survival rate. 80%~90% of the cells are identified as living cells by trypan blue dyeing method. The cell appearances are varied, well about 70%~80% of them are just like the earthworms and bananas; also other shapes as ellipse, round and irregularity. Sizes of the cells are not at equal. The average diameter of them is about 12~16vm, and the length about 60~100vm. The edge of the cells is distinct; the membrane surface is smooth; endoparticle is few.2 Compared with 0μM propofol group, 300μM propofol can decrease the intracellular calcium peak intensity obviously (P<0.05).3 After treated by Pro+2-APB, compared with their corresponding groups in the control group , there are obvious decrease in 0μM and 30μM Pro+2-APB group(P<0.05); and no differences in 300μM Pro+2-APB group(P>0.05).4 After treated by Pro+RyD, compared with their corresponding groups in the control group , the intracellular calcium peak intensity in each group has an obvious decrease(P<0.05). Compared with 0μM Pro+RyD group , the intracellular calcium peak intensity decreased obviously in 300μM Pro+RyD group(P<0.05).5 Compared with 0μM ketamine group , there is obvious decrease in 1000μM ketamine group(P<0.05).6 After treated by ket+2-APB, Compared with their corresponding groups in the control group, the intracellular calcium peak intensity in 0μM and 100μM ketamine+2-APB groups have obvious reduction(P<0.05) . No obvious decrease in 1000μM ketamine+2-APB group(P>0.05).7 After treated by ket+RyD, compared with their corresponding groups in the control group, the intracellular calcium peak intensity have obvious decrease in each ket+RyD group(P<0.05); compared with 0μM ket+RyD group, 1000μM ket+RyD group has obvious decrease(P<0.05) . Conclusions: 1 300μM propofol can decrease the intracellular calcium, while 30μM cannot.2 Propofol maybe block the IP3 way to decrease the intracellular calcium ,while no relationship with ryanodine way.3 1000μM ketamine can decrease the intracellular calcium, while 100μM cannot.4 Ketamine could block the IP3 way to inhibit the calcium release from endoplasmic reticulum to decrease intracellular calcium .while the ryanodine way cannot be blocked by ketamine.