[Dextran sedimentation for study of neutrophil polarization].

OBJECTIVE: To determine the optimal method for separating neutrophils for studying neutrophil polarization. METHODS: Human neutrophil was separated from healthy human peripheral blood by Percoll density gradient centrifugation and Dextran sedimentation. The cell polarization, purity and activity of the neutrophils were determined, and F-actin polymerization and [Ca2+]i were analyzed. RESULTS: No significant difference was found in cell polarization, purity and activity of the human neutrophils separated by Dextran sedimentation and Percoll density gradient centrifugation (P>0.05), but F-actin polymerization was inhibited in PMNs separated by Dextran sedimentation, and the peak value of [Ca2+]i was decreased by 25% in PMNs separated by Dextran sedimentation compared to the cells separated by Percoll density gradient centrifugation. CONCLUSIONS: Both Percoll density gradient centrifugation and Dextran sedimentation can be used for isolating human neutrophils to study cell polarization, but the former method allows better isolation. Dextran sedimentation can be considered when a large number of neutrophils need to be separated.

[Modification of vascular endothelial cell treatment for patch clamp study].

A modified protocol for quick and economic treatment of cultured human umbilical vein endothelial cells has been established, which result in high viability of the cells to allow better performance in patch-clamp studies. The electrophysiological properties of Ca (2+)-activated K(+) (KCa) channel of the cultured cells were investigated with a cell-attached configuration. With this modified treatment method, the cultured cells appear fusiform in shape under microscope, and KCa channel currents could be detected readily, suggesting their eligibility for patch-clamp studies.

[Investigation of IL-8Rbeta mRNA expression profile in single human neutrophil].

AIM: To validate the abundance of Interleukin 8 receptor beta (IL-8Rbeta) mRNA in single human neutrophil. METHODS: Human neutrophils were isolated and purified from volunteers, total RNA was extracted and a regular RT-PCR aiming at IL-8Rbeta mRNA was performed to ascertain its expression profile in human neutrophils and optimize the reaction conditions for the following single-cell RT-PCR procedures. Subsequently, single neutrophil or the cellular content was harvested to conduct reverse transcription and two-round PCR with the same primer pairs used before. Serial dilution of single neutrophil cDNA pool was carried out at the same time with the exact two-round PCR followed. The specificity of this single-cell RT-PCR procedure was verified by the BamHI restriction endonuclease digestion on the final cDNA products. RESULTS: Regular RT-PCR indicated IL-8Rbeta mRNA expression in human neutrophils. While single-cell RT-PCR was sensitive enough to detect trace IL-8Rbeta mRNA as predicted cDNA product could be amplified from a 10 000 times diluted intracellular specimen from single neutrophil, which indicated an abundant expression of this mRNA in human neutrophil. Moreover, BamHI digestion on the final cDNA product clarified the specificity of this single-cell RT-PCR procedure. CONCLUSION: This simplified semi-quantitative single-cell RT-PCR procedure specifically confirmed that IL-8Rbeta mRNA was highly expressed in human neutrophil, which also provided the possibility of comparing mRNA abundance at single cell level.

[Electrophysiological study on rat conduit pulmonary artery smooth muscle cells under normoxia and acute hypoxia].

The present study was designed to investigate the electrophysiological characteristics of rat conduit pulmonary artery smooth muscle cells (PASMCs) and the response to acute hypoxia. PASMCs of the 1st to 2nd order branches in the conduit pulmonary arteries were obtained by enzymatic isolation. The PASMCs were divided into acute hypoxia preconditioned group and normoxia group. Hypoxia solutions were achieved by bubbling with 5% CO2 plus 95% N2 for at least 30 min before cell perfusion. Potassium currents were compared between these two groups using whole-cell patch clamp technique. The total outward current of PASMCs was measured under normoxia condition when iBTX [specific blocking agent of large conductance Ca-activated K(+) (BK(Ca)) channel] and 4-AP [specific blocking agent of delayed rectifier K(+) (K(DR)) channel] were added consequently into bath solution. PASMCs were classified into three types according to their size, shape and electrophysiological characteristics. Type I cells are the smallest with spindle shape, smooth surface and discrete perinuclear bulge. Type II cells show the biggest size with banana-like appearance. Type III cells have the similar size with type I, and present intermediary shape between type I and type II. iBTX had little effect on the total outward current in type I cells, while 4-AP almost completely blocked it. Most of the total outward current in type II cells was inhibited by iBTX, and the remaining was sensitive to 4-AP. In type III cells, the total outward current was sensitive to both iBTX and 4-AP. Acute hypoxia reduced the current in all three types of cells: (1614.8+/-62.5) pA to (892.4+/-33.6) pA for type I cells (P<0.01); (438.3+/-42.8) pA to (277.5+/-44.7) pA for type II cells (P<0.01); (1 042.0+/-37.2) pA to (613.6+/-23.8) pA for type III (P<0.01), and raised the resting membrane potentials (E(m)) in all these three types of cells: (-41.6+/-1.6) mV to (-18.6+/-1.5) mV (P<0.01), (-42.3+/-3.8) mV to (-30.6+/-3.0) mV (P<0.01), (-43.3+/-1.6) mV to (-28.4+/-1.4) mV (P<0.01), for type I, II, III cells, respectively. These results suggest that acute hypoxia suppresses the potassium current and improves the E(m) in PASMCs. These effects may be involved in the modulation of constriction/relaxation of conduit artery under acute hypoxia. Different distribution of K(DR) and BK(Ca) channels in these three types of PASMCs might account for their different constriction/relaxation response to acute hypoxia.

[Effect of 4 degrees C pretreatment on the membrane current and cell polarity in human neutrophils].

To investigate the role of ion channels in the coupling responses of neutrophils to extracellular stimulus, it is necessary to study the membrane ion channel activities using patch-clamp technique. However, little has been known about the ion channel activities in neutrophils due to the difficulties in forming giga-seal with pipettes because of small diameter of neutrophils and the easily developed polarization. Some studies indicated that favorable results could be achieved through pretreatment at low temperature before electrophysiological recordings. But it remains unclear whether the pretreatment affects the membrane current and why the seal rate increases after low temperature pretreatment. The purpose of this study was to investigate the effects of 4 degrees C pretreatment on the membrane current and cell polarity in human neutrophils. In the experiments, human neutrophils were isolated from fresh peripheral blood of healthy volunteers and divided into two groups (room temperature group and 4 degrees C pretreatment group). Voltage-dependent K(+) (Kv) currents were recorded in whole-cell voltage-clamp mode and large-conductance Ca(2+)-activated K(+) (BK(Ca)) currents were recorded using inside-out patches. The results showed that 4 degrees C pretreatment significantly inhibited cell polarity (P<0.05), and it took more time for neutrophils to form a polarity-cycle [(534+/-32) s, n=20] compared with those at room temperature [(257+/-24) s, n=20]. Meanwhile, seal rate significantly increased in 4 degrees C pretreatment group (64%) compared with that in the room temperature group (27.5%). The seal rate and cell polarity rate during 0 approximately 1 min after 4 degrees C pretreatment were significantly different from those at room temperature, while no significant difference was found during 9 approximately 10 min between the two groups. Our results suggest that 4 degrees C pretreatment can inhibit cell polarity and increase seal rate, but has no effects on membrane currents. It is also suggested that 0 approximately 1 min after 4 degrees C pretreatment is a more suitable time for electrophysiological recording in neutrophils.

[Effect of doxofylline on calcium-activated potassium channels in human peripheral blood eosinophils in asthma].

OBJECTIVE: To study the effects of doxofylline on calcium-activated potassium (K(Ca)) channels of human peripheral blood eosinophils in asthma. METHODS: Peripheral blood eosinophils from patients with asthma were isolated and divided equally into two groups, a control group and a doxofylline incubated group. The data were recorded using cell-attached configuration of patch-clamp technique and the kinetic changes of K(Ca) channels activated by 0.2 micromol/L platelet activating factor (PAF) were compared. RESULTS: As compared with the control group, the open probability of the K(Ca) channels decreased from 0.135 +/- 0.021 to 0.044 +/- 0.018, the open time from (5.75 +/- 0.40) ms to (2.39 +/- 0.13) ms, while the close time increased from (2.17 +/- 0.50) ms to (23.73 +/- 2.50) ms in the doxofylline incubated group. The differences were significant between the two groups (all P < 0.05). CONCLUSION: Doxofylline could decrease the open probability of the channels as a result of both the shortening of open period and the prolongation of close time.

[Effects of temperature on the burst opening of voltage-dependent K+ channels in hypothalamic neurons].

AIM AND METHODS: To observe the effect of temperature on burst opening of voltage-dependent K+ channels(Kv) in hypothalamic neurons by cell-attached mode of patch-clamp technique. RESULTS: With temperature raising, the number of burst opening increased, so did its average burst duration. B1 and B2 raised from 1.5 ms and 6.6 ms at 32 degrees C to 8.1 ms and 83.2 ms (P < 0.05) respectively while the open number of inter-burst, from 1-2 to 8 (P < 0.05) too. Instead of SB, CB displayed predominantly a kind of burst opening. CONCLUSION: More burst opening of Kv in hypothalamic neurons with temperature raising, this was benefited to the body temperature regulation of neurons on hypothalamus.