The Effects of Protein Kinase C (PKC) on the Tension of Normal and Passively Sensitized Human Airway Smooth Muscle and the Activity of Voltage-dependent Delayed Rectifier Potassium Channel (Kv)

The effects of protein kinase C （PKC） on the tension and the activity of voltage-dependent delayed rectifier potassium channel （K,,） were examined in normal and passively sensitized human airway smooth muscle （HASM）, by measuring tones and whole-cell patch clamp techniques, and the Kv activities and membrane potential （Em） were also detected. The results showed that phorbol 12-myristate 13-acetate （PMA）, a PKC activator, caused a concentration-dependent constriction in normal HASM rings. The constriction of the passively sensitized muscle in asthma serum group was significantly higher than that of the normal group （P〈0.05）, and the constrictions of both groups were completely abolished by PKC inhibitor Ro31-8220 and calcium channel inhibitor nifedipine. Kv activities of HASM cells were significantly inhibited by PMA, and the Em became more positive, as compared with the DMSO （a PMA menstruum）-treated group （P〈0.01）. This effect could be blocked by Ro31-8220 （P〈0.01 ）. It was concluded that activation of PKC could increase the tones of HASM, which might be related to the reduced Kv activity. In passively sensitized HASM rings, this effect was more notable.

The role of potassium channels in the nitric oxide-induced relaxation of human airway smooth muscle of passively sensitization by serum from allergic asthmatic patients

Objective： To investigate the role of large Ca^2＋-activated, delayed-rectifier and ATP-sensitive potassium channel in regulating the relaxation induced by nitric oxide （NO） in normal and passively sensitized human airway smooth muscle （HASM） with serum from asthmatic patients. Methods： The effects of NO or/and potassium channel blockers on the tensions of normal and passively sensitized HASM were measured by using nitric oxide donor and potassium blockers, with the isometric tension recording technique. Results： Showed that（1）In the control group and passively sensitized group, Kv blocker（4-AP） cause concentration-dependent augmentation in the contraction induced by histamine （ 1 ×10^-4 mol/L ）, （P 〈 0.05）, but Glib （ 1 × 10^-2 mol/L ） and TEA （1×10^-4 mol/L） have no significant effects on the contraction induced by histamine （1×10^-4 mol/L）. The maximum tension induced by histamine in passively sensitized group is higher than that in the control group （P 〈 0.05）. （2） NO-donor Sodium Nitroprusside （SNP） bring about significant relaxation in normal and passively sensitized HASM rings （P 〈 0.05）. Relaxations of passively sensitized airway rings [ （29.4 ± 3.3）% ] were significant less than those of normal HASM rings [ （44.1 ± 10.2）% ], （P 〈 0.05）.（3） Glib（1×10^-2 mol/L）have no significant effect on the relaxations induced by SNP（1×10^-4 mol/L）. 4-AP（1×10^-2 mol/L） inhibited relaxation induced by SNP （1×10^-4 mol/L）, （P 〈 0.01）. TEA （1×10^-3 tool/L） inhibited relaxation induced by SNP （1×10^-4 mol/L） （P 〈 0.05）, and the inhibiting effect in passively sensitized HASM rings were significant less than in normal HASM, （P 〈 0.05）. Conclusion： It was concluded that SNP（NO-donor） relaxed the contraction of HASM partly via BKca channel opening. In passively sensitized HASM in vitro, the relaxation of SNP decreased compared with control group, which might be associated with the down-regulating activity of BKca in passively sensitized HASM.

Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation

Background Airway smooth muscle proliferation plays an important role in airway remodeling in asthma. But little is known about the intracellular signal pathway in the airway smooth muscle cell proliferation in asthma. The objective of this paper is to investigate the contribution of protein kinase C (PKC) and its alpha isoform to passively sensitized human airway smooth muscle cells (HASMCs) proliferation. Methods HASMCs in culture were passively sensitized with 10% serum from asthmatic patients,with non-asthmatic human serum treated HASMCs used as the control. The proliferation of HASMCs was examined by cell cycle analysis,3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT) colorimetric assay and proliferating cell nuclear antigen (PCNA) immunofluorescence staining. The effect of PKC agonist phorbol 12-myristate 13-acetate (PMA) and PKC inhibitor Ro-31-8220 on the proliferation of HASMCs exposed to human asthmatic serum and non-asthmatic control serum was also examined by the same methods. The protein and mRNA expression of PKC-α in passively sensitized HASMCs were detected by immunofluorescence staining and reverse transcription-polymerase chain reaction. Results The percentage of S phase,absorbance (value A) and the positive percentage of PCNA protein expression in HASMCs passively sensitized with asthmatic serum were (16.30±2.68)%,0.430±0.060 and (63.4±7.4)% respectively,which were significantly increased compared with HASMCs treated with control serum [(10.01±1.38)%,0.328±0.034 and (37.2±4.8)%,respectively] ( P <0.05). After HASMCs were passively sensitized with asthmatic serum,they were treated with PMA,the percentage of S phase,value A and the positive percentage of PCNA protein expression were (20.33±3.39)%,0.542±0.065 and (76.0±8.7)% respectively,which were significantly increased compared with asthmatic serum sensitized HASMCs without PMA( P <0.05). After HASMCs passively sensitized with asthmatic serum were treated with Ro-31-8220,the percentage of S phase,value A and the positive percentage of PCNA protein expression were (11.21±1.56)%,0.331±0.047 and (38.8±6.0)% respectively,which were significantly decreased compared with asthmatic serum sensitized HASMCs without Ro-31-8220 ( P <0.05). The relative ratio of value A of PKC-α mRNA and the positive percentage of PKC-α protein expression in passively sensitized HASMCs were 1.23±0.10 and (61.1±9.4)% respectively, which were significantly increased compared with HASMCs treated with control serum [1.05±0.09 and (34.9±6.7)%,respectively] ( P <0.05). Conclusions The proliferation of HASMCs passively sensitized with human asthmatic serum is increased. PKC and its alpha isoform may contribute to this proliferation.