Mechanism Underlying Ca²⁺ Oscillation Frequency-dependent Regulation Of Transcriptional Factor Activation Induced By Histamine

The free calcium can be an important second messager in cytoplasm ,which has a critical role in modulate cell functions. Ca²⁺ works diversity in regulation cell activities,including cell adhesion,proliferation,differentiation,and gene expression. However, how can a singal Ca²⁺ achieve its specificity? This is a hot subject in studing Ca²⁺ signaling. The rise of [Ca²⁺]i can exhibit several forms: Ca²⁺ transient,continous [Ca²⁺]i in a high level,calcium oscillation. Calcium oscillation , which is a interesting biological phenomenon, is a general signal pattern in cytoplasm. Considering the movement forms , it can perform multiple parameters: amplitude,frequency,duration. In 1998, Lewis proved firstly that Ca²⁺ oscillation frequency regulate transcriptional factors NF-κB,NF-AT,OCT/OAP with specificity through applying Ca²⁺ clamp, and also enhance its efficiency. The perspective that calcium oscillation– dependent specificity in modulating cell functions has been accepted generallly. However, what is the mechanism underlying frequency-dependent? through observed carefully, we found that the nature of frequency changing is the times of up and down of [Ca²⁺]i. in unit time. The higer frequency, the more times of up and down in [Ca²⁺]i per minute, and also the longer time accumulation of [Ca²⁺]i in a concentration leval. When oscillation in a lower leval, the opposite condition occures. Thus we guess that the nature of frequency has a close relation with total wave widthes(the acummulated time of Ca²⁺ in a certain leval ). So in this experiment, we use histamine to stimulate human bronchial epithelial cells 16HBE intermitently in the base of the principle of calcium clamp, establishing the model of same amplitude and frequency but different total wave widthes,same amplitude and total wave widthes but different frenquency, also detecting the activition of transcriptional factor NF-κB. The result show different transcriptional factor activition in the condition of same amplitude and total wave widthes but different frequency, however, in another model, same transcriptional factor activity appears. Thus it confirms the inferation that the nature of calcium oscillation frequency-dependent is total wave widthes.Objective:To establish the calcium oscillation model of same amplitude and frequency but different total wave widthes,same amplitude and total wave widthes but different frequency, we use histamine to stimulate human bronchial epithelial cells 16HBE inmittently, and detect transcriptional factor NF-κB activities for investigating the mechanism underling calcium oscillation frequency-depend transcriptional factor activition.Methods:1.A computer-controlled micro perfusion system is used to switch rapidly between histamine-containing solution and histamine-free solution flowing into a champer and over cells that were loaded with Ca2+ fluorescent indicator Fura-2. At the same time, we use calcium imaging system to meature intracelluler calcium level. In the base of above,we can establish calcium oscillation model.2.To simulate calcium oscillation model, a computer-controlled micro perfusion system is used to switch rapidly between histamine-containing solution and histamine-free solution flowing into a champer and over cells that were loaded with Ca2+ fluorescent indicator Fura-2. we detect NF-κB activity using ELASA based method.Results:1. 10μmmol histamine-containing solution is applied to cells 20s,40s,60s per 100s,200s,300s for 45 minutes. The amplitude of the three calcium oscillations above is (467.1±57.1)nmol/L(n=21),(466.8±84.6)nmol/L(n=28),(467.9±73.2)nnmol/L(n=38)respectively, these amplitudes have no significant differences (p>0.05). The frequency of all is 0.6(min-1),0.3(min-1),0.2(min-1)respectively. The singal wave widths is (18.1±1.6)s(n=13),(36.0±3.0)s(n=17),(54.6±2.5)s(n=17). The total wave widths of that three calcium oscillations is same, which is 486s in 45 minutes; also the same for the total time of hitamine stimulated. Then changing the perfusion condition, 10μmmol histamine-containing solution is applied to cell 30s in100s for 45 minute. The calcium concentration in solution applied above is 1.0mmol,1.5mmol,2.0mmol respectively. The amplitude of all is (466.7±58.6)nmol/L(n=29),(465.3±52.8)nmol/L(n=31),(466.8±84.5)nmol/L(n=28)respectively, these amplitudes have no significant differences (p>0.05). The singal wave widths is (15.3±1.9)s(n=18),(20.4±4.1)s(n=24),(29.6±4.7)s(n=28), the wave width has significance differences (p<0.05). the frequency of the three calcium oscillation above is same 0.6 min-1. In the base of above, the model of same amplitude and frequency but different total wave widths is established. Then we establish same amlitude and frenquency but different total wave widths in the same way, and the frequency is 0.3 min-1,0.2min-1 in every model.2. 1)The activity of NF-κB regulated by the calcium oscillation of same amplitude and total wave widths but different frequency: 10μmmol histamine-containing solution is applied to cells 20s,40s,60s per 100s,200s,300s for 45minutes. The activities of NF-κB above is (184.7±22.7)%,(157.8±9.1)%,(158.3±12.7)% respectively, and the blank control is 100%. The activities of NF-κB of these three groups are higher than the blank control group, but there is no significanc diffrences among three groups (p>0.05).(2)The activity of NF-κB regulated by the calcium oscillation of same amplitude and frequency but different total wave widths: 10μmmol histamine-containing solution is applied to cells 30s in 100s for 45 minutes, the calcium concentration in solution applied above is 1.0mmol,1.5mmol,2.0mmol respectively. The activities of NF-κB above is (144.4±6.1)%,(209.1±22.2)%,(394.3±65.4)% respectively, and the blank control is 100%, The activities of NF-κB of these three groups are higher than the blank control group(p<0.05), but there is significanc diffrences among three groups (p<0.05). 10μmmol histamine-containing solution is applied to cells 30s in 200s for 45 minutes, the calcium concentration in solution applied above is 1.0mmol,1.5mmol,2.0mmol respectively. The activities of NF-κB above is (126.7±12.2)%,(147.6±13.9)%,(198.6±26.3)% respectively, and the blank control is 100%, The activities of NF-κB of these three groups are higher than the blank control group(p<0.05). Among them, the activities of NF-κB have no significant differences between group of 1.0mmol and 1.5mmol(p>0.05); but there is significant differences betwen the group of 1.5mmol and 2.0mmol(p<0.05), and also the 1.0mmol group and 2.0mmol group(p<0.05). 10μmmol histamine-containing solution is applied to cells 30s in 300s for 45 minutes, the calcium concentration in solution applied above is 1.0mmol,1.5mmol,2.0mmol respectively. The activities of NF-κB above is (111.4±7.0)%,(113.0±3.2)%,(119.6±9.8)% respectively, and the blank control is 100%, The activities of NF-κB of these three groups are higher than the blank control group(p<0.05).The activities of NF-κB have no significant differences among these three groups(p>0.05). Free histamine solution is applied to cells for the control between group, the calcium concentration in solution applied above is 1.0mmol,1.5mmol,2.0mmol respectively, .The activities of NF-κB have no significant differences among these three groups(p>0.05).Conclusions:We have established successively the model of same amplitude and frequency but different total wave widths, also the same amplitude and total wave widths but different frequency, which lay a foundation for investigating the mechanism underling calcium oscillation-dependent transciptional factor activition.The activity of NF-κB is regulated by calcium oscillation, when in the model of same amplitude and total wave widths but different frequency, there is no significant differences in NF-κB activity; but when in the model of same amplitude and frequency but different total wave widths, the activity of NF-κB has significant differences. So we proved that the nature of calcium oscillation frequency-dependent activition of transcriptional factor is total wave widths.