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First published online 15 July 2008
doi: 10.1242/jcs.031997

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Ca²⁺ oscillation frequency regulates agonist-stimulated gene expression in vascular endothelial cells

Liping Zhu^1,2,*, Yougen Luo^1,2,*, Taoxiang Chen^1,3,*, Fengrong Chen^1,2, Tao Wang¹ and Qinghua Hu^1,2,

¹ Key Laboratory of Pulmonary Diseases of Ministry of Health of China
² Department of Pathophysiology, Tongji Medical College, Huazhong Science and Technology University, Wuhan 430030, China
³ Department of Physiology, Wuhan University School of Medicine, Wuhan 430072, China

View larger version (89K): [in this window] [in a new window]   Fig. 1. Generation of frequency-manipulated [Ca2+]i oscillations during agonist stimulation. [Ca2+]i oscillations are generated by alternately exposing intracellular-Ca2+-store-depleted EC monolayers to Ca2+-free/EGTA or Ca2+-containing (1.5 mM) HBS (see Materials and Methods) in the absence of (left-hand column) and presence of (right-hand column) 1 µM histamine. Average [Ca2+]i was determined from 30-40 ECs with the same amplitude of oscillation of 0.9 µM and spike duration of 30 seconds but the varied oscillation frequencies of 0.1, 0.3, 0.5 or 0.7 oscillations/minute (A-D, respectively).

View larger version (9K): [in this window] [in a new window]   Fig. 2. Agonist stimulation increases the efficiency of VCAM1 gene expression that is regulated by [Ca2+]i oscillation frequency. EC monolayers were exposed to conditions that generated [Ca2+]i oscillations with the same amplitude of 0.9 µM and four different oscillation frequencies of 0.1, 0.3, 0.5 and 0.7 oscillations/minute, in the presence or absence of 1 µM histamine stimulation. Then, total RNA was isolated for subsequent real-time RT-PCR for determination of VCAM1 mRNA expression. Agonist stimulation shifts the frequency–gene-expression curve to the left and decreases the optimal [Ca2+]i oscillation frequency from 0.45 oscillations/minute in the absence of histamine to 0.3 oscillations/minute in the presence of histamine stimulation (non-linear Lorentzian regression analysis, P<0.05, n=3 for each).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Intracellular ROS contributes to [Ca2+]i-oscillation-regulated VCAM1 gene expression during agonist stimulation. (A) Intracellular-Ca2+-store-depleted EC monolayers were exposed to 1 µM histamine stimulation in Ca2+-free/EGTA HBS for 60 minutes. This condition, under which histamine does not trigger any [Ca2+]i signal, still upregulates VCAM1 mRNA expression (*P<0.05 vs intracellular-Ca2+-store-depleted control ECs, n=3 for each). The histamine-stimulated VCAM1 mRNA expression in intracellular-Ca2+-store-depleted ECs is abolished by NADPH-oxidase inhibition through Rac–/– expression and this is reversed by an external application of 10 µM H2O2. H2O2 alone at 10 µM induces VCAM1 mRNA expression in intracellular-Ca2+-store-depleted ECs. (B) Rac–/–-transfected or control-vector-transfected ECs were pre-treated to deplete the intracellular Ca2+ store and were then exposed to the conditions generating 0.1, 0.3, 0.5 and 0.7 [Ca2+]i oscillations/minute in the concomitant presence of histamine stimulation for 60 minutes. Total RNA was then isolated for real-time RT-PCR analysis. The bell-shaped regulation of VCAM1 mRNA expression by [Ca2+]i oscillation frequency during histamine stimulation was shifted to the right when H2O2 generation was inhibited in Rac–/–-expressing ECs versus vector-control ECs (n=3 for each).

View larger version (8K): [in this window] [in a new window]   Fig. 4. H2O2 cooperates with [Ca2+]i oscillation frequency to optimize agonist-stimulated gene expression. Intracellular-Ca2+-store-depleted ECs were exposed to the conditions generating 0.1, 0.3, 0.5 and 0.7 [Ca2+]i oscillations/minute in the presence of 10 µM H2O2, or in the concomitant presence of 10 µM H2O2 and 1 µM histamine stimulation, in Rac–/–-expressing ECs for 60 minutes. Total RNA was isolated for real-time RT-PCR analysis. An external application of 10 µM H2O2 reverses the altered bell-shaped graph of histamine-stimulated VCAM1 expression versus [Ca2+]i oscillation frequency that is induced by Rac–/– expression (see Fig. 3) to that of vector controls. In the absence of histamine stimulation, the external application of 10 µM H2O2 cooperates with [Ca2+]i oscillation frequency to optimize VCAM1 gene expression.

View larger version (12K): [in this window] [in a new window]   Fig. 5. NF-B inhibitor blocks histamine-upregulated VCAM1 gene expression. (A) Intracellular-Ca2+-store-depleted EC monolayers were exposed to 1 µM histamine stimulation in the presence of an NF-B-specific inhibitor, SN50 (1 µM), or its inactive control, SN50M (1 µM), in Ca2+-free/EGTA HBS for 60 minutes. Total RNA was then isolated for real-time RT-PCR analysis. The histamine-stimulated VCAM1 mRNA expression in intracellular-Ca2+-store-depleted ECs is almost completely blocked by SN50, but not by SN50M (* and , P<0.05 vs intracellular-Ca2+-store-depleted control ECs and SN50-treated ECs, respectively, n=3 for each). Note that related data from Fig. 3A is included here for comparison. (B) Intracellular-Ca2+-store-depleted EC monolayers were exposed to the conditions generating 0.3 [Ca2+]i oscillations/minute in the presence of 1 µM histamine and 1 µM SN50 or SN50M for 60 minutes. Total RNA was then isolated for real-time RT-PCR analysis. The histamine-upregulated VCAM1 mRNA expression under this condition is significantly inhibited by SN50, but not by SN50M (* and , P<0.05 vs intracellular-Ca2+-store-depleted control ECs and SN50-treated ECs, respectively, n=3 for each). Note that related data from Fig. 2 is included here for comparison.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Agonist stimulation increases the efficiency of [Ca2+]i-oscillation-frequency-regulated NF-B transcriptional activation. (A) EC monolayers were exposed to conditions that generated [Ca2+]i oscillations with the same amplitude of 0.9 µM and four different oscillation frequencies of 0.1, 0.3, 0.5 or 0.7 oscillations/minute, in the presence or absence of 1 µM histamine stimulation. Then, cell lysates were prepared for subsequent ELISA-based assay for determination of endogenous NF-B transcriptional activity. Histamine stimulation shifts the frequency–NF-B-transcriptional-activity curve to the left and decreases the optimal [Ca2+]i oscillation frequency from 0.45 oscillations/minute in the absence of histamine to 0.3 oscillations/minute in the presence of histamine stimulation (n=3 for each). (B) Rac–/–-transfected or control-vector-transfected ECs were pre-treated to deplete the intracellular Ca2+ store and were then exposed to the conditions generating 0.1, 0.3, 0.5 or 0.7 oscillations/minute in the concomitant presence of histamine stimulation for 60 minutes. Cell lysates were then isolated for subsequent ELISA-based assay for determination of endogenous NF-B transcriptional activity. The bell-shaped regulation curve of NF-B transcriptional activity versus [Ca2+]i oscillation frequency during histamine stimulation was shifted to the right when H2O2 generation was inhibited in Rac–/–-expressing ECs versus vector-control ECs (n=3 for each). (C) Intracellular-Ca2+-store-depleted ECs were exposed to the conditions generating 0.1, 0.3, 0.5 and 0.7 oscillations/minute in the presence of 10 µM H2O2, or in the concomitant presence of 10 µM H2O2 and 1 µM histamine stimulation, in Rac–/–-expressing ECs for 60 minutes. Then, cell lysates were isolated for subsequent ELISA-based assay for determination of endogenous NF-B transcriptional activity. An external application of 10 µM H2O2 reverses the altered bell-shaped graph of histamine-stimulated NF-B transcriptional activity versus [Ca2+]i oscillation frequency that is induced by Rac–/– expression (see B) to that of vector controls. In the absence of histamine stimulation, the external application of 10 µM H2O2 cooperates with [Ca2+]i oscillation frequency to optimize NF-B transcriptional activation.

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