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

A physiological membrane-receptor agonist typically stimulates oscillations, of varying frequencies, in cytosolic Ca2+ concentration ([Ca2+]i). Whether and how [Ca2+]i oscillation frequency regulates agonist-stimulated downstream events, such as gene expression, in non-excitable cells remain unknown. By precisely manipulating [Ca2+]i oscillation frequency in histamine-stimulated vascular endothelial cells (ECs), we demonstrate that the gene expression of vascular cell adhesion molecule 1 (VCAM1) critically depends on [Ca2+]i oscillation frequency in the presence, as well as the absence, of histamine stimulation. However, histamine stimulation enhanced the efficiency of [Ca2+]i-oscillation-frequency-regulated VCAM1 gene expression, versus [Ca2+]i oscillations alone in the absence of histamine stimulation. Furthermore, a [Ca2+]i oscillation frequency previously observed to be the mean frequency in histamine-stimulated ECs was found to optimize VCAM1 mRNA expression. All the above effects were abolished or attenuated by blocking histamine-stimulated generation of intracellular reactive oxygen species (ROS), another intracellular signaling pathway, and were restored by supplementary application of a low level of H2O2. Endogenous NF-κB activity is similarly regulated by [Ca2+]i oscillation frequency, as well as its co-operation with ROS during histamine stimulation. This study shows that [Ca2+]i oscillation frequency cooperates with ROS to efficiently regulate agonist-stimulated gene expression, and provides a novel and general strategy for studying [Ca2+]i signal kinetics in agonist-stimulated downstream events.

Introduction membrane-receptor occupation are (patho-)physiologically relevant; however, it is hard to fully manipulate the kinetics, such as frequency, of these oscillations. 'Artificial' [Ca 2+ ] i oscillations isolate the Ca 2+ signal from very complicated cell-signaling networks and enable its kinetics to be precisely manipulated. However 'artificial' [Ca 2+ ] i oscillation itself appears to not be physiologically relevant in most circumstances. In the current study, we attempt to directly combine membrane-receptor occupation and 'artificial' [Ca 2+ ] i oscillations to generate a reliable strategy. This novel experimental design fully exploits the advantages of both [Ca 2+ ] i oscillations stimulated by membrane-receptor occupation and those generated 'artificially'.
A Ca 2+ agonist at low, physiologically relevant concentrations usually stimulates [Ca 2+ ] i oscillations through its membranereceptor occupation. Histamine at a concentration of 1 μM stimulates [Ca 2+ ] i oscillations in many types of cells (Ambler et al., 1988;Hu et al., 1999;Jacob et al., 1988;Matsu-ura et al., 2006), including ECs employed in the present studies, with an ~0.9 μM amplitude and heterogeneous frequencies between 0.1 and 0.5 oscillations/minute with a mean level of 0.3 oscillations/minute (Hu et al., 1999). As mentioned above, whether an alteration of [Ca 2+ ] i oscillation frequency regulates agonist-stimulated downstream events such as gene expression is unknown. The gene expression of vascular cell adhesion molecule 1 (VCAM1) in ECs was previously reported to be dependent on [Ca 2+ ] i signaling (Nakada et al., 1998;Quinlan et al., 1999). Using the 'calcium clamp' method, we generated [Ca 2+ ] i oscillations with a series of frequenciesincluding 0.1, 0.3, 0.5 and 0.7 oscillations/minute for 60 minutes -in ECs exposed to 1 μM histamine. The subsequent real-time reverse transcriptase (RT)-PCR analysis revealed a frequencydependent regulation of VCAM1 mRNA expression.
We also explored the potential mechanism underlying the difference in [Ca 2+ ] i -oscillation-frequency-regulated VCAM1 gene expression in the presence versus absence of histamine stimulation. We found that the cooperation between [Ca 2+ ] i oscillations and intracellular reactive oxygen species (ROS) enhanced the efficiency of [Ca 2+ ] i -oscillation-frequency-regulated VCAM1 mRNA expression during histamine stimulation versus [Ca 2+ ] i oscillations alone. Additionally, the mechanistic importance of the NF-κB transcriptional pathway in the above process is also revealed in this study.

Results
To generate frequency-manipulated [Ca 2+ ] i oscillations in physiologically relevant circumstances, we applied 'artificial' [Ca 2+ ] i oscillations (Dolmetsch et al., 1998) to ECs during histamine stimulation. The 'calcium clamp' method was employed here to generate [Ca 2+ ] i oscillations with the same amplitude but different frequencies. Intracellular-Ca 2+ -store-depleted ECs in the presence of histamine in the perfusion medium were alternatively exposed to Ca 2+ -free and Ca 2+ -containing buffer to enable us to manipulate [Ca 2+ ] i oscillations in agonist-stimulated conditions to give the same amplitude of ~0.9 μM Ca 2+ and the varied oscillation frequencies of 0.1, 0.3, 0.5 or 0.7 oscillations/minute (shown in Fig. 1A-D, respectively). It is noted that the spike duration remained consistent among different [Ca 2+ ] i oscillation frequencies (spike duration = 28.99±2.30, 29.08±0.92, 29.11±0.56 and 29.01±0.35 seconds for 0.1, 0.3, 0.5 or 0.7 oscillations/minute, respectively; P = not significant, n=30-40 for each).
To determine whether [Ca 2+ ] i oscillation frequency regulates agonist-stimulated gene expression, EC monolayers were subjected to conditions (as determined in Fig. 1) that generated [Ca 2+ ] 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. Although the [Ca 2+ ] i oscillations, either with or without concomitant histamine stimulation, upregulated VCAM1 gene expression in a frequencyoptimized manner (Fig. 2) ] i oscillation frequency within or beyond the physiological range to 0.5 or 0.7 oscillations/minute decreased VCAM1 mRNA expression (4.49±0.38-and 3.52±0.45-fold for 0.5 and 0.7 oscillations/minute, respectively, P<0.05 vs VCAM1 mRNA level at frequency of 0.3 oscillations/minute, n=3 for each). As compared with [Ca 2+ ] i oscillations alone (in the absence of histamine stimulation), the bell-shaped curve of [Ca 2+ ] i oscillation frequency against VCAM1 mRNA level shifts to the left in the presence of histamine and the optimal frequency for VCAM1 gene expression shifts to 0.3 oscillations/minute in the presence of histamine stimulation vs 0.45 oscillations/minute in the absence of histamine stimulation. In other words, histamine stimulation increases the efficiency of [Ca 2+ ] i -oscillation-frequency-regulated VCAM1 gene expression. It is also noted that histamine-stimulated VCAM1 mRNA expression is optimized at a [Ca 2+ ] i oscillation frequency of 0.3 oscillations/minute, which is the mean [Ca 2+ ] i oscillation frequency in this type of cells upon 1 μM histamine stimulation (Hu et al., 1999;Hu et al., 2002). The distinct [Ca 2+ ] i -oscillationfrequency dependence of gene expression in the presence and absence of agonist stimulation suggests that, in addition to [Ca 2+ ] i oscillations alone, other intracellular signaling pathway(s) are also involved in agonist-stimulated gene expression.
To test this hypothesis, we exposed intracellular-Ca 2+ -storedepleted EC monolayers to 1 μM histamine stimulation in Ca 2+free/EGTA HEPES-buffered saline (HBS) for 60 minutes. It was found that histamine does not trigger any [Ca 2+ ] i signal in Ca 2+free/EGTA HBS in intracellular-Ca 2+ -depleted EC monolayers (not shown). However, it still upregulated VCAM1 mRNA expression (3.53±0.46-fold for histamine, P<0.05 vs control, n=3 for each, Fig.  3A). This result confirms that, in addition to [Ca 2+ ] i signal alone, another intracellular signaling pathway(s) is actually involved in histamine-stimulated VCAM1 gene expression. We therefore designed the following experiments to elucidate whether intracellular ROS cooperate with the [Ca 2+ ] i signal to regulate the agonist-stimulated gene expression.
Previous studies, including our own, prove that intracellular ROS are also generated as cellular signaling molecules during membrane-receptor occupation, e.g. hydrogen peroxide (H 2 O 2 ) in histaminestimulated ECs (Hu et al., 2002). Additionally, the gene expression of VCAM1 has been shown to be sensitive to intracellular ROS (Shimozawa et al., 2004), especially NADPH-oxidase-derived H 2 O 2 (Wautier et al., 2001). Hence, we explored whether intracellular ROS are involved in this process. We transfected ECs with a dominant-negative Rac1 (Rac -/-) construct to inhibit  Materials and Methods) in the absence of (left-hand column) and presence of (right-hand column) 1 μM histamine. Average [Ca 2+ ] 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).

Fig. 2.
Agonist stimulation increases the efficiency of VCAM1 gene expression that is regulated by [Ca 2+ ] i oscillation frequency. EC monolayers were exposed to conditions that generated [Ca 2+ ] 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 [Ca 2+ ] 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).
histamine-stimulated H 2 O 2 generation from NADPH oxidase, as was previously established (Hu et al., 2002), and then exposed the cells to [Ca 2+ ] i oscillations in the presence of histamine stimulation. It was found that Rac -/abolishes histamine-induced VCAM1 mRNA expression in intracellular-Ca 2+ -store-depleted ECs in Ca 2+free/EGTA HBS (1.00±0.10-fold, P>0.05 vs vector control, n=3 for each, Fig. 3A). Supplying 10 μM H 2 O 2 , a concentration that was shown in our previous study to compensate NADPH-oxidase inhibition without interfering with the [Ca 2+ ] i level (Hu et al., 2002), restores histamine-stimulated VCAM1 gene expression in Rac -/-transfected ECs (3.82±0.61-fold, P<0.05 vs control, n=3 for each, Fig. 3A). Furthermore, 10 μM H 2 O 2 alone similarly induced VCAM1 mRNA expression in intracellular-Ca 2+ -store-depleted ECs in Ca 2+ -free/EGTA HBS (4.23±0.25-fold, P<0.05 vs control, n=3 for each, Fig. 3A). H 2 O 2 , not a superoxide generation system, was purposely used in the present study. Our previous study showed that the increased sensitivity of intracellular Ca 2+ stores to Ins(1,4,5)P 3 stimulated by NADPH-oxidase activity was blocked by catalase but was unaffected by superoxide dismutase in human aortic endothelial cells (HAECs) (Hu et al., 2000). In our previous study, upon 1 μM histamine stimulation, an extracellular exposure of H 2 O 2 at 10 μM was also established to revert the diminished [Ca 2+ ] i oscillations in HAECs in which endogenous H 2 O 2 generation was inhibited, without interfering with the [Ca 2+ ] i level (Hu et al., 2002), indicating a physiological relevance in HAECs stimulated with 1 μM histamine.
From the above, we show that intracellular ROS are involved in histamine-stimulated VCAM1 gene expression. Our next question in the present study was therefore: how does H 2 O 2 cooperate with [Ca 2+ ] i oscillation frequency to regulate agonist-stimulated gene expression? It was found that the bell-shaped regulation of VCAM1 mRNA expression by [Ca 2+ ] i oscillation frequency during histamine stimulation was shifted to the right when H 2 O 2 generation was inhibited in Rac -/--expressing ECs, as compared with vectorcontrol ECs (Fig. 3B) To reveal any signaling pathway through which the above signals cooperate in the regulation of histamine-simulated VCAM1 gene expression, we explored the potential importance of NF-κB transcription. We co-exposed intracellular-Ca 2+ -store-depleted EC monolayers to the membrane-permeable NF-κB-specific inhibitor Journal of Cell Science 121 (15) Fig. 3. Intracellular ROS contributes to [Ca 2+ ] ioscillation-regulated VCAM1 gene expression during agonist stimulation. (A) Intracellular-Ca 2+ -storedepleted EC monolayers were exposed to 1 μM histamine stimulation in Ca 2+ -free/EGTA HBS for 60 minutes. This condition, under which histamine does not trigger any [Ca 2+ ] i signal, still upregulates VCAM1 mRNA expression (*P<0.05 vs intracellular-Ca 2+ -store-depleted control ECs, n=3 for each). The histamine-stimulated VCAM1 mRNA expression in intracellular-Ca 2+ -store-depleted ECs is abolished by NADPH-oxidase inhibition through Rac -/expression and this is reversed by an external application of 10 μM H 2 O 2 . H 2 O 2 alone at 10 μM induces VCAM1 mRNA expression in intracellular-Ca 2+ -store-depleted ECs. (B) Rac -/--transfected or control-vector-transfected ECs were pre-treated to deplete the intracellular Ca 2+ store and were then exposed to the conditions generating 0.1, 0.3, 0.5 and 0.7 [Ca 2+ ] 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 [Ca 2+ ] i oscillation frequency during histamine stimulation was shifted to the right when H 2 O 2 generation was inhibited in Rac -/--expressing ECs versus vector-control ECs (n=3 for each).  (Fig. 5B). These results indicate that the NF-κB transcriptional pathway mainly mediates histamine-stimulated VCAM1 gene expression in both Ca 2+ -dependent and Ca 2+ -independent manners. We then pursued whether NF-κB transcriptional activity is regulated by signal cooperation between [Ca 2+ ] i oscillations and intracellular ROS. Using the same experimental strategy employed above for VCAM1 gene expression, and a sensitive ELISA-based assay that we recently improved (Jin et al., 2005), to analyze endogenous NF-κB activity, we found that [Ca 2+ ] i oscillation frequency also dually regulates histamine-induced endogenous NF-κB transcriptional activation (Fig. 6A) (Hajjar and Bonventre, 1991;Jacob et al., 1988), hypoxia (Meng et al., 2007), cell aging (Igarashi et al., 1997;Takahashi et al., 2003), extracellular Ca 2+ concentration (Kawanishi et al., 1989;Rooney et al., 1989) and temperature (Hajjar and Bonventre, 1991). Pro-inflammatory-stimuli-enhanced expression of adhesion molecules on the surface of endothelium and the subsequent leukocyte adhesion to endothelium are pathogenic processes in the development of many cardiovascular diseases.
[Ca 2+ ] i oscillation frequency has been proven to be an important parameter in the regulation of transcriptional activation and gene expression in previous studies using 'artificial' [Ca 2+ ] i -oscillation models (i.e. in the absence of agonist stimulation) (Dolmetsch et al., 1998;Li et al., 1998;Tomida et al., 2003). These studies showed that [Ca 2+ ] i oscillation frequency increases the efficiency and specificity of nuclear transcription, or in other words, optimizes transcriptional activation. In an early attempt of ours, we also observed a parallel decline in NF-κB transcriptional activity while [Ca 2+ ] i oscillation frequency was decreased by Ins(1,4,5)P 3 -receptor inhibition in agonist-stimulated ECs (Hu et al., 1999).
In order to clearly address the complex role of [Ca 2+ ] i oscillation frequency in determining agonist-stimulated downstream biological effects, one major obstacle must be overcome: one must devise a method to precisely control the [Ca 2+ ] i oscillation frequency during agonist stimulation rather than by an 'artificial' oscillation model alone. A feasible strategy we considered was the direct combination of membrane-receptor occupation and the 'artificial' [Ca 2+ ] ioscillation model. In this new experimental strategy, the advantages of both membrane-receptor-occupation-mediated [Ca 2+ ] i oscillations and 'artificially' generated [Ca 2+ ] i oscillations are fully exploited.
After we established 'artificial' [Ca 2+ ] i oscillations and observed frequency-dependent dual regulation of VCAM1 gene expression, we exposed ECs concomitantly to membrane-receptor stimulation and 'artificial' [Ca 2+ ] i oscillations. In this way, we found that [Ca 2+ ] i oscillation frequency regulates VCAM1 gene expression A B Fig. 5. NF-κB inhibitor blocks histamine-upregulated VCAM1 gene expression. (A) Intracellular-Ca 2+ -store-depleted EC monolayers were exposed to 1 μM histamine stimulation in the presence of an NF-κB-specific inhibitor, SN50 (1 μM), or its inactive control, SN50M (1 μM), in Ca 2+ -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-Ca 2+ -store-depleted ECs is almost completely blocked by SN50, but not by SN50M (* and Δ, P<0.05 vs intracellular-Ca 2+ -storedepleted 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-Ca 2+ -store-depleted EC monolayers were exposed to the conditions generating 0.3 [Ca 2+ ] 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 Δ, P<0.05 vs intracellular-Ca 2+store-depleted control ECs and SN50-treated ECs, respectively, n=3 for each). Note that related data from Fig. 2 (Li et al., 1998). It is worthy to point out that 0.3 oscillations/minute is exactly the mean value of varied [Ca 2+ ] i oscillation frequencies in histamine-stimulated ECs (Hu et al., 1999). This might imply that a cell is able to efficiently and cooperatively use intracellular signaling cascades to regulate gene expression. The result also suggests that another signal, in addition to [Ca 2+ ] i oscillations, contributes to histamine-stimulated VCAM1 gene expression, and this is confirmed by histamine-upregulated VCAM1 gene expression in the absence of Ca 2+ signal, as shown in Fig. 3A. It is well-established in many types of cells, including histamine-stimulated ECs in the present study, that agonist-stimulated intracellular ROS serve as a signaling molecule (D'Autreaux and Toledano, 2007;Finkel, 2006;Hu et al., 2002;Saito et al., 2007;Schmidt et al., 1995;Sundaresan et al., 1995). Our experiments, as also shown in Fig. 3A, using Rac -/to inhibit NADPH oxidase and subsequently block ROS generation to abolish histaminestimulated VCAM1 upregulation, confirm that ROS is involved in histamine-stimulated VCAM1 expression. To further reveal how this ROS cooperates with [Ca 2+ ] i oscillations in the induction of VCAM1 gene expression, we generated 'artificial' [Ca 2+ ] i oscillations with different frequencies in the concomitant presence of histamine stimulation in Rac -/transfected ECs. We found that ROS inhibition abolishes histamine-stimulation-enhanced efficiency in the induction of VCAM1 gene expression as compared with vector control (Fig. 3B) and this can be completely reversed by an external application of a low level of H 2 O 2 (Fig. 4). Furthermore, 'artificial' [Ca 2+ ] i oscillations in the presence of H 2 O 2 stimulate VCAM1 gene expression in the same way as in histamine-stimulated ECs (Fig. 4).
To further delineate the signaling pathway through which H 2 O 2 and Ca 2+ oscillations cooperate in the regulation of agoniststimulated VCAM1 gene expression, we explored the potential role of NF-κB transcriptional activation in this process. VCAM1 gene expression has been well-documented to depend on NF-κB transcriptional activity in vascular ECs and we have previously shown that histamine activates NF-κB transcription in human aortic ECs (Hu et al., 1999), the type of cell that we used in the present study. Additionally, NF-κB is a [Ca 2+ ] i -oscillation-frequencysensitive transcription factor (Dolmetsch et al., 1998;Hu et al., 1999). Furthermore, NF-κB has been well-known to be sensitive to redox status in many types of cells, including vascular ECs (Bowie and O'Neill, 2000;Chakrabarti et al., 2007;Foncea et al., 2000;Henderson and Tyagi, 2006;Sulciner et al., 1996).

Journal of Cell Science 121 (15)
A B C Fig. 6. Agonist stimulation increases the efficiency of [Ca 2+ ] i -oscillationfrequency-regulated NF-κB transcriptional activation. (A) EC monolayers were exposed to conditions that generated [Ca 2+ ] 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-κB transcriptional activity. Histamine stimulation shifts the frequency-NF-κB-transcriptional-activity curve to the left and decreases the optimal [Ca 2+ ] 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 Ca 2+ 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-κB transcriptional activity. The bell-shaped regulation curve of NF-κB transcriptional activity versus [Ca 2+ ] i oscillation frequency during histamine stimulation was shifted to the right when H 2 O 2 generation was inhibited in Rac -/--expressing ECs versus vectorcontrol ECs (n=3 for each). (C) Intracellular-Ca 2+ -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 H 2 O 2 , or in the concomitant presence of 10 μM H 2 O 2 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-κB transcriptional activity. An external application of 10 μM H 2 O 2 reverses the altered bell-shaped graph of histamine-stimulated NF-κB transcriptional activity versus [Ca 2+ ] 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 H 2 O 2 cooperates with [Ca 2+ ] i oscillation frequency to optimize NF-κB transcriptional activation.
We found in our experiments that the NF-κB-specific inhibitor SN50, but not its inactive control, SN50M, completely blocks or largely inhibits histamine-upregulated VCAM1 gene expression under different experimental conditions (Fig. 5B), suggesting that NF-κB mainly mediates the VCAM1 gene-expression process. This is highly consistent with previous studies showing the dependence of VCAM1 gene expression on NF-κB transcriptional activity in vascular ECs (Wolle et al., 1995;Yoshimura et al., 2001). It is of particular interest to note that VCAM1 mRNA expression in human aortic ECs has previously been shown to depend on the oxidantsensitive activation of NF-κB (Wolle et al., 1995).
We next wanted to investigate whether NF-κB transcriptional activity is regulated by the cooperation between [Ca 2+ ] i oscillations and intracellular ROS, as it is for VCAM1 gene expression (Figs  2-4). Our experiments showed that [Ca 2+ ] i oscillation frequency also dually regulates histamine-induced endogenous NF-κB transcriptional activation (Fig. 6A). Not only is this consistent in general with previous studies showing dependence of this transcriptional factor on [Ca 2+ ] i oscillation frequency (Dolmetsch et al., 1998;Hu et al., 1999), but it also further verifies that the regulation of NF-κB transcriptional activity by [Ca 2+ ] i oscillation frequency is physiologically relevant, as we previously established using a membrane-permeable inhibitor of the Ins(1,4,5)P 3 receptor (Hu et al., 1999). Finally, our experiments using Rac -/or applying 10 μM extracellular H 2 O 2 to manipulate or mimic endogenous H 2 O 2 generation (Fig. 6B,C) revealed that histamine-induced endogenous NF-κB transcriptional activation is significantly enhanced by the frequency-dependent cooperation between [Ca 2+ ] i oscillations and ROS. It is worthy to note that the dynamical importance of [Ca 2+ ] i oscillation frequency and its cooperation with intracellular ROS in the regulation of NF-κB transcriptional activity highly coincides with that in the regulation of VCAM1 gene expression during histamine stimulation (Figs 2-4 versus Fig. 6). All these results indicate a mechanistically pivotal role of the NF-κB transcriptional pathway in the signaling cooperation between [Ca 2+ ] i oscillation frequency and ROS in the induction of histamine-stimulated VCAM1 gene expression in ECs.
From the above, our results strongly suggest that intracellular ROS cooperates with [Ca 2+ ] i oscillation frequency to efficiently regulate gene expression during agonist stimulation. Additionally, the novel experimental strategy that combines a (patho)physiologically relevant agonist stimulation and kinetically manipulated [Ca 2+ ] i oscillations is expected to be generally applicable.

Cell culture and [Ca 2+ ] i measurements
ECs cultured from human aorta were purchased from Lonza (Walkersville, MD 21793). The maintenance of ECs in culture and [Ca 2+ ] i measurements were performed as previously described in detail (Hu et al., 1999;Hu et al., 2002). Briefly, ECs were grown to passage 5-9 at complete confluence on gelatin-coated 25-mm-diameter circular glass coverslips (VWR Scientific, West Chester, PA 19380). EC [Ca 2+ ] i were measured after loading with 10 μM of the acetoxymethyl ester form of Fura-2 (Invitrogen-Molecular Probes, Carlsbad, CA 92008) for 30 minutes at room temperature. The coverslips were washed and the cells were maintained for at least 30 minutes before experimentation in indicator-free HBS of the following composition (in mM): NaCl 137, KCl 4.9, CaCl 2 1.5, MgSO 4 1.2, NaH 2 PO 4 1.2, D-glucose 15 and HEPES 20 (pH adjusted to 7.40 at room temperature with NaOH). The fluorescence of Fura-2 was recorded from an EC monolayer on coverslips in a perfusion chamber mounted on the stage of a modified Olympus inverted epifluorescence microscope after excitation at 340±10 and 380±10 nm using a xenon short-arc lamp (Ushio), corresponding to the Ca 2+ -bound and Ca 2+ -free forms of the indicator, respectively. Bandpass interference filters (Omega Optical, Brattleboro, VT 05301) selected wavelength bands of emitted fluorescence at 510±10 nm. Emitted Fura-2 fluorescence was collected and measured using a spectrofluorometer (PTI, Deltascan). Autofluorescence from unloaded ECs was generally <5% of Fura-2-loaded ECs and was subtracted automatically from Fura-2-fluorescence recordings.
To determine [Ca 2+ ] i from Fura-2-fluorescence ratios, the intracellular minimum and maximum ratios (R min and R max , respectively) were determined as previously described. To determine R min , Fura-2-loaded ECs on the glass coverslips were perfused with a solution containing (in mM): NaCl 137, KCl 5.0, MgSO 4 1.2, NaH 2 PO 4 1.2, D-glucose 16, HEPES 10 and EGTA 2, pH 7.40. ECs were then exposed to a solution of similar composition except with 10 mM EGTA and 0.05% Triton X-100. An intracellular R max value was determined by first perfusing ECs with a solution containing 132 mM KCl, 10 mM K-HEPES, 1 mM MgSO 4 , 2 μM rotenone (Sigma), 2 μM carbonyl cyanide p-trifluoromethoxyphenylhydrozone (Sigma) and 10 ng/ml valinomycin (Calbiochem, La Jolla, CA). ECs were then exposed to a similar solution containing 2 μM ionomycin (Sigma), 69.2 mM CaCl 2 and 100 mM HEPES (free [Ca 2+ ] of 5900 nM). The values of intracellular R min and R max were used to calculate [Ca 2+ ] i according to the following formula: where K d is the dissociation constant of Fura-2, and S f2 and S b2 are the fluorescence intensities at ~510 nm of the Ca 2+ -free and Ca 2+ -saturated indicator, respectively. All [Ca 2+ ] i measurements were performed at room temperature.
[Ca 2+ ] i clamp Fura-2-loaded EC monolayers on coverslips in the perfusion chamber were placed on the stage of a modified Olympus inverted microscope. [Ca 2+ ] i was clamped using a computer-controlled solenoid valve (General Valve) perfusion system as previously described (Dolmetsch et al., 1998). At the start of each experiment, ECs were treated with 3 nM thapsigargin (Sigma, St Louis, MO) in Ca 2+ -free/EGTA (Calbiochem, San Diego, CA) HBS for 10 minutes to deplete intracellular Ca 2+ stores and irreversibly activate store-operated Ca 2+ -entry channels. The computer-controlled solenoid valve was used to switch rapidly and repetitively between the Ca 2+ -containing and Ca 2+free HBS flowing into the chamber to clamp [Ca 2+ ] i spike amplitude at ~1 μM and frequencies at 0.1, 0.3, 0.5 and 0.7 oscillations/minute.

ECs transiently expressing the dominant-negative allele of Rac1
An adenovirus encoding dominant-negative Rac1 cDNA containing a substitution at position 17 (Rac1 N17 ) was used as described previously (Hu et al., 2002).

Assessment of VCAM1 mRNA expression by real-time RT-PCR
EC monolayers were subjected to the above conditions generating [Ca 2+ ] i oscillations with 1 μM spike amplitude and different frequencies in the presence and absence of 1 μM histamine at room temperature, then total RNA was isolated using a phenolfree total-RNA isolation kit, RNAqueous, according to the manufacturer's protocol (Ambion, Austin, TX). For each sample, 100 ng total RNA was used to generate cDNA using Superscript 1st Strand System (Invitrogen, Carlsbad, CA). Briefly, each RNA/primer was gently mixed with 10 μL cDNA Synthesis Mix (Invitrogen) and incubated at 25°C for 10 minutes, followed by a 50 minutes incubation at 50°C. The reactions were terminated by 5 minutes incubation at 85°C and then chilled on ice. Rnase H (1 μl; Invitrogen) was added to each tube and the reactions were incubated at 37°C for 20 minutes to digest any remaining RNA. Real-time PCR was performed on Sequence Detection Systems 7700 (Applied Biosystems, Foster City, CA) using TagMan Universal PCR Master Mix (Roche, Indianapolis, IN) and specific primers for VCAM1 and β-actin from Applied Biosystems. The thermal-cycle conditions were 2 minutes at 50°C, 10 minutes at 95°C and a 40 cycle loop: 15 seconds at 95°C and 1 minute at 60°C. Duplicate measurements were conducted for each sample and the VCAM1 mRNA level (ΔΔCT) was normalized by β-actin and then expressed as a fold of the corresponding control for each condition.
Our pilot microarray experiments in EC monolayers subjected to conditions generating 0.3 [Ca 2+ ] i oscillations/minutes in the continuous presence of 1 μM histamine (Sigma) for a series of time durations (from 10 to 120 minutes) revealed that 60 minutes was an optimal time point at which to monitor gene expression in the current study.

Measurement of endogenous NF-κB transcriptional activity and blockage of NF-κB transcriptional activation
An ELISA-based assay was performed to measure endogenous NF-κB transcriptional activity in treated and untreated EC monolayers using a modified procedure that we recently established with greatly improved sensitivity and specificity (Jin et al., 2005).

Statistical analysis
Data are reported as mean ± s.e.m. Statistical comparisons were made using oneway ANOVA followed by a Holm-Sidak test. A difference was considered significant at P<0.05.