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J Appl Physiol 89: 1657-1662, 2000;
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Vol. 89, Issue 4, 1657-1662, October 2000

HIGHLIGHTED TOPICS
Cellular Responses to Mechanical Stress
Invited Review: Effects of flow on vascular endothelial intracellular calcium signaling of rat aortas ex vivo

Chauying J. Jen, Shuo-Ju Jhiang, and Hsiun-Ing Chen

Department of Physiology, National Cheng-Kung University Medical College, Tainan, Taiwan 701, Republic of China


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To study the effects of flow on in situ endothelial intracellular calcium concentration ([Ca2+]i) signaling, rat aortic rings were loaded with fura 2, mounted on a tissue flow chamber, and divided into control and flow-pretreated groups. The latter was perfused with buffer at a shear stress of 50 dyns/cm2 for 1 h. Endothelial [Ca2+]i responses to ACh or shear stresses were determined by ratio image analysis. Moreover, ACh-induced [Ca2+]i elevation responses were measured in a calcium-free buffer, or in the presence of SKF-96365, to elucidate the role of calcium influx in the flow effects. Our results showed that 1) ACh increased endothelial [Ca2+]i in a dose-dependent manner, and these responses were incremented by flow-pretreatment; 2) the differences in ACh-induced [Ca2+]i elevation between control and flow-pretreated groups were abolished by SKF-96365 or by Ca2+-free buffer; and 3) in the presence of 10-5 M ATP, shear stress induced dose-dependent [Ca2+]i elevation responses that were not altered by flow-pretreatment. In conclusion, flow-pretreatment augments the ACh-induced endothelial calcium influx in rat aortas ex vivo.

flow pretreatment; calcium image; acetylcholine; shear stress; endothelial cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIAL CELLS CAN PRODUCE at least three kinds of endothelium-derived relaxing factors (EDRF) to modulate vascular tone, i.e., nitric oxide (NO), endothelium-derived hyperpolarization factor, and prostacyclin (37). Numerous factors, including shear stress (or flow) and receptor-mediated agonists such as ACh, are capable of stimulating EDRF release and/or causing vasodilation (11, 12, 17, 26). Although shear stress has been known to affect many endothelial parameters, such as K+ channel opening (27) and intracellular pH (39), this study focuses on its effect on the intracellular calcium concentration ([Ca2+]i) elevation. Recent animal studies have reported that flow or ACh increased endothelial [Ca2+]i levels and dilated isolated arterioles in rats or in rabbits (9, 25). Studies on cultured endothelial cells or on an isolated arteriole of the rat have shown that fluid shear stress causes elevation of endothelial [Ca2+]i (8, 31, 33). Previous studies using cultured human umbilical vein endothelial cells also indicate that histamine-stimulated EDRF release requires calcium influx (18, 19). In addition, receptor-regulated endothelial NO synthase (NOS) translocation and activation need endothelial [Ca2+]i elevation as well (29). Therefore, the endothelial [Ca2+]i signaling should play an important role in these endothelial functions.

Previous studies have indicated that exercise enhances agonist-stimulated, endothelium-dependent vasorelaxation responses (2-4, 7). Interestingly, these exercise effects have been observed in aortas and pulmonary arteries but not in carotid arteries (3). Regional increases in blood flow during exercise may thus play an important role. As mentioned previously, calcium signaling is important in mediating endothelium-dependent vasodilating responses. Whether exercise effects are mediated by changes in agonist-evoked endothelial [Ca2+]i responses cannot be easily ascertained using cultured endothelial cells, because these cells fail to respond to muscarinic agonist administration with either an increase in [Ca2+]i or a release of EDRF (20, 28). Besides, cultured endothelial cells express different muscarinic receptor mRNAs (35). Recently, our laboratory developed an in situ calcium imaging method, with single-cell resolution, to examine the endothelial [Ca2+]i signaling in rat aortas (14). Moreover, a previous study from our laboratory showed that exercise increased ACh-induced endothelial [Ca2+]i responses by facilitating calcium influx (5). Therefore, the present study is designed to investigate the effect of flow-pretreatment, which simulates the flow condition in exercise, on endothelial cell [Ca2+]i responses to ACh.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and vessel preparation. This study was conducted in conformity with the Guiding Principles in the Care and Use of Animals. Six- to eight-week-old male Sprague-Dawley rats were purchased from National Cheng-Kung University Animal Center (Tainan, Taiwan). The rats were anesthetized with ether anesthesia and killed by decapitation. The thoracic aorta was then isolated and cut into 5-mm-long vessel rings. After removal, the aorta was placed in an organ chamber containing Krebs-Ringer solution bubbled with 95% O2-5% CO2 (22°C, pH 7.4). This solution had the following composition (in mM): 118.0 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 24 NaHCO3, 0.03 Na2-EDTA, and 11.0 glucose.

Aortic rings were fluorescently labeled by incubating with 10 µM of fura 2 AM and 0.025% pluronic F-127 in Krebs-Ringer solution for 1 h (36). Extracellular fura 2 AM was washed out afterward. After fura 2 loading, vessel rings were divided into control and flow-pretreated groups, then cut open and pinned to the baseplate of a flow chamber (5, 14). We made the dent on the cover plate deeper to accommodate the extra thickness of tissue. Although this gap, strictly speaking, was not fixed, it was estimated to be within 10% of 0.15 mm. After tissue mounting, the true gap thickness was monitored under a microscope, and the flow rate was adjusted accordingly to be 50 dyn/cm2 for each flow-pretreated specimen.

The flow-pretreated vessel segments were perfused with Krebs-Ringer solution at a shear stress of 50 dyn/cm2 for 1 h, whereas the controls were not pretreated with any flow before the experiments detailed below.

Measurement of in situ endothelial [Ca2+]i. The setup for endothelial [Ca2+]i image analysis described in previous studies by our laboratory was used (5, 14). The flow chamber, mounted with either control or flow-pretreated vessel segment, was placed on an inverted microscope with epifluorescence attachments (Diaphot 300, Nikon, Tokyo, Japan). The excitation light from a xenon lamp was filtered with a high-speed rotating filter wheel (Lambda 10-2, Sutter, Novato, CA) to provide wavelengths of 340 and 380 nm. The fluorescence images at 510 nm were recorded by a high-sensitivity SIT camera (model C2400-08, Hamamatsu, Hamamatsu, Japan). Axon image workbench software (Axon Instruments, Foster City, CA) was used to acquire, digitize, and store the experimental results for off-line processing. Depending on the objective magnification, calcium images for up to 400 endothelial cells could be recorded simultaneously. In some experiments, 100 cells were randomly picked to calculate the histogram of [Ca2+]i. The average value of [Ca2+]i in each preparation was also calculated by monitoring a large area, covering about 0.12 mm2 tissue surface or >200 cells.

At the end of each experiment, the calcium concentration was calibrated by applying ionomycin (5 µM) in the presence of 4 mM EGTA, followed by 10 mM CaCl2. All signals were corrected for autofluorescence, determined by exposing the tissue to 5 mM manganese to quench cytosol fura 2 at 360 nm excitation wavelength. Endothelial [Ca2+]i was estimated after subtracting background and autofluorescence using the following equation (13)
[Ca<SUP>2+</SUP>]<SUB>i</SUB><IT>=K</IT><SUB>d</SUB>[(R<IT>−</IT>R<SUB>min</SUB>)<IT>/</IT>(R<SUB>max</SUB><IT>−</IT>R)]B
where Kd is the dissociation constant (~224 nM); R is the ratio of 340 over 380 during measurements; Rmax is the ratio of 340 to 380 in the presence of saturating calcium levels; Rmin is the ratio in calcium-free solution; and B is the ratio of the fluorescence at 380 nm with calcium-free solution to that of saturated CaCl2 solution. All experiments were conducted at room temperature.

[Ca2+]i elevation responses to ACh. After the vascular endothelial cells had been focused properly, fresh Krebs-Ringer buffer was perfused through the chamber at a flow rate of 0.05 ml/min. At the same flow rate, dose responses of ACh-induced [Ca2+]i elevation were determined by subsequent applications of ACh (from 10-8 to 10-5 M). Between each ACh application, the chamber was washed with fresh buffer for ~4 min to recover the basal [Ca2+]i level. The results between control and flow-pretreated groups were compared by off-line image analysis.

Role of calcium influx in ACh-evoked [Ca2+]i response. The ACh (10-6 M)-evoked [Ca2+]i response was evaluated in the presence or absence of 30 µM of SKF-96365, a membrane calcium channel blocker. Calcium-free buffer containing 0.1 mM EGTA was used as the perfusion buffer in some experiments to evaluate the role of calcium influx in ACh-induced [Ca2+]i response.

[Ca2+]i elevation responses to shear stress in the presence of ATP. Previous reports have shown that flow or shear stress can increase [Ca2+]i in cultured endothelial cells if the solution contains ATP (16, 24). Our preliminary results showed that shear stress alone was unable to evoke endothelial [Ca2+]i responses, which is consistent with results using an almost identical preparation (21). The minimal ATP concentration required for evoking significant [Ca2+]i responses to shear stress was ~10-5 M. We then compared the direct shear-induced endothelial [Ca2+]i responses in the presence of 10-5 M ATP between control and flow-pretreated vessel segments. Fresh ATP-containing buffer was perfused through the vessel-mounted chamber at a flow rate of 0.05 ml/min. After the flow had ceased for 4 min to reach the basal endothelial [Ca2+]i level, ATP-containing buffer was perfused through the flow chamber with different shear stresses ranging from 0.1 to 50 dyn/cm2. Each shear stress was maintained for ~1-3 min to obtain peak [Ca2+]i values, and the flow was stopped for ~2-3 min to allow the recovery of basal [Ca2+]i levels before the next application of shear stress.

Reagents. All chemicals for the preparation of Krebs-Ringer solution were purchased from Merck (Darstadt, Germany). Other reagents were obtained from Sigma Chemical (St. Louis, MO), except SKF-96365, which was purchased from Biomol Research Laboratories (Plymouth, PA).

Statistical analysis. Results are expressed as means ± SE with sample sizes indicated by n. Dose responses of ACh-induced or shear stress-evoked [Ca2+]i elevation were analyzed by repeated-measures ANOVA. Differences between control and flow-pretreated groups were compared by using an unpaired Student's t-test, with P < 0.05 considered to be statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelial [Ca2+]i responses to ACh. ACh reversibly induced endothelial [Ca2+]i elevation in the rat aortic endothelium, and this elevation increased with increasing ACh concentrations (Fig. 1). It was noticed that flow-pretreatment of dissected vessel segments enhanced their endothelial [Ca2+]i responses to ACh. The average values of dose responses of ACh-stimulated [Ca2+]i elevation in control and flow-pretreated groups are shown in Fig. 2.


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  		Fig. 1.   Examples of ACh-induced intracellular calcium concentration ([Ca2+]i) elevation response tracings in control (A) and flow-pretreated (B) groups. Signals were obtained from areas covering ~250 endothelial cells in the mainstream region of rat thoracic aortas.



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  		Fig. 2.   Comparison of dose-response relations of ACh-induced [Ca2+]i elevation responses between control (open circle ; n = 10) and flow-pretreated (; n = 10) groups. Results were analyzed by repeated-measures ANOVA (# P < 0.05), followed by unpaired Student's t-test at given doses of ACh (* P < 0.05).

The basal endothelial [Ca2+]i level was quite homogeneous, between 50 and 150 nM in most cells. When the histograms of [Ca2+]i responses in 100 individual endothelial cells from one set of experiments were analyzed, it was clear that individual endothelial cells on the same endothelium had heterogeneous responses to ACh (Fig. 3). However, whereas certain cells responded to ACh application with a several-fold increase in [Ca2+]i level, some cells were relatively unresponsive to ACh. The higher the concentration of ACh, the more widespread the cell number was distributed. This histogram shift was more pronounced in the flow-pretreated group than the control; i.e., more cells had greater ACh-stimulated [Ca2+]i elevation responses than the controls.


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  		Fig. 3.   Histograms of basal or ACh-induced [Ca2+]i levels in 100 endothelial cells from 1 control (A) and 1 flow-pretreated (B) specimen.

Role of calcium influx in ACh-evoked [Ca2+]i responses. SKF-96365, a calcium influx blocker, alone did not influence the basal endothelial [Ca2+]i (without SKF-96365: 125 ± 14 nM control, 128 ± 25 nM flow-pretreated; with SKF-96365: 130 ± 11 nM control; 123 ± 15 nM flow-pretreated, n = 5 for each group). ACh-evoked [Ca2+]i responses in the presence or absence of SKF-96365 were compared between control and flow-pretreated groups. Table 1 demonstrates that flow-pretreatment increased ACh-evoked endothelial [Ca2+]i responses and that this effect disappeared after administration of SKF-96365. If a calcium-free buffer substituted the Krebs-Ringer solution, minimal ACh-induced calcium elevation was observed in both groups (Table 1). Therefore, the enhancement of ACh-evoked endothelial [Ca2+]i responses by flow-pretreatment was mainly due to an increase in calcium influx.

                              
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  		Table 1.   Effects of SKF-96365 or calcium-free solution on ACh-induced [Ca2+]i elevation responses in control and flow-pretreated groups

[Ca2+]i elevation responses to shear stress in the presence of ATP. Figure 4 demonstrates that, in the presence of 10-5 M ATP, different levels of shear stress directly evoked different extents of endothelial cell [Ca2+]i elevation. The average "dose-response" curves of control and flow-pretreated groups are shown in Fig. 5. There was no significant difference between these two groups.


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  		Fig. 4.   Examples of shear stress-induced [Ca2+]i elevation response tracings in the presence of ATP (10-5 M) for control (A) and flow-pretreated (B) groups.



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  		Fig. 5.   Comparison of dose-response relations of shear stress-induced [Ca2+]i elevation responses between control (open circle , n = 5) and flow-pretreated (, n = 6) groups.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous reports of endothelial cell calcium signaling in response to agonists or to flow were mainly based on studies using cultured cells (16, 18, 19, 24, 31, 33). However, cultured endothelial cells may lose their inherent properties and behave differently from the vascular endothelium (20, 28, 35). We therefore developed an in situ calcium imaging method (with single-cell resolution) to study endothelial cell calcium signaling. By analyzing calcium images of rat thoracic aortic vascular endothelium ex vivo, we are the first to report that 1) flow pretreatment enhances ACh-induced [Ca2+]i elevation; 2) the differences between control and flow-pretreated groups in ACh-evoked [Ca2+]i responses were abolished by SKF-96365 or by Ca2+-free buffer; 3) in the presence of ATP, shear stress also induced [Ca2+]i elevation; and 4) this shear effect was not altered by flow-pretreatment.

It is well known that both physical (e.g., blood flow) and chemical factors (e.g., hormones in the bloodstream) change drastically during exercise. In principle, either type of factor could mediate the exercise effects on vascular function. Recently, our laboratory applied this in situ endothelial [Ca2+]i imaging technique to compare the aortas from exercised animals and controls. The results indicate that exercise sensitizes the endothelial [Ca2+]i responses to ACh (5). Because flow-pretreatment also enhances ACh-evoked endothelial [Ca2+]i elevation (Figs. 1-3), it apparently mimics these exercise effects. Furthermore, exercise has been shown to affect the vascular responsiveness in rabbit aortas and pulmonary arteries but not in carotid arteries (3). Therefore, it is very likely that the exercise effects on vasculature are, at least partially, caused by elevated blood flows in vivo.

It has been proposed that agonist-induced NO release or vasorelaxation is mediated by endothelial calcium signaling (9, 18, 19, 25). Our results show that SKF-96365, an inhibitor of receptor-mediated calcium entry (23), diminished ACh-induced [Ca2+]i elevation and abolished the difference between control and flow-pretreated groups. In cultured human endothelial cells, SKF-96365 has been reported to double [Ca2+]i and to increase Ca2+ influx at comparable or higher concentrations (15, 32). However, these adverse effects may not happen in our present study, because this inhibitor alone at a dose of 3 × 10-5 M did not influence the basal endothelial [Ca2+]i. In addition, the calcium-free condition almost completely inhibited ACh-induced [Ca2+]i elevation in both control and flow-pretreated groups. Taken together, our results support that ACh-induced [Ca2+]i elevation is mainly due to calcium influx and that the effect of flow-pretreatment is caused by an increase in calcium influx. Consistently, calcium influx has been reported to play a critical role in the receptor-stimulated release of NO or prostacyclin (18, 19). Therefore, it is plausible to assume that the enhancement of ACh-stimulated NO release and vasorelaxation by acute exercise may be, at least in part, due to an increase of calcium influx caused by flow during exercise.

The underlying mechanisms for the enhancement of ACh-induced endothelial calcium responses by flow-pretreatment are unclear at the present time. However, one can speculate that the modulation of either calcium channel activity or ACh receptors (i.e., M3 receptors) may account for this effect. If the calcium channel activity itself was altered by flow pretreatment, one would expect to see an increase in shear-induced endothelial calcium responses as well. However, because our flow and ATP results (Figs. 4, 5) are against this viewpoint, it is likely that M3 receptors are upregulated instead. The previous study by our laboratory showed that acute exercise enhances ACh-evoked endothelium-dependent vasorelaxation by M3 receptor upregulation in rat aortas (4). Whether flow-pretreatment also upregulates endothelial M3 receptors is still unknown. In 1997, Takada et al. (34) reported that cultured human umbilical vein endothelial cells exposed to fluid shear stress increased gene expression of the G protein-coupled receptors EDG1 and FEG1. In their study, the level of EDG1 mRNA began to increase as early as 1 h after exposure to shear stress. Because the muscarinic receptor is one of the G protein-coupled receptors (38), we favor the possibility of upregulating muscarinic receptors by flow-pretreatment.

In this study, we also confirmed that ACh evoked endothelial [Ca2+]i elevation in a dose-dependent manner, which is consistent with previous animal studies (9, 25). Nonetheless, Falcone et al. (9) and Muller et al. (25) reported that shear stress alone (in the absence of exogenous ATP) increased endothelial [Ca2+]i in rat cremaster arterioles or in rabbit coronary arterioles. In the present study, shear, by itself, did not induce endothelial [Ca2+]i elevation in rat aorta, which is consistent with a previous report that also used the rat aorta (21). It appears that the direct shear effect only occurs in small arterioles, not in large vessels. The concept of a regional difference in endothelial [Ca2+]i signaling is supported by our laboratory's recent report, in which endothelial heterogeneity between branch and nonbranch regions was observed (14). Besides, when cultured endothelial cells from large vessels are used, shear evokes significant endothelial [Ca2+]i elevation only if the perfusion solution contains ATP (16, 24).

Complicated forms of [Ca2+]i signaling, such as single transients and repeated spikes with variable frequencies, have been reported in studies that mainly used cultured cells (1). However, unlike a previous study on cultured single bovine aortic endothelial cells (33), we rarely observed [Ca2+]i oscillations in single vascular endothelial cells in situ, either under shear (present study) or with agonist application (14). Therefore, the physiological significance of endothelial [Ca2+]i oscillation remains to be elucidated. A previous study indicated that administration of atropine or acetylcholinesterase would inhibit flow-induced NO release and vasorelaxation (22). The results of that study imply that besides the flow-mediated mechanotransduction (6), local ACh release from the endothelial cells may also mediate the flow effect. It would be interesting to examine the role of endothelial [Ca2+]i in this aspect.

Despite the fact that flow-pretreatment did not alter direct effects of the shear stress-induced endothelial [Ca2+]i elevation, we cannot rule out the possibility that flow-pretreatment increases NO release by activating NOS in a Ca2+-independent manner or enhancing NOS gene expression. Recent studies have shown that fluid shear stress can activate NOS either by rapid caveolin dissociation and calmodulin association (30), or in a Ca2+-independent, but tyrosine phosphatase inhibitor-sensitive, manner (10). Besides, exposure to flow for a period of time can upregulate endothelial NOS mRNA in cultured endothelial cells (26).

In conclusion, flow-pretreatment, used to simulate the flow condition in exercise, enhances the ACh-induced endothelial [Ca2+]i elevation in situ by causing a greater calcium influx. On the contrary, flow-pretreatment does not change shear stress-induced endothelial [Ca2+]i responses.


    ACKNOWLEDGEMENTS

We thank Tung-Yi Huang and Tzu-Fang Chu for helpful discussions and technical assistance.


    FOOTNOTES

This study was supported by grants from the National Sciences Council of Taiwan and the National Health Research Institute in Taiwan, ROC Grants NSC89-2320-B006-045, NSC89-2320-B006-046, and NHRI-GT-EX89S834L.

Address for reprint requests and other correspondence: H.-I. Chen, Dept. of Physiology, NCKU Medical College, Tainan, Taiwan 701, ROC (E-mail: hichen{at}mail.ncku.edu.tw).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 22 February 2000; accepted in final form 16 May 2000.


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DISCUSSION
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J APPL PHYSIOL 89(4):1657-1662
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