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Heterogeneity of [Ca²⁺]_i signaling in intact rat aortic endothelium

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

¹Correspondence: Department of Physiology, College of Medicine, National Cheng-Kung University Medical College, #1, Ta-Hsiue Rd., Tainan 701, Taiwan. E-mail: jen{at}mail.ncku.edu.tw

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Most existing knowledge about [Ca²⁺]_i signaling in vascular endothelium has been based on studies using endothelial cells cultured in vitro. To examine how endothelial cells behave in situ, we have developed a method to monitor single-cell [Ca²⁺]_i from Fura-2-loaded rat aortic segments. Fluorescence ratio images from large numbers of endothelial cells were acquired by using a flow chamber mounted on a dual-wavelength fluorescence microscope. Our results showed that either acetylcholine or histamine reversibly activated the vascular endothelium by eliciting M₃ or H₁ receptor-mediated [Ca²⁺]_i increases, respectively. The acetylcholine-evoked endothelial [Ca²⁺]_i elevation at the branch site (intercostal orifice) was much more pronounced than that at the non-branch area. However, endothelium at the branch site was relatively insensitive to histamine. Both acetylcholine-sensitive and histamine-sensitive endothelial cells were arranged in belts aligned along flow lines and were intercalated with each other. Data analyzed from 400 endothelial cells located at the non-branch site showed drastically heterogeneous [Ca²⁺]_i responses to a fixed concentration of either acetylcholine or histamine, differing by two orders of magnitude in individual cells. As a conclusion, vascular endothelial cells appear to have their own characteristic [Ca²⁺]_i ‘fingerprint’ to various agonists and they may function coordinately in situ.—Huang, T.-Y., Chu, T.-F., Chen, H.-i., Jen, C. J. Heterogeneity of [Ca²⁺]_i signaling in intact rat aortic endothelium.

Key Words: atherosclerosis • acetylcholine • histamine • blood vessel • endothelial cells

	INTRODUCTION

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VASCULAR ENDOTHELIUM PLAYS an important role in regulating many important physiological functions, such as angiogenesis, hemostasis, vascular tone, and immune defense. Calcium ions are one of the most common signal transduction elements in numerous cell types, including endothelial cells. For example, endothelial cells respond to various stimuli by elevating intracellular calcium ion level ([Ca²⁺]_i), followed by releasing NO, prostacyclin, platelet activating factor, tissue plasminogen activator, etc. (for a review, see ref 1 ). Most existing knowledge about the endothelial signal transduction mechanisms comes from studies on cultured cells, which may behave differently from vascular endothelium. Besides, the culture conditions not only alter some of the cellular characteristics, such as diminishing the expression of surface M₃ muscarinic receptors (2) , but also selectively maintain the fastest growing clone from a cell population. This latter shortcoming strongly hampers our understanding of endothelial heterogeneity.

Although vascular heterogeneity among different vessel types or different organs has been well appreciated, the information regarding local endothelial heterogeneity is largely lacking at the present time. The acquisition and maintenance of specialized properties in endothelial cells is crucial in the functional homeostasis of different organs in our body (3 , 4) . Moreover, embryonic arteries and veins express complementary surface molecules that serve as bi-directional signaling receptors and ligands in angiogenic interactions (5) . Whether such heterogeneity also exists between the mainstream artery and its adjacent branch, or even among individual endothelial cells in the same area, has not been studied before.

Perhaps due to technical difficulties, single-cell [Ca²⁺]_i measurements in endothelium of excised vessels have not been very popular. The involvement of calcium in endothelium-dependent vascular dilation by flow and agonists has been ascertained by fluorescence calcium imaging of whole arterioles (6) . A setup, consisting of optical fibers connecting to a spectrofluorimeter to excite and record fluorescence, has been used to monitor a group of 250–560 endothelial cells as a whole from the luminal face of rat aorta (7) . Due to insufficient spatial resolution, neither study addressed the issue of endothelial heterogeneity. On the other hand, patch-clamp labeling of endothelial cells in isolated rat aorta provides a way to examine both membrane potential and [Ca²⁺]_i in single cells (8) . According to that study, individual endothelial cells show different sensitivity to acetylcholine. However, only a few cells have been selected for characterization, and their relative positions in situ are unknown. Because of these limitations, the distribution and correlation of these heterogeneously responsive cells can not be elucidated from their study. Therefore, we have developed a method to monitor single-cell [Ca²⁺]_i from intact rat aortic endothelium. This method allowed simultaneous visualization of large numbers of endothelial cells with single-cell resolution. Using this approach we studied the changes of [Ca²⁺]_i in individual aortic endothelial cells in situ, which were stimulated with either acetylcholine or histamine. Individual endothelial cells in the aortic mainstream areas as well as those cells located at the intercostal orifice were compared directly. Our findings indicated that local endothelial heterogeneity was very pronounced, i.e., the relative response to these two agonists varied by two orders of magnitude and the intercostal orifice being very sensitive to acetylcholine, but not to histamine.

	MATERIALS AND METHODS

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Vessel preparation and Fura-2 loading
This study was conducted in conformity with the policies and procedures detailed in the Guide for animal Care and Use of Laboratory Animals. Male Wistar rats (6–8 wk old) were anesthetized with ether inhalation, and their thoracic aortas were immediately obtained. After the residual blood had been flushed out with heparin containing Krebs-Ringer solution (10 U/ml heparin), these vessels were then carefully trimmed to remove excess adventitial tissue. Finally, they were cut into rings (5 mm long) and stored 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 at room temperature (7) . Extracellular Fura-2 AM was washed out afterward.

Measurement of in situ endothelial [Ca²⁺]_i
The basic setup for endothelial [Ca²⁺]_i imaging was similar to our previous one that has been used for single-platelet [Ca²⁺]_i measurements (9) , except that the flow chamber (10) was modified to accommodate vessel mounting (Fig. 1 ). After Fura-2 loading, vessel rings were then longitudinally opened and pinned to the base plate of the flow chamber with the proximal end facing flow inlet. There was a 0.15 mm gap between the vessel lumen and the cover glass to allow flow passage. The chamber was mounted on an inverted microscope with epifluorescence attachments (Diaphot 300, Nikon, Tokyo, Japan). The excitation light from a xenon lamp was filtered to provide wavelengths of 340 and 380 nm using a high-speed rotating filter wheel (Lambda 10–2, Sutter, Novato, Calif.). 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, Calif.) was used to acquire, digitize, and store the experimental results for off-line image processing. Depending on the objective lens magnification, calcium images for up to 700 endothelial cells could be recorded simultaneously. The average value of [Ca²⁺]_i in each preparation was calculated by monitoring a large area, covering at least 0.15 mm² tissue surface, or more than 200 cells. Results from randomly picked cells were used to construct the histogram of single-cell [Ca²⁺]_i levels. At the end of each experiment, the calcium concentration was calibrated by applying ionomycin (5 µM) in the presence of 5 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 fluorescence. Endothelial [Ca²⁺]_i was estimated after subtracting background and autofluorescence according to the established formula (11) . All experiments were conducted at room temperature. At the end of some experiments, the specimens were fixed with formaldehyde (3.5%) and stained with silver nitrate (12) to verify the integrity of endothelium.

View larger version (27K): [in this window] [in a new window]   Figure 1. The experimental setup.

[Ca²⁺]_i elevation responses to agonists
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. The flow effect on endothelial [Ca²⁺]_i was minimal under these experimental conditions. At the same flow rate, dose responses of agonist-induced [Ca²⁺]_i elevation were determined by subsequent applications of acetylcholine (from 10^-8 M to 10^-5 M) or histamine (from 10^-7 M to 10^-4 M). Between each application, the chamber was washed with fresh buffer for at least 3.5 min to recover the basal [Ca²⁺]_i level. In certain experiments, the specimens were pretreated with receptor antagonists for 3 min before adding either agonist. The results were compared by off-line image analysis.

Reagents
All reagents for preparing Krebs-Ringer solution were purchased from Merck (Darstadt, Germany). Other reagents were obtained from Sigma (St. Louis, Mo.), except that receptor antagonists (4-diphenylacetoxy-N-methylpiperidine methiodide [4DAMP], cimetidine, and diphenhydramine) were purchased from RBI (Natick, Mass.).

Statistical analysis
Results were expressed as mean ± SE. Sample sizes were indicated by n. Differences between two locations of the same vessel segments were compared by using paired Student’s t test with P < 0.05 considered statistically significant.

	RESULTS

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Figure 2 shows that the rat aortic endothelium, but not the smooth muscle cells underneath, was labeled under our experimental conditions. Fluorescence images from individual endothelial cells are clearly visible under our experimental conditions. When the luminal surface of specimen was gently scraped, a dark area devoid of endothelial cells was observed, indicating little fluorescence signal coming from the underlying smooth muscle layer. The fluorescence ratio value, which could be converted to [Ca²⁺]_i level, from intact areas became reversibly elevated in response to acetylcholine treatment. In comparison, the smooth muscle agonist, phenylephrine, was totally ineffective. Fluorescence signals in the denuded areas remained minimal and unchanged in the presence of either agonist.

View larger version (65K): [in this window] [in a new window]   Figure 2. The rat aortic endothelium and its fluorescence ratio image. A rat aortic segment was labeled with the fluorescence dye Fura-2 AM (10 µM), mounted on a flow chamber, and mechanically injured by gently scraping a line on the endothelium. a) The fluorescence ratio image was taken at excitation wavelengths 340 nm/380 nm and emission wavelength 510 nm. Note: the denuded area is devoid of fluorescence. b) The specimen was subsequently fixed and stained with silver nitrate to show the endothelial cell integrity. Bar equals 100 µm. c) The intact endothelium showed reversible [Ca2+]i elevation in response to acetylcholine (ACh), but not to phenylephrine (Phe).

When a specimen covering the intercostal orifice was mounted on the flow chamber, we could focus either on the non-branch areas or at the branch site (orifice) (Fig. 3 ). We then applied either acetylcholine or histamine to the specimens. Both agonists reversibly induced endothelial [Ca²⁺]_i elevation in a concentration-dependent manner. Although the endothelium at either mainstream area or at the intercostal orifice responded to both agonists, cells at these two locations showed drastically different extent of evoked-[Ca²⁺]_i elevation (Fig. 4 ). Comparing to endothelium at the mainstream areas, endothelium at the branch site showed large acetylcholine-evoked [Ca²⁺]_i response. However, the histamine-evoked [Ca²⁺]_i response at the same location was relatively small.

View larger version (105K): [in this window] [in a new window]   Figure 3. Endothelium fluorescence image from the site covering an intercostal orifice. Images were focused on either the mainstream area (a) or the branch orifice (b). The fluorescence images were taken in the absence of agonists, at excitation wavelength 340 nm and emission wavelength 510 nm. Bar equals 100 µm.

View larger version (26K): [in this window] [in a new window]   Figure 4. Acetylcholine-evoked and histamine-evoked endothelial [Ca2+]i responses from either mainstream region (non-branch) or intercostal orifice (branch). Results were calculated from averaging areas either covering more than 200 cells in the mainstream region, or covering the endothelium-intact region within the branch orifice. The left panels represent results from acetylcholine (ACh) treatments and the right panels for histamine (His) results (n=4). *P < 0.05 (paired Student’s t test).

Pictures in Fig. 5 show calcium images from a typical specimen that was either untreated (basal), or treated with agonists in the presence or absence of various receptor antagonists. The entire endothelium in untreated specimen was relatively quiescent. Calcium images were much brighter in agonist-treated specimens than in the control. Moreover, the intercostal orifice was very bright when acetylcholine, but not histamine, was present. Besides, these [Ca²⁺]_i elevations could be blocked by receptor-specific antagonists. These agonist-induced responses mentioned above could be blocked either by the M₃ receptor antagonist 4-DAMP (for acetylcholine treatment), or by the H₁ receptor antagonist diphenhydramine (for histamine treatment), respectively (Fig. 5) . On the contrary, the H₂ receptor antagonist cimetidine was ineffective. These results indicate that rat aortic endothelium mainly expresses M₃ and H₁ receptors.

View larger version (103K): [in this window] [in a new window]   Figure 5. Blockage of agonist-evoked endothelial [Ca2+]i responses by receptor antagonists. The concentrations of agonists and antagonists used are (in M): 5 x 10-5 histamine (His), 10-5 diphenhydramine (DPH), 10-4 cimetidine (Cim), 5 x 10-7 acetylcholine (ACh), and 3 x 10-7 4-diphenylacetoxy-N-methylpiperidine (4DAMP). All pictures were focused on mainstream areas from the same specimen.

To investigate the cell-to-cell heterogeneity, individual endothelial cells were randomly selected from the non-branch area and analyzed for single-cell [Ca²⁺]_i levels. Their basal [Ca²⁺]_i levels were consistently low (87.0±1.3 nM, n=400). The agonist-induced [Ca²⁺]_i change varied drastically among individual cells. Figure 6 shows a few examples. Although these response curves differed markedly in amplitude, they were all biphasic, i.e., consisting of an initial peak, followed by a plateau that lasted as long as agonists were present. In general, the initial [Ca²⁺]_i peaks were sharper in histamine-evoked responses than in acetylcholine-evoked responses. Although prolonged histamine incubation made cells refractory to subsequent histamine treatments, cells could withstand repeated acetylcholine treatments. Very few cells (almost none in most animals and less than 10% cells in one particular animal) showed repeated [Ca²⁺]_i spikes when the aortic endothelium was stimulated with acetylcholine (data not shown).

View larger version (26K): [in this window] [in a new window]   Figure 6. Agonist-evoked endothelial [Ca2+]i responses in five single endothelial cells from the mainstream areas. The concentrations of agonists applied were 10-6 M acetylcholine (upper panel) and 5 x 10-5 M histamine (lower panel). Same symbols represent same cells.

We then compared [Ca²⁺]_i responses from 400 individual cells treated with either 5 x 10^-7 M acetylcholine or 5 x 10^-5 M histamine. Under these two specific concentrations, the agonists-evoked responses were not saturated and the averaged [Ca²⁺]_i response values were similar in magnitude, 553 and 440 nM for acetylcholine and histamine treatments, respectively. Histograms in Fig. 7 showed the heterogeneity of these agonist-induced heterogeneous [Ca²⁺]_i responses in individual endothelial cells, ranging from trivial values to a few µM. There was no correlation between an endothelial cell’s basal [Ca²⁺]_i level and its evoked response, i.e., the correlation coefficients (R²) were 0.025 and 0.144 for acetylcholine and histamine treatments, respectively. When the acetylcholine-evoked and histamine-evoked [Ca²⁺]_i responses were compared in each of these 400 cells, it became clear that their relative responsiveness to these two agonists were very heterogeneous as well. Although many cells responded similarly to either agonist, some cells selectively responded to either acetylcholine or histamine. Figure 8 shows the distribution pattern of these cells in the same specimen. Acetylcholine-sensitive but histamine-insensitive endothelial cells were not randomly distributed in the mainstream areas. Instead, they seemed to be arranged in intercalating belts aligned along flow lines. Moreover, all cells at the intercostal orifice were relatively acetylcholine-sensitive but histamine-insensitive. That is, their relative responsiveness ratio values were 9.3 ± 0.7.

View larger version (23K): [in this window] [in a new window]   Figure 7. Histogram of control or agonist-evoked endothelial [Ca2+]i responses in 400 randomly selected cells from the mainstream area. The concentrations of agonists were 5 x 10-7 M acetylcholine and 5 x 10-5 M histamine.

View larger version (182K): [in this window] [in a new window]   Figure 8. The distribution of relatively acetylcholine-sensitive and histamine-insensitive endothelial cells in the mainstream area. We took the ratio between acetylcholine-evoked response image and histamine-evoked response image from the same specimen to make this composite picture. A bright cell in this picture indicates that it has a relatively high [Ca2+]i response ratio between acetylcholine (5 x 10-7 M) and histamine (5 x 10-5 M) treatments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Using a newly developed method, we have successfully acquired single-cell [Ca²⁺]_i images from large areas of intact rat aortic endothelium. Since the agonist-evoked responses were reversible and some acetylcholine-responsive cells were histamine-insensitive and vise versa, these facts ruled out the possible technical aberration resulting from uneven staining or incomplete perfusion. Our results showed that both acetylcholine and histamine evoked heterogeneous receptor-mediated [Ca²⁺]_i responses. Moreover, the heterogeneity of [Ca²⁺]_i responses to the same agonist was present not only among neighboring individual cells but also between mainstream areas and the intercostal orifice.

Although the basal [Ca²⁺]_i levels in individual cells of the same endothelium were similar, their acetylcholine-evoked or histamine-evoked [Ca²⁺]_i responses varied by more than 100-fold. Moreover, there were no apparent correlation between an endothelial cell’s basal [Ca²⁺]_i level and the magnitude of its agonist-evoked [Ca²⁺]_i response. These results suggest that endothelial cells in situ are intrinsically different in terms of their responses toward either agonist. This cell-to-cell difference is in agreement with the previous findings from a patch-clamp study using isolated rat aortic endothelium (8) . That is, acetylcholine stimulates heterogeneous changes of both membrane potential and [Ca²⁺]_i in a small number of endothelial cells examined.

The distribution of agonist-responsive cells reported in this study revealed possible influence of blood flow in vivo. Both acetylcholine-sensitive cells and histamine-sensitive cells were grouped in‘belts’oriented along the longitudinal axis of the aorta. Similarly, von Willebrand factor-positive endothelial cells in situ also arrange in groups oriented parallel to the longitudinal axis of the rat aorta (13) . It is well known that arterial endothelial cells in situ are elongated and aligned in the direction of flow, and that in vitro laminar shear stress induces the alignment of cultured endothelial cell (14) . This flow-induced cell shape change and alignment are thought to minimize the shear effect on cells (15) . Consequently, a vascular endothelial cell might arrange its mitotic spindle along the flow direction to minimize the flow shear during its division, and make its progeny cells arranged in longitudinal‘belts’in situ.

The characteristics of endothelial cells near the branch site appear to be related to the local blood flow as well, only more complicated than in the mainstream areas. Previous in vivo studies demonstrated that intimal thickening and atherosclerotic plaques predominantly occur in flow regions of low wall shear stress and recirculation zones (16 , 17) . We showed that the cells located in the intercostal tributaries, not the cells surrounding the orifices, were highly acetylcholine-sensitive but relatively inert to histamine treatment. In comparison, endothelial cells surrounding the intercostal orifices show strong and uniform staining of von Willebrand factor (13) . Although acetylcholine stimulates the release of nitric oxide, which is known to be a vasorelaxant and platelet inhibitor (18) , von Willebrand factor is a procoagulant factor that facilitates platelet adhesion/aggregation (19) . Since mechanical forces in vitro regulate the endothelial signal transduction pathways and the expression of a variety of genes (20 , 21) , it is conceivable that local blood flow disturbances in situ may also cause endothelial adaptation in the gene expression level. This type of differential expression of various genes, along with the direct physical effect of local blood flow pattern, such as altered convective transport of cellular and soluble components, may eventually make certain locations in the circuitry atherosclerosis-prone.

Our results from antagonist experiments showed that both M₃ muscarinic receptors and H₁ receptors were present in the rat aortic endothelium, and they were responsible for acetylcholine- and histamine-evoked [Ca²⁺]_i responses. M₃ muscarinic receptors have been reported to be present in various animal vessels, including rat aorta (22) . Furthermore, this study also showed that the aortic endothelium was rather insensitive to histamine, indicating a high inflammatory threshold in large arteries. This notion is supported by an early study reporting that the density of histamine receptors are high in venules, but low in arterioles, veins, capillaries, and lowest in aorta (23) .

However, in addition to the different types or numbers of receptors expressed by each cell, other mechanisms may be involved in the observed heterogeneous [Ca²⁺]_i responses. For example, the calcium mobilization machinery could be different among individual endothelial cells in situ. To clarify the possible role of calcium influx in agonist-evoked responses, we performed acetylcholine experiments either under calcium-free conditions, or in the presence of calcium channel blocker. Our preliminary results indicated that whereas the endothelial [Ca²⁺]_i responses were reduced to almost minimum at the mainstream area, it was partially reduced to ~40% at the branch site. Moreover, the calcium influx during exogenous calcium replenishment was much more pronounced in the mainstream area than at the branch site (data not shown). Taken together, it appeared that there were multiple causes responsible for the endothelial heterogeneity in situ.

Results in this study are in general agreement with previous observations showing rapid endothelial [Ca²⁺]_i elevation on agonist application. However, there are important differences between endothelium in vessel segments and endothelial cells in culture. This study showed that both acetylcholine and histamine induced synchronous [Ca²⁺]_i responses in single endothelial cells in situ, indicating either individual endothelial cells had similar agonist-evoked [Ca²⁺]_i response times or they were well connected through gap junctions. Moreover, the agonist-evoked [Ca²⁺]_i responses were mostly biphasic, consisting of an initial peak, followed by a sustained plateau. It has been reported that acetylcholine evokes a similar [Ca²⁺]_i response in groups of 250–560 endothelial cells in intact rat aortas (7) . In contrast, cultured endothelial cells behave quite differently, for example, they were not responsive to acetylcholine (24) . This discrepancy may be explained by the fact that cultured endothelial cells lack M₃ muscarinic receptor expression (2) .

In histamine treatments, we rarely observed any repeated [Ca²⁺]_i spikes in individual aortic endothelial cells in situ. They were either inert when treated with low doses (submicromolar) histamine, or responded with a sharp [Ca²⁺]_i peak, followed by a low plateau at concentrations higher than 10 µM. On the contrary, culture conditions may either enhance or suppress cell responses to histamine. In cultured human umbilical endothelial cells, histamine at low doses evokes pronounced [Ca²⁺]_i spikes, whereas high doses (more than 2–3 µM) cause a maintained elevated [Ca²⁺]_i level (25) . On the other hand, histamine at doses up to 100 µM fail to elicit any [Ca²⁺]_i increase in cultured bovine aortic endothelial cells (26) . Thus one should be cautious in appraising the physiological significance of studies solely using cultured cells.

Although acetylcholine in the submicromolar range is known to cause significant vasorelaxation (27) , the corresponding [Ca²⁺]_i response in vascular endothelium was relatively small. There are several reasons to explain this. First, unlike most vessel contraction/relaxation experiments, our current study was carried out in the absence of vasoconstricting agents. It has been reported that endothelial muscarinic and ₂-adrenergic receptors in rabbit cerebral arteries cross talk each other (28) . Indeed, the acetylcholine-evoked [Ca²⁺]_i elevation in our system was larger when norepinephrine was present (data not shown). Second, there is a Ca²⁺-independent pathway that leads to endothelial NO synthase activation (29) . It would be interesting to investigate the relative importance between the Ca²⁺-dependent and Ca²⁺-independent vasorelaxation in our system. Currently we are trying to measure the endothelial [Ca²⁺]_i responses along with concomitant vasorelaxation. Finally, it is possible that a local elevation in endothelial [Ca²⁺]_i, especially near the plasma membrane (30) , is sufficient to generate vasorelaxation. Additional endothelial [Ca²⁺]_i elevation may serve other functions, such as to generate extra amounts of NO or PGI₂. This last possibility is plausible if NO or PGI₂ are to be effective in preventing platelet activation in the bloodstream.

In conclusion, vascular endothelial cells appear to have their own characteristic [Ca²⁺]_i response to various agonists, and this heterogeneous population of cells may function coordinately in situ. Perhaps we should modify the current view regarding vascular endothelium as a homogeneous layer of endothelial cells that interact with local environment in a uniform way.

	ACKNOWLEDGMENTS

 
This work was supported by grants from the National Science Council and National Health Research Institute in Taiwan, ROC (grant numbers NSC88–2314-B006–068, 070 and DOH88-HR-834).

	FOOTNOTES

 
Received for publication June 7, 1999. Revised for publication November 5, 1999.

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