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Am J Physiol Heart Circ Physiol 286: H2322-H2331, 2004. First published February 12, 2004; doi:10.1152/ajpheart.00006.2004
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Localized transient increases in endothelial cell Ca2+ in arterioles in situ: implications for coordination of vascular function

Tasmia Duza1 and Ingrid H. Sarelius2

1Department of Biomedical Engineering and 2Department of Pharmacology and Physiology, University of Rochester, Rochester, New York 14642

Submitted 8 January 2004 ; accepted in final form 9 February 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intracellular Ca2+ transients were identified in endothelial cells (ECs) in intact blood-perfused arterioles. ECs in cremaster muscle arterioles (diameter ~45 µm) in anesthetized mice were loaded with the Ca2+ indicator fluo 4-AM by intraluminal perfusion, after which blood flow was reestablished. Confocal microscopy was used to visualize Ca2+ as a function of fluo-4 intensity in real time. Separate sets of experiments were performed under the following conditions: control, ischemia, during inhibition of P2x or P1 purinoreceptors, and with the application of exogenous adenosine. In controls, spontaneous EC Ca2+ transients displayed a wide range of activity frequency (1–32 events/min) and about one-third of these transient events were synchronized between adjacent ECs. The increase in Ca2+ remained localized and did not spread to encompass the entire cell body. Ca2+ transient activity decreased significantly with ischemia (from 9.9 ± 0.6 to 3.1 ± 0.3 events/min, n = 135) but was unaffected by P2x or P1 receptor inhibition. Exogenous adenosine significantly increased the frequency of Ca2+ transients (to 12.8 ± 0.9 events/min) and increased synchronization so that 50% of all Ca2+ events were synchronized between ECs. This response to adenosine was not due to an increase in shear stress. These data indicate that localized Ca2+ transients are sensitive to flow conditions and, separately, to metabolically active pathways (exogenous adenosine), although the basal activity occurs independently of P2x or P1 receptors. These transients may represent a mechanism by which individual EC responses are integrated to result in coordinated arteriolar responses in situ.

oscillations; fluorescence imaging; microvascular communication

INTRACELLULAR Ca2+ plays a ubiquitous role as a signaling intermediate in many physiological responses. In endothelial cells (ECs), changes in Ca2+ are implicated in both acute responses (e.g., alterations in vascular tone and permeability) and more long-term effects (e.g., changes in cell morphology and gene expression). These established roles of EC Ca2+ changes are supported by direct measurements of steady-state whole cell Ca2+, primarily in isolated systems (28, 40). However, a key property of Ca2+ as a signaling molecule is that it often involves highly localized and/or transient changes (5, 32, 43). Thus spatially averaged measurements of steady-state Ca2+ changes are not necessarily representative of localized signaling events within individual cells. Transitory increases in cell Ca2+ can remain confined within localized regions of the cytoplasm (sparks, blips, and puffs) or, alternatively, spread throughout the cell body (waves, oscillations) (6, 24, 37). Furthermore, Ca2+ waves/oscillations can spread from one cell to the next and, in a variety of systems, are responsible for the coordinated activity of cells (5, 32). In this study, we report the occurrence of spontaneous transitory increases in EC Ca2+ (which we refer to as Ca2+ transients) in intact blood-perfused arterioles. Understanding the physiological role of these transients requires, as an initial step, the capability to consistently detect the events, clearly define baseline activity in resting arterioles, and demonstrate that these signals can be modified by relevant stimuli. To this end, we have established a methodology for real-time quantification of transient Ca2+ signals in arteriolar ECs in situ, have investigated their involvement in the coordination of signaling activity in neighboring ECs, and have tested whether specific mechanisms that have been implicated in alteration of EC Ca2+ in isolated cell systems can also affect these Ca2+ transients in ECs in situ.

The collective findings of many studies indicate that Ca2+ is a messenger molecule for flow-induced EC responses, which are most commonly ascribed to the influence of shear stress on endothelium. Numerous studies (1, 2, 11, 13, 15, 41) conducted in isolated cell systems demonstrate that EC Ca2+ is sensitive, although not invariably (25, 35, 42), to the introduction or cessation of flow and is also sensitive to changes in flow rate. However, the roles of EC Ca2+-dependent signaling processes in response to flow have been studied primarily in macrovascular EC lines, where changes in Ca2+ brought about by step changes in shear stress/flow have typically been the subject of investigation. This is strikingly different from the hemodynamic environment of ECs in situ, where the cells are continuously exposed to dynamic flow conditions. Thus what effects flow might have on Ca2+ changes in ECs in intact blood perfused microvessels are not known, although it is clearly reasonable to expect that flow may modulate EC Ca2+ in situ. In this study, we therefore tested the hypothesis that spontaneously occuring Ca2+ transients in arteriolar ECs are sensitive to flow.

The specific mechanisms that mediate flow-induced EC Ca2+ changes are still being defined. Purines have been targeted as one class of candidate molecules, although their role in mechanotransduction is controversial. Several (2, 15, 41) but not all (11, 26) studies in isolated cell systems indicate that shear stress-dependent Ca2+ increases require extracellular ATP. In particular, P2x purinoceptors have been proposed as shear transducers in the response of human umbilical vein ECs to fluid flow (41). In this study, we therefore tested the hypothesis that spontaneous EC Ca2+ transients (which we report are indeed flow sensitive) are dependent on P2x receptor-mediated signaling.

We have recently shown that in small arterioles in situ, endothelium-dependent dilations to purines [both ATP and adenosine (Ado)] occur via a P1 (as opposed to P2) receptor pathway, and require an increase in whole EC Ca2+ for both radially and axially communicated components of the dilator signal (12). Because purines have been identified as key participants of oscillatory Ca2+ activity in several systems (14, 31, 34), we tested for the involvement of P1 receptors during basal EC Ca2+ transients and also determined whether exogenous Ado influences the Ca2+ activity.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. All protocols were approved by the Animal Care and Use Committee of the University of Rochester and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996).

Male C57BL/6J mice (wt 25–30 g) were anesthetized with pentobarbital sodium (75 mg/kg ip) and tracheotomized to maintain a patent airway. A jugular venous catheter was placed for introduction of flow markers and administration of supplemental pentobarbital sodium as needed throughout the experiment. The depth of anesthesia was assessed by monitoring the animal's reflex withdrawal to a tail pinch. Mouse body temperature was maintained at 37°C via convective heat. The right cremaster was exteriorized and prepared for intravital microscopy, as described previously (21, 22). During surgery and experimental protocols, the muscle preparation was continuously superfused with a bicarbonate-buffered physiological salt solution warmed to 36°C containing (in mM) 131.9 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, and 30.0 NaHCO3, equilibrated with 5% CO2-95% N2 to maintain pH 7.40 ± 0.05. Tubocurarine (4 µM) was added to the superfusion solution to eliminate the spontaneous twitching of skeletal muscles and associated tissue movement. At the completion of all experimental protocols, the animals were administered a lethal intravenous dose of pentobarbital sodium.

Dye loading of arteriolar ECs. Arterioles (maximum diameter ~50 µm) were cannulated as previously described (12) with micropipettes containing 5 µM fluo 4-AM indicator solution. Briefly, the ends of the cannulating micropipettes were triple beveled to produce sharp tips (diameter 7–10 µm) for smooth penetration through skeletal muscle cells and the vessel wall. Once the pipette had entered the vessel lumen it was pressurized with the use of a manometer to initiate flow out of the pipette. A blunted, curved glass occluding rod was gently placed upstream of the cannulating pipette to temporarily inhibit blood flow and allow fluo 4-AM to intraluminally perfuse the downstream microvascular network. Areas of perfusion could be verified visually as blood was completely cleared from the vessel by flow of the dye. After 15 min of dye perfusion (total volume {approx}10 µl) the pipette and occluding rod were removed and blood flow was allowed to resume. No fluorescence of smooth muscle cells (SMCs) was visible under these loading conditions. As shown in Fig. 1 (see http://ajpheart.physiology.org/cgi/content/full/00006.2004/DC1 for a video), fluo 4-loaded cells were easily identified as ECs due to their characteristic axially oriented morphology (23). As has been established for both isolated (13) and in situ (27) arterioles, luminal perfusion of AM forms of dyes for a short duration confines indicator loading to ECs. A 10- to 15-min period was allowed for intracellular deesterification of the dye and reestablishment of vessel tone prior to data collection. Fluo-4-loaded arterioles exhibited normal vessel tone and retained their ability to respond appropriately to vasoactive constrictors and dilators (verified by responses to topical application of phenylephrine and Ado, respectively).



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  		Fig. 1. A: schematic of the volume sampled at the "top" of the vessel using confocal microscopy. Arrow indicates the direction of blood flow. Not drawn to scale. B: typical confocal image of fluo-4-loaded endothelial cells (ECs) in blood-perfused cremaster muscle arterioles in situ. Scale bar is 20 µm. The direction of flow is from left to right. As an example of the spatial orientation of adjacent EC pairs in the synchronization analysis, two cells are marked A and B.

 
Ca2+ imaging. Arterioles were visualized with a x20 water-immersion objective [numerical aperture (NA) 0.5] with the use of an imaging system that consisted of an Olympus BX50WI microscope fitted with a Nipkow disk scanning confocal head (Yokogawa) coupled to an intensified charge-coupled device video camera (XR-Mega 10, Solamere Technology Group). This optical configuration resulted in a spatial resolution of 1.8 pixel/µm. Fluo 4 was excited at 488 nm with a 20-mW argon laser and emissions > 516 nm were collected. Images of vessel cross sections along their long axis, where the top plane of the vessel was in clear focus in the confocal plane (see Fig. 1), were recorded on 3/4-in. videotape (VO9600, Sony).

The sensitivity of the imaging system was determined in vitro with the use of 5 µM fluo 4 (K+ salt) and a Ca2+ calibration buffer kit; this established that nanomolar Ca2+ concentrations could be discriminated in our system. We ascertained that the system responds linearly (r2 = 0.98) for fluo-4 emission intensity changes over the range of 0–600 nM Ca2+. Furthermore, the span of emissions intensities detected from ECs in situ was comparable to that of the linear range of the in vitro calibration. Tissue autofluorescence was negligible and dye leakage and bleaching were insignificant over the time frame (<2 s) of the Ca2+ transients characterized in this study. Thus a change in Ca2+ could be measured as a function of relative change in fluorescence emissions intensity. In view of the uncertainties regarding the applicability of in vitro calibrations of absolute Ca2+ concentration to in situ cell systems, we report relative changes in Ca2+ from baseline, rather than absolute concentrations.

Measurement of Ca2+ transients. Images recorded on videotape were digitized at 5 or 30 Hz for off-line analysis using a CG7 Scion Image frame grabber and NIH Image software. The average fluorescence intensity (grayscale range: 0–255; 0 = black, 255 = white) was measured in a small area of interest (AOI; ~15–25 µm2) positioned on individual ECs. Usually, there was a site within each cell that could be detected visually during review of the images as being where the Ca2+ increase originated (designated as "the 1° site"). The AOI was placed at this site for all analyses unless specified otherwise. Artifactual changes in intensity (e.g., due to slight tissue movement during breathing or drift in focus) were clearly distinguishable from Ca2+ transients by visual inspection. Measurement of background fluorescence was made by positioning an identical AOI adjacent to the vessel in the avascular tissue space: background was subtracted from all intensity measurements. We established in preliminary analyses using images digitized at 30 Hz that digitization of images at 5 Hz was sufficient to discern the Ca2+ transients. We also determined that the standard deviation of the raw signal (attributed to noise inherent in imaging this optically complex system) ranged between 2 and 4%. To rule out the possibility that the transient Ca2+ signals resulted from random noise, we therefore defined, conservatively, that to qualify as the peak of a transient, fluorescence intensity had to be greater than twice the maximum standard deviation (i.e., 8%) relative to the immediately before and after 0.6 or 0.8 s (i.e., during a Ca2+ transient both the rise and fall in fluorescence intensity had to occur within a total of 1.2 to 1.6 s). The peak change in amplitude of the fluorescence signal during the transients was also measured for comparison of the relative magnitude of the change in Ca2+ during transients under control conditions and with Ado.

Localization of Ca2+ signal and synchronization of Ca2+ activity. The extent to which the Ca2+ transient spread was quantified in two ways. First, as a function of cell length (measured at 1-µm intervals in the axial direction along the cell body) over which fluo 4 emission intensity (raw single pixel value) increased by >20% of baseline during a Ca2+ transient. Second, by simultaneous measurement of Ca2+ change in two AOIs, one positioned at the 1° site (AOI1) and the other (AOI2) positioned at a distant site within the same cell.

Synchronization of Ca2+ signals between neighboring cells has been postulated to underlie coordinated cell behavior in a variety of systems (20, 32). To determine whether endothelial Ca2+ transients are coordinated in arterioles either at rest or during dilations to Ado, we defined Ca2+ events in two separate cells as "synchronized" if transients occurred in both cells within 1 s of each other. To quantify this, the relative frequency of synchronized versus isolated (unsynchronized) Ca2+ events was determined for adjacent cell pairs (e.g., Fig. 1B, cell A and cell B). As a control, two ECs from separate arterioles were randomly paired for an analogous analysis. The time course of Ca2+ activity in the adjacent and randomly selected cell pairs was compared and events were scored as "synchronized" if transients occurred in both cells within <1 s of each other and as "isolated" otherwise. Because an isolated event was counted if a transient occurred in either cell, whereas a synchronized event was scored only when transients occurred in both cells, the probability of encountering an isolated event was twice that of a synchronized event. Therefore, the final number of isolated events was halved, to equalize the probability of encountering each type of event.

Experimental protocols. Vessel segments with fluo 4-loaded ECs were continuously observed for 2 min using four different protocols. Protocols 1 and 4 were performed in the same preparations, whereas protocols 2 and 3 were each performed in separate preparations.

Protocol 1: control. To capture basal EC Ca2+ activity, control data were collected during exposure of tissue preparations to control superfusion solution only.

Protocol 2: ischemia. After dye loading, ischemic conditions were produced using a previously described method (22). Briefly, two miniature surgical clamps were placed across the proximal region of the muscle, thus occluding blood flow in the entire preparation. Stagnant red blood cells with no significant movement verified ischemia. Recordings of EC Ca2+ were made under these stopped-flow conditions, after which the clamps were removed and flow was allowed to reestablish. EC Ca2+ measurements were made again in the same vessel segments for paired flow recovery data. The superfusion solution in these experiments was supplemented with 10 mM glucose and 1 mM pyruvate and equilibrated with gas containing 19% O2-5% CO2-balance N2 to maintain tissue viability in the absence of blood flow, thus under these conditions the tissue preparation was ischemic but not hypoxic. The modified buffer, which did not affect EC Ca2+ baseline activity, continued to bathe the tissue during collection of flow recovery data.

Protocol 3: purinergic receptor inhibition. The cremaster preparation was exposed to 10–5 M pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS; P2x antagonist) or (in separate preparations) 10–5 M xanthine amine congener (XAC; P1 antagonist) by adding the agent to the superfusion solution. These treatments are sufficient to inhibit vasomotor responses via the targeted receptors in arterioles in situ (12). After 30 min of exposure to the antagonist, EC Ca2+ activity was recorded in the continued presence of the blocker.

Protocol 4: Ado application. To test whether exogenous Ado alters EC Ca2+ transients, observations were made in the same cells before and during application of Ado. EC Ca2+ activity was recorded continuously during 1 min of control, followed by 1 min of exposure to 10–4 M Ado (added to the solution superfusing the tissue).

At the end of all four of the above protocols, we observed EC Ca2+ responses to 10–4 M ACh [an agonist known to maximally increase intracellular Ca2+ in ECs (13, 27)] to confirm that there had been dye uptake by all ECs and also to verify that the system had the capacity to detect changes in Ca2+ throughout the entire cell (and not just in localized regions displaying Ca2+ transients, for example, due to compartmentalization of the fluorophore).

Measurement of hemodynamic parameters. In an intact vascular network, dilation produced by the application of Ado to the tissue does not necessarily increase blood flow across the entire tissue and therefore is not necessarily associated with an increase in shear stress at the endothelial wall (33). We therefore measured changes in shear rate, which is a good index of shear stress (21), to determine whether wall shear stress had in fact changed during Ado-induced dilations. To do this, measurements of diameter and blood velocity were made in the same arterioles before and during application of Ado to the tissue and were used to calculate wall shear rate as described elsewhere (21). Briefly, fluorescent polyethylene beads (0.2–0.5 µm diameter) injected via a jugular catheter were used as flow markers. Bead velocities were sampled for 5–10 s during control and Ado application, to estimate average blood velocity (Vavg). Blood flow was calculated from the product of Vavg and vessel cross-sectional area, and wall shear rate was calculated as 8 Vavg/D, where D is diameter. Changes in hemodynamic parameters with Ado are reported as the relative changes from control values measured in the same arterioles.

Materials. A 5-µl aliquot of 10–3 M fluo 4-AM (Molecular Probes; Eugene, OR; dissolved in 100% DMSO) and 2 µl of 12.5 mg/ml Pluronic-127 (TEF Labs; Austin, TX; made in 100% DMSO) stock solutions were mixed and diluted in 1 ml 0.9% NaCl for a final concentration of 5 µM fluo 4-AM (indicator solution). Aliquots (5 µl) of 10–3 M fluo 4 K+ (Molecular Probes) were added to 1 ml Ca2+ standards from a calibration buffer kit (Molecular Probes) for a final concentration of 5 µM fluo 4 (K+ salt) in various known Ca2+ concentration solutions. Fluorescent polyethylene beads were purchased from Polysciences (Warrington, PA). All other reagents were obtained from Sigma (St. Louis, MO). Stock XAC (10–2 M) was made in 0.1 N NaOH in saline and then added to the superfusion solution for a final concentration of 10–5 M XAC. Solutions composed of (in M) 10–4 Ado, 10–4 ACh, 10–5 PPADS, and 10–4 phenylephrine were made directly in superfusion solution. Solutions were prepared fresh daily.

Statistical analyses. Data for each experiment set were collected from a minimum of three animals. Multiple vessel segments (usually between 3 and 6) from one or two arterioles were studied in each animal. Typically, 5–10 ECs were observable (in clear focus in the confocal plane for the entire 2-min observation period) in each segment. The reported number of observations (n) refers to the number of ECs studied unless specified otherwise. A brief waiting time (~5–10 min) was maintained between successive Ado exposures to allow vessel diameter to return to control. All data are reported as means ± SE. Data were compared by paired Student's t-test, repeated-measures ANOVA, or correlation coefficient analysis, as appropriate. Statistical significance was determined at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ca2+ transients are present in ECs under basal conditions. Under control conditions, the resting diameter of arterioles was stable and did not change during the 2-min observation period. Spontaneously occurring transient increases in Ca2+ were detected in all ECs. However, the span of frequencies of the Ca2+ activity was widely distributed (range 1–32 events/min, Fig. 2A). An equal fraction of ECs increased (29 cells) and decreased (29 cells) the frequency of their Ca2+ activity between minute 1 and 2 (with no change in four cells); however, overall, there was no difference in the average number of transients observed in minute 1 versus minute 2 (P > 0.05) or in the distribution of frequencies between minute 1 and minute 2 (P > 0.05), confirming that a 1-min observation period was sufficient for quantitative characterization of this spontaneous activity. These findings indicate that while the capacity for Ca2+ transients is inherent in all arteriolar ECs, all cells, importantly, do not function identically under resting conditions in their native environment (see http://ajpheart.physiology.org/cgi/content/full/00006.2004/DC1 for video supplement 1). The time course of changes in Ca2+ activity in two representative cells is shown in Fig. 2B. Overall, there were 11.0 ± 0.6 transients·cell–1·min–1 under basal conditions (n = 62; Fig. 3).



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  		Fig. 2. A: frequency distribution histogram of EC Ca2+ transients during minute 1 vs. minute 2 of observation under control conditions (n = 62). B: representative time courses of EC Ca2+ transients are shown as a function of changes in fluo 4 emissions intensity over 1 min in two representative ECs (left and right). The obvious upward spikes in EC fluo 4 emission intensity, shown in gray, represent EC Ca2+ transients. Each time point (digitized at 5 Hz) was tested to see whether it qualifies as the peak of a Ca2+ transient (as defined in METHODS). There are no changes in corresponding background fluorescence signals, shown in black, measured in areas of interest (AOIs) placed outside the lumen in the avascular tissue space.

 


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  		Fig. 3. EC Ca2+ transients during control (n = 62), ischemia (n = 135), and flow recovery (n = 115), inhibition of P2x (n = 89) or P1 purinoreceptors (n = 91), and with adenosine (Ado; n = 62). Values are means ± SE. *P < 0.05, significantly different from control.

 
ACh elevated Ca2+ throughout the cytosol in all cells (see http://ajpheart.physiology.org/cgi/content/full/00006.2004/DC1 for video supplement 2). This confirmed that the heterogeneity in transients observed in localized regions of the EC body was not due to artifacts resulting from, for example, nonuniform dye uptake by individual cells or compartmentalization of the dye in intracellular organelles. Note that although fluorescence levels recorded in some of the ECs during basal conditions were not much higher than background, their response to ACh demonstrated clearly that the fluorophore was adequately loaded and capable of reporting a change in cytosolic Ca2+ concentration should it have occurred. This observation also gives further support to our conclusion that Ca2+ activity is not uniform among ECs during resting conditions.

Spatial and temporal characteristics of Ca2+ transients. Figure 4A shows a montage of a single representative Ca2+ transient in one EC for image frames consecutive at 33-ms intervals. In Fig. 4B, the Ca2+ transient is shown as raw fluo-4 emissions intensity averaged in a small AOI (Fig. 4A, first panel) over 1 s. This increase in Ca2+ did not encompass the whole cell but rather remained localized, extending ~10 µm along the long axis of the cell. The direction, distance, and duration of the Ca2+ increase is illustrated in Fig. 4C.



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  		Fig. 4. A: montage of a single representative Ca2+ transient. Image frames are consecutive at 33-ms intervals (left to right, top to bottom). Fluo-4 emission intensity is shown in gray scale (low [Ca2+] = black, high [Ca2+] = white). B: Ca2+ transient shown as raw fluo-4 emissions intensity averaged in a small area of interest (outlined in white in A, first panel) for every frame in A (total time 1 s). C: two-dimensional projection of the cell in A at the peak of the Ca2+ increase is shown on the left. The approximate area of the cell is outlined in black. The position of the line along the axial length of the cell over which spread of Ca2+ increase is measured is shown in white. The intensity scale is shown in pseudocolor. The direction and distance of the spread in Ca2+ along the length of the cell (measured at ~1-µm intervals) is shown over time (frames are the same as in A and B) on the right. White indicates increase in Ca2+ from baseline, blue indicates no change.

 
To further explore the local nature of the Ca2+ transients, we compared the change in fluorescence at two distinct AOIs within the same cell, positioned 42.3 ± 2.8 µm apart (n = 14 cells). Approximately 30% fewer transients (9.6 ± 1.4 vs. 15.7 ± 1.5 events/min in AOI1) were detected at AOI2 relative to AOI1, which was the 1° site of Ca2+ activity. There was a significant correlation between the times when Ca2+ increased at AOI2 and was also elevated at AOI1 (r2 = 0.6; P < 0.05). This is consistent with the conclusion that activity at AOI2 originated at AOI1 rather than originating as a second independent Ca2+ transient. These data also clearly show that Ca2+ does not increase uniformly throughout the cell body during the transients.

ECs are synchronized with respect to their Ca2+ activity. We also asked whether these Ca2+ transients are spatially and temporally synchronized. We reasoned that during comparison of the time course of Ca2+ activity in two cells, some events might be scored "synchronized" simply due to the inherent frequency dependence of the Ca2+ transients. To control for this, ECs from arterioles in separate animals were randomly paired. Analysis of the synchronization in these randomly paired cells showed that there are 2.9 ± 0.3 synchronized events/min for every 6.4 ± 0.3 isolated events/min (n = 72 cell pairs; Fig. 5). This level of synchronized activity occurs by chance and is clearly independent of the spatial relationships between cells. Thus for Ca2+ activity to be spatially coordinated, the relative occurrence of synchronized events must be higher than that determined for the random pairing. Analysis of synchronization conducted for pairs of adjacent ECs within the same arteriole showed that in these adjacent cells, the frequency of synchronized events is significantly higher than in the randomly paired cells (4.8 ± 0.8 events/min, n = 48 cell pairs; P < 0.05), whereas the frequency of isolated events in each cell remains no different (P > 0.05) from that in randomly sampled isolated cells (Fig. 5; see http://ajpheart.physiology.org/cgi/content/full/00006.2004/DC1 for video supplement 3). The finding that isolated Ca2+ transients in individual ECs occur more frequently than synchronized transients occur in adjacent cells clearly indicates that arteriolar ECs do not act as a single functional unit under the basal conditions of these experiments. There is, however, some synchronization of Ca2+ activity, indicating that there is indeed some communication and/or integration of signaling activities between adjacent ECs in situ.



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  		Fig. 5. Relative number of synchronized and isolated Ca2+ transients in randomly paired cells from arterioles in separate animals (left bars) and adjacent cell pairs within the same arteriole (right bars). Values are means ± SE (n = 72 for random cell pairs, n = 48 for adjacent cell pairs). *P < 0.05, significantly different from random synchronized events; #P < 0.05, significantly different from synchronized events by each respective method of pairing. Cell pairs are indicated by an asterisk.

 
Ischemia inhibits Ca2+ transients in ECs. Under ischemic conditions, fewer Ca2+ events (3.1 ± 0.3 transients·cell–1· min–1, n = 135, P < 0.05 compared with control) were detected (Fig. 3). EC Ca2+ transients increased to 9.9 ± 0.6 transients·cell–1·min–1 after reestablishment of flow, which was not different from control values (n = 115, P < 0.05 compared with ischemia, P > 0.05 compared with control). In contrast to controls, where Ca2+ transients were detected in 100% of ECs, no transients were detected in 8% of cells during ischemia. The frequency distribution histogram of EC Ca2+ transients during ischemia was markedly left shifted (Fig. 6), again illustrating the overall decrease in frequency of Ca2+ transients during ischemia. Thus these data indicate that spontaneous transients in EC Ca2+ are sensitive to the presence of blood flow.



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  		Fig. 6. Frequency-distribution histogram of transitory EC Ca2+ events during ischemia (n = 135) and after reestablishment of flow (n = 115) in the same tissue preparations.

 
Purinergic receptors do not mediate spontaneous EC Ca2+ transients. To investigate the contribution of purinergic receptors to the transitory Ca2+ activity, EC Ca2+ measurements were made in preparations treated with selective inhibitors of P2x or P1 receptors. The frequency of the Ca2+ transients was 11.9 ± 0.9 events·cell–1·min–1 (n = 89, P > 0.05 compared with control) with PPADS and 10.8 ± 0.5 events·cell–1· min–1 (n = 91, P > 0.05 compared with control) with XAC treatment (Fig. 3). Thus neither inhibitor changed the frequency of Ca2+ transients from that observed in control conditions, indicating that P2x and P1 purinergic receptors do not play a role in the generation of these basal EC Ca2+ transients.

Ado increases Ca2+ transients in ECs. Despite our finding that purines are not involved in the maintenance of the baseline transitory Ca2+ activity, they may still initiate or modify the Ca2+ transients. To determine whether exogenous Ado alters the Ca2+ activity, observations were made during 1 min of control and in the same ECs during 1 min of exposure to Ado. As expected, Ado dilated fluo 4-loaded arterioles from 29.9 ± 3.4 µm (control) to 42.2 ± 2.7 µm (n = 10 arterioles, P < 0.05 vs. control). Overall, Ado produced a small but significant increase in the frequency (12.8 ± 0.9 transients·cell–1·min–1; P < 0.05 compared with control; Fig. 3), but not amplitude (P > 0.05 compared with control) of the transitory Ca2+ signal (n = 62). Both increases and decreases in the number of Ca2+ transients were seen in response to exogenous Ado; however, in the presence of Ado approximately twice as many cells (38 cells) increased the frequency of their Ca2+ activity relative to the number (20 cells) that decreased their Ca2+ activity. Ado produced a dramatic rightward shift in the frequency distribution histogram of the population of cells that responded with increased Ca2+ activity compared with their baseline behavior (Fig. 7A). In contrast, there was little change in the activity pattern of cells that did not increase the number of transients (Fig. 7B). Thus these data suggest that there may be both an Ado-sensitive (60%) and an Ado-insensitive (40%) population of cells: mechanisms underlying such differences in the EC population remain to be determined. One possibility is that Ado may have significantly activated a unique population of ECs based on their baseline activity frequency or, alternatively, Ado may have produced a slight elevation in the activity of a majority of the cells. Either of these alternatives could result in the significant increase in Ca2+ transients seen overall. To distinguish between these two possibilities, the number of events during baseline and Ado exposure were compared for each individual cell. This comparison revealed that, whereas Ado produced an increase in the activity frequency of at least 100% in a few cells (n = 10), most cells (n = 28) responded with a small increase in the number of Ca2+ transients. Moreover, there was no correlation between baseline activity frequency and whether the cell became activated (Fig. 7C). Thus cells displaying a specific frequency of basal Ca2+ activity do not appear to be preferential targets of Ado stimulation.



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  		Fig. 7. Frequency distribution histogram of EC Ca2+ transients during control vs. in the presence of Ado in the population of cells that increased (A; n = 38) and did not increase (B; n = 24) their Ca2+ activity on exposure to Ado compared with their baseline behavior. C: dependence of EC Ca2+ sensitivity to Ado on resting activity frequency (n = 62). Number of Ca2+ transients during exposure to Ado (minute 2) in each individual EC as a function of its transitory activity during baseline (minute 1). D: relative number of synchronized and isolated Ca2+ transients in the presence of Ado in randomly paired cells from arterioles in separate animals (left bars) and adjacent cell pairs within the same arteriole (right bars). Values are means ± SE (n = 72 for random cell pairs, n = 48 for adjacent cell pairs). *P < 0.05 and #P < 0.05, significantly different from random synchronized events.

 
Ado increases synchronization of EC Ca2+ transients. We also asked whether Ado alters the synchronization pattern of Ca2+ activity relative to control conditions (Fig. 7D). Because we found that the total number of transient Ca2+ events is higher (compared with control) in the presence of Ado, the Ca2+ transient activity in Ado-exposed ECs was analyzed as described earlier, by comparing the frequency of transient Ca2+ events in randomly sampled versus paired cells. Randomly paired cells had 3.1 ± 0.3 synchronized events/min for every 6.4 ± 0.2 isolated events/min during Ado exposure (n = 72 cell pairs), which was not different (P > 0.05) from the behavior of randomly paired cells under control conditions that we described earlier. However, when the comparison was made for adjacent ECs (n = 48 cell pairs) in the presence of Ado, the frequency of synchronized events was dramatically higher (5.9 ± 0.7/min, P < 0.05) than that for paired control ECs, whereas the frequency of isolated events in the Ado-exposed cells was unchanged (P > 0.05). Furthermore, during Ado exposure, the likelihood of encountering isolated events in adjacent cell pairs was no longer different from that of encountering synchronized events (P > 0.05). To further confirm that the increased synchronization of Ca2+ transients in adjacent cells in the presence of Ado was not an artifact related to the total number of events, transients were expressed as the fraction of synchronized relative to isolated events, normalized for total number of events. This analysis showed that even after accounting for the total number of Ca2+ transient events, the fraction of synchronized events in adjacent cell pairs was significantly higher during control conditions than predicted by the random pairing analysis (0.72 ± 0.12 vs. 0.50 ± 0.05; P < 0.05). The fraction of synchronized Ca2+ events in adjacent cell pairs was further increased by Ado (to 0.89 ± 0.10; P < 0.05).

Ado increases blood flow but not shear stress. Ado increased arteriolar diameter 52.9 ± 16.1% from control (P < 0.05, n = 17 arterioles) but did not change average blood velocity (83.7 ± 10.2 µm/s in controls vs. 88.6 ± 9.7 µm/s with Ado; P > 0.05). This resulted in a 4.8 ± 1.7-fold increase in flow (P < 0.05 compared with control) but a 17.1 ± 8.8% decrease in shear rate (P < 0.05 compared with control) in the presence of Ado. Because wall shear rate is a good index of wall shear stress in these vessels (21), these data indicate that Ado did not increase the level of shear stress experienced by ECs in these in situ arterioles, despite an increase in tissue perfusion, indicating that the increase in frequency of EC Ca2+ activity with Ado was unlikely to be due to increased flow and was therefore most likely due to a P1 receptor-mediated event.


   DISCUSSION
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This study shows that Ca2+ changes spontaneously in ECs of skeletal muscle arterioles that are blood perfused and exhibit resting tone in situ. Using a confocal imaging system, we have imaged rapid Ca2+ transients from regions of micrometer dimensions in endothelium of intact blood perfused arterioles. During these transitory events, the increase in Ca2+ does not spread uniformly throughout the cell body, but rather remains localized. Importantly, ECs in these arterioles do not behave identically to each other with respect to transitory Ca2+ activity and display a wide range of frequencies of Ca2+ events. Some, but not all, of the basal transients are synchronized in neighboring ECs, indicating that while some degree of communication occurs between ECs during basal conditions, most appear to be functioning independently of each other, at least with respect to Ca2+-dependent pathways. Furthermore, we show that this Ca2+ activity is closely associated with blood flow: it decreases in frequency with ischemia and returns to control levels on reestablishment of flow. The spontaneous Ca2+ transients are not dependent on P1 or P2x purinergic receptor pathways, although exogenous Ado produces an increase in their frequency and further synchronizes the Ca2+ activity among neighboring ECs. Our findings also indicate that the increase in Ca2+ transients with Ado is unlikely to operate via a shear stress-sensitive pathway. We conclude that direct receptor-ligand interactions or flow effects unrelated to shear stress must underlie the response in EC Ca2+ transients in the presence of Ado.

Our study indicates that in the native vascular environment there are spontaneously occurring elevations in endothelial Ca2+ in localized regions of the cell body. Typically, successive Ca2+ spikes arose from the same location within each cell, as has been reported for wavelike Ca2+ activity (7, 19, 37, 39). The transients that we observed are very rapid, each lasting <2 s, which differs from Ca2+ oscillations reported in ECs in isolated systems, where the time frame over which Ca2+ is elevated is strikingly longer (30 s to several minutes) (18, 29, 42). Short transients, such as we observed here, have also been reported recently in ECs in arterioles in isolated rat ureter preparations (7). The structural integrity of the native microvasculature thus appears to have a critical influence on spatially and temporally variant signaling behaviors. Slower Ca2+ events typically (15, 42) though not necessarily (16, 29) encompass the entire cell body, unlike the intracellularly localized Ca2+ activity characterized in the present report. Whether Ca2+ transients of longer durations also occur in these arterioles in situ is not known, as the experimental design of the current study, as discussed later, was not suitable for quantification of slower Ca2+ events. The spatially restricted Ca2+ transients that we report resemble elementary Ca2+ events classified as puffs, and also share features reported for repetitive Ca2+ waves (6, 7, 24). It is postulated that Ca2+ puffs/waves are initiated at specific regions of cells where inositol 1,4,5-trisphosphate receptors are located (3, 6, 24, 37). Identification of the specific Ca2+ channels involved and the source of Ca2+ (influx from the extracellular space vs. release from internal stores) for the signals observed in the intact system used in our study is likely to provide important clues regarding the mechanisms by which these transients are generated, but was beyond the scope of the current work.

In isolated systems, heterogeneous behavior of Ca2+ activity within a population of ECs has been reported in response to exposure to a single agonist (7, 13, 15, 18, 29). Similarly, in our study, we also found that EC Ca2+ transients display a wide range of frequencies under any given condition, even between adjacent cells within the same arteriole. In our experiments, the field of view was such that during a single recording period, observations were made in vascular segments with a maximum length of 200 µm. It is reasonable to expect arteriolar tone to be uniform over this length of vessel, making the level of tone alone unlikely to account for differences in the frequency of Ca2+ transients. Variations in local forces defined by cell surface geometry have, however, been shown to produce significant differences in the local hydrodynamic forces both within and between individual cells, likely contributing to heterogeneous endothelial responses to fluid flow (4, 8). Thus a plausible source of the variability in the localized Ca2+ responses could be the heterogeneous topography of ECs at the luminal surface of the vessel wall. Alternatively, the endothelium within our observation area may have been subject to differing levels of plasma proteins, myoendothelial communication etc. at the microscopic level, which could potentially contribute to variable Ca2+ signals in ECs (10, 28, 40). We speculate that the wide range of frequencies of Ca2+ transients observed in situ is a reflection of cell-specific differences in sensitivity to various stimuli, including but not limited to mechanical forces.

Our examination of the time course of Ca2+ activity showed that Ca2+ transients can be synchronized between adjacent cells, illustrating that despite heterogeneity in the apparent sensitivity threshold of individual cells, the potential exists for there to be significant cell-cell coupling and/or integrated activity in arterioles in situ, although we cannot rule out the possibility that the synchronization that we report in fact reflects the identical response of two adjacent cells to the same localized stimulus. Importantly, we found that synchronization of these Ca2+ transients could be modulated by vasoactive agents (Ado), demonstrating that these Ca2+ signals have the potential to participate in the coordination of vascular function and communication between different parts of the vascular wall. Both gap junction and paracrine-mediated signaling pathways have been implicated in intercellular Ca2+ waves in EC monolayers (9, 42) and presumably are also involved in the synchronous Ca2+ transients that occur between ECs in situ.

Many stimuli that could affect Ca2+ activity of the endothelium are present within the intact vascular system. One stimulus that is continually present and has been repeatedly identified as an initiator of Ca2+-dependent signals in vascular endothelium is flow, and indeed our study shows that EC Ca2+ transients are highly contingent on the presence of flow. It is unlikely that the decreased Ca2+ activity that occurred during ischemia was due to metabolic alterations during the flow stoppage because the tissue was not allowed to become hypoxic; no relationship was found between changes in transitory Ca2+ activity and the time elapsed after flow was either stopped or reestablished (data not shown). Interestingly, despite the acute dependence of transitory Ca2+ activity on the presence of flow, neither the starting location nor the direction of Ca2+ spread appeared to correlate with the direction of blood flow. For example, Ca2+ transients in neighboring cells were seen to originate and spread simultaneously in both upstream and downstream axial directions. Customarily, it is the increase in shear stress brought about by an increase in flow rate that is thought to underlie the EC Ca2+ response to flow. It is therefore likely that the reduction in the frequency of Ca2+ transients under ischemic conditions is related to the decrease in wall shear stress to virtually 0 dyn/cm2. However, an alternate possibility is that the reduction in Ca2+ transients was a result of the depletion of luminally available endogenous Ca2+ agonists (e.g., hormones, nutrients) or modifications in shear independent aspects of flow sensitivity (discussed below).

Reports indicating that P2x receptors are involved in shear stress-sensitive Ca2+ influx in human umbilical vein ECs (41) targeted this receptor as a likely candidate for regulation of Ca2+ transients in situ. However, in the presence of the P2x receptor antagonist PPADS, EC Ca2+ transients were not different from those observed in control preparations. We (12) have shown elsewhere that treatment with PPADS effectively inhibits P2x receptor-mediated vasomotor responses, indicating that P2x receptors are present in the cells of the arteriolar wall and are indeed sensitive to the antagonist. Thus the lack of P2x receptor involvement on basal Ca2+ transients found in the present work suggests that either these events are not shear dependent or alternatively, that a different signaling pathway mediates shear sensitivity in situ.

Inhibition of P1 receptors also failed to modify the frequency of basal Ca2+ transients, indicating that as for P2x receptors, stimulation of this pathway does not underlie the spontaneous EC Ca2+ signals. However, our results with exogenous Ado show that indeed P1 receptors can modulate the transitory EC Ca2+ activity. The effects of Ado were twofold, comprising increases in both the frequency of events and also in the synchronization of Ca2+ activity between cells. We hypothesize that synchronization of Ca2+ transients in arterioles in situ may drive ECs to act as a functional unit, the final outcome of which is an integrated alteration of vascular resistance. While P1 purinergic receptors on SMCs have traditionally been considered the primary target for vasomotor changes produced by Ado, there is a growing body of evidence that Ado can act via an endothelial pathway (12, 38). The findings of the current study clearly substantiate the latter notion. Our comparison of shear rate in the absence and presence of Ado indicates that the increase in frequency of Ca2+ transients with Ado is not a result of exposure to augmented levels of shear stress, thus biochemical signals initiated by direct receptor-ligand interactions are a more plausible pathway for the EC Ca2+ response to Ado. However, because volumetric flow increased during Ado exposure, we cannot rule out some other (shear independent) effect of flow as grounds for the increased Ca2+ activity. For example, circumferential stretch imposed by pressure acting normal to the vessel wall may have altered during arteriolar dilation. Integrins, extracellular matrix proteins, cell adhesion molecules, and ion channels have each been proposed as possible mechanosensitive mediators of responses to fluid flow (17, 30, 36), and any of these could be indirectly responsible for the Ado-induced increase in Ca2+ transients. Further studies will be necessary to identify the specific pathway(s) underlying the Ado-induced response and differential sensitivity in EC Ca2+ transients in arterioles in situ.

In our study, the perinuclear region of ECs typically had higher fluorescence intensity relative to the rest of the cell body but was not necessarily associated with the 1° site of Ca2+ transients. Although background tissue fluorescence remained close to the noise floor of the camera, there was a gradual decrease in cell brightness over the duration of data collection (~1 h after dye loading in each preparation). This is most likely indicative of dye leakage from the cells as has previously been reported for several fluorescent Ca2+ indicators at body temperature (39). Indeed, to facilitate dye retention, Ca2+ fluorophores are often used at room temperature in isolated cell systems, whereas our in situ system required that the skeletal muscle preparation be maintained at its physiological temperature. We circumvented the potential for introduction of artifacts associated with dye leakage and bleaching of the fluorophore over long exposure times (several minutes) by limiting the study only to identification of short-lived Ca2+ transients ({approx}2 s), a time frame over which dye leakage and bleaching were insignificant. For this reason also, we chose to analyze responses to the various treatment conditions primarily via effects on the frequency of Ca2+ transients, and not their amplitude. Furthermore, data included in the analysis were from preparations that maintained the ability to report changes in EC Ca2+ as verified using topical application of ACh at the conclusion of data collection. It is, however, possible that the frequency and spread of the Ca2+ transients may have been underestimated due to the conservative criteria that were used to define the events.

In summary, we report transient increases in endothelial Ca2+ in real time in intact blood perfused arterioles: these spontaneous localized Ca2+ transients are spatially coordinated and occur in ECs in arterioles that exhibit resting tone in situ. This spontaneous activity is contingent on the presence of blood flow. Although the basal activity occurs independently of P1 and P2x purinergic receptors, stimulation with Ado increases the frequency of local Ca2+ events, and also amplifies the synchronization of Ca2+ activity between adjacent cells. The changes in Ca2+ activity during exposure to Ado are via pathways that are not dependent on increases in shear stress. The novel spatial and temporal characteristics of Ca2+ mobilization in arteriolar endothelium described here may indeed underlie mechanisms by which individual cell responses are integrated to result in coordinated vascular responses to vasoactive stimuli. These rapid and synchronous Ca2+ transients should be taken into consideration in the understanding of both individual and communicated (EC-EC and EC-SMC) endothelial signaling processes in the blood vessel wall.


   GRANTS
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 ABSTRACT
 METHODS
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 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants HL-56574 and HL-18208.

 


   ACKNOWLEDGMENTS
 
We thank Drs. Michael B. Kim and David I. Yule for helpful discussions and Patricia A. Titus for skilled technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. H. Sarelius, Dept. of Pharmacology and Physiology, Univ. of Rochester Medical Center, Box 711, Rochester, NY 14642 (E-mail: ingrid_sarelius{at}urmc.rochester.edu).

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.


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