INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

SMOOTH MUSCLE CELL (SMC) and endothelial cell (EC) functions in the arterial wall are linked by complex intercellular signaling processes. The close apposition of the two cell types enables a signal derived from one cell to rapidly diffuse to neighboring cells not only of the same type (homocellular communication) but also of the other type (heterocellular communication). For example, agents that stimulate a rise in the intracellular calcium concentration ([Ca²⁺]_i) of ECs can cause the release of vasodilators such as the endothelium-derived hyperpolarizing factor and the endothelium-derived relaxing factor (32). Endothelial cell products can also modulate the magnitude of the arterial diameter response to a vasoconstrictor, as evinced by the greater constriction after endothelium removal or nitric oxide (NO) synthesis blockade (3, 4, 14). Interestingly, such effects have been reported during stimulation with agents such as phenylephrine (PE) and KCl, which are thought to have little or no direct effect on ECs. Because SMCs and ECs are electrically and chemically coupled (23, 35), a possible signaling pathway is the diffusion through myoendothelial gap junctions (22).

The goal of the present study was to analyze the intercellular calcium communication between SMCs and ECs by simultaneously monitoring artery diameter and [Ca²⁺]_i in a perfused artery in vitro under physiological conditions. This is motivated by the fact that the contractile state of an artery depends very much on the [Ca²⁺]_i not only of the SMCs but also of the ECs (18). It was thus interesting to see whether the calcium dynamics in one type of cells can influence that of the other and in what way. If a calcium increase in one type of cells also generates a calcium rise in the other, then this could indicate an intrinsic regulatory feedback mechanism within the arterial wall. Indeed, if calcium rises in the SMCs, this contracts the artery; on the other hand, an increase of calcium in the ECs leads to the formation of NO and/or other endothelium-derived relaxing factors and thus tends to relax the vessel.

So far, there has not been a widely established method for monitoring calcium dynamics together with diameter for both SMCs and ECs in vessels perfused under physiological conditions. Many studies (5, 18) have already been carried out on cultured and/or isolated cells or even in coculture, but even though this has already given a very good idea about the different intracellular mechanisms, the natural environment and morphology of the cells was altered and thus likely their behavior and function. It is thus essential to measure the ubiquitous calcium also in an intact tissue, preserving in this way the natural environment of the cells. This also has the advantage of allowing us to relate the microscopic calcium dynamics to macroscopic quantities like the vessel diameter, pressure, and flow. Moreover, because calcium dynamics can be monitored separately in SMCs and ECs, this enables the study of intercellular communication between the two types of cells in their natural environment.

Calcium measurement in intact arteries has until now been limited (12, 13, 19, 21, 24, 25, 31, 33, 36), giving generally only an overall mean calcium concentration in the SMCs but not in both the SMCs and ECs. To the best of our knowledge, only two studies (13, 36) have attempted this, but the method was not adapted to vessels with their diameter free to vary on change of vascular smooth muscle tone. In the present study, we have developed a new method, which allows the simultaneous measurement of intracellular calcium dynamics together with vessel diameter for both SMCs and ECs, pressure, and flow rate in an arterial segment perfused under physiological conditions.

A typical application of this method could be the study of vasomotion. Indeed, vasomotion (i.e., the spontaneous oscillation of vessel diameter, uncorrelated with either heartbeat or any other physiological rhythm) is thought to be due to ionic fluxes across the cell membrane (1, 15, 16, 29; Oishi H, Schuster A, Stergiopulos N, Meister J-J, and Bény J-L, unpublished observations) and especially Ca²⁺.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Preparation of arteries. Male Wistar rats weighing 200-300 g were anesthetized with halothane, and the mesenteric arcade was excised and placed in a modified Krebs-Ringer solution [physiological saline solution (PSS)] at 4°C containing (in mM) 145 NaCl, 5 KCl, 1 CaCl₂, 0.5 MgSO₄, 1 Na₂HPO₄, 20 HEPES, and 23 Tris base. Second-order mesenteric arteries were then cleaned from extracellular tissue and cannulated at both ends using stainless steel cannulas in a homebuilt micro-organ cannulation chamber (capacity 0.5 ml; Fig. 1). After cannulation, vessels were pressurized at 50 mmHg and stretched until they appeared straight (passive diameter 400 ± 40 µm). Only vessels without leaks were used. Perfusion with PSS at a flow rate of 50 µl/min was induced using a syringe pump. The temperature of the cannulation chamber was then slowly raised to and kept at 37°C with a continuous superfusion of the same PSS at a rate of 1.5 ml/min.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Schematic view of setup (not to scale). The artery is secured with 8-0 nylon filaments on two stainless steel cannulas and perfused at 50 µl/min with physiological saline solution (PSS). The distal end is linked to a pressure column of 50 mmHg. A superfusion system brings PSS at 37°C into the cannulation chamber and is then discarded. Light emitted from a Xe lamp passes through an excitation filter (340 and 380 nm, respectively) and shutter before being deflected by the dichroic mirror through the inverted objective. This excites the dye present in the arterial wall [either smooth muscle cells (SMCs) or endothelial cells (ECs)], thus inducing fluorescence, which is recovered by the objective and passed onto a charge-coupled device (CCD) camera linked to a computer. The entire system is controlled by the software Openlab (Improvision, UK).

Diameter measurement. Images of the artery were taken at regular intervals (~4 images/s) with a digital image acquisition system (Openlab, Improvision, UK), which then gave the diameter and fluorescence images. The diameter is accurate to within pixel resolution (images are 512 × 512 pixels), thus yielding a precision of ~2 µm.

Measurement of [Ca²⁺]_i fluorescence imaging. Fura 2-acetoxymethyl ester (AM) (50 µg) was dissolved in 50 µl DMSO containing 2% pluronic solution and suspended in 5 ml of PSS. When entering a cell, the fura 2-AM is deesterified by enzymes, thus releasing a free fura 2 molecule. When a calcium ion then binds to this molecule, it increases its fluorescence when excited at 340 nm and decreases it when excited at 380 nm. The ratio obtained when dividing the fluorescence obtained when excited by 340 nm by that obtained for the 380-nm excitation is then proportional to the free cytosolic calcium concentration. For the measurement of SMC intracellular calcium, this loading solution was put into the superfusion for 1.5 h at room temperature, followed by a washout period of 30 min at 37°C. For the measurement of EC intracellular calcium, the loading solution was perfused for 25 min at room temperature, followed by a washout period of 30 min. Excitation was achieved by fluorescence microscopy using a 100-W xenon light source and a filter wheel rotating at ~4 Hz and containing 340- and 380-nm interference filters. Care was taken to limit the amount of exposure time to the fluorescence light. The emitted fluorescence was collected by a ×10 Fluar objective (numerical aperture 0.50) mounted on a Zeiss Axiovert S 100 TV inverted microscope before passing onto a charged-coupled device camera, permitting subsequent image analysis with the Openlab software for calculation of the ratio images and diameter. Calcium concentration was expressed as the 340-to-380-nm fluorescence intensity ratio. The imaging and measurements, carried out at a rate of ~4 Hz, allows us to select a very small region on the vessel (0.25 mm²) or even several different regions, thus providing a tool to measure local calcium concentration and even calcium wave propagation along the vessel.

Chemicals. All vasoactive agents acting on the SMCs [PE and/or norepinephrine (NE)] were applied by changing the superfusion solution; acetylcholine (ACh), which acts directly on ECs, was added to the perfusion. The gap junction uncouplers palmitoleic acid and 18-glycyrrhetinic acid were simultaneously added to both the superfusion and perfusion solutions. All chemicals were obtained from Sigma (Buchs, Switzerland) except the dyes, which were obtained from Molecular Probes (Leiden, The Netherlands).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Validation of experimental technique: simultaneous calcium and diameter measurements. Superfusion of the vessel with fura 2 from the adventitial side loads the SMCs (Fig. 2A) but not the ECs; only the outline of the SMCs is visible on the fluorescence images. Similarly, perfusion of the dye through the lumen of the vessel loads the ECs but not the SMCs (Fig. 2B), thus allowing a good separation of the signals from the two types of cells.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   A: SMCs perpendicular to the vessel axis of a fura 2-loaded artery squeezed between two coverslips at the end of an experiment. Only cells parallel to the vessel axis can be seen, even when varying the focal plane throughout the vessel wall, which confirms that only SMCs and no ECs have been loaded. B: ECs parallel to vessel axis of a fura 2-loaded artery perfused at 50 µl/min and pressurized at 50 mmHg. Only the outlines of cells parallel to the vessel axis can be seen, even when varying the focal plane throughout the vessel wall, which confirms that only ECs and no SMCs have been loaded.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   A: response to ACh; calcium level of ECs. Fluorescence signals for excitation at 340 and 380 nm and the 340-to-380-nm (340/380) fluorescence ratio and diameter are shown. Vessel was perfused at 50 µl/min and pressurized at 50 mmHg. B: calcium level of ECs. When ACh is no longer added to the PSS, the calcium level in ECs decreases, but the diameter of the artery remains unchanged. Time 0 corresponds to the moment when the ACh is no longer added. C: calcium level in SMCs. When ACh is no longer added, the artery begins to recontract very slowly and gradually, but the calcium level in SMCs remains unchanged. au, Arbitrary units.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Diameter (solid line) and fluorescence (dashed line) dynamics as a response to phenylephrine (PE) or ACh. A: 0.5 µM PE stimulation on SMCs. The half-maximal increase in calcium (time t50[Ca]) precedes the half-maximal diameter variation (time t50[d]) by ~248 s. B: 1.0 µM ACh stimulation on SMCs in the presence of 0.5 µM PE. The half-maximal changes in calcium and diameter nearly coincide, with diameter slightly preceding calcium by ~5 s. C: 0.5 µM PE stimulation on ECs. Calcium precedes the diameter variation by ~34 s. D: 1.0 µM ACh stimulation on ECs in the presence of 0.5 µM PE. The oscillations observed after a slight recontraction are not motion artifacts but correspond to "spontaneous" vasomotion, which occurs sometimes but not always after ACh stimulation in the presence of PE; calcium precedes diameter by ~16 s. C and D are from a different arterial segment than A and B.

Heterocellular calcium fluxes. To establish whether there is a heterocellular calcium flux between the SMCs and ECs, we used the gap junction uncouplers 18-glycyrrhetinic acid and palmitoleic acid. In the presence of 18-glycyrrhetinic acid, PE still gave an important increase of calcium level in ECs (Fig. 5C) of an average of +18.4 ± 6.0% (n = 6), which is significantly greater (P < 0.01) than the increase caused by PE in the absence of the uncoupler (Fig. 4C; +7.7 ± 3.0%, n = 19). On the other hand, palmitoleic acid nearly completely abolished this increase of calcium in ECs during PE stimulation (Fig. 5D) to +2.4 ± 1.5% [significantly less (P < 0.01) than in the absence of the uncoupler]. Concerning the calcium in SMCs, there is no significant difference (P > 0.05) between the presence (Fig. 5A; +14.9 ± 0.7%, n = 3) or the absence (Fig. 4A; +17.7 ± 2.9%, n = 6) of 18-glycyrrhetinic acid during PE stimulation. On the other hand again, there still is an increase of calcium in SMCs of +2.7 ± 0.8% (n = 6) during PE stimulation in the presence of palmitoleic acid (Fig. 5B), but it is also significantly decreased (P < 0.01) with respect to the PE stimulation in the absence of the uncoupler (+17.7 ± 2.9%, n = 6).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Diameter (solid line) and fluorescence (dashed line) dynamics as a response to PE in the presence of a gap junction uncoupler. A: calcium level in SMCs together with diameter; action of PE in the presence of the gap junction uncoupler 18-glycyrrhetinic acid (GA). The half-maximal increase in calcium (at t50[Ca]) precedes the half-maximal diameter (at t50[d]) variation by ~9 s. B: same as A but with the gap junction uncoupler palmitoleic acid (PA) instead of GA. The calcium variation precedes the diameter variation by ~203 s. Notice the relatively late response of the vessel to the PE stimulation compared with the absence of PA; C and D: same as A and B, respectively, but with fluorescence of ECs instead of SMCs. Phase lags between calcium and diameter are ~5 and 85 s, respectively. We can see that in the presence of PA, the calcium increase in the ECs transmitted from the SMCs through gap junctions is strongly reduced, whereas it is still quite significant in the presence of GA. This suggests that PA is more efficient in uncoupling the gap junctions than GA. Each figure comes from a different arterial segment.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Diameter (solid line) and fluorescence (dashed line) dynamics as a response to the gap junction uncouplers (in the absence of any other stimulus). A: calcium level in SMCs together with diameter; action of the gap junction uncoupler GA. B: same as A but with the gap junction uncoupler PA instead of GA. C and D: same as A and B, respectively, but with calcium of ECs instead of fluorescence of SMCs. The half-maximal increase in calcium (at t50[Ca]) precedes the half-maximal diameter (at t50[d]) variation by ~44 and 1.5 s, respectively. In each case, the gap junction uncoupler relaxes the vessel slightly, with a slight decrease of the calcium level in SMCs, and a transient increase in the fluorescence of ECs. Each figure comes from a different arterial segment.

Vasomotion. Vasomotion induced by 0.5 µM PE or a combination of 0.5 µM PE and 1.0 µM ACh is, in itself, still another proof for the validity of the method, because the observed "anti-phase shift" between the calcium and diameter signals (the delay between the maximum of the calcium oscillations and the minimum of the diameter oscillations) of ~1.7 ± 0.3 s rules out any possibility of motion artifacts. Indeed, it is first the calcium that increases in the SMCs (and also in the ECs), then the diameter that decreases, and then the calcium that decreases before the diameter relaxes again (Fig. 7, A and B).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Diameter (solid line) and fluorescence (dashed line) dynamics during vasomotion. A: calcium level in SMCs together with diameter. Example of vasomotion induced by 0.5 µM PE; mean period (n = 4) observed: 5.9 ± 3.1 s; "anti-phase" shift: 1.7 ± 0.3 s. B: same as A but with the calcium level in ECs; mean period (n = 4): 5.4 ± 0.6 s; anti-phase shift: 2.1 ± 0.4 s. Each figure comes from a different arterial segment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

To the best of our knowledge, this is the first study reporting simultaneous measurements of calcium dynamics together with the resulting diameter response for both SMCs and ECs in an intact artery perfused in vitro. The study reveals an important calcium coupling between SMCs and ECs, suggesting a heterocellular calcium flux via gap junctions. This coupling is also present during vasomotion.

One of the main advantages of this new experimental method is its ability to distinguish between the calcium concentration of ECs and that of SMCs. Because calcium variations on these two types of cells have often opposite tendencies, a signal that would contain both simultaneously would be difficult to interpret. However, the method described allows us to load the two types of cells separately with fura 2, one at a time, leaving the other unloaded. The point here is that when the SMCs are loaded, one can see only SMCs (outlines of cells perpendicular to the vessel axis) and no ECs at all (no outlines of cells parallel to the vessel axis), even when the focal plane is varied throughout the whole thickness of the vessel wall. On the other hand, when using the EC loading protocol, only cells whose alignment is parallel to the vessel axis are loaded, and no SMCs (outlines of cells perpendicular to the vessel axis) can be seen, even when the focal plane is changed throughout the vessel wall. It is thus the orientation of the loaded cells with respect to the vessel axis that allows us to determine whether the outline of the observed cells corresponds to SMCs or ECs.

One could think that once the dye is in one type of cells, it could perhaps diffuse through gap junctions to the other type of cells. However, whereas fura 2 is indeed permeable through gap junctions comprised of connexin (Cx)43, this connexin is extremely rare in ECs, which contain mostly Cx37 and Cx40. Only at very specific points, like arterial bifurcations, can some Cx43 be found in ECs. It is thus not very probable that homotypic Cx43-Cx43 gap junctions form between SMCs and ECs. The exact structure of the heterocellular gap junction is as yet unknown. A new connexin (Cx45) has recently been discovered in ECs, but its properties are not yet known. On the other hand, Lucifer yellow staining has revealed that there is no heterocellular dye transfer between the two types of cells (figures not shown, see also Ref. 30).

The use of ratiometric dyes like fura 2 is essential in the study of calcium dynamics in vessels because of the natural movement of the vessel (contraction/dilation, vasomotion). For example, on relaxation due to an increase of [Ca²⁺] in the ECs, the fluorescence at 340 nm (per unit vessel volume) increases and that at 380 nm (per unit vessel volume) decreases, but, because the fluorescence that reaches the objective comes from a smaller vessel volume when the vessel dilates (less volume in focal plane and less surface concentration of the dye within the focal plane), both fluorescence signals decrease, with a stronger decrease of the signal at 380-nm excitation compared with the one at 340-nm excitation (Fig. 3A). The ratio, however, is independent of the volume providing the fluorescence and thus accurately reflects the calcium dynamics independently of any motion of the vessel. It is also independent of local dye concentration differences and gives thus the same result no matter where it is measured in the vessel wall.

The intracellular calcium level was expressed as an uncalibrated fluorescence ratio in the present study, with always the same parameters (threshold for background subtraction, range for the fluorescence ratio) for the data analysis, thus providing comparable but only qualitative measurements. A calibration has not been carried out mainly due to time considerations. Also, the commonly used calibration methods are based on a hypothetical dissociation constant of the fura 2-calcium complex, which actually varies according to the conditions of the vessel, thus leading to new uncertainties (26).

The results of the different tests of the method are summarized in Table 1. ACh induces Ins(1,4,5)P₃ formation in ECs (10), thus leading to Ca²⁺ release from intracellular stores. The increase of intracellular [Ca²⁺] in the ECs catalyses NO formation (34), thus inducing a decrease of [Ca²⁺]_i in the SMCs and an endothelium-dependent relaxation (7). The results obtained agree again very well with this scenario: [Ca²⁺]_i increasing in the endothelium and decreasing in the SMCs on ACh stimulation (Fig. 4, B and D). The delay between the increase of Ca²⁺ in the ECs and its decrease in the SMCs (~32 ± 14 s) reflects the time necessary for NO formation and diffusion to the SMCs. Kanai et al. (20) found that the NO formation in the ECs takes ~2 s after calcium increase, which means that the observed delay corresponds mainly to the time necessary for NO diffusion from the ECs to the SMCs and the initiation of the relaxation pathway in the SMCs.

The time lags observed in each case between the EC₅₀ changes of calcium and diameter show again that the observed signals are not motion artifacts, and because calcium generally precedes the diameter variation (except for ACh stimulation on SMCs, where calcium very slightly follows the diameter), this confirms that the diameter changes result from calcium changes.

PE, an ₁-agonist, induces Ca²⁺ release in the SMCs, thus causing a rise in [Ca²⁺]_i (Fig. 4A). Since there are no ₁-receptors on the ECs in the vessels studied (14), PE acts only on the SMCs. Nevertheless, we also observed a slight increase in the [Ca²⁺] of the ECs during PE stimulation (Fig. 4C), which suggests an intercellular propagation of Ca²⁺ from the SMCs to the ECs through gap junctions (6, 13). Indeed, when [Ca²⁺]_i changes in the SMCs, this ion will tend to equilibrate its concentration by diffusion through heterocellular gap junctions coupling the ECs to the SMCs (11). In this way, the change of [Ca²⁺]_i in the SMCs is reflected in the ECs. Because PE stimulation increases [Ca²⁺]_i through the production of Ins(1,4,5)P₃, it is nevertheless not clear whether it is calcium itself or Ins(1,4,5)P₃ that diffuses through gap junctions to influence directly or indirectly the [Ca²⁺]_i of ECs. In any case, the molecule, Ca²⁺ or Ins(1,4,5)P₃, must diffuse through gap junctions. Therefore, the possible implication of heterocellular gap junctions in the control of calcium in the ECs in an intact artery was tested using molecules known to uncouple the cells linked by gap junctions: 18-glycyrrhetinic acid and palmitoleic acid.

Concerning these commonly used gap junction uncouplers 18-glycyrrhetinic acid and palmitoleic acid, we have observed that the latter is much more effective in uncoupling gap junctions than the former. Indeed, even though both uncouplers cause a transient increase in calcium of ECs by direct action (Fig. 6, C and D), showing that they had some effect, the calcium of ECs increase on SMC stimulation was not reduced by 18-glycyrrhetinic acid, whereas it was nearly completely abolished by palmitoleic acid (Fig. 5, C and D). This fact is confirmed by a study on coronary artery strips, where we used scrape dye loading to determine the efficiency of the uncouplers (Budel S, Chabaud F, Schuster A, and Bény J-L; unpublished results). In this study, the dye remained within the scrape-loaded cells in the case of palmitoleic acid uncoupling but was still able to spread to a few adjacent cells when 18-glycyrrhetinic acid was used as the uncoupler.

The results obtained are in agreement with those of Dora et al. (13, 14) who observed an increased NO synthesis on PE or KCl stimulation of the SMCs, thus also indicating a rise of calcium in the ECs on SMC calcium increase. This increase was hypothesized to be due to a calcium flux from the SMCs to the ECs, but another signaling molecule diffusing through the gap junctions, like Ins(1,4,5)P₃ or a still unrecognized smooth muscle-derived relaxing factor, could not be excluded. Even though it was not clear then whether the signals diffused through gap junctions or through a different pathway, we can now conclude that the signal passes indeed through the gap junctions. The hypothesis that the signal is calcium itself, and not Ins(1,4,5)P₃ or another molecule, is supported by the following considerations. Because the relative volume of the ECs compared with the SMCs is small, there needs to be only a rather small calcium flux to change the calcium concentration of ECs significantly. Furthermore, the distance the calcium needs to diffuse is relatively small (estimated at ~2 µm), due to the reduced size of the ECs (diameter ~1 µm) and the thickness of the internal elastic lamina (~1.6 µm). On the basis of the diffusion coefficient for Ca²⁺ in cytosolic extracts (2), this yields an estimate of the diffusion time from a SMC to an adjacent EC of <100 ms, a period within our present detection limit. It must be noted that the reported diffusion coefficient for Ins(1,4,5)P₃ is even greater (2), which means that an Ins(1,4,5)P₃ diffusion from SMCs to ECs cannot be excluded.

The direct effect of palmitoleic acid on the calcium of ECs was not really expected but can be explained in the following way: ECs generally possess less active cell membrane calcium extrusion pumps than SMCs, which is shown by the fact that ionomycin (which permeabilizes the cell membrane) does not cause a calcium increase in cultured porcine coronary artery SMCs but causes saturation of the calcium dye in ECs (8). This could mean that to decrease the calcium in ECs, calcium needs to be extruded partially via the SMCs through gap junctions. When the gap junctions are blocked, this extrusion can no longer take place, and the calcium concentration in the ECs increases. The fact that this increase is only transient (Fig. 6, C and D) could be explained by the enhancement of the Ca²⁺ membrane pump activity at very high [Ca²⁺]_i and/or the activation of another extrusion mechanism after a certain time of elevated [Ca²⁺]_i. It is quite likely that, after a prolonged high calcium level in the ECs, calcium sequestration through organelles takes place, thus reducing the free cytosolic calcium concentration.

Vasomotion is a phenomenon that has been thought to be due to calcium fluxes through the cell membrane or to calcium release from intracellular stores, most probably a combination of the two mechanisms (28), but this is the first time that calcium has actually been measured during vasomotion not just in the SMCs but also the ECs. The results strongly suggest a calcium coupling between the two cell types, because vasomotion originates in the SMCs and is thus reflected only in the ECs. One would expect there to be a certain phase shift between the calcium oscillations in the SMCs and ECs, but the time resolution of our system (~4 images/s) has not allowed us to detect any statistically significant differences. However, there is clearly a phase shift between the calcium and the diameter oscillations, which reflects the time needed for the contractile apparatus to develop the necessary tone on calcium increase. Furthermore, this phase shift also confirms that we have been successful in ruling out any motion artifacts with our ratioing technique, because motion artifacts would result in simultaneous variations of diameter and fluorescence.

The method developed so far can be extended to add the measurement of membrane potential using electrophysiology (Oishi H, Schuster A, Stergiopulos N, Meister JJ, and Bény J-L; unpublished results), thus allowing the simultaneous monitoring of intracellular calcium, membrane potential, and diameter.

Several groups have already done some calcium imaging on intact vessels but always with some limitations. The main advantages of this method with respect to existing methods are the following: 1) it does not require artificial immobilization of the vessel, thus allowing correlation of diameter measurements to intracellular calcium dynamics; 2) it allows us to follow calcium dynamics in the SMCs or ECs selectively; 3) it can be used for a large range of vessel sizes; and 4) it does not present the problems associated with dual probes, like colocalization, similar binding dynamics, or different loading speeds for the two dyes.

In conclusion, we have established a method that allows the simultaneous measurement of intracellular calcium together with the diameter of an intact arterial segment perfused in vitro under physiological conditions for both SMCs and ECs. The study has revealed an important calcium coupling between SMCs and ECs, suggesting a heterocellular calcium flux via gap junctions. This method can be a useful experimental tool for the study of arterial physiology and is especially adapted to measurements where a change in smooth vascular tone can give rise to important diameter variations, like during vasomotion and vasospasm.