INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

THE VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) family of polypeptides is a series of powerful chemokines that act on the microcirculation to increase delivery of nutrients to tissues (1). They perform this function through three main mechanisms of action. Acutely, they act as vasodilators and hence decrease vascular resistance and increase blood flow to tissue (24). They also result in increased microvascular permeability, both acutely over a period of a few minutes and chronically over a period of days. In addition, they are highly angiogenic and result in the formation of new blood vessels from an existing microvasculature (1). All three of these mechanisms that result in increased solute delivery occur through direct activation of microvascular endothelial cells in arterioles (vasodilatation), capillaries (angiogenesis and increased permeability), and venules (increased permeability). These growth factors have been shown to be critically upregulated in a variety of pathological conditions associated with angiogenesis and permeability, including all solid tumors so far investigated, diabetic retinopathy, psoriasis, and rheumatoid arthritis (1). They are currently being investigated as angiogenic stimulators to enable revascularization of underperfused tissue after myocardial infarction and in peripheral ischemia (20).

The mechanisms of actions of VEGF are now beginning to be elucidated. VEGFs have been shown to bind to three receptors: flt-1 (VEGFR-1), flk1/KDR (VEGFR-2), and flt-4 (VEGFR-3, found only on lymphatic endothelial cells) (30). Stimulation of these receptor tyrosine kinases results in phosphorylation and activation of phospholipase C- (PLC-), both in vivo (28) and in vitro (36), and so results in production of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (7) in large-vessel endothelial cells in vitro. VEGF has been shown to increase endothelial intracellular calcium concentration ([Ca²⁺]_i) by increasing Ca²⁺ influx across the plasma membrane in endothelial cells in vitro (7), and this Ca²⁺ influx across the plasma membrane is responsible for the increase in permeability seen in vivo (4). Increased [Ca²⁺]_i has previously been shown to result in increased microvascular permeability (16) through Ca²⁺-dependent activation of nitric oxide (NO) synthase (15) and subsequent production of NO and cGMP (17), and both of these have also been shown to be produced after VEGF stimulation (24).

Many of the signaling pathways stimulated by VEGF in endothelial cells in culture and some of those in vivo have therefore been identified. It is far less clear which of these pathways actually results in the increase in permeability and increased [Ca²⁺]_i. We have previously shown that the VEGF-mediated increase in permeability is dependent on stimulation of Ca²⁺ influx across the plasma membrane (4), but the mechanism that results in increased [Ca²⁺]_i after VEGF stimulation has not been shown. One hypothesis is that stimulation of receptor tyrosine kinases results in PLC- activation, which produces IP₃. This acts on IP₃ receptors on the endoplasmic reticulum to release Ca²⁺ from internal stores (5). This may be great enough to result in a transient increase in [Ca²⁺]_i. A second possibility is that release of Ca²⁺ from internal stores generates a Ca²⁺ release-activated Ca²⁺ (CRAC) influx across the plasma membrane, by capacitative Ca²⁺ entry (12), and it is the subsequent influx of Ca²⁺ that results in increased [Ca²⁺]_i. This pathway has been shown to result in Ca²⁺ influx into endothelial cells in culture when stimulated with ATP, but it is not currently known whether this pathway is the mechanism by which [Ca²⁺]_i increases in endothelial cells in vivo or whether it is responsible for the increases in permeability that result from exposure to ATP or VEGF. It has not even been shown that VEGF does cause Ca²⁺ release from intracellular stores in endothelial cells in vivo or whether the increase in permeability is a result of this Ca²⁺ release. A third hypothesis is that VEGF acts to increase Ca²⁺ influx across the plasma membrane through a store-independent mechanism, possibly by acting on receptor-operated Ca²⁺ channels. To determine which, if any, of these three hypotheses is correct, we measured the increase in [Ca²⁺]_i and permeability brought about by VEGF and ATP under conditions where the Ca²⁺ stores have been depleted and therefore do not release Ca²⁺. Ca²⁺ stores may be depleted by perfusion of vessels with thapsigargin, an irreversible inhibitor of the endoplasmic reticulum Ca²⁺-ATPase (26). The sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA) pumps Ca²⁺ from the cytoplasm into the endoplasmic reticulum. There is a slow but steady leak of Ca²⁺ from the stores into the cytoplasm, so inhibition of this protein by use of thapsigargin results in a steady depletion of Ca²⁺ stores. Although thapsigargin has been extensively used in endothelial (and other) cells in culture, there has been only one study to date investigating the effect of thapsigargin on [Ca²⁺]_i of endothelial cells of the microvasculature in vivo (37). Therefore, we also investigated the effect of thapsigargin on endothelial [Ca²⁺]_i and permeability. Some of these results have previously been published as abstracts (2, 34).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Frog preparation. All experiments were carried out on male leopard frogs (20-35 g). Hydraulic conductivity (L_p) measurements were made in Rana temporaria supplied by Blades, and Ca²⁺ measurements were made in Rana pipiens supplied by J. M. Hazen, V.T. All chemicals were purchased from Sigma unless otherwise specified. ATP was perfused at 30 µM and VEGF at 1 nM, because these doses have previously been shown to give a reproducible increase in both [Ca²⁺]_i and L_p. Thapsigargin (Calbiochem) was perfused at 100 nM, because this has been shown to effectively inhibit SERCA in a variety of animal species and does not result in inhibition of other Ca²⁺ pumps.

Measurement of L_p. Frogs were anesthetized by immersion in 1 mg/ml MS-222 (3-aminobenzoic acid ethyl ester) in water, and anesthesia was maintained by superfusion of the gut with 0.1-0.25 mg/ml MS-222 in frog Ringer solution (111 mM NaCl, 2.4 mM KCl, 1 mM MgSO₄, 1.1 mM CaCl₂, 0.20 mM NaHCO₃, 2.63 mM HEPES acid, and 2.37 mM HEPES sodium salt). The pH of this solution was 7.40 ± 0.02 at room temperature. The animal was laid supine, and the limbs were secured lightly. A small incision (8-10 mm) was made in the right lateral skin and muscular body wall. The distal ileum was floated out and carefully draped over a 1-cm-diameter transparent quartz pillar. The microvessels in the mesentery were visualized under a inverted microscope (Leica DMIL). A video camera (Panasonic WVBP32, 8 mm) was attached to the top of the microscope to allow for binocular visualization and simultaneous recording of a 270-µm segment of the vessel (out of a total length of 800-2,000 µm). The video was connected through an electronic timer (ForA VT33) to a video cassette recorder (Panasonic AG7350; Panasonic, Bracknell, UK). The upper surface of the mesentery was kept continuously superfused with frog Ringer solution during the entire time that it was exposed. All experiments were carried out at room temperature (20-22°C). At the end of the experiment, the frog was killed by destruction of the cranium.

7

1

1

Calculation of L_p. The transcapillary water flow per unit area of capillary wall (J_v/S) was calculated from the initial velocity of the red blood cells (dl/dt, change in length over change in time) after occlusion, the capillary radius (r), and the length between the marker cell and the point of occlusion (l), all of which were measured offline from the videotape
The L_p was calculated from the Starling equation
where P is the effective hydrostatic and oncotic pressure difference between the capillary and the interstitium. The capillary pressure was set at 30 cmH₂O, so P was 26.4 cmH₂O (1% BSA has an effective oncotic pressure of 3.6 cmH₂O), with the assumption that tissue pressure was negligible, and tissue oncotic pressure was equivalent to that in the superfusate (zero).

Measurement of [Ca²+]_i. [Ca²⁺]_i was measured in frog mesenteric microvessels as previously described (4). In brief, the frog was pithed and laid supine, and the limbs were secured to a supporting tray. The abdominal cavity was opened by making incisions on both sides of the trunk and across the midline. The body wall flap was then lifted back over the upper body to expose the viscera, which were held in place by cotton wool soaked in Ringer. The mesentery was floated over a glass coverslip attached to the supporting tray and held in place by pinning of the gut to the edge of the coverslip. This allowed for observation of a perfused microvessel with the short working distance lens necessary for fluorescence measurement. The upper surface of the mesentery was continuously superfused with Ringer, and the temperature of the superfusate was kept at 20-22°C. Vessels chosen for cannulation were postcapillary venules (first-, second-, and third-order venules) of diameter 25-40 µm. Vessels were visualized under an epifluorescence microscope (Leitz Diavert) equipped with quartz optics in the excitation pathway, a photomultiplier tube (Leitz MPV) and excitation filter changer (Kinetek) under computer control, and a 100-W mercury lamp. A selected vessel was cannulated and perfused with 1% BSA in Ringer. Fluorescence intensity (I_f), collected by a dry Fluotar lens (20×, numerical aperture 0.75), was measured from a window 150-µm long and 40-µm wide that was placed ~200 µm downstream of the cannulation site. I_f values at excitation wavelengths of 340 ± 5 and 380 ± 5 nm (selected by 2 narrow band interference filters) and emission at 500 ± 35 nm were collected by use of a 0.25-s exposure to give an initial background I_f for each vessel that could be used to estimate fura loading. All I_f values were measured during perfusion at a pressure of 30 cmH₂O. The vessel was then perfused with frog Ringer containing 5 µM fura 2-AM and 1% BSA for 60-120 min in the dark. The vessel was briefly examined during this time by illumination with 380-nm light to check that even loading was occurring. Once the I_f had reached 6-10× background, the vessel was perfused for 10 min with 1% BSA to give a baseline Ca²⁺ reading.

Calculation of [Ca²+]_i. [Ca²⁺]_i was calculated from the equation (16, 35)
where R is the normalized ratio of I_f calculated as
where R_exp = (I_f 340  B₃₄₀)/(I_f 380  B₃₈₀). I_f 340 is the I_f with excitation at 340 nm, I_f 380 is the I_f with excitation at 380 nm, and B₃₄₀ and B₃₈₀ are the background I_f values at excitations of 340 and 380 nm, respectively (measured as the I_f after Mn²⁺ quenching). R_max is the in vitro ratio at saturating Ca²⁺ concentration normalized to R_min. R_min is the in vitro ratio for zero Ca²⁺ concentration, and K is the product of the effective dissociation constant for fura 2 and the ratio of the in vitro I_f 380 for zero and saturating Ca²⁺. K was estimated from an in vitro calibration curve for fura 2, as previously described (4).

Data analysis and statistics. To compare responses of different vessels, individual readings from single vessels were normalized relative to baseline and then time averaged in 15-s (for L_p measurement) or 10-s (for [Ca²⁺]_i measurement) bins, starting immediately after perfusion with agonist. The means ± SE of the time-averaged measurements for all the vessels within that group were then calculated. The peak of the time-averaged data was therefore always lower than the actual peak value for the vessel, by definition. The mean peak values for the vessel will therefore be greater than the time-averaged and vessel-averaged [Ca²⁺]_i and L_p.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effect of inhibition of Ca²+ influx on VEGF-induced changes in endothelial cell [Ca²⁺]_i. Four vessels were perfused with 1 nM VEGF, and [Ca²⁺]_i was measured. To investigate the contribution of Ca²⁺ influx across the plasma membrane on VEGF-mediated increased [Ca²⁺]_i, we used 5 mM NiCl₂ to inhibit Ca²⁺ influx into endothelial cells in vivo, as previously described (13). Time-averaged data are shown in Fig. 1 for vessels perfused with and without NiCl₂. In four vessels perfused with 1 nM VEGF, a significant 108 ± 26 nM transient increase in [Ca²⁺]_i was observed from 79 ± 10 to 187 ± 33 nM (P < 0.01). When 5 mM NiCl₂ was included in the perfusate and superfusate, there was no significant increase in [Ca²⁺]_i. After washout of nickel with normal Ringer, perfusion with 1 nM VEGF again caused a significant 190 ± 24 nM transient rise in [Ca²⁺]_i from 95 ± 15 to 285 ± 19 nM (P < 0.001). This was significantly (P < 0.001) higher than the increase in [Ca²⁺]_i in the presence of nickel. The increase in Ca²⁺ was therefore significantly attenuated by perfusion and superfusion with 5 mM NiCl₂

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effect of Ni2+ on the increase in intracellular calcium concentration ([Ca2+]i) brought about by vascular endothelial growth factor (VEGF). Time-averaged Ca2+ measurement of 4 vessels during 1 nM VEGF perfusion in the absence () or presence () of 5 mM NiCl2 is shown. Values are means ± SE. There was no significant increase in Ca2+ during VEGF perfusion with Ni2+ (P > 0.05).

Effect of thapsigargin on [Ca²+]_i and L_p. To investigate the effect of thapsigargin on endothelial [Ca²⁺]_i, 16 vessels were perfused with 100 nM thapsigargin, and [Ca²⁺]_i was measured (see Fig. 2A). Perfusion with thapsigargin resulted in a significant transient increase in [Ca²⁺]_i from 91 ± 11 to 278 ± 28 nM (P < 0.001), usually within 5 min and in all cases within 15 min. [Ca²⁺]_i then returned to control values until it reached a significantly lower (P < 0.001) sustained concentration of 105 ± 14 nM after an additional 5 min, which was not significantly different from the concentration before thapsigargin (not significant, Fig. 2).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effect of thapsigargin (TG) on [Ca2+]i and hydraulic conductivity (Lp). A: [Ca2+]i during perfusion of a vessel with 100 nM TG. B: Lp measurements on a single vessel during perfusion with TG. , Lp during 1 occlusion of the vessel. C: mean ± SE baseline (before TG, solid bars), peak (open bars), and sustained (hatched bars) [Ca2+]i (left) and Lp (right) during TG perfusion. ** P < 0.01 vs. baseline. ++ P < 0.01 vs. sustained. Neither sustained [Ca2+]i nor sustained Lp was significantly greater than respective baseline values.

7

1

1

7

1

1

Effect of thapsigargin on the increase in L_p brought about by VEGF. Measurements of L_p were made on nine vessels perfused with 1 nM VEGF (Table 1). Perfusion with VEGF caused an immediate and transient 5.4 ± 1.1-fold increase in L_p, as previously described (P < 0.05, paired t-test). VEGF was then washed out for 20 min to prevent further responses being masked by the tachyphylaxis previously described. After 20-min perfusion with thapsigargin, L_p was not significantly different from control. Subsequent perfusion with 1 nM VEGF in the presence of thapsigargin caused a rapid 7.5 ± 2.7-fold increase in L_p, which was on average not significantly different from the response to VEGF before thapsigargin perfusion (P > 0.05). The time-averaged responses to VEGF in the presence and absence of thapsigargin are shown in Fig. 3A.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effect of TG pretreatment on VEGF- and ATP-mediated Lp increase. A: time-averaged Lp measurements on 9 vessels during 1 nM VEGF perfusion before () and after () 20-min pretreatment with 1 nM TG. B: time-averaged Lp measurements on 7 vessels during perfusion with 30 µM ATP before () and after () 20-min pretreatment with 1 nM TG. The increase in Lp is calculated as the Lp during agonist perfusion divided by the mean baseline Lp during perfusion immediately before agonist perfusion. Values are means ± SE.

7

1

1

7

1

1

7

1

1

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Effect of store depletion on VEGF- and ATP-mediated increase in Lp. A: Lp measurements on a vessel with 1% BSA and 1 nM VEGF before and after 20-min pretreatment with 100 nM TG. Perfusate composition is shown by boxes. B: Lp increased during perfusion with 30 µM ATP. After 20-min perfusion with TG, 30 µM ATP did not increase Lp. Subsequent perfusion with 1 nM VEGF resulted in a typical transient increase in Lp. C: Lp measurements on a vessel after pretreatment with 30 µM cyclopiazonic acid (CPA), a chemically distinct sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor. Subsequent perfusion with 30 µM ATP resulted in no increase in Lp, but perfusion with 1 nM VEGF increased permeability. D: Lp measurements on a vessel during perfusion with 1% BSA in Ringer and then 1 nM VEGF. The vessel was then perfused with 1 µM ionomycin (a Ca2+ ionophore) and 100 nM TG to ensure depletion of Ca2+ stores. Once Lp had returned to control values, the vessel was perfused with 1 nM VEGF, which increased permeability despite store depletion.

Effect of thapsigargin on the increase in [Ca²+]_i brought about by VEGF. Measurements of [Ca²⁺]_i were made in nine vessels perfused with 1 nM VEGF (Table 1). Perfusion with VEGF stimulated a 111 ± 24 nM rapid, transient rise in [Ca²⁺]_i from 71 ± 12 to 182 ± 20 nM (P < 0.001). After vessels were perfused with thapsigargin, perfusion with 1 nM VEGF and thapsigargin produced a significant rapid, transient 211 ± 44 nM increase in [Ca²⁺]_i, from 85 ± 20 to 296 ± 90 nM (P < 0.001), which was, on average, not significantly different from that before thapsigargin (P > 0.05). The time-averaged increase in Ca²⁺ across all nine vessels is shown in Fig. 5A (time-averaged data will not show the full extent of the increase in Ca²⁺ because each measurement in each vessel is the average of 4 Ca²⁺ measurements, only 1 of which could have been the peak response). It can be seen that there was no significant difference in the VEGF response whether thapsigargin was present or absent.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Effect of TG pretreatment on ATP- and VEGF-mediated increase in [Ca2+]i. A: time-averaged [Ca2+]i measurements on 9 vessels during perfusion with 1 nM VEGF before () and after () pretreatment with 1 nM TG. B: time-averaged [Ca2+]i measurements on 7 vessels during perfusion with 30 µM ATP before () and after () pretreatment with 1 nM TG. The increase in [Ca2+]i is calculated as the [Ca2+]i during agonist perfusion minus the mean baseline [Ca2+]i during perfusion with 1% BSA (with or without TG) immediately before agonist perfusion. Values are means ± SE.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Effect of store depletion on VEGF- and ATP-mediated increase in [Ca2+]i in single vessels. A: [Ca2+]i measurements were made on a single vessel during perfusion with 1% BSA and 1 nM VEGF. The vessel was perfused for 20 min with 100 nM TG and then with 1 nM VEGF and 100 nM TG, and [Ca2+]i was measured again. Once [Ca2+]i had returned to control, the vessel was perfused with ATP. No increase in [Ca2+]i was seen. The vessel was perfused with 100 nM TG and then with 1 nM VEGF again. VEGF repeatedly increased [Ca2+]i in the presence of TG, but ATP did not. Composition of the perfusate is shown by boxes. B: [Ca2+]i measurements were made on a single vessel during perfusion with 1 nM VEGF and 30 µM ATP, as in A. The vessel was perfused with 100 nM TG to deplete intracellular stores. The vessel was then alternately perfused with 30 µM ATP and 1 nM VEGF. This resulted in a repeated increase in [Ca2+]i, despite the Ca2+ stores being emptied when VEGF was perfused, but no increase when ATP was perfused. C: [Ca2+]i measurements on a single vessel after pretreatment with 30 µM CPA. Subsequent perfusion with 30 µM ATP resulted in no increase in [Ca2+]i, but perfusion with 1 nM VEGF increased [Ca2+]i.

VEGF stimulates Mn²+ influx in the presence of thapsigargin. As part of the methodology for the measurement of [Ca²⁺]_i in endothelial cells in vivo by use of fluorescent indicator dyes such as fura 2, it was necessary to determine the background I_f by quenching the Ca²⁺-sensitive fura 2. To do this, at the end of the experiment, the vessels were perfused with Mn²⁺, which enters cells through Ca²⁺ channels and quenches the Ca²⁺-sensitive indicator. The rate of quench of the indicator is a measure of the rate of Mn²⁺ influx (and, by inference, Ca²⁺ influx across the plasma membrane). We noticed that the rate of quench with thapsigargin appeared to be slower than usual. We therefore investigated the change in the rate of quench stimulated by VEGF and ATP to determine whether VEGF or ATP could indeed stimulate quenching of fura 2. At the end of the experiments to measure Ca²⁺ changes, the vessels were perfused with frog Ringer, which was nominally Ca²⁺ free and contained 5 mM Mn²⁺ and 1% BSA. The I_f 360 (the isosbestic point for fura 2, the wavelength at which there is no Ca²⁺ sensitivity) was then measured for 10-20 s. Four vessels were perfused with 1% BSA with Mn²⁺ and then 1 nM VEGF with 1% BSA and Mn²⁺. In another four vessels, vessels were perfused with the 1% BSA and Mn²⁺; then with 1% BSA, Mn²⁺, and 30 µM ATP; and then with 1% BSA, Mn²⁺, and 1 nM VEGF. I_f 360 was measured for 10-20 s after each cannulation.

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Effect of VEGF and ATP perfusion on the rate of quench by Mn2+. Fluorescence intensity measurements were made of a fura-loaded vessel excited at 360 nm. The vessel had already been perfused with TG for over 1 h and had already been exposed to VEGF and ATP. The relative fluorescence intensity (If/I0) was calculated as the fluorescence intensity (If) relative to the initial fluorescence intensity (I0) immediately after start of perfusion with the agonist. The vessel was perfused with 1% BSA in Ca2+-free Ringer containing 5 mM Mn2+ (dashed line, ). After 10-20 s, the pipette was removed, and the vessel was perfused with the same solution, except that it contained 30 µM ATP (dotted line, ). After an additional 10-20 s, the vessel was then perfused with 1 nM VEGF (solid line, ). The slope of the quench was 0.021 in 1% BSA, 0.028 in ATP, and 0.4 s1 with VEGF.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The ability to increase [Ca²⁺]_i was one of the first effects of VEGF described during its initial purification (8). Although VEGF has been shown to result in increased [Ca²⁺]_i and permeability in a variety of systems (4, 7), the mechanisms by which these are brought about have not been extensively investigated. The experiments described here are the first investigation of the mechanism of that increase in [Ca²⁺]_i. We proposed three hypotheses for the mechanism of increased [Ca²⁺]_i. These were 1) the release of Ca²⁺ from intracellular stores, 2) Ca²⁺ release-activated Ca²⁺ influx across the plasma membrane, or 3) store-independent Ca²⁺ influx across the plasma membrane. Our data are consistent only with the third hypothesis, that VEGF increases [Ca²⁺]_i by a store-independent mechanism of Ca²⁺ influx across the plasma membrane. The permeability effects of both VEGF and ATP have been shown to be dependent on Ca²⁺ influx in vivo (4). It has been assumed, therefore, that they increase permeability through the same mechanism. The experiments described here show that VEGF-induced increases in permeability are not affected by store depletion with SERCA inhibitors. This finding was in marked contrast to the increase in permeability mediated by ATP, which was significantly attenuated by store depletion. These data support the novel hypothesis that VEGF increases [Ca²⁺]_i and microvascular permeability through a mechanism distinct from the increases evoked by ATP.

The mechanism of VEGF-stimulated Ca²+ influx. Of the three likely explanations for the VEGF-mediated increase in [Ca²⁺]_i, our data are only consistent with the hypothesis that the increase in [Ca²⁺]_i is due to Ca²⁺ influx rather than store release. This hypothesis is also consistent with previous data showing that the increase in permeability brought about by VEGF can also be inhibited by perfusion with nickel (4). However, thapsigargin- and CPA-insensitive Ca²⁺ stores have been described in endothelial cells in vitro. It has been shown that [Ca²⁺]_i may increase by release of Ca²⁺ from mitochondrial Ca²⁺ stores (23). If VEGF stimulated significant Ca²⁺ release from mitochondria, inhibition of Ca²⁺ influx might not be expected to affect the increase in [Ca²⁺]_i so dramatically. We cannot rule out a contribution from mitochondria store, however, even though inhibition of Ca²⁺ influx with nickel greatly reduces the magnitude of [Ca²⁺]_i increase after VEGF.

VEGF and ATP act through different mechanisms. ATP is known to stimulate P_2Y purinoreceptors, which results in IP₃ production and release of Ca²⁺ from endoplasmic reticulum (33). It is this release of Ca²⁺ from intracellular stores that stimulates Ca²⁺ entry through a passive conductance pathway, possibly by hyperpolarization of the endothelial cell by stimulation of Ca²⁺-activated potassium channels (K_Ca) (9). Our data suggest that the Ca²⁺ influx resulting from stimulation of endothelial cells with ATP in vivo is brought about by release of Ca²⁺ from intracellular stores, in the same manner as it is in vitro. It has previously been shown that the permeability increase caused by ATP is also dependent on activation of Ca²⁺ influx. The increase in permeability is dependent on extracellular Ca²⁺ and can be inhibited by reducing the driving force for Ca²⁺ influx (18). It may be hypothesized that inhibition of the stimulus for Ca²⁺ influx, the release of Ca²⁺ from intracellular stores, would inhibit the permeability increase. Figure 3B shows that perfusion with thapsigargin for 20 min successfully inhibited the permeability increase brought about by ATP. This shows that the conditions described here effectively blocked store-dependent, Ca²⁺ influx-mediated permeability increases. They serve as useful controls to differentiate between store-dependent and -independent increases in permeability.

Effect of thapsigargin on [Ca²+]_i and vascular permeability. Thapsigargin has been extensively used in many studies of endothelial [Ca²⁺]_i regulation. However, all of the studies where endothelial [Ca²⁺]_i has been measured during exposure to thapsigargin have been carried out in cultured endothelial cells (usually from large arteries or veins), with the exception of one study, which measured [Ca²⁺]_i in endothelial cells of the lung microvasculature in situ (37). This study unfortunately did not measure Ca²⁺ changes during thapsigargin perfusion but showed that Ca²⁺ waves were inhibited by thapsigargin perfusion. The data presented here are therefore the first description of the effects of SERCA inhibitors on [Ca²⁺]_i in intact endothelial cells of vessels in vivo. Figure 2 shows that thapsigargin perfusion into microvessels causes a transient increase in [Ca²⁺]_i. This is similar in some respects to the effect of thapsigargin on endothelial cells in vitro (11) but also differs in one important aspect. Application of thapsigargin to endothelial cells in vitro results in a transient increase in [Ca²⁺]_i, which peaks at approximately the same level as described here (400 ± 110 nM) and does not return to the control values but maintains a high sustained level (11). This sustained increase is 70% of the peak increase. This is significantly greater than the sustained level measured in this study, which is only 8 ± 10% of the peak increase (P < 0.05). This finding implies that Ca²⁺ store depletion does not result in a sustained [Ca²⁺]_i increase in endothelial cells in vivo. Possible differences between in vivo and in vitro settings include the extent to which Ca²⁺ extrusion is activated and the contribution of K_Ca to the regulation of membrane hyperpolarization and hence part of the driving force for Ca²⁺ entry (14). The finding that store depletion does not result in a sustained increase in [Ca²⁺]_i is somewhat contradictory to our expectation, and this question deserves further attention. However, the mechanisms linking the thapsigargin-induced Ca²⁺ release to the time course of change in [Ca²⁺]_i and L_p are not the focus of this investigation.