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Am J Physiol Heart Circ Physiol 284: H1468-H1478, 2003. First published January 2, 2003; doi:10.1152/ajpheart.00585.2002
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Vol. 284, Issue 4, H1468-H1478, April 2003

Role of endothelial Ca2+ stores in the regulation of hydraulic conductivity of Rana microvessels in vivo

C. A. Glass and D. O. Bates

Microvascular Research Laboratories, Department of Physiology, Preclinical Veterinary School, University of Bristol, Bristol BS2 8EJ, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Vascular permeability is regulated by endothelial cytosolic Ca2+ concentration ([Ca2+]i). To determine whether vascular permeability is dependent on extracellular Ca2+ influx or release of Ca2+ from stores, hydraulic conductivity (Lp) was measured in single perfused frog mesenteric microvessels in the presence and absence of Ca2+ influx and store depletion. Prevention of Ca2+ reuptake into stores by sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) inhibition increased Lp in the absence of extracellular Ca2+ influx. Lp was further increased when Ca2+ influx was restored. Depletion of the Ca2+ stores with ionomycin and SERCA inhibition increased Lp in the presence and the absence of extracellular Ca2+ influx. However, store depletion in itself did not significantly increase Lp in the absence of active Ca2+ release from stores into the cytoplasm. There was a significant positive correlation between baseline permeability and the magnitude of the responses to both Ca2+ store release and Ca2+ influx, indicating that the Ca2+ regulating properties of the endothelial cells may regulate the baseline Lp. To investigate the role of Ca2+ stores in regulation of Lp, the relationship between SERCA inhibition and store release was studied. The magnitude of the Lp increase during SERCA inhibition significantly and inversely correlated with that during store release by Ca2+ ionophore, implying that the degree of store depletion regulates the size of the increase on Lp. These data show that microvascular permeability in vivo can be increased by agents that release Ca2+ from stores in the absence of Ca2+ influx. They also show that capacitative Ca2+ entry results in increased Lp and that the size of the permeability increase can be regulated by the degree of Ca2+ release.

calcium influx; store calcium release; endothelium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE MAIN SITES OF SOLUTE and fluid exchange between the plasma and interstitium are the capillaries and postcapillary venules. Microvessels are formed from a heterogeneous population of endothelial cells (23) that form the main barrier to fluid filtration lying on a secreted basement membrane. Homeostasis and tissue function are maintained by the endothelial cell regulation of microvascular permeability, which is increased during wound healing and inflammation. When vascular permeability is chronically raised, it is usually pathological; i.e., in shock, burns, and tumors.

Microvascular permeability is assumed to be regulated by cytosolic Ca2+ concentration ([Ca2+]i) of the endothelial cells forming the vessel walls, although other cell types such as pericytes and mast cells may also play a role. He and Curry (12) have shown that the Ca2+ ionophore ionomycin (IM) increased hydraulic conductivity (Lp), and this increase was attenuated under conditions of reduced Ca2+ influx brought about by depolarizing the membranes of endothelial cells with high-potassium Ringer solutions. This implies that extracellular Ca2+ influx is required for increased vascular permeability. He and Curry also demonstrated that removal of extracellular Ca2+ (<1 µM) greatly attenuates the Ca2+ response. However, a small transient increase remains, presumably due to Ca2+ release from intracellular stores in the presence of IM (12). IM is known to stimulate release of Ca2+ from intracellular stores in endothelial cells (9). Bates and Curry (5) showed that the addition of the cation channel blocker Ni2+ (5 mM) prevents Ca2+ influx and completely blocks the Ca2+ responses to VEGF. However, VEGF increases Lp through a Ca2+ store-independent mechanism, as demonstrated by the inability of the sarco(endo)plasmic reticulum (ER) Ca2+-ATPase (SERCA) inhibitor thapsigargin (TG) to block the response by first emptying stores (25).

It is not clear whether increased permeability mediated by increased [Ca2+]i can result solely from intracellular store release or if Ca2+ influx is necessary. Here we describe experiments testing the hypothesis that SERCA inhibition or Ca2+ release from the intracellular stores without Ca2+ influx is sufficient to increase vascular permeability and to determine if the increase in Lp stimulated by IM is by Ca2+ release-activated Ca2+ entry (CRAC) stimulated by increasing free cytosolic Ca2+ or by capacitative Ca2+ entry (CCE) stimulated by depleted intracellular Ca2+ stores. In these experiments we have used IM to release Ca2+ from intracellular stores, Ni2+ or SKF-96365 (SKF) to inhibit Ca2+ entry into the cytoplasm across the plasma membrane, and TG or cyclopiazonic acid (CPA) to inhibit reuptake of Ca2+ into the calcium store (i.e., cause store depletion).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

Male frogs (Rana temporaria) were supplied by Blades. Rat erythrocytes were collected by cardiac puncture from 5% halothane-anesthetized male Wistar rats that were then killed by cervical dislocation. All chemicals were purchased from Sigma, with the exception of CPA and TG, which were purchased from Calbiochem.

In Vivo Mesenteric Microvascular Preparation

Frogs were anesthetized by immersion in 1 mg/ml 3-aminobenzoic acid ethyl ester (MS-222) in water. At the end of the experiment, the frogs were euthanized by destruction of the brain and central nervous system. Anesthesia was maintained by superfusing the mesentery with 0.25 mg/ml MS-222 in physiological frog Ringer (FR) solution composed of (in mM) 111 NaCl, 2.4 KCl, 1 MgSO4, 1.1 CaCl2, 0.2 NaHCO3, 5 glucose, 2.63 HEPES acid, and 2.37 HEPES sodium salt, pH corrected to 7.40 ± 0.02 with 0.115 M NaOH. An incision was made through the body wall, and the ileum was gently teased out with a moist cotton ball and then draped over a transparent Perspex pillar so that the mesentery could be visualized through a Leitz inverted microscope. Experiments were recorded with the use of a video camera (Forn) connected through an electronic timer (Panasonic) to a video recorder (Panasonic). All experiments were performed at room temperature (20-22°C).

Measurement of Lp

Lp was measured using the Landis-Michel technique (18), as previously described by this laboratory (3, 24). A relatively straight true capillary or postcapillary venule with diameter 15-40 µm was selected that had flowing blood, was free of side branches for at least 800 µm, and had no leukocytes adhering to the vessel wall. The vessel was cannulated with a beveled glass micropipette connected to a manometer and perfused with 1% bovine serum albumin (BSA) in physiological FR pH 7.40 ± 0.02 containing rat erythrocytes as flow markers (baseline solution). Baseline was measured for all vessels before the experiment was performed. Vessels with a baseline Lp > 10 × 10-7 cm · s-1 · cmH2O-1 were excluded unless otherwise stated. The pipette was refilled with test solutions as previously described (14) to prevent washout of the drugs between applications. Pressures between 30 and 40 cmH2O were used. Any drugs used were made as stock solutions in DMSO vehicle or water before being diluted in the baseline solution (final DMSO concentration, in %: 0.02 CPA, 0.01 TG, and 0.05 IM). Downstream from the cannulation site a pulled glass micropipette was used to occlude the vessel for at least 5 s. The vessel was allowed to flow freely for at least 15 s between occlusions.

Calculation of Lp

The radius of the vessel (r), velocity of the marker cells ~2 s after occlusion (dl/dt), the length (l) between the marker cell and the occlusion site were measured offline from the video recording. Solute flux per unit area (Jv/A) was calculated as
Jv/<IT>A</IT> = (d<IT>l</IT>/d<IT>t</IT>) · (<IT>r</IT>/2<IT>l</IT>) (1)
Lp was calculated from the Starling equation, where Delta P is the hydrostatic pressure difference, Delta pi is the oncotic pressure difference between the capillary lumen and the interstitium, and sigma  is the oncotic reflection coefficient. The effective oncotic pressure (sigma Delta pi ) of 1% BSA is 3.6 cmH2O.
L<SUB>p</SUB><IT>=</IT>(<IT>J</IT>v/<IT>A</IT>)/(&Dgr;P − &sfgr;&Dgr;&pgr;) (2)

Statistics

Baseline values are expressed as the mean Lp during the time perfused. All other perfusion treatments are expressed as the peak (highest) Lp value. Peaks and baselines were compared on the basis of repeated-sample measures analysis (2). Where there is more than one transient increase in Lp, the peak of the largest transient increase from the baseline was used. Pooled data are expressed as means ± SE. A nonparametric paired Wilcoxon test was used to compare Lp measurements and the nonparametric Spearman rank test to look for correlations between data groups unless otherwise stated.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Does Inhibition of Ca2+ Reuptake Into ER Increase Lp in Absence of Extracellular Ca2+ Influx and Does CCE Increase Lp?

Effects of TG. To determine whether Ca2+ store release was sufficient to increase Lp in the absence of Ca2+ influx, the irreversible SERCA inhibitor 100 nM TG was perfused with the nonspecific cation channel blocker 5 mM NiCl2 (Ni2+). TG (100 nM) has previously been shown in this laboratory to transiently increase Lp in the presence of Ca2+ influx (25). Vessels were perfused with 1% BSA in FR and rat erythrocytes for a variable time period until the baseline Lp stabilized and then perfused with TG and Ni2+ for 20 min. During all Ni2+ perfusions, 5 mM Ni2+ was also added to the superfusate. To determine whether CCE induced by depleted Ca2+ stores could stimulate increased Lp, the vessels were then perfused with 1% BSA in normal FR (and normal FR superfusion) to wash out the Ni2+ and allow Ca2+ influx for a further 20 min (with continued SERCA inhibition).

Figure 1A shows an example of the effects of TG perfusion on Lp in the presence and absence of Ca2+ influx in a single microvessel. The Lp transiently increased during perfusion with TG in the presence of Ni2+ and reached a maximum (peaked) at 10 min, returning to baseline after 15 min. Lp increased again when Ni2+ was removed to allow CCE. Approximately 10 min after the removal of Ni2+ the Lp peaked and then recovered to baseline over a further 10 min. In 11 vessels, when perfused with TG and Ni2+, the Lp increased from a means ± SE 0.9 ± 0.2 × 10-7 to 3.6 ± 1.0 × 10-7 cm · s-1 · cmH2O-1 (P < 0.05, n = 11). When Ni2+ was washed out in these vessels, the mean value of the highest (peak) ± SE Lp was 6.3 ± 1.9 × 10-7 cm · s-1 · cmH2O-1 (P < 0.005, n = 10), as summarized in Fig. 1B. No Lp increase was measured over 60 min when vessels were perfused with either 1% BSA (baseline solution) or 0.05% DMSO vehicle in baseline solution. Interestingly, the baseline Lp significantly correlated with peak Lp during perfusion with TG and Ni2+ (r = 0.73, P < 0.005, n = 12, Spearman rank correlation coefficient, Fig. 1C), suggesting that the greater the leak of Ca2+ from intracellular stores, the greater the permeability. This was still true if the extreme data point was removed (r = 0.61, P < 0.05, n = 11, Fig. 1C, inset). This correlation appeared to be more specific for vessels with baseline Lp of >1. Furthermore, the baseline Lp also correlated with the peak Lp during perfusion with TG in the absence of Ni2+ (r = 0.87, P = 0.0005, n = 10, Fig. 1D). Again this was true if the extreme data point is removed (r = 0.76, n = 9, Fig. 1D, inset). This suggests that CCE stimulated a greater increase in vessels with a higher baseline Lp. There was no significant correlation found between the peak Lp during TG and Ni2+ perfusion and TG alone.


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  		Fig. 1.   Permeability can be increased both by store depletion and capacitative Ca2+ entry (CCE). A: hydraulic conductivity (Lp) measurements from a single microvessel perfused with 100 nM thapsigargin (TG) and 5 mM Ni2+ for 20 min, and then TG alone for at least 10 min. The peak values taken for comparison between vessels are indicated. A transient Lp increase was measured in the presence of TG and Ni2+ and a second larger transient increase in Lp in the presence of TG alone. B: mean baseline Lp [bovine serum albumin (BSA)] and peak Lp with TG/Ni2+ and TG alone. Values are means ± SE. *P < 0.05, ****P < 0.005. Only significant differences are shown. C: correlation between BSA baseline Lp and peak TG/Ni2+ Lp (r = 0.73, P < 0.005, n = 13). Inset: relationship between baseline and peak when baseline was low. D: correlation between BSA baseline Lp and peak TG Lp (r = 0.87, P = 0.0005, n = 10). Inset: relationship between baseline and peak when baseline was low.

Effects of CPA. The same protocol was used with the reversible SERCA inhibitor CPA (30 µM) to confirm the findings in Fig. 1. Vessels were perfused with 1% BSA in FR and rat erythrocytes until the baseline Lp stabilized and were then perfused with CPA and 5 mM Ni2+ for 20 min. The vessels were then perfused with CPA and superfused with normal FR for a further 20 min to wash out the Ni2+ and allow Ca2+ influx.

Figure 2A shows a typical example of the Lp responses to CPA in a single microvessel. During perfusion with CPA and Ni2+ a small transient increase in Lp was measured during the first 2-3 min. There was a second, smaller increase in Lp after ~12 min. When Ni2+ was removed, the Lp immediately increased but recovered toward baseline by 20 min. Figure 2B summarizes data from six vessels. During CPA and Ni2+ perfusion, the Lp increased from 2.2 ± 0.9 × 10-7 to 20.8 ± 13.8 × 10-7 cm · s-1 · cmH2O-1 (P < 0.05, n = 6). This confirms that inhibition of Ca2+ store reuptake can increase Lp in the absence of Ca2+ influx. On Ni2+ removal the Lp again increased to 21.8 ± 7.7 × 10-7 cm · s-1 · cmH2O-1 (P < 0.05, n = 6), again showing that CCE increased vascular permeability. One different finding from the TG data was that this time, there was no significant correlation between either the peak Lp with CPA and Ni2+ or the peak Lp with CPA alone compared with the baseline Lp. However, there was a significant correlation between the peak response with CPA and Ni2+ and the peak with CPA alone (CCE) (r = 0.94, P < 0.05, n = 6) (Fig. 2C). This relationship appeared to be best fit by an exponential (see Fig. 2C), indicating that a more significant store depletion (i.e., a greater Lp increase) resulted in a greater Lp response to CCE. Although further experiments may be necessary to define the actual relationship between permeability due to CCE and because of store depletion, an estimate is given in Fig. 2C. The possible reasons for the differences between this and the TG experiments are discussed later.


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  		Fig. 2.   The size of the increase in Lp due to CCE correlates with that due to store depletion. A: Lp measurements from a single microvessel perfused with 30 µM cyclopiazonic acid (CPA) and 5 mM Ni2+ for 20 min, followed by perfusion with CPA alone for 20 min. There was an immediate transient increase in Lp in the presence of CPA and Ni2+ and an immediate increase in Lp when the Ni2+ was removed that returned toward basal levels after 20 min. B: mean baseline Lp (BSA) and peak Lp with CPA/Ni2+ and CPA alone. Values are means ± SE. *P < 0.05. C: correlation between the peak CPA/Ni2+ Lp and peak CPA Lp (r = 0.94, P < 0.05, n = 6). Only significant differences are shown.

To exclude the effect of Ni2+ on other mechanisms of Ca2+ regulation a specific cation channel blocker, 100 µM SKF (17, 28), was used instead of Ni2+. Vessels were perfused with TG and SKF for 20 min once a baseline with 1% BSA had been established. SKF was washed out by a further 10 min perfusion with 1% BSA.

Figure 3A shows the Lp measured in two vessels. The top trace shows an experimental vessel treated as described by the protocol above (solid diamond), and the bottom trace shows the Lp measurements taken with a control vessel treated with only SKF (solid square). The Lp increased transiently and peaked after 5-min perfusion with SKF and TG in the experimental vessel, and on SKF removal the Lp transiently increased for a second time returning to basal levels within 5 min. There was no difference between the time to peak in the presence of TG/SKF compared with TG/Ni. The Lp in the control vessel does not significantly change during a 60-min perfusion (40 min shown). A second example of a single vessel is shown in Fig. 3B. This vessel perfusion with TG and SKF resulted in a cyclic alteration in Lp. This type of response was seen in 4 of 10 vessels tested with this protocol (two vessels were excluded due to a high-baseline Lp).


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  		Fig. 3.   Store depletion also increases Lp in the presence of a pharmacological Ca2+ channel inhibitor. A: Lp measurements taken from a single microvessel perfused with 100 µM SKF-96365 (SKF) and 100 nM TG for 20 min, followed by TG alone for at least 10 min (black-lozenge ). A transient Lp increase was measured when perfused with TG and SKF that returned to baseline after 20 min. A second transient Lp increase occurred on removal of SKF. The lower trace shows a different microvessel perfused with SKF alone for 40 min () that did not significantly change throughout this time. B: another example of measurements taken from a single microvessel. Four of ten vessels tested displayed cyclic behavior when subjected to this protocol. C: mean BSA baseline Lp, peak SKF/TG Lp, and peak TG Lp. Values are means ± SE. *P < 0.05, **P < 0.01; only significant differences are shown.

In eight vessels in which these responses were measured, Lp significantly increased from 2.4 ± 0.6 × 10-7 to 15.1 ± 6.5 × 10-7 cm · s-1 · cmH2O-1 (P < 0.01, n = 8) while perfused with SKF and TG. When the SKF was washed out, Lp peaked at 12.5 ± 2.7 × 10-7 cm · s-1 · cmH2O-1 in the presence of TG alone (P < 0.01, n = 8) as shown in Fig. 3C. This was not significantly different to the peak Lp during perfusion with SKF and TG (P > 0.1, n = 8). In general, therefore, the effects of store depletion in the absence of Ca2+ entry were broadly similar irrespective of the combination of agonist used (TG/Ni, TG/SKF, and CPA/Ni). However, subtle changes in the responses are interpreted in the discussion.

Does Store Depletion Increase Lp in Absence of Extracellular Ca2+ Influx?

After it was determined that SERCA inhibition was sufficient to increase Lp in the absence and presence of extracellular Ca2+ influx, 5 µM IM was used to determine whether the stores were fully depleted by SERCA inhibition after a 20-min perfusion. If the stores were not fully depleted, an increase in Lp would be expected in the presence of IM. Vessels were perfused with 1% BSA until the baseline Lp stabilized and then with Ni2+ for at least 10 min to establish a new baseline. The pipette was refilled with Ni2+ and 30 µM CPA and perfused for another 20 min, followed by perfusion for 20 min with Ni2+, CPA, and IM. To determine whether CCE was still able to stimulate Lp increases, the vessels were perfused with CPA and IM in the absence of Ni2+ to allow Ca2+ influx.

Figure 4A shows an example of the effect of store depletion on vascular permeability in the absence of Ca2+ influx in a single vessel. During perfusion with CPA and Ni2+, the Lp increased slightly and returned to baseline in 3 min (point i). When IM was coperfused with CPA and Ni2+, the Lp increased after ~10 min (point ii). The Lp increased further when the Ni2+ was washed out (points iii and iv). This contrasted strongly with vessels that were perfused with IM alone (an example is shown in Fig. 4B). In this case perfusion with IM resulted in a transient increase in Lp that peaked between 3 and 5 min and returned to baseline within 10 min and was maintained at a low Lp. He and Curry (12) have previously performed experiments using the same technique looking at the effect of IM on Lp in the presence of Ni2+. They measured an attenuated response to IM that recovered fully in the presence of Ni2+.


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  		Fig. 4.   Store release increases permeability; combined Ca2+ release, depletion, and entry most potently increases permeability; and the size of the increase in Lp induced by store depletion correlates with the baseline Lp. A: Lp measurements taken from a single microvessel perfused with 5 mM Ni2+ for 10 min, Ni2+ and 30 µM CPA for 20 min, Ni2+, CPA, and 5 µM ionomycin (IM) for 20 min, then CPA and IM for 10 min. During perfusion with CPA and Ni2+, the Lp increased slightly and returned to baseline (point i). When perfused with CPA, Ni2+, and IM, Lp increased after 10 min (point ii). When Ni2+ was removed the Lp increased further (points iii to iv). B: Lp measurements taken from a single microvessel during perfusion of 5 µM IM for 15 min demonstrating that IM induces a transient Lp response. C: mean Ni2+ baseline Lp, peak Ni2+/CPA Lp, peak Ni2+/CPA/IM Lp and peak CPA/IM Lp. Values are means ± SE. *P < 0.05, **P < 0.01; only significant differences are shown. D: correlation between mean Ni2+ Lp (baseline) and peak Ni2+/CPA Lp (r = 0.83, P < 0.02, n = 8).

When the effects of IM, CPA, and Ni2+ were examined in eight vessels, the baseline did not significantly change with Ni2+ perfusion alone (from 6.6 ± 2.3 × 10-7 to 2.9 ± 0.5 × 10-7 cm · s-1 · cmH2O-1, P > 0.1, n = 8, Fig. 4C). CPA and Ni2+ coperfusion slightly, but significantly, increased Lp to 5.4 ± 0.9 × 10-7 cm · s-1 · cmH2O-1 (P < 0.01 vs. Ni2+ baseline, n = 8). Coperfusion of Ni2+, CPA, and IM maintained an increase in Lp to × 10-7 cm · s-1 · cmH2O-1 (P < 0.01 vs. Ni2+ baseline, n = 8) 7.4 ± 2.1 but this was not significantly greater than perfusion with Ni2+ and CPA alone (P > 0.5, n = 8), implying that the stores were depleted during a 20-min perfusion. However, Ni2+ removal during perfusion with CPA and IM dramatically increased Lp to 27.4 ± 18.8 × 10-7 cm · s-1 · cmH2O-1 (P < 0.05 vs. Ni2+ baseline, n = 6), indicating that CCE could result in a significant increase in permeability. Interestingly, there was again a significant positive correlation between perfusion with Ni2+ (baseline, no Ca2+ influx) and perfusion with CPA and Ni2+ (store depletion, r = 0.83, P < 0.02, n = 8, see Fig. 4D).

Does Active Store Release Increase Lp in Absence of Extracellular Ca2+ Influx?

To determine the effect of actively releasing Ca2+ from stores, we measured the permeability when the IM was added at the same time as SERCA inhibition in the absence of Ca2+ influx (this time using TG and SKF). After the Lp stabilized with perfusion with 1% BSA, SKF was perfused for 10 min to establish a new baseline. SKF, TG, and IM were co-perfused for 20 min to ensure the Ca2+ stores were released. This was followed by a 10-min perfusion with IM to wash out the SKF and allow Ca2+ influx.

Figure 5A shows an example of the effect of Ca2+ store release in the absence of Ca2+ influx and the effect of depleted Ca2+ stores in the presence of Ca2+ influx in a single vessel. When SKF, TG, and IM were coperfused, there was an immediate large transient increase in Lp that returned to basal levels within 5 min. When the SKF was removed and Ca2+ influx allowed, there was a second smaller transient increase in Lp between 4-8 min.


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  		Fig. 5.   Actively releasing Ca2+ from intracellular stores increases Lp. A: Lp measurements from a single microvessel perfused with 100 µM SKF for 10 min (baseline), SKF, 100 nM TG, and 5 µM IM for 20 min, and TG and IM for a further 10 min. A large transient increase in Lp was measured when perfused with SKF, TG, and IM. With SKF removal there was a second smaller transient increase in Lp. B: mean SKF baseline Lp, peak SKF/TG/IM Lp and peak TG/IM Lp. Values are means ± SE. *P < 0.05; only significant differences are shown.

This experiment was carried out in eight vessels, and the mean data are shown in Fig. 5B. In these vessels, perfusion of SKF did not significantly change the baseline Lp (from 3.1 ± 0.8 × 10-7 to 2.1 ± 0.8 × 10-7 cm · s-1 · cmH2O-1, P > 0.1, n = 8). The presence of SKF, TG, and IM significantly and transiently increased Lp to 17.2 ± 9.4 × 10-7 cm · s-1 · cmH2O-1 (P < 0.02 vs. SKF baseline, n = 8). When SKF was washed out to allow Ca2+ entry, a second transient increase in Lp to 14.3 ± 3.5 × 10-7 cm · s-1 · cmH2O-1 was measured (P < 0.05 vs. SKF baseline, n = 7). There was no correlation between the time to peak and the baseline Lp in the presence of either of the influx inhibitors.

Does SERCA Inhibition with CPA Deplete Intracellular Ca2+ Stores in Presence of Ca2+ Influx?

To determine whether SERCA inhibition with CPA resulted in depletion of intracellular stores, and hence an inhibition of store release-mediated increase in Lp, we investigated the relationship between the permeability increase stimulated by store depletion (SERCA inhibition, reduced-store Ca2+) and that stimulated by active release of Ca2+ from the store (stimulated by IM, defined as store release). If the permeability increase in the presence of Ca2+ influx was dependent on the amount or rate of Ca2+ released from stores, then we would predict an inverse relationship between the permeability increase caused by CPA perfusion (store depletion) and that caused by CPA and IM perfusion (store release). Thus if CPA causes store depletion and release, then CPA will increase permeability, but subsequent IM will not (i.e., there will be little or no Ca2+ in the stores for IM to release). However, if CPA results in SERCA inhibition but not store depletion, then there will be a small effect of CPA but a large effect of ionomycin (i.e., the stores will be full before IM is added and empty afterward).

To test this prediction, 22 vessels were perfused with CPA for 20 min after baseline measurement. In 11 of these, this was followed by perfusion with CPA and IM for at least 10 min. Figure 6, A-C, gives examples of Lp measurements in three different vessels. Figure 6A shows a small transient increase in Lp in the first 2-5 min of CPA perfusion and a large immediate transient increase in Lp when perfused with CPA and IM that declined toward baseline within 10 min. Figure 6B shows a vessel that responded transiently with both CPA perfusion and CPA and IM coperfusion to a similar magnitude. Figure 6C shows a vessel that responded with a larger transient Lp increase with CPA perfusion and a smaller transient Lp increase in the presence of CPA and IM. Taking all 22 vessels, CPA perfusion transiently increased Lp from a mean of 2.0 ± 0.4 × 10-7 to 10.9 ± 2.0 × 10-7 cm · s-1 · cmH2O-1 (P < 0.0001, n = 22, Fig. 6D). In the 11 vessels, in which this was followed by co-perfusion of CPA and IM, Lp transiently increased to 30.4 ± 9.9 × 10-7 cm · s-1 · cmH2O-1 (P = 0.002, n = 11, Fig. 6D).


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  		Fig. 6.   The size of the increased permeability depends on the filling state of the stores. A-C: Lp measurements from single microvessels perfused with 30 µM CPA for 20 min then CPA and 5 µM IM for at least 10 min. D: mean BSA baseline Lp, peak CPA Lp and peak CPA/IM Lp. Values are means ± SE. ***P < 0.005, ****P < 0.0005. Only significant differences are shown. E. correlation between the fold increase from baseline during CPA perfusion and fold increase during CPA/IM (r = -1.0, P < 0.005, n = 11). F: correlation between mean BSA Lp and peak CPA Lp (r = 0.55, P < 0.01, n = 22). G: correlation data in F interpreted as two populations, vessels with a basal Lp below (black-lozenge ) or above () 3.6 × 10-7 cm · s-1 · cmH2O-1 (below, r = 0.45, P = 0.056, n = 19 above, r = 1.0, Spearman rank, r = 0.995, Pearson, P < 0.005, n = 4). One vessel is included that was previously excluded due to a basal Lp > 10 × 10-7 cm · s-1 · cmH2O-1.

Interestingly, as predicted, there was a highly significant negative correlation between the Lp increase with CPA perfusion (fold increase from BSA) and that with CPA and IM perfusion (fold increase from recovery after CPA) [r = -1.0, Spearman rank correlation coefficient (nonparametric), P < 0.005, r = -0.95, Pearson correlation coefficient (parametric), P < 0.005, see Fig. 6E]. This showed that the degree of store emptying was variable across vessels and is consistent with the hypothesis that the size of the IM response was proportional to degree of filling of the Ca2+ stores.

Furthermore, there was again a significant correlation between the baseline Lp and peak Lp during perfusion with CPA (r = 0.55, P < 0.01, Fig. 6F). This provides further evidence for the hypothesis that the baseline Lp is determined by the leak of Ca2+ from the stores. On closer examination two populations of vessels were found, those with a basal Lp <3.6 × 10-7 cm · s-1 · cmH2O-1 and those with a basal Lp >3.6 × 10-7cm · s-1 · cmH2O-1 as demonstrated in Fig. 6G. There is a slight but not significant correlation between the basal Lp and the peak CPA Lp when the baseline is <3.6 × 10-7 cm · s-1 · cmH2O-1 (r = 0.45, P = 0.056, n = 19). However, when the basal Lp is >3.6 × 10-7 cm · s-1 · cmH2O-1 (including one vessel with a baseline Lp > 10 × 10-7 cm · s-1 · cmH2O-1), the correlation became highly significant (r = 1.0, Spearman rank, r = 0.995, Pearson, P < 0.005, n = 4). This implies that some vessels have a high store leak of Ca2+, and this endows them with a high permeability.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Release of Ca2+ from Intracellular Stores is Sufficient to Increase Vascular Permeability

Vascular permeability is significantly increased by SERCA inhibition using either TG or CPA in the absence of Ca2+ influx (by inhibition of plasmalemmal cation channels by Ni2+ or SKF). TG has been shown to irreversibly block all isoforms of SERCA in endothelial cells in culture (8). This SERCA inhibition appears to be sufficient to significantly increase Lp in the absence of Ca2+ influx (Figs. 1-3). These data support the observations of He and Curry (12), who have previously shown that Ni2+ significantly attenuates but does not completely block the permeability response to release of Ca2+ from stores by IM. However, they state that the permeability responses remained attenuated even after Ni2+ was washed out in their experiments, although no data were shown. In our experiments a large increase in Lp was demonstrated on removal of Ni2+ (Figs. 1 and 2) or SKF (Fig. 3). The main difference between our experiments and those performed by He and Curry (12) was that we used TG or CPA to inhibit SERCA, whereas He and Curry used IM to release Ca2+ from the stores. Therefore it is likely that the stores were still depleted when the Ni2+ or SKF were removed in the experiments described here, whereas Ca2+ released during the IM-mediated increase in permeability could be reuptaken as SERCA was unaffected.

To date, the signaling mechanism for store-dependent Ca2+ influx or "capacitative Ca2+ entry" as described by Putney (26) remains unknown, as does the identity of the cation channel(s) involved (22). However, it is known that IM stimulates the release of Ca2+ from intracellular stores in endothelial cells, rather than by a direct action on plasma membrane Ca2+ channels, (21) at least in vitro. In cultured bovine pulmonary artery endothelial cells Ca2+ influx was a graded response determined by the degree of Ca2+ store depletion and that maximal Ca2+ influx did not require complete Ca2+ store depletion (29). Furthermore, Ca2+ entry has been shown to be regulated by the degree of store filling in endothelial cells in culture (16). It is possible that a small amount of Ca2+ in the ER is required to signal extracellular Ca2+ influx. This theory is supported by the differences seen between our experiments and those of He and Curry (12) when Ni2+ is removed. Interestingly, the H2 receptor agonist dimaprit transiently increased the permeability of occluded rat brain venular capillaries in the presence of SKF or zero extracellular Ca2+ supporting the hypothesis that permeability increases can be brought about by store-mediated Ca2+ release (27).

It is interesting to note that the data described here showed an increase in permeability in response to both CPA and TG that took some minutes to occur. We have assumed that this is due to a delayed increase in [Ca2+]i in response to these agonists, because previous work (25), where [Ca2+]i and permeability were measured in the same model (although in different vessels), showed that there was a distinct time lag between the application of TG to the cells and the increase in both [Ca2+]i and permeability. This is in contrast to many studies of endothelial cells in culture where TG results in an immediate transient increase in intracellular Ca2+, followed by a sustained increased [Ca2+]i (8). It is clear that the ability of endothelial cells to regulate their [Ca2+]i in response to such agonists is different in vitro than it is in vivo.

Degree of Store Release Regulates Permeability

He et al. (13) demonstrated a positive correlation between the peak Lp response to IM and the [Ca2+]i when the [Ca2+]i was >130 nM, which lies just outside of the range of an unstimulated endothelial cell (60-110 nM). They suggest that 130 nM may be the threshold level for Ca2+-dependent processes (13). It has therefore been assumed that the size of the Lp increase is proportional to the size of the Ca2+ increase. In our experiments there was a significant correlation between baseline Lp and peak Lp during store depletion (TG and Ni2+, r = 0.73). Furthermore, there was a correlation between baseline Lp and peak Lp with CCE, and this was greater when inhibition of Ca2+ influx by Ni2+ (r = 0.87) was removed than when SKF was removed (r = 0.52). These findings support the hypothesis that, assuming [Ca2+]i and permeability are correlated, the degree of leak of Ca2+ from intracellular stores regulates the baseline permeability, in the same manner that the rate (or amount) of release or influx regulates the agonist-stimulated increase in permeability (12). In the absence of Ca2+ influx, SERCA inhibition would be expected to result in a greater increase in [Ca2+]i if the leak from stores was greater. The correlation with Ni2+ but not SKF would be explained if Ni2+ blocks other means of Ca2+ regulation such as the plasma membrane Ca2+ ATPase (PMCA) as well as the Na2+/Ca2+ exchanger (15). A high-baseline leak from stores would result in an increase in the signal for CCE to a greater extent with Ni2+ because Ca2+ buffering or extrusion would be limited. This would then result in increased Ca2+ entry on removal of the block to influx. This extrusion mechanism has not been extensively studied in endothelial cells in vivo, and its importance may well have been underestimated. With SKF moreover, the Ca2+ regulation would not be affected, and extrusion of Ca2+ by the PMCA should reset the Ca2+ release-activated Ca2+ entry signal.

If the baseline Lp was determined by the degree of Ca2+ store leak, it might follow that with a higher baseline, a larger response would be expected when SERCA and Ca2+ influx are inhibited. The measured significant correlation between baseline Lp and the peak with TG and Ni2+ (Fig. 1C) suggests that the magnitude of Ca2+ release and Ca2+ influx is indeed determined by the degree of Ca2+ leak from the ER under baseline (nonstimulated) conditions. If the response was large enough to deplete the stores, there would be a stronger signal to stimulate capacitative Ca2+ influx and hence a greater response would be observed when the Ca2+ influx inhibitor was removed.

The responses were heterogeneous, as has been previously described both within a vessel (23) and between vessels (4, 10), which would be expected if responses were dependent on the degree of Ca2+ store leak in each endothelial cell and that leak was different from cell to cell or vessel to vessel.

Does Store Emptying or Active Release Increase Lp in Absence of Extracellular Ca2+ Influx?

SERCA inhibition by CPA was able to significantly increase the Lp in the absence of Ca2+ influx and also correlated to the baseline Lp (Fig. 4). When IM was also added (still in the absence of Ca2+ influx), the Lp only increased slightly compared with Ni2+ and CPA treatment, and although significantly higher than the baseline, it was not significantly different to Ni2+ and CPA treatment. This suggests that either CPA was able to empty the stores or that the leak from the stores had sufficiently run down the store in 20 min to prevent a larger response with IM. A run down of stores has previously been shown to occur in endothelial cells in culture when stimulated with an agonist such as histamine (16).

To further investigate the effect of emptying stores on the permeability, a similar protocol to that in Fig. 4 was used with SKF and TG as the inhibitors. This time IM was added to the perfusate at the same time point as TG to ensure the stores were not depleted (Fig. 5). A large significant increase in the Lp was measured with SKF, TG, and IM treatment, implying that the reason why there was no increase in Lp with IM after CPA treatment was because CPA reduced the degree of Ca2+ filling of the store, as expected (20). Once Ca2+ influx was permitted the Lp increased again. Interestingly, both responses were transient when SKF was used but sustained when Ni2+ was used (Fig. 4A vs. Fig. 5A). It appeared that Ni2+ had an effect on the inactivation of the permeability response (the recovery to baseline). One interpretation of this is that Ni2+ may have nonspecifically blocked the PMCA analagous to its inhibition of the Na/Ca exchanger (15), preventing Ca2+ efflux as well as influx across the plasma membrane. Therefore it appears that active release of Ca2+ from stores increases permeability, in the absence of influx, more effectively than the lack of Ca2+ in the store itself.

CPA Increased Lp Without Completely Depleting Intracellular Ca2+ Stores in Presence of Ca2+ Influx

CPA transiently increased Lp in the presence of Ca2+ influx and showed a correlation to the baseline Lp (Fig. 6). When IM was subsequently added to the perfusate, the Lp response on average increased above that seen with CPA alone indicating that CPA did not deplete the stores entirely in the presence of Ca2+ influx (despite doing so in the absence of influx). However, the relative responses to CPA and CPA co-perfusion with IM differed between vessels as shown by the three examples (Fig. 6, A-C). Some vessels responded to a larger extent with CPA (store depletion) than they did to CPA and IM perfusion (store release) (Fig. 6C) and some responded more during the CPA and IM perfusion than they did to CPA perfusion alone (Fig. 6A), whereas others responded moderately to both treatments (Fig. 6B). When the data were pooled and averaged, they showed that store release (with CPA and IM coperfusion) increased the Lp twice as much as store depletion (with CPA alone).

The leak of Ca2+ from the stores has been suggested to have a positive feedback on the inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor as the local [Ca2+] rises during SERCA inhibition due to phospholipase C activation (11). As the stores begin to run down, CCE is triggered and the global [Ca2+] in the cytoplasm will increase (26). When the global cytosolic [Ca2+] increases, Ca2+ itself will inhibit the Ins(1,4,5)P3 receptor with an IC50 of 300 nM preventing further loss of Ca2+ from the stores (1, 7). Therefore, when IM was included in the perfusate, we propose that there was sufficient release of store Ca2+ to further increase the Lp probably by signaling Ca2+ influx.

A highly significant inverse correlation between CPA perfusion (SERCA inhibition) and CPA and IM coperfusion (store release) was shown. This again demonstrated that CPA did not completely empty the stores and that the size of the increase in Lp was proportional to the intracellular Ca2+ store depletion. Alternately, IM could be releasing Ca2+ from organelles other than the ER, such as the golgi (19) and mitochondria, which have been shown to be up to 25% of the Ca2+ store in vitro (31).

A significant correlation between baseline Lp and the Lp during CPA perfusion (with Ca2+ influx) was demonstrated (Fig. 6F). However, on closer examination, the data can be interpreted as two populations of vessels as shown in Fig. 6G. The first population of vessels has been defined as having a baseline Lp <3.6 × 10-7 cm · s-1 · cmH2O-1 and the second population having a baseline >3.6 × 10-7cm · s-1 · cmH2O-1. Interestingly, the slope of the line is the same for both populations but is shifted to the right when the baseline is above 3.6 × 10-7 cm · s-1 · cmH2O-1. He et al. (13) have demonstrated a positive correlation between the peak Lp response to IM and the [Ca2+]i when the [Ca2+]i was >130 nM (13). There did not appear to be any correlation between the baseline Lp and [Ca2+]i in their experiments. However, they did not test whether there was a relationship between the baseline Lp and the Ca2+ loading of the stores, and this relationship, suggested by the experiments above, awaits further study.

Permeability is Increased by CCE and CRAC

CCE is the stimulation of Ca2+ influx due to a depletion of Ca2+ from the intracellular store. We tested the hypothesis that CCE could stimulate permeability increases by measuring the effect of removing inhibitors of Ca2+ entry once the stores had been depleted. This resulted in a significant, transient increase in Lp, suggesting that CCE was able to stimulate increased permeability (Fig. 5A). In addition, we also tested the hypothesis that CRAC entry could stimulate permeability increases by perfusing vessels with a Ca2+ ionophore under conditions of varying store filling. As predicted, there was a significant negative correlation between store filling and the size of the response to IM (see Fig. 6E). Therefore, it appears that it is not the mechanism of increased Ca2+ that determines the permeability of the vessel, but either the [Ca2+]i, or the rate of increase of [Ca2+]i in the cytoplasm.

Is Permeability Regulated by Rate of Ca2+ Increase or Endothelial [Ca2+]i?

We have shown that microvascular permeability in vivo can be increased in the absence of extracellular Ca2+ influx although not to the same magnitude as when Ca2+ influx is present. Our findings are therefore consistent with those of He and Curry (12), although we show a more significant increase in permeability in the absence of Ca2+ influx. Because there are several sources of Ca2+, it is likely that they have differing rates of flux into the cytosol. This has been demonstrated using Ca2+ fluorescent dyes in HeLa cell cultures (6). It is possible that these different rates of flux are responsible for the differing degrees of permeability while Ca2+ is continuously recycled across the membranes. Interestingly, baseline Lp is not determined by the absolute [Ca2+]i (13), despite the fact that during stimulation the permeability measurements closely track the [Ca2+]i. Because we found that the magnitude of the permeability responses correlated with the baseline permeability, we propose that this is linked to the degree of store filling because the rate of Ca2+ leak into the cytosol from stores would be expected to be greater if the stores had a higher [Ca2+] (a higher concentration gradient would exist between the ER store and the cytosol).

The net Ca2+ leak, possibly through the Ins(3)P receptor, which is itself determined by the [Ca2+] gradient between the ER store and the cytoplasm, is continually compensated for by Ca2+ reuptake through SERCA or Ca2+ flux across the plasmalemma (30) and extrusion by the PMCA. We propose that it is the balance between these Ca2+ movements that regulates Lp as the plasma and store membranes are in close proximity to each other. The expected order for rate of flux would be the following: untreated Ca2+ leak < SERCA inhibition < Ins(1,4,5)P3 receptor activation < capacitative Ca2+ influx < agonist-mediated Ca2+ influx. We predict therefore that this would also be the order for the magnitude of the permeability changes in a single vessel. However, it has not yet been possible to determine the rate of change of cytosolic [Ca2+] and the permeability in the same vessel at the same time.

In summary, we have shown that permeability is increased by release of Ca2+ from intracellular stores in the absence of Ca2+ influx, and by CCE in the presence of Ca2+ influx, and provided evidence supporting the hypothesis that the baseline permeability is set by the degree of leak of Ca2+ from stores.


    ACKNOWLEDGEMENTS

The authors thank Rebecca Foster and Rachel Perrin for technical assistance and support.


    FOOTNOTES

This study was supported by British Heart Foundation Grants FS98027 and BB2000003 (to D. O. Bates) and FS2000057 (to C. A. Glass).

Address for reprint requests and other correspondence: D. O. Bates, Microvascular Research Laboratories, Dept. of Physiology, Preclinical Veterinary School, Southwell St., Univ. of Bristol, Bristol BS2 8EJ, United Kingdom (E-mail: dave.bates{at}bristol.ac.uk).

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.

First published January 2, 2003;10.1152/ajpheart.00585.2002

Received 11 July 2002; accepted in final form 10 December 2002.


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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 284(4):H1468-H1478
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