Cytosolic Ca2+ concentration and rate of increase of the cytosolic Ca2+ concentration in the regulation of vascular permeability in Rana in
1 Microvascular Research Laboratories, Department of Physiology, Preclinical Veterinary School, Southwell Street, University of Bristol, Bristol BS2 8EJ, UK
2 Department of Physiology and Membrane Biology, University of California at Davis, Davis, CA 95616, USA
Abstract
Vascular permeability is assumed to be regulated by the cytosolic Ca2+ concentration ([Ca2+]c) of the endothelial cells. When permeability is increased, however, the maximum [Ca2+]c appears to occur after the maximum permeability increase, suggesting that Ca2+-dependent mechanisms other than the absolute Ca2+ concentration may regulate permeability. Here we investigate whether the rate of increase of the [Ca2+]c (d[Ca2+]c/dt) may more closely approximate the time course of the permeability increase. Hydraulic conductivity (Lp) and endothelial [Ca2+]c were measured in single perfused frog mesenteric microvessels in vivo. The relationships between the time courses of the increased Lp, [Ca2+]c and d[Ca2+]c/dt were examined. Lp peaked significantly earlier than [Ca2+]c in all drug treatments examined (Ca2+ store release, store-mediated Ca2+ influx, and store-independent Ca2+ influx). When Lp was increased in a store-dependent manner the time taken for Lp to peak (3.6 ± 0.9 min during store release, 1.2 ± 0.3 min during store-mediated Ca2+ influx) was significantly less than the time taken for [Ca2+]c to peak (9.2 ± 2.8 min during store release, 2.1 ± 0.7 min during store-mediated influx), but very similar to that for the peak d[Ca2+]c/dt to occur (4.3 ± 2.0 min during store release, 1.1 ± 0.5 min during Ca2+ influx). Additionally, when the increase was independent of intracellular Ca2+ stores, Lp (0.38 ± 0.03 min) and d[Ca2+]c/dt (0.30 ± 0.1 min) both peaked significantly before the [Ca2+]c (1.05 ± 0.31 min). These data suggest that the regulation of vascular permeability by endothelial cell Ca2+ may be regulated by the rate of change of the [Ca2+]c rather than the global [Ca2+].
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Introduction
Exchange of fluid, nutrients and waste products between the plasma and interstitium occurs in the capillaries and postcapillary venules. This exchange is regulated by the endothelial cell barrier of the microvascular wall. Increased vascular permeability may occur under pathological situations or be induced by growth factors and inflammatory mediators. These mediators result in a breakdown of the integrity of the wall, increased macromolecular transport by diffusion and convection, and increased fluid flow down the Starling pressure gradient. This increase in permeability results in oedema and impaired tissue function, probably due to inefficient oxygen and glucose delivery, but also allows the inflammatory process access to the interstitium and enables tissue remodelling. The increase in permeability brought about by inflammatory mediators and growth factors is a regulated process, dependent on endothelial cell surface receptor activation and subsequent downstream signalling.
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Many studies have shown that when vascular permeability is increased, it is usually accompanied by an increase in cytosolic Ca2+ concentration ([Ca2+]c) (He & Curry, 1991; He et al. 1996; Pocock et al. 2000a). This increase in [Ca2+]c is required for the permeability to increase in response to many cytokines. These cytokines include growth factors such as vascular endo-thelial growth factor (VEGF; Pocock et al. 2000a), and classical inflammatory mediators such as histamine (Sarker et al. 1998), ATP (He et al. 1996; Pocock et al. 2000a) and serotonin (Olesen, 1985). Moreover, exposure of vessels to Ca2+ ionophores, such as A23187, results in an increase in vessel permeability (Michel & Phillips, 1989; He et al. 1990; Neal & Michel, 1995) and as the changes in permeability closely track changes in the [Ca2+]c, and inhibition of the increase in [Ca2+]c inhibits the permeability increase (He & Curry, 1991; Sarker et al. 1998; Glass & Bates, 2003b), it has been assumed that the [Ca2+]c regulates permeability (He et al. 1990). However, the mechanisms by which this increase in Ca2+ translates to increased permeability are still not known.
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There have been a few studies attempting to measure [Ca2+]c and vascular permeability in vivo using the same preparation (although in different animals). He & Curry have shown that the time courses of increases in hydraulic conductivity (Lp), a measure of permeability, and the [Ca2+]c responses to the Ca2+ ionophore, ionomycin, were closely correlated, and indeed overlapping (with a time resolution of 15–30 s), and that the size of the increase in permeability was directly correlated with the size of the increase in Ca2+ (He et al. 1990). They showed that the time courses for these responses were not significantly different from each other. It therefore appeared that the increase in permeability was stimulated by an increase in [Ca2+]c.
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Whilst investigating the role of Ca2+ stores in the regulation of vascular permeability, we showed that Lp increases differ in magnitude when [Ca2+]c is increased by Ca2+ from different sources (Glass & Bates, 2003b). We suggested that different sources of Ca2+ result in different rates of increase of [Ca2+]c (Glass & Bates, 2003b). We hypothesized that differing rates of Ca2+ entry into the cytoplasm might explain the differences in magnitude of the increased Lp, This would be consistent with He & Curry's findings that the permeability was correlated with [Ca2+]c, as a higher rate would be needed to reach that higher concentration, if this occurs with the same time course. However, this would require the Lp to reach its peak coincident with the maximum rate of Ca2+ entry, i.e. slightly before the [Ca2+]c reached its peak. Since the time resolution of the experiments by He & Curry was not sufficient to determine whether this was the case, we have investigated whether the time taken for the maximum Lp and [Ca2+]c to occur is the same, irrespective of the source of the increased Ca2+, or if the rate of change of the [Ca2+]c is more closely linked to the peak Lp value.
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Some of these experiments have previously been published as an abstract of a conference presentation (Glass & Bates, 2003a).
Methods
Materials
Male frogs (Rana temporaria) were supplied by Blades, UK. All regulated procedures were carried out under licence from the Home Office in accordance with the Animals (Scientific Procedures) Act 1986. Rat erythrocytes were collected by cardiac puncture from male Wistar rats that had been anaesthetized with 5% halothane and killed by cervical dislocation. All chemicals were purchased from Sigma unless otherwise stated.
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Any drugs used were made as stock solutions in water or DMSO vehicle before being diluted in 1% (w/v) bovine serum albumin (BSA) solution (final DMSO concentrations as percentages (v/v) were 0.01 and 0.05 in thapsigargin and ionomycin, respectively). VEGF-Aics was used.
In vivo mesenteric microvascular preparation
Frogs were anaesthetized by immersion in 1 mg ml–1 MS222 (3-aminobenzoic acid ethyl ester) in water. At the end of the experiment they were killed by destruction of the brain and central nervous system. Anaesthesia was maintained by superfusing the mesentery with 0.25–0.40 mg ml–1 MS222 in physiological frog Ringer solution (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 bud and draped over a transparent quartz pillar so that the mesentery could be visualized through a Leica inverted microscope (DMIL). Experiments were recorded using a video camera (Pulnix) connected through an electronic timer (ForA) to a video recorder (Panasonic). All experiments were performed at room temperature (20–22°C).
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Measurement of hydraulic conductivity (Lp)
Lp was measured using the Landis-Michel technique (Michel et al. 1974), as previously described by this laboratory (Pocock & Bates, 2001a). 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 leucocytes adhering to the vessel wall. The vessel was cannulated with a bevelled glass micropipette connected to a manometer and perfused with 1% BSA in physiological frog Ringer solution, pH 7.40 ± 0.02, containing rat erythrocytes as flow markers (baseline solution). To measure Lp a pulled glass micropipette was used to occlude the vessel for approximately 5 s at least 800 μm downstream from the cannulation site. The vessel was allowed to flow freely for at least 8 s between occlusions. Baseline Lp was measured for all vessels before the experiment was performed. Vessels with a baseline Lp > 10 x 10–7 cm s–1 cmH2O–1 were excluded, since these lie more than 2 standard deviations from the mean of unstimulated frog microvessels, and were therefore considered to be a separate population of vessels. The pipette was refilled with test solutions as previously described (Hillman et al. 2001). Pressures between 30 and 40 cmH2O were used. Any drugs used were made as stock solutions in water or vehicle before being diluted in the baseline solution.
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Calculation of Lp
The radius of the vessel (r), the initial velocity of the marker cells (dl/dt) within a few seconds of occlusion and the length (l) between the marker cell and the occlusion site were measured offline from the video recording. Filtration rate per unit area (Jv/A) was calculated as:
Hydraulic conductivity (Lp) was calculated from the Starling equation, where P was the hydrostatic pressure difference, the oncotic pressure difference between the capillary lumen and the interstitium, and the oncotic reflection coefficient. (The effective oncotic pressure () of 1% BSA is 3.6 cmH2O.)
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Measurement of [Ca2+]cin vivo
The frog was anaesthetized and an incision made in the lateral body wall as described. For [Ca2+]c measurement the frog tray was modified to accommodate the short working distance required for the objectives used. The quartz pillar was replaced with a glass coverslip with a ‘horseshoe’-shaped layer of Sylgaard glued around the edge. The mesentery was spread over the coverslip and held in place by pins placed through the avascular portions of the mesentery and into the supporting Sylgaard layer. The mesentery was superfused with physiological frog Ringer solution throughout the experiments.
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Vessels were visualized under an epifluorescence microscope (Leica DM IRB) equipped with a photomultiplier tube (PMT) (Cairn). The excitation wavelengths were controlled by a filter wheel (Cairn) controlled in turn by a spectrophotometer (Cairn) that also controlled the PMT so that the fluorescence signals could be synchronized with the passage of the filters through the light beam. Thus, excitation at wavelengths of 340 ± 5, 360 ± 5 and 380 ± 5 nm could be delineated using fast cycle speeds (e.g. 50 Hz). The spectrophotometer voltages at each filter position were displayed on a computer through a PowerLab/4SP (ADInstruments).
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Vessels were cannulated and perfused at 30–40 cmH2O pressure with 10–25 μM Fura-2 AM (acetoxymethyl ester form of Fura-2; Molecular Probes) (0.01–0.25% final DMSO vehicle) in baseline solution (1% BSA in frog Ringer solution). The vessels were perfused in the dark for 60–120 min at 20–22°C. Fluorescence intensities (If) were measured from a window 150 μm long and 50 μm wide that was placed approximately 200 μm downstream of the cannulation site. If at excitation wavelengths () of 340 ± 5 nm and 380 ± 5 nm and emission at 510 ± 35 nm were collected. An initial background estimate was measured from an area of mesentery two vessel widths away from the perfused vessel. The vessel was briefly examined under 360 nm light to ensure even loading had occurred. If the Fura-2 loading was not even or if extravascular Fura-2 was observed the vessels were rejected. Once the If reached a level 3–10 times greater than the background, the vessel was perfused for 10 min with 1% BSA to provide a baseline [Ca2+]c measurement. The If emitted when Fura-2 was excited at 340, 380 and 360 nm was recorded using Chart V3.6 MacLab system for later analysis.
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The values of If emitted when Fura-2 was excited at 340 nm and 380 nm were measured and the ratio (R) was calculated as
where If is the fluorescence intensity and B is the background fluorescence measured following Mn2+ quench of the Fura-2. The ratio was normalized as
where Rexp is the experimental ratio and Rmin is the ratio from an in vitro calibration buffer with < 1 nM[Ca2+].
When measuring the If emissions, a sampling frequency of two measurements per second was used and the rate of increase of Ca2+ was calculated as the slope of the [Ca2+]c plotted against time, averaged over three measurements.
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Data analysis and statistics
The time taken for the peak Lp, peak [Ca2+]c (taken from the time for the Rnorm to reach a maximum) and peak rate of increase of the [Ca2+]c (d[Ca2+]c/dt, taken from the rate of change of Rnorm) to occur following drug exposure are expressed as mean ±S.E.M. ANOVA followed by Bonferroni's post hoc test was used to compare the time taken for Lp to reach a maximum with either the time taken for [Ca2+]c to reach a maximum or for the rate of increase of [Ca2+]c to reach a maximum.
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Results
Effects of store release
To determine whether the time taken for the maximum Lp and [Ca2+]c to occur is the same during release of calcium from intracellular stores, vessels were perfused with agents that release calcium from intracellular stores under conditions of inhibited calcium influx. It has previously been shown that 5 μM ionomycin releases Ca2+ from intracellular stores in endothelial cells (Morgan & Jacob, 1994), 100 nM thapsigargin depletes calcium stores and SKF 96365 inhibits calcium entry across the plasma membrane through non-specific cation channels in endothelial cells of intact frog microvessels (Pocock et al. 2000b; Bates et al. 2001). Basal [Ca2+]c was measured during 1% BSA perfusion. The vessels were then perfused for 10 min with 100 μM SKF 96365 (SKF), followed by SKF, 100 nM thapsigargin (TG) and 5 μM ionomycin (IM) for at least 20 min to release Ca2+ from the intracellular stores without Ca2+ influx. Figure 1 shows an example of a single microvessel that has been treated as described above. Figure 1A shows the fluorescence intensity (If) measured when Fura-2 was excited at 340 (upper trace) and 380 nm (lower trace). Figure 1B shows Rnorm, calculated as described above. During perfusion with SKF, the [Ca2+]c slightly increased from basal levels but remained stable. When Ca2+ was released from the Ca2+ stores of the endothelial cells that line the vessel by perfusion with TG and IM, a transient increase in Rnorm was measured that returned to basal levels after 5 min exposure. In the continued presence of SKF, TG and IM, Rnorm fell below basal levels.
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Fluorescence measurements from a single microvessel. A, If340 (upper trace) and If380 (lower trace) during basal conditions (bovine serum albumin (BSA) solution), during Ca2+ influx inhibition (SKF 96365; SKF) and during total store release without Ca2+ influx (SKF/TG/IM). B, Rnorm, the ratio of If340 to If380, normalized to the minimum ratio for zero calcium (Rmin), for the same experiment. Time of drug applications are indicated by the bars. Inhibition of Ca2+ influx resulted in a small increase in the baseline Rnorm (point A). Application of thapsigargin (TG) and ionomycin (IM) (point B) resulted in a significant transient increase in Rnorm that returned to below baseline within 10 min (point C).
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The mean changes in fluorescence ratios from eight experiments are shown in Fig. 2A. During Ca2+ influx inhibition (SKF), the [Ca2+]c did not significantly change from a mean ±S.E.M.Rnorm of 1.0 ± 0.2 under basal conditions with 1% BSA (n= 8) to an Rnorm of 1.1 ± 0.2 (n= 6). Perfusion with SKF, TG and IM (store release without Ca2+ influx) significantly increased the ratio 1.5 ± 0.2 fold from an Rnorm of 1.1 ± 0.2 to 1.7 ± 0.5 (P < 0.05, n= 8). There were no significant differences between Rnorm values under basal conditions with BSA or SKF (P > 0.05). There was a significant correlation between the Rnorm under basal conditions with SKF and the peak Rnorm during store release (SKF/TG/IM) (r= 0.94, P < 0.02, Spearman, Fig. 2B).
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A, mean ±S.E.M.Rnorm under basal conditions (BSA), during Ca2+ influx inhibition (SKF) and during total store release without Ca2+ influx (SKF/TG/IM). * Significant difference versus SKF. B, [Ca2+]c during Ca2+ influx inhibition (SKF) significantly correlated with [Ca2+]c during total store release (peak SKF/TG/IM).
The time course of the response was compared with experiments performed in other vessels where the Lp had been measured under the same conditions. To try and compare separate vessels, in Fig. 3A we have illustrated the two fastest responses we have recorded for calcium and permeability. This figure shows an example of a microvessel in which the Lp has been measured during perfusion with SKF, TG and IM to induce Ca2+ store release. The Lp (upper panel) reaches a maximum value within a minute of the start of the perfusion with IM and TG (continuous arrow). A second measurement reveals that the Lp values have started to return to control values. The peak Lp must have occurred before the second of these two points (shown by the dashed arrow). The lower panel shows the [Ca2+]c measured in a separate microvessel, which gave the most rapid response to TG and IM. At the time of maximum [Ca2+]c (dashed arrow) the Lp response in other vessels had already started to subside. In fact the Lp was at its maximum measured value in most vessels before the [Ca2+]c had even approached its maximum value. The differential of the [Ca2+]c trace was also plotted on the bottom graph showing the rate of change of [Ca2+]c. The maximum rate of change of Ca2+ occurs before the peak Lp. These examples suggest that the peak Lp during store release occurs before the peak [Ca2+]c but after the peak rate of increase of [Ca2+]c. This suggests that the absolute [Ca2+]c is unlikely subsequently to regulate the magnitude of the permeability increase, but that the rate of increase of the [Ca2+]c might.
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A, the top graph shows an individual microvessel where Lp was measured during total store release without Ca2+ influx (SKF/TG/IM added at time point zero). The bottom graph shows Rnorm in a separate microvessel treated with the same protocol as the top graph. The rate of change of Rnorm is shown. The time frame before the dashed arrow indicates when the Lp will be maximum. These two vessels gave the fastest permeability and calcium responses. The highest Lp value was reached before the highest fluorescence ratio was reached. B, mean ±S.E.M. time to peak Lp, time to peak [Ca2+]c and time to peak rate of change of [Ca2+]c during total store release in the absence of Ca2+ influx (SKF/TG/IM). * Significant difference versus time to peak Lp. In general, the permeability peaks before the calcium, but at a similar time to the rate of change of calcium.
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As the experiments shown in Fig. 3A are in separate vessels the time course of the responses may not be representative of the Lp and Ca2+ response that occur in a single vessel. In order to address this problem we have measured the responses in several vessels and used the average time for each response to reach a peak as a measure for the time course. Figure 3B shows the average time taken for the Lp to peak from 10 vessels and the average time taken for the [Ca2+]c to peak from 8 vessels during Ca2+ store release. The Lp peaked after 3.6 ± 0.9 min perfusion with SKF, TG and IM whereas the time taken for [Ca2+]c to peak was significantly greater: 9.2 ± 2.8 min (P < 0.05), i.e. more than two standard errors higher than the time for Lp to peak. The time taken for the maximum rate of change of [Ca2+]c to occur, however, was 4.3 ± 2.0 min, very similar to the time taken for the Lp to peak (P > 0.05).
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Effects of store-mediated calcium influx
We determined whether this relationship between Lp and the rate of increase of the [Ca2+]c was also true for permeability increased by a physiological agonist that increases Lp by releasing endoplasmic reticulum (ER) store Ca2+ and subsequently inducing Ca2+ influx. ATP, at a concentration of 30 μM, has been shown to stimulate store-dependent calcium entry in endothelial cells of frog microvessels in vivo (Pocock et al. 2000b). Lp was measured in vessels that were perfused with 30 μM ATP following perfusion with 1% BSA to establish a baseline (n= 12, Fig. 4). The same experiment was performed whilst measuring [Ca2+]c in a separate set of vessels (n= 6). A summary of the results is shown in Fig. 4. The average time for the Lp to peak was 1.2 ± 0.3 min (n= 12). This was lower than the average time taken for the [Ca2+]c to peak, 2.1 ± 0.7 min (n= 6, P < 0.05), but very similar to the average time taken for the peak rate of increase of [Ca2+]c to occur (1.1 ± 0.5 min, n= 6). These results support the experiments using SKF, TG and IM.
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Mean ±S.E.M. time to peak Lp, time to peak [Ca2+]c and time to peak rate of change of [Ca2+]c with ATP. * Significant difference versus time to peak Lp.
Effects of store-independent calcium influx
Both sets of experiments described above increase Lp in a store-dependent manner. VEGF (1 nM) has been shown to increase intracellular calcium in endothelial cells of frog microvessels by receptor tyrosine kinase-mediated activation of phospholipase C (PLC), and activation of a non-specific cation channel by 1,2-diacylglycerol (DAG) activation, probably the canonical transient receptor TRPC3 or TRPC6 (Pocock & Bates, 2001b; Pocock et al. 2004). To determine if the maximum rate of increase of [Ca2+]c occurs at the same time as the maximum Lp during a store-independent increase in permeability, previous experiments using VEGF to increase Lp and [Ca2+]c in a store-independent manner were re-analysed (Bates & Curry, 1996, 1997; Pocock et al. 2000a). The time to peak Lp was taken from the first 19 vessels in which the Lp response to VEGF was measured and the [Ca2+]c in the first 18 vessels and peak rate of increase in 14 vessels. The data are summarized in Fig. 5. The time taken for the Lp to peak was 22.7 ± 1.8 s (0.38 ± 0.03 min). The time to the peak Lp was again significantly less than the time to the peak [Ca2+]c, 63 ± 18 s (1.05 ± 0.31 min, P < 0.01), but not significantly different from the time to the peak rate of increase of [Ca2+]c, 18 ± 5.6 s (0.30 ± 0.1 min, P > 0.05).
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A, measurement of calcium and rate of change of calcium in vessel exposed to 1 nM VEGF. The [Ca2+]c reaches a maximum at around 90 s (point i), by which time all the permeability responses measured have returned to control. The maximum rate of change, however, occurs within 30 s (point ii), similar to the peak permeability measurement. B, mean ±S.E.M. time to peak Lp, time to peak [Ca2+]c and time to peak rate of change of [Ca2+]c with VEGF. * Significant difference versus time to peak Lp.
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The experiments described so far have measured either Lp or [Ca2+]c but not both simultaneously in the same vessel. These experiments are technically very difficult but nonetheless possible when combining infrared illumination, a 700 nm dichroic mirror in front of the photomultiplier tube, and an infrared camera connected to a video, time generator and monitor as described for the Lp experiments. Figure 6 shows the results from the only two microvessels in which we have successfully measured both Lp and Ca2+ during perfusion with 30 μM ATP (to induce store-dependent Ca2+ influx). [Ca2+]c was continually measured except for the brief periods when Lp was measured (dashed line). The graph shows that Lp reaches a maximum value before [Ca2+]c in both vessels. Furthermore, peak Lp corresponds with the steepest part of the [Ca2+]c trace indicating that Lp is maximum when the rate of change of [Ca2+]c is maximum.
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ATP (30 μM) was added at time zero to induce store-dependent Ca2+ influx. Each filled square is a Lp measurement. The thin lines show the change in Lp and the thick interrupted line indicates [Ca2+]c as represented by Rnorm. Note that Lp reaches a maximum before Rnorm.
If it is the rate of change of calcium that regulates the permeability, rather than the absolute [Ca2+]c during stimulation, then there should be a positive correlation between the time for d[Ca2+]c/dtmax and the [Ca2+]max. Figure 7 demonstrates that there was a highly significant correlation between these two parameters.
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The time taken for the [Ca2+]c to peak was highly significantly correlated to the time taken for the maximum rate of increase of the [Ca2+]c to occur when the [Ca2+]c was increased by 1 nM VEGF (), ATP () and thapsigargin ().
Discussion
The mechanisms that underlie agonist-mediated increases in permeability are poorly understood. Although Ca2+ is implicated in most permeability responses, the mechanism by which Ca2+ regulates the increase in permeability is not understood. It has been assumed, and data have been presented supporting the hypothesis, that the magnitude of the increase in permeability is dependent on the size of the increase in [Ca2+]i in the endothelial cell (He et al. 1990; He & Curry, 1991; Sarker et al. 1998; Glass & Bates, 2003b). However, previous studies have shown that it is possible for Lp to be increased over a sustained period of time without increased intracellular [Ca2+], under certain conditions. Perfusion of vessels without albumin (i.e. Ringer perfusion) (He & Curry, 1993), and the chronic sustained increase seen with VEGF and ATP 24 h after exposure (Bates et al. 2001), all have a normal baseline Ca2+, despite increased Lp. The results here show that the measured [Ca2+]c in endothelial cells of mesenteric microvessels reaches a maximum after the permeability reaches a maximum. This rules out the possibility that the size of the increase in Ca2+ is directly responsible for the magnitude of the increase in permeability, contrary to our expectations, and previously published interpretations (He et al. 1996). Thus, the global Ca2+ concentration may not be the only key factor by which permeability is regulated. These results force us to investigate more subtle methods of permeability regulation by endothelial cell Ca2+.
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We have previously shown that both the magnitude of the increase in Ca2+ and the baseline permeability are dependent on the state of filling of the Ca2+ store (Glass & Bates, 2003b). Since the rate of flux of Ca2+ from the store to the cytoplasm will be greater when stores are relatively full, we hypothesized that perhaps in endothelial cells of vessels that have a normal baseline permeability, the rate of Ca2+ entry into the cytoplasm may be more critical for the regulation of the magnitude of the permeability response than the global Ca2+ concentration. Furthermore it has been shown that, under conditions of altered membrane potential, the quantitative relation between the initial change in Ca2+ concentration and the calculated Ca2+ influx using the Goldman–Hodgkin–Katz equation for Ca2+ influx correlates well with the peak Lp under conditions of high extracellular potassium (depolarization) or Ca2+ (Curry, 1992). Additionally, Pagakis et al. show examples of single vessels in which regions of interest within three different vessels indicate a correlation between the initial rate of change of Ca2+ and peak permeability values (Pagakis & Curry, 1994). The results shown here clearly indicate that the maximum permeability is more closely correlated with the rate of change than the absolute concentration during store-mediated Ca2+ entry. To test this hypothesis further we determined whether this was also true for the increase in Ca2+ and permeability stimulated by increasing Ca2+ entry across the plasma membrane, and found that indeed the permeability reached its maximum before the [Ca2+]c did, whereas the peak rate of Ca2+ rise again matched the permeability response.
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Nevertheless, a close correlation does not show that rate of change determines Lp, and there are other, equally valid, possibilities for regulating the permeability. One alternative hypothesis is that the kinetics of the shift in fluorescence of Fura-2 is so slow that the calcium response occurs significantly before the change in fluorescence signal is measured. However, it is clear from studies on the kinetics of calcium binding to Fura-2 that the time taken for a change in [Ca2+]c to be detected, and subsequently signal through the system described above, should be at least an order of magnitude faster than the time course described (Kao & Tsien, 1988). A second explanation is that the presence of Fura-2 significantly delays the calcium response, since the experiments where hydraulic conductivity alone was measured were not carried out in the presence of Fura-2. To address this aim, we have measured Lp and [Ca2+]c in the same capillary at the same time, and in both examples where we were successful, the peak Lp increase did precede the [Ca2+]c peak, suggesting that the presence of Fura-2 did not affect the time course of the permeability response.
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Is permeability determined by the rate of increase of [Ca2+]c
There is supporting evidence for the hypothesis that the rate of change of [Ca2+]c rather than the absolute [Ca2+]c regulated permeability from endothelial cell culture experiments. At least two different Ca2+-dependent mechanisms may be regulated by Ca2+ influx. In pulmonary endothelial cells, the rate of Ca2+ influx into the cell has been implicated in the indirect regulation of permeability/barrier integrity, as it regulates cAMP (Stevens et al. 1995). Stevens et al. demonstrated that cAMP production was increased when Ca2+ influx was blocked with La3+ (Stevens et al. 1995). Furthermore, they showed that IM decreased cAMP production. However, although they suggested that the rate of Ca2+ influx might regulate cAMP production, they did not distinguish between the rate of Ca2+ influx and the [Ca2+]c. They did, however, state that La3+ decreased cation influx by more than 90%, whereas the [Ca2+]c was only decreased by 40%. This led them to conclude that the rate of Ca2+ influx might be more important in regulating cAMP production than the absolute [Ca2+]c. cAMP has been shown to inhibit increased permeability by activating protein kinase A, which phosphorylates myosin light chain kinase (MLCK) (Garcia et al. 1995). When MLCK is phosphorylated it has a lower affinity for Ca2+–calmodulin and reduces the ability of the cell to contract (Garcia et al. 1995; He et al. 2000). There may also be additional cAMP-dependent mechanisms that regulate permeability because recent evidence suggests that inflammatory mediators such as platelet-activating factor and bradykinin increase permeability independently of MLC phosphorylation (Adamson et al. 2003).
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Nitric oxide synthase (NOS) may also be regulated by the rate of Ca2+ influx. NOS is associated with caveolin-1 in the caveolae on the plasma membrane (Segal et al. 1999; Goligorsky et al. 2002). The Ca2+ influx channels are also located in the caveolae (Isshiki & Anderson, 1999) and many IP3 receptors on the ER membrane are closely apposed to the caveolae (Isshiki et al. 2002). Hence, NOS is in the appropriate location to be regulated by the rate of Ca2+ influx. When NOS is inhibited, agonists such as ATP are no longer able to increase the permeability effectively (He et al. 1997a), demonstrating that NO regulates agonist-mediated increases in permeability. We also note that, in vessels that are not exposed to inflammatory agents, inhibitors of NOS increase microvessel permeability (He et al. 1997b) suggesting it can play a stabilizing role similar to cAMP under some conditions.
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In this study we have measured the global [Ca2+]c under conditions of intracellular Ca2+ store release using the same drug combination (SKF, TG and IM) and protocol as in a separate set of permeability experiments. From the [Ca2+]c trace, the rate of change of [Ca2+]c could be calculated as the differential of the [Ca2+]c trace. This, however, is the rate of change of [Ca2+]c rather than the rate of Ca2+ flux into the cytosol, as Ca2+ extrusion from the cell by the plasma membrane Ca2+-ATPase pump or the Na+–Ca2+ exchanger has not been blocked (Hinde et al. 1999). The relationship between the time courses of the permeability and rate of change of the [Ca2+]c responses appears to occur in the absence or presence of stores, and in the absence or presence of Ca2+ influx across the plasma membrane. However, at least one of these processes is necessary since removing store-dependent Ca2+ release in the absence of Ca2+ influx, or removing Ca2+ influx in the absence of store release (Pocock et al. 2000a), blocks the permeability response. Thus the nature of the mechanisms linking calcium to permeability are likely to be cytoplasmic, and common to different Ca2+ sources. One explanation for this would be that the local site of increase in [Ca2+] is a regulating mechanism for the magnitude of the permeability response.
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Rate of flux, or site of flux
Agents such as ATP activate G-protein-coupled receptors linked to PLC- that generates diacylglycerol (DAG) and IP3 (Ward et al. 2003). IP3 then releases ER store Ca2+ and increases permeability via the NO–cGMP pathway (He et al. 1997a). If, however, the ER store is depleted with TG, extracellular ATP can no longer increase the permeability (Pocock et al. 2000a). VEGF increases permeability through a different signalling cascade. VEGF receptor-2 is a tyrosine kinase receptor that phosphorylates PLC- (Guo et al. 1995). DAG and IP3 are generated as with ATP stimulation, but ER store depletion with TG does not inhibit VEGF-stimulated permeability increases (Pocock et al. 2000a). VEGF in vivo acutely increases permeability through a PLC-dependent, but Ca2+ store-, mitogen activated protein kinase (MAPK) and extracellular signal related Kinase (ERK) Kinase (MEK)-, PKC- and DAG lipase-independent pathway (Pocock & Bates, 2001a). Recent evidence using TRP activators, inhibitors and heterologous expression systems, and the finding that the membrane-permeable DAG analogue OAG increases permeability, suggests that DAG directly induces Ca2+ influx through the activation of second messenger-mediated Ca2+ channels such as TRPC3, 6 or 7 channels (Pocock & Bates, 2002).
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The fact that ATP (store dependent) and VEGF (store independent) both increase permeability at the same rate as they increase Ca2+ influx, but VEGF acts much more quickly, might be due to the localization of the different signalling cascades. There is significant evidence to show that both the ATP- and VEGF-stimulated signalling pathways are linked to caveolae, which appear to act as regulatory localized subdomains for signal transduction in endothelial cells (Shaul & Anderson, 1998; Isshiki & Anderson, 1999; Lockwich et al. 2001; Labrecque et al. 2003). Distinct populations of caveolae in the plasma membrane might contain different signalling proteins and channels. As ATP and VEGF activate different PLC isoforms this could suggest that the signalling cascade for each originates in a separate caveolar population. Therefore, not only is the signalling different but the localization of the receptors and effector molecules is also likely to vary, which may account for the different time courses of the responses with ATP and VEGF.
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If the differences seen between the ATP and VEGF Lp responses are due to different populations of caveolae, either the rate of Ca2+ influx or the [Ca2+] within the microdomain could be regulators of permeability. Current data are unable to distinguish between these two possibilities, but merit further research. A third possibility is that a threshold [Ca2+]c may regulate the initiation of the permeability increase. We were unable to establish if there is a minimum increase in [Ca2+]c required before the permeability increases, as the Ca2+ had already started to increase by the time of the first permeability measurement. The peak [Ca2+]c may follow the peak Lp because a threshold [Ca2+]c sufficient to increase the permeability occurred first. The amount of time that the endothelial cells are above a threshold might thereby regulate the permeability. The magnitude of the [Ca2+]c increase would in this case still correlate with the magnitude of the permeability increase, since a threshold would be reached faster when the magnitude is higher. He et al. suggested that there is a direct correlation between the magnitude of Lp and the [Ca2+]c when the [Ca2+]c was greater than 130 nM (He et al. 1990). Once the global [Ca2+]c was 130 nM, the Lp would increase, and the rate at which the threshold was reached would control the magnitude of the response. The correlation demonstrated by He et al. (1990) implies that the magnitude of the Lp increase is related to [Ca2+]c once the Lp response had already been initiated, but the threshold at which that is initiated and the rate of change by which that threshold is reached could still regulate the magnitude of the response.
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In summary, we have shown here, that contrary to previous interpretations, in response to inflammatory mediators and growth factors, the Ca2+ concentration in endothelial cells reaches a peak after the permeability does so, and therefore cannot be responsible for the magnitude of the increase. The maximum rate of Ca2+ influx, however, appears to match closely with the magnitude of the permeability response when the permeability increase is either store mediated, or store independent. These findings lead us to the conclusion that it is either the local Ca2+ concentration by the membrane, or the rate of flux of Ca2+ into the cytosol that regulates the permeability, and that store-independent Ca2+ increases can result in a more rapid permeability increase. This implies that the local concentration of Ca2+ immediately underneath the membrane is likely to regulate the permeability response, raising the possibility that store-dependent and independent permeability increases may act through separate populations of membrane microdomains.
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Ward PD, Ouyang H & Thakker DR (2003). Role of phospholipase C-beta in the modulation of epithelial tight junction permeability. J Pharmacol Exp Ther 304, 689–698., 百拇医药(C. A. Glass, T. M. Pocock)
2 Department of Physiology and Membrane Biology, University of California at Davis, Davis, CA 95616, USA
Abstract
Vascular permeability is assumed to be regulated by the cytosolic Ca2+ concentration ([Ca2+]c) of the endothelial cells. When permeability is increased, however, the maximum [Ca2+]c appears to occur after the maximum permeability increase, suggesting that Ca2+-dependent mechanisms other than the absolute Ca2+ concentration may regulate permeability. Here we investigate whether the rate of increase of the [Ca2+]c (d[Ca2+]c/dt) may more closely approximate the time course of the permeability increase. Hydraulic conductivity (Lp) and endothelial [Ca2+]c were measured in single perfused frog mesenteric microvessels in vivo. The relationships between the time courses of the increased Lp, [Ca2+]c and d[Ca2+]c/dt were examined. Lp peaked significantly earlier than [Ca2+]c in all drug treatments examined (Ca2+ store release, store-mediated Ca2+ influx, and store-independent Ca2+ influx). When Lp was increased in a store-dependent manner the time taken for Lp to peak (3.6 ± 0.9 min during store release, 1.2 ± 0.3 min during store-mediated Ca2+ influx) was significantly less than the time taken for [Ca2+]c to peak (9.2 ± 2.8 min during store release, 2.1 ± 0.7 min during store-mediated influx), but very similar to that for the peak d[Ca2+]c/dt to occur (4.3 ± 2.0 min during store release, 1.1 ± 0.5 min during Ca2+ influx). Additionally, when the increase was independent of intracellular Ca2+ stores, Lp (0.38 ± 0.03 min) and d[Ca2+]c/dt (0.30 ± 0.1 min) both peaked significantly before the [Ca2+]c (1.05 ± 0.31 min). These data suggest that the regulation of vascular permeability by endothelial cell Ca2+ may be regulated by the rate of change of the [Ca2+]c rather than the global [Ca2+].
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Introduction
Exchange of fluid, nutrients and waste products between the plasma and interstitium occurs in the capillaries and postcapillary venules. This exchange is regulated by the endothelial cell barrier of the microvascular wall. Increased vascular permeability may occur under pathological situations or be induced by growth factors and inflammatory mediators. These mediators result in a breakdown of the integrity of the wall, increased macromolecular transport by diffusion and convection, and increased fluid flow down the Starling pressure gradient. This increase in permeability results in oedema and impaired tissue function, probably due to inefficient oxygen and glucose delivery, but also allows the inflammatory process access to the interstitium and enables tissue remodelling. The increase in permeability brought about by inflammatory mediators and growth factors is a regulated process, dependent on endothelial cell surface receptor activation and subsequent downstream signalling.
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Many studies have shown that when vascular permeability is increased, it is usually accompanied by an increase in cytosolic Ca2+ concentration ([Ca2+]c) (He & Curry, 1991; He et al. 1996; Pocock et al. 2000a). This increase in [Ca2+]c is required for the permeability to increase in response to many cytokines. These cytokines include growth factors such as vascular endo-thelial growth factor (VEGF; Pocock et al. 2000a), and classical inflammatory mediators such as histamine (Sarker et al. 1998), ATP (He et al. 1996; Pocock et al. 2000a) and serotonin (Olesen, 1985). Moreover, exposure of vessels to Ca2+ ionophores, such as A23187, results in an increase in vessel permeability (Michel & Phillips, 1989; He et al. 1990; Neal & Michel, 1995) and as the changes in permeability closely track changes in the [Ca2+]c, and inhibition of the increase in [Ca2+]c inhibits the permeability increase (He & Curry, 1991; Sarker et al. 1998; Glass & Bates, 2003b), it has been assumed that the [Ca2+]c regulates permeability (He et al. 1990). However, the mechanisms by which this increase in Ca2+ translates to increased permeability are still not known.
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There have been a few studies attempting to measure [Ca2+]c and vascular permeability in vivo using the same preparation (although in different animals). He & Curry have shown that the time courses of increases in hydraulic conductivity (Lp), a measure of permeability, and the [Ca2+]c responses to the Ca2+ ionophore, ionomycin, were closely correlated, and indeed overlapping (with a time resolution of 15–30 s), and that the size of the increase in permeability was directly correlated with the size of the increase in Ca2+ (He et al. 1990). They showed that the time courses for these responses were not significantly different from each other. It therefore appeared that the increase in permeability was stimulated by an increase in [Ca2+]c.
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Whilst investigating the role of Ca2+ stores in the regulation of vascular permeability, we showed that Lp increases differ in magnitude when [Ca2+]c is increased by Ca2+ from different sources (Glass & Bates, 2003b). We suggested that different sources of Ca2+ result in different rates of increase of [Ca2+]c (Glass & Bates, 2003b). We hypothesized that differing rates of Ca2+ entry into the cytoplasm might explain the differences in magnitude of the increased Lp, This would be consistent with He & Curry's findings that the permeability was correlated with [Ca2+]c, as a higher rate would be needed to reach that higher concentration, if this occurs with the same time course. However, this would require the Lp to reach its peak coincident with the maximum rate of Ca2+ entry, i.e. slightly before the [Ca2+]c reached its peak. Since the time resolution of the experiments by He & Curry was not sufficient to determine whether this was the case, we have investigated whether the time taken for the maximum Lp and [Ca2+]c to occur is the same, irrespective of the source of the increased Ca2+, or if the rate of change of the [Ca2+]c is more closely linked to the peak Lp value.
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Some of these experiments have previously been published as an abstract of a conference presentation (Glass & Bates, 2003a).
Methods
Materials
Male frogs (Rana temporaria) were supplied by Blades, UK. All regulated procedures were carried out under licence from the Home Office in accordance with the Animals (Scientific Procedures) Act 1986. Rat erythrocytes were collected by cardiac puncture from male Wistar rats that had been anaesthetized with 5% halothane and killed by cervical dislocation. All chemicals were purchased from Sigma unless otherwise stated.
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Any drugs used were made as stock solutions in water or DMSO vehicle before being diluted in 1% (w/v) bovine serum albumin (BSA) solution (final DMSO concentrations as percentages (v/v) were 0.01 and 0.05 in thapsigargin and ionomycin, respectively). VEGF-Aics was used.
In vivo mesenteric microvascular preparation
Frogs were anaesthetized by immersion in 1 mg ml–1 MS222 (3-aminobenzoic acid ethyl ester) in water. At the end of the experiment they were killed by destruction of the brain and central nervous system. Anaesthesia was maintained by superfusing the mesentery with 0.25–0.40 mg ml–1 MS222 in physiological frog Ringer solution (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 bud and draped over a transparent quartz pillar so that the mesentery could be visualized through a Leica inverted microscope (DMIL). Experiments were recorded using a video camera (Pulnix) connected through an electronic timer (ForA) to a video recorder (Panasonic). All experiments were performed at room temperature (20–22°C).
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Measurement of hydraulic conductivity (Lp)
Lp was measured using the Landis-Michel technique (Michel et al. 1974), as previously described by this laboratory (Pocock & Bates, 2001a). 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 leucocytes adhering to the vessel wall. The vessel was cannulated with a bevelled glass micropipette connected to a manometer and perfused with 1% BSA in physiological frog Ringer solution, pH 7.40 ± 0.02, containing rat erythrocytes as flow markers (baseline solution). To measure Lp a pulled glass micropipette was used to occlude the vessel for approximately 5 s at least 800 μm downstream from the cannulation site. The vessel was allowed to flow freely for at least 8 s between occlusions. Baseline Lp was measured for all vessels before the experiment was performed. Vessels with a baseline Lp > 10 x 10–7 cm s–1 cmH2O–1 were excluded, since these lie more than 2 standard deviations from the mean of unstimulated frog microvessels, and were therefore considered to be a separate population of vessels. The pipette was refilled with test solutions as previously described (Hillman et al. 2001). Pressures between 30 and 40 cmH2O were used. Any drugs used were made as stock solutions in water or vehicle before being diluted in the baseline solution.
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Calculation of Lp
The radius of the vessel (r), the initial velocity of the marker cells (dl/dt) within a few seconds of occlusion and the length (l) between the marker cell and the occlusion site were measured offline from the video recording. Filtration rate per unit area (Jv/A) was calculated as:
Hydraulic conductivity (Lp) was calculated from the Starling equation, where P was the hydrostatic pressure difference, the oncotic pressure difference between the capillary lumen and the interstitium, and the oncotic reflection coefficient. (The effective oncotic pressure () of 1% BSA is 3.6 cmH2O.)
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Measurement of [Ca2+]cin vivo
The frog was anaesthetized and an incision made in the lateral body wall as described. For [Ca2+]c measurement the frog tray was modified to accommodate the short working distance required for the objectives used. The quartz pillar was replaced with a glass coverslip with a ‘horseshoe’-shaped layer of Sylgaard glued around the edge. The mesentery was spread over the coverslip and held in place by pins placed through the avascular portions of the mesentery and into the supporting Sylgaard layer. The mesentery was superfused with physiological frog Ringer solution throughout the experiments.
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Vessels were visualized under an epifluorescence microscope (Leica DM IRB) equipped with a photomultiplier tube (PMT) (Cairn). The excitation wavelengths were controlled by a filter wheel (Cairn) controlled in turn by a spectrophotometer (Cairn) that also controlled the PMT so that the fluorescence signals could be synchronized with the passage of the filters through the light beam. Thus, excitation at wavelengths of 340 ± 5, 360 ± 5 and 380 ± 5 nm could be delineated using fast cycle speeds (e.g. 50 Hz). The spectrophotometer voltages at each filter position were displayed on a computer through a PowerLab/4SP (ADInstruments).
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Vessels were cannulated and perfused at 30–40 cmH2O pressure with 10–25 μM Fura-2 AM (acetoxymethyl ester form of Fura-2; Molecular Probes) (0.01–0.25% final DMSO vehicle) in baseline solution (1% BSA in frog Ringer solution). The vessels were perfused in the dark for 60–120 min at 20–22°C. Fluorescence intensities (If) were measured from a window 150 μm long and 50 μm wide that was placed approximately 200 μm downstream of the cannulation site. If at excitation wavelengths () of 340 ± 5 nm and 380 ± 5 nm and emission at 510 ± 35 nm were collected. An initial background estimate was measured from an area of mesentery two vessel widths away from the perfused vessel. The vessel was briefly examined under 360 nm light to ensure even loading had occurred. If the Fura-2 loading was not even or if extravascular Fura-2 was observed the vessels were rejected. Once the If reached a level 3–10 times greater than the background, the vessel was perfused for 10 min with 1% BSA to provide a baseline [Ca2+]c measurement. The If emitted when Fura-2 was excited at 340, 380 and 360 nm was recorded using Chart V3.6 MacLab system for later analysis.
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The values of If emitted when Fura-2 was excited at 340 nm and 380 nm were measured and the ratio (R) was calculated as
where If is the fluorescence intensity and B is the background fluorescence measured following Mn2+ quench of the Fura-2. The ratio was normalized as
where Rexp is the experimental ratio and Rmin is the ratio from an in vitro calibration buffer with < 1 nM[Ca2+].
When measuring the If emissions, a sampling frequency of two measurements per second was used and the rate of increase of Ca2+ was calculated as the slope of the [Ca2+]c plotted against time, averaged over three measurements.
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Data analysis and statistics
The time taken for the peak Lp, peak [Ca2+]c (taken from the time for the Rnorm to reach a maximum) and peak rate of increase of the [Ca2+]c (d[Ca2+]c/dt, taken from the rate of change of Rnorm) to occur following drug exposure are expressed as mean ±S.E.M. ANOVA followed by Bonferroni's post hoc test was used to compare the time taken for Lp to reach a maximum with either the time taken for [Ca2+]c to reach a maximum or for the rate of increase of [Ca2+]c to reach a maximum.
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Results
Effects of store release
To determine whether the time taken for the maximum Lp and [Ca2+]c to occur is the same during release of calcium from intracellular stores, vessels were perfused with agents that release calcium from intracellular stores under conditions of inhibited calcium influx. It has previously been shown that 5 μM ionomycin releases Ca2+ from intracellular stores in endothelial cells (Morgan & Jacob, 1994), 100 nM thapsigargin depletes calcium stores and SKF 96365 inhibits calcium entry across the plasma membrane through non-specific cation channels in endothelial cells of intact frog microvessels (Pocock et al. 2000b; Bates et al. 2001). Basal [Ca2+]c was measured during 1% BSA perfusion. The vessels were then perfused for 10 min with 100 μM SKF 96365 (SKF), followed by SKF, 100 nM thapsigargin (TG) and 5 μM ionomycin (IM) for at least 20 min to release Ca2+ from the intracellular stores without Ca2+ influx. Figure 1 shows an example of a single microvessel that has been treated as described above. Figure 1A shows the fluorescence intensity (If) measured when Fura-2 was excited at 340 (upper trace) and 380 nm (lower trace). Figure 1B shows Rnorm, calculated as described above. During perfusion with SKF, the [Ca2+]c slightly increased from basal levels but remained stable. When Ca2+ was released from the Ca2+ stores of the endothelial cells that line the vessel by perfusion with TG and IM, a transient increase in Rnorm was measured that returned to basal levels after 5 min exposure. In the continued presence of SKF, TG and IM, Rnorm fell below basal levels.
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Fluorescence measurements from a single microvessel. A, If340 (upper trace) and If380 (lower trace) during basal conditions (bovine serum albumin (BSA) solution), during Ca2+ influx inhibition (SKF 96365; SKF) and during total store release without Ca2+ influx (SKF/TG/IM). B, Rnorm, the ratio of If340 to If380, normalized to the minimum ratio for zero calcium (Rmin), for the same experiment. Time of drug applications are indicated by the bars. Inhibition of Ca2+ influx resulted in a small increase in the baseline Rnorm (point A). Application of thapsigargin (TG) and ionomycin (IM) (point B) resulted in a significant transient increase in Rnorm that returned to below baseline within 10 min (point C).
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The mean changes in fluorescence ratios from eight experiments are shown in Fig. 2A. During Ca2+ influx inhibition (SKF), the [Ca2+]c did not significantly change from a mean ±S.E.M.Rnorm of 1.0 ± 0.2 under basal conditions with 1% BSA (n= 8) to an Rnorm of 1.1 ± 0.2 (n= 6). Perfusion with SKF, TG and IM (store release without Ca2+ influx) significantly increased the ratio 1.5 ± 0.2 fold from an Rnorm of 1.1 ± 0.2 to 1.7 ± 0.5 (P < 0.05, n= 8). There were no significant differences between Rnorm values under basal conditions with BSA or SKF (P > 0.05). There was a significant correlation between the Rnorm under basal conditions with SKF and the peak Rnorm during store release (SKF/TG/IM) (r= 0.94, P < 0.02, Spearman, Fig. 2B).
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A, mean ±S.E.M.Rnorm under basal conditions (BSA), during Ca2+ influx inhibition (SKF) and during total store release without Ca2+ influx (SKF/TG/IM). * Significant difference versus SKF. B, [Ca2+]c during Ca2+ influx inhibition (SKF) significantly correlated with [Ca2+]c during total store release (peak SKF/TG/IM).
The time course of the response was compared with experiments performed in other vessels where the Lp had been measured under the same conditions. To try and compare separate vessels, in Fig. 3A we have illustrated the two fastest responses we have recorded for calcium and permeability. This figure shows an example of a microvessel in which the Lp has been measured during perfusion with SKF, TG and IM to induce Ca2+ store release. The Lp (upper panel) reaches a maximum value within a minute of the start of the perfusion with IM and TG (continuous arrow). A second measurement reveals that the Lp values have started to return to control values. The peak Lp must have occurred before the second of these two points (shown by the dashed arrow). The lower panel shows the [Ca2+]c measured in a separate microvessel, which gave the most rapid response to TG and IM. At the time of maximum [Ca2+]c (dashed arrow) the Lp response in other vessels had already started to subside. In fact the Lp was at its maximum measured value in most vessels before the [Ca2+]c had even approached its maximum value. The differential of the [Ca2+]c trace was also plotted on the bottom graph showing the rate of change of [Ca2+]c. The maximum rate of change of Ca2+ occurs before the peak Lp. These examples suggest that the peak Lp during store release occurs before the peak [Ca2+]c but after the peak rate of increase of [Ca2+]c. This suggests that the absolute [Ca2+]c is unlikely subsequently to regulate the magnitude of the permeability increase, but that the rate of increase of the [Ca2+]c might.
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A, the top graph shows an individual microvessel where Lp was measured during total store release without Ca2+ influx (SKF/TG/IM added at time point zero). The bottom graph shows Rnorm in a separate microvessel treated with the same protocol as the top graph. The rate of change of Rnorm is shown. The time frame before the dashed arrow indicates when the Lp will be maximum. These two vessels gave the fastest permeability and calcium responses. The highest Lp value was reached before the highest fluorescence ratio was reached. B, mean ±S.E.M. time to peak Lp, time to peak [Ca2+]c and time to peak rate of change of [Ca2+]c during total store release in the absence of Ca2+ influx (SKF/TG/IM). * Significant difference versus time to peak Lp. In general, the permeability peaks before the calcium, but at a similar time to the rate of change of calcium.
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As the experiments shown in Fig. 3A are in separate vessels the time course of the responses may not be representative of the Lp and Ca2+ response that occur in a single vessel. In order to address this problem we have measured the responses in several vessels and used the average time for each response to reach a peak as a measure for the time course. Figure 3B shows the average time taken for the Lp to peak from 10 vessels and the average time taken for the [Ca2+]c to peak from 8 vessels during Ca2+ store release. The Lp peaked after 3.6 ± 0.9 min perfusion with SKF, TG and IM whereas the time taken for [Ca2+]c to peak was significantly greater: 9.2 ± 2.8 min (P < 0.05), i.e. more than two standard errors higher than the time for Lp to peak. The time taken for the maximum rate of change of [Ca2+]c to occur, however, was 4.3 ± 2.0 min, very similar to the time taken for the Lp to peak (P > 0.05).
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Effects of store-mediated calcium influx
We determined whether this relationship between Lp and the rate of increase of the [Ca2+]c was also true for permeability increased by a physiological agonist that increases Lp by releasing endoplasmic reticulum (ER) store Ca2+ and subsequently inducing Ca2+ influx. ATP, at a concentration of 30 μM, has been shown to stimulate store-dependent calcium entry in endothelial cells of frog microvessels in vivo (Pocock et al. 2000b). Lp was measured in vessels that were perfused with 30 μM ATP following perfusion with 1% BSA to establish a baseline (n= 12, Fig. 4). The same experiment was performed whilst measuring [Ca2+]c in a separate set of vessels (n= 6). A summary of the results is shown in Fig. 4. The average time for the Lp to peak was 1.2 ± 0.3 min (n= 12). This was lower than the average time taken for the [Ca2+]c to peak, 2.1 ± 0.7 min (n= 6, P < 0.05), but very similar to the average time taken for the peak rate of increase of [Ca2+]c to occur (1.1 ± 0.5 min, n= 6). These results support the experiments using SKF, TG and IM.
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Mean ±S.E.M. time to peak Lp, time to peak [Ca2+]c and time to peak rate of change of [Ca2+]c with ATP. * Significant difference versus time to peak Lp.
Effects of store-independent calcium influx
Both sets of experiments described above increase Lp in a store-dependent manner. VEGF (1 nM) has been shown to increase intracellular calcium in endothelial cells of frog microvessels by receptor tyrosine kinase-mediated activation of phospholipase C (PLC), and activation of a non-specific cation channel by 1,2-diacylglycerol (DAG) activation, probably the canonical transient receptor TRPC3 or TRPC6 (Pocock & Bates, 2001b; Pocock et al. 2004). To determine if the maximum rate of increase of [Ca2+]c occurs at the same time as the maximum Lp during a store-independent increase in permeability, previous experiments using VEGF to increase Lp and [Ca2+]c in a store-independent manner were re-analysed (Bates & Curry, 1996, 1997; Pocock et al. 2000a). The time to peak Lp was taken from the first 19 vessels in which the Lp response to VEGF was measured and the [Ca2+]c in the first 18 vessels and peak rate of increase in 14 vessels. The data are summarized in Fig. 5. The time taken for the Lp to peak was 22.7 ± 1.8 s (0.38 ± 0.03 min). The time to the peak Lp was again significantly less than the time to the peak [Ca2+]c, 63 ± 18 s (1.05 ± 0.31 min, P < 0.01), but not significantly different from the time to the peak rate of increase of [Ca2+]c, 18 ± 5.6 s (0.30 ± 0.1 min, P > 0.05).
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A, measurement of calcium and rate of change of calcium in vessel exposed to 1 nM VEGF. The [Ca2+]c reaches a maximum at around 90 s (point i), by which time all the permeability responses measured have returned to control. The maximum rate of change, however, occurs within 30 s (point ii), similar to the peak permeability measurement. B, mean ±S.E.M. time to peak Lp, time to peak [Ca2+]c and time to peak rate of change of [Ca2+]c with VEGF. * Significant difference versus time to peak Lp.
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The experiments described so far have measured either Lp or [Ca2+]c but not both simultaneously in the same vessel. These experiments are technically very difficult but nonetheless possible when combining infrared illumination, a 700 nm dichroic mirror in front of the photomultiplier tube, and an infrared camera connected to a video, time generator and monitor as described for the Lp experiments. Figure 6 shows the results from the only two microvessels in which we have successfully measured both Lp and Ca2+ during perfusion with 30 μM ATP (to induce store-dependent Ca2+ influx). [Ca2+]c was continually measured except for the brief periods when Lp was measured (dashed line). The graph shows that Lp reaches a maximum value before [Ca2+]c in both vessels. Furthermore, peak Lp corresponds with the steepest part of the [Ca2+]c trace indicating that Lp is maximum when the rate of change of [Ca2+]c is maximum.
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ATP (30 μM) was added at time zero to induce store-dependent Ca2+ influx. Each filled square is a Lp measurement. The thin lines show the change in Lp and the thick interrupted line indicates [Ca2+]c as represented by Rnorm. Note that Lp reaches a maximum before Rnorm.
If it is the rate of change of calcium that regulates the permeability, rather than the absolute [Ca2+]c during stimulation, then there should be a positive correlation between the time for d[Ca2+]c/dtmax and the [Ca2+]max. Figure 7 demonstrates that there was a highly significant correlation between these two parameters.
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The time taken for the [Ca2+]c to peak was highly significantly correlated to the time taken for the maximum rate of increase of the [Ca2+]c to occur when the [Ca2+]c was increased by 1 nM VEGF (), ATP () and thapsigargin ().
Discussion
The mechanisms that underlie agonist-mediated increases in permeability are poorly understood. Although Ca2+ is implicated in most permeability responses, the mechanism by which Ca2+ regulates the increase in permeability is not understood. It has been assumed, and data have been presented supporting the hypothesis, that the magnitude of the increase in permeability is dependent on the size of the increase in [Ca2+]i in the endothelial cell (He et al. 1990; He & Curry, 1991; Sarker et al. 1998; Glass & Bates, 2003b). However, previous studies have shown that it is possible for Lp to be increased over a sustained period of time without increased intracellular [Ca2+], under certain conditions. Perfusion of vessels without albumin (i.e. Ringer perfusion) (He & Curry, 1993), and the chronic sustained increase seen with VEGF and ATP 24 h after exposure (Bates et al. 2001), all have a normal baseline Ca2+, despite increased Lp. The results here show that the measured [Ca2+]c in endothelial cells of mesenteric microvessels reaches a maximum after the permeability reaches a maximum. This rules out the possibility that the size of the increase in Ca2+ is directly responsible for the magnitude of the increase in permeability, contrary to our expectations, and previously published interpretations (He et al. 1996). Thus, the global Ca2+ concentration may not be the only key factor by which permeability is regulated. These results force us to investigate more subtle methods of permeability regulation by endothelial cell Ca2+.
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We have previously shown that both the magnitude of the increase in Ca2+ and the baseline permeability are dependent on the state of filling of the Ca2+ store (Glass & Bates, 2003b). Since the rate of flux of Ca2+ from the store to the cytoplasm will be greater when stores are relatively full, we hypothesized that perhaps in endothelial cells of vessels that have a normal baseline permeability, the rate of Ca2+ entry into the cytoplasm may be more critical for the regulation of the magnitude of the permeability response than the global Ca2+ concentration. Furthermore it has been shown that, under conditions of altered membrane potential, the quantitative relation between the initial change in Ca2+ concentration and the calculated Ca2+ influx using the Goldman–Hodgkin–Katz equation for Ca2+ influx correlates well with the peak Lp under conditions of high extracellular potassium (depolarization) or Ca2+ (Curry, 1992). Additionally, Pagakis et al. show examples of single vessels in which regions of interest within three different vessels indicate a correlation between the initial rate of change of Ca2+ and peak permeability values (Pagakis & Curry, 1994). The results shown here clearly indicate that the maximum permeability is more closely correlated with the rate of change than the absolute concentration during store-mediated Ca2+ entry. To test this hypothesis further we determined whether this was also true for the increase in Ca2+ and permeability stimulated by increasing Ca2+ entry across the plasma membrane, and found that indeed the permeability reached its maximum before the [Ca2+]c did, whereas the peak rate of Ca2+ rise again matched the permeability response.
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Nevertheless, a close correlation does not show that rate of change determines Lp, and there are other, equally valid, possibilities for regulating the permeability. One alternative hypothesis is that the kinetics of the shift in fluorescence of Fura-2 is so slow that the calcium response occurs significantly before the change in fluorescence signal is measured. However, it is clear from studies on the kinetics of calcium binding to Fura-2 that the time taken for a change in [Ca2+]c to be detected, and subsequently signal through the system described above, should be at least an order of magnitude faster than the time course described (Kao & Tsien, 1988). A second explanation is that the presence of Fura-2 significantly delays the calcium response, since the experiments where hydraulic conductivity alone was measured were not carried out in the presence of Fura-2. To address this aim, we have measured Lp and [Ca2+]c in the same capillary at the same time, and in both examples where we were successful, the peak Lp increase did precede the [Ca2+]c peak, suggesting that the presence of Fura-2 did not affect the time course of the permeability response.
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Is permeability determined by the rate of increase of [Ca2+]c
There is supporting evidence for the hypothesis that the rate of change of [Ca2+]c rather than the absolute [Ca2+]c regulated permeability from endothelial cell culture experiments. At least two different Ca2+-dependent mechanisms may be regulated by Ca2+ influx. In pulmonary endothelial cells, the rate of Ca2+ influx into the cell has been implicated in the indirect regulation of permeability/barrier integrity, as it regulates cAMP (Stevens et al. 1995). Stevens et al. demonstrated that cAMP production was increased when Ca2+ influx was blocked with La3+ (Stevens et al. 1995). Furthermore, they showed that IM decreased cAMP production. However, although they suggested that the rate of Ca2+ influx might regulate cAMP production, they did not distinguish between the rate of Ca2+ influx and the [Ca2+]c. They did, however, state that La3+ decreased cation influx by more than 90%, whereas the [Ca2+]c was only decreased by 40%. This led them to conclude that the rate of Ca2+ influx might be more important in regulating cAMP production than the absolute [Ca2+]c. cAMP has been shown to inhibit increased permeability by activating protein kinase A, which phosphorylates myosin light chain kinase (MLCK) (Garcia et al. 1995). When MLCK is phosphorylated it has a lower affinity for Ca2+–calmodulin and reduces the ability of the cell to contract (Garcia et al. 1995; He et al. 2000). There may also be additional cAMP-dependent mechanisms that regulate permeability because recent evidence suggests that inflammatory mediators such as platelet-activating factor and bradykinin increase permeability independently of MLC phosphorylation (Adamson et al. 2003).
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Nitric oxide synthase (NOS) may also be regulated by the rate of Ca2+ influx. NOS is associated with caveolin-1 in the caveolae on the plasma membrane (Segal et al. 1999; Goligorsky et al. 2002). The Ca2+ influx channels are also located in the caveolae (Isshiki & Anderson, 1999) and many IP3 receptors on the ER membrane are closely apposed to the caveolae (Isshiki et al. 2002). Hence, NOS is in the appropriate location to be regulated by the rate of Ca2+ influx. When NOS is inhibited, agonists such as ATP are no longer able to increase the permeability effectively (He et al. 1997a), demonstrating that NO regulates agonist-mediated increases in permeability. We also note that, in vessels that are not exposed to inflammatory agents, inhibitors of NOS increase microvessel permeability (He et al. 1997b) suggesting it can play a stabilizing role similar to cAMP under some conditions.
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In this study we have measured the global [Ca2+]c under conditions of intracellular Ca2+ store release using the same drug combination (SKF, TG and IM) and protocol as in a separate set of permeability experiments. From the [Ca2+]c trace, the rate of change of [Ca2+]c could be calculated as the differential of the [Ca2+]c trace. This, however, is the rate of change of [Ca2+]c rather than the rate of Ca2+ flux into the cytosol, as Ca2+ extrusion from the cell by the plasma membrane Ca2+-ATPase pump or the Na+–Ca2+ exchanger has not been blocked (Hinde et al. 1999). The relationship between the time courses of the permeability and rate of change of the [Ca2+]c responses appears to occur in the absence or presence of stores, and in the absence or presence of Ca2+ influx across the plasma membrane. However, at least one of these processes is necessary since removing store-dependent Ca2+ release in the absence of Ca2+ influx, or removing Ca2+ influx in the absence of store release (Pocock et al. 2000a), blocks the permeability response. Thus the nature of the mechanisms linking calcium to permeability are likely to be cytoplasmic, and common to different Ca2+ sources. One explanation for this would be that the local site of increase in [Ca2+] is a regulating mechanism for the magnitude of the permeability response.
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Rate of flux, or site of flux
Agents such as ATP activate G-protein-coupled receptors linked to PLC- that generates diacylglycerol (DAG) and IP3 (Ward et al. 2003). IP3 then releases ER store Ca2+ and increases permeability via the NO–cGMP pathway (He et al. 1997a). If, however, the ER store is depleted with TG, extracellular ATP can no longer increase the permeability (Pocock et al. 2000a). VEGF increases permeability through a different signalling cascade. VEGF receptor-2 is a tyrosine kinase receptor that phosphorylates PLC- (Guo et al. 1995). DAG and IP3 are generated as with ATP stimulation, but ER store depletion with TG does not inhibit VEGF-stimulated permeability increases (Pocock et al. 2000a). VEGF in vivo acutely increases permeability through a PLC-dependent, but Ca2+ store-, mitogen activated protein kinase (MAPK) and extracellular signal related Kinase (ERK) Kinase (MEK)-, PKC- and DAG lipase-independent pathway (Pocock & Bates, 2001a). Recent evidence using TRP activators, inhibitors and heterologous expression systems, and the finding that the membrane-permeable DAG analogue OAG increases permeability, suggests that DAG directly induces Ca2+ influx through the activation of second messenger-mediated Ca2+ channels such as TRPC3, 6 or 7 channels (Pocock & Bates, 2002).
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The fact that ATP (store dependent) and VEGF (store independent) both increase permeability at the same rate as they increase Ca2+ influx, but VEGF acts much more quickly, might be due to the localization of the different signalling cascades. There is significant evidence to show that both the ATP- and VEGF-stimulated signalling pathways are linked to caveolae, which appear to act as regulatory localized subdomains for signal transduction in endothelial cells (Shaul & Anderson, 1998; Isshiki & Anderson, 1999; Lockwich et al. 2001; Labrecque et al. 2003). Distinct populations of caveolae in the plasma membrane might contain different signalling proteins and channels. As ATP and VEGF activate different PLC isoforms this could suggest that the signalling cascade for each originates in a separate caveolar population. Therefore, not only is the signalling different but the localization of the receptors and effector molecules is also likely to vary, which may account for the different time courses of the responses with ATP and VEGF.
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If the differences seen between the ATP and VEGF Lp responses are due to different populations of caveolae, either the rate of Ca2+ influx or the [Ca2+] within the microdomain could be regulators of permeability. Current data are unable to distinguish between these two possibilities, but merit further research. A third possibility is that a threshold [Ca2+]c may regulate the initiation of the permeability increase. We were unable to establish if there is a minimum increase in [Ca2+]c required before the permeability increases, as the Ca2+ had already started to increase by the time of the first permeability measurement. The peak [Ca2+]c may follow the peak Lp because a threshold [Ca2+]c sufficient to increase the permeability occurred first. The amount of time that the endothelial cells are above a threshold might thereby regulate the permeability. The magnitude of the [Ca2+]c increase would in this case still correlate with the magnitude of the permeability increase, since a threshold would be reached faster when the magnitude is higher. He et al. suggested that there is a direct correlation between the magnitude of Lp and the [Ca2+]c when the [Ca2+]c was greater than 130 nM (He et al. 1990). Once the global [Ca2+]c was 130 nM, the Lp would increase, and the rate at which the threshold was reached would control the magnitude of the response. The correlation demonstrated by He et al. (1990) implies that the magnitude of the Lp increase is related to [Ca2+]c once the Lp response had already been initiated, but the threshold at which that is initiated and the rate of change by which that threshold is reached could still regulate the magnitude of the response.
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In summary, we have shown here, that contrary to previous interpretations, in response to inflammatory mediators and growth factors, the Ca2+ concentration in endothelial cells reaches a peak after the permeability does so, and therefore cannot be responsible for the magnitude of the increase. The maximum rate of Ca2+ influx, however, appears to match closely with the magnitude of the permeability response when the permeability increase is either store mediated, or store independent. These findings lead us to the conclusion that it is either the local Ca2+ concentration by the membrane, or the rate of flux of Ca2+ into the cytosol that regulates the permeability, and that store-independent Ca2+ increases can result in a more rapid permeability increase. This implies that the local concentration of Ca2+ immediately underneath the membrane is likely to regulate the permeability response, raising the possibility that store-dependent and independent permeability increases may act through separate populations of membrane microdomains.
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