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Microvascular Research Laboratories, Department of Physiology, Preclinical Veterinary School, Southwell Street, University of Bristol, Bristol BS2 8EJ, UK

	Abstract

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Microvascular permeability is regulated by changes in intracellular calcium concentration. The mechanism by which this increase in calcium determines permeability under normal conditions and during stimulation with agonists remains to be elucidated. In order to determine whether calcium release from intracellular stores could contribute towards the regulation of vascular permeability, hydraulic conductivity (L_p) was measured in frog mesenteric microvessels during stimulation of the endothelial cells of these vessels with agonists that release calcium from the intracellular stores. ATP (which acts through activation of inositol 1,4,5-trisphosphate (IP₃) receptors) increased L_p in the absence of calcium influx across the plasma membrane 2.3 ± 0.3 fold (mean ±S.E.M.., P < 0.01, n= 8), which was less than the increase in the presence of calcium influx (3.1 ± 1.1 fold). Caffeine (which acts through activation of ryanodine receptors) also increased L_p in the absence of calcium influx across the plasma membrane 3.8 ± 1.0 fold (P < 0.01, n= 9), but by at least as much as it does in the presence of calcium influx (2.8 ± 0.5 fold). It is surprising that there was a strong positive correlation between the size of the response during store release and the baseline permeability (r = 0.91 for ATP, r = 0.75 for caffeine). This suggests that the filling state of the stores may regulate the baseline permeability of the microvessels.

(Received 2 December 2003; accepted after revision 18 February 2003; first published online 16 March 2004)
Corresponding author D. Bates: Microvascular Research Laboratories, Department of Physiology, Preclinical Veterinary School, Southwell Street, University of Bristol, Bristol BS2 8EJ, UK. Email: dave.bates{at}bristol.ac.uk

	Introduction

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Regulation of vascular permeability by endothelial cells results from controlled endothelial intracellular signalling. Increased microvascular permeability is stimulated by a signalling cascade that usually involves increased cytoplasmic calcium concentration ([Ca²⁺]_c). Neither the mechanisms by which Ca²⁺ increases microvascular permeability, nor its role in regulating normal vessel permeability have yet been elucidated, although a variety of pathways have been proposed, including actin–myosin-mediated contraction(Garcia et al. 1995), activation of protein kinase G through activation of endothelial nitric oxide synthase (eNOS) (Huang & Yuan, 1997), and induction of rho kinase(Wojciak-Stothard et al. 2001). The increase in cytosolic Ca²⁺ concentration is brought about usually by the activation of Ca²⁺ influx across the plasma membrane, either through store-activated Ca²⁺ channels or by store-independent Ca²⁺ channels. For instance, agents that increase IP₃ production and subsequent Ca²⁺ release from the endoplasmic reticulum (ER), such as thrombin and histamine, increase solute leakage out of microvessels (Al-Naemi & Baldwin, 2000; Tiruppathi et al. 2002), increase albumin leakage across endothelial cell monolayers (Moore et al. 2000; Sandoval et al. 2001; Borbiev et al. 2001) and decrease transendothelial monolayer electrical resistance (Sandoval et al. 2001; Tiruppathi et al. 2002), which are markers of increased vascular permeability in vitro or ex vivo.

The dependence of permeability regulation on intracellular Ca²⁺ has also been investigated in vivo. Sarker et al. (1998, 2000) have demonstrated that bradykinin and histamine, both of which increase intracellular Ca²⁺ concentration in endothelial cells, increased the permeability of pial venular capillaries of rat. Furthermore, He & Curry (1991) have shown that the Ca²⁺ ionophore, A23187 [GenBank] , increased the hydraulic conductivity (L_p) of frog mesenteric microvessels and increased intracellular Ca²⁺ concentration. The L_p response to A23187 [GenBank] was attenuated under conditions of reduced Ca²⁺ influx brought about by depolarizing the membranes of the endothelial cells with high potassium-containing Ringer solutions (He & Curry, 1991). These experiments demonstrate an involvement of Ca²⁺ in the regulation of microvascular permeability in vivo. However, they did not show that activation of signalling pathways that stimulate release of Ca²⁺ from intracellular stores can increase permeability by increasing intracellular Ca²⁺ concentration directly.

Pocock et al. (2000) demonstrated that ATP increased L_p and [Ca²⁺]_c in frog mesenteric microvessels in vivo. ATP stimulates release of Ca²⁺ from the ER through IP₃ receptor (IP₃R) activation. If vessels were pretreated with the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor, thapsigargin, to deplete the ER stores, ATP no longer increased L_p. This demonstrated that ATP increased L_p by first releasing Ca²⁺ from the ER before stimulating Ca²⁺ influx. As thapsigargin had already depleted the ER store, ATP was unable to release further Ca²⁺ by stimulating the IP₃Rs via IP₃ production.

We have previously shown that the Ca²⁺ ionophore, ionomycin, increased L_p in the absence of extracellular Ca²⁺ influx by releasing Ca²⁺ from the intracellular stores (Glass & Bates, 2003). Ca²⁺ influx was inhibited using the cation channel inhibitor SK & F96365 (1-{ß-[3-(4-methoxy-phenyl)propoxyl]-4-methoxyphenethyl}-1H-imidazole hydrochloride) (Merritt et al. 1990). This compound has been shown to be an inhibitor of receptor-mediated Ca²⁺ entry in non-excitable cells lacking voltage-gated Ca²⁺ channels. It inhibits Ca²⁺ entry through the transient receptor potential (TRP) family of non-specific cation channels (Inoue et al. 2001) and capacitative Ca²⁺ entry (Ju et al. 2003). Although SK & F96365 does inhibit voltage-gated Ca²⁺ entry, in endothelial cells, which lack these channels (Nilius et al. 1997), it is specific for Ca²⁺ entry and does not block Ca²⁺ store release (Merritt et al. 1990). Our previous study showed that increasing [Ca²⁺]_c simply by releasing Ca²⁺ from intracellular stores could increase vascular permeability (Glass & Bates, 2003). However, ionomycin is a synthetic Ca²⁺ ionophore and is not involved in the normal regulation of Ca²⁺ release from intracellular stores. ATP, on the other hand acts on plasma membrane purinergic receptors to stimulate IP₃ production from the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP₂) by phospholipase C. IP₃ activates IP₃Rs on ER stores to result in release of Ca²⁺ from these stores. The Ca²⁺ concentration around the IP₃Rs increases and activates nearby ryanodine receptors (RyRs) by a cascade commonly referred to as Ca²⁺-induced Ca²⁺ release or CICR (Lee et al. 1996; Murayama et al. 2000). This results in a Ca²⁺ wave forming that runs along the ER where the RyRs are located, similar to that observed in the T-tubules of muscle cells (Murayama et al. 2000). To determine whether agonist-mediated store release can regulate increased vascular permeability, we examined the role of agonist-mediated release of Ca²⁺ from ER Ca²⁺ stores by both IP₃Rs (through ATP-mediated activation of purinergic receptors) and RyRs (by activation of RyRs by caffeine) in the regulation of permeability both in the presence and absence of extracellular Ca²⁺ influx.

	Methods

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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 local and national ethical guidelines and within the Animals (Scientific Procedures) Act 1986. Erythrocytes were collected by cardiac puncture from male Wistar rats under halothane anaesthesia (5%). The rats were then killed by cervical dislocation. All chemicals were purchased from Sigma.

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 mg ml^–1 MS222 in physiological frog Ringer solution containing (mM): NaCl 111, KCl 2.4, MgSO₄1, CaCl 1.1 ₂, NaHCO₃ 0.2, Glucose 5, Hepes acid 2.63 and Hepes sodium salt 2.37; 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 Perspex pillar so that the mesentery could be visualized through a Leitz inverted microscope. 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).

Measurement of hydraulic conductivity (L_p)

L_p was measured using the Landis-Michel technique (Michel et al. 1974), as we have previously described (Pocock & Bates, 2001). 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% bovine serum albumin (BSA) in physiological frog Ringer solution (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 L_p > 10 x 10^–7cm s^–1 cmH₂O^–1 were excluded. 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 before being diluted in the baseline solution. Downstream from the cannulation site a pulled glass micropipette was used to occlude the vessel for approximately 5 s. The vessel was allowed to flow freely for at least 8 s between occlusions.

To measure L_p during perfusion with ATP, vessels were perfused with 1% BSA until a stable baseline L_p was achieved and then perfused with 30 µM ATP (as previously used in this laboratory; Pocock et al. 2000) for 10 min to stimulate the IP₃Rs and release Ca²⁺ from the ER. The vessels were subsequently perfused with 1% BSA for 15 min to wash out the ATP and allow the ER stores to refill and recover to baseline values. The vessels were then perfused with 100 µM SK & F 96365 (SKF) for 10 min to block Ca²⁺ influx, followed by 10 min perfusion with SKF and ATP. Half of the vessels were perfused with ATP alone, washed out and then ATP was tested in the presence of SKF while the others were perfused in the reverse order, i.e. SKF and ATP, washed out and followed by ATP alone. We have previously shown that SKF blocks Ca²⁺ influx to the same extent as Ni²⁺ in this system (Glass & Bates, 2003).

Before using caffeine with a Ca²⁺ influx blocker, the L_p response to caffeine alone was characterized to ensure that 100 µM caffeine was effective in increasing L_p. Vessels were perfused with 1% BSA until the baseline L_p was stable and followed by 10 min perfusion with 100 µM caffeine. Having shown that 100 µM caffeine was a suitable concentration to induce an increase in L_p, the experiments were repeated in the absence of extracellular Ca²⁺ influx. Unfortunately, it was not technically possible to make reproducible measurements during a second successive perfusion with caffeine. As a result, two sets of vessels were compared (i.e. the experimental and control vessels). Vessels were perfused with the Ca²⁺ influx blocker, 100 µM SKF, for 10 min once a stable baseline was achieved with 1% BSA. The vessels were then perfused with SKF and 100 µM caffeine for a further 10 min to release ER store Ca²⁺ without Ca²⁺ influx.

Calculation of L_p

The radius of the vessel (r), velocity of the marker cells (dL/dt) approximately 2 s after occlusion and the length (l) between the marker cell and the occlusion site were measured offline from the video recording. Solute flux per unit area (J_v/A) was calculated as:

(1)

Hydraulic conductivity (L_p) 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)

(2)

Statistics

Baseline values are expressed as the mean L_p during the time perfused. All other perfusion treatments are expressed as the peak (highest) L_p value. Where there was more than one transient increase in L_p the peak of the largest transient increase from the baseline was used. To compare L_p measurements for single comparisons paired t tests were used, as the data were normally distributed. For multiple comparisons one way ANOVA followed by Student–Newmann–Keuls (SNK), or Buonferroni post hoc tests as appropriate. The Spearman correlation test was used to determine the significance of correlation between data groups.

	Results

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Does release of the endoplasmic reticulum Ca²⁺ store through ryanodine receptors increase L_p in the absence of extracellular Ca²⁺ influx?

Active release of Ca²⁺ from the ER (ER store release) was investigated using 100 µM caffeine. Caffeine acts as an agonist at the amphibian - and ß-ryanodine receptor (RyR) isoforms and stimulates the release of Ca²⁺ from the ER (Murayama & Ogawa, 2001). Figure 1A shows an example of a single vessel perfused with caffeine. There was an immediate transient increase in L_p during ER store release (with the addition of caffeine) that returned to basal levels within 4 min. As the time course of the response with caffeine was highly reproducible the data could be sorted into 15 s time bins as shown in Fig. 1B. The pipette was refilled at time point zero. The L_p increased 2.0 ± 0.5 fold (mean ±S.E.M.) by the first 15 s of ER store release and rapidly returned to baseline within 120 s (diamonds). The lower trace shows the time-averaged data from the control when the pipette was refilled with 1% BSA instead of caffeine. Figure 1C shows the summary of data from nine vessels. The L_p transiently increased 2.8 ± 0.5 fold during ER store release from 1.9 ± 0.5 x 10^–7 to 4.3 ± 1.1 x 10^–7 cm s^–1 cmH₂O^–1 when perfused with caffeine (P < 0.01), and then decreased below baseline to 0.8 ± 0.25 x 10^–7cm s^–1 cmH₂O^–1 (P < 0.01 versus peak L_p with caffeine). There was a significant correlation between the baseline L_p and the L_p during ER store release (the peak L_p with caffeine) (r = 0.75, P < 0.05) as shown in Fig. 2A. This implies that the determinants of increases in store-dependent L_p also determine the basal L_p. Furthermore, there were correlations between the L_p following the caffeine-induced transient and the baseline L_p with 1% BSA (r = 0.76, P < 0.05) and the peak caffeine-induced L_p (r = 0.73, P < 0.05) shown in Fig. 2B and C.

View larger version (15K): [in this window] [in a new window]   Figure 1.  RyR mediated ER store release increases Lp A, an example of an individual microvessel is shown. ER store release (caffeine) induced an immediate transient increase in Lp. Time of caffeine application is shown. B, Lp (mean ±S.E.M.) from nine vessels time-averaged into 15 s time bins. The pipettes were refilled with either caffeine or 1% BSA at time point zero. C, Lp under basal conditions (BSA), during store release (caffeine peak) and following store release in the continued presence of caffeine (caffeine min). **P < 0. 01versus BSA, P < 0.01versus peak caffeine peak, one way ANOVA, SNK post hoc test.

View larger version (21K): [in this window] [in a new window]   Figure 2.  Basal Lp correlates with Lp during ER store release A, Lp during ER store release (peak caffeine) and B, Lp following store release (min caffeine) positively correlate with Lp under basal conditions (1% BSA). C, Lp during ER store release positively correlates with Lp following store release. SCC, Spearman correlation coefficient.

View larger version (15K): [in this window] [in a new window]   Figure 3.  ER store release increases Lp in the absence of Ca2+ influx A, Lp immediately transiently increases during ER store depletion (caffeine) in the absence of Ca2+ influx (SKF). An example of an individual vessel is shown. Time of application of caffeine and SKF are indicated. B, Lp(mean ±S.E.M.) from nine vessels time-averaged into 15 s time bins. The pipettes were refilled at time point zero. C, Lp under control conditions (BSA), during Ca2+ influx inhibition (SKF) and during RyR-mediated ER store release without Ca2+ influx (SKF/caffeine). **P < 0.01versus BSA, P < 0.01versus SKF, P < 0.001 ANOVA, SNK post hoc test.

The mean effect of caffeine on permeability in the absence of Ca²⁺ influx is shown in Fig. 3C. L_p decreased from a baseline (1% BSA) of 3.8 ± 0.7 x 10^–7 to 2.2 ± 0.4 x 10^–7 cm s^–1 cmH₂O^–1 when Ca²⁺ influx was blocked with SKF. ER store release without Ca²⁺ influx resulted in a transient 3.8 ± 1.0 fold increase in L_p to 7.0 ± 1.2 x 10^–7 cm. s^–1 cmH₂O^–1 (P < 0.01versus BSA, P= 0.001versusSKF, n= 9). These data were compared to those with ER store release and Ca²⁺ influx (caffeine alone, from Fig. 1C). The increase in L_p during ER store release with Ca²⁺ influx (2.8 ± 0.5 fold) was no different from the increase in L_p with ER store release in the absence of Ca²⁺ influx (3.8 ± 1.0 fold, n= 9, P > 0.05, unpaired t test with Welch's correction for unequal variances, Fig. 4A). Figure 4B shows the scatter of the data from Fig. 4A and a horizontal line indicates the median of each data set. There was no correlation between the baseline L_p and the peak L_p during perfusion with caffeine and SKF. Perfusion of three vessels with the acetoxymethyl ester form of BAPTA (BAPTA-AM), which chelates cytoplasmic Ca²⁺, abolished the response to caffeine (mean increase 1.37 ± 0.11 fold).

View larger version (12K): [in this window] [in a new window]   Figure 4.  RyR-mediated store release increases Lp in the presence or absence of Ca2+ influx A, increases in Lp. There was no significant difference between Lp during RyR-mediated ER store release in the presence of Ca2+ influx (caffeine) and in the absence of Ca2+ influx (SKF/caffeine), (P > 0.05, t test) 1 equals no change. B, the scatter of the data shown in A. Horizontal bar, median.

Figure 5A shows an example of a single vessel perfused with SKF then SKF plus ATP for 10 min, washed out for 15 min with 1% BSA and perfused with ATP for a second time without SKF. During IP₃-mediated ER store release without Ca²⁺ influx (SKF + ATP) there was a transient increase in the L_p, which peaked after approximately 3 min (point i). A second peak occurred approximately 1 min later (point ii) then the L_p returned to baseline. During IP₃-mediated ER store release with Ca²⁺ influx (ATP alone) a transient increase in the L_p occurred that peaked after about 2 min (point iii) followed by a second transient increase after another 3 min (point iv). After perfusion with SKF plus ATP 3 of 8 vessels displayed a double transient L_p response, compared to 4 of 7 vessels perfused with ATP alone. The peak L_p with ATP alone was greater than the peak L_p with ATP plus SKF suggesting that part of the increase in L_p induced by ATP was due to Ca²⁺ influx. The second peak with SKF was smaller than the first possibly due to gradual loss of Ca²⁺ from the cells when Ca²⁺ influx was blocked, whereas in the absence of SKF the size of the peaks was similar.

View larger version (14K): [in this window] [in a new window]   Figure 5.  IP3-mediated ER store release induced Lp is attenuated in the absence of Ca2+ influx A, an individual perfused microvessel is shown. ER store release without Ca2+ influx (SKF/ATP) and with Ca2+ influx (ATP alone) both resulted in two transient increases in Lp (points i–iv). B, Lp under basal conditions (BSA), during Ca2+ influx inhibition (SKF), during ER store release with (ATP) and without (SKF/ATP) Ca2+ influx. *P < 0.05versus BSA, P < 0.01versus SKF. C, increase in Lp during ER store release in the presence (ATP alone) and absence of Ca2+ influx (SKF/ATP). 1 equals no change.

There was a significant positive correlation between the baseline L_p in the absence of Ca²⁺ influx (SKF) and the peak L_p during ER store release without Ca²⁺ influx (SKF + ATP) (r = 0.91, P < 0.005, n= 8, Spearman correlation test) as shown in Fig. 6A. However, there was no correlation between the baseline L_p with 1% BSA and the peak L_p during ER store release with Ca²⁺ influx (ATP alone) (r =–0.04, P > 0.05, n= 7, Spearman correalation test, Fig. 6B).

View larger version (21K): [in this window] [in a new window]   Figure 6.  Lp during ER store release correlates to the basal Lp in the absence of Ca2+ influx A, Lp during ER store release (peak SKF/ATP) in the absence of Ca2+ influx positively correlates with the Lp under basal conditions (SKF). B, Lp during ER store release in the presence of Ca2+ influx (peak ATP) did not correlate with basal Lp (BSA).

	Discussion

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Ca²⁺ release from the ER store increases vascular permeability

In exchange vessels, in vivo, such as the mesentery capillary bed, ATP acts through stimulation of metabotropic purinergic (P2Y) receptors. Agonist-mediated release of Ca²⁺ from the ER through IP₃ production due to P2Y receptor activation or by activation of RyRs with caffeine was able to increase vascular permeability when extracellular Ca²⁺ influx was blocked by SKF. These results support our previous work showing that Ca²⁺ influx is not required to increase permeability (Glass & Bates, 2003). Sarker et al. (1998) showed that the histamine H₂ receptor agonist, dimaprit, which releases Ca²⁺ from the store via IP3, transiently increased the permeability of occluded rat brain venular capillaries in the presence of SKF or zero extracellular Ca²⁺. The permeability increases that can be brought about by store-mediated Ca²⁺ release are therefore not restricted to visceral vessels. In contrast, He et al. (1996) showed that L_p increases induced by a submaximal dose of ATP (10 µM) were abolished under conditions of zero extracellular Ca²⁺ influx. Submaximal release of Ca²⁺ from the intracellular stores or a slower release of store Ca²⁺ may therefore require Ca²⁺ influx to increase permeability.

Permeability increased 3-fold when the vessels were perfused with ATP alone but only increased 2-fold when Ca²⁺ influx was blocked with SKF. This suggests that ATP increases the permeability both by Ca²⁺ release from the ER store and by subsequent Ca²⁺ influx. In contrast to ATP perfusion, there was no difference between the L_p responses to caffeine with or without SKF. Therefore, caffeine appears to be able to increase L_p without Ca²⁺ influx even when the cation channels are not blocked by SKF. Caffeine is a RyR agonist, whereas ATP activates IP₃Rs. IP₃Rs are not restricted to the ER membranes and are also found in the caveolae regions of the plasma membrane and the Golgi (Bush et al. 1994; El-Daher et al. 2000; Van Baelen et al. 2001). The caveolae are the site at which IP₃ is produced from PIP₂ (Shaul & Anderson, 1998). So, IP₃ is colocalized to the caveolae where plasma membrane IP₃Rs are located and activated directly by IP₃, allowing extracellular Ca²⁺ influx independently or in addition to the release of Ca²⁺ from ER stores. Therefore, the increased ATP response in the presence of Ca²⁺ influx could either be due to Ca²⁺-induced Ca²⁺ influx, or direct activation of plasma membrane IP₃Rs. RyRs on the other hand are only found on intracellular stores. Kohler et al. (2001) have reported that the ryanodine receptor subtype RyR-3, but not RyR-1 or RyR-2, was present in freshly isolated human mesenteric arteries. It is less clear whether or not RyRs are expressed in the microvasculature. They also found that the expression of RyR-3 was down-regulated in cultured cells, making the link between their expression in cultured cells and the in vivo situation more difficult to establish (Kohler et al. 2001).

Pocock et al. (2000) have shown that the ATP-induced increases in L_p can be completely blocked by pretreating the vessels with thapsigargin for 20 min to deplete the ER Ca²⁺ store, suggesting that plasma membrane IP₃Rs, if involved in ATP-induced permeability increases, are store dependent. A possible explanation for these differences would be the requirement of Ca²⁺ ions in addition to IP₃ to activate IP₃Rs (Irvine, 1990; Finch et al. 1991). Ca²⁺ signals would be localized to the domain around the ER as SERCA and the mitochondria are localized close to the Ca²⁺-release sites (Nassar & Simpson, 2000). Moreover, although cultured and large vessel endothelial cells have recently been shown to express ionotropic P2X purinergic receptors (Ray et al. 2002; Schwiebert et al. 2002; Wang et al. 2002), this cannot be the case in exchange vessels in vivo, as ATP-mediated Ca²⁺ increases are store dependent.

Determinants of the basal permeability also determine the magnitude of the permeability increases evoked by releasing Ca²⁺ from the ER

A significant positive correlation was found between the baseline L_p and the peak L_p induced by Ca²⁺ release from the ER. This was true for both caffeine and ATP. However, calcium influx was necessary for this correlation when the release was stimulated by ryanodine receptor activation, whereas this was not true for IP₃ receptor activation. He et al. (1990) showed that there was no correlation between the [Ca²⁺]_c and Lp in mesenteric microvessels under basal conditions, despite the fact that during stimulation the permeability closely tracks the [Ca²⁺]_c, suggesting that basal permeability is not determined by the absolute [Ca²⁺]_c. As the basal permeability is not set by [Ca²⁺]_c, but does correlate with the size of the store-dependent response, it is possible that the basal permeability and the size of the response are both dependent on the same thing, i.e. the state of filling of the stores. By changing the filling state of stores with varying exposure to thapsigargin, Missiaen et al. (1992) showed that in A7r5 smooth muscle cells, the amount of releasable Ca²⁺ was lowered and the magnitude of agonist-mediated Ca²⁺ increases was decreased. This suggests that the rate of release of Ca²⁺ from the ER with IP₃ was greater when the stores were full compared with stores that had been partially depleted with thapsigargin. In BHK-1 fibroblasts, Hofer et al. (1996) have shown that under certain conditions (thapsigargin, store depletion and 100 µm [Ca²⁺]_c), the Ca²⁺ leak changed direction and Ca²⁺ moved from the cytoplasm into the ER. This suggests that the rate of Ca²⁺ release from the ER during agonist stimulation is in part determined by the concentration gradient between the ER and cytosol, i.e. the filling of the store. Consequently, the rate of Ca²⁺ leak is determined by the loading of the ER store and the concentration gradient between the ER and cytoplasm under basal conditions. During agonist stimulation, the concentration gradient set by the loading of the ER store then determines the rate of Ca²⁺ release. It is possible therefore that both basal and store-mediated permeability are linked to the Ca²⁺ concentration in the ER ([Ca²⁺]_ER). The rate of Ca²⁺ leak from the ER is determined by the conductance of the leak channels, the number of channels and the concentration difference between the ER and cytoplasm. During agonist stimulation, the increased rate of Ca²⁺ release from the ER due to increased channel conductance will depend on the same concentration gradient between stores and cytoplasm that was present during basal conditions. These data therefore suggest that it is the loading of the stores that sets both of these values – the size of the response and the basal permeability.

In summary, Ca²⁺ release from the ER store increased L_p in the absence of extracellular Ca²⁺ influx. Furthermore, an increase in L_p was achieved by stimulating either the IP₃Rs with ATP or by stimulating the RyRs with caffeine. The L_p increases induced by Ca²⁺ release from the ER were correlated with the basal L_p implying that the determinants of the basal permeability also determine ER store-mediated permeability increases, and suggesting that the basal permeability is determined, at least in part by the degree of filling of the endothelial cell Ca²⁺ stores.

	References

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