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(Circulation Research. 1995;77:37-42.)
© 1995 American Heart Association, Inc.

Articles

Acetylcholine-Sensitive Intracellular Ca²⁺ Store in Fresh Endothelial Cells and Evidence for Ryanodine Receptors

Xiaodong Wang, Frankie Lau, Li Li, Akiyoshi Yoshikawa, Cornelis van Breemen

From the Department of Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, Canada.

Correspondence to Dr Casey van Breemen, The University of British Columbia, Department of Pharmacology and Therapeutics, Faculty of Medicine, 2176 Health Sciences Mall, Vancouver, BC V6T 1Z3 Canada.

	Abstract

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Abstract In a freshly isolated endothelial cell preparation from rabbit aorta, the regulation of the acetylcholine (ACh)–sensitive intracellular Ca²⁺ store and the effects of the Ca²⁺-induced Ca²⁺ release agonists ryanodine and caffeine were studied using fura 2 imaging fluorescence microscopy. ACh (10 µmol/L) caused a transient release of Ca²⁺ from an intracellular store, presumably via an inositol tris-phosphate–sensitive mechanism. This ACh response could be repeated in the presence of extracellular Ca²⁺ but was obtained only once in Ca²⁺-free bathing solution, which shows that a depleted intracellular Ca²⁺ store can be rapidly refilled from the extracellular space. Refilling can be prevented by the endoplasmic reticulum Ca²⁺-ATPase inhibitor cyclopiazonic acid (10 µmol/L), implying that Ca²⁺ enters the cytoplasm before accumulation in the endoplasmic reticulum. Ionomycin (10 µmol/L) caused a large Ca²⁺ release even after the ACh-releasable store had been emptied, indicating the existence of other ACh-insensitive stores, perhaps including the mitochondria. In one third of the cells studied, ACh induced oscillations in [Ca²⁺]_i that were dependent on extracellular Ca²⁺. Also investigated were the effects of caffeine and ryanodine. In this cell preparation neither caffeine nor ryanodine induced a Ca²⁺ transient but instead slowly increased [Ca²⁺]_i. It was observed that both caffeine and ryanodine were able to slowly deplete the ACh-sensitive store. These results indicate the presence of functional ryanodine receptors in native endothelial cells and demonstrate overlap between the caffeine and agonist-sensitive Ca²⁺ stores. We also found that caffeine was able to directly inhibit the process of ACh-induced Ca²⁺ release. It is hypothesized that endothelial endoplasmic reticulum contains both inositol tris-phosphate receptors and ryanodine receptors but that the former class are more densely distributed.

Key Words: ryanodine • caffeine • Ca²⁺-induced Ca²⁺ release • acetylcholine

	Introduction

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Hormonal and mechanical stimulation of vascular endothelial cells induce secretion of vasodilator/anticoagulant¹ and vasoconstrictor/procoagulant substances such as endothelium-derived relaxing factor (nitric oxide [NO]), endothelium-derived hyperpolarizing factor, prostacyclin, endothelin, von Willebrand factor, and factor VIII.² Ca²⁺ signaling appears to link stimulation to secretion, and it may play a role in endothelial cell proliferation. The essential role of Ca²⁺ in the release of NO^{3 4 5 6} is due to the presence of constitutive Ca²⁺-sensitive NO synthase in the endothelium.⁷ The exact mechanisms linking elevated [Ca²⁺]_i to release of the other secretions listed above are not known. A central question in the area of endothelial physiology relates to how a single intracellular messenger, Ca²⁺, can differentially regulate several secretory processes. The possibility exists that the spatial and temporal characteristics of the Ca²⁺ signal contribute to its informational content. Although data on endothelial Ca²⁺ signaling are rapidly accumulating, little is known about the contribution by intracellular compartments in the native cell.

It has been established that upon stimulation with various agonists, such as acetylcholine (ACh), bradykinin, ATP, and histamine, cultured endothelial cells release Ca²⁺ from the endoplasmic reticulum (ER), probably through opening of inositol tris-phosphate (IP₃)–sensitive Ca²⁺ channels.^{8 9} Unfortunately, the process of tissue culture has been shown to alter the nature of Ca²⁺ signaling in endothelial cells. Especially with respect to agonist responses, cultured endothelial cells produce inconsistent results. Most cultured endothelial cell preparations fail to respond to ACh, a response that is diagnostic in the laboratory for normal, healthy intact endothelium. This is apparently caused by a loss of expression of m1 and m3 muscarinic receptors during cell culture.¹⁰ There have been relatively few studies showing that ACh induced Ca²⁺ transients in fresh endothelial cells or primary culture, and these did not focus on intracellular Ca²⁺ release.^{11 12 13 14}

There has been controversy regarding the functional presence of ryanodine receptors in endothelial cells.^{15 16} However, Lesh et al¹⁷ have demonstrated binding of anti–ryanodine receptor antibodies to vascular endothelial cells. Most recently, Ziegelstein et al¹⁸ showed in cultured endothelial cell lines that ryanodine had some effect on Ca²⁺ regulation and concluded that functional ryanodine receptors do exist in endothelial cells. A similar conclusion was reached by Graier et al¹⁹ for endothelial cells isolated from porcine coronary artery. These recent findings make it necessary to explore the role of functional ryanodine receptors in Ca²⁺ regulation in native endothelial cells.

In the present study we used a fresh endothelial cell preparation from rabbit aorta to investigate ACh-induced intracellular Ca²⁺ release. We showed that a single dose of ACh completely releases an intracellular Ca²⁺ store, which is refilled by the cyclopiazonic acid (CPA)–sensitive Ca²⁺-ATPase SERCA (sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase) and which is slowly depleted by ryanodine and caffeine.

	Materials and Methods

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Isolation of the Rabbit Aortic Endothelial Cell
Endothelial cells were dispersed from thoracic aortas of New Zealand White rabbits (weighing 2 to 2.5 kg) according to a method described previously by Sakai.²⁰ In brief, each rabbit (UBC Animal Centre) was killed by CO₂ asphyxiation and exsanguinated, and the thoracic aorta was removed and placed in normal physiological saline solution (PSS). After careful removal of the surrounding fat and connective tissue, the aorta was placed in a test tube containing nominally 0 mmol/L Ca²⁺ PSS with 0.01% collagenase and 0.01% elastase. After 40 minutes to 1 hour of enzyme treatment at 37°C, the aorta was placed in normal PSS solution containing 1 mg/mL bovine serum albumin. Endothelial cells were dispersed by trituration by use of a Pasteur pipette and kept in this solution at 4°C for up to 8 hours. The final preparation consisted of single cells and small clusters of cells, which maintained their typical tilelike morphology. Before the experiment the cells were seeded on a glass coverslip at a room temperature of 23°C and used within 4 hours.

Fura 2 Digital Fluorescence Imaging Microscopy
The endothelial cells on the coverslip were loaded with 0.75 µmol/L fura 2-AM (acetoxymethylester) in normal PSS (1 mmol/L stock in dimethyl sulfoxide) for 30 minutes at room temperature. The coverslip chamber was mounted on an inverted microscope (Nikon, Diaphot). A Nikon x20 phase/fluor objective was used to visualize the cells. A glass tube was used to infuse (by gravity) the chamber with fresh experimental solution (total volume of the chamber is 0.5 mL, which is continually maintained by using vacuum suction at the surface of the fluid). A total of 3 mL of solution was used for each solution change. The endothelial cells were exposed to alternating 340-nm and 380-nm wavelengths (bandwidth, 10 nm) of UV light, and emission light was passed through a 510-nm (bandwidth, 40 nm) filter prior to acquisition by an ICCD camera (intensified charge-coupled device, 4810 series, Cohu). A Sun Sparc workstation and INOVISION IMAGE software (Inovision Corp) were used to record and analyze the fluorescence ratio (340 nm/380 nm). Multiple cells (up to 64 maximal) can be recorded simultaneously. In the experiment each individual cell is counted as n=1, and maximally 8 cells from the same tissue are selected, so that most data are collected by use of multiple tissues. Before measurement, background fluorescence at 340 and 380 nm was measured by bringing the cells out of the focus. Ratio images were collected every 10 seconds. Because of uncertainties in calibrating the fura 2 signals in intact cells, no attempt was made to calibrate [Ca²⁺]_i, and all results were instead reported as changes in the 340 nm/380 nm signal ratio. All experiments were performed at a room temperature of 23°C.

Chemicals and Composition of the Solutions
The fluorescence indicator fura 2-AM was purchased from Molecular Probes, Inc. All other chemicals and drugs used in this study were from Sigma Chemical Co. The collagenase used was type IV.

The solutions used were constituted as follows (mmol/L): (1) nominally 0 mmol/L Ca²⁺ PSS for enzyme treatment: NaCl 126, KCl 5, MgCl₂ 5, HEPES 10, and D-glucose 10, pH 7.4. (2) normal PSS: NaCl 126, KCl 5, MgCl₂ 5, HEPES 10, D-glucose 10, and CaCl₂ 1, pH 7.4. (3) Ca²⁺-free PSS: NaCl 126, KCl 5, MgCl₂ 5, HEPES 5, D-glucose 10, and EGTA 1, pH 7.4. (4) N-methyl-D-glucamine (NMDG) solution: 70 mmol/L NaCl of the normal PSS was replaced by NMDG-Cl.

Statistics
Where applicable, the results for multiple experiments are reported as mean±SEM. Unpaired Student's t test was performed, and a value of P>.05 was considered statistically nonsignificant.

In total, 350 cells from 16 animals were used. All figures shown are average tracings from the cells with identical experimental protocols except for Figs 1 and 6, which are single examples.

View larger version (13K): [in this window] [in a new window]   Figure 1. Tracing shows that acetylcholine (ACh) (10 µmol/L) induced a Ca2+ transient in freshly dispersed endothelial cells. In the presence of extracellular Ca2+ (solid line), the ACh-releasable intracellular store was refilled after 7 minutes. ACh was washed out after 100 seconds, which was sufficient to deplete most of the inositol trisphosphate–sensitive Ca2+ store. Dashed line indicates repeated ACh application in Ca2+-free physiological saline solution. In the first application, ACh was washed out after 100 seconds. The second ACh application failed to cause an intracellular Ca2+ transient. The times of exposure in Ca2+-free physiological saline solution were the same in both experiments.

View larger version (15K): [in this window] [in a new window]   Figure 6. Tracings show that acetylcholine (ACh) induced Ca2+ oscillations in intact endothelial cells. Top, Oscillations appear after washout of ACh (10 µmol/L). The cells were perfused with 0 mmol/L Ca2+ physiological saline solution (PSS) at 1200 seconds, which arrests the oscillations. Bottom, ACh was applied in 0 mmol/L Ca2+ PSS. Oscillations appear after cells were replaced in normal PSS (900 seconds).

	Results

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ACh-Sensitive Ca²⁺ Store in Fresh Endothelial Cells
ACh- and Agonist-Induced Ca²⁺ Transient
ACh (10 µmol/L) consistently induced transient increases in intracellular Ca²⁺ in this endothelial cell preparation. Fig 1 shows a typical ACh response in PSS (solid line) and in Ca²⁺-free PSS (dashed line). ACh was repeatedly applied to the cells. ACh was washed out after about 100 seconds, and the fluorescence ratio returned to baseline. If ACh was applied for a longer period, a maintained plateau was seen only in PSS, which suggests that the plateau is derived from Ca²⁺ influx from the extracellular space (data not shown). The time span of 100 seconds was sufficient to empty most of the IP₃-sensitive store (see below). If the cells were allowed 7 minutes for recovery in normal PSS, a subsequent application of ACh induced a similar Ca²⁺ transient. If ACh was applied repeatedly in Ca²⁺-free PSS, only one initial intracellular Ca²⁺ transient was obtained (Fig 1, dashed line). The ACh-induced change in the fluorescence ratio in normal PSS was from 0.37±0.01 (average in baseline) to 0.82±0.03 (average in peak) (n=181). The peak value of the ACh response in Ca²⁺-free PSS was not statistically significantly different from that in normal PSS (baseline, 0.38±0.01; peak, 0.75±0.04 [n=66]).

Refilling of the ACh-Sensitive Store Is Prevented by CPA
The results in Fig 1 suggest that extracellular Ca²⁺ is necessary for the refilling of the ACh-sensitive Ca²⁺ store.

To determine the mechanism of the refilling, a 10-µmol/L solution of the ER Ca²⁺-ATPase inhibitor CPA was applied to the bath. CPA was chosen because its effect is reversible and because there is evidence that there might be two isoforms of ER ATPase, both of which are inhibited by CPA, whereas thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) are more specific.²¹ As shown in Fig 2, in the presence of CPA the second ACh application failed to cause a Ca²⁺ transient despite the presence of extracellular Ca²⁺ (n=11, top tracing). The bottom tracing in Fig 2 illustrates the time course of the effects of CPA on the intracellular Ca²⁺ level. CPA caused release of the Ca²⁺ store(s) and maintained an elevated baseline [Ca²⁺]_i compared with the baseline prior to blockade of the ER Ca²⁺ pump (before blockade, 0.29±0.04; after blockade, 0.40±0.04 [n=9]). CPA-induced Ca²⁺ release showed a delayed time course in contrast to the rapid ACh-induced transient release.

View larger version (18K): [in this window] [in a new window]   Figure 2. Tracings show that cyclopiazonic acid (CPA) prevents the refilling of the acetylcholine (ACh)–depleted intracellular Ca2+ store. Top, 10 µmol/L CPA was applied to the bath. Ten minutes after the first ACh application, a second ACh application failed to stimulate a peak [Ca2+]i response (compare with results shown in Fig 1). The whole experiment was done in normal physiological saline solution. Bottom, Effect of CPA alone on intracellular Ca2+. CPA induced a slow intracellular Ca2+ transient followed by an elevated baseline.

ACh-Sensitive Store Is Part of the Total Organellar Ca²⁺
In Fig 3B, ionomycin (10 µmol/L) was applied after two ACh stimulations. The cells were in Ca²⁺-free PSS so that no refilling of the intracellular store from external Ca²⁺ was possible. The lack of a second ACh response indicated that the ACh-sensitive store was depleted. Nevertheless, ionomycin caused an additional Ca²⁺ release that may have originated from other ACh-insensitive unidentified Ca²⁺ stores (n=25). If ionomycin was applied first, no ACh response was seen, as expected from interaction of ionomycin with all organellar membranes (Fig 3A; n=7).

View larger version (42K): [in this window] [in a new window]   Figure 3. Tracings show that acetylcholine (ACh) releases a fraction of organellar Ca2+. A, No ACh response was seen after ionomycin induced release. B, After the ACh-sensitive store was discharged in Ca2+-free physiological saline solution, ionomycin induced Ca2+ release from other intracellular stores. C, Ionomycin induced transient Ca2+ release after exposure to CPA (at 200 seconds) and ACh (at 500 seconds) in the absence of extracellular Ca2+. D, Ionomycin induced transient Ca2+ release after exposure to ACh (at 100 seconds) and cyclopiazonic acid (CPA) (at 250 seconds) in the absence of extracellular Ca2+.

In Fig 3C and 3D the results of experiments in which ionomycin was applied after ACh and CPA exposure are shown. In both cases the ionomycin-induced transients are about half of the first ACh transient. ACh induced a transient even after previous 6-minute exposure of the cells to 10 µmol/L CPA (Fig 3C).

Evidence for Ryanodine Receptors in Endothelial Cells
In this cell preparation, the Ca²⁺-induced Ca²⁺ release agonist caffeine did not induce a Ca²⁺ transient like ACh (80 cells tested with 4 or 20 mmol/L caffeine); instead, 4 mmol/L caffeine and 30 µmol/L ryanodine slowly increased [Ca²⁺]_i (see Fig 4A, middle and bottom tracings). Caffeine (20 mmol/L) did not induce an intracellular Ca²⁺ transient but had some suppressing effect on the fura 2 fluorescence (data not shown).

View larger version (32K): [in this window] [in a new window]   Figure 4. Tracings show evidence for the existence of ryanodine receptors in the intact endothelial cells. A, Cells exposed for 350 seconds to 0 mmol/L Ca2+ physiological saline solution (PSS) without caffeine responded to acetylcholine (ACh) with an intracellular Ca2+ transient (top). After 350 seconds of pretreatment with caffeine in 0 mmol/L Ca2+ PSS and a 50-second washout of caffeine, 10 µmol/L ACh failed to induce a significant Ca2+ transient (middle). Ryanodine (30 µmol/L) in 0 mmol/L Ca2+ PSS depleted the ACh-sensitive store (bottom). B, Ionomycin induced a Ca2+ transient after exposure to caffeine (4 mmol/L, at 100 seconds) and cyclopiazonic acid (CPA) (at 450 seconds) in 0 mmol/L Ca2+ solution.

Fig 4A shows the results of an experiment in which pretreatment with caffeine (4 mmol/L, middle tracing) or ryanodine (30 µmol/L, bottom tracing) for 300 seconds attenuated the subsequent ACh response. Caffeine/ryanodine was washed out 50 seconds before the ACh application. As is apparent in Fig 5, washout of caffeine removes its direct inhibitory effects within seconds (see below), such that the inhibition seen in Fig 4A reflects depletion of the ACh-sensitive store. The top tracing in Fig 4A shows that simply leaving the cells in 0 mmol/L Ca²⁺ PSS for 300 seconds did not deplete the ACh-sensitive store. Fig 4B shows that after 4 mmol/L caffeine exposure followed by CPA, ionomycin still induced an intracellular Ca²⁺ transient. This shows that during a 50-second exposure to CPA the ER Ca²⁺ leak is not sufficient for depletion of the caffeine- and ACh-resistant store. Unfortunately, ionomycin is not reversible, so we cannot know whether CPA blocks refilling of this second store.

View larger version (12K): [in this window] [in a new window]   Figure 5. Tracing shows that caffeine directly inhibits the acetylcholine (ACh) response. Caffeine (20 mmol/L) was first applied with 10 µmol/L ACh in normal physiological saline solution, and no response was seen. Immediately after washout of caffeine, the remaining ACh caused a Ca2+ transient. After the cells were allowed to recover in the absence of drugs in physiological saline solution for 6 minutes, a second ACh response of the same magnitude was elicited.

Direct Effects of Caffeine on the ACh-Releasable Store
While studying the interaction between ACh-sensitive and caffeine-sensitive Ca²⁺ release, we found that caffeine has a direct inhibitory effect on ACh-mediated Ca²⁺ release (n=21). If ACh was applied in the presence of caffeine (4 or 20 mmol/L), the usual Ca²⁺ transient was abolished. Immediately after washout of caffeine, the remaining ACh caused a Ca²⁺ transient similar to the control response observed after 6 minutes of washout in PSS (Fig 5). Because the usual ACh-induced transient returned to the baseline within 100 seconds, the possibility that this inhibitory effect is caused by caffeine's suppressing the fura 2 fluorescence can be excluded.

ACh-Induced [Ca²⁺]_i Oscillations
In 68 of 247 cells exposed to ACh, we observed oscillations in [Ca²⁺]_i after washout of ACh from the bathing solution. Fig 6 (top tracing) shows an example of spikelike fluctuations in [Ca²⁺]_i. The oscillations were abolished after removal of external Ca²⁺. The bottom tracing in Fig 6 shows another example in which ACh was applied in the absence of Ca²⁺. Oscillation occurred immediately after replenishment of external Ca²⁺.

	Discussion

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In this study we present [Ca²⁺]_i measurements in a native rabbit aortic endothelial preparation. This cell preparation responds to ACh with a transient Ca²⁺ release from an intracellular store, the refilling of which is blocked by the ER Ca²⁺-ATPase inhibitor CPA. In addition, we found evidence for the existence of ryanodine receptors in this native endothelial cell preparation that confirms the recent finding in cultured endothelial cells.^{18 19} This study provides functional data in native endothelial preparation consistent with the ryanodine receptor binding study by Lesh et al.¹⁷

ACh (10 µmol/L) induced a transient Ca²⁺ release–induced [Ca²⁺]_i peak followed by a low plateau dependent on Ca²⁺ influx in >300 cells from 16 animals. The transient nature of the initial response was not related to hydrolysis of ACh because 2 to 10 µmol/L carbachol induced identical changes in [Ca²⁺]_i (data not shown). Removal of external Ca²⁺ abolished the plateau phase but had no effect on the peak value of the ACh response.

In about one third of the cells, the peak [Ca²⁺]_i response was followed by oscillations in [Ca²⁺]_i. The latter were dependent on the presence of extracellular Ca²⁺ and may be causally related to the ACh-induced oscillations of the membrane potential reported by Marchenko and Sage.²² The two phenomena are probably linked through the Ca²⁺-dependent K⁺ channel, as previously described.^{20 23} Because the [Ca²⁺]_i oscillations are dependent on Ca²⁺ influx, they may be driven by periodic Ca²⁺-induced Ca²⁺ release²⁴ or fluctuations in plasmalemma Ca²⁺ permeability.²⁵

The ACh-sensitive Ca²⁺ store appears to be of limited capacity, because a single 100-second exposure to a maximal dose of ACh in Ca²⁺-free solution prevents subsequent responses. Repetitive intracellular Ca²⁺ transients were obtained in normal Ca²⁺-containing PSS, indicating that Ca²⁺ influx is essential for store refilling.

However, refilling was also dependent on an active ER Ca²⁺ pump, because 10 µmol/L of the ER Ca²⁺-ATPase blocker CPA abolished this process. Various agonists have been shown to elevate endothelial cell levels of IP₃,²⁶ and this second messenger has been shown to release ER Ca²⁺ in saponin-permeabilized cultured endothelial cells.²⁷ We thus postulate that ACh indirectly releases Ca²⁺ by stimulating phospholipase C and activating IP₃ receptors. The ER is subsequently replenished by the two-step process of Ca²⁺ entry into the cytoplasm and subsequent uptake by the ER Ca²⁺ pump (SERCA), a process similar to that described for smooth muscle.²⁸

Alternatively, it is possible that in the presence of extracellular Ca²⁺ part of the Ca²⁺ released by ACh is recycled from the cytosol into the ER after washout of the agonist through a CPA-sensitive ATPase. By removal of the extracellular Ca²⁺ the gradient of Ca²⁺ is changed in favor of extrusion, so further refilling of the ER becomes impossible.

Blockade of SERCA uncovers a substantial leak of Ca²⁺ from the ER. The initial CPA-induced loss of Ca²⁺ from the ER is sufficient to produce a transient increase in [Ca²⁺]_i, which is of lower magnitude and longer duration (250 seconds with CPA) than the one induced by ACh (90 seconds). Subsequently, [Ca²⁺]_i is maintained at a suprabasal level for as long as CPA is present in the bath. This temporal pattern of [Ca²⁺]_i elevation is very similar to the CPA-induced increase in vascular cGMP and endothelium-dependent relaxation reported by Moritoki et al.²⁹ Chen et al³⁰ have shown that changes in the steady state Ca²⁺ after blocking of the ER Ca²⁺ transport requires interaction between the ER and plasmalemma membrane. Two possible mechanisms could explain the elevated [Ca²⁺]_i baseline after CPA: ER Ca²⁺ depletion may stimulate Ca²⁺ entry,^{31 32 33} or ER-mediated Ca²⁺ extrusion could be inhibited by CPA.^{28 30 34 35} Recently evidence for Ca²⁺ depletion–dependent Ca²⁺ entry in endothelial cells has been presented.³⁶

The results of the ionomycin experiment raised the possibility of different Ca²⁺ stores in endothelial cells. Our data suggest that in addition to the ACh-sensitive store there is another compartment of the ER that is sensitive to CPA but insensitive to ACh, because after exposure to both CPA and ACh in 0 mmol/L Ca²⁺ PSS, the ionomycin-induced transient was further reduced.

In striated and smooth muscle, Ca²⁺-induced Ca²⁺ release via the ryanodine receptor plays an important role in [Ca²⁺]_i regulation.^{34 37 38} Only recently Lesh et al¹⁷ showed that antibodies to ryanodine receptors bind to vascular endothelium. Most recently, Ziegelstein et al¹⁸ demonstrated for the first time in cultured endothelial cell lines that ryanodine depletes the bradykinin- and histamine-sensitive stores. We explored the existence of functional ryanodine receptors in this intact endothelial cell preparation, which contains functional ACh receptors that lead to intracellular Ca²⁺ release and Ca²⁺ influx.

In the endothelial cell preparation used in the present study, caffeine (4 and 20 mmol/L) failed to induce a Ca²⁺ transient. Instead, caffeine and ryanodine both increased the intracellular Ca²⁺ level with a very slow time course. Subsequent application of ACh after caffeine or ryanodine failed to induce the usual transient response, indicating that activation of ryanodine receptors caused depletion of the ACh-sensitive Ca²⁺ store.

The failure of caffeine to induce rapid transient Ca²⁺ release is not due to slow action by the low concentration of caffeine, because 20 mmol/L caffeine did not induce a Ca²⁺ transient either. Although 20 mmol/L caffeine has some suppressing effect on the fura 2 signal, such a maintained effect would not prevent the recording of a transient elevation of [Ca²⁺]. This has been shown in the studies from our laboratory³⁹ and another laboratory,¹⁹ in which 10 or 20 mmol/L caffeine transiently increased the fura 2 ratio in endothelial or smooth muscle cells despite its mild suppressing effect on the fura 2 signals.

Our observations also show that the ER of native endothelium contains both ryanodine receptors and IP₃ receptors. Experiments on intact and skinned muscles and on ryanodine receptors incorporated in lipid bilayers indicate that these channels have a high conductance and lead to rapid release of Ca²⁺ from the sarcoplasmic reticulum. The failure of caffeine to produce an intracellular Ca²⁺ transient under control conditions could be explained by a sparsity of ryanodine receptors that are, under these conditions, unable to support regenerative Ca²⁺ release. Furthermore, it implies that the rapid ACh-induced Ca²⁺ release is effected mainly through activation of more abundant IP₃ receptors. It is not clear at this stage how caffeine and ryanodine cause depletion of the ACh-sensitive store. Our data indicate that the ER may constitute one intracellular Ca²⁺ compartment that contains a high density of IP₃ receptors and a low density of ryanodine receptors. This is consistent with the study by Lesh et al,¹⁷ who showed a continuous ER network in the endothelial cells by using special stains. However, it is not possible to rule out that endothelial ER contains multiple stores (see Iino et al,⁴⁰ van Breemen and Saida,²⁸ and Graier et al¹⁹ ) that interact with each other by means of unknown mechanisms. Our data also suggest the presence of an intracellular Ca²⁺ store that is depleted by ionomycin but is insensitive to ACh and caffeine. It cannot be ruled out that other actions of caffeine such as inhibition of phosphodiesterase may influence the results.

The present study also revealed a direct inhibitory effect by caffeine on ACh-induced Ca²⁺ release. This blocking effect dissipated immediately upon washout of caffeine and could therefore be differentiated from ER depletion. Although the direct inhibitory effect of caffeine is new for endothelial cells, there have been several reports of similar findings in other cells.^{41 42 43 44} By use of IP₃ receptors incorporated in the lipid bilayer it was shown that caffeine was able to directly block the IP₃-sensitive Ca²⁺ release channel in the ER.⁴⁴

It may be concluded from the data presented here that this freshly dispersed endothelial cell preparation is especially well suited for the study of ACh-induced Ca²⁺ release and may represent a good model system for studying the physiology of native endothelium.

Received October 20, 1994; accepted March 31, 1995.

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