This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Corda, S. Articles by Ziegelstein, R. C. Search for Related Content PubMed PubMed Citation Articles by Corda, S. Articles by Ziegelstein, R. C.

(Circulation Research. 1995;77:927.)
© 1995 American Heart Association, Inc.

Articles

Endoplasmic Reticulum Ca²⁺ Depletion Unmasks a Caffeine-Induced Ca²⁺ Influx in Human Aortic Endothelial Cells

Stefano Corda, Harold A. Spurgeon, Edward G. Lakatta, Maurizio C. Capogrossi, Roy C. Ziegelstein

From the Laboratory of Cardiovascular Science (S.C., H.A.S., E.G.L., M.C.C.), Gerontology Research Center, National Institute on Aging, National Institutes of Health, and the Department of Medicine (M.C.C., R.C.Z.), Division of Cardiology, Johns Hopkins University, Baltimore, MD.

Correspondence to Roy C. Ziegelstein, MD, Johns Hopkins Bayview Medical Center, Cardiology B-1 South, 4940 Eastern Ave, Baltimore, MD 21224. E-mail rziegels@welchlink.welch.jhu.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Intracellular Ca²⁺ pools contribute to changes in cytosolic [Ca²⁺] ([Ca²⁺]_i), which play an important role in endothelial cell signaling. Recently, endothelial ryanodine-sensitive Ca²⁺ stores were shown to regulate agonist-sensitive intracellular Ca²⁺ pools. Since caffeine binds the ryanodine Ca²⁺ release channel on the endoplasmic reticulum in a variety of cell types, we examined the effect of caffeine on [Ca²⁺]_i in human aortic endothelial cell monolayers loaded with the fluorescent probe indo 1. Under baseline conditions, 10 mmol/L caffeine induced a small increase in [Ca²⁺]_i from 86±10 to 115±17 nmol/L (mean±SEM); this effect was similar to that of 5 µmol/L ryanodine and was unaffected by buffer Ca²⁺ removal. After depletion of an intracellular Ca²⁺ store by the irreversible endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (1 µmol/L), ryanodine did not affect [Ca²⁺]_i. In contrast, caffeine induced a large rapid increase in [Ca²⁺]_i (176±19 to 338±35 nmol/L, P<.001) after thapsigargin exposure; this effect of caffeine was only observed when extracellular Ca²⁺ was present. A similar increase in [Ca²⁺]_i was induced by caffeine after depletion of ryanodine- and histamine-sensitive Ca²⁺ stores or after pretreatment with the endoplasmic reticulum Ca²⁺-ATPase inhibitor cyclopiazonic acid (10 µmol/L). Thus, under baseline conditions the effect of caffeine on [Ca²⁺]_i is similar to that of ryanodine and appears to be due to the release of an intracellular store. However, after depletion of an endoplasmic reticulum Ca²⁺ store, caffeine, but not ryanodine, stimulates Ca²⁺ influx, resulting in a large increase in [Ca²⁺]_i. The data suggest that caffeine-induced Ca²⁺ influx is controlled by the status of Ca²⁺ loading of intracellular Ca²⁺ stores in human aortic endothelial cells.

Key Words: cell calcium • endothelium • caffeine • thapsigargin • indo 1

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intracellular Ca²⁺ plays an important role in endothelial cell signaling. In endothelial cells, stimulation of histamine and bradykinin receptors increases [Ca²⁺]_i through InsP₃-mediated mobilization of an ER Ca²⁺ store^{1 2} and through stimulation of extracellular Ca²⁺ influx.^{3 4} The increase in endothelial [Ca²⁺]_i in turn triggers the release of vasoactive substances such as prostacyclin⁵ and endothelium-derived relaxing factor/nitric oxide.^{6 7} Because of this and other critical roles in modulating the functional properties of the vascular endothelium, considerable attention has recently been placed on characterizing endothelial cell intracellular Ca²⁺ regulation.

The ER Ca²⁺-ATPase inhibitor thapsigargin^{8 9} has recently been used as a pharmacological probe of intracellular Ca²⁺ pools in endothelial cells. Thapsigargin largely depletes the InsP₃- and agonist-sensitive Ca²⁺ pools in many cell types,¹⁰ including vascular endothelial cells,^{3 11} without activation of the InsP₃ pathway.⁸ Thapsigargin also promotes extracellular Ca²⁺ entry in vascular endothelial cells,¹¹ resulting in a sustained elevation in [Ca²⁺]_i in the presence of buffer Ca²⁺.

The plant alkaloid ryanodine has also been used to study intracellular Ca²⁺ pools in several cell types. Ryanodine binds to the SR Ca²⁺ release channel of muscle cells.

At nanomolar concentrations in experiments using isolated SR vesicles, ryanodine maintains the Ca²⁺ release channel in an open substate conductance, whereas at micromolar or higher concentrations, ryanodine appears to inhibit channel activation.¹² The Ca²⁺ release channel of muscle cells is also sensitive to caffeine,¹³ which opens SR Ca²⁺ channels and promotes discharge of Ca²⁺ stores.¹⁴ The presence of the ryanodine receptor was recently demonstrated in vascular and endocardial endothelium.¹⁵ Endothelial ryanodine-sensitive Ca²⁺ stores appear to be involved in the regulation of agonist-sensitive intracellular Ca²⁺ pools.¹⁶ Whether ryanodine and caffeine act on the same Ca²⁺ pool and similarly affect [Ca²⁺]_i in endothelial cells is unknown.

A recent study using patch-clamp techniques in bovine aortic endothelial cells¹⁷ demonstrated that caffeine releases Ca²⁺ from an intracellular store that is pharmacologically distinct from the InsP₃-releasable Ca²⁺ store. Caffeine-induced Ca²⁺ release from intracellular stores was also recently documented by whole-cell membrane current recordings in freshly dissociated aortic endothelial cells.¹⁸ The present study further characterizes the effect of caffeine on [Ca²⁺]_i in HAECs loaded with the membrane-permeant (acetoxymethyl ester) form of indo 1. Our results demonstrate that under baseline conditions, caffeine induces a small increase in [Ca²⁺]_i both in the presence and absence of buffer Ca²⁺. This increase in [Ca²⁺]_i appears to be due to release of an intracellular store and is similar to the effect elicited by ryanodine.¹⁶ However, after ER Ca²⁺ depletion in the presence of buffer Ca²⁺, caffeine induces a rapid and marked increase in [Ca²⁺]_i due to extracellular Ca²⁺ influx. In contrast, ryanodine does not stimulate Ca²⁺ influx under these conditions. We propose that caffeine-stimulated Ca²⁺ influx is controlled by the status of ER Ca²⁺ stores in HAECs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
HAECs (Clonetics) were obtained as cryopreserved cells and grown to confluence to passages 4 to 7 in endothelial cell growth medium (Clonetics) with 2% fetal bovine serum, 50 µg/mL gentamicin, 50 ng/mL amphotericin B, and 12 µg/mL bovine brain extract at 37°C in a humidified atmosphere of 95% air/5% CO₂. After treatment with 0.25% trypsin in a Ca²⁺- and Mg²⁺-free solution, cells were plated on glass coverslips or in 1-mm² glass capillary tubes (Vitro Dynamics) precoated with 1% gelatin (Sigma Chemical Co) 1 to 2 days before experimental use.

Measurement of Endothelial [Ca²⁺]_i
HAEC monolayers were loaded with 8 µmol/L of the ester derivative (acetoxymethyl ester form) of the fluorescent Ca²⁺ probe indo 1 (Molecular Probes) in a 95% air/5% CO₂ incubator at room temperature for 30 minutes. Monolayers were then gently washed in indicator-free HEPES-buffered saline solution and maintained an additional 30 to 60 minutes to allow for deesterification of the indicator before the experiment. HAEC monolayers were studied on the stage of a modified inverted fluorescence microscope (Zeiss IM-35) as previously described.¹⁹ Briefly, indo 1 fluorescence collected from a field of 15 cells was excited at 350±5 nm by using a xenon strobe lamp (model FX 193, EG & G Electro-Optics). Bandpass interference filters (Andover) selected wavelength bands of emitted fluorescence at 391 to 434 nm (410 channel) and 457 to 507 nm (490 channel), corresponding to the Ca²⁺-bound and Ca²⁺-free forms of the indicator, respectively. The 410- to 490-nm ratio was used as an index of [Ca²⁺]_i. Autofluorescence of a comparable field of unloaded cells was <5% of indo 1–loaded cells.

Previous work has shown that caffeine quenches indo 1 fluorescence by the same factor at both emission wavelengths without affecting either the indo 1 ratio, the K_d of indo 1 for Ca²⁺, or cell autofluorescence.²⁰ In a prior study using the fluorescence system in our laboratory, caffeine decreased the fluorescence at each wavelength by 15% in isolated cardiac myocytes.²¹ In the present study, indo 1 fluorescence decreased by 20% in HAECs during caffeine superfusion. The comparability of the indo 1 fluorescence ratios and the intracellular Ca²⁺ transients in the presence and absence of caffeine in cardiac myocytes was affected only to the extent to which cell autofluorescence contributed to total fluorescence.²¹ Since endothelial cell autofluorescence is negligible compared with that of cardiac myocytes under our experimental conditions, the effect of caffeine on indo 1 fluorescence would not be expected to have a significant effect on determinations of [Ca²⁺]_i.

To approximate [Ca²⁺] values from indo 1 fluorescence ratios, intracellular R_min and R_max values were determined as previously described.²² Briefly, to determine an intracellular R_min, indo 1-loaded HAEC monolayers were superfused with a solution containing (mmol/L) NaCl 137.0, KCl 5.0, MgSO₄ 1.2, NaH₂PO₄ 1.2, D-glucose 16, HEPES 10, and EGTA 2 (pH 7.40±0.01). The monolayers were then exposed to a solution of similar composition with 10 mmol/L EGTA and 0.05% Triton X-100, resulting in a rapid decrease in the indo 1 ratio before appreciable loss of indicator occurred, determined by simultaneously recording absolute fluorescence at each wavelength as well as by the 410- to 490-nm ratio. To determine the intracellular R_max, different monolayers were exposed to a solution containing 132 mmol/L KCl, 10 mmol/L K-HEPES, 1 mmol/L MgSO₄, 2 µmol/L rotenone, 2 µmol/L carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (Sigma), and 10 ng/mL valinomycin (Calbiochem). HAEC monolayers were then exposed to a similar solution containing 2 µmol/L ionomycin, 69.2 mmol/L CaCl₂, and 100 mmol/L N-hydroxyethylethyl-enediaminetriacetic acid (free [Ca²⁺] of 5900 nmol/L). In this system, the intracellular R_min was 0.40 and the R_max was 2.64; these were used to provide approximations of [Ca²⁺]_i according to the following formula: [Ca²⁺]=K_d(R-R_min/R_max-R)(S_f2/S_b2),²³ where K_d is the effective dissociation constant of indo 1, and S_f2 and S_b2 are the fluorescence intensities at 490 nm of Ca²⁺-free and Ca²⁺-saturated dyes, respectively. K_d was extrapolated from the published determination by using the Chelex assay by Lattanzio.²⁴

Experimental Protocol
HAEC monolayers were exposed to thapsigargin (Calbiochem), CPA (Sigma), caffeine (Sigma), histamine (Sigma), or ryanodine (Penick Corp) in a buffer containing (mmol/L) NaCl 137.0, KCl 4.9, MgSO₄ 1.2, NaH₂PO₄ 1.2, HEPES 20, D-glucose 15, and CaCl₂ 1.5 (pH 7.40). In some experiments, the same solution without added Ca²⁺ and with 1 mmol/L EGTA was used to achieve Ca²⁺-free conditions. All experiments were performed at 23°C, since more rapid and significant intracellular loss of indo 1 occurs at 37°C than at 23°C.¹⁹

Data Analysis and Statistics
Data are reported as mean±SEM. Statistical comparisons of endothelial [Ca²⁺]_i values among treatments of a single endothelial monolayer were performed by paired Student’s t test. Statistical comparisons of endothelial [Ca²⁺]_i values among different monolayers were analyzed by unpaired Student’s t test. A difference was considered significant at P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of Caffeine on HAEC [Ca²⁺]_i Under Baseline Conditions
To determine the effect of caffeine on [Ca²⁺]_i under baseline conditions, indo 1–loaded HAEC monolayers were exposed to 10 mmol/L caffeine for 16 minutes. Caffeine produced a sustained increase in the indo 1 ratio, corresponding to an increase in [Ca²⁺]_i from 86±10 to 115±17 nmol/L (n=8, P<.05); this effect was similar to that reported previously¹⁶ when HAEC monolayers were exposed for 15 minutes to 5 µmol/L ryanodine. The increase in [Ca²⁺]_i upon caffeine exposure was no different when extracellular Ca²⁺ influx was inhibited by implementing experiments in Ca²⁺-free buffer (Fig 1; increase in [Ca²⁺]_i, 29±13 nmol/L in Ca²⁺-containing buffer [n=8] and 46±12 nmol/L in Ca²⁺-free buffer [n=7]; P=NS). Thus, under baseline conditions, caffeine, like ryanodine,¹⁶ induces a small increase in HAEC [Ca²⁺]_i that appears to be due to mobilization of an intracellular store.

View larger version (13K): [in this window] [in a new window]   Figure 1. Effect of caffeine (CAF) on human aortic endothelial [Ca2+]i in Ca2+-free buffer. Representative example of fluorescence measurements obtained from an indo 1–loaded HAEC monolayer superfused for 16 minutes with 10 mmol/L CAF in Ca2+-free HEPES buffer with 1 mmol/L EGTA. A small increase in [Ca2+]i that was reversible upon washout of caffeine in Ca2+-free buffer was observed (n=7). For this and subsequent figures, breaks in fluorescence traces represent time between files; these breaks were necessary because of the design limitations of the computer program. These breaks occur at regularly spaced intervals in most of the figures.

Effect of Caffeine on HAEC [Ca²⁺]_i After ER Ca²⁺ Depletion by Thapsigargin
Since, under baseline conditions, caffeine appeared to mobilize an intracellular Ca²⁺ store, the effect of prior depletion of an ER Ca²⁺ store on the effect of caffeine was examined. For these experiments, HAECs were pretreated with the irreversible ER Ca²⁺-ATPase inhibitor thapsigargin, which has been shown to deplete ER Ca²⁺ stores in endothelial cells.^{3 11} Under baseline conditions in the absence of thapsigargin, histamine (100 µmol/L) induces a large rapid increase in [Ca²⁺]_i (112±7 to 929±278 nmol/L, n=5). Whereas pretreatment with thapsigargin abolished the initial rapid increase in [Ca²⁺]_i during exposure to 100 µmol/L histamine (Fig 2), the application of caffeine following thapsigargin pretreatment resulted in a large and prolonged increase in [Ca²⁺]_i (233±48 to 512±49 nmol/L, n=4). Upon washout of caffeine and return to control buffer, [Ca²⁺]_i returned to levels observed before caffeine exposure over 10 minutes. The effect of caffeine on HAEC [Ca²⁺]_i after a more brief exposure to thapsigargin was also determined while recording indo 1 fluorescence. Superfusion of monolayers with thapsigargin for 10 minutes increased [Ca²⁺]_i from 83±9 to 206±16 nmol/L (n=11). After a brief thapsigargin superfusion, caffeine produced an additional increase in [Ca²⁺]_i (176±19 to 338±35 nmol/L, n=10, P<.001, Fig 3), which was approximately fourfold to fivefold greater than the caffeine-induced increase in [Ca²⁺]_i under baseline conditions. The caffeine-induced increase in [Ca²⁺]_i following this brief thapsigargin exposure was of shorter duration than that observed following prolonged pretreatment with thapsigargin (Fig 2). This effect was not specific to thapsigargin-induced ER Ca²⁺-ATPase inhibition, as it was also observed when monolayers were stimulated by caffeine after ER Ca²⁺-ATPase inhibition by CPA, an agent without apparent structural similarity to thapsigargin.³ After a 10-minute exposure to CPA (10 µmol/L), caffeine stimulated a large increase in [Ca²⁺]_i (167±48 to 332±64 nmol/L, P<.01, n=8), which was similar to that observed after brief thapsigargin pretreatment. When monolayers were exposed to caffeine after a 10-minute pretreatment with either 0.1 or 1 µmol/L CPA, there was a trend toward a greater increase in [Ca²⁺]_i compared with the effect of caffeine under baseline conditions (107±15 to 163±26 nmol/L after 0.1 µmol/L CPA and 155±28 to 211±43 nmol/L after 1 µmol/L CPA, n=6 for each), but these changes were not significantly different from each other or from that initiated by caffeine under control conditions (P=NS). No additional increase in [Ca²⁺]_i was stimulated by caffeine after pretreatment with higher concentrations of CPA (data not shown). The different durations of the caffeine-induced increase in [Ca²⁺]_i (Figs 2 and 3) may relate to the extent of ER Ca²⁺ depletion produced by the different durations of thapsigargin pretreatment, since it has previously been shown³ that pharmacological inhibition of the ER Ca²⁺-ATPase by thapsigargin, CPA, or BHQ causes time-dependent depletion of the endothelial internal Ca²⁺ store. Alternatively, the sustained caffeine-stimulated increase in [Ca²⁺]_i may be simply a function of time after pool depletion. The transient increase in [Ca²⁺]_i initiated by caffeine was not observed after exposures to thapsigargin of >7.5 minutes in experiments using 20- and 30-minute thapsigargin pretreatment (data not shown).

View larger version (12K): [in this window] [in a new window]   Figure 2. Effect of histamine (HIS) and caffeine (CAF) on human aortic endothelial [Ca2+]i after thapsigargin pretreatment. A representative example of indo 1 fluorescence was obtained from an endothelial monolayer that was pretreated for 2 hours with 1 µmol/L thapsigargin in HEPES buffer with 1.5 mmol/L Ca2+ in order to deplete ER Ca2+ stores. Although the rapid increase in [Ca2+]i due to HIS (100 µmol/L) was abolished after thapsigargin pretreatment, CAF (10 mmol/L) induced a rapid and prolonged increase in [Ca2+]i (n=4) that returned to baseline upon return to control buffer.

View larger version (10K): [in this window] [in a new window]   Figure 3. Effect of thapsigargin (TG) and caffeine (CAF) on human aortic endothelial [Ca2+]i. A representative example of indo 1 fluorescence was obtained from an HAEC monolayer in HEPES buffer with 1.5 mmol/L Ca2+ exposed to 10 mmol/L CAF after a 7.5-minute exposure to 1 µmol/L TG. Under these conditions, CAF induced a large and rapid increase in [Ca2+]i (n=10).

To estimate the extent of ER Ca²⁺ depletion produced by thapsigargin or by different concentrations of CPA, monolayers were first pretreated with thapsigargin or CPA in the absence of extracellular Ca²⁺ and were then exposed to ionomycin in Ca²⁺-free solution to release residual Ca²⁺ from the intracellular pool. Under baseline conditions without CPA or thapsigargin pretreatment, ionomycin (1 µmol/L in Ca²⁺-free buffer with 1 mmol/L EGTA) increased [Ca²⁺]_i by 105±39 nmol/L (n=5). This was similar to the increase in [Ca²⁺]_i stimulated by 1 µmol/L thapsigargin in Ca²⁺-free buffer (109±68 nmol/L, n=3, P=NS versus ionomycin). After a 7.5-minute pretreatment with 1 µmol/L thapsigargin in Ca²⁺-free buffer, no further increase in [Ca²⁺]_i was elicited by ionomycin (-9±4 nmol/L, n=5), suggesting that this concentration and duration of thapsigargin pretreatment fully depleted intracellular Ca²⁺ stores. Similarly, ionomycin failed to increase [Ca²⁺]_i (-3±3 nmol/L, n=3) after a 10-minute pretreatment with 10 µmol/L CPA. In contrast, ionomycin increased [Ca²⁺]_i after pretreatment with lower concentrations of CPA. After a 10-minute exposure to either 1 µmol/L CPA (n=6) or to 0.1 µmol/L CPA (n=4), ionomycin stimulated increases in [Ca²⁺]_i of 16±5 and 23±12 nmol/L, respectively (P<.05 versus 10 µmol/L CPA for each).

Thus, the intracellular pool appears to be fully depleted by either a 7.5-minute pretreatment with 1 µmol/L thapsigargin or a 10-minute pretreatment with 10 µmol/L CPA, since ionomycin failed to increase [Ca²⁺]_i after either method of pool depletion. In separate experiments performed in buffer with 1.5 mmol/L Ca²⁺, the caffeine-stimulated increase in [Ca²⁺]_i was similar after either of these pretreatment protocols (160 nmol/L). In contrast, after pretreatment with lower concentrations of CPA, which do not fully deplete the intracellular pool, there was a trend toward a greater caffeine-stimulated [Ca²⁺]_i increase in buffer with 1.5 mmol/L Ca²⁺ compared with baseline conditions, but these changes were not significant.

Effect of Ryanodine on HAEC [Ca²⁺]_i After ER Ca²⁺ Depletion by Thapsigargin
The presence of the ryanodine receptor was recently demonstrated in vascular and endocardial endothelium,¹⁵ and ryanodine-sensitive Ca²⁺ stores were found in several endothelial cell types, including HAECs.¹⁶ However, it is unknown if ryanodine and caffeine bind the same ER Ca²⁺ release channel in endothelial cells or whether they have similar effects on endothelial [Ca²⁺]_i. Therefore, experiments were performed to examine whether ryanodine, like caffeine, increases HAEC [Ca²⁺]_i after brief exposure to thapsigargin. In contrast to caffeine, under these experimental conditions ryanodine did not increase [Ca²⁺]_i (data not shown). In other experiments performed after thapsigargin ER Ca²⁺ depletion, the same HAEC monolayer was first exposed to 5 µmol/L ryanodine for 15 minutes and then to 10 mmol/L caffeine; whereas ryanodine had no effect on [Ca²⁺]_i, caffeine induced a rapid and reversible increase in [Ca²⁺]_i (165±24 to 318±128 nmol/L, n=8, Fig 4). Thus, whereas ryanodine and caffeine have similar effects on HAEC [Ca²⁺]_i under baseline conditions, distinct effects on HAEC [Ca²⁺]_i are revealed by prior exposure to thapsigargin. The more sustained increase in [Ca²⁺]_i stimulated by caffeine after brief thapsigargin pretreatment combined with ryanodine exposure (Fig 4) is in contrast to the more transient caffeine-stimulated increase in [Ca²⁺]_i following brief thapsigargin pretreatment alone (Fig 3). This sustained increase in [Ca²⁺]_i was also observed after combined thapsigargin and histamine pretreatment (data not shown). As noted above, this difference in the duration of the caffeine response may relate either to the time after pool depletion or to the extent of prior ER Ca²⁺ depletion.

View larger version (16K): [in this window] [in a new window]   Figure 4. Effect of ryanodine (RY) and caffeine (CAF) on human aortic endothelial [Ca2+]i following exposure to thapsigargin (TG). A representative example of indo 1 fluorescence was obtained from an HAEC monolayer during exposure to 1 µmol/L TG, 5 µmol/L RY, and 10 mmol/L CAF in HEPES buffer with 1.5 mmol/L Ca2+. Although RY did not affect [Ca2+]i after TG exposure, CAF initiated a large increase in [Ca2+]i in these endothelial monolayers (n=8).

Effect of Caffeine on HAEC [Ca²⁺]_i After Depletion of Ryanodine- and Agonist-Sensitive ER Ca²⁺ Pools
To determine whether the caffeine-induced increase in HAEC [Ca²⁺]_i following thapsigargin administration was stimulated by depletion of an ER Ca²⁺ store or by an effect specific to thapsigargin, monolayers were exposed to caffeine after exposure to ryanodine and histamine. HAECs were exposed for 20 minutes to 5 µmol/L ryanodine in order to deplete a ryanodine-sensitive intracellular Ca²⁺ store. This concentration and duration of exposure were chosen on the basis of prior work,¹⁶ which showed that the ryanodine-induced increase in HAEC [Ca²⁺]_i reached a plateau after 15 to 20 minutes. In contrast to the effect of caffeine after thapsigargin, after a 20-minute ryanodine exposure only a small increase in [Ca²⁺]_i was stimulated by caffeine (142±40 to 164±40 nmol/L, n=5, P<.05). Whether caffeine and ryanodine bind the same ER Ca²⁺ release channel in endothelial cells or whether caffeine acts on a ryanodine-insensitive intracellular pool as occurs in hepatocytes²⁵ is unknown. The small increase in [Ca²⁺]_i stimulated by caffeine under these conditions (1.5 mmol/L buffer Ca²⁺) may also be due to extracellular influx.

In other experiments, after release of the ryanodine-sensitive Ca²⁺ pool, monolayers were exposed for 5 minutes to 100 µmol/L histamine, which mobilizes Ca²⁺ from an InsP₃-sensitive intracellular store.^{26 27 28} Histamine stimulation after ryanodine exposure (Fig 5) resulted in only a small increase in [Ca²⁺]_i (166±21 to 184±23 nmol/L, n=8, P=NS), consistent with an effect of ryanodine on agonist-sensitive intracellular Ca²⁺ stores as previously reported.¹⁶ After release of the ryanodine- and histamine-sensitive intracellular Ca²⁺ pools, exposure to caffeine resulted in a rapid increase in [Ca²⁺]_i (175±22 to 254±24 nmol/L, n=8, P<.01). Thus, the effect of caffeine following depletion of an ER Ca²⁺ store is not specific to thapsigargin-induced ER Ca²⁺-ATPase inhibition, although the increase in [Ca²⁺]_i initiated by caffeine stimulation following ryanodine and histamine exposure was smaller than that observed upon caffeine stimulation following thapsigargin exposure (80±22 nmol/L [n=8] versus 162±27 nmol/L [n=10], P<.05).

View larger version (15K): [in this window] [in a new window]   Figure 5. Effect of caffeine (CAF) on human aortic endothelial [Ca2+]i following exposure to ryanodine (RY) and histamine (HIS). Representative example of fluorescence measurements was obtained from an indo 1–loaded HAEC monolayer in HEPES buffer with 1.5 mmol/L Ca2+ that was exposed for 20 minutes to 5 µmol/L RY in order to deplete a ryanodine-sensitive intracellular Ca2+ store (see text; only the final minute shown). HAECs were then exposed for 6 minutes to 100 µmol/L HIS, resulting in only a small increase in [Ca2+]i. Subsequent exposure to 10 mmol/L CAF resulted in a large and rapid increase in [Ca2+]i (n=8).

Effect of Caffeine on HAEC [Ca²⁺]_i After ER Ca²⁺ Depletion by Thapsigargin in Ca²⁺-Free Buffer
To determine the contribution of extracellular Ca²⁺ influx to the caffeine-induced [Ca²⁺]_i increase following thapsigargin exposure, experiments were performed in Ca²⁺-free/EGTA buffer. When HAEC monolayers were exposed to thapsigargin in Ca²⁺-free buffer for 5 minutes (Fig 6, top), [Ca²⁺]_i increased and then gradually returned to control levels over a period of 5 to 10 minutes. The sustained increase in [Ca²⁺]_i that occurred during thapsigargin application in the presence of extracellular Ca²⁺ was abolished under these experimental conditions; this appears to be due to Ca²⁺ influx.¹¹ In contrast to its effect when extracellular Ca²⁺ was present, caffeine did not increase [Ca²⁺]_i after thapsigargin exposure in the absence of buffer Ca²⁺ (Fig 6, bottom). Thus, the caffeine-induced increase in [Ca²⁺]_i after thapsigargin exposure is dependent on extracellular Ca²⁺.

View larger version (19K): [in this window] [in a new window]   Figure 6. Effect of caffeine (CAF) on human aortic endothelial [Ca2+]i following exposure to thapsigargin (TG) in the absence of extracellular Ca2+. Top, Representative example of fluorescence measurements obtained from an indo 1–loaded HAEC monolayer in HEPES buffer without added Ca2+ and with 1 mmol/L EGTA exposed to 1 µmol/L TG (n=3). TG produced a transient increase in [Ca2+]i, which gradually returned to baseline levels. Bottom, Representative indo 1 fluorescence obtained from an HAEC monolayer in Ca2+-free HEPES buffer with 1 mmol/L EGTA exposed to 10 mmol/L CAF after a 5-minute exposure to 1 µmol/L TG. CAF did not induce an increase in [Ca2+]i under these conditions (n=5).

Effect of Caffeine on HAEC [Ca²⁺]_i After ER Ca²⁺ Depletion in Ca²⁺-Free Buffer Followed by a Brief Period of Ca²⁺ Repletion
The dependence on extracellular Ca²⁺ of the caffeine-induced [Ca²⁺]_i increase following ER Ca²⁺ depletion suggests that caffeine stimulates Ca²⁺ influx under these conditions. Alternatively, ER Ca²⁺ depletion may stimulate extracellular Ca²⁺ influx and thereby refill a caffeine-sensitive intracellular Ca²⁺ pool only in the presence of buffer Ca²⁺. In this way, the observed dependence on extracellular Ca²⁺ of the caffeine-induced [Ca²⁺]_i increase may actually reflect release of an intracellular store rather than caffeine-stimulated Ca²⁺ influx. To explore this possibility, HAECs were grown in 1-mm² glass capillary tubes (total volume, 50 µL) to facilitate the rapid exchange of buffer solutions (perfusion rate, 1.1 mL/min). Monolayers were first exposed to thapsigargin in Ca²⁺-free buffer in order to deplete ER Ca²⁺ under conditions that prevent extracellular Ca²⁺ influx. The subsequent exposure to caffeine in Ca²⁺-free buffer did not increase [Ca²⁺]_i (Fig 7). The perfusion solution was then rapidly changed to HEPES buffer with 1.5 mmol/L Ca²⁺ for 2 minutes in an attempt to refill both the cytosol and intracellular Ca²⁺ stores not irreversibly depleted by thapsigargin. This attempt would likely be facilitated by the Ca²⁺ concentration gradient between the cytosolic and intracellular compartments. Changing to a Ca²⁺-containing solution resulted in a rapid increase in [Ca²⁺]_i as extracellular Ca²⁺ influx filled the cytosol. If the repleted cytosolic compartment then filled the caffeine-sensitive Ca²⁺ pool, the subsequent exposure to caffeine, even in the absence of extracellular Ca²⁺, would be expected to increase [Ca²⁺]_i by releasing a caffeine-sensitive intracellular store. When monolayers were exposed to caffeine in Ca²⁺-free buffer at this time (Fig 7A), no increase in [Ca²⁺]_i was observed; in fact, [Ca²⁺]_i rapidly decreased from the higher level in buffer with 1.5 mmol/L Ca²⁺. In contrast, when caffeine exposure occurred in buffer with 1.5 mmol/L Ca²⁺ (Fig 7B), a further increase in [Ca²⁺]_i occurred (326±95 nmol/L, n=5). The data suggest that after ER Ca²⁺ depletion, caffeine stimulates extracellular Ca²⁺ influx.

View larger version (21K): [in this window] [in a new window]   Figure 7. Extracellular Ca2+–dependent effect of caffeine (CAF) on human aortic endothelial [Ca2+]i following exposure to thapsigargin. A, Representative example of indo 1 fluorescence obtained from an endothelial monolayer after exposure to 1 µmol/L thapsigargin for 5 minutes in Ca2+-free HEPES buffer with 1 mmol/L EGTA. Exposure to thapsigargin is not shown on the tracing. After thapsigargin exposure, the monolayer was exposed to 10 mmol/L CAF in Ca2+-free buffer. Only a small decrease in [Ca2+]i was observed in the Ca2+-free buffer; CAF did not increase [Ca2+]i. Ca2+-replete HEPES buffer (1.5 mmol/L) was then rapidly reintroduced for 2 minutes to refill an intracellular Ca2+ pool that might previously have been emptied and not irreversibly depleted by thapsigargin. The repletion of extracellular Ca2+ resulted in a rapid increase in [Ca2+]i. After this increase, the monolayer was exposed again to CAF (10 mmol/L) in Ca2+-free buffer. No increase in [Ca2+]i was seen under these conditions (n=5). B, Representative example of indo 1 fluorescence obtained from an endothelial monolayer after exposure to 1 µmol/L thapsigargin for 5 minutes in Ca2+-free HEPES buffer with 1 mmol/L EGTA. Exposure to thapsigargin is not shown on the tracing. After thapsigargin exposure, 10 mmol/L caffeine in Ca2+-free buffer did not increase [Ca2+]i. Extracellular Ca2+ (1.5 mmol/L) was then rapidly reintroduced for 2 minutes to refill an intracellular Ca2+ pool, resulting in a rapid increase in [Ca2+]i. After this increase, the monolayer was exposed again to CAF (10 mmol/L) in HEPES buffer with 1.5 mmol/L Ca2+, which resulted in a further increase in [Ca2+]i (n=5).

Comparison of the Effect of Histamine With That of Caffeine on HAEC [Ca²⁺]_i After ER Ca²⁺ Depletion in Ca²⁺-Free Buffer Followed by a Brief Period of Ca²⁺ Repletion
The large rapid Ca²⁺ influx following ER Ca²⁺ depletion was observed with caffeine but not with ryanodine. To determine whether this effect is seen upon histamine stimulation, experiments were performed under conditions similar to those previously described in Fig 7. HAEC monolayers grown in glass capillary tubes were superfused with Ca²⁺-free buffer and then exposed to thapsigargin in order to deplete the ER Ca²⁺ store (Fig 8). The solution was then rapidly changed to one with 1.5 mmol/L Ca²⁺ for 2 minutes, resulting in a rapid increase in [Ca²⁺]_i. When HAECs were then exposed to histamine in Ca²⁺-free buffer, [Ca²⁺]_i rapidly returned to levels noted before exposure to Ca²⁺-containing buffer. An increase in [Ca²⁺]_i would have been expected if the histamine (InsP₃)–sensitive intracellular Ca²⁺ store had been refilled during exposure to buffer with 1.5 mmol/L Ca²⁺. Thus, although exposure to Ca²⁺-containing buffer rapidly filled the cytosol, it did not fill the histamine-sensitive intracellular Ca²⁺ store, which had most likely been irreversibly depleted by thapsigargin.

View larger version (21K): [in this window] [in a new window]   Figure 8. Comparison of the effect of histamine (HIS) and caffeine (CAF) on human aortic endothelial [Ca2+]i following exposure to thapsigargin (TG). A representative example of indo 1 fluorescence was obtained from an endothelial monolayer exposed to 1 µmol/L TG in Ca2+-free HEPES buffer with 1 mmol/L EGTA. TG resulted in a transient increase in [Ca2+]i. The perfusion solution was then rapidly changed for 2 minutes to one with 1.5 mmol/L Ca2+, resulting in a rapid increase in [Ca2+]i. The monolayer was then exposed to 100 µmol/L HIS in Ca2+-free/EGTA buffer, resulting in a decrease in [Ca2+]i to baseline. The perfusion solution was changed a second time to one with 1.5 mmol/L Ca2+, again rapidly increasing [Ca2+]i. Exposure to 100 µmol/L HIS in Ca2+-containing buffer did not increase [Ca2+]i; instead, [Ca2+]i decreased to baseline levels in Ca2+-replete solution. Subsequent exposure to 10 mmol/L CAF resulted in a further rapid increase in [Ca2+]i (n=4).

HAEC monolayers were then exposed again to Ca²⁺-containing buffer (Fig 8) and then to histamine in this same buffer solution. No increase in [Ca²⁺]_i was observed when monolayers were exposed to histamine at this time; indeed [Ca²⁺]_i decreased to a "new" baseline in the Ca²⁺-containing solution. However, when monolayers were exposed to caffeine after return to the new baseline [Ca²⁺]_i, a rapid increase in [Ca²⁺]_i (110±6 to 306±62 nmol/L, n=4, P<.05) was observed. Thus, caffeine, but not histamine, induces extracellular Ca²⁺ influx under these experimental conditions.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study documents the existence of a caffeine-stimulated Ca²⁺ influx in human endothelial cells that is regulated by the status of Ca²⁺ loading of ER Ca²⁺ stores. Under baseline conditions, when ER Ca²⁺ stores are full, caffeine induces a small increase in HAEC [Ca²⁺]_i due to release of an intracellular Ca²⁺ store. This effect is similar to that of ryanodine.¹⁶ In contrast, when ER Ca²⁺ stores have been emptied, caffeine promotes a rapid increase in HAEC [Ca²⁺]_i due principally to extracellular Ca²⁺ influx. This effect on Ca²⁺ influx is not observed with ryanodine or with histamine.

Although the effects of caffeine on [Ca²⁺]_i in excitable cells are well established, the role of caffeine in endothelial cell Ca²⁺ regulation has only recently been examined. In the present study, the increase in HAEC [Ca²⁺]_i stimulated by caffeine under baseline conditions was not altered when extracellular Ca²⁺ was absent (Fig 1). This suggests the existence of a caffeine-sensitive intracellular Ca²⁺ store and is consistent with the findings of other recent studies.^{17 18} By recording the activity of Ca²⁺-activated K⁺ channels in bovine aortic endothelial cell monolayers, Thuringer and Sauve¹⁷ demonstrated caffeine-induced Ca²⁺ release from an InsP₃-insensitive intracellular store. Application of caffeine opened Ca²⁺-activated K⁺ channels in cell-attached patch-clamp experiments in both Ca²⁺-free and Ca²⁺-containing external solutions.¹⁷ In another study of freshly dissociated single rabbit aortic endothelial cells,¹⁸ caffeine induced a transient rise in [Ca²⁺]_i in fura 2–loaded cells in the presence and absence of extracellular Ca²⁺. A dose-dependent increase in unitary current activity superimposed on a large prolonged transient outward current upon application of caffeine was documented by whole-cell membrane currents recorded under voltage-clamp conditions.¹⁸ The caffeine-induced transient outward current was not affected by heparin, an InsP₃ receptor antagonist. Thus, caffeine appears to release an InsP₃-insensitive intracellular Ca²⁺ store, which may be an important source of Ca²⁺ for activation of K⁺ channels in endothelial cells.

In muscle cells, both caffeine and ryanodine bind the SR Ca²⁺ release channel^{12 13} but appear to have different mechanisms of action. At low (nanomolar) concentrations in experiments using isolated SR vesicles, ryanodine maintains the Ca²⁺ release channel in an open substate conductance, whereas at high concentrations, ryanodine appears to inhibit channel activation.¹¹ Caffeine opens SR Ca²⁺ release channels,¹² promotes discharge of Ca²⁺ stores,¹³ and activates Ca²⁺-induced Ca²⁺ release. Caffeine appears to increase the affinity of the activation site of a Ca²⁺-permeable SR channel for Ca²⁺²⁹ and to increase the number and duration of open-channel events.³⁰ The present study shows that in HAECs under baseline conditions, caffeine results in an increase in [Ca²⁺]_i similar to that induced by ryanodine in this cell type.¹⁶ However, caffeine produces an additional small increase in HAEC [Ca²⁺]_i after ryanodine stimulation. Since this experiment was performed in the presence of extracellular Ca²⁺, it is uncertain whether this effect is due to Ca²⁺ release from a ryanodine-insensitive intracellular pool as occurs in hepatocytes²⁵ or from extracellular influx. The endothelial ER was recently found to contain a protein having homology with the ryanodine receptor/Ca²⁺ release channel of muscle.¹⁵ The pattern of immunofluorescence staining of endothelial cells labeled with a specific anti–ryanodine receptor antibody was consistent with the distribution of the ER.¹⁵ However, whether caffeine and ryanodine bind the same site on the ER of endothelial cells is not known.

When thapsigargin was used to deplete ER Ca²⁺ in the present study, caffeine did not increase [Ca²⁺]_i when buffer Ca²⁺ was not present. Thus, thapsigargin appears to deplete the caffeine-sensitive Ca²⁺ store in HAECs, suggesting that the caffeine-sensitive intracellular Ca²⁺ store is a part of the ER Ca²⁺ store. This may not be the case in all cell types. In Jurkat T-lymphocytes,³¹ caffeine (15 mmol/L) increased [Ca²⁺]_i when added after thapsigargin (1 µmol/L) in the absence of extracellular Ca²⁺. Under these conditions, thapsigargin abolished the response to the monoclonal anti-CD3 antibody OKT3, which releases Ca²⁺ from an InsP₃-sensitive pool in Jurkat T-lymphocytes,³¹ suggesting that the caffeine-sensitive Ca²⁺ pool in this cell type is not part of the ER Ca²⁺ store.

The endothelial caffeine-sensitive intracellular Ca²⁺ store appears to require access, either directly or indirectly, to extracellular Ca²⁺. Recently, a small transient caffeine-induced hyperpolarization was recorded with microelectrodes from an intact guinea pig coronary artery preparation.³² When buffer Ca²⁺ was removed, the hyperpolarization decreased progressively with each successive application of caffeine, suggesting that the caffeine-sensitive pool was dependent on extracellular Ca²⁺ for its repletion. Similarly, exposure to caffeine produced repetitive [Ca²⁺]_i spikes in fura 2–loaded human umbilical vein endothelial cells³³ but only in the presence of extracellular Ca²⁺.

In the present study, the caffeine-sensitive intracellular Ca²⁺ store was depleted by the ER Ca²⁺-ATPase inhibitor thapsigargin. In Ca²⁺-free solution, caffeine stimulation did not increase [Ca²⁺]_i after exposure to thapsigargin. If monolayers were then superfused with Ca²⁺-containing solution, a rapid increase in [Ca²⁺]_i occurred as extracellular Ca²⁺ influx filled the cytosol (Fig 7). This might be expected to fill an intracellular store not irreversibly depleted by thapsigargin, because of the Ca²⁺ concentration gradient between the cytolic and intracellular compartments. The absence of [Ca²⁺]_i increase when monolayers were subsequently exposed to caffeine in Ca²⁺-free buffer (Fig 7A) suggests that the caffeine-sensitive intracellular Ca²⁺ store was still empty at this time. In contrast, when caffeine exposure occurred in buffer with 1.5 mmol/L Ca²⁺, a further increase in [Ca²⁺]_i occurred, consistent with caffeine-stimulated extracellular Ca²⁺ influx. This caffeine-stimulated Ca²⁺ influx was not observed with either ryanodine (Fig 4) or with histamine (Fig 8). Although increases in [Ca²⁺]_i assessed by changes in indo 1 fluorescence could reflect either Ca²⁺ influx or inhibition of Ca²⁺ efflux, the absence of a caffeine effect in Ca²⁺-free buffer following intracellular store depletion suggests that Ca²⁺ influx is responsible for the effect of caffeine following store depletion in Ca²⁺-containing solution.

There are several possible mechanisms of the caffeine-stimulated Ca²⁺ influx after intracellular pool depletion in HAECs. One possibility is that caffeine modulates the depletion-dependent Ca²⁺ influx described by other investigators.^{3 11} In the present study, the magnitude of Ca²⁺ influx stimulated by caffeine correlates, at least in part, with the extent of prior ER Ca²⁺ depletion, since the increase in [Ca²⁺]_i initiated by caffeine was greater after pretreatment with concentrations of thapsigargin or CPA that appeared to fully deplete intracellular Ca²⁺ pools than it was after pretreatment with concentrations of CPA that did not fully deplete intracellular stores. On the basis of experiments performed with ionomycin administration following thapsigargin or CPA pretreatment in Ca²⁺-free buffer, the intracellular Ca²⁺ store appears to be emptied by either a 7.5-minute pretreatment with 1 µmol/L thapsigargin or a 10-minute pretreatment with 10 µmol/L CPA. In separate experiments performed in buffer with 1.5 mmol/L Ca²⁺, the caffeine-stimulated increase in [Ca²⁺]_i was similar (160 nmol/L) after each method of store depletion. Lower concentrations of CPA appeared to only partially deplete the intracellular store, since ionomycin caused an additional increase in [Ca²⁺]_i after a 10-minute pretreatment with either 0.1 or 1 µmol/L CPA. After pretreatment with either of these partially depleting concentrations of CPA, there was a trend toward a greater caffeine-stimulated increase in [Ca²⁺]_i than under baseline conditions, but these changes were not significantly different from control.

The relation of plasmalemmal Ca²⁺ influx to the degree of filling of intracellular Ca²⁺ stores has been described by Putney’s "capacitative" pathway.³⁴ Evidence in support of this model was recently provided by patch-clamp recordings in bovine aortic endothelial cells.³⁵ By use of different concentrations of the ER Ca²⁺-ATPase inhibitor BHQ, inward Ca²⁺ currents were found to vary with the extent of depletion of intracellular Ca²⁺ stores. Although the signal responsible for communicating the level of the intracellular store to the plasmalemmal influx pathway was not identified in that or the present study, it has been suggested that the intracellular store and the influx pathway are spatially in close association.³⁵ Whether the caffeine-sensitive intracellular compartment in endothelial cells is in close association with an influx pathway is unknown. Alternatively, caffeine may stimulate the production of a second messenger that signals the opening of plasmalemmal Ca²⁺ channels only when intracellular stores are depleted.

Although capacitative Ca²⁺ influx may be involved in the effect of caffeine described in the present study, other processes that stimulate Ca²⁺ entry in endothelial cells may be enhanced by caffeine. Extracellular Ca²⁺ influx may occur by at least four other mechanisms, which have recently been reviewed by Adams et al.¹⁸ These include a passive Ca²⁺ "leak," mechanosensitive (stretch-activated) Ca²⁺ channels, Na⁺-Ca²⁺ exchange, and receptor-mediated Ca²⁺ influx through receptor-operated or second messenger (eg, inositol 1,3,4,5-tetrakis-phosphate)–activated channels. The effect of caffeine is unlikely to involve dihydropyridine-sensitive L-type Ca²⁺ channels, since electrophysiological and unidirectional ⁴⁵Ca²⁺ flux measurements indicate an absence of these channels in cultured endothelial cells.^{18 36 37} Although an effect of caffeine on [Ca²⁺]_i mediated by an increase in cAMP through its inhibition of phosphodiesterase cannot be excluded, this appears to be an unlikely mechanism of the rapid caffeine-stimulated Ca²⁺ influx described in the present study. In concentrations up to 10 mmol/L, caffeine is only a weak inhibitor of endothelial phosphodiesterases, with a potency significantly less than that of other inhibitors of this enzyme, such as 3-isobutyl-1-methylxanthine (W.F. Graier, unpublished data, 1995), which has no effect on endothelial [Ca²⁺]_i.³⁸

The data from the present study and other work suggest that the caffeine-sensitive intracellular Ca²⁺ store, like the ryanodine- and InsP₃-sensitive stores, is part of the ER Ca²⁺ store in endothelial cells. Although the caffeine-sensitive and InsP₃-sensitive stores may be distinct,^{17 18} further investigation is necessary to determine whether ryanodine and caffeine bind the same putative Ca²⁺ release channel in endothelial cells. The present report shows that caffeine stimulates Ca²⁺ entry in endothelial cells and that this Ca²⁺ influx is regulated by the status of ER Ca²⁺ stores such that caffeine promotes Ca²⁺ entry when the stores are depleted, regardless of the mechanism of pool depletion. Although caffeine, ryanodine, and histamine each affect intracellular Ca²⁺ pools, the rapid large influx of Ca²⁺ following thapsigargin exposure is initiated only by caffeine and not by the other two agonists. The functional role of caffeine-stimulated Ca²⁺ influx requires further study.

	Selected Abbreviations and Acronyms

 

BHQ = 2',5'-di(tert-butyl)-1,4-benzohydroquinone CPA = cyclopiazonic acid ER = endoplasmic reticulum HAEC = human aortic endothelial cell InsP3 = inositol 1,4,5-tris-phosphate Rmax = maximum fluorescence ratio Rmin = minimum fluorescence ratio SR = sarcoplasmic reticulum

Received October 20, 1994; accepted July 21, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Colden-Stanfield M, Schilling WP, Ritchie AK, Eskin SG, Navarro LT, Kunze DL. Bradykinin induced increases in cytosolic calcium and ionic currents in cultured bovine aortic endothelial cells. Circ Res.. 1987;61:632-640. [Abstract/Free Full Text]

2. Pollock WK, Wreggett KA, Irvine RF. Inositol phosphate production and Ca²⁺ mobilization in human umbilical vein endothelial cells stimulated by thrombin and histamine. Biochem J.. 1988;256:371-376. [Medline] [Order article via Infotrieve]

3. Schilling WP, Cabello OA, Rajan L. Depletion of the inositol 1,4,5-trisphosphate sensitive intracellular Ca²⁺ store in vascular endothelial cells activates the agonist sensitive Ca²⁺ influx pathway. Biochem J.. 1992;284:521-530.

4. Hallam T, Jacob R, Merritt JE. Evidence that agonists stimulate bivalent cation influx into human endothelial cells. Biochem J.. 1988;255:179-184. [Medline] [Order article via Infotrieve]

5. Hallam TJ, Pearson JD, Needham LA. Thrombin stimulated elevation of human endothelial cell cytoplasmic free calcium concentration causes prostacyclin production. Biochem J.. 1988;251:243-249. [Medline] [Order article via Infotrieve]

6. Palmer RMJ, Ferridge AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature.. 1987;327:524-526. [Medline] [Order article via Infotrieve]

7. Long CJ, Stone TW. The release of endothelium derived relaxant factor is calcium dependent. Blood Vessels.. 1985;22:205-208. [Medline] [Order article via Infotrieve]

8. Thastrup OP, Cullen PJ, Drobak MR, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca²⁺ stores by specific inhibition of the endoplasmic reticulum Ca²⁺-ATPase. Proc Natl Acad Sci U S A.. 1990;87:2466-2470. [Abstract/Free Full Text]

9. Sagara Y, Inesi G. Inhibition of the sarcoplasmic reticulum Ca²⁺ transport ATPase by thapsigargin at subnanomolar concentrations. J Biol Chem.. 1991;266:13503-13506. [Abstract/Free Full Text]

10. Bian J, Ghosh TK, Wang JC, Gill DL. Identification of intracellular Ca²⁺ pools: selective modification by thapsigargin. J Biol Chem.. 1991;266:8801-8806. [Abstract/Free Full Text]

11. Dolor JR, Hurtwitz LM, Mirza Z, Strauss HC, Whorton AR. Regulation of extracellular Ca²⁺ entry in endothelial cells: role of intracellular Ca²⁺ pool. Am J Physiol.. 1992;262:C171-C181. [Abstract/Free Full Text]

12. Lai FA, Misra M, Xu L, Smith HA, Meissner G. The ryanodine receptor-Ca²⁺ release channel complex of skeletal muscle sarcoplasmic reticulum. J Biol Chem.. 1989;264:16776-16785. [Abstract/Free Full Text]

13. Endo M. Calcium release from the sarcoplasmic reticulum. Physiol Rev.. 1977;57:71-108. [Free Full Text]

14. Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol.. 1983;245:C1-C14. [Abstract/Free Full Text]

15. Lesh RE, Marks AR, Somlyo AV, Fleischer S, Somlyo AP. Antiryanodine receptor antibody binding sites in vascular and endocardial endothelium. Circ Res.. 1993;72:481-488. [Abstract/Free Full Text]

16. Ziegelstein RC, Spurgeon HA, Pili R, Passaniti A, Cheng L, Corda S, Lakatta EG, Capogrossi MC. A functional ryanodine-sensitive intracellular Ca²⁺ store is present in vascular endothelial cells. Circ Res.. 1994;74:151-156. [Abstract/Free Full Text]

17. Thuringer D, Sauve R. A patch-clamp study of the Ca²⁺ mobilization from internal stores in bovine aortic endothelial cells, II: effect of thapsigargin on the cellular Ca²⁺ homeostasis. J Membr Biol.. 1992;130:139-148. [Medline] [Order article via Infotrieve]

18. Adams DJ, Rusko J, van Slooten G. Calcium signaling in vascular endothelial cells: Ca²⁺ entry and release. In: Weir EK, Hume JR, Reeves JT, eds. Ion Flux in Pulmonary Vascular Control. New York, NY: Plenum Publishing Corp; 1993:259-275.

19. Spurgeon HA, Stern MD, Baartz G, Raffaeli S, Hansford RG, Talo A, Lakatta EG, Capogrossi MC. Simultaneous measurement of Ca²⁺, contraction, and potential in cardiac myocytes. Am J Physiol.. 1990;258:H574-H586. [Abstract/Free Full Text]

20. O’Neill SC, Donoso P, Eisner DA. The role of [Ca²⁺]_i and [Ca²⁺] sensitization in the caffeine contracture of rat myocytes: measurement of [Ca²⁺]_i and [caffeine]. J Physiol (Lond).. 1990;425:55-70. [Abstract/Free Full Text]

21. Janczewski AM, Lakatta EG. Buffering of calcium influx by sarcoplasmic reticulum during the action potential in guinea-pig ventricular myocytes. J Physiol (Lond).. 1993;471:343-363. [Abstract/Free Full Text]

22. Ziegelstein RC, Cheng L, Blank PS, Spurgeon HA, Lakatta EG, Hansford RG, Capogrossi MC. Modulation of calcium homeostasis in cultured rat aortic endothelial cells by intracellular acidification. Am J Physiol.. 1993;265:H1424-H1433. [Abstract/Free Full Text]

23. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem.. 1985;260:3440-3450. [Abstract/Free Full Text]

24. Lattanzio FA Jr. The effects of pH and temperature on fluorescent calcium indicators as determined with Chelex-100 and EDTA buffer systems. Biochem Biophys Res Commun.. 1990;171:102-108. [Medline] [Order article via Infotrieve]

25. McNulty TJ, Taylor CW. Caffeine-stimulated Ca²⁺ release from the intracellular stores of hepatocytes is not mediated by ryanodine receptors. Biochem J.. 1993;291:799-801.

26. Resink TJ, Grigorian GY, Moldabaeva AK, Danilov SM, Bühler FR. Histamine-induced phosphoinositide metabolism in cultured human umbilical vein endothelial cells: association with thromboxane and prostacyclin release. Biochem Biophys Res Commun.. 1987;144:438-446. [Medline] [Order article via Infotrieve]

27. Lo WWY, Fan TPD. Histamine stimulates inositol phosphate accumulation via the H₁-receptor in cultured human endothelial cells. Biochem Biophys Res Commun.. 1987;148:47-53. [Medline] [Order article via Infotrieve]

28. Pollock WK, Wreggett KA, Irvine RF. Inositol phosphate production and Ca²⁺ mobilization in human umbilical-vein endothelial cells stimulated by thrombin and histamine. Biochem J.. 1988;256:371-376.

29. Nagasaki K, Kasai M. Channel selectivity and gating specificity of calcium-induced calcium release channel in isolated sarcoplasmic reticulum. J Biochem.. 1984;96:1769-1775. [Abstract/Free Full Text]

30. Rousseau E, LaDine J, Liu Q-Y, Meissner G. Activation of the Ca²⁺ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds. Arch Biochem Biophys.. 1988;267:75-86. [Medline] [Order article via Infotrieve]

31. Guse AH, Roth E, Emmrich F. Intracellular Ca²⁺ pools in Jurkat T-lymphocytes. Biochem J.. 1993;291:447-451.

32. Chen G, Cheung DW. Pharmacological distinction of the hyperpolarization response to caffeine and acetylcholine in guinea-pig coronary endothelial cells. Eur J Pharmacol.. 1992;223:33-38. [Medline] [Order article via Infotrieve]

33. Neylon CB, Irvine RF. Synchronized repetitive spikes in cytoplasmic calcium in confluent monolayers of human umbilical vein endothelial cells. FEBS Lett.. 1990;275:173-176. [Medline] [Order article via Infotrieve]

34. Putney JW. A model for receptor-regulated calcium entry. Cell Calcium.. 1986;7:1-12. [Medline] [Order article via Infotrieve]

35. Vaca L, Kunze DL. Depletion and refilling of intracellular Ca²⁺ stores induce oscillations of Ca²⁺ current. Am J Physiol.. 1993;264:H1319-H1322. [Abstract/Free Full Text]

36. Takeda K, Klepper M. Voltage-dependent and agonist-activated ionic currents in vascular endothelial cells: a review. Blood Vessels.. 1990;27:169-183. [Medline] [Order article via Infotrieve]

37. Takeda K, Schini V, Stoeckel H. Voltage-activated potassium, but not calcium currents in cultured bovine aortic endothelial cells. Pflugers Arch.. 1987;410:385-393. [Medline] [Order article via Infotrieve]

38. Graier WF, Schmidt K, Kukovetz WR. Effect of sodium fluoride on cytosolic free Ca²⁺-concentrations and cGMP-levels in endothelial cells. Cell Signal.. 1990;2:369-375. ]CG[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				J. Satriano, R. Cunard, O. W. Peterson, T. Dousa, F. B. Gabbai, and R. C. Blantz Effects on kidney filtration rate by agmatine requires activation of ryanodine channels for nitric oxide generation Am J Physiol Renal Physiol, April 1, 2008; 294(4): F795 - F800. [Abstract] [Full Text] [PDF]

				Y.-a. Zhang, R. A. Tuft, L. M. Lifshitz, K. E. Fogarty, J. J. Singer, and H. Zou Caffeine-activated large-conductance plasma membrane cation channels in cardiac myocytes: characteristics and significance Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2448 - H2461. [Abstract] [Full Text] [PDF]

				B. Nilius and G. Droogmans Ion Channels and Their Functional Role in Vascular Endothelium Physiol Rev, October 1, 2001; 81(4): 1415 - 1459. [Abstract] [Full Text] [PDF]

				Q. Hu and R. C. Ziegelstein Hypoxia/Reoxygenation Stimulates Intracellular Calcium Oscillations in Human Aortic Endothelial Cells Circulation, November 14, 2000; 102(20): 2541 - 2547. [Abstract] [Full Text] [PDF]

				J. Kitayama, T. Kitazono, S. Ibayashi, M. Wakisaka, Y. Watanabe, M. Kamouchi, T. Nagao, M. Fujishima, and F. M. Faraci Role of Phosphatidylinositol 3-Kinase in Acetylcholine-Induced Dilatation of Rat Basilar Artery Editorial Comment Stroke, October 1, 2000; 31(10): 2487 - 2493. [Abstract] [Full Text] [PDF]

				M. Frieden and W. F Graier Subplasmalemmal ryanodine-sensitive Ca2+ release contributes to Ca2+-dependent K+ channel activation in a human umbilical vein endothelial cell line J. Physiol., May 1, 2000; 524(3): 715 - 724. [Abstract] [Full Text] [PDF]

				J. Paltauf-Doburzynska, K. Posch, G. Paltauf, and W. F Graier Stealth ryanodine-sensitive Ca2+ release contributes to activity of capacitative Ca2+ entry and nitric oxide synthase in bovine endothelial cells J. Physiol., December 1, 1998; 513(2): 369 - 379. [Abstract] [Full Text] [PDF]

				T. M. Moore, P. M. Chetham, J. J. Kelly, and T. Stevens Signal transduction and regulation of lung endothelial cell permeability. Interaction between calcium and cAMP Am J Physiol Lung Cell Mol Physiol, August 1, 1998; 275(2): L203 - L222. [Abstract] [Full Text] [PDF]

				G. Faury, Y. Usson, M. Robert-Nicoud, L. Robert, and J. Verdetti Nuclear and cytoplasmic free calcium level changes induced by elastin peptides in human endothelial cells PNAS, March 17, 1998; 95(6): 2967 - 2972. [Abstract] [Full Text] [PDF]

				Q. Hu, S. Corda, J. L. Zweier, M. C. Capogrossi, and R. C. Ziegelstein Hydrogen Peroxide Induces Intracellular Calcium Oscillations in Human Aortic Endothelial Cells Circulation, January 27, 1998; 97(3): 268 - 275. [Abstract] [Full Text] [PDF]

				B. J. Buckley and A. R. Whorton Tunicamycin increases intracellular calcium levels in bovine aortic endothelial cells Am J Physiol Cell Physiol, October 1, 1997; 273(4): C1298 - C1305. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Corda, S. Articles by Ziegelstein, R. C. Search for Related Content PubMed PubMed Citation Articles by Corda, S. Articles by Ziegelstein, R. C.