INTRODUCTION

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THE SYNTHESIS and/or release of endothelium-derived relaxing and contracting factors in response to stimulation by vasoactive agents is triggered by an increase in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) (12). The agonist-induced increase in [Ca²⁺]_i is biphasic: a rapid, transient Ca²⁺ increase due to Ca²⁺ release from intracellular stores and a plateau phase due to a slow sustained Ca²⁺ influx across the plasma membrane (for reviews see Refs. 1 and 19). The increases in [Ca²⁺]_i are generally accompanied by changes in membrane potential due to activation of K⁺ conductances (23, 32).

Caffeine, a methylxanthine, stimulates the release of Ca²⁺ from ryanodine-sensitive intracellular stores by enhancing Ca²⁺-induced Ca²⁺ release (CICR) from the endoplasmic reticulum (ER) (8, 41). Vascular endothelial cells have been shown to possess inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores as well as functional ryanodine-sensitive Ca²⁺ stores (4, 33, 39, 46). Caffeine has also been shown to inhibit cAMP phosphodiesterase activity, increasing intracellular cAMP concentrations, which subsequently increases Ca²⁺ reuptake by the stores (18). Furthermore, caffeine has been reported to inhibit plasma membrane Ca²⁺ channels and to suppress transient outward Ca²⁺-independent K⁺ currents in smooth muscle cells (29).

The aim of the present study was to investigate the effects of caffeine on intracellular Ca²⁺ mobilization in vascular endothelial cells and determine the temporal relationship between global changes in [Ca²⁺]_i measured fluorometrically and the activation of ionic currents and changes in membrane potential. Recent studies suggest that measurements of [Ca²⁺]_i using Ca²⁺ indicator dyes such as fura 2 are insensitive to changes in [Ca²⁺]_i adjacent to the plasma membrane (14, 21, 38). These changes in local Ca²⁺ concentration have been proposed to underlie the occurrence of spontaneous increases in [Ca²⁺]_i ("Ca²⁺ sparks") in myocytes (for review see Ref. 31) and the occurrence of spontaneous transient outward and inward currents (STOCs and STICs) in smooth muscle cells (44). These events are attributed to the spontaneous release of Ca²⁺ from caffeine-sensitive CICR channels in the ER. The mobilization of intracellular Ca²⁺ in freshly isolated endothelial cells was studied by simultaneous measurement of caffeine-induced cytosolic Ca²⁺ transients and membrane currents. The physiological relevance of these changes was assessed by comparing results from similar measurements made during agonist stimulation of cells using the vasoactive substance ACh.

	MATERIALS AND METHODS

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Endothelial cell preparation. Endothelial cells from rabbit arterial vessels were isolated using an enzymatic dissociation procedure described previously (22, 32). Briefly, 40 male New Zealand White rabbits (2-3 kg) were killed by cervical dislocation, and the thoracic aorta and pulmonary arteries were dissected out and placed in physiological salt solution (Na⁺-saline) with the following composition (mM): 125.4 NaCl, 5.9 KCl, 1.5 CaCl₂, 1.2 MgCl₂, 11.5 glucose, and 10 HEPES, adjusted to pH 7.35 with NaOH. Connective and adipose tissue from the arteries were removed, the vessels were cut longitudinally, and thin sheets of endothelium were carefully peeled off. The endothelium sheets were transferred to a high-K⁺ solution (K⁺-saline) containing (mM) 126 KCl, 5.3 NaCl, 1.5 CaCl₂, 1.2 MgCl₂, 11.5 glucose, and 10 HEPES, adjusted to pH 7.35 with KOH, which also contained 0.45 mg/ml papain (7 U/mg) and 0.4 mg/ml dithiothreitol, and were incubated at 37°C for ~40 min. The tissue was then washed with K⁺-saline containing 0.5% BSA and gently triturated. The resulting suspension was filtered (62-µm nylon mesh filter) and centrifuged at 2,500 rpm for 5 min. The pellet of endothelial cells was resuspended in K⁺-saline, plated on glass coverslips, and maintained at 4°C for 1 h, allowing the cells to reequilibrate and adhere to the glass. The coverslips were placed in a recording chamber (0.5 ml volume), mounted on the stage of a Nikon Diaphot inverted microscope equipped with ultraviolet transparent optics, and visualized at ×400 magnification with phase-contrast optics. The chamber was continuously perfused at a rate of ~10 ml/min. Experiments were performed at room temperature (21 ± 2°C). Freshly dissociated cells were positively identified as endothelial cells, as described previously (22, 32).

Microfluorometric measurements. After adhesion to the coverslips, the cells were incubated for 1 h at room temperature in ~0.3 ml of Na⁺-saline solution containing 5 µM fura 2-AM (1 mM fura 2-AM in DMSO stock solution), 0.02% Pluronic F-127, and 0.5% BSA. After incubation with the dye, the cells were washed in Na⁺-saline and allowed 30 min to recover before experiments were begun. A 75-W xenon arc lamp supplied alternating (model OC-4000 Optical Chopper, Photon Technology International, South Brunswick, NJ) 340- and 380-nm illumination via a fiber-optic cable, a 450-nm dichroic mirror (model DM 400, Nikon), and a ×40 oil immersion objective (Fluor 40/1.3 NA, Nikon). Emission fluorescence (510-nm band-pass filter) was collected by a photomultiplier tube (model R928, Hamamatsu) through a variable aperture set around the cell image. The output of the photomultiplier tube was digitized using a Photon Technology International interface and sampled at 5 Hz using Felix 1.1 software (Photon Technology International) run on a Pentium 133-MHz personal computer.

Electrical recording. Membrane currents and potentials were recorded using the perforated-patch whole cell recording configuration (30). Patch electrodes (1-4 M) were fabricated from borosilicate glass (model GC150TF, Clark Electromedical Instruments, Reading, UK). The tip of the pipette was dipped in standard pipette solution and backfilled with the same solution containing amphotericin B. Filled pipettes were mounted on the head stage of a patch-clamp amplifier (model EPC-7, List-Medical, Darmstadt, Germany). Pipette resistance and gigaseal formation were determined by the current responses to brief 1-mV voltage steps (pCLAMP 6, Axon Instruments, Foster City, CA). Capacitance and series resistance (R_S) compensation were applied to determine cell capacitance and access resistance. The reduction in R_S of cell-attached patches induced by amphotericin B insertion into the membrane began within seconds of seal formation, and experiments were initiated when R_S became <30 M, which generally occurred within 10 min. R_S was monitored and adjusted throughout the course of the experiment. Membrane potentials were corrected for liquid junction potentials, which, by theoretical calculations (2), had values of 8.1 mV (Na⁺-saline and K⁺-saline) and 16.5 mV (SO²₄-saline) at 22°C.

Solutions and reagents. Freshly isolated endothelial cells were perfused with normal Na⁺-saline. In a series of experiments the external solution was changed to a high-K⁺ solution (K⁺-saline), with Na⁺ isosmotically replaced by K⁺, or to a low-Cl solution (SO²₄-saline) composed of (mM) 75 Na₂SO₄, 55 NaCl, 5.9 KCl, 1.5 CaCl₂, 1.2 MgCl₂, 11.5 glucose, and 10 HEPES, adjusted to pH 7.35 with NaOH.

	RESULTS

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Fluorometric and electrophysiological experiments were performed on freshly isolated endothelial cells enzymatically dissociated from rabbit aorta (n > 270) and pulmonary arteries (n = 7). Fura 2-loaded cells were simultaneously patch clamped using the perforated-patch whole cell recording configuration to correlate changes in [Ca²⁺]_i to membrane currents and voltages. The mean resting potential for cells studied was 35 ± 3 mV (n = 15), ranging from 58 to 25 mV, and the mean resting [Ca²⁺]_i was 65 ± 10 nM (n = 22).

Temporal relationship between [Ca²⁺]_i and membrane response when endothelial cells are activated by ACh or caffeine. Intracellular Ca²⁺ transients and whole cell currents or membrane potential changes, elicited by bath application of caffeine or ACh, were highly variable between individual cells. In 44 of 60 (73%) cells responding to 5 mM caffeine, 27 responded with measurable changes in [Ca²⁺]_i and membrane current or potential, whereas 3 exhibited a change in membrane potential or current without a corresponding change in [Ca²⁺]_i and 14 cells responded with an increase in [Ca²⁺]_i without measurable changes in membrane current or potential. Similarly, in 14 of 30 (42%) cells that responded to 100 µM ACh, 6 responded with measurable changes in [Ca²⁺]_i and membrane current or potential, whereas 3 exhibited a change in membrane potential or current with no change in [Ca²⁺]_i and 5 responded with an increase in [Ca²⁺]_i without an apparent change in membrane current or potential. Despite this heterogeneity, it was possible to characterize cellular responses in freshly dissociated endothelial cells to ACh and caffeine application.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Temporal relationship between intracellular Ca2+ concentration ([Ca2+]i) and membrane current (I) or membrane potential (V) in response to stimulation by caffeine or ACh. Fura 2-loaded cells were voltage clamped at 0 mV (A and B) or current clamped (C) using perforated-patch whole cell recording configuration. Cells were perfused with Na+-saline containing caffeine or 100 µM ACh at indicated times. Top traces of [Ca2+]i responses are temporally aligned to bottom traces of membrane current (A and B) or membrane potential (C), which were recorded simultaneously with fura 2 fluorescence.

Overlap of ACh- and caffeine-sensitive intracellular Ca²⁺ stores. Although caffeine and ACh elicited similar changes in [Ca²⁺]_i and current or membrane potential, caffeine and ACh have been shown to affect distinct ER Ca²⁺ release channels (41). Figure 2A shows the [Ca²⁺]_i response and the corresponding changes in membrane potential in a fura 2-loaded cell successively exposed to 100 µM ACh and 5 mM caffeine. Independent of the order of agonist application, the cell responded to ACh and caffeine. The magnitudes and time courses of the [Ca²⁺]_i responses, however, depended on which agonist was applied first. When ACh was applied before caffeine, the total [Ca²⁺]_i increased 2.1-fold and the peak membrane hyperpolarization was 1.6-fold larger than when ACh was applied after caffeine. Similarly, application of caffeine before ACh caused a biphasic [Ca²⁺]_i increase, which was 1.7-fold greater than that elicited when caffeine was applied after ACh. In the latter condition, caffeine evoked only an increase in voltage noise, whereas caffeine application before ACh evoked a 10-mV hyperpolarization (Fig. 2B). Figure 2C shows [Ca²⁺]_i responses to 1 mM ACh in the absence and presence of a maximally effective concentration of caffeine. The mean peak [Ca²⁺]_i response to ACh in the presence of 20 mM caffeine was 233 ± 53 nM (n = 5), which was not statistically different from the maximal [Ca²⁺]_i response to ACh alone, 254 ± 97 nM (P = 0.85), measured in the same cells.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Overlap between ACh- and caffeine-induced changes in [Ca2+]i and membrane potential (Vm). Fura 2-loaded cells were perfused with Na+-saline containing ACh and/or caffeine at indicated times and concentrations. [Ca2+]i (A) and membrane potential (B) were recorded as functions of time. C: ACh-induced [Ca2+]i responses obtained in absence and presence of 20 mM caffeine. ACh (1 mM) alone increased [Ca2+]i to 610 nM, whereas in presence of caffeine, ACh increased peak [Ca2+]i to 236 nM. Breaks in fluorescence and voltage records are 5-min intervals to allow cell to recover from prior ACh/caffeine stimulation. Membrane potential traces are temporally aligned to fluorescence traces.

Concentration dependence of caffeine-induced [Ca²⁺]_i response and current activation. The [Ca²⁺]_i transient and membrane current induced by agonists such as ACh, bradykinin, and histamine have been shown to be concentration dependent in vascular endothelial cells (5, 24, 27). Caffeine-induced Ca²⁺ release from intracellular stores and activation of outward currents in endothelial cells have been described as a regenerative process with all-or-nothing activation (34) or as a dose-dependent process, with the magnitudes of the responses increasing with increasing caffeine concentration (4). Figure 3 shows a dose-dependent increase in [Ca²⁺]_i and outward current by caffeine. Changes in [Ca²⁺]_i obtained in response to increasing concentrations (0.5-5 mM) of caffeine are shown in Fig. 3A, top traces. The kinetics of the [Ca²⁺]_i response were also dependent on the caffeine concentration, whereby the rate of initial rise in [Ca²⁺]_i increased with increasing caffeine concentration (from 17 nM/s at 0.5 mM caffeine to 580 nM/s at 5 mM caffeine). The caffeine-induced membrane outward currents obtained from the same cell held at 0 mV are shown in Fig. 3A, bottom traces. The peak current amplitude and rate of activation exhibit a dose dependence on caffeine similar to that observed for changes in [Ca²⁺]_i (Fig. 3A, top traces). Repeated applications of caffeine at >2 mM attenuated subsequent [Ca²⁺]_i responses. Although desensitization of caffeine responses may underestimate the magnitude of the saturating [Ca²⁺]_i increase or current with increasing caffeine concentration, the half-maximal increase in [Ca²⁺]_i was obtained with 1.2 mM caffeine (n = 7-18) and 1.1 mM caffeine (n = 4-8) for half-maximal current activation (Fig. 3B). These data indicate that near-maximal [Ca²⁺]_i and current activation can be obtained with 5 mM caffeine.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Concentration dependence of caffeine-induced changes in [Ca2+]i and membrane current amplitude. A: fura 2-loaded cell voltage clamped at 0 mV and perfused with Na+-saline containing 0.5, 1.0, 2.0, and 5.0 mM caffeine. [Ca2+]i (top traces) and membrane current (bottom traces) were recorded simultaneously and are plotted as functions of time. Breaks in traces are 5-min intervals to allow cell to recover from previous caffeine stimulation. Current traces are temporally aligned to fluorescence traces above. B: dose-response curves for caffeine-induced [Ca2+]i increases ([Ca2+]i, n > 7) and current activation (I, n > 4). Lines drawn are best fits to Hill equation with calculated EC50 of 1.2 and 1.1 mM and slopes of 1.1 and 2.3 for [Ca2+]i and I, respectively. Data points obtained for [Ca2+]i at 20 mM caffeine and I at 10 mM caffeine were not used in fits.

Effect of inhibition of reuptake of Ca²⁺ by internal stores on responses to caffeine. The IP₃-sensitive intracellular Ca²⁺ store in endothelial cells has been reported to be rapidly refilled after Ca²⁺ influx from the extracellular space during the plateau phase of the [Ca²⁺]_i response (6, 36). The refilling of the intracellular Ca²⁺ store can be blocked by the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor CPA (20, 45). The caffeine-induced [Ca²⁺]_i transient has been shown to be independent of external Ca²⁺, suggesting that emptying of the caffeine-sensitive Ca²⁺ stores does not activate influx of Ca²⁺ from the extracellular space (33, 34). The mobilization of intracellular Ca²⁺ and hyperpolarization of vascular endothelial cells by caffeine and the subsequent extrusion of cytoplasmic Ca²⁺ were examined after SERCA inhibition by CPA or thapsigargin.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Cyclopiazonic acid (CPA) inhibition of Ca2+ reuptake by caffeine depleted intracellular stores. Fura 2-loaded cells were perfused with Na+-saline containing caffeine and 30 µM CPA at indicated times and concentrations. [Ca2+]i and membrane potential are plotted as functions of time. Dashed lines, resting [Ca2+]i. A: fura 2-loaded cell perfused with Na+-saline containing 5 mM caffeine in absence and presence of 30 µM CPA. Membrane potential is temporally aligned with [Ca2+]i. B: cell perfused with Na+-saline containing 20 mM caffeine in absence and presence of 30 µM CPA in a Ca2+-free solution with 1 mM EGTA (physiological salt solution, PSS).

I-V relationship and ionic dependence of caffeine-induced currents. When caffeine-induced currents were recorded at hyperpolarized membrane potentials, caffeine-induced [Ca²⁺]_i transients were accompanied by small inward currents of 17 ± 5 (n = 9) and 13 ± 7 pA (n = 12) at 80 and 60 mV, respectively. Given that the K⁺ equilibrium potential (E_K) is 78 mV, these data suggest that a charge carrier other than K⁺ contributes to the caffeine-activated current. The ionic dependence of caffeine-induced currents was investigated by ion substitution. [Ca²⁺]_i transients and membrane currents were recorded simultaneously from a fura 2-loaded endothelial cell held at 0 mV in response to 5 mM caffeine in Na⁺-saline, K⁺-saline, and SO²₄-saline (Fig. 5A). Replacement of external Na⁺ with K⁺ did not affect the caffeine-induced [Ca²⁺]_i response but reversed the direction of the caffeine-induced membrane current from outward to inward (n = 3). There was, however, no change in the caffeine-induced current amplitude when external Na⁺ was replaced with Li⁺ or N-methyl-D-glucamine (n = 3, data not shown). Similarly, replacement of external Cl with SO²₄ did not markedly change the caffeine-induced [Ca²⁺]_i transient but reduced the amplitude of the caffeine-activated outward current by ~50% (Fig. 5A).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Ionic and voltage dependence of caffeine-evoked membrane currents. A: fura 2-loaded cell voltage clamped at 0 mV and perfused with Na+-saline, K+-saline, and SO24-saline. Caffeine (5 mM) (solid bars) was applied as indicated. [Ca2+]i (top traces) and membrane current (bottom traces) in response to caffeine are shown as functions of time. Dashed line, zero-current level. Current traces are temporally aligned with fluorescence traces above. B: membrane currents measured in response to voltage ramps from a different cell perfused with Na+-saline, K+-saline, and SO24-saline containing 5 mM caffeine. Voltage ramps (300 mV/s) were applied before and during peak of caffeine-induced response. Current-voltage (I-V) relationships represent net caffeine-induced currents obtained by subtracting currents recorded in absence of caffeine from those recorded in presence of caffeine. Membrane potentials are corrected for liquid junction potentials. Reversal potentials of caffeine-induced currents are 47.9, 0.3, and 22.3 mV in presence of Na+-saline [K+ equilibrium potential (EK) = 78 mV; Cl equilibrium potential (ECl) = 25 mV], K+-saline (EK = 0 mV; ECl = 25 mV), and SO24-saline (EK = 78 mV; ECl = 0 mV), respectively.

2

2

Sensitivity of caffeine-induced changes in [Ca²⁺]_i and membrane potential to external La³⁺, Zn²⁺ and TEA. The actions of the putative ion channel blockers TEA, La³⁺, and Zn²⁺ on the caffeine-evoked currents were used to further characterize the ionic dependence of the caffeine response. In the absence of caffeine, 5 mM TEA reduced the outward current in response to voltage ramps from 120 to +60 mV (n = 3), whereas 1 mM La³⁺ (n = 2) and 0.1-0.2 mM Zn²⁺ (n = 5) reduced outward and inward currents (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effects of tetraethylammonium (TEA) and La3+ on caffeine-induced changes in [Ca2+]i and membrane potential. A fura 2-loaded cell was perfused with Na+-saline containing 5 mM caffeine (solid bars) in absence or presence of 5 mM TEA or 1 mM La3+. [Ca2+]i (A) and membrane potential (B) were recorded simultaneously and plotted as functions of time. Dashed line, initial resting membrane potential. Membrane potential traces are temporally aligned to fluorescence traces above and corrected for liquid junction potentials. Breaks in traces represent 5-min recovery periods after caffeine stimulation.

	DISCUSSION

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These results describe quantitatively the relationship between the increase in [Ca²⁺]_i and the activation of ionic currents in arterial endothelial cells in response to ACh and caffeine. In 7% of the cells that exhibited caffeine-activated currents, no apparent changes in [Ca²⁺]_i were observed, and 32% of cells that responded to caffeine with increases in [Ca²⁺]_i had no corresponding changes in membrane current or voltage. These observations are most likely due to the fact that single- cell photometry of fura 2 fluorescence measures the average change in [Ca²⁺]_i throughout the bulk cytoplasm, whereas Ca²⁺-activated currents respond to [Ca²⁺]_i changes adjacent to the plasma membrane. Fura 2 measurements are unlikely to resolve localized [Ca²⁺]_i changes in small, restricted volumes of the cell as postulated to exist between the ER and the plasma membrane (9, 14). The occurrence of STOCs and STICs in vascular smooth muscle cells have been proposed to be due to local release of Ca²⁺ and the concomitant activation of Ca²⁺-activated K⁺ and Cl currents, respectively (43, 44). Similarly, freshly dissociated endothelial cells exhibit STOCs that have been suggested to be due to the release of Ca²⁺ from CICR channels in the ER adjacent to the plasma membrane and activation of K⁺ channels sensitive to TEA and charybdotoxin (32). The occurrence of STOCs or spontaneous transient hyperpolarizations in rabbit aortic endothelial cells was not accompanied by measurable changes in fura 2 fluorescence (Figs. 1 and 4). Agonist-induced currents without global changes in [Ca²⁺]_i suggest that cells can respond to increases in [Ca²⁺]_i in the restricted space adjacent to the plasma membrane (3). Similarly, changes in [Ca²⁺]_i that occurred ~1-2 s after current activation may be due to local changes in [Ca²⁺]_i, which activated the current preceding or even stimulating the increase in global [Ca²⁺]_i.

Although ACh and caffeine elicit similar increases in [Ca²⁺]_i and changes in membrane current and potential, our results suggest that the intracellular Ca²⁺ stores sensitive to ACh and caffeine may be distinct. Fura 2 fluorescence measurements indicate that, in endothelial cells, application of caffeine does not completely empty ACh-sensitive Ca²⁺ stores (Fig. 2), suggesting that the IP₃- and ryanodine-sensitive Ca²⁺ stores do not overlap entirely. The degree of store overlap is difficult to quantitate because of desensitization of responses; however, Fig. 2 indicates that only a fraction of the Ca²⁺ stores overlap or interact. Similar observations have been reported for the caffeine- and ACh-induced changes in membrane potential, which were associated with [Ca²⁺]_i transients, and suggest the existence of partially overlapping IP₃- and ryanodine-sensitive Ca²⁺ stores (40, 45). Our results would also be consistent with a CICR mechanism, wherein Ca²⁺ released from the IP₃-sensitive store could impact on the ryanodine-sensitive Ca²⁺ store and vice versa. A potential concern is that caffeine may attenuate the ACh-induced [Ca²⁺]_i response, given that caffeine (10 mM) has been shown to inhibit IP₃-gated channel activity in lipid bilayers (7).

Mobilization of intracellular Ca²⁺ and membrane current activation by caffeine was dose dependent, desensitized with repeated applications of high concentrations of caffeine, and highly variable from cell to cell, consistent with previous studies in vascular endothelial cells (4, 15, 39). The magnitude of the increase in [Ca²⁺]_i, as well as the kinetics of the response, was dependent on the caffeine concentration, with the initial rate of rise in [Ca²⁺]_i and the peak [Ca²⁺]_i increasing with caffeine concentration. Half-maximal caffeine concentrations for increasing [Ca²⁺]_i and current activation were similar, ~1.1 mM. Similar dose-response relations obtained for caffeine-induced [Ca²⁺]_i increases and outward current activation suggest that the events are unlikely to be independent and that caffeine-induced Ca²⁺ mobilization activates Ca²⁺-sensitive membrane currents. Previous studies on rabbit aortic endothelial cells, however, have suggested that [Ca²⁺]_i mobilization and current responses to caffeine, measured independently, were regenerative (34).

The initial phase of the agonist-induced [Ca²⁺]_i transient in endothelial cells has been shown to be independent of external Ca²⁺ and is due to the release of Ca²⁺ from internal Ca²⁺ stores (for reviews see Refs. 1 and 19). This initial rapid rise in [Ca²⁺]_i is assumed to be closely related to the membrane hyperpolarization due to the activation of Ca²⁺-dependent K⁺ current by Ca²⁺ released from the IP₃-sensitive Ca²⁺ stores (27, 32, 35, 37). The sustained phase of the agonist (ACh)-induced [Ca²⁺]_i increase is dependent on external Ca²⁺ (for reviews see Refs. 1 and 19), and the influx of divalent cations from the extracellular space has been directly demonstrated by Mn²⁺ quench of the fura 2 signal (6). Activation of Ca²⁺-sensitive cation currents (16) and associated membrane depolarizations observed in the presence of external TEA are consistent with cation influxes during the plateau phase of the [Ca²⁺]_i increase. In contrast, the [Ca²⁺]_i transient induced by caffeine is not dependent on external Ca²⁺, and it is not accompanied by Mn²⁺ quench of the fura 2 signal (33, 34). Thus the caffeine-induced transient depolarization blocked by La³⁺ (Fig. 6) appears to differ from the sustained depolarization observed with bradykinin or thapsigargin activation of cultured bovine aortic endothelial cells (40, 42) or ACh-stimulated endothelium of intact rat aorta (26). Furthermore, La³⁺-induced depolarization of a resting cell (Fig. 6B) suggests that La³⁺ may block an ionic current other than the Ca²⁺ release-activated Ca²⁺ current. Substitution of external Na⁺ by K⁺, reduction of external Cl, or change in the membrane potential had no appreciable effect on caffeine-induced [Ca²⁺]_i responses, also suggesting that the [Ca²⁺]_i increase was due to release of intracellular Ca²⁺ and not Ca²⁺ influx across the plasma membrane (15, 42).

Incubation of cells with CPA or thapsigargin caused relatively small increases in basal [Ca²⁺]_i levels (50 nM) and did not appreciably change the membrane potential or the holding current (cf. Ref. 13). In the presence of CPA, however, the magnitude of the caffeine-induced [Ca²⁺]_i response was decreased by ~50%. The caffeine-induced [Ca²⁺]_i response was further reduced in Ca²⁺-free external solutions containing CPA, suggesting that Ca²⁺ influx contributes to the response or that removal of external Ca²⁺ affects the state of the internal Ca²⁺ stores and thereby reduces the amount of Ca²⁺ available for release by caffeine. The [Ca²⁺]_i response to caffeine in the absence of CPA and external Ca²⁺ was not significantly different from that in the presence of external Ca²⁺. Taken together, these results suggest that loss of Ca²⁺ from internal stores in the absence of external Ca²⁺ is balanced, in part, by Ca²⁺ reuptake, which can be blocked by CPA (Fig. 4B).

In the majority of caffeine-induced Ca²⁺ responses (84%), the rate of recovery to initial [Ca²⁺]_i levels was sensitive to the presence of caffeine, whereby washout of caffeine increased the rate of [Ca²⁺]_i decline 2.7-fold. This effect was independent of the presence of CPA, suggesting that the increased rate of [Ca²⁺]_i decline is not due to an inhibitory effect of caffeine on reuptake of Ca²⁺ into internal stores. Elevated [Ca²⁺]_i or the state of the internal stores has been proposed to increase the rate of Ca²⁺-ATPase activity (25); however, this mechanism cannot explain the increased rate of [Ca²⁺]_i decline on washout of caffeine. If caffeine maintains an elevated [Ca²⁺]_i by preventing reuptake of Ca²⁺ into the internal stores, then, in the presence of SERCA inhibitors, washout of caffeine would have no effect on the rate at which [Ca²⁺]_i declines. Reuptake, however, is an important mechanism for lowering [Ca²⁺]_i to initial levels after agonist stimulation. In the presence of caffeine or after its washout, CPA increased the time constant of decay of the caffeine-induced [Ca²⁺]_i response four- to fivefold.

The shift of reversal potential of the caffeine-evoked current after replacement of external Na⁺ with K⁺ and the inhibition of caffeine-induced hyperpolarizations by TEA, an inhibitor of K⁺ channels (32), suggest that K⁺ contributes to the caffeine-induced outward current. However, the reversal potential (48 mV) of caffeine-induced currents is positive to E_K, and at holding potentials of no more than 60 mV caffeine evoked an inward current, indicating the activation of an ionic current with a charge carrier other than K⁺. In lowered external Cl concentration, caffeine-induced outward current amplitudes were reduced and the reversal potential was shifted to more positive potentials, consistent with a decrease in the driving force for inward Cl movement. Caffeine-activated currents were also partially inhibited by external Zn²⁺, an inhibitor of Cl channels (11, 33). Taken together, these results indicate that Ca²⁺-activated Cl and K⁺ conductance increases underlie caffeine-induced currents, consistent with previous studies suggesting that agonist- or caffeine-induced release of intracellular Ca²⁺ activates Ca²⁺-dependent K⁺ currents (16, 33), Ca²⁺-dependent Cl currents (16, 28, 33), and/or nonselective cation currents (42).

In conclusion, in freshly dissociated endothelial cells, ACh and caffeine activate increases in [Ca²⁺]_i from intracellular Ca²⁺ stores, which, if distinct, must be interactive. Also in these cells, ACh and caffeine activate outward currents, which transiently cause membrane hyperpolarization. The caffeine-induced hyperpolarization is due to a dose-dependent increase in [Ca²⁺]_i and the activation of Ca²⁺-sensitive K⁺ and Cl conductances; however, the peak current was not always observed to occur after the peak increase in [Ca²⁺]_i measured by fura 2 fluorescence. The lack of a clear temporal relationship between agonist-induced changes in membrane current and increases in [Ca²⁺]_i reflects the inability of single-cell photometry to resolve local agonist-induced [Ca²⁺]_i increases measured electrophysiologically. This conclusion, from results obtained in rabbit arterial endothelial cells, is consistent with recently reported results in bovine coronary arterial smooth muscle (38) and porcine coronary endothelial cells (14).