Autocrine action and its underlying mechanism of nitric oxide on intracellular Ca2+ homeostasis in vascular endothelial cells.

The rise in cytosolic Ca(2+) concentration (Ca(2+)(i)) in vascular endothelial cells (ECs) activates the production and release of nitric oxide (NO). NO modifies Ca(2+)(i) homeostasis in many types of nonendothelial cells. However, its effect on endothelial Ca(2+)(i) homeostasis at basal and excited states remains unclear. In the present study, to elucidate the effect of NO on basal Ca(2+)(i), inositol 1,4,5-trisphosphate-induced Ca(2+)(i) release (IICR) was blocked by expressing an antisense against type-1 inositol 1,4,5-trisphosphate receptors or by microinjecting heparin to individual ECs, and the effects of NO that was released by and diffused from adjacent IICR-intact ECs were recorded. After ATP or bradykinin stimulation, IICR-inhibited ECs showed a marked reduction of basal Ca(2+)(i), which was abolished by N(G)-monomethyl-l-arginine monoacetate pretreatment. The reduction disappeared in sparsely seeded ECs. Exogenous NO gas mimicked the effect of ATP or bradykinin to reduce basal Ca(2+)(i). Blocking plasma membrane Ca(2+)-ATPase (PMCA), but not Na(+)-Ca(2+) exchange or sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, suppressed the reduction, indicating that the reduction resulted from a NO-dependent potentiation of PMCA. To elucidate the effect of NO on elevated Ca(2+)(i), ATP-, bradykinin-, or thapsigargin-evoked Ca(2+)(i) response in the presence and absence of NO production was compared in adjacent IICR-intact ECs. NO was found to potentiate PMCA, which, in turn, greatly attenuated agonist-evoked Ca(2+)(i) elevation. NO also potentiated Ca(2+) influx, which markedly increased the sustained phase of Ca(2+)(i) elevation and possibly NO production. NO did not affect other Ca(2+)(i)-elevating and Ca(2+)(i)-sequestrating components. Thus, NO-dependent potentiation of PMCA is crucial for Ca(2+)(i) homeostasis over a wide Ca(2+)(i) range.


Vascular endothelial cells (ECs)
play an important role in the regulation of blood pressure and local blood flow. ECs respond to physical stimuli and blood-borne chemical signals with the production and release of vasoactive substances that directly affect the tone of vascular smooth muscle cells (VSMCs) (1)(2)(3)(4). Cytosolic free Ca 2ϩ concentration (Ca 2ϩ i ) in ECs plays a crucial role in these processes. For example, Ca 2ϩ i elevation activates endothelial nitric-oxide synthase to produce nitric oxide (NO) (5,6), which is the most potent substance to decrease VSMC tone and proliferation. In ECs, a typical Ca 2ϩ i elevation evoked by G-protein-coupled receptor agonists consists of an initial spike and a subsequent sustained phase (7)(8)(9). The initial spike mainly originates from inositol 1,4,5-trisphosphate (IP 3 )-induced Ca 2ϩ release (IICR) from the endoplasmic reticulum, and the following sustained phase results from capacitative Ca 2ϩ entry (CCE) across into the plasma membrane (10,11). In addition to these Ca 2ϩ i -elevating parts, Ca 2ϩ i is also regulated by Ca 2ϩ i -sequestrating components, including Ca 2ϩ i uptake by sarcoplasmic/endoplasmic reticulum Ca 2ϩ -ATPase (SERCA) (12,13), Ca 2ϩ i extrusion by plasma membrane Ca 2ϩ ATPase (PMCA) (14 -17), and Na ϩ -Ca 2ϩ exchange (NCX) (16 -18). Our previous studies have shown that IICR is essential for the induction of CCE in ECs and that CCE plays an important role in the continuous production of NO (7,8). On the other hand, in a series of nonendothelial cell types, NO has been reported to modify almost all Ca 2ϩ i -elevating and Ca 2ϩ i -sequestrating components (19,20). NO attenuates IICR at several steps, by inhibiting G-protein in VSMCs (21) and platelets (22), by inhibiting phospholipase ␤ in PC12 cells (23), and by inhibiting IP 3 receptors in VSMCs (24). The effects of NO on CCE vary among cell types (19,20,(25)(26)(27)(28)(29)(30); NO potentiates CCE in pancreatic acinar cells (25)(26)(27) and colonic epithelial cells (28), does not affect CCE in Jurkat T-lymphocytes (29) and embryonic kidney cells (30), and inhibits CCE in platelets (31) and VSMCs (32). Regarding the effects on Ca 2ϩ i sequestration, NO potentiates PMCA (33,34), NCX (35)(36)(37), and SERCA (32) in VSMCs, platelets, and astrocytes. However, in ECs, the effect of NO on Ca 2ϩ i homeostasis including Ca 2ϩ i -elevating and Ca 2ϩ i -sequestrating components remains unclear. On the other hand, most of the published results so far have been obtained by using exogenous NO. The effects on Ca 2ϩ i homeostasis varied among NO donor, NO gas, and endogenous NO, even in the same cell type (19,20,34,38,39). In the present study, endogenous NO released from bovine aortic ECs was used to clarify its effects on Ca 2ϩ i homeostasis by a twostep protocol. First, to observe the effect of NO on basal Ca 2ϩ i , IICR response was inhibited in individual ECs by expressing antisense against type 1 IP 3 receptor (IP 3 R 1 ) or by microinjecting heparin. Then the effect of NO that was released by and diffused from adjacent cells on basal Ca 2ϩ i of IICR-inhibited ECs was recorded after ATP or bradykinin (BK) stimulation. As a result, NO caused a marked reduction in basal Ca 2ϩ i . The reduction was a result of a NO-dependent potentiation of Ca 2ϩ i extrusion by PMCA, indicating that this action is crucial for Ca 2ϩ i homeostasis at the resting state. Second, to observe the effect of NO on Ca 2ϩ i dynamics, ATP-or BK-evoked Ca 2ϩ i response in the presence and absence of NO production was compared in adjacent IICR-intact cells. It was found that endogenous NO potentiated PMCA and CCE but did not affect IP 3 production, NCX, and SERCA. The potentiation of PMCA greatly attenuated agonist-evoked Ca 2ϩ i elevation, while the potentiation of CCE markedly increased the sustained phase of Ca 2ϩ i elevation and possibly NO production.

MATERIALS AND METHODS
Culture of ECs-ECs were enzymatically isolated from bovine aorta, cultured, and identified as described previously (8). To avoid fluctuation of results due to differences in cell number and growth cycles, cells that reached confluence were subjected to starvation of fetal bovine serum (1%) for 24 h before experiments. Cells of 4 -12 passages were used in this study.
Preparation of Antibodies to Isoforms of IP 3 Receptor and Immunoblotting-Three peptides were synthesized according to the amino acid sequence of the cytosolic C-terminal domain of human type 1 (GHP-PHMNVNPQQ(C)), type 2 ((C)LGSNTPHVNHHMPPH), and type 3 ((C)RQRLGFVDVQNCISR) IP 3 receptors. All of them were synthesized with an additional cysteine at their N-or C-terminal to facilitate coupling reaction. The sequence for IP 3 R 1 is preserved in most species including mouse and Xenopus (40). The sequences of the corresponding domain in rats are identical except for substitutions of the underlined residues. Antibodies to each of the peptides were purified from rabbit serum by affinity chromatography, and specificities were determined by immunoblotting. After solubilizing the crude homogenate of cultured ECs, SDS-polyacrylamide gel electrophoresis was performed in 6% gel. Samples were then electrotransferred to nitrocellulose membranes using a semidry blotter. Membranes were incubated with antibodies (ϫ 200) against type 1, 2, or 3 IP 3 receptor. Subsequently, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Antigens recognized by antibodies were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech).
Partial Cloning of Bovine IP 3 R 1 Gene, Construction of Vector, and Transient Transfection of ECs-Total RNA from cultured bovine aortic ECs was used as a template to generate first stand cDNA using an oligo(dT) 18 primer. A two-strand DNA fragment of the bovine IP 3 R 1 gene was amplified by polymerase chain reaction from cDNA using primers (upper, 5Ј-catcctaacggaacgagctc-3Ј; lower, 5Ј-catagcttaaagaggcagtct-3Ј). The single fragment obtained was inserted into the pCR TM II TA cloning vector (Invitrogen) and sequenced (Hitachi-5500). The fragment spanned 379 bases (Ϫ197 to ϩ182) shared 94% homology with human and 93% homology with mouse in the corresponding domain of the IP 3 R 1 gene. The fragment was then subcloned into pEGFPC1 (CLONTECH), and antisense (pG.IP 3 R 1 -AS) or sense (pG.IP 3 R 1 -S) orientation of the insert was confirmed by sequencing. To reduce the intercellular distance between transfectants and adjacent untransfected cells and to decrease the transfection rate, we used 100% confluent ECs. Transfection was done in a serum-free medium using a mixture of 1.33 g of pG.IP 3 R 1 -AS and 3.3 l of Lipofectin (Life Technologies, Inc.) per 1.2-mm diameter dish. This method resulted in a low transfection rate of 4 -6%. pG.IP 3 R 1 -S was used as the control. 12-24 h after transfection, the medium was changed to Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Cells were used for experiments 24 h after the medium was changed.
Buffer Solutions-HEPES buffer solution, which contained 145 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 0.5 mM MgCl 2 , 10 mM D-glucose, and 10 mM Hepes (pH 7.4), was utilized as the extracellular medium. The Ca 2ϩ -free medium consisted of HEPES buffer solution in which CaCl 2 was replaced with EGTA (1 mM). To block PMCA, we employed a buffer consisting of 115 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 20 mM MgCl 2 , 10 mM D-glucose, and 10 mM Hepes (pH 8.8) (33). For blockade of NCX, NaCl in the HEPES buffer was totally replaced with 145 mM choline chloride. For blockade of both PMCA and NCX, La 3ϩ (125 M) was added to the Na ϩ -free HEPES buffer.
Cell Microinjection-After loading fura-2/AM (4 M, Molecular Probes, Inc., Eugene, OR) for 40 min, ECs were rinsed and incubated in the Ca 2ϩ -free medium. Heparin (M r ϭ 5000; Wako) dissolved in the vehicle (48 mM K 2 HPO 4 , 14 mM Na 2 HPO 4 , 4.5 mM KH 2 PO 4 , and 400 M unesterified fura-2, pH 6.9) was injected into the perinuclear cytoplasm (Eppendorf 5171 micromanipulator and 5246 transjector, Hamburg). The injection volume into a cell was controlled by varying heparin concentration and injecting pressure (5-80 mmH 2 O). Within 5 min after the injection, the medium was changed to HEPES buffer solution containing 1 mM Ca 2ϩ and incubated further for 10 min to stabilize the injected cells.
Measurement of Ca 2ϩ i and Mn 2ϩ influx-ECs were stimulated by ATP, BK, ionomycin (IM), or thapsigargin (TG). All of the four agonists were obtained from Sigma. Ca 2ϩ i responses of transfected and untransfected cells as well as heparin-injected and -uninjected cells in the same observation field were individually analyzed by a two-dimensional Ca 2ϩ i imaging system, as reported previously (8,41). The absolute Ca 2ϩ i was calculated by comparing the fluorescence ratios at both wavelengths obtained at maximum Ca 2ϩ i (achieved by lysing the cells and saturating fura-2 with Ca 2ϩ ) and minimum Ca 2ϩ i (achieved by chelating all free Ca 2ϩ with EGTA) using the following equation: Ca 2ϩ . K d is the dissociation constant (224 nM for fura-2), and R min and R max are the F 340 /F 380 ratios of the Ca 2ϩ -free and Ca 2ϩ -bound forms. Sf2/Sb2 is the ratio of the fluorescence values at 380-nm excitation determined at R min and R max , respectively. Mn 2ϩ (0.5 mM)-induced quenching of fura-2 fluorescence was recorded at the excitation wavelength of 360 nm (F 360 ) for measurement of the cation influx rate. The percentage Mn 2ϩ quenching was obtained from the dynamic F 360 divided by the basal F 360 .
Immunocytostaining-ECs grown on CELLocate coverslips (Eppendorf) were rinsed with phosphate-buffered saline (pH 7.4) (8) and then fixed in the same buffer plus 3.6% paraformaldehyde. All subsequent steps were performed in Ca 2ϩ /Mg 2ϩ -free phosphate-buffered saline; cells were washed once for 2 min, permeabilized with 0.5% Triton X-100 for 10 min, and washed three times (5 min/wash). Endogenous peroxidase activity was blocked by MetOH containing 0.3% H 2 O 2 for 10 min. Nonspecific staining was reduced by incubating with 2% skim milk for 15 min before cells were subjected to the antibody against IP 3 R 1 (ϫ 200) in the presence of 10% goat serum for 1 h at room temperature. Then cells were incubated with biotinylated goat anti-rabbit IgG, and peroxidase labeling was carried out with an avidin-biotin complex kit (Vector Laboratories) and visualized by 0.05% 3,3Ј-diaminobenzidine with 0.01% H 2 O 2 (42). NO 2 Ϫ Determination and Preparation of NO Gas Solution-ECs were rinsed, and 0.2 ml of each medium was sampled before and 3 min after the administration of BK (500 nM) or 8 min after the addition of TG (1 M). The amount of NO 2 Ϫ produced was assayed as NO 2 Ϫ , which was determined by colorimetry after the Griess reaction, as reported previously (8,43). NO gas solution was prepared according to the method described by Shikano (44). In brief, 50 ml of 50 mM Tris-HCl, pH 7.4, was placed in a rubber-stoppered glass tube (300 ml). The tube was evacuated under vacuum for 20 min at 25°C and then flushed with O 2 -free N 2 for 20 min on ice; NO gas (99.99%) was flushed through the tube to saturate the atmosphere, and then the tube was vortexed and placed on ice. After 20 min, atmospheric NO was removed by flushing with N 2 for 20 min. Saturated NO in buffer (about 3.3 mM) was diluted into degassed buffer and was freshly prepared immediately before use.
Measurement of IP 3 Production-ECs were incubated for 1 h in serum-free HEPES buffer solution and then stimulated with 10 M ATP or 500 nM BK for 15 and 120 s. Then the medium was removed, and an equal volume of ice-cold 15% (v/v) trichloroacetic acid was added. The lysate was collected and centrifuged for 15 min at 2000 ϫ g at 4°C. The supernatants were extracted three times with 10 volumes of watersaturated diethyl ether and neutralized to pH 7.5 with NaHCO 3 . The remaining IP 3 was quantified according to the protocol of the IP 3 [ 3 H]assay system (Amersham Pharmacia Biotech).

Effects of NO on Basal Ca 2ϩ
i in IICR-inhibited Transfectants-Immunoblotting revealed that the antibody against IP 3 R 1 reacted with a single 260-kDa band in the crude homogenate of cultured bovine aortic ECs (Fig. 1A). In contrast,

Endogenous Nitric Oxide and Ca 2ϩ Signal in Endothelial Cells
neither type 2 nor type 3 IP 3 receptor was detected. A pharmacological study of Ca 2ϩ i dynamics revealed that ECs responded weakly (⌬Ca 2ϩ i rise ϭ 11 nM) to caffeine (3-60 mM), with or without ryanodine pretreatment (10 -60 M; data not shown). These results suggest that IICR mediated by IP 3 R 1 , but not Ca 2ϩ -induced Ca 2ϩ i release, could be the major mechanism involved in Ca 2ϩ i release in ECs. Of the 4 -6% of cells that were successfully transfected with pG.IP 3 R 1 -AS, 16% showed reduced IP 3 R 1 expression (data not shown), and 12% showed almost complete inhibition of IP 3 R 1 expression by immunocytostaining (Fig. 1B). On the other hand, the surrounding untransfected cells and cells transfected with pG.IP 3 R 1 -S demonstrated normal staining, indicating that the reduction or complete inhibition of IP 3 R 1 expression was a specific effect of pG.IP 3 R 1 -AS (Fig. 1, B and C).
Individual transfectants surrounded by untransfected cells were selected to measure Ca 2ϩ i handling. Of all of the pG.IP 3 R 1 -AS transfectants, about 15 and about 12% demonstrated attenuated and complete inhibition of Ca 2ϩ i response to ATP or BK, respectively. The incompletely (data not shown) or completely (Fig. 1B) inhibited Ca 2ϩ i response is consistent with immunocytostaining results. Ca 2ϩ i dynamics in cells with incomplete IICR inhibition exhibited a delayed and shortened initial Ca 2ϩ i spike, with subsequent Ca 2ϩ i reduction below the basal level after ATP (⌬Ca 2ϩ i ϭ 74 Ϯ 5 nM, Fig

complete IICR inhibition demonstrated not only no initial Ca 2ϩ
i spike but also marked Ca 2ϩ i reduction immediately after ATP (⌬Ca 2ϩ i ϭ 84 Ϯ 5 nM; Figs. 1B and 2C) or BK (⌬Ca 2ϩ i ϭ 73 Ϯ 4 nM; Fig. 1B) stimulation. After a wash-out of the first ATP solution followed by a 30-min equilibration period, these Ca 2ϩ i reductions were reproducible by secondary ATP stimulation (Fig. 2, A and C). On the other hand, in pG.IP 3 R 1 -S transfectants, ATP and BK induced a normal Ca 2ϩ i response (Fig. 1C).
Our previous study has shown that NO is produced upon stimulation with ATP or BK, and ATP-induced NO production is reproducible and can be blocked by L-NMMA in bovine aortic ECs (8). Therefore, Ca 2ϩ i reduction with or without NO production was compared in the same cell. Cells that exhibited marked Ca 2ϩ i reduction after the first ATP stimulation were pretreated with L-NMMA (1 mM; Calbiochem) for 30 min. This procedure dramatically attenuated Ca 2ϩ i reduction (⌬Ca 2ϩ i ϭ 16 Ϯ 4 nM) at the second ATP stimulation (Figs. 2, B and D), suggesting that Ca 2ϩ i reduction was caused by endogenous NO. However, Ca 2ϩ i reduction was unchanged by pretreatment with indomethacin (25 M) for 30 min (data not shown).
Effects of NO on Basal Ca 2ϩ i in IICR-inhibited ECs by Microinjection of Heparin-Since IICR inhibition induced by pG.IP 3 R 1 -AS is a relatively slow process, the effects of NO were further investigated in ECs where IICR was directly inhibited by heparin. As shown in Fig. 3, cell 1, injected with 50 mg/ml of heparin at 10 mm H 2 O, exhibited an incomplete inhibition of the initial Ca 2ϩ i spike, with a subsequent Ca 2ϩ i reduction below the basal level in response to ATP stimulation. Cell 2 and cell 3, injected with 200 mg/ml of heparin at 20 mm H 2 O, exhibited a complete inhibition of the initial Ca 2ϩ i spike and, moreover, marked Ca 2ϩ i reduction (⌬Ca 2ϩ i ϭ 103 Ϯ 4 nM), similar to that occurring in pG.IP 3 R 1 -AS transfectants. The reduction of Ca 2ϩ i in cells with complete IICR inhibition was reproducible in the second ATP stimulation (⌬Ca 2ϩ i ϭ 102 Ϯ 4 nM, Fig. 3, B and D). BK also induced Ca 2ϩ i reduction in IICR-inhibited cells (⌬Ca 2ϩ i ϭ 98 Ϯ 4 nM). As shown in Fig. 4A, microinjection of vehicle or heparin (200 mg/ml) had no effect on basal Ca 2ϩ i during a 60-min follow up period. Ca 2ϩ i reduction was induced only by the application of an agonist, and it was unlikely that the nonspecific effects of heparin were involved in the process. Cells that exhibited complete IICR inhibition after the first ATP stimulation were pretreated with L-NMMA for 30 min. This procedure completely abolished the marked Ca 2ϩ i reduction and led to nearly no Ca 2ϩ i rise at the second ATP stimulation (⌬Ca 2ϩ i ϭ 2 Ϯ 4 nM; Fig. 4B). When heparin was injected into ECs that were plated at a low density, no Ca 2ϩ i reduction was observed after ATP (Fig. 4C) stimulation. Furthermore, in sparsely seeded ECs, treatment with NO gas solution (3 M) induced Ca 2ϩ i reduction in cells injected with heparin (⌬Ca 2ϩ i ϭ 38 Ϯ 7 nM) as well as the adjacent IICR-intact cells (⌬Ca 2ϩ i ϭ 19 Ϯ 5 nM; Fig. 4D). These findings demonstrated that the reduction of basal Ca 2ϩ i caused by NO also occurred in heparin-injected cells.
Changes of Ca 2ϩ Influx, Internal Ca 2ϩ i Stores, and Ca 2ϩ i Extrusion in IICR-inhibited ECs-The reduction of basal Ca 2ϩ i was reversible naturally (Fig. 5, A, a). Mn 2ϩ quenching showed that ATP-induced (Fig. 5A, c) or BK-induced (data not shown) Ca 2ϩ influx was almost absent during the reduction. After removal of the agonist, the reduction was restored relatively rapidly ( Fig. 5A, b), during which Ca 2ϩ influx increased mildly (Fig. 5A, c). Next, the Ca 2ϩ i sequestration pathway through which NO reduced basal Ca 2ϩ i was examined. First, we investigated whether the reduction of Ca 2ϩ i was a result of a NO-dependent potentiation of SERCA. In the absence of Ca 2ϩ , ATP-or BKinduced Ca 2ϩ i release is caused by IICR, and further Ca 2ϩ i release induced by IM represents the residual content of internal Ca 2ϩ i stores. In pG.IP 3 R 1 -AS transfectants and cells injected with heparin, no significant increase in IM-releasable stores was found in between cells that showed marked Ca 2ϩ i reduction after ATP stimulation and cells that were directly stimulated by IM (Fig. 5B). Cells used in the study of direct IM stimulation demonstrated an ATP-induced Ca 2ϩ i reduction 30 min prior to IM addition. Furthermore, at 20 s after TG addition, ATP (Fig. 5C, a) or BK (data not shown) still induced a reduction of basal Ca 2ϩ i . The reduction, however, is attenuated compared with the case without TG. In IICR-inhibited cells, TG induced Ca 2ϩ i elevation in the Ca 2ϩ -free medium (data not shown). TG-ATP-induced (Fig. 5C, b) or TG-induced (Fig. 5C, c) Mn 2ϩ quenching increased, compared with ATP-induced quenching (Fig. 5A, c). Thus, the attenuation of Ca 2ϩ i reduction might be caused by the counteraction of the reduction by TGinduced Ca 2ϩ leakage from ER and TG-induced Ca 2ϩ influx. These results proved that Ca 2ϩ i reduction in IICR-inhibited cells was not caused by SERCA stimulation.
Second, we investigated whether the reduction of basal Ca 2ϩ  Fig. 6B). Blockade of both PMCA and NCX by 125 M La 3ϩ and 0 mM Na ϩ also eliminated the reduction (⌬Ca 2ϩ i ϭ 82 Ϯ 4 nM; 11 Ϯ 6 nM; Fig. 6C). To rule out the possibility that these blockades might decrease NO production in adjacent IICR-intact cells, NO 2 Ϫ production was assayed. Even with the blockade of PMCA and/or NCX, NO was produced during BK stimulation (Fig. 6D). Thus, Ca 2ϩ i reduction in IICR-inhibited cells was caused by the NO-dependent potentiation of Ca 2ϩ i extrusion by PMCA.
Effects of NO on Agonist-evoked Ca 2ϩ i Elevation in Adjacent IICR-intact ECs-The effects of NO on agonist-evoked Ca 2ϩ i elevation were investigated by comparing Ca 2ϩ i response in the presence and absence of NO production. Inhibition of NO production by L-NMMA significantly potentiated the initial spike in the presence and absence of 1 mM Ca 2ϩ (Figs. 7, A and B, and  8, A and B, and Table I). Our previous study has proven that NO is produced in the Ca 2ϩ -free medium after ATP or BK stimulation, although the amount produced is less than that produced in the presence of 1 mM Ca 2ϩ (8).
First, the mechanism of NO-dependent attenuation of the initial Ca 2ϩ i spike was investigated. The effect of NO on ATPor BK-induced IP 3 production was measured by radioimmunoassay. The basal IP 3 concentration was 0.7 Ϯ 0.1 M in ECs. In the Ca 2ϩ -free medium, ATP-and BK-induced IP 3 concentrations were 2.8 Ϯ 0.3 and 2.9 Ϯ 0.3 at 15 s and 2.7 Ϯ 0.4 and 2.9 Ϯ 0.4 M at 120 s after stimulation, respectively (n ϭ 4). After L-NMMA pretreatment, ATP-and BK-induced IP 3 con-centrations were 2.9 Ϯ 0.4 and 3.1 Ϯ 0.3 at 15 s and 2.8 Ϯ 0.4 and 3.0 Ϯ 0.7 M at 120 s after stimulation, respectively (n ϭ 4). Therefore, IP 3 concentration was not significantly changed in cells with and without pretreatment with L-NMMA after both ATP and BK stimulus. This indicates that NO did not inhibit IP 3 production in ECs.
Even after the initial Ca 2ϩ i spikes were attenuated by NO in the Ca 2ϩ -free medium, IM-induced residual Ca 2ϩ release was not significantly changed (Fig. 8, Table I). Furthermore, TG (1 M) was used at 20 s before treatment with ATP or BK in the Ca 2ϩ -free medium. Under the inhibition of SERCA, L-NMMA pretreatment still potentiated the agonist-evoked initial Ca 2ϩ i spike (Fig. 9A, Table I), suggesting that NO did not potentiate SERCA.
The following studies were addressed to investigate the NOdependent potentiation of Ca 2ϩ i extrusion. In the presence of 1 mM Ca 2ϩ , the effect of NO on BK-induced Ca 2ϩ dynamics was observed after the blockade of PMCA or NCX, while the SERCA mechanism remained operational. Blockade of PMCA by 20 mM Mg 2ϩ (pH 8.8) significantly raised BK-induced Ca 2ϩ i elevation, decreased Ca 2ϩ influx, and slightly attenuated NO production ( Figs. 6D and 7, B and F, and Table I). Under the blockade of PMCA, L-NMMA pretreatment did not markedly influence these effects (Fig. 7, B and F, and Table I). In contrast, blockade of NCX by 0 mM Na ϩ demonstrated no prominent effect on the initial spike, the sustained phase, and the Ca 2ϩ influx (Figs. 7,  C and G). Under the blockade of NCX, L-NMMA pretreatment mildly potentiated Ca 2ϩ i elevation. Blockade of both PMCA and NCX by 0 mM Na ϩ and 125 M La 3ϩ raised BK-induced Ca 2ϩ i elevation, decreased Ca 2ϩ influx, and attenuated NO production (Fig. 7, D and H, Table I). Under the blockade of both PMCA and NCX, L-NMMA pretreatment did not markedly influence these effects. These results indicated that the NO-de- i spike (Fig. 9A, Table I). Under the blockade of PMCA, further inhibition of NO production did not change the potentiation of the BK-induced Ca 2ϩ i spike. On the other hand, blockade of NCX showed no significant effect on the BK-induced Ca 2ϩ i spike (Fig. 9B, Table I). Under the blockade of NCX by 0 mM Na ϩ , further inhibition of NO production mildly potentiated the BK-induced Ca 2ϩ i spike. Blockade of both PMCA and NCX by 0 mM Na ϩ and 125 M La 3ϩ markedly raised the Ca 2ϩ i spike (Fig. 9C). Under the blockade of both PMCA and NCX, L-NMMA pretreatment did not markedly influence these effects. Similar results were obtained by using TG only, to inhibit SERCA and to stimulate Ca 2ϩ i rise and NO production in the Ca 2ϩ -free medium (Fig. 9, D-F). NO was produced during TG stimulation even in the presence of PMCA and/or NCX blockade (Fig. 10). These results strongly indicate that the attenuation in the initial Ca 2ϩ i spike is caused by the NO-dependent potentiation of PMCA, which plays an important role in attenuating Ca 2ϩ i signal in the excited state. Second, in contrast to attenuating the initial Ca 2ϩ i spike, NO significantly increased the sustained Ca 2ϩ i phase (Fig. 7, A   results from NO that was released by and diffused from adjacent IICR-intact ECs. 3) NO reduces basal Ca 2ϩ i by potentiation of Ca 2ϩ i extrusion by PMCA. 4) This potentiation also markedly attenuates agonist-evoked Ca 2ϩ i elevation in adjacent IICR-intact cells. 5) Endogenous NO markedly promotes agonist-evoked CCE, which maintains Ca 2ϩ i elevation and possibly NO production in ECs.
Crucial Role of NO-dependent Potentiation of PMCA in Basal Ca 2ϩ i Homeostasis-NO donor or NO gas has been widely used to mimic the effect of endogenous NO (19,20). However, their effect on basal Ca 2ϩ i has been controversial because of conflicting results. In platelets, Johansson et al. (34)   i elevation in all ECs and consequently led to a decrease in NO production. The effect of NO may only be analyzed in individual ECs in which agonist-evoked Ca 2ϩ i elevation is inhibited. In the present study, by using individual ECs in which agonist-evoked Ca 2ϩ i elevation was inhibited by the antisense or heparin and by using adjacent excited ECs as a source of NO production, the potent effect of endogenous NO on reducing basal Ca 2ϩ i was successfully elucidated. The reduction of basal Ca 2ϩ i by endogenous NO has been substantiated, including the partial mimicry by exogenous NO gas. Our studies suggested that endogenous NO is the most ideal source for exploring the physiological effect of NO. NO donor or even NO gas does not always act in the same way as endogenous NO because of difficulty in maintaining the narrow range of effective concentration and mimicking the diffusion pattern of NO in vivo.
The mechanism involved in the reduction of basal Ca 2ϩ i was clarified. Our results excluded the involvement of NO-dependent potentiation of SERCA, since there was no consequent increase in IM-releasable Ca 2ϩ i stores after the reduction of basal Ca 2ϩ i (Fig. 5B), and ATP or BK still induced Ca 2ϩ i reduction after SERCA was blocked (Fig. 5C). After blocking SERCA, the attenuation of Ca 2ϩ i reduction could be explained by the counteraction by TG-induced Ca 2ϩ leakage from internal stores and TG-induced CCE. The remaining hypothesis is that NO potentiates Ca 2ϩ i extrusion by PMCA or NCX. Recently, there is increasing evidence that PMCA is important for Ca 2ϩ i extrusion, while NCX plays a minor role (14 -18). Thus, the effects of blocking PMCA or NCX on Ca 2ϩ i reduction in IICR-inhibited cells were investigated. It was reported that extracellular increase in pH to 8.8 blocks the activity of PMCA in squid axons and red cells, does not alter the activity of NCX and SERCA, and minimally changes intracellular pH (33,35,48,49). Increasing extracellular Mg 2ϩ concentration to 20 -30 mM can also block the activity of PMCA in red cells and VSMCs (33,35,49). The combination of 20 mM Mg 2ϩ and pH 8.8 blocks PMCA-mediated Ca 2ϩ i efflux by up to 80% in VSMCs, whereas it did not affect NCX (33,35,50). That PMCA can be blocked by 20 mM Mg 2ϩ (pH 8.8) from the side opposite to the ATP-binding site of the membrane holds true not only for the case in VSMCs but is a general property of the membrane Ca 2ϩ pump ATPase (33, 35, 48 -51). Furthermore, extracellular La 3ϩ at concentrations of 60 -250 M specifically blocks PMCA but spares NCX in red cells and VSMCs (IC 50 ϭ 50 -65 M; Refs. 33 and 52). Low concentrations of La 3ϩ (20 -100 M) inhibit PMCA activity by displacing Mg 2ϩ from the site at which it combines to accelerate dephosphorylation (53). Therefore, these methods were used to block PMCA of ECs in the present study. On the other hand, NCX was blocked by a Na ϩ -free buffer. The extracellular Na ϩ -free buffer would induce changes in intracellular conditions, which, in turn, could affect the activity of PMCA indirectly. However, a previous study showed that the extracellular Na ϩ -free buffer caused a decrease in intracellular Na ϩ concentration by 30% in VSMCs (33). Such a decrease in Na ϩ concentration did not affect the activity of PMCA significantly, since intracellular Na ϩ and K ϩ substituted for each other in activating PMCA (54). Several studies have revealed the function of PMCA under the inhibition of NCX using the same method (32, 33, 35, 48 -51).
Consequently, Ca 2ϩ i reduction was suppressed after the blockade of PMCA but still occurred after the blockade of NCX in IICR-inhibited cells (Fig. 6). These blockades did not significantly affect BK-induced NO production by adjacent IICRintact cells. Thus, Ca 2ϩ i reduction results from the NO-dependent potentiation of Ca 2ϩ i extrusion by PMCA, which may be a major and potent regulator of basal Ca 2ϩ i homeostasis in ECs. Our results are consistent with previous reports for VSMCs and platelets. In VSMCs, Furukawa et al. (33) reported that SNP or 8-Br-cGMP markedly accelerated the Ca 2ϩ i extrusion by PMCA, especially at basal Ca 2ϩ i level. In platelets, Johansson et al. (34) reported that both SNP (10 M) and dibutyryl-cGMP (1 mM) significantly increased V m of PMCA without affecting its K m or Hill coefficient. SNP or dibutyryl-cGMP, however, did not change the rate of NCX that has a minor contribution to basal Ca 2ϩ i extrusion. As a result of potentiation of PMCA, SNP or dibutyryl-cGMP decreased the basal Ca 2ϩ i and attenuated the ionomycin-induced Ca 2ϩ i elevation. These effects resulted from a cGMP-induced phosphorylation of PMCA (34).
NO Potentiates PMCA as Well as CCE during Agonist Stimulation-NOdependent potentiation of PMCA is also important for Ca 2ϩ i extrusion during the BK-or TG-evoked Ca 2ϩ i elevation (Fig. 9). As indicated by its action on the basal Ca 2ϩ i (70 -150 nM), the TG-evoked Ca 2ϩ i elevation (200 -260 nM), and the ATP-or BK-evoked Ca 2ϩ i elevation (ϳ600 nM), NO-dependent potentiation of PMCA functions to extrude Ca 2ϩ i over a wide Ca 2ϩ i range. Therefore, the potentiation may be crucial for the normal function of ECs. As regards NCX, our results indicated that it also plays a minor role during agonist-evoked
The present study also showed the significant potentiation of CCE by NO. The lifetime of NO spans only about 6 s (57), suggesting that the potentiation was not due to the NO produced during initial Ca 2ϩ i spike but rather to the NO produced during the sustained Ca 2ϩ i phase. As shown in Fig. 7, the potentiation is necessary for the maintenance of Ca 2ϩ i elevation during the sustained phase, where IICR is decreased and PMCA still functions to decrease Ca 2ϩ i . Our group and others have reported the importance of CCE in NO production (7,8,58). The mechanism was explained by the ability of endothelial nitric-oxide synthase to be directly activated by Ca 2ϩ influx, based on immunostaining findings that endothelial nitric-oxide synthase is locally linked to caveolin beneath the plasma membrane (5). The potentiation of CCE by endogenous NO may contribute to the continuous NO production during the sustained phase. By summing up these results, we extrapolate that the Ca 2ϩ i elevation and continuous NO production during the sustained phase might be maintained by a positive feedback mechanism between NO production and Ca 2ϩ influx. The feedback mechanism explains clearly the continuous NO production during the sustained phase (7,8,58).
In conclusion, the present study revealed that endogenous NO modifies Ca 2ϩ i signal in ECs by potentiating PMCA and CCE. At the basal state and during the initial Ca 2ϩ i spike after agonist stimulation, NO potentiates PMCA, which contributes to keep the basal Ca 2ϩ i level low and buffer the rapid Ca 2ϩ i rise induced by agonists. During the sustained Ca 2ϩ i phase, apart from potentiating PMCA, NO mainly potentiates CCE, which keeps the Ca 2ϩ i level elevated and possibly enhances NO production.