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J. Biol. Chem., Vol. 275, Issue 37, 28739-28749, September 15, 2000

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Autocrine Action and Its Underlying Mechanism of Nitric Oxide on Intracellular Ca²⁺ Homeostasis in Vascular Endothelial Cells^*

Jie Chen, Yuepeng Wang, Yue Wang, Toshiaki Nakajima, Kuniaki Iwasawa, Hisako Hikiji^§, Mie Sunamoto^¶, Dong-Kug Choi^**, Yutaka Yoshida, Yoshiyuki Sakaki^**, and Teruhiko Toyo-oka

From the Second Department of Internal Medicine, the ^§ Department of Oral and Maxillofacial Surgery, and the ^** Human Genome Center, Institute of Medical Science, University of Tokyo, Tokyo 113-0033, Japan, the ^¶ Molecular Chemistry Laboratory, Takeda Chemical Industries, Osaka 532-0024, Japan, and the  Department of Pharmacology, Niigata University, Niigata 951-8122, Japan

Received for publication, February 3, 2000, and in revised form, June 6, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The rise in cytosolic Ca²⁺ concentration (Ca²⁺_i) in vascular endothelial cells (ECs) activates the production and release of nitric oxide (NO). NO modifies Ca²⁺_i homeostasis in many types of nonendothelial cells. However, its effect on endothelial Ca²⁺_i homeostasis at basal and excited states remains unclear. In the present study, to elucidate the effect of NO on basal Ca²⁺_i, inositol 1,4,5-trisphosphate-induced Ca²⁺_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 Ca²⁺_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 Ca²⁺_i. Blocking plasma membrane Ca²⁺-ATPase (PMCA), but not Na⁺-Ca²⁺ exchange or sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase, suppressed the reduction, indicating that the reduction resulted from a NO-dependent potentiation of PMCA. To elucidate the effect of NO on elevated Ca²⁺_i, ATP-, bradykinin-, or thapsigargin-evoked Ca²⁺_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 Ca²⁺_i elevation. NO also potentiated Ca²⁺ influx, which markedly increased the sustained phase of Ca²⁺_i elevation and possibly NO production. NO did not affect other Ca²⁺_i-elevating and Ca²⁺_i-sequestrating components. Thus, NO-dependent potentiation of PMCA is crucial for Ca²⁺_i homeostasis over a wide Ca²⁺_i range.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Vascular endothelial cells (ECs)¹ 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) (1-4). Cytosolic free Ca²⁺ concentration (Ca²⁺_i) in ECs plays a crucial role in these processes. For example, Ca²⁺_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 Ca²⁺_i elevation evoked by G-protein-coupled receptor agonists consists of an initial spike and a subsequent sustained phase (7-9). The initial spike mainly originates from inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release (IICR) from the endoplasmic reticulum, and the following sustained phase results from capacitative Ca²⁺ entry (CCE) across into the plasma membrane (10, 11). In addition to these Ca²⁺_i-elevating parts, Ca²⁺_i is also regulated by Ca²⁺_i-sequestrating components, including Ca²⁺_i uptake by sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) (12, 13), Ca²⁺_i extrusion by plasma membrane Ca²⁺ ATPase (PMCA) (14-17), and Na⁺-Ca²⁺ 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 Ca²⁺_i-elevating and Ca²⁺_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 IP₃ receptors in VSMCs (24). The effects of NO on CCE vary among cell types (19, 20, 25-30); NO potentiates CCE in pancreatic acinar cells (25-27) 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 Ca²⁺_i sequestration, NO potentiates PMCA (33, 34), NCX (35-37), and SERCA (32) in VSMCs, platelets, and astrocytes. However, in ECs, the effect of NO on Ca²⁺_i homeostasis including Ca²⁺_i-elevating and Ca²⁺_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 Ca²⁺_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 Ca²⁺_i homeostasis by a two-step protocol. First, to observe the effect of NO on basal Ca²⁺_i, IICR response was inhibited in individual ECs by expressing antisense against type 1 IP₃ receptor (IP₃R₁) or by microinjecting heparin. Then the effect of NO that was released by and diffused from adjacent cells on basal Ca²⁺_i of IICR-inhibited ECs was recorded after ATP or bradykinin (BK) stimulation. As a result, NO caused a marked reduction in basal Ca²⁺_i. The reduction was a result of a NO-dependent potentiation of Ca²⁺_i extrusion by PMCA, indicating that this action is crucial for Ca²⁺_i homeostasis at the resting state. Second, to observe the effect of NO on Ca²⁺_i dynamics, ATP- or BK-evoked Ca²⁺_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 IP₃ production, NCX, and SERCA. The potentiation of PMCA greatly attenuated agonist-evoked Ca²⁺_i elevation, while the potentiation of CCE markedly increased the sustained phase of Ca²⁺_i elevation and possibly NO production.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 IP₃ Receptor and Immunoblotting-- Three peptides were synthesized according to the amino acid sequence of the cytosolic C-terminal domain of human type 1 (GHPPHMNVNPQQ(C)), type 2 ((C)LGSNTPHVNHHMPPH), and type 3 ((C)RQRLGFVDVQNCISR) IP₃ receptors. All of them were synthesized with an additional cysteine at their N- or C-terminal to facilitate coupling reaction. The sequence for IP₃R₁ 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 (× 200) against type 1, 2, or 3 IP₃ 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 IP₃R₁ 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 IP₃R₁ gene was amplified by polymerase chain reaction from cDNA using primers (upper, 5'-catcctaacggaacgagctc-3'; lower, 5'-catagcttaaagaggcagtct-3'). The single fragment obtained was inserted into the pCR^TMII TA cloning vector (Invitrogen) and sequenced (Hitachi-5500). The fragment spanned 379 bases (197 to +182) shared 94% homology with human and 93% homology with mouse in the corresponding domain of the IP₃R₁ gene. The fragment was then subcloned into pEGFPC1 (CLONTECH), and antisense (pG.IP₃R₁-AS) or sense (pG.IP₃R₁-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 µg of pG.IP₃R₁-AS and 3.3 µl of Lipofectin (Life Technologies, Inc.) per 1.2-mm diameter dish. This method resulted in a low transfection rate of 4-6%. pG.IP₃R₁-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 CaCl₂, 0.5 mM MgCl₂, 10 mM D-glucose, and 10 mM Hepes (pH 7.4), was utilized as the extracellular medium. The Ca²⁺-free medium consisted of HEPES buffer solution in which CaCl₂ was replaced with EGTA (1 mM). To block PMCA, we employed a buffer consisting of 115 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 20 mM MgCl₂, 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, La³⁺ (125 µM) was added to the Na⁺-free HEPES buffer.

Cell Microinjection-- After loading fura-2/AM (4 µM, Molecular Probes, Inc., Eugene, OR) for 40 min, ECs were rinsed and incubated in the Ca²⁺-free medium. Heparin (M_r = 5000; Wako) dissolved in the vehicle (48 mM K₂HPO₄, 14 mM Na₂HPO₄, 4.5 mM KH₂PO₄, and 400 µM 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 mmH₂O). Within 5 min after the injection, the medium was changed to HEPES buffer solution containing 1 mM Ca²⁺ and incubated further for 10 min to stabilize the injected cells.

Measurement of Ca²⁺_i and Mn²⁺ influx-- ECs were stimulated by ATP, BK, ionomycin (IM), or thapsigargin (TG). All of the four agonists were obtained from Sigma. Ca²⁺_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 Ca²⁺_i imaging system, as reported previously (8, 41). The absolute Ca²⁺_i was calculated by comparing the fluorescence ratios at both wavelengths obtained at maximum Ca²⁺_i (achieved by lysing the cells and saturating fura-2 with Ca²⁺) and minimum Ca²⁺_i (achieved by chelating all free Ca²⁺ with EGTA) using the following equation: Ca²⁺_i = K_d × (R  R_min)/(R_max  R) × Sf2/Sb2. K_d is the dissociation constant (224 nM for fura-2), and R_min and R_max are the F₃₄₀/F₃₈₀ ratios of the Ca²⁺-free and Ca²⁺-bound forms. Sf2/Sb2 is the ratio of the fluorescence values at 380-nm excitation determined at R_min and R_max, respectively. Mn²⁺ (0.5 mM)-induced quenching of fura-2 fluorescence was recorded at the excitation wavelength of 360 nm (F₃₆₀) for measurement of the cation influx rate. The percentage Mn²⁺ quenching was obtained from the dynamic F₃₆₀ divided by the basal F₃₆₀.

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 Ca²⁺/Mg²⁺-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% H₂O₂ for 10 min. Nonspecific staining was reduced by incubating with 2% skim milk for 15 min before cells were subjected to the antibody against IP₃R₁ (× 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% 3,3'-diaminobenzidine with 0.01% H₂O₂ (42).

NO₂ 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 µM). The amount of NO₂ produced was assayed as NO₂, 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 O₂-free N₂ 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 N₂ 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 IP₃ Production-- ECs were incubated for 1 h in serum-free HEPES buffer solution and then stimulated with 10 µM 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 × g at 4 °C. The supernatants were extracted three times with 10 volumes of water-saturated diethyl ether and neutralized to pH 7.5 with NaHCO₃. The remaining IP₃ was quantified according to the protocol of the IP₃ [³H]assay system (Amersham Pharmacia Biotech).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effects of NO on Basal Ca²⁺_i in IICR-inhibited Transfectants-- Immunoblotting revealed that the antibody against IP₃R₁ reacted with a single 260-kDa band in the crude homogenate of cultured bovine aortic ECs (Fig. 1A). In contrast, neither type 2 nor type 3 IP₃ receptor was detected. A pharmacological study of Ca²⁺_i dynamics revealed that ECs responded weakly (Ca²⁺_i rise = 11 nM) to caffeine (3-60 mM), with or without ryanodine pretreatment (10-60 µM; data not shown). These results suggest that IICR mediated by IP₃R₁, but not Ca²⁺-induced Ca²⁺_i release, could be the major mechanism involved in Ca²⁺_i release in ECs.

View larger version (47K): [in this window] [in a new window]   Fig. 1.   Subtypes of IP3 receptor expression (A), IP3R1 expression, two-dimensional images of Ca2+i response, and Ca2+i dynamics induced by ATP or BK in transfectants of pG.IP3R1-AS (B) and pG.IP3R1-S (C). A, the crude homogenates of cultured bovine aortic ECs (lanes 1, 3, and 5), A7r5 cells (lanes 2 and 6), and Chinese hamster ovary cells (lane 4) were electrophoresed on 6% SDS-polyacrylamide gel. Each of the two strips was immunoblotted with specific antibodies against type 1 (lanes 1 and 2), 2 (lanes 3 and 4), and 3 (lanes 5 and 6) IP3 receptors. B and C, a-f show transfectants expressing reporter EGFP (a), fura-2-loaded cells (b), two-dimensional images of basal Ca2+i level (c), peak Ca2+i response induced by ATP (10 µM; d), and BK (500 nM; e) in the presence of 1 mM Ca2+ and the immunocytostaining of IP3R1 (f). After wash-out of ATP for 30 min, cells were stimulated by BK. g-h show Ca2+i dynamics in transfectants and adjacent untransfected cells in response to ATP (arrows; g) and BK (arrows; h). Bar, 50 µm.

Of the 4-6% of cells that were successfully transfected with pG.IP₃R₁-AS, 16% showed reduced IP₃R₁ expression (data not shown), and 12% showed almost complete inhibition of IP₃R₁ expression by immunocytostaining (Fig. 1B). On the other hand, the surrounding untransfected cells and cells transfected with pG.IP₃R₁-S demonstrated normal staining, indicating that the reduction or complete inhibition of IP₃R₁ expression was a specific effect of pG.IP₃R₁-AS (Fig. 1, B and C).

Individual transfectants surrounded by untransfected cells were selected to measure Ca²⁺_i handling. Of all of the pG.IP₃R₁-AS transfectants, about 15 and about 12% demonstrated attenuated and complete inhibition of Ca²⁺_i response to ATP or BK, respectively. The incompletely (data not shown) or completely (Fig. 1B) inhibited Ca²⁺_i response is consistent with immunocytostaining results. Ca²⁺_i dynamics in cells with incomplete IICR inhibition exhibited a delayed and shortened initial Ca²⁺_i spike, with subsequent Ca²⁺_i reduction below the basal level after ATP (Ca²⁺_i = 74 ± 5 nM, Fig. 2A) or BK (Ca²⁺_i = 70 ± 7 nM) stimulation. Ca²⁺_i dynamics in cells with complete IICR inhibition demonstrated not only no initial Ca²⁺_i spike but also marked Ca²⁺_i reduction immediately after ATP (Ca²⁺_i = 84 ± 5 nM; Figs. 1B and 2C) or BK (Ca²⁺_i = 73 ± 4 nM; Fig. 1B) stimulation. After a wash-out of the first ATP solution followed by a 30-min equilibration period, these Ca²⁺_i reductions were reproducible by secondary ATP stimulation (Fig. 2, A and C). On the other hand, in pG.IP₃R₁-S transfectants, ATP and BK induced a normal Ca²⁺_i response (Fig. 1C).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   ATP-induced Ca2+i dynamics in pG.IP3R1-AS transfectants in the presence of 1 mM Ca2+. Shown are ATP (10 µM)-induced Ca2+i dynamics in cells with incomplete (A and B) and complete (C and D) IICR inhibition with (B and D) and without (A and C) pretreatment with L-NMMA (1 mM) for 30 min between two ATP applications (1st and 2nd, arrows). L-NMMA was added to the medium throughout ATP stimulation (n = 5-6).

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, Ca²⁺_i reduction with or without NO production was compared in the same cell. Cells that exhibited marked Ca²⁺_i reduction after the first ATP stimulation were pretreated with L-NMMA (1 mM; Calbiochem) for 30 min. This procedure dramatically attenuated Ca²⁺_i reduction (Ca²⁺_i = 16 ± 4 nM) at the second ATP stimulation (Figs. 2, B and D), suggesting that Ca²⁺_i reduction was caused by endogenous NO. However, Ca²⁺_i reduction was unchanged by pretreatment with indomethacin (25 µM) for 30 min (data not shown).

Effects of NO on Basal Ca²⁺_i in IICR-inhibited ECs by Microinjection of Heparin-- Since IICR inhibition induced by pG.IP₃R₁-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 H₂O, exhibited an incomplete inhibition of the initial Ca²⁺_i spike, with a subsequent Ca²⁺_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 H₂O, exhibited a complete inhibition of the initial Ca²⁺_i spike and, moreover, marked Ca²⁺_i reduction (Ca²⁺_i = 103 ± 4 nM), similar to that occurring in pG.IP₃R₁-AS transfectants. The reduction of Ca²⁺_i in cells with complete IICR inhibition was reproducible in the second ATP stimulation (Ca²⁺_i = 102 ± 4 nM, Fig. 3, B and D). BK also induced Ca²⁺_i reduction in IICR-inhibited cells (Ca²⁺_i = 98 ± 4 nM). As shown in Fig. 4A, microinjection of vehicle or heparin (200 mg/ml) had no effect on basal Ca²⁺_i during a 60-min follow up period. Ca²⁺_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 Ca²⁺_i reduction and led to nearly no Ca²⁺_i rise at the second ATP stimulation (Ca²⁺_i = 2 ± 4 nM; Fig. 4B). When heparin was injected into ECs that were plated at a low density, no Ca²⁺_i reduction was observed after ATP (Fig. 4C) stimulation. Furthermore, in sparsely seeded ECs, treatment with NO gas solution (3 µM) induced Ca²⁺_i reduction in cells injected with heparin (Ca²⁺_i = 38 ± 7 nM) as well as the adjacent IICR-intact cells (Ca²⁺_i = 19 ± 5 nM; Fig. 4D). These findings demonstrated that the reduction of basal Ca²⁺_i caused by NO also occurred in heparin-injected cells.

View larger version (61K): [in this window] [in a new window]   Fig. 3.   Two-dimensional images of Ca2+i responses and Ca2+i dynamics induced by ATP in heparin-injected ECs in the presence of 1 mM Ca2+. A, cell position and time-serial two-dimensional images of Ca2+i responses to first ATP (1st ATP, 10 µM) stimulation. Cell 1 (1), injected with 50 mg/ml heparin, showed a delayed and attenuated Ca2+i rise. Cell 2 (2) and cell 3 (3), injected with 200 mg/ml heparin, showed marked Ca2+i reduction. Shown is the localization of ECs and Ca2+i responses at 10, 20, 35, and 90 s after ATP stimulation. B, fura-2-loaded cells and time-serial images of Ca2+i responses to second ATP (2nd ATP; 10 µM) stimulation. Ca2+i rise was partially recovered in cell 1 but remained suppressed in cell 2 and cell 3. Shown are images of fura-2-loaded cells and Ca2+i responses at 5, 10, 35, and 90 s after ATP stimulation. C and D, Ca2+i dynamics of cell 1 and cell 2 after ATP stimulation (arrow), respectively. Ca2+i dynamics of cell 3, which is similar to that of cell 2, is omitted (n = 4). Bar, 50 µm.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Basal Ca2+i level and ATP- and NO gas solution-induced Ca2+i response in confluent or sparse ECs injected with heparin (200 mg/ml). A, in confluent cells, after microinjection (arrow) of vehicle (a; n = 6) or heparin (b; n = 4), basal Ca2+i was monitored for 60 min and compared with that of adjacent uninjected cells (c; n = 16). B, in confluent cells, cell 1 (1), injected with heparin, showed marked Ca2+i reduction after ATP (10 µM) stimulation (b). Following a wash-out of ATP, cells were pretreated with L-NMMA (1 mM) for 30 min. Ca2+i reduction was abolished and recovered to the basal line after the second ATP stimulation (c). a-d exhibit two-dimensional images of fura-2-loaded cells (a), the Ca2+i rise at 25 s after the first (b) and second (c) ATP stimulation, and Ca2+i dynamics (d; n = 4). C, in sparsely seeded ECs, cell 2 (2), injected with heparin, showed no Ca2+i reduction after ATP stimulation. a-c exhibit two-dimensional images of fura-2-loaded cells (a), the Ca2+i rise at 25 s (b) after ATP stimulation, and Ca2+i dynamics (c; n = 2). D, in sparsely seeded ECs, cell 3 (3), injected with heparin, shows Ca2+i reduction after treatment with NO gas solution (3 µM). a-c exhibit two-dimensional images of fura-2-loaded cells (a), the peak reduction of basal Ca2+i at 10 s (b) after treatment with NO, and Ca2+i dynamics (c; n = 3). Bar, 100 µm.

Changes of Ca²⁺ Influx, Internal Ca²⁺_i Stores, and Ca²⁺_i Extrusion in IICR-inhibited ECs-- The reduction of basal Ca²⁺_i was reversible naturally (Fig. 5, A, a). Mn²⁺ quenching showed that ATP-induced (Fig. 5A, c) or BK-induced (data not shown) Ca²⁺ influx was almost absent during the reduction. After removal of the agonist, the reduction was restored relatively rapidly (Fig. 5A, b), during which Ca²⁺ influx increased mildly (Fig. 5A, c).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Recovery process of Ca2+i reduction (A), ionomycin-releasable Ca2+i stores (B), and Ca2+i reduction after inhibition of SERCA (C). A, in heparin-injected cells, ATP (10 µM)-evoked Ca2+i reduction shows a reversible process, naturally (a) or by ATP removal (w.o.) (b). Coinciding with a and b, c shows changes in Mn2+ (0.5 mM) quenching of IICR-inhibited cells (traces 1 and 2) and adjacent IICR-intact cells (trace 3) with (traces 2 and 3) and without (trace 1) the removal (arrows) of ATP. Note that after removal of ATP, fresh medium contains 0.5 mM Mn2+. B, heparin-injected cells show no difference in Ca2+i release between direct IM application (thin line) and IM application after ATP-induced Ca2+i reduction (thick line, a). In b, each column denotes the mean ± S.E. of Ca2+i reduction (black columns) and Ca2+i rise induced by direct IM application (open columns) and IM application after ATP-induced Ca2+i reduction (stippled columns) in both pG.IP3R1-AS transfectants and heparin-injected cells. C, the transfectants of pG.IP3R1-AS (a, thin line) with complete IICR inhibition and adjacent IICR-intact cells (thick line) were stimulated by ATP at 20 s after the addition of TG (1 µM). Note that there was still an ATP-evoked Ca2+i reduction. Coinciding with a, b denotes changes in Mn2+ quenching after TG-ATP stimulation for IICR-inhibited cells (thin line) and adjacent IICR-intact cells (thick line). c denotes changes in Mn2+ quenching after TG stimulation for IICR-inhibited cells (thin line) and adjacent IICR-intact cells (thick line). NS, no significance; n = 4-7.

Next, the Ca²⁺_i sequestration pathway through which NO reduced basal Ca²⁺_i was examined. First, we investigated whether the reduction of Ca²⁺_i was a result of a NO-dependent potentiation of SERCA. In the absence of Ca²⁺, ATP- or BK-induced Ca²⁺_i release is caused by IICR, and further Ca²⁺_i release induced by IM represents the residual content of internal Ca²⁺_i stores. In pG.IP₃R₁-AS transfectants and cells injected with heparin, no significant increase in IM-releasable stores was found in between cells that showed marked Ca²⁺_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 Ca²⁺_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 Ca²⁺_i. The reduction, however, is attenuated compared with the case without TG. In IICR-inhibited cells, TG induced Ca²⁺_i elevation in the Ca²⁺-free medium (data not shown). TG-ATP-induced (Fig. 5C, b) or TG-induced (Fig. 5C, c) Mn²⁺ quenching increased, compared with ATP-induced quenching (Fig. 5A, c). Thus, the attenuation of Ca²⁺_i reduction might be caused by the counteraction of the reduction by TG-induced Ca²⁺ leakage from ER and TG-induced Ca²⁺ influx. These results proved that Ca²⁺_i reduction in IICR-inhibited cells was not caused by SERCA stimulation.

Second, we investigated whether the reduction of basal Ca²⁺_i was a result of NO-dependent Ca²⁺_i extrusion by PMCA (Na⁺-independent Ca²⁺_i efflux) or NCX (Na⁺-dependent Ca²⁺_i efflux). Blockade of PMCA by 20 mM extracellular Mg²⁺ (pH 8.8; Fig. 6A) almost completely abolished the NO-induced Ca²⁺_i reduction (Ca²⁺_i = 82 ± 4 nM; 8 ± 5 nM) in IICR-inhibited cells. Blockade of NCX by replacing extracellular Na⁺ with choline did not affect the reduction (Ca²⁺_i = 82 ± 4 nM; 80 ± 6 nM; Fig. 6B). Blockade of both PMCA and NCX by 125 µM La³⁺ and 0 mM Na⁺ also eliminated the reduction (Ca²⁺_i = 82 ± 4 nM; 11 ± 6 nM; Fig. 6C). To rule out the possibility that these blockades might decrease NO production in adjacent IICR-intact cells, NO₂ production was assayed. Even with the blockade of PMCA and/or NCX, NO was produced during BK stimulation (Fig. 6D). Thus, Ca²⁺_i reduction in IICR-inhibited cells was caused by the NO-dependent potentiation of Ca²⁺_i extrusion by PMCA.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Effects of PMCA or NCX blockade on the Ca2+i reduction in IICR-inhibited cells and NO production from adjacent IICR-intact cells in the presence of 1 mM Ca2+. A, pG.IP3R1-AS transfectants showed Ca2+i reduction upon ATP (10 µM, thick line) stimulation. After wash-out of ATP for 30 min, cells were stimulated by BK (500 nM, thin line) under the blockade of PMCA by 20 mM Mg2+ (pH 8.8). B, pG.IP3R1-AS transfectants showed Ca2+i reduction upon ATP (thick line) stimulation. After wash-out of ATP, cells were stimulated by BK (thin line) under the blockade of NCX by 0 mM Na+. C, under the blockade of NCX by 0 mM Na+, pG.IP3R1-AS transfectants showed Ca2+i reduction upon ATP (thick line) stimulation. After wash-out of ATP, cells were stimulated by BK (thin line) under the blockade of both PMCA and NCX by 125 µM La3+ and 0 mM Na+. D, effect of PMCA or NCX blockade on BK-induced NO production in IICR-intact cells. Each column denotes the mean ± S.E. *, p < 0.05 between the presence and absence of L-NMMA (1 mM) (n = 4-7).

Effects of NO on Agonist-evoked Ca²⁺_i Elevation in Adjacent IICR-intact ECs-- The effects of NO on agonist-evoked Ca²⁺_i elevation were investigated by comparing Ca²⁺_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 Ca²⁺ (Figs. 7, A and B, and 8, A and B, and Table I). Our previous study has proven that NO is produced in the Ca²⁺-free medium after ATP or BK stimulation, although the amount produced is less than that produced in the presence of 1 mM Ca²⁺ (8).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Effects of NO on the sustained phase of Ca2+i dynamics and Ca2+ influx with and without blockade of PMCA or NCX in IICR-intact ECs in the presence of 1 mM Ca2+. The upper panel shows Ca2+i dynamics in response to ATP (10 µM; A) and BK (500 nM; B-D) with (thin solid line) or without (thick solid line) pretreatment with L-NMMA (1 mM). Coinciding with the upper panel, the lower panel shows Ca2+ influx in response to ATP (E) and BK (F-H) with (thin solid line) or without (thick solid line) pretreatment with L-NMMA. Furthermore, in B and F, cells were stimulated under the blockade of PMCA by 20 mM Mg2+ (pH 8.8) with (thin dotted line) or without (thick dotted line) pretreatment with L-NMMA. In C, D, G, and H, cells were stimulated under the blockade of NCX by 0 mM Na+ with (thin dotted line) or without (thick dotted line) pretreatment with L-NMMA. In D and H, cells were stimulated under the blockade of both PMCA and NCX by 125 µM La3+ and 0 mM Na+ with (thin gray solid line) or without (thick gray solid line) pretreatment with L-NMMA. Mn2+ (0.5 mM) was simultaneously applied to cells with stimuli (arrows) (n = 6-10).

View this table: [in this window] [in a new window]   Table I Modification of intracellular Ca2+i dynamics of ECs by NO Agonist actions were examined in the presence of 1 mM extracellular Ca2+ (Ca2+ (+) or after chelating with 1 mM EGTA without the addition of Ca2+ (Ca2+ ()). The peak Ca2+i (Peak) and Ca2+i level at 90, 210, or 240 s after the addition of an agonist is expressed in nM. Mn2+ quenching at 210 s (Mn2+-210 s) is expressed as the percentage of change from the basal level. After ATP or BK stimulation in Ca2+ (), the peak of the residual Ca2+i release induced by ionomycin (Peak-IM) is expressed in nM. Each value indicates the mean ± S.E. (n = 7-14). L-NMMA was applied 30 min prior to stimulation. TG-ATP and TG-BK, cells were treated with TG at 20 s before ATP and BK stimulation, respectively.

First, the mechanism of NO-dependent attenuation of the initial Ca²⁺_i spike was investigated. The effect of NO on ATP- or BK-induced IP₃ production was measured by radioimmunoassay. The basal IP₃ concentration was 0.7 ± 0.1 µM in ECs. In the Ca²⁺-free medium, ATP- and BK-induced IP₃ concentrations were 2.8 ± 0.3 and 2.9 ± 0.3 at 15 s and 2.7 ± 0.4 and 2.9 ± 0.4 µM at 120 s after stimulation, respectively (n = 4). After L-NMMA pretreatment, ATP- and BK-induced IP₃ concentrations were 2.9 ± 0.4 and 3.1 ± 0.3 at 15 s and 2.8 ± 0.4 and 3.0 ± 0.7 µM at 120 s after stimulation, respectively (n = 4). Therefore, IP₃ 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 IP₃ production in ECs.

Even after the initial Ca²⁺_i spikes were attenuated by NO in the Ca²⁺-free medium, IM-induced residual Ca²⁺ release was not significantly changed (Fig. 8, Table I). Furthermore, TG (1 µM) was used at 20 s before treatment with ATP or BK in the Ca²⁺-free medium. Under the inhibition of SERCA, L-NMMA pretreatment still potentiated the agonist-evoked initial Ca²⁺_i spike (Fig. 9A, Table I), suggesting that NO did not potentiate SERCA.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Effects of NO on initial Ca2+i spike and IM-releasable residual Ca2+i stores in IICR-intact cells in Ca2+-free medium. Shown is the initial Ca2+i spike in response to ATP (10 µM; A) or BK (500 nM; B) and then to ionomycin (1 µM) (n = 4-10).

View larger version (23K): [in this window] [in a new window]   Fig. 9.   Effects of NO on Ca2+i extrusion mediated by PMCA or NCX after blockade of SERCA in IICR-intact cells in Ca2+-free medium. Shown is the initial Ca2+i spike evoked by TG and BK (upper panel) or TG only (lower panel) after blockade of PMCA by 20 mM Mg2+ (pH 8.8; A and D), blockade of NCX by 0 mM Na+ (B and E), and blockade of both PMCA and NCX by 125 µM La3+ and 0 mM Na+ (C and F). In the upper panel, cells were treated with TG (1 µM) at 20 s before BK (500 nM) stimulation (n = 6-10).

The following studies were addressed to investigate the NO-dependent potentiation of Ca²⁺_i extrusion. In the presence of 1 mM Ca²⁺, the effect of NO on BK-induced Ca²⁺ dynamics was observed after the blockade of PMCA or NCX, while the SERCA mechanism remained operational. Blockade of PMCA by 20 mM Mg²⁺ (pH 8.8) significantly raised BK-induced Ca²⁺_i elevation, decreased Ca²⁺ 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⁺ demonstrated no prominent effect on the initial spike, the sustained phase, and the Ca²⁺ influx (Figs. 7, C and G). Under the blockade of NCX, L-NMMA pretreatment mildly potentiated Ca²⁺_i elevation. Blockade of both PMCA and NCX by 0 mM Na⁺ and 125 µM La³⁺ raised BK-induced Ca²⁺_i elevation, decreased Ca²⁺ 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-dependent potentiation of PMCA might be involved in Ca²⁺_i extrusion while Ca²⁺_i is elevated by agonist stimulation.

To prove this hypothesis, we investigated the effect of NO after further inhibition of SERCA by TG (1 µM) at 20 s before BK treatment in the Ca²⁺-free medium. Blockade of PMCA by 20 mM Mg²⁺ (pH 8.8) significantly raised the BK-induced initial Ca²⁺_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 Ca²⁺_i spike. On the other hand, blockade of NCX showed no significant effect on the BK-induced Ca²⁺_i spike (Fig. 9B, Table I). Under the blockade of NCX by 0 mM Na⁺, further inhibition of NO production mildly potentiated the BK-induced Ca²⁺_i spike. Blockade of both PMCA and NCX by 0 mM Na⁺ and 125 µM La³⁺ markedly raised the Ca²⁺_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 Ca²⁺_i rise and NO production in the Ca²⁺-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 Ca²⁺_i spike is caused by the NO-dependent potentiation of PMCA, which plays an important role in attenuating Ca²⁺_i signal in the excited state.

View larger version (27K): [in this window] [in a new window]   Fig. 10.   TG-induced NO production in IICR-intact cells in Ca2+-free medium. Shown is the effect of PMCA or NCX blockade on TG (1 µM)-induced NO production. Each column denotes the mean ± S.E. *, p < 0.05 between the presence and absence of L-NMMA (1 mM). (n = 4-6).

Second, in contrast to attenuating the initial Ca²⁺_i spike, NO significantly increased the sustained Ca²⁺_i phase (Fig. 7, A and B, Table I). As shown by Mn²⁺ quenching of fluorescence, NO markedly potentiated Ca²⁺ influx (from 17 ± 3 to 30 ± 3% at 210 s after ATP stimulation, p < 0.05; from 18 ± 3 to 27 ± 4% at 210 s after BK stimulation, p < 0.05; Fig. 7, E and F, and Table I). The potentiated Ca²⁺ influx coincided with the sustained phase of Ca²⁺_i transients. These results suggest that endogenous NO maintains Ca²⁺_i elevation during the sustained phase by promoting CCE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The major findings of the present study are as follows. 1) There is marked reduction of basal Ca²⁺_i after ATP or BK stimulation in individual ECs in which IICR and the subsequent CCE are inhibited by expressing an antisense against IP₃R₁ or microinjecting heparin. 2) The reduction of Ca²⁺_i results from NO that was released by and diffused from adjacent IICR-intact ECs. 3) NO reduces basal Ca²⁺_i by potentiation of Ca²⁺_i extrusion by PMCA. 4) This potentiation also markedly attenuates agonist-evoked Ca²⁺_i elevation in adjacent IICR-intact cells. 5) Endogenous NO markedly promotes agonist-evoked CCE, which maintains Ca²⁺_i elevation and possibly NO production in ECs.

Crucial Role of NO-dependent Potentiation of PMCA in Basal Ca²⁺_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 Ca²⁺_i has been controversial because of conflicting results. In platelets, Johansson et al. (34) reported that sodium nitroprusside (SNP; 20 µM) reduces basal Ca²⁺_i by 20-23 nM. In contrast, in ECs, Volk et al. (38) reported that SNP (10-1000 µM) significantly increases Ca²⁺_i up to about 200% of the basal level (Ca²⁺_i = 300 nM), similar to the Ca²⁺_i rise induced by 10 µM ATP. Actually, our group also confirmed that 100-300 µM SNP, and not 1-30 µM SNP, mildly increases basal Ca²⁺_i by 10-20 nM in both ECs and VSMCs. However, its nonspecific Ca²⁺_i-raising effect, which is produced by its intermediate or metabolized compounds, could not be excluded from the results, since the Ca²⁺_i rise could not be inhibited by methemoglobin (50 µM).² In platelets, Sang et al. (39) reported that a high (5 µM) and not a low concentration of NO gas solution (0.1 µM) increases basal Ca²⁺_i by 10 nM.

On the other hand, our previous studies have shown that after ATP stimulation, endogenous NO that is released by and diffused from ECs decreases basal Ca²⁺_i in adjacent VSMCs by about 40-90 nM in the coculture of both cells (8, 41). The results are consistent with the physiological effect of NO on VSMCs. However, a similar analysis of the effect of endogenous NO on basal Ca²⁺_i in ECs is difficult because agonist-evoked Ca²⁺_i elevation is needed for NO production. Although antagonists against receptor (45), G-protein (46), or phospholipase  (47) are able to inhibit Ca²⁺_i elevation, they are inadequate for the analysis, since these treatments in a culture dish inhibited Ca²⁺_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 Ca²⁺_i elevation is inhibited. In the present study, by using individual ECs in which agonist-evoked Ca²⁺_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 Ca²⁺_i was successfully elucidated. The reduction of basal Ca²⁺_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 Ca²⁺_i was clarified. Our results excluded the involvement of NO-dependent potentiation of SERCA, since there was no consequent increase in IM-releasable Ca²⁺_i stores after the reduction of basal Ca²⁺_i (Fig. 5B), and ATP or BK still induced Ca²⁺_i reduction after SERCA was blocked (Fig. 5C). After blocking SERCA, the attenuation of Ca²⁺_i reduction could be explained by the counteraction by TG-induced Ca²⁺ leakage from internal stores and TG-induced CCE. The remaining hypothesis is that NO potentiates Ca²⁺_i extrusion by PMCA or NCX. Recently, there is increasing evidence that PMCA is important for Ca²⁺_i extrusion, while NCX plays a minor role (14-18). Thus, the effects of blocking PMCA or NCX on Ca²⁺_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 Mg²⁺ 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 Mg²⁺ and pH 8.8 blocks PMCA-mediated Ca²⁺_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 Mg²⁺ (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 Ca²⁺ pump ATPase (33, 35, 48-51). Furthermore, extracellular La³⁺ at concentrations of 60-250 µM specifically blocks PMCA but spares NCX in red cells and VSMCs (IC₅₀ = 50-65 µM; Refs. 33 and 52). Low concentrations of La³⁺ (20-100 µM) inhibit PMCA activity by displacing Mg²⁺ 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⁺-free buffer. The extracellular Na⁺-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⁺-free buffer caused a decrease in intracellular Na⁺ concentration by 30% in VSMCs (33). Such a decrease in Na⁺ concentration did not affect the activity of PMCA significantly, since intracellular Na⁺ and K⁺ 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, Ca²⁺_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 IICR-intact cells. Thus, Ca²⁺_i reduction results from the NO-dependent potentiation of Ca²⁺_i extrusion by PMCA, which may be a major and potent regulator of basal Ca²⁺_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 Ca²⁺_i extrusion by PMCA, especially at basal Ca²⁺_i level. In platelets, Johansson et al. (34) reported that both SNP (10 µM) 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 Ca²⁺_i extrusion. As a result of potentiation of PMCA, SNP or dibutyryl-cGMP decreased the basal Ca²⁺_i and attenuated the ionomycin-induced Ca²⁺_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 Ca²⁺_i extrusion during the BK- or TG-evoked Ca²⁺_i elevation (Fig. 9). As indicated by its action on the basal Ca²⁺_i (70-150 nM), the TG-evoked Ca²⁺_i elevation (200-260 nM), and the ATP- or BK-evoked Ca²⁺_i elevation (~600 nM), NO-dependent potentiation of PMCA functions to extrude Ca²⁺_i over a wide Ca²⁺_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 Ca²⁺_i elevation, consistent with the results of previous studies (16, 55, 56).

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 Ca²⁺_i spike but rather to the NO produced during the sustained Ca²⁺_i phase. As shown in Fig. 7, the potentiation is necessary for the maintenance of Ca²⁺_i elevation during the sustained phase, where IICR is decreased and PMCA still functions to decrease Ca²⁺_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 Ca²⁺ 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 Ca²⁺_i elevation and continuous NO production during the sustained phase might be maintained by a positive feedback mechanism between NO production and Ca²⁺ 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 Ca²⁺_i signal in ECs by potentiating PMCA and CCE. At the basal state and during the initial Ca²⁺_i spike after agonist stimulation, NO potentiates PMCA, which contributes to keep the basal Ca²⁺_i level low and buffer the rapid Ca²⁺_i rise induced by agonists. During the sustained Ca²⁺_i phase, apart from potentiating PMCA, NO mainly potentiates CCE, which keeps the Ca²⁺_i level elevated and possibly enhances NO production.

	ACKNOWLEDGEMENTS

We are grateful to Dr. Masamitsu Iino (Department of Pharmacology) and Dr. Katsuhiko Mikoshiba (Department of Molecular Neurobiology, Institute of Medical Science) of the University of Tokyo for expert assistance during the course of this study.

	FOOTNOTES

^* This work was supported by grants-in-aid from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare of Japan, the Research Foundation for Health Science, the Japanese-Chinese Medical Research Collaboration Foundation, the Research Foundation of the Japan Society for the Promotion of Science, the Kanehara-ichiro Foundation, and the Uehara Memorial Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: The Second Dept. of Internal Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5449-5625; Fax: 81-3-5449-5445; E-mail: srwang-tky@umin.ac.jp.

Published, JBC Papers in Press, June 13, 2000, DOI 10.1074/jbc.M000910200

² J. Chen, Y. Wang, Y. Wang, T. Nakajima, K. Iwasawa, H. Hikiji, M. Sunamoto, D. K. Choi, Y. Yoshida, Y. Sakaki, and T. Toyo-oka, unpublished data.

	ABBREVIATIONS

The abbreviations used are: EC, endothelial cell; BK, bradykinin; Ca²⁺_i, intracellular Ca²⁺; CCE, capacitative Ca²⁺ entry; IICR, inositol 1,4,5-trisphosphate-induced Ca²⁺_i release; IM, ionomycin; IP₃, inositol 1,4,5-trisphosphate; IP₃R₁, type 1 IP₃ receptor; L-NMMA, N^G-monomethyl-L-arginine monoacetate; NCX, Na⁺-Ca²⁺ exchange; NO, nitric oxide; PMCA, plasma membrane Ca²⁺-ATPase; SERCA, sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase; TG, thapsigargin; VSMC, vascular smooth muscle cell.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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