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CARDIOVASCULAR

Distinct Ca²⁺ Requirement for NO Production between Proteinase-Activated Receptor 1 and 4 (PAR₁ and PAR₄) in Vascular Endothelial Cells

Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences (K.H., N.N., M.H., F.M., A.H., H.K.) and 21st Century Center of Excellence Program (H.K.), Kyushu University, Fukuoka, Japan

Received February 6, 2007; accepted May 8, 2007.

	Abstract

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Proteinase-activated receptors 1 and 4 (PAR₁ and PAR₄) are the major receptors mediating thrombin-induced NO production in endothelial cells. The intracellular signaling following their activation still remains to be elucidated. The present study provides the first evidence for the distinct Ca²⁺ requirement for the NO production between PAR₁ and PAR₄. The activation of PAR₁ by the activating peptide (PAR₁-AP) elevated cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and activated NO production in porcine aortic and human umbilical vein endothelial cells, whereas it had little effect on bovine aortic endothelial cells. PAR₄ activation by PAR₄-AP consistently induced NO production without an appreciable [Ca²⁺]_i elevation in three types of endothelial cells. The PAR₁-mediated NO production was significantly inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), whereas the PAR₄-mediated NO production was resistant. NO production following the PAR₁ and PAR₄ activation was significantly inhibited by pertussis toxin, but it was resistant to a G_q/11 inhibitor, YM254890 [(1R)-1-{(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl}-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. However, YM254890 abrogated the PAR₁-mediated Ca²⁺ signal. PAR₄-mediated NO production was substantially inhibited by the inhibitors of phosphotidylinositol-3 kinase (PI3K) and Akt, as well as by the dominant negative mutant of Akt. The PAR₁-mediated NO production was relatively resistant to inhibitors of PI3K. An immunoblot analysis revealed a transient increase in the phosphorylation of Akt and endothelial NO synthase following the PAR₄ stimulation. In conclusion, PAR₁ and PAR₄ engage distinct signal transduction mechanisms to activate NO production in vascular endothelial cells. PAR₄ preferably activates G_i/o and induced NO production in a manner mostly independent of Ca²⁺ but dependent on the PI3K/Akt pathway, whereas PAR₁ activates both the Ca²⁺-dependent and -independent mechanisms.

We have previously reported that thrombin induced NO production with a slight elevation of cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) in the endothelial cells of the porcine aortic valve (Mizuno et al., 2000b). Thrombin was also found to induce the greater NO production for a given elevation of [Ca²⁺]_i than the other stimulants we tested: ATP, bradykinin, and ionomycin (Mizuno et al., 2000b). Thus, our observations suggested that thrombin activated the Ca²⁺-independent and the Ca²⁺-dependent mechanism of NO production. We also reported that PAR₄ induced NO production without any appreciable elevation of [Ca²⁺]_i in the cultured bovine aortic endothelial cells (BAEC) (Momota et al., 2006). There is a possibility that PAR₄ mediates the Ca²⁺-independent component of the thrombin-induced NO production. However, BAEC were found to be unique in that they were well responsive to PAR₄-AP but not PAR₁-AP (Momota et al., 2006). Therefore, the Ca²⁺ independence of the PAR₄-mediated NO production still waits for the evaluation in other types of endothelial cells, which are responsive to both PAR₁ and PAR₄ activation. Furthermore, the mechanism for the PAR₄-mediated Ca²⁺-independent NO production still remains to be elucidated.

In the present study, using diaminorhodamine-4M (DAR-4M) (Kojima et al., 2001; Momota et al., 2006) and fura-2 fluorometry, we aimed to elucidate the signal transduction pathways, especially in terms of Ca²⁺ signal, from the activation of PAR₁ and PAR₄ to the production of NO in three difference types of endothelial cells: BAEC, porcine aorta endothelial cells (PAEC), and human umbilical vein endothelial cells (HUVEC). BAEC are taken to represent the endothelial cells, which predominantly respond to PAR₄ stimulation, whereas PAEC and HUVEC are taken to represent the cells, which respond to both PAR₁ and PAR₄ stimulation (see under Results). We consistently found that the PAR₄ activation induced the NO production in a manner independent of Ca²⁺ signal in three different cell types. The mechanism for the PAR₄-mediated Ca²⁺-independent NO production was further elucidated using BAEC. Thus, the present study provides the first evidence for the differential requirement of Ca²⁺ signal for the endothelial NO production between PAR₁ and PAR₄.

	Materials and Methods

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Materials. DAR-4M was purchased from Daiichi Pure Chemicals (Tokyo, Japan). Fura-2-acetoxymethyl ester (AM) and BAPTA-AM were purchased from Dojin (Kumamoto, Japan). Anti-phospho-Akt (Ser473) and anti-phospho-endothelial NO synthase (eNOS) (Ser1179) antibodies were purchased from Cell Signaling Technologies (Beverly, MA); anti-Akt and anti-eNOS antibodies were from BD Bioscience (San Jose, CA); anti-(His)₆ antibody was from QIAGEN (Hilden, Germany); and antitubulin antibody was from Serotec (Oxford, UK). Thrombin (bovine plasma, 1880 NIH U/mg protein, 1 U/ml =10 nM), bradykinin, aminoguanidine, and ionomycin were purchased from Sigma (St. Louis, MO). TFLLR-NH₂ (PAR₁-AP) and AYPGKF-NH₂ (PAR₄-AP) were purchased from Bachem (Bubendorf, Switzerland). The negative control peptides for PAR₁-AP (FTLLR-NH₂) and PAR₄-AP (YAPGKF-NH₂) were synthesized by Rapid Multiple Peptide Synthesis Service, University of Calgary (Calgary, AB, Canada). LY294002, wortmannin, SH-6 (an Akt inhibitor), 1400W, AG1478, AG1024, AG538, KN-93, GF109203X, and H-89 were purchased from Calbiochem (San Diego, CA). MAHMA-NONOate was purchased from Alexis Biochemicals (Lausen, Switzerland). N-nitro-L-arginine methyl ester and N-nitro-L-arginine were purchased from Wako Pure Chemicals (Tokyo, Japan). PD98059 was purchased from Biomol (Plymouth Meeting, PA). Pertussis toxin was purchased from Seikagaku Co. (Tokyo, Japan). YM254890 was donated by Astellas Pharma Inc. (Takasaki et al., 2004).

Cell Culture. BAEC, PAEC, and A7r5 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as described previously (Hirano et al., 2001, 2004b; Eto et al., 2003). HUVEC were cultured in MCDB104 supplemented with 5% fetal bovine serum and endothelial cell growth supplements (Nissui Pharmaceutical, Tokyo, Japan), as described previously (Nakayama et al., 2004). For the measurement of NO production, the cells were plated on Cell Desk LF1 (Sumitomo Bakelite, Tokyo, Japan) coated with type 1-P collagen (Nitta Gelatin, Osaka, Japan). Unless otherwise stated, the cells were plated on culture dishes. The cells were used at confluence (days 3–4).

DAR-4M Fluorometry. The fluorescence intensity of 10 µM DAR-4M in 1 ml of HEPES-buffered saline (HBS) (10 mM HEPES, pH 7.4, 135 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 5.5 mM D-glucose) was first measured in a quartz cuvette at 25°C with a fluorescence spectrophotometer 650-40 (Hitachi, Tokyo, Japan) using a prescan mode, which automatically set the full-scale amplitude for the recoding (17.1 ± 0.15 fluorescence units, n = 31) so that the maximal intensity obtained with 10 µM DAR-4M was 70% of the full scale. Cell Desk LF1 containing the cells was then inserted into the cuvette, and the changes in the fluorescence intensity (excitation at 540 ± 5 nm; emission at 580 ± 10 nm) were recorded under such a full-scale setting. The fluorescence data obtained with the cells were normalized by multiplying with the full-scale amplitude predetermined for each measurement and thus expressed in arbitrary units. The prestimulation level was assigned to be zero fluorescence. When the effects of various inhibitors on NO production were studied, the DAR-4M fluorescence data were expressed as percentage of the control value. The calibration curve for DAR-4M fluorescence was obtained using MAHMA-NONOate as a standard in the absence of the cells (Fig. 1A). The indicated concentrations of MAHMA-NONOate were added to the HBS containing 10 µM DAR-4M, and then the excitation spectrum of the DAR-4M fluorescence was recorded after the incubation period sufficient for the half-life of NO release of MAHMA-NONOate (3 min at room temperature). The calibration curve was constructed with the values obtained at the peak of fluorescence intensity of the excitation spectrum.

View larger version (34K): [in this window] [in a new window]   Fig. 1. NO production induced by thrombin, PAR1-AP, PAR4-AP, and bradykinin in BAEC. A, standard curve for the relationship between MAHMA-NONOate concentrations and DAR-4M fluorescence. The data are the mean ± S.E.M. (n = 7). B, representative recording of the change in the DAR-4M fluorescence intensity induced by 30 µM PAR4-AP. C–F, concentration-dependent NO production by thrombin (C), PAR4-AP (D), PAR1-AP (E), and bradykinin (F). The zero concentration indicates the value obtained by the buffer change without any stimulation. G, H, effects of NOS inhibitors (100 µM N-nitro-L-arginine methyl ester for thrombin; 1 mM N-nitro-L-arginine for bradykinin and PAR4-AP) and iNOS inhibitors (50 and 100 µM 1400W and 1 mM aminoguanidine) on the NO production induced by 0.3 U/ml thrombin, 30 µM PAR4-AP, and 10 nM bradykinin. Because N-nitro-L-arginine interfered with the DAR-4M fluorometry on thrombin, we used N-nitro-L-arginine methyl ester to examine whether the thrombin-induced changes in the DAR-4M fluorescence depended on the activity of NOS. The data are the mean ± S.E.M. (n = 4). *, p < 0.05; n.s., not significantly different versus NO production in the absence of NOS or iNOS inhibitors.

The following observations validate DAR-4M fluorometry as a method to estimate the NO production: 1) the linear relationship between the DAR-4M fluorescence intensity and the concentration of MAHMA-NONOate (Fig. 1); 2) the concentration dependence in the agonist-induced increases in DAR-4M fluorescence (Fig. 1); 3) the inhibition of the agonist-induced increases in the DAR-4M fluorescence by NOS inhibitors (Fig. 1); and 4) no increase in the DAR-4M fluorescence by thrombin in the smooth muscle cell line A7r5 (thrombin did induce [Ca²⁺]_i elevation in fura-2 fluorometry) (data not shown).

Fura-2 Fluorometry. The cells on 35-mm dishes were loaded with fura-2 in an acetoxymethyl ester form as described previously (Eto et al., 2003). When the fura-2 fluorometry was conducted in the BAPTA-loaded cells, BAPTA-AM was added during the last 30 min of the fura-2 loading period. After loading with fura-2 and/or BAPTA, the cells were equilibrated in HBS at room temperature for 30 min, and then fluorometry was started. The changes in fura-2 fluorescence (excitation at 340 ± 10 and 380 ± 10 nm; emission at 500 ± 10 nm) were monitored in HBS at 25°C using a front-surface fluorometer as described previously (Eto et al., 2003; Kanaide, 2006). The response to 50 µM ionomycin was recorded as a reference response at the end of each recording. The fluorescence ratio data were expressed as a percentage, while assigning the values at rest and at the peak [Ca²⁺]_i elevation induced by 50 µM ionomycin to be 0 and 100%, respectively.

The effect of BAPTA loading on the resting level of [Ca²⁺]_i was evaluated in a separate experiment, according to the protocol that we have described previously (Kanaide, 2006). In brief, the resting level of [Ca²⁺]_i was first recorded, and then the maximal and minimal fluorescence ratio was sequentially recorded in HBS containing 50 µM ionomycin, and then in HBS containing 2 mM EGTA but no Ca²⁺.

Treatment with Pertussis Toxin. The cells were treated with 100 ng/ml pertussis toxin in the growth media for 24 h as previously reported (Momota et al., 2006). When the cells were loaded with fura-2, fura-2-AM was added during the last 1 h of the pertussis toxin treatment. The cells were then washed and equilibrated in HBS at room temperature for at least 30 min and then subjected to fura-2 or DAR-4M fluorometry.

Recombinant Proteins. A dominant negative mutant of Akt with (TATHA-Akt) and without [(His)₆-Akt] a cell-penetrating peptide, as well as a RhoA-inhibitory protein with a cell-penetrating peptide (TATHA-RB), was expressed in bacteria and prepared as described previously (Hirano et al., 2004b; Koga et al., 2004; Shiga et al., 2005; Yufu et al., 2005). TATHA-Akt consisted of a hexahistidine tag, a cell-penetrating peptide of Tat, a hemagglutinin (HA) epitope, and the N-terminal 147 amino acids of Akt1 (Accession no. M63167 [GenBank] ), whereas (His)₆-Akt lacks a cell-penetrating peptide and an HA epitope (Koga et al., 2004). TATHA-RB consisted of a hexahistidine tag, a cell-penetrating peptide, an HA epitope, and the RhoA-binding region of Rho kinase (Hirano et al., 2004b; Shiga et al., 2005). The recombinant proteins were affinity-purified through Ni²⁺-loaded Hi-Trap chelating column on Akta Prime (GE Healthcare, Tokyo, Japan). The protein concentration of the recombinant proteins was estimated with Coomassie protein assay reagent with bovine serum albumin as a standard (Pierce, Rockford, IL).

Immunoblot Analysis of Protein Transduction. The transduction of TATHA-Akt and TATHA-RB into BAEC was confirmed by an immunoblot analysis as described previously (Hirano et al., 2004b; Koga et al., 2004; Yufu et al., 2005). In brief, the cells on 100-mm dishes were harvested and resuspended in phosphate-buffered saline (PBS) at room temperature. TATHA-RB, TATHA-Akt, and (His)₆-Akt were added to the cell suspensions at a concentration of 0.3 µM. The cells were exposed to proteins for 30 min and then were rapidly and thoroughly washed three times in ice-cold PBS. The cells were once frozen at –80°C and then lysed in the buffer (50 mM Tris-HCl, pH 7.2, 0.5 M NaCl, 10 mM MgCl₂, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µM 4-aminidophenylmethane sulfonyl fluoride), as described previously (Hirano et al., 2004b). The protein concentration of the lysates was estimated with Coomassie protein assay reagent. The lysates (25-µg proteins) were then separated with SDS-polyacrylamide gel electrophoresis on 7.5 to 20% gradient polyacrylamide gel, followed by transfer to polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The membranes were blocked overnight with 5% nonfat dry milk in PBS containing 0.1% Tween 20. The recombinant proteins were detected with anti-(His)₆ antibody (x500 dilution), horseradish peroxidase-conjugated secondary antibody, and enhanced chemiluminescence technique (Amersham, Buckinghamshire, UK). Tubulin was detected to validate the equal loading of the cell extract. The luminescence signal was detected and analyzed with a ChemiDoc XRS-J image analysis system (Bio-Rad, Tokyo, Japan).

Immunoblot Analysis of Phosphorylation of Akt and eNOS. The cells on a 60-mm culture dish were stimulated with 30 µM PAR₄-AP in the growth media, and then the cells were immediately washed twice in ice-cold PBS. PBS was thoroughly aspirated, and then the cells were lysed by scraping in 150 µl of lysis buffer (50 mM Tris-HCl, pH 7.2, 0.5 M NaCl, 10 mM MgCl₂, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µM 4-aminidophenylmethane sulfonyl fluoride, 5 µM microcystin-LR, 20 µM NaF, 2 mM Na₃VO₄, and 5 mM sodium pyrophosphate). The cell lysates were snap-frozen in liquid N₂ and kept at –80°C. When the effects of various inhibitors were examined, the cells were treated with inhibitors 30 min before and during the stimulation with PAR₄-AP. The cell lysates (50-µg proteins) were then subjected to an immunoblot analysis as described above. The polyvinylidene difluoride membranes were blocked overnight with 5% nonfat dry milk in Tris-buffered saline (20 mM Tris-HCl and 150 mM NaCl, pH 7.5) containing 0.05% Tween 20. The antibodies were diluted (x250 for anti-phospho-Akt and anti-phospho-eNOS antibodies; x1000 for anti-Akt and anti-eNOS antibodies; x1000 for horseradish peroxidase-conjugated secondary antibodies) in an immunoreaction enhancer solution named Can-Get-Signal (Toyobo, Osaka, Japan). The luminescence signal was detected and analyzed with a ChemiDoc XRS-J image analysis system (Bio-Rad). The level of phosphorylation of Akt and eNOS was normalized by the total amount of Akt and eNOS, respectively, and then the value obtained with the unstimulated cells was assigned a value of 1.

Statistical Analysis. The data are the mean ± S.E.M. The experimental number (n) indicates the number of independent experiments using different cell preparations. The analysis of variance was used to evaluate statistical significance. A value of p < 0.05 was considered to be significantly different.

	Results

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NO Production and [Ca²⁺]_i Elevation in Response to Thrombin, PAR₁-AP, PAR₄-AP, and Bradykinin in BAEC. The standard curve for the relationship between the DAR-4M fluorescence intensity and the concentrations of MAHMA-NONOate was first obtained (Fig. 1A). The linear relationship was observed within the range of 0 to 100 nM MAHMA-NONOate. The change in 10 arbitrary units of DAR-4M fluorescence was approximately equivalent to the changes in 10 nM MAHMA-NONOate. In the presence of BAEC, the buffer change from HBS to HBS induced a negligible change, if any, in the DAR-4M fluorescence (Fig. 1B). The subsequent change to HBS containing 30 µM PAR₄-AP increased the DAR-4M fluorescence intensity (Fig. 1B). The fluorescence level thereafter remained sustained with no further increase (Fig. 1B), whereas washing out PAR₄-AP in HBS containing 10 µM DAR-4M reverted the fluorescence level to that seen before stimulation (data not shown). Because DAR-4M is converted to a fluorescence triazole by reaction with NO (Kojima et al., 2001), this observation indicated a transient nature of the NO production induced by PAR₄-AP, which lasted for a few minutes.

Thrombin, PAR₁-AP, PAR₄-AP, and bradykinin all induced a concentration-dependent production of NO in BAEC (Fig. 1, C–F). The maximal NO production obtained with thrombin was comparable with that obtained with PAR₄-AP, whereas PAR₁-AP induced only a small production of NO in BAEC. Thus, these findings suggested that PAR₄ may play a major role in the thrombin-induced NO production in BAEC. The NO production seen with thrombin and PAR₄-AP in BAEC was estimated to be approximately 80 to 120 nM (Fig. 1), which was consistent with that obtained in vivo (Vallance et al., 1995). The NOS inhibitors almost completely abolished the NO production induced by thrombin (0.3 U/ml), PAR₄-AP (30 µM), and bradykinin (10 nM) (Fig. 1G). However, inducible NO synthase (iNOS) inhibitors had no significant effect on the PAR₄-AP-induced NO production (Fig. 1H).

Bradykinin induced a transient elevation of [Ca²⁺]_i in a concentration-dependent manner in BAEC (Fig. 2). On the other hand, thrombin, PAR₁-AP, and PAR₄-AP induced no increase in [Ca²⁺]_i in BAEC (Fig. 2).

View larger version (29K): [in this window] [in a new window]   Fig. 2. Effect of thrombin, PAR1-AP, PAR4-AP, and bradykinin on [Ca2]i in BAEC. Representative traces (A) and the concentration-response curves (B) showing the changes in the fura-2 fluorescence ratio induced by thrombin, PAR1-AP, PAR4-AP, and bradykinin. The unit concentration of thrombin was converted to the molar concentration, while assigning 1 U/ml to be 10 nM. The data are the mean ± S.E.M. (n = 4).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Effect of BAPTA loading on the [Ca2+]i increase and NO production in BAEC. A, concentration-dependent inhibition of the 10 nM bradykinin-induced [Ca2+]i elevation by BAPTA loading. The data are the mean ± S.E.M. (n = 4). B, NO production induced by 10 nM bradykinin, 0.3 U/ml thrombin, and 30 µM PAR4-AP in BAEC untreated (–) and treated (+) with 50 µM BAPTA-AM 30 min before the measurement. The data are the mean ± S.E.M. (n = 4). *, p < 0.05; n.s., not significantly different.

View larger version (31K): [in this window] [in a new window]   Fig. 4. NO production induced by thrombin, PAR1-AP, and PAR4-AP in PAEC. A, [Ca2+]i elevation and NO production induced by thrombin, PAR1-AP, and PAR4-AP in PAEC. The data are the mean ± S.E.M. (n = 4). B, effect of BAPTA loading on the NO production induced by 3 U/ml thrombin, 30 µM PAR1-AP, and 30 µM PAR4-AP in PAEC. The NO production obtained without BAPTA loading was assigned to be 100%. The values obtained with the buffer change alone are also shown (buffer). The data are the mean ± S.E.M. (n = 7 for thrombin, n = 6 for PAR1, n = 4 for PAR4). *, p < 0.05 versus BAPTA-AM (–).

Effect of Thrombin, PAR₁-AP, and PAR₄-AP on [Ca²⁺]_i and NO Production in PAEC and HUVEC. In PAEC, both thrombin and PAR₁-AP induced a concentration-dependent elevation of [Ca²⁺]_i and the production of NO (Fig. 4A). However, in PAEC, PAR₄-AP also induced NO production (Fig. 4A) without any appreciable elevation of [Ca²⁺]_i (data not shown). Similar results were also observed in HUVEC (Fig. 5). In HUVEC, the NO production induced by PAR₄-AP was not associated with any [Ca²⁺]_i elevation, whereas that seen with thrombin and PAR₁-AP was associated with a significant elevation of [Ca²⁺]_i. The negative control peptides for PAR₁-AP and PAR₄-AP had no effects on either BAEC or PAEC, as evaluated by the Ca²⁺ response. In PAEC, BAPTA-AM, at 50 µM, almost completely abolished the [Ca²⁺]_i elevation induced by PAR₁-AP and thrombin (data not shown). The NO production induced by PAR₁-AP was partially, but significantly, inhibited by BAPTA loading (Fig. 4B). However, the NO production induced by PAR₄-AP was resistant to BAPTA (Fig. 4B). BAPTA did not significantly inhibit the thrombin-induced NO production in PAEC (Fig. 4B).

View larger version (18K): [in this window] [in a new window]   Fig. 5. NO production induced by thrombin, PAR1-AP, and PAR4-AP in HUVEC. [Ca2+]i elevation and NO production induced by thrombin, PAR1-AP, and PAR4-AP in HUVEC. The data are the mean ± S.E.M. (n = 4). *, p < 0.05 versus the basal level.

Involvement of G_i/o in the NO Production following the PAR₁ and PAR₄ Activation. We examined the effect of pertussis toxin on the NO production as reported previously (Momota et al., 2006) and thereby evaluated the involvement of G_i/o in the NO production. In BAEC, the NO production induced by 0.1 U/ml thrombin and 30 µM PAR₄-AP was significantly inhibited by the treatment with pertussis toxin, whereas that seen with 1 µM ionomycin was resistant to pertussis toxin (Fig. 6A). Thus, these observations are consistent with those of our previous report (Momota et al., 2006). In PAEC, the NO production induced by not only thrombin and PAR₄-AP but also PAR₁-AP was significantly inhibited by pertussis toxin (Fig. 6B). The NO production induced by ionomycin in PAEC was resistant to pertussis toxin (Fig. 6B). The observed inhibitory effect of pertussis toxin on the NO production induced by thrombin and PAR₁-AP was not associated with the inhibition of the Ca²⁺ signal in PAEC (Fig. 5C).

View larger version (24K): [in this window] [in a new window]   Fig. 6. Effects of pertussis toxin on the NO production and [Ca2+]i elevation induced by thrombin, PAR1-AP, and PAR4-AP in BAEC and PAEC. A and B, NO production induced by 0.1 U/ml thrombin, 30 µM PAR1-AP, 30 µM PAR4-AP, and 1 µM ionomycin in BAEC (A) and PAEC (B) with (+) and without (–) pertussis toxin treatment. C, [Ca2+]i elevation induced by 3 U/ml thrombin and 30 µM PAR1-AP in PAEC with (+) and without (–) pertussis toxin treatment. The cells were treated with 100 ng/ml pertussis toxin in the cultured media for 24 h, and then they were subjected to the measurement of [Ca2+]i and NO production in the absence of pertussis toxin. The data are the mean ± S.E.M. (n = 10–14 for A, n = 4–7 for B, n = 3–4 for C). *, p < 0.05; n.s., not significantly different.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Effects of YM254890 on the NO production and [Ca2+]i elevation induced by thrombin, PAR1-AP, and PAR4-AP in BAEC and PAEC. A, concentration-dependent effects of YM254890 on the [Ca2+]i elevation induced by 100 nM bradykinin in BAEC. B, effects of YM254890 on the [Ca2+]i elevation induced by 3 U/ml thrombin and 30 µM PAR1-AP in PAEC. The cells were treated with YM254890 30 min before and during the stimulations. C and D, NO production induced by 0.1 U/ml thrombin, 30 µM PAR1-AP, 30 µM PAR4-AP, and 1 µM ionomycin in BAEC (C) and PAEC (D) with (+) and without (–) the treatment with 30 nM YM254890. The data are the mean ± S.E.M. (n = 3 for A and B; n = 3–5 for C; n = 3–4 for D). *, p < 0.05; n.s., not significantly different.

Thrombin, PAR₁-AP, and PAR₄-AP in BAEC (Fig. 2) and PAR₄-AP in PAEC (Fig. 3) induced no appreciable elevation of [Ca²⁺]_i. The effects of pertussis toxin or YM254890 on the [Ca²⁺]_i elevation were not examined under these situations.

Involvement of the Phosphotidylinositol-3 Kinase/Akt Pathway in the PAR₄-Mediated Ca²⁺-Independent NO Production. We investigated the involvement of the phosphotidylinositol-3 kinase (PI3K)-Akt pathway in the PAR₄-mediated Ca²⁺-independent NO production in BAEC (Fig. 8). The PAR₄-AP-induced NO production was almost completely inhibited by 30 µM LY294002, 10 µM wortmannin, and 10 µM SH6, an Akt inhibitor (Fig. 8A). In contrast, the PAR₁-AP-induced NO production in PAEC was resistant to wortmannin. However, it was significantly inhibited by LY294002 but to a lesser extent than that seen with PAR₄-AP (Fig. 8B). All the inhibitors had no significant effect on the bradykinin-induced [Ca²⁺]_i elevation and NO production (Fig. 8C) in BAEC.

View larger version (37K): [in this window] [in a new window]   Fig. 8. The effect of inhibitors of the PI3K-Akt pathway on the NO production induced by PAR4-AP, PAR1-AP, and bradykinin. A–C, NO production induced by 30 µM PAR4-AP in BAEC (A), 30 µM PAR1-AP in PAEC (B), and 10 nM bradykinin in BAEC (C) in the presence and absence of LY294002 (LY), wortmannin (WM), or SH-6. In C, the effect of the PI3K-Akt inhibitors on the bradykinin-induced [Ca2+]i elevation is also shown. The inhibitors were added at the indicated concentrations 30 min before and during the stimulations. D, NO production induced by 30 µM PAR4-AP in BAEC untreated and treated with 0.3 µM TATHA-Akt, 0.3 µM TATHA-RB, or 0.3 µM (His)6-Akt. The recombinant proteins were added to the cells 30 min before and during the stimulation with PAR4-AP. E, immunoblot detection (IB) of the recombinant proteins using anti-(His)6 antibody in the extract of BAEC, untreated and treated with 0.3 µM TATHA-Akt, 0.3 µM TATHA-RB, or 0.3 µM (His)6-Akt for 30 min. The purified recombinant proteins (50 ng) were loaded as a positive control for immunoblot detection. Tubulin was detected to validate the equal loading of the cell extract (25-µg proteins). The data are the mean ± S.E.M. (n = 4). *, p < 0.05; n.s., not significantly different versus the NO production seen without inhibitors or in untreated cells.

The involvement of Akt in the PAR₄-mediated NO production in BAEC was then further investigated using a dominant negative mutant of Akt (TATHA-Akt) (Fig. 8, D and E). The RhoA inhibitory protein (TATHA-RB) was used to investigate any possible involvement of RhoA in NO production. TATHA-Akt and TATHA-RB were introduced to the cells by using a cell-penetrating peptide-mediated protein transduction technique (Hirano et al., 2004a,b; Koga et al., 2004). TATHA-Akt, but not TATHA-RB, significantly inhibited the PAR₄-AP-induced NO production in BAEC. The dominant negative mutant of Akt without a cell-penetrating peptide [(His)₆-Akt] had no significant effect (Fig. 8D). The intracellular transduction of TATHA-Akt and TATHA-RB, but not (His)₆-Akt, was detected by an immunoblot analysis (Fig. 8E).

Insulin has been reported to activate the PI3K-Akt pathway (Hemmings, 1997; Saltiel and Pessin, 2002; Wymann et al., 2003) and induce NO production with no increase in [Ca²⁺]_i (Montagnani et al., 2001). In BAEC, insulin did induce a concentration-dependent NO production, with the maximal production at 100 nM (Fig. 9A). However, insulin induced no [Ca²⁺]_i elevation (data not shown). An inhibitor of epidermal growth factor receptor tyrosine kinase (AG1478) and inhibitors of insulin-like growth factor 1 and insulin receptor kinase (AG1024 and AG538) significantly inhibited the insulin-induced NO production (Fig. 9B). However, none of these inhibitors of receptor tyrosine kinases had any significant effect on the PAR₄-AP-induced NO production in BAEC (Fig. 9B).

View larger version (31K): [in this window] [in a new window]   Fig. 9. Effects of the receptor tyrosine kinase inhibitors on the NO production induced by insulin and PAR4-AP in BAEC. A, concentration-dependent NO production by insulin in BAEC. B, NO production induced by 100 nM insulin and 30 µM PAR4-AP in BAEC, in the presence or absence of AG1478, AG1024, or AG538 at the indicated concentrations. The NO production obtained without the receptor tyrosine kinase inhibitors was assigned a value of 100%. The data are the mean ± S.E.M. (n = 3–4). *, p < 0.05 versus the NO production seen without inhibitors.

View larger version (35K): [in this window] [in a new window]   Fig. 10. Increases in the phosphorylation of Akt and eNOS by PAR4-AP in BAEC. A, time course of the phosphorylation of Akt at Ser473 and eNOS at Ser1179 induced by 30 µM PAR4-AP in BAEC. The data are the mean ± S.E.M. (n = 7). B, representative immunoblot findings and the summary of the effects of wortmannin (10 µM), LY294002 (30 µM), or SH-6 (10 µM) on the phosphorylation of Akt and eNOS at 1 min after the stimulation with PAR4-AP in BAEC. The data are the mean ± S.E.M. (n = 4 for Akt; n = 11 for eNOS). C, effects of KN-93 (1 µM), GF109203X (1 µM), H-89 (10 µM), PD98059 (10 µM), and LY294002 (30 µM) on the PAR4-AP-induced phosphorylation of Akt and eNOS in BAEC. The data are the mean ± S.E.M. (n = 7). The cells were either untreated or treated with inhibitors for 30 min and then were stimulated with 30 µM PAR4-AP in the continuous presence of the inhibitors. The level of phosphorylation seen in the unstimulated cells was assigned a value of 1. , p < 0.05 versus unstimulated control; *, p < 0.05 versus PAR4-AP.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The intracellular signal transduction following the PAR₁ activation has been comprehensively studied (Coughlin, 2000; Macfarlane et al., 2001; Hollenberg and Compton, 2002), whereas the PAR₄-initiated cell signaling still remains largely unknown (Steinberg, 2005). Nevertheless, the mechanisms underlying the NO production mediated by PAR₁ and PAR₄ remain to be investigated. The present study provides the first evidence for the distinct Ca²⁺ requirement for the NO production following the activation of PAR₁ and PAR₄ in vascular endothelial cells. The PAR₄ activation induced NO production without an appreciable elevation of [Ca²⁺]_i and also in a manner resistant to BAPTA. Importantly, the PAR₄-mediated Ca²⁺-independent NO production was consistently observed in three different types of endothelial cells. Thus, the present study advanced our previous study in BAEC (Momota et al., 2006) and could draw a conclusion that PAR₄ preferably elicits some type of signal transduction other than the Ca²⁺ signal in vascular endothelial cells, thereby activating the NO production in a manner independent of Ca²⁺. On the other hand, PAR₁ activation induced NO production with a concomitant elevation of [Ca²⁺]_i, and such NO production was significantly but partly inhibited by BAPTA. Thus, these observations suggest PAR₁ to induce the NO production via both Ca²⁺-dependent and -independent mechanisms.

In PAEC, which respond to both the activation of PAR₁ and PAR₄, the thrombin-induced NO production was similar to that seen with PAR₁-AP and PAR₄-AP, and no apparent additive effect of PAR₁-AP and PAR₄-AP was observed. Although BAPTA inhibited the PAR₁-mediated NO production, thrombin-induced NO production was relatively resistant to BAPTA. These observations suggest that thrombin-induced NO production was mediated mainly by PAR₄ when the PAR₁ signaling was blocked. There may be some redundancy between PAR₁ and PAR₄ in NO production.

Our observations, which suggest the existence of the distinct signal transduction mechanisms between PAR₁ and PAR₄ in vascular endothelial cells, are consistent with those of the previous studies in human platelets (Ma et al., 2005; Holinstat et al., 2006). However, our observations contrast with those of a recent report that showed the PAR₄-mediated human platelet aggregation to be abolished by BAPTA, whereas the PAR₁-mediated aggregation was resistant (Holinstat et al., 2006). Instead, our findings of the poor coupling of PAR₄ to the Ca²⁺ signal are consistent with those in mouse cardiomyocytes (Sabri et al., 2003). In cardiomyocytes, PAR₄ was shown to be a weak activator of phospholipase C, which can be linked to a Ca²⁺ signal by a generation of inositol trisphosphate (Sabri et al., 2003). As a result, the specificity of receptor coupling to the downstream signaling pathways seems to vary depending on the cell type and/or species. However, the mechanism underlying the cell type- or species-specific coupling still remains to be elucidated.

iNOS is a Ca²⁺-independent NO synthase (Alderton et al., 2001). However, its contribution to the PAR₄-induced NO production has been ruled out. Thus, PAR₄ is suggested to activate eNOS in a Ca²⁺-independent manner. The Ca²⁺-independent activation of eNOS and NO production has been reported to be accompanied by the Akt-catalyzed phosphorylation of eNOS at Ser1179 (Ser1177 in humans) (Sessa, 2004). Our observations suggested the major contribution of the PI3K-Akt pathways to the PAR₄-induced Ca²⁺-independent NO production. Both the observation of an increase in the phosphorylation level of Akt and eNOS and the inhibition of this phosphorylation by the PI3K inhibitor further support the involvement of PI3K-Akt in the PAR₄-induced NO production. In contrast, the PI3K-Akt pathway was not suggested to play a major role in the PAR₁-induced NO production. Thus, the present study also provides the first evidence that PAR₄-mediated signaling is distinct from that of PAR₁ regarding PI3K-Akt and the Ca²⁺ signal.

Insulin induced NO production in a Ca²⁺-independent manner in BAEC, as previously reported (Montagnani et al., 2001), and this NO production was significantly inhibited by the receptor tyrosine kinase inhibitors. However, the PAR₄-induced NO production was resistant to these inhibitors, thus ruling out the involvement of insulin receptor activation or the transactivation of the receptor tyrosine kinases (Zwick et al., 1999) in the PAR₄-induced NO production. On the other hand, G protein-coupled receptors can directly activate the PI3K-Akt pathway through interactions between the subunit of G proteins and a type of PI3K (Wymann et al., 2003). This mechanism is consistent with our findings that the pertussis toxin significantly inhibited the PAR₄-induced NO production. Collectively, our results suggest that PAR₄ is preferentially coupled to G_i/o and then activates the PI3K-Akt pathway, thereby inducing the NO production mostly in a Ca²⁺-independent manner.

Our results further showed the differential coupling of G proteins to the [Ca²⁺]_i elevation and the NO production. Our observations suggest that G_q/11 plays a major role in the PAR₁-mediated generation of a Ca²⁺ signal, whereas G_i/o is suggested to be involved in the NO production following the activation of not only PAR₄ but also PAR₁. It should be noted that the inhibition of G_q/11 signaling by YM254890 had no significant effect on the PAR₁-mediated NO production, whereas it abolished a transient [Ca²⁺]_i elevation. On the other hand, the inhibition of Ca²⁺ signaling by BAPTA significantly inhibited the PAR₁-mediated NO production. These results suggest that the transient elevation of [Ca²⁺]_i is not necessary for the PAR₁-mediated NO production, although a part of the PAR₁-mediated NO production is Ca²⁺-dependent. It is conceivable that YM254890 inhibited an agonist-induced elevation of [Ca²⁺]_i without affecting the resting level of [Ca²⁺]_i. In contrast, BAPTA not only inhibited the [Ca²⁺]_i elevation but also decreased the resting [Ca²⁺]_i level. Thus, the basal level of [Ca²⁺]_i is considered to be sufficient to support the PAR₁-mediated Ca²⁺-dependent NO production.

It should also be noted that YM254890 had no significant effect on the NO production induced by PAR₁-AP and PAR₄-AP, whereas it significantly inhibited the thrombin-induced NO production in both BAEC and PAEC. These observations simply suggest that a part of the thrombin-induced NO production was mediated by some new receptor other than PAR₁ and PAR₄ (Hamilton et al., 1998). However, it is also possible that the coupling of PAR to the downstream signaling pathways may differ depending on the mode of receptor activation, either proteolytic or nonproteolytic (Blackhart et al., 2000; Al-Ani et al., 2004; Kim et al., 2004), the state of receptor oligomerization (Leger et al., 2006), or the involvement of the transactivation of PAR₂ following the proteolytic activation of PAR₁ (O'Brien et al., 2000). These possibilities still remain to be elucidated.

In conclusion, the present study showed the distinct mechanisms underlying the NO production induced by the activation of PAR₁ and PAR₄ in vascular endothelial cells; PAR₄ preferentially activates the PI3K-Akt pathway and induced the NO production in a manner mostly independent of Ca²⁺. In contrast, PAR₁ activates both Ca²⁺-dependent and -independent NO production. G_i/o plays an important role in mediating the NO production, whereas G_q/11 is coupled to the Ca²⁺ signal. The intracellular signal transduction elicited by PAR₁ has been intensively investigated, whereas the PAR₄-induced signal transduction still remains largely unknown. Thus, our study is considered to shed some light on how PAR₄ contributes to the NO production in vascular endothelial cells. Therefore, PAR₄ may play an important role in mediating the Ca²⁺-independent component of the thrombin-induced NO production.

	Acknowledgements

 
We thank Brian Quinn for linguistic comments and help with the manuscript.

	Footnotes

 
This study was supported in part by a grant from the 21st Century COE Program, a Grant-in-aid for Scientific Research (17590744) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.121038.

ABBREVIATIONS: PAR, proteinase-activated receptor(s); PAR-AP, proteinase-activated receptor-activating peptide(s); [Ca²⁺]_i, cytosolic Ca²⁺ concentration(s); BAEC, bovine aortic endothelial cell(s); DAR-4M, diaminorhodamine-4M; PAEC, porcine aorta endothelial cell(s); HUVEC, human umbilical vein endothelial cell(s); AM, acetoxymethyl ester; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; eNOS, endothelial NO synthase; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; 1400W, n-[3-(aminomethyl) benzyl] acetamidine; AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline; AG1024, 3-bromo-5-t-butyl-4-hydroxy-benzylidenemalonitrile; AG538, -cyano-(3,4-dihydroxy)cinnamoyl-(3',4'-dihydroxyphenyl)ketone; KN-93, 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; GF109203X, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; MAHMA-NONOate, (Z)-1-(N-methyl-N-[6-(N-methylammoniohexyl)amino])-diazen-1-ium-1,2-diolate; PD98059, 2'-amino-3'-methoxyflavone; YM254890, (1R)-1-{(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,-20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl}-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate; HBS, HEPES-buffered saline; HA, a hemagglutinin tag; PBS, phosphate-buffered saline; iNOS, inducible NO synthase; PI3K, phosphotidylinositol-3 kinase.

Address correspondence to: Hideo Kanaide, Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. E-mail: kanaide{at}molcar.med.kyushu-u.ac.jp

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