Bradykinin, thapsigargin, SKF-96365, ruthenium red, Gd³⁺, La³⁺, and okadaic acid were purchased from Sigma (St. Louis, MO, USA). Bovine PRL (BIO grade) was obtained from the National Hormone and Pituitary Program (NHPP, Torrance, CA, USA). (Z)-1-[N-(2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate or DETA-NONOate was purchased from Alexis Corporation (San Diego, CA, USA). Recombinant human vasoinhibins (corresponding to both 16 and 18 kDa fragments of prolactin) were produced by site-directed mutagenesis using a baculovirus expression system as described [25]. Negative controls included aliquots of recombinant vasoinhibins that were inactivated by incubation for 30 min at 90 °C, followed by centrifugation for 60 min at 2500 × g to remove denatured proteins.

BUVEC obtained and cultured as described [26] were routinely grown in F12K medium (A1963, AppliChem GMBH, Darmstadt, Germany) supplemented with 10% FBS and 50 U/mL penicillin/streptomycin. For all experiments, cells were incubated in 0.5% FBS-culture medium for 24 h prior treatments.

Cell proliferation was first assessed by [³H]thymidine incorporation (Amersham International plc, Cardiff, UK), and once validated, we used the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an index of proliferation to avoid radioisotopes. The ³H-thymidine incorporation assay consisted in seeding cells in 48-well plates (Nunc) at an initial density of 5000 cells/well and subjecting them to a series of treatments (all in 200 μL of complete culture medium containing 10% FBS) that included a 48 h-incubation with BK (10 μM) combined or not with increasing doses of vasoinhibins (10, 20, 40, and 60 nM), and in the presence or the absence of DETA-NONOate (10 μM), as indicated. When required, treatments were performed in Ca²⁺-free medium obtained by adding 10 mM EDTA to the standard F12K medium, which normally contains 0.4 mM Ca²⁺. Twenty-four h before ending the treatment, 1 μL of ³H-thymidine per well was added. After the 48-h treatment, wells were washed thrice in 5% trichloroacetic acid, with the last wash for 20 min at 4 °C. Next, 250 μL boiling 0.25 N NaOH was added, and samples were transferred to scintillation vials for quantification. For the MTT assay, cells were seeded in 96-well plates (Nunc) at an initial density of 2500 cells/well and treated for 48 h with 10% FBS-culture medium supplemented or not with BK (10 μM), vasoinhibins (60 nM), and the nonselective TRP channel blockers SKF-96365 (10 μM) [27,28], ruthenium red (10 mM), or Gd³⁺ (1 μM), as indicated. La³⁺ (10 μM), known to enhance the activity of TRPC4 and 5 [20], was also tested. In another group of experiments, BUVEC were incubated with vasoinhibins (60 nM) and BK (10 μM) in the presence or in the absence of okadaic acid (50 nM) for 48 h. Next, cells were incubated with MTT (500 mg/mL, Sigma-Aldrich) at 37 °C for 4 h, and the formazan precipitate was solubilized with 0.4 N HCl-10 % SDS for 30 min at room temperature and quantified by measuring absorbance at 570 nm.

PLC activity was measured according to the method of Hofmann and Majerus [29]. Reaction mixtures contained 280 μM phosphatidylinositol, 30,000 dpm of PIP₂-(myo-inositol-2-³H(N)), 1 mg/mL sodium deoxycholate, 1 mM CaCl₂, 50 mM HEPES (pH 7.0), and BUVEC extracts. After incubation for 15 min at 37 °C, BUVEC extracts were incubated for 5 min with vasoinhibins (60 nM) prior to a 2-min incubation with BK (10 μM). A two-min incubation with BK, vasoinhibins, or basal medium (ctl) was also tested. The reaction was stopped with 1 mL of chloroform/methanol (1:2 v/v), followed by 0.3 mL of 1 N HCl containing 5 mM EGTA. After centrifugation for 10 min at 3000 × g, the supernatant was removed for liquid scintillation counting. Protein content was measured using bicinchoninic acid (TM) (American Radiolabeled Chemicals, Inc., St. Louis, MO, USA).

The accumulation of IP₃ was measured by the receptor displacement assay described by Challiss et al. [30]. Briefly, BUVEC were incubated for 2 min with basal medium (ctl), vasoinhibins (60 nM) or BK (10 μM), this latter being preceded or not by a 5-min incubation with vasoinhibins. The reaction was stopped after periods of 1 and 2 min by the addition of ice-cold perchloric acid to extract IP₃. Following a 20-min incubation on ice, the tubes were centrifuged at 2000 × g for 15 min; the supernatants were neutralized with 5 N KOH. IP₃ content was assayed using the inositol-1,4,5-triphosphate [³H] radioreceptor assay procedure (Perkin Elmer Life Sciences, Inc., Boston, MA, USA), a competitive ligand binding assay.

Intracellular Ca²⁺ was measured using an Aminco-Bowman Series-2 luminescence spectrometer with a 150-W xenon source (Rochester, NY, USA). BUVEC were mechanically dispersed after a 2-min incubation with 0.01% trypsin-EDTA solution, centrifuged, and resuspended at a final density of 10⁶ cells/mL in HBSS (Hanks balanced salt solution) containing (in mM): 140 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 0.3 Na₂HPO₄, 0.4 KH₂PO₄, 4 NaHCO₃, 5 glucose, and 10 HEPES (adjusted to pH 7.4 with NaOH). Cells were loaded with Fura-2 by exposure to 2 μM Fura-2 AM (Molecular probes, Eugene, OR, USA) at 37 °C for 1 h in HBSS. After incubation, cells were washed three times in HBSS. Cell suspensions were then incubated under constant perfusion with Ca²⁺-free or 2 mM Ca²⁺-containing HBSS containing BK (10 μM) or thapsigargin (Tg, 1 μM), as indicated. Ca²⁺-free medium was prepared by omitting CaCl₂ and adding 10 mM EGTA. Prior to BK perfusion, BUVEC were incubated for 2 min with vasoinhibins (60 nM), heat-inactivated vasoinhibins, or prolactin (10 nM). Similarly, prior to Tg perfusion, BUVEC were incubated for 2 min with vasoinhibins (60 nM). Fura-2 fluorescence, at an emission wavelength of 510 nm, was recorded by exciting the probe alternately at 340 and 380 nm. Cytosolic [Ca²⁺] was derived from the 340 nm/380 nm signal ratio using the Grynkiewicz equation [31]. All reagents were diluted to their final concentration in HBSS and applied with a perfusion system. Data were accumulated from at least three measurements for each experiment.

NO production was assessed by measuring the accumulation of nitrites, a stable end product of NO metabolism, in the supernatant of BUVEC. Cells were grown in 12-well plates until 80% confluence and then incubated for 1 h with BK (10 μM) combined or not with vasoinhibins (60 nM), and in the presence or the absence of La³⁺ (10 μM). The culture media were collected. The amount of nitrites was determined spectrophotometrically by the Griess reagent. A 100-μL aliquot of sample was added to 100 μL of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride, and 2% phosphoric acid). After a 10-min incubation at room temperature, the absorbance was measured at 550 nm with a Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA), and the NO concentration in samples was determined using a curve calibrated with NaNO₂ standards.

eNOS activity was determined by L-citrulline assay in BUVEC grown in 12-well plates until 80% confluence that were then pretreated with okadaic acid (50 nM) for 10 min, then vasoinhibins (60 nM) for 1 h and followed or not by BK (10 μM) for 10 min. Protein concentration was evaluated using the Bradford assay (Bio-Rad). The reaction was carried out in reaction buffer (50 mM HEPES, 1 mM NADPH, 1.25 mM CaCl₂, 1 μM FAD, 1 μM FMN, and 10 μg/mL calmodulin) and initiated by adding 1 μCi/mL [³H]l-arginine and 50 μg of BUVEC protein in a final volume of 100 μL. After 1 h, ice-cold stop medium (50 mM HEPES, pH 5.5, and 4 mM EDTA) was added for 10 min. BUVEC extracts were then applied onto 1-mL columns of Dowex AG50WX8. [³H]L-citrulline was eluted with 1 mL water and quantified by liquid scintillation counting.

Total RNA from BUVEC was isolated using TRIzol (GIBCO/BRL, Life Technologies, Breda, The Netherlands) prior to reverse transcription using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). cDNA was amplified using TRPC5 (Forward primer 5′-TGCAACTGTGTGGAGTGTGT-3′, reverse primer 5′-TGTGGTCATCTCGATGGTTGA-3′), TRPC6 (Forward primer 5′-GTCATGAATGCAGCTGACAGA-3′, reverse primer 5′-CTTTACATTCAGCCCATATCAT-3′), TRPM6 (Forward primer 5′-CCAAGCACCTTTTCCAAATTCT-3′, reverse primer 5′-TCCCAAGCCATTGCCAGATT-3′), TRPM7 (Forward primer 5′-GCCCCGTGAGGAGAATGTC-3′, reverse primer 5′-GTATTTCATGGCAAGACTTGCA-3′), actin (Forward primer 5′-CCATCATGAAGTGTGACGTTG-3′, reverse primer 5′-ACAGAGTACTTGCGCTCAGGA-3′), and L19 (Forward primer 5′-CGAAATCGCCAATGCCAACTC-3′, reverse primer 5′-TGCTCCATG AGAATCCGCTTG-3′), and subsequently analyzed by agarose gel electrophoresis. Because identical amounts of cDNA were added to the PCR reaction, we estimated the changes in transcript expression levels relative to those of housekeeping genes.

BUVEC were pretreated with vasoinhibins (60 nM) for 1 h followed by incubation with BK (10 μM) for 1, 5, or 10 min. Protein samples were denatured by incubation in Laemmli buffer, and then subjected to SDS-PAGE. Immunoblots were incubated with polyclonal anti-phospho-Ser¹¹⁷⁹-eNOS antibody (1:1000 dilution; catalog 9571; Cell Signaling Technology, Danvers, MA, USA) overnight, and the antigen-antibody complex was detected using HRP-coupled secondary antibodies (catalog 111-035-003; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) and enhanced chemiluminescence (ECL; Super-Signal West Pico Chemiluminescent Substrate; Pierce Biotechnology, Rockford, IL, USA). Membranes were then stripped and probed with monoclonal anti-eNOS (1:1,000 dilution; catalog 33–4500, Zymed Laboratories Inc., Invitrogen, Carlsbad, CA, USA) and the alkaline phosphatase secondary antibody kit (Bio-Rad, Hercules, CA, USA).

Values are expressed as mean ± SEM. Statistical significance between groups was determined by analysis of variance (ANOVA) followed by unpaired Student's T-test. Differences in means with P < 0.05 were considered statistically significant. The statistical analysis was performed using the Sigma Stat 7.0 software (Systat Software Inc., San Jose, CA, USA).

Compared to untreated control cultures, BK induced a 50% increase in BUVEC proliferation that was significantly diminished by vasoinhibins at a dose of 60 nM (Figure 1A). The NO donor DETA-NONOate stimulated BUVEC proliferation to similar levels as BK, but it did not enhance BK action (Figure 1B) confirming first, that NO is a major mediator of BUVEC proliferation and second, that BK acts by promoting NO production. In contrast, DETA-NONOate prevented the vasoinhibin blockage of BK-stimulated BUVEC proliferation (Figure 1B). Vasoinhibins alone had no effect, and they could not prevent the stimulatory action of DETA-NONOate (Figure 1B), indicating that vasoinhibins signal upstream of NO production. Previous studies have unveiled eNOS activation as a target of vasoinhibin actions [5,14].

Inhibition of intracellular Ca²⁺ mobilization was postulated to mediate vasoinhibin blockage of eNOS activation [14], and BK mobilizes Ca²⁺ from intracellular stores by generating IP₃ [15]. We show that vasoinhibins reduced BK-stimulated PLC activity (Figure 2A) and IP₃ production (Figure 2B) to basal levels, but vasoinhibins alone had no effect on either parameter. Whether vasoinhibins interfere with BK stimulation of Gαq, Gαi, or B2 receptor proteins has not been determined yet. In this context, it is worth mentioning that vasoinhibins specifically bind to high-affinity saturable sites in endothelial cells [32] but their molecular nature remains to be unveiled so as to provide a proximal mechanism for vasoinhibin action.

IP₃ can promote extracellular Ca²⁺ entry by depleting Ca²⁺ stores, a process known as capacitative Ca²⁺ entry [33,34], but also by an independent pathway [34,35]. In particular, BK can stimulate both capacitative [34] and non-capacitative Ca²⁺ entry in endothelial cells [21,36-38]. To define the action of vasoinhibins in BK-induced extracellular Ca²⁺ entry, we quantified the amount of Ca²⁺ released from the endoplasmic reticulum and measured the magnitude of the Ca²⁺ entry in response to the addition of 2 mM Ca²⁺ to the bath solution. Vasoinhibins clearly reduced the depletion of intracellular Ca²⁺ stores in response to BK, as previously shown [14], and they also reduced the BK-induced capacitative Ca²⁺ entry (Figures 3A,B). This effect was specific since heat-denatured vasoinhibins (Figure 3C) and full-length prolactin (Figure 3D) lacked action. Importantly, BUVEC cell perfusion with thapsigargin, a well-known inhibitor of sarcoplasmic and endoplasmic reticulum Ca²⁺ ATPase that depletes intracellular Ca²⁺ stores without affecting IP₃ production, activated a Ca²⁺ influx through store-operated channels that was insensitive to vasoinhibins (Figures 3E,F). These data suggest that vasoinhibins can regulate the activity of plasma membrane channels independently of Ca²⁺ store depletion. These latter may be the BK-activated cation channels reported to mediate non-capacitative Ca²⁺ entry in endothelial cells [38].

The contribution of Ca²⁺ influx to vasoinhibin blockage of BK-induced BUVEC proliferation was examined next. Using the basal culture medium that contains 0.4 mM Ca²⁺, we can measure cell events that involve both Ca²⁺ entry and intracellular Ca²⁺ mobilization. The use of Ca²⁺-depleted medium eliminates Ca²⁺ influx, thereby helping to identify its contribution when compared to basal culture medium conditions. Figure 4 shows that BK required extracellular Ca²⁺ to exert its maximal stimulatory effect on BUVEC proliferation, but IP₃-induced Ca²⁺ mobilization of intracellular stores was sufficient for BK to increase BUVEC proliferation. Notably, vasoinhibins reduced the BK-induced endothelial cell proliferation to basal levels, both in the absence and in the presence of extracellular Ca²⁺ (Figure 4) indicating first, that vasoinhibins interfere with IP₃-induced Ca²⁺ mobilization and second, that they also affect extracellular Ca²⁺ influx to exert their inhibitory effect. We examined next the role of TRPC channels in the Ca²⁺ mobilization initiated by BK.

In the absence of specific inhibitors of TRPC channels, we tested SKF-96365, ruthenium red, and Gd³⁺, which have been extensively used to investigate the role of TRPCs [19,27,39]. These inhibitors have off-targets [39] including voltage-gated Ca²⁺ channels, which appear to be absent, however, in endothelial cells [19,38]. All three agents completely inhibited the proliferation of BUVEC in response to BK, supporting the contribution of TRPC channels to this phenomenon (Figure 5A). Under these conditions, vasoinhibins had no effect (Figure 5A). Notably, micromolar concentrations of La³⁺, known to inhibit TRPC1, 3, 6, and 7 channels [40] but to potentiate TRPC4 and 5 [20,41], enhanced the BK effect and prevented the inhibitory effect of vasoinhibins (Figure 5A). Furthermore, we observed that vasoinhibins blocked BK-induced NO production and that this effect disappeared in the presence of La³⁺ (Figure 5B). La³⁺ alone had no effect on either basal or BK-stimulated NO levels. These data suggest the involvement of TRPC4/5 in the BK-induced BUVEC proliferation. This is not unexpected since TRPC4 and TRPC5 have been shown to form nonselective cation channels, which integrate G-protein-coupled receptor signaling pathways that are activated independently of store depletion [20,42].

Based on sequence, function, pharmacology, and regulatory similarities, the seven members of the mammalian TRPC family are grouped in four subfamilies; TRPC4/5, TRPC1, TRPC3/6/7, and TRPC2 [19,20]. All of them except TRPC2 have been found in endothelial cells, but their expression pattern varies according to the vascular bed [19]. Notably, TRPC6 contributes to BK-induced mobilization of Ca²⁺ in capillary- and aorta-derived endothelial cells [21], and TRPC6-deleted mice are hypertensive [43]. Also, TRPC5 [44,45], but not TRPC4 [46], is a vascular target of NO. There is no report of TRP channel expression in BUVEC. RT-PCR analysis in BUVEC showed the expression of TRPC5 but not of TRPC6 (Figure 6A). Notably, the 48-h treatment of BUVEC with BK induced an apparent increase in TRPC5 expression that was reduced by the concomitant administration of vasoinhibins (Figure 6B). Interestingly, vasoinhibins alone had no effect on TRPC5 expression. Human umbilical vein endothelial cells (HUVEC) contain other types of TRP channels, known as members of the melastatin family (TRPM), specifically TRPM7 and its closest homologue TRPM6 [47]. Because silencing TRPM7 in HUVEC increases eNOS expression and NO production [48], we examined TRPM6 and TRPM7 mRNA expression in BUVEC (Figure 6C). The expression of TRPM6 and TRPM7 was detected in BUVEC but was not modified by adding BK alone or combined with vasoinhibins (Figure 6D). Together these observations suggest that TRPC5 channels may be a specific target for vasoinhibins in reducing BK vascular effects.

Phosphorylation of eNOS at Ser¹¹⁷⁹ renders eNOS more sensitive to Ca²⁺/calmodulin binding [23,24] and contributes to eNOS-vascular functions [49,50] and to BK signaling [22]. Western-blot analysis showed that BK enhanced eNOS phosphorylation at Ser¹¹⁷⁹ in BUVEC as compared to untreated cells, whereas incubation with vasoinhibins for 1 h prior to BK addition blocked BK-induced eNOS phosphorylation (Figure 7A). Phosphorylation levels in the presence of vasoinhibins alone were similar to those of untreated controls. Figure 7B shows quantification of phosphorylated eNOS after normalizing for the amount of total eNOS on the gel. Phosphorylation of eNOS occurred within 1 min after addition of BK, and the level of phosphorylation did not further increase with longer exposure to BK (5 and 10 min).

Because protein phosphatase-2A (PP2A) dephosphorylates eNOS at Ser¹¹⁷⁹ [15], and vasoinhibins activate PP2A in BUVEC [5], we used okadaic acid (OA), a PP2A inhibitor [51], to investigate whether the inhibition by vasoinhibins of BK-induced eNOS activity and BUVEC proliferation is mediated by PP2A. Consistent with the inhibitory effect of vasoinhibins on NO production and eNOS phosphorylation, vasoinhibins blocked BK-induced eNOS activity in BUVEC, and this effect was prevented by OA (Figure 7C). OA alone had no effect on either basal eNOS activation or BK-induced eNOS stimulation (Figure 7C). Similarly, OA abrogated the vasoinhibin effect on BK-induced BUVEC proliferation, and OA alone did not modify basal growth (Figure 7D). Altogether these data show that vasoinhibins prevent the BK-induced BUVEC growth via PP2A-mediated inactivation of eNOS.