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Am J Physiol Heart Circ Physiol 290: H948-H958, 2006. First published September 23, 2005; doi:10.1152/ajpheart.00868.2005
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Positive cooperativity between the thrombin and bradykinin B2 receptors enhances arachidonic acid release

Claudie Hecquet,1 Dauren Biyashev,1 Fulong Tan,1,2 and Ervin G. Erdös1,2

Departments of 1Pharmacology and 2Anesthesiology, University of Illinois College of Medicine at Chicago, Chicago, Illinois

Submitted 12 August 2005 ; accepted in final form 20 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Bradykinin (BK) or kallikreins activate B2 receptors (R) that couple G{alpha}i and G{alpha}q proteins to release arachidonic acid (AA) and elevate intracellular Ca2+ concentration ([Ca2+]i). Thrombin cleaves the protease-activated-receptor-1 (PAR1) that couples G{alpha}i, G{alpha}q, and G{alpha}12/13 proteins. In Chinese hamster ovary cells stably transfected with human B2R, thrombin liberated little AA, but it significantly potentiated AA release by B2R agonists. We explored mechanisms of cooperativity between constitutively expressed PAR1 and B2R. We also examined human endothelial cells expressing both Rs constitutively. The PAR1 agonist hexapeptide (TRAP) was as effective as thrombin. Inhibitors of components of G{alpha}i, G{alpha}q, and G{alpha}12/13 signaling pathways, and a protein kinase C (PKC)-{alpha} inhibitor, Gö-6976, blocked potentiation, while phorbol, an activator, enhanced it. Several inhibitors, including a RhoA kinase inhibitor, a [Ca2+]i antagonist, and an inositol-(1,3,4)-trisphosphate R antagonist, reduced mobilization of [Ca2+]i by thrombin and blocked potentiation of AA release by B2R agonists. Because either a nonselective inhibitor (isotetrandrine) of phospholipase A2 (PLA2) or a Ca2+-dependent PLA2 inhibitor abolished potentiation of AA release by thrombin, while a Ca2+-independent PLA2 inhibitor did not, we concluded that the mechanism involves Ca2+-dependent PLA2 activation. Both thrombin and TRAP modified activation and phosphorylation of the B2R induced by BK. In lower concentrations they enhanced it, while higher concentrations inhibited phosphorylation and diminished B2R activation. Protection of the NH2-terminal Ser1-Phe2 bond of TRAP by an aminopeptidase inhibitor made this peptide much more active than the unprotected agonist. Thus PAR1 activation enhances AA release by B2R agonists through signal transduction pathway.

protease-activated-receptor-1; thrombin receptor activator peptide; protein kinases; potentiation; calcium; kallikrein

THROMBIN, the potent physiological activator of platelet aggregation, is a major factor in myocardial infarction and other thrombotic processes (15, 22, 32). In addition to promoting coagulation, thrombin plays a central role in the cellular response to injury or inflammation and wound repair. Thrombin initiates many of these complex cellular events by cleaving the Arg41-Ser42 bond of the protease-activated receptor-1 (PAR1) to expose a new NH2 terminus. This terminus then acts as a tethered ligand (13, 38) and activates PAR1. Various guanine nucleotide-binding (G) protein {alpha}-subunits can then couple with PAR1. When coupled to G{alpha}q protein, PAR1 generates diacylglycerol (DAG) and inositol-(1,4,5) trisphosphate (IP3), which in turn binds to IP3 receptors (Rs) on the endoplasmic reticulum to trigger intracellular calcium concentration [Ca2+]i release. Interaction of IP3R with transient R potential channel 1 (TRPC1) is required to activate store depletion-induced Ca2+ entry (21, 27, 31). In addition to G{alpha}q, the stimulated PAR1 couples G{alpha}i/0 (14, 25, 37) and G{alpha}12/13 proteins (12, 17, 27). Activation of the G{alpha}i/o protein leads to release of the prostaglandin precursor arachidonic acid (AA).

Activation of B2Rs for bradykinin (BK) can cause among others hypotension, bronchoconstriction, pain, or inflammation (3, 16) and releases vascular mediators such as NO, or prostaglandins, and others (3, 8–11). The PAR1–4 group of thrombin and trypsin Rs, as their name indicates, were thought to be unique protease-activated Rs. However, we showed that the human BK B2R can be activated by certain proteases as well (20). Kallikreins and some other human or bacterial serine proteases, including cathepsin G or endoarginase C, can activate B2 BK Rs directly (19, 20). Furthermore, activation of the ubiquitous B2R by BK or proteolytic enzymes (20) recruits both G{alpha}i and G{alpha}q proteins, as indicated by the release of AA and elevation of [Ca2+]i (18, 26).

Burch and colleagues (5–7) found that BK and thrombin synergized prostaglandin synthesis in fibroblasts, and after prolonged stimulation, thrombin amplified responses to BK. After we discovered that kallikrein and other proteases directly activate the BK B2Rs (19, 20), we used cultured cells to determine if thrombin would enhance AA release by kallikrein via B2R and if PAR1 activation potentiates kallikrein via signal transduction pathways. We established that the two proteases acted on a single R, either PAR1 or B2, because we obtained the same results with peptide agonists specific for the individual Rs. We used inhibitors of the major factors in signal transduction to point out the steps in the potentiation process.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chinese hamster ovary (CHO) cells were purchased from American Type Culture Collection (Rockville, MD). Human pulmonary artery endothelial (HPAE) cells were obtained from Clonetics (Walkersville, MD). The cDNA encoding the human BK B2R was donated by Dr. K. Jarnigan, Syntex (Palo Alto, CA), and the human regulator of G protein signaling type 10 (RGS10) cDNA was from the University of Missouri-Rolla (Rolla, MO). Mammalian expression vectors pcDNA3 were from Invitrogen (San Diego, CA); lipofectin and geneticin (G418) were from GIBCO-BRL (Gaithersburg, MD). [5,6,8,9,11,12,14,15-3H(N)]AA (100 Ci/mmol) and [3H]BK were purchased from American Radiolabeled Chemicals (St. Louis, MO). The fura-2 AM, anti-green fluorescent protein (anti-GFP) polyclonal antibodies were obtained from Molecular Probes (Eugene, OR), and D-valyl-leucyl-arginyl-aminomethyl-coumarine (D-Val-Leu-Arg-AMC) was from Enzyme Systems Products (Livermore, CA). Bromoenol lactone (BEL) was obtained from Cayman Chemical Company (Ann Arbor, MI), and cytosolic phospholipase A2 (cPLA2) inhibitor (N-{(2S,4R)-4-(biphenyl-2-ylmethyl-isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolidin-2-ylmethyl}-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-phenyl]acrylamide, HCl), Gö-6976, Y-27632, H-89 dihydrochloride, 2-aminoethoxydiphenyl borate (2-APB), and 8-(N,N-diethylamino)octoyl-3,4,5-trimethoxybenzoate (TMB-8) were from Calbiochem (La Jolla, CA). Human thrombin and human plasma kallikrein were from Enzyme Research Lab (South Bend, IN), BK, porcine pancreatic kallikrein, thermolysin, trypsin, genistein, culture media, penicillin, phorbol-12-myristate 13-acetate (PMA), thrombin R activator peptide (TRAP) and scrambled TRAP (scTRAP), amastatin, and other peptides and chemicals were purchased from Sigma Chemical (St. Louis, MO). HOE-140 was a gift from Dr. B. A. Schölkens, Novartis (Frankfurt, Germany). The Pro-Q diamond phosphoprotein gel stain kit was from Molecular Probes (Eugene, OR).

Cell culture and transfection. CHO cells were grown in Ham's F-12 culture medium supplemented with L-glutamine, penicillin-streptomycin, HEPES buffer, and 10% fetal bovine serum (FBS). One day before transfection, cells were seeded into 60-mm dishes at 30–40% confluence. The human B2R and human B2R COOH terminally tagged with the GFP (B2-GFPct) were inserted into a pCDNA3 vector, and CHO cells were transfected with human BK B2R (CHO/B2), the B2-GFPct (CHO/B2-GFPct), or cotransfected with human BK B2R and RGS10 (CHO/B2 + RGS10) using lipofectin. After 2 h incubation at 37°C, cells were thoroughly washed with DMEM or Ham's F-12 medium, and 2 ml of the same medium containing geneticin (G418) was added to start the selection of stably B2 or tagged B2R-transfected cells. Blasticidin was added to select the RGS10 cotransfected cells. Different clones were selected and propagated using cloning rings. HPAE cells, which constitutively express BK B2Rs, were grown to confluence (4–6 passages).

[3H]BK saturation binding assay. To select the transfected clone with the highest expression of B2 or B2-GFPct Rs and to quantitate the effect of thrombin on BK binding to its R, we used a [3H]BK saturation binding assay (20). We found that both human BK B2 and B2-GFPct were expressed on CHO plasma membrane; we estimated the B2R to be 5.8 x 104 per cell and the B2-GFPct to be 1.4 x 105 per cell.

Ca2+ mobilization. We assessed the effectiveness of specific Ca2+ release inhibitors by measuring [Ca2+]i levels in CHO/B2 cells with a microspectrofluorometer (PTI Deltascan, Princeton, NJ) coupled to an inverted microscope and the Ca2+-sensitive fluorescent dye fura-2 AM (20). The excitation wavelengths were 340 and 380 nm, and the emission wavelength was 510 nm. The signals are represented in figures as a ratio of bound/free Ca2+, F340/F380.

[3H]AA release. Endothelial and transfected CHO cells were grown to confluence in six-well dishes. The medium was replaced with 1 ml of Ham's F-12 medium containing 0.5 µCi/ml of [3H]AA, and cells were incubated for 16 h at 37°C. The labeled cells were then serum starved for 3 h before each assay. After washing with incubation medium (Ham's F-12 medium plus 0.1% bovine serum albumin, BSA), they were incubated for 30 min at 37°C with either medium alone, 1–50 nM BK, or enzymes with or without the B2R blocker HOE-140 (0.5 µM). The medium was removed and its [3H]AA content was measured by scintillation counting. The amount of released [3H]AA was expressed after subtracting the background (spontaneous release) (19). The experiments were carried out routinely in triplicate.

Thrombin and kallikrein assay. A putative direct effect of TRAP on human plasma kallikrein activity and inactivation of thrombin by diisopropyl fluorophosphate (DFP) were followed fluorometrically by measuring cleavage of D-Val-Leu-Arg-AMC substrate by the two enzymes (1, 36). Plasma kallikrein (100 nM) was preincubated with increasing concentrations of TRAP from 1 µM to 300 µM for 15 min. Fifty microliters of kallikrein solution were then mixed with 445 µl of 0.1 M Tris·HCl (pH 8.0). Addition of 5 µl of AMC substrate to final concentration of 0.1 mM initiated the reaction. The liberated fluorogenic coumarin derivative was measured in a spectrofluorometer with excitation at 380 nm and emission at 460 nm wavelength; the increase in fluorescence was monitored with a recording spectrofluorometer. We used thrombin irreversibly inhibited by DFP as an additional control.

Human BK B2 immunoprecipitation and phosphorylation. CHO/B2-GFPct cultures in six-well culture dishes were treated with 1 µM BK in the presence or absence of increasing concentrations of thrombin (3 nM–1 µM) for 30 min at 37°C. Cells were washed with ice-cold PBS and lysed with 0.4 ml 0.5% deoxycholate (DOC) buffer (pH 7.5) containing 1% NP-40, 0.1% SDS, 1 mM PMSF, 50 mM Tris, 150 mM NaCl, and 10 µl protease inhibitor mixture. After being shaken for 10 min at 4°C, the lysates were sonicated and then centrifuged at 16,000 g for 15 min at 4°C. Supernatants were collected and diluted with 390 µl of 50 mM Tris buffer (pH 7.5) containing 150 mM NaCl and protease inhibitors. Samples were then incubated with 1 µg rabbit anti-GFP antibody overnight at 4°C. B2-GFPct R immune complexes were precipitated with protein A-Sepharose beads (Sigma) at 4°C for 2 h. The beads were then washed five times with lysis buffer, and the precipitated proteins were eluted by boiling the beads in sample buffer [80 mM Tris (pH 6.8), 3% SDS, 15% glycerol, 0.01% bromophenol blue, 5% DTT]. Proteins were then separated on a 4–20 % gradient SDS-PAGE gel. The phosphorylated R was selectively stained with a Pro-Q diamond phosphoprotein gel stain kit, and its 555/580 nm excitation/emission maxima were detected with a visible-light-scanning instrument (Molecular Imager FX Pro Plus, Bio-Rad). Density of the band was quantified using Labworks software, and data were expressed as the ratio of phosphorylated protein density/total protein density.

Statistical analysis. Means and SEs were calculated for all experiments, and statistical significance of differences between means was tested by a paired t-test (Microsoft Excel).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Thrombin enhances [3H]AA release by BK B2R agonists. BK or kallikrein released [3H]AA from labeled CHO cells that were stably transfected to express human B2R (CHO/B2). Although these cells express thrombin PAR1 constitutively, direct activation of PAR1 by 10 nM thrombin for 30 min at 37°C released relatively little labeled AA. If the amount released spontaneously was taken as baseline to equal 1.0, the value for AA was 1.76 ± 0.4-fold over the baseline. Figure 1A shows that the amount of AA released by thrombin was unaffected by 0.5 µM HOE-140, an inhibitor of the BK B2R. However, when thrombin activated PAR1 first, subsequent stimulation of B2R with porcine pancreatic kallikrein nearly doubled [3H]AA release from 6.4 ± 1.5 to 12.6 ± 2 (relative units). Similar results were obtained with human plasma kallikrein (5.9 ± 0.8 to 11.0 ± 0.7) and BK (6.3 ± 1.7 to 11.8 ± 2.6), where thrombin-activated cells reacted to the agonists with a significant increase in release of AA. HOE-140 (0.5 µM) completely blocked the effects of thrombin receptor activation. We consistently recorded thrombin potentiation of B2 agonists in numerous experiments: n = 21 for BK (1 nM) and n = 17 for tissue kallikrein (1–10 nM). HPAE cells that constitutively express both receptors confirmed positive cooperativity (P < 0.01) between PAR1 and BK B2Rs (Fig. 1A, right). Human thrombin (10 nM) increased basal release of AA by endothelial cells slightly (1.4 ± 0.1) but also potentiated the effect of BK and plasma kallikrein on the BK B2R significantly.


Figure 1
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  		Fig. 1. Protease-activated receptor-1 (PAR1) activation by human thrombin and thrombin R activator peptide (TRAP) potentiates bradykinin (BK) B2 receptor (R) agonists. Chinese hamster ovary (CHO) cells transfected with human B2Rs (CHO/B2) and human pulmonary artery endothelial (HPAE) cells were incubated 30 min at 37°C with 1 nM BK, 10 nM human plasma kallikrein (KLK), or porcine tissue KLK in the presence or absence of 10 nM human thrombin (thr) (A), 30 µM TRAP, or 30 µM scrambled TRAP {scrTRAP: [3H]arachidonic acid ([3H]AA) release is expressed on the ordinate in relative units; B}. Thrombin and TRAP enhanced [3H]AA release by B2R agonists; this response was blocked by the B2 antagonist HOE-140. Solid bars, agonist alone; diagonally hatched bars, combination of B2R agonist and PAR1 agonist; vertically hatched bars, combination of B2R agonist and PAR1 agonist in the presence of 0.5 µM HOE-140. All data are means ± SE of 3–5 separate determinations. *P < 0.05 vs. control agonist alone. C: thrombin and TRAP did not potentiate wild-type (WT) CHO cells that express PAR1 but lack B2R. Solid bars, agonist alone; diagonally hatched bars, PAR1 agonist alone; vertically hatched bars, combination of B2R agonist and PAR1 agonist. Data are means ± SE from 3–5 separate experiments.

 
The synthetic thrombin R activating peptide SFLLRN (TRAP) is composed of the first six amino acids of the new NH2-terminal tethered ligand that is revealed when thrombin cleaves PAR1. This peptide functions as a PAR1 agonist that activates the R independently of thrombin and proteolysis (12, 38). TRAP was used as a control for two reasons: it is specific for the PAR1 and it ensures that enhanced B2 agonist activity results from PAR1 activation, not from thrombin acting on B2R (Fig. 1B). Like thrombin, TRAP (30 µM) released trace amounts of [3H]AA from CHO/B2 cells (2.4 ± 0.8), and the B2R blocker HOE-140 (2.4 ± 0.1) did not affect it. TRAP doubled [3H]AA released by either plasma kallikrein (6.4 ± 1.5 to 13.0 ± 2) or BK (6.3 ± 1.7 to 12.9 ± 2.0), a significant elevation over control cells blocked by HOE-140. In separate experiments in vitro, TRAP did not inhibit substrate hydrolysis by kallikrein (not shown). Reaction to the PAR1 peptide agonist was specific, because 30 µM of a scrambled TRAP peptide (scrTRAP, LFRSLN) neither increased basal [3H]AA nor potentiated [3H]AA release induced by kallikrein or BK (Fig. 1B, right). As an additional control, thrombin inhibited by 1 mM DFP failed to activate PAR1 and did not modify [3H]AA release by B2R agonists. Together these data show that activation of the thrombin PAR1 leads to a positive cooperativity with BK R agonists, resulting in increased receptor-mediated activities.

We did additional experiments with wild-type (WT) CHO cells that have native PAR1 Rs but lack the B2Rs. Figure 1C shows that treatment of WT CHO cells with BK, porcine pancreatic, and human plasma kallikreins did not release [3H]AA. Basal release of [3H]AA induced by 10 nM thrombin (1.2 ± 0.7) or TRAP peptide (1.2 ± 0.2) was not significantly increased by added BK, tissue kallikrein, or plasma kallikrein in three separate experiments. These data confirm that expression of both receptors is required for receptor-to-receptor crosstalk and the positive cooperativity reflected by enhanced release of labeled AA.

Inhibitors of PKC{alpha} block potentiation. To determine the mechanisms of thrombin potentiation, we used selective inhibitors of G protein (G{alpha}i and G{alpha}q) signaling pathways, phospholipases (PLA2, PLCbeta) and protein kinases that regulate G protein coupled R activity. Because the PKC{alpha} subtype phosphorylates transient receptor potential Cl (TRPC1) channel and regulates store-operated Ca2+ entry in endothelial cells on PAR1 activation (2), we inhibited PKC{alpha} to see if it could block potentiation of AA release by thrombin. CHO/B2 cells were pretreated for 30 min with either a nonspecific PKC inhibitor, calphostin C (100 nM), or a specific inhibitor of PKC{alpha}, Gö-6976 (100 nM) (30). PKC is not essential for regulation of the B2R activity (4), and calphostin C and Gö-6976 did not inhibit AA release by either kallikrein or BK significantly (Fig. 2). Addition of either calphostin C or Gö-6976 inhibited thrombin potentiation without significantly affecting basal B2R activity because it was only slightly less than in untreated cells (Fig. 2A). Thrombin-potentiated AA release by BK was significantly reduced by calphostin C (7.3 ± 0.9) and Gö-6976 (9.4 ± 1.1) compared with control cells (15.9 ± 1.4). The thrombin-enhanced response to kallikrein was similarly blocked by calphostin C (4.1 ± 0.3) or Gö-6976 (6.1 ± 0.9) vs. control (10.2 ± 1.1) (Fig. 2B). Gö-6976 also inhibited the potentiation by TRAP (30 µM) and reduced the augmentation of either BK (15.1 ± 1.3 vs. 9.7 ± 1.2) or kallikrein (10.3 ± 0.8 vs. 5.5 ± 0.8) responses. Phorbol 12-myristate 13-acetate (PMA, 100 nM) had opposite effects; it enhanced thrombin potentiation of both BK (from 15.9 ± 1.4 in controls to 21.2 ± 1.3) and kallikrein (from 10.2 ± 1.1 to 16.3 ± 1.2) (Fig. 2).


Figure 2
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  		Fig. 2. Inhibitors of protein kinase C (PKC)-{alpha} blocked thrombin-induced potentiation. CHO/B2 cells were incubated with 100 nM Gö-6976, calphostin C (Calph C) or phorbol 12-myristate 13-acetate (PMA) for 30 min; then 1 nM BK (A) or 10 nM KLK (B) was added in the presence or absence of 10 nM human thrombin or 30 µM TRAP. Ordinate as in Fig 1. Solid bars, agonist alone; diagonally hatched bars, PAR1 agonist alone; vertically hatched bars, combination of B2R agonist and PAR1 agonist. Data are means ± SE from 3–5 separate experiments. *P < 0.05 vs. control untreated cells. Phorbol enhanced the potentiation by PAR1 agonists, but PKC inhibitors blocked it.

 
Because different signaling pathways resulting from PAR1 activation might be involved in enhancement of the BK B2R agonists, we applied additional inhibitors of serine/threonine kinases: a PKG inhibitor, RQIKIWFQNRRMKWKKLRKKKKH (0.5 µM); a PKA inhibitor, H-89 dihydrochloride (0.5 µM); an IP3 kinase inhibitor, wortmannin (0.5 µM); and a tyrosine kinase inhibitor, genistein (100 µM). None of these agents significantly modified the thrombin potentiation of BK on the B2R (Table 1), indicating that it does not require PKG, PKA, IP3 kinase, or tyrosine kinase. Methyl-beta-cyclodextrin (MCD) can be used to deplete cholesterol from cell membranes and lipid rafts. Because pretreatment of CHO/B2 cells with 5 mM of MCD for 30 min at 37°C had no effect, it is highly unlikely that thrombin potentiates B2R by heterodimerization with PAR1 (Table 1).


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  		Table 1. Negative inhibitors of B2R potentiation

 
Role of G{alpha}12/13 in potentiation. Inhibition of PKC{alpha}, a downstream effector of G{alpha}q, blocked potentiation of B2R agonists by thrombin. Stimulation of G{alpha}q generates DAG and IP3, and IP3 then binds to IP3R in the endoplasmic reticulum to trigger [Ca2+]i release. The interaction of IP3R with TRPC1 channels activates store depletion-induced Ca2+ entry (21, 27, 31). Together with DAG, Ca2+ mobilized from intracellular stores activates PKC{alpha}. Figure 3 shows that activation of the IP3R and the subsequent [Ca2+]i release are required for the thrombin potentiation phenomenon. CHO/B2 cells were pretreated for 30 min with either 75 µM of 2-APB to inhibit the R of IP3 or 20 µM of TMB-8, a [Ca2+]i antagonist. 2-APB blocked the IP3R dependent [Ca2+]i mobilization (Fig. 3A) and the thrombin-induced potentiation of B2 agonists (4.7 ± 0.6 compared with 9.9 ± 0.8) (Fig. 3B). Potentiation by BK was also reduced in cells pretreated with the [Ca2+]i antagonist TMB-8 (7 ± 0.6 compared with 9.9 ± 0.8), a weaker inhibitor of Ca2+ flux (Fig. 3A).


Figure 3
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  		Fig. 3. Role of G{alpha}12/13 in potentiation. A: inhibition of thrombin-induced intracellular calcium concentration ([Ca2+i]) release. Fura-2 AM-loaded CHO/B2 cells were incubated with 75 µM 2-aminoethoxydiphenyl borate (2-APB), 20 µM 8-(N,N-diethylamino)octoyl-3,4,5-trimethoxybenzoate (TMB-8), or 10 µM Y-27632 for 30 min to inhibit inositol-(1,3,4) trisphosphate (IP3) R, [Ca2+]i release, or RhoA kinase. Cells were then exposed to 10 nM thrombin. Abscissa: time in s. Ordinate: relative [Ca2+]i level. Experiments were repeated 3 times with similar results. B and C: [3H]AA-loaded CHO/B2 cells were treated with 75 µM 2-APB, 20 µM TMB-8, or 10 µM Y27632 for 30 min at 37°C and then exposed to 1 nM BK (B) or 10 nM plasma kallikrein (C) with or without 10 nM thrombin. Solid bars, agonist alone; diagonally hatched bars, PAR1 agonist alone; vertically hatched bars, combination B2R agonist and PAR1 agonist. Data are means ± SE from 3–5 separate experiments. *P < 0.03 comparing B2R agonists in thrombin-stimulated control cells. 2-APB blocks Ca2+ entry into cells; the 2 other agents reduced it. Inhibitors abolished or reduced potentiation by thrombin. TMB-8 blocked potentiation of BK but not that of kallikrein.

 
PAR1 agonists can activate other heterotrimeric G proteins, the G{alpha}i (14, 25, 37) and the G{alpha}12/13 (13, 17, 27) that initiate cascading downstream processes. The released {alpha}-subunit of G12/13 activates P115 Rho guanine nucleotide exchange factor (p115RhoGEF) and thereby also Rho. To determine whether thrombin potentiates BK effects through the G{alpha}12/13 pathway as well, we pretreated CHO/B2 cells for 30 min with 10 µM Y-27632, a Rho kinase inhibitor. This inhibitor decreased extracellular Ca2+ entry into thrombin-activated cells (Fig. 3A) and consequently lessened the potentiation of B2 agonist (6.8 ± 0.7 vs. untreated cells 9.95 ± 0.8). It follows that the mechanism of potentiation at the B2R requires Rho acting via IP3R to induce [Ca2+]i release and activate the subsequent signaling cascade. Based on our experiments with inhibitors, thrombin appears to potentiate plasma kallikrein differently than BK. Even though 2-APB completely blocked the thrombin-induced potentiation of kallikrein just as for BK, neither TMB-8 nor the RhoA kinase blocker Y-27632 blocked it (Fig. 3C). These data thus suggest that influencing the R of IP3 is a focal point of interaction between the B2R and PAR1 signaling pathway. Additional experiments indicated that kallikrein and BK regulate [Ca2+]i mobilization through different mechanisms (D. Biyashev, unpublished observations).

Thrombin enhances cytosolic PLA2 activation. Among the different isoforms of PLA2, cPLA2 is a major factor for receptor-regulated release of AA from membrane phospholipids (24). The activity of cPLA2 is Ca2+ dependent, and increased [Ca2+]i triggers translocation from the cytosol to plasma membranes (34, 35) to enhance its activity. Because the thrombin potentiation effect requires mobilization of [Ca2+]i, we investigated the role of cPLA2. We preincubated CHO/B2 cells for 30 min with 20 µM of isotetrandrine, a nonselective inhibitor that blocks both Ca2+-dependent and Ca2+-independent PLA2s. When both Ca2+-dependent and Ca2+-independent PLA2s were inhibited, basal release of [3H]AA by 1 nM BK or by 10 nM plasma kallikrein was blunted (2.5 ± 0.1 vs. 5.9 ± 0.3 and 3.9 ± 0.5 vs. 7.4 ± 0.9), as was potentiation of these agonists by thrombin. A more specific inhibitor of cPLA2 (20 nM; see MATERIALS AND METHODS) similarly reduced the effects of B2R agonists or plasma kallikrein on untreated cells as well as their potentiation by thrombin. In cells where cPLA2 was inhibited, release of AA fell to approximately one-half that in uninhibited cells. We used BEL, a specific Ca2+-independent PLA2 (iPLA2) inhibitor, as a control. Figure 4 shows that BEL (20 µM) after 30 min preequilibration inhibited the release of [3H]AA by BK by 32% (4.0 ± 0.5 vs. 5.9 ± 0.3) but not that of plasma kallikrein. However, the relative potentiation by thrombin of either B2R agonist was not abolished by BEL. Even though the release of AA by BK in the presence of thrombin decreased, the ratio of [3H]AA released by BK in presence of thrombin (7.9 ± 0.7) over that released in absence of thrombin (4.0 ± 0.5) was similar to the ratio in control cells (9.9 ± 0.8 over 5.9 ± 0.3) (Fig. 4).


Figure 4
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  		Fig. 4. Thrombin enhances cytosolic phospholipase A2 (cPLA2) activation by B2 agonists to release AA. CHO/B2 cells were treated with 20 µM of isotetrandrine (isot), a specific cPLA2 inhibitor (cPLA2 inhib; 100 nM), or with 20 µM bromoenol lactone (BEL) for 30 min before incubation with agonists for a further 30 min. Solid bars, BK (1 nM; A) or plasma kallikrein (10 nM; B); diagonally hatched bars, thrombin (10 nM); open bars, thrombin + B2R agonist. Data are means ± SE from 3–4 separate experiments. *P < 0.01, **P < 0.005, significant differences with untreated cells stimulated with B2R agonists and thrombin. The cPLA2 inhibitors reduced AA release and abolished potentiation (*), but BEL did not. The expression of regulator of G protein signaling type 10 (RGS10), a negative modulator of G{alpha}i/o, completely blocked B2R agonists even in presence of thrombin.

 
Effect of transfected G{alpha}i negative regulator. We repeated our thrombin potentiation experiments in CHO/B2 cells that were cotransfected with a regulator of G protein signaling type 10 (RGS10), which negatively modulates G{alpha}i/o signaling on Rs activation. Figure 4 shows that expression of RGS10 completely blocked B2R agonists, even in the presence of thrombin. RGS10 serves as a GTPase-activating protein for G{alpha}i/o, but it may also regulate the G{alpha}q subunit (33). Data from these experiments suggest that thrombin mobilizes [Ca2+]i to activate the Ca2+-dependent cPLA2 and thus potentiates the activity of BK B2R agonists as reflected by a doubling of AA release.

Thrombin modifies the phosphorylation of the BK R. To determine how PAR1 agonists augment B2 agonists, we investigated their actions on B2R phosphorylation. We used 1 µM BK to detect the marked phosphorylated R better. Figures 5A and 6A show changes in [3H]AA release from CHO/B2 cells. The response to BK alone was taken as 100% and plotted vs. results from increased concentrations of PAR1 agonists. Thrombin potentiated either 1 µM or 1 nM BK, but the response was more prominent at the lower BK concentration (Fig. 5A). Figure 5B shows the precipitation of B2R with antibodies to GFP in CHO/B2-GFP-transfected cells. CHO/B2-GFPct cells were incubated for 30 min with 1 µM BK (37°C), and increasing concentrations of thrombin were added. We established that the GFP tag attached to the COOH terminus of the B2R affected neither release AA nor mobilization of [Ca2+]i (19). Furthermore, this manipulation did not influence potentiation by thrombin (data not shown). The phosphorylated R was quantified and normalized to the total B2 protein level using the ratio of phosphorylated protein density/total protein density (Fig. 5B). The increased basal phosphorylation of the receptor after BK challenge (4.3 ± 0.5-fold) was taken as 100%. Figure 5B shows how B2R phosphorylation changed with increasing amounts of thrombin. In the absence of added BK, thrombin did not phosphorylate the BK B2R (data not shown), but it enhanced BK-induced phosphorylation of B2-GFPct R in a concentration-dependent manner, reaching a maximum of 166% at 30 nM. Higher concentrations of thrombin decreased it. The phosphorylation of B2R that thrombin augmented was inhibited in cells pretreated with either 75 µM of 2-APB or 100 nM of Gö-6976 (Fig. 5B).


Figure 5
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  		Fig. 5. Effect of thrombin on stimulated B2R phosphorylation. A: CHO/B2-transfected cells were incubated with 1 nM or 1 µM BK in the presence or absence of increasing concentrations of thrombin (abscissa). The increase in [3H]AA is expressed as % on the ordinate. The effect of BK without thrombin is taken as 100%. B: thrombin modified BK-induced B2R phosphorylation (P) in a concentration-dependent manner. CHO/B2-COOH-terminally tagged with green fluorescent protein (B2GFPct)-transfected cells untreated (+) or treated with 100 nM Gö-6976 ({square}) or 75 µM 2-APB({blacktriangleup}) were incubated with 1 µM BK (30 min) and increasing concentrations of thrombin. Data are from 3 separate determinations. BK increased the basal level of phosphorylation of the B2R by 4.3 ± 0.5-fold (n = 3) taken as 100%. Thrombin in low concentrations enhanced phosphorylation of B2GFP by BK; higher concentrations blocked it. Effects were abolished by 2-APB or Gö-6976.

 

Figure 6
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  		Fig. 6. Effect of aminopeptidase inhibitor on [3H]AA release and B2R phosphorylation stimulated by TRAP. A: TRAP potentiates stimulation of CHO/B2 transfected cells by 1 nM BK (bullet) or 1 µM BK ({blacktriangleup}) to release AA in a concentration-dependent manner. Effects of TRAP were increased by 50 µM amastatin ({circ} for 1 nM BK and {triangleup} for 1 µM BK). Ordinate and abscissa as in Fig. 5. B: TRAP modifies BK-induced B2R phosphorylation in a concentration-dependent manner. CHO/B2-GFPct transfected cells were incubated with 1 µM BK (30 min) and increasing concentration of TRAP ranging from 1 to 300 µM with or without 50 µM amastatin. Details as in Fig. 5. Aminopeptidase inhibitor amastatin blocked cleavage of NH2 terminus of TRAP and enhanced its activity.

 
Similarly to thrombin, TRAP potentiated either 1 nM or 1 µM BK on CHO/B2-transfected cells. [3H]AA was released in a concentration-dependent manner with maximum at 10–30 nM of peptide. BK alone increased the basal level of phosphorylation; this increase was taken as 100%, and data from additional treatments are expressed relative to this (Fig. 6A). Because an aminopeptidase on the cell surface rapidly degrades TRAP, we repeated this experiment in the presence of amastatin (50 µM), an aminopeptidase inhibitor. When hydrolysis of the NH2-terminal Ser of TRAP was blocked by amastatin, as little as 3 µM TRAP potentiated the BK response. Increasing amounts of TRAP augmented B2R phosphorylation by BK (1 µM); maximum phosphorylation (192% of control) was obtained with 30–100 µM TRAP in the absence of the aminopeptidase inhibitor. When 50 µM amastatin was added to the reaction mixture, the effect of TRAP resembled that of thrombin (Figs. 5B and 6B). TRAP potentiation of BK-induced B2R phosphorylation reached a maximum (232%) between 3 and 10 µM concentration of the peptide, and higher concentrations decreased it (Fig. 6B).


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We found that activation of PAR1 by either thrombin or the hexapeptide TRAP potentiates BK B2R agonists to enhance release of [3H]AA. AA is the precursor of prostaglandins, such as prostacyclin, a coronary vasodilator, and prostaglandin E2, which prevents platelet aggregation. It is also a precursor of thromboxane (8, 35). Although thrombin releases AA from many cell types, we used CHO cells in most of our experiments because they express native PAR1. Thrombin or TRAP released relatively small amounts of AA above baseline, but these agents both potentiated B2R agonists. In experiments repeated all together 35–40 times with human plasma kallikrein (10 nM) or BK (1 nM), we found that thrombin and TRAP consistently doubled AA release by these agonists. We obtained qualitatively similar results in experiments with human arterial endothelial cells that constitutively express both PAR1 and B2Rs.

Besides BK, we also tested human plasma kallikrein because kallikreins affect B2R differently than BK. It is clear that kallikrein action at the B2R does not depend on release of a kinin (19, 20). As with BK, stimulation of the B2R by kallikrein was blocked by HOE-140, and both prokallikrein and inhibited kallikrein were inactive. Kallikrein and BK both desensitized the R, but there was no cross-desensitization. [3H]AA release by BK was reduced 40% by addition of carboxypeptidase M, which removes Arg9. Carboxypeptidase M did not influence R activation by kallikreins and other proteases. Site-directed mutagenesis of the Arg169 residue of the B2R further emphasized differences in the mechanisms of activation by the proteases and BK. The mutation decreased R activation by BK but not by kallikrein. In addition, our study shows that B2R activation by BK depends, at least in part, on Ca2+-independent PLA2 activation while activation by kallikrein does not.

Potentiation at the B2R is not simply due to direct activation of PAR1 by the protease action of kallikrein, because we obtained similar results with the peptide agonist BK. We further ruled out a proteolytic action of thrombin at the B2R because TRAP, a specific agonist of PAR1, potentiated both BK and kallikrein. DFP-inhibited thrombin (inactive on PAR1) failed to enhance B2R activation by agonists. A scrambled peptide sequence of TRAP was equally inactive, indicating that the potentiation mechanism is initiated and mediated through activation of PAR1. TRAP did not affect substrate hydrolysis by kallikrein (not shown).

Lipid rafts are believed to be essential for BK B2R and epidermal growth factor R crosstalk (23), and MCD blocks this interaction by depleting cholesterol, which disrupts complex formation. We found that 5 mM MCD failed to modify thrombin-induced potentiation in CHO/B2-transfected cells. Thus it seems unlikely that the potentiation phenomenon depends on PAR1/B2R dimer formation, even though B2Rs can form heterodimers with angiotensin-converting enzyme (ACE; Z. Chen and E. G. Erdös, unpublished observations and Ref. 28). In addition, because of the discrepancy in the ratio of PAR1 to B2 expression in CHO/B2-transfected cells, potentiation cannot be due simply to colocalization; it is presumably a consequence of enhanced signal transduction.

In a comparable system, thrombin first activates G{alpha}q/PKC{alpha} and the G{alpha}12/13/Rho pathways to mobilize [Ca2+]i in endothelial cells (21, 31). On stimulation, PAR1 coupled to G{alpha}q protein generates DAG and IP3; the latter then binds to IP3 Rs on the endoplasmic reticulum to trigger [Ca2+]i release. The interaction of IP3 R with transient receptor potential channel 1 (TRPC1) activates Ca2+ entry by store depletion (21, 27, 31). Yet, besides G{alpha}q, stimulated PAR1 couples to G{alpha}12/13 proteins (13, 17, 27). The released {alpha}-subunit of G12/13 induces P115 Rho guanine nucleotide exchange factor (p115RhoGEF) activation that, in turn, activates Rho. On activation of G{alpha}12/13/Rho signaling cascade, activated Rho associates with IP3 R and TRPC1 by actin filament polymerization to provoke [Ca2+]i store depletion (31). Following emptying of stored Ca2+, the Rho/IP3 R/TRPC1 complex translocates to the plasma membrane to trigger extracellular Ca2+ entry (21, 31). The phosphorylation of p115RhoGEF depends on PKC{alpha} (21), which is activated via G{alpha}q signaling pathway stimulation. These experiments with endothelial cells show that increased [Ca2+]i upon PAR1 activation requires cooperative interaction of both G{alpha}12/13 and G{alpha}q/PKC{alpha} pathways.

Our results are consistent with those findings and indicate that [Ca2+]i release indeed triggers thrombin-mediated potentiation of BK. We found that enhancement of AA release by thrombin was attenuated after inhibition of either G{alpha}q/PKC{alpha} or G{alpha}12/13 /Rho pathways. The inactivation of the IP3 R-dependent [Ca2+]i mobilization in CHO/B2 cells by 2-APB, the inhibition of PKC{alpha} by Gö-6976, and inhibition of Rho by a Rho kinase inhibitor Y-27632 all blocked potentiation of BK. Together these findings suggest that PAR1 R stimulation causes formation of the Rho/TRPC1/IP3 R complex and that the resulting [Ca2+]i release initiates signaling events leading to increased [3H]AA release.

However, interestingly thrombin potentiates the effects of plasma kallikrein and BK on the B2R differently. While an inhibitor of the IP3 R completely blocked the potentiation of kallikrein by thrombin to release [3H]AA, the selective inhibitor of [Ca2+]i release (TMB-8) or that of Rho kinase (Y-27632) did not modify it. Thus, both the IP3 R activation and the resulting elevation of [Ca2+]i level are involved in potentiation of BK, but only the IP3 R activation is necessary for potentiation of kallikrein by thrombin. Altogether, these data are showing that IP3 R is indeed the critical point of interaction between the pathways leading to positive cooperativity. Because PAR1 initiates enhancement of [3H]AA release by B2R agonists and Ca2+ stimulates cPLA2, cPLA2 is likely involved in potentiation. Indeed, a specific inhibitor of Ca2+-dependent PLA2 blocked the potentiation by thrombin, while an inhibitor of the Ca2+-independent isoform was ineffective, implying that [Ca2+]i mediates enhanced B2R activity by agonists. Both thrombin and TRAP modified B2R phosphorylation by agonists in a concentration-dependent manner. Once activated by agonist, B2R undergoes phosphorylation and desensitization. ACE inhibitors decreased the phosphorylation of B2R by BK and thus enhanced the activity of the peptide agonist (29). Now we find that low concentrations of thrombin (10–30 nM) or TRAP (30–100 µM without addition of the aminopeptidase inhibitor amastatin and 1–10 µM with amastatin) elevate BK B2R activity and increase phosphorylation of the R. Higher amounts of enzyme or peptide reversed the process and inhibited BK-initiated phosphorylation. Blocking of these effects by the IP3 R inhibitor 2-APB and by the PKC{alpha} inhibitor Gö-6976 can be explained by coactivation of G{alpha}12/13 and G{alpha}q /PKC{alpha} pathways by thrombin and the subsequent [Ca2+]i mobilization that modulates B2R phosphorylation and activity. Increased phosphorylation is not likely due to increased expression of B2R, because we found no concomitant increase in B2-GFP total protein with the increase in R phosphorylation (Fig. 5). In addition, inhibitors would unlikely alter protein synthesis and receptor expression during the brief time course of our experiments, but only modify activity. Additional controls where we measured [3H]BK binding to the cell membrane indicated no modification by thrombin (data not shown).

Figure 7 provides a scheme that summarizes pathways potentially involved in the interaction of PAR1 and B2R agonists.


Figure 7
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  		Fig. 7. Scheme for the enhanced AA release. On thrombin activation, G{alpha}q and G{alpha}12/13 signaling pathways converge to empty Ca2+ stores and redirect subsequent PKC{alpha}-dependent Ca2+ entry. The elevation of [Ca2+]i enhances Ca2+-dependent PLA2 activation initiated by B2R agonists and increases release of AA. Kallikrein, kallikrein of human plasma; DAG, diacylglycerol; cPLA2 inhib, Ca2+-dependent phospholipase A2 inhibitor; Y-27362, Rho kinase inhibitor; |Figure 7 or Figure 7|, inhibition.

 
We conclude that PAR1 R activation enhances the effects of BK B2R agonists in signal transduction, which then results in augmented release of AA. Because AA is a precursor of several vasoactive mediators, regulation of these pathways influences many diverse physiological functions. The cooperativity between the two Rs that we described for signal transduction pathways involved in AA release may underlie the cardioprotective nature of kinins in vivo.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-36473 and HL-68580.


   ACKNOWLEDGMENTS
 
We thank Drs. C. Tirrupathi and D. Mehta of UIC College of Medicine for assistance in the project and Dr. P. Deddish for collaborating in the calculation of expression levels.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. G. Erdös, U. Illinois College of Medicine at Chicago, Dept. of Pharmacology (M/C 868), 835 S. Wolcott Ave., Rm. E403, Chicago, IL 60612 (e-mail: egerdos{at}uic.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abe M, Nakamura F, Tan F, Deddish PA, Colley KJ, Becker RP, Skidgel RA, and Erdös EG. Expression of rat kallikrein and epithelial polarity in transfected Madin-Darby canine kidney cells. Hypertension 26: 891–898, 1995.[Abstract/Free Full Text]
  2. Ahmmed GU, Mehta D, Vogel S, Holinstat M, Paria BC, Tiruppathi C, and Malik AB. Protein kinase C{alpha} phosphorylates the TRPC1 channel and regulates store-operated Ca2+ entry in endothelial cells. J Biol Chem 279: 20941–20949, 2004.[Abstract/Free Full Text]
  3. Bhoola KD, Figueroa CD, and Worthy K. Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmacol Rev 44: 1–80, 1992.[Web of Science][Medline]
  4. Blaukat A, Pizard A, Breit A, Wernstedt C, Alhenc-Gelas F, Muller-Esterl W, and Dikic I. Determination of bradykinin B2 receptor in vivo phosphorylation sites and their role in receptor function. J Biol Chem 21: 21, 2001.[Medline]
  5. Burch RM. Diacylglycerol in the synergy of bradykinin and thrombin stimulation of prostaglandin synthesis. Eur J Pharmacol 168: 39–42, 1989.[Medline]
  6. Burch RM and Axelrod J. Dissociation of bradykinin-induced prostaglandins formation, from other phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A2. Proc Natl Acad Sci USA 84: 6374–6378, 1987.[Abstract/Free Full Text]
  7. Burch RM, Ma AL, and Axelrod J. Phorbol esters and diacylglycerols amplify bradykinin-stimulated prostaglandin synthesis in Swiss 3T3 fibroblasts. Possible independence from protein kinase C. J Biol Chem 263: 4764–4767, 1988.[Abstract/Free Full Text]
  8. Campbell WB and Halushka PV. Lipid-derived autacoids: eicosanoids and platelet-activating factor. In: Goodman & Gilman's The Pharmacological Basis of Therapeutics (9th ed.), edited by Hardman JC, Limberd LE, Molinoff PB, Ruddon RW, and Gilman AG. New York: McGraw-Hill, 1996, p. 602–616.
  9. Carretero O, Nasjletti A, and Fasciolo JC. The kinin content of human blood at rest and during vasodilatation. Experientia 21: 141–142, 1965.[Medline]
  10. Carretero OA, Carbini LA, and Scicli AG. The molecular biology of the kallikrein-kinin system. I. General description, nomenclature and the mouse gene family. J Hypertens 11: 693–697, 1993.[CrossRef][Web of Science][Medline]
  11. Carretero OA and Scicli AG. Kinin as regulators of blood flow and blood pressure. In: Hypertension: Pathophysiology, Diagnosis and Management, edited by Laragh JH and Brenner BM. New York: Raven, 1990, p. 805–817.
  12. Coughlin SR. How the protease thrombin talks to cells. Proc Natl Acad Sci USA 96: 11023–11027, 1999.[Abstract/Free Full Text]
  13. Coughlin SR, Vu TK, Hung DT, and Wheaton VI. Characterization of a functional thrombin receptor. Issues and opportunities. J Clin Invest 89: 351–355, 1992.[Web of Science][Medline]
  14. Crouch MF, Belford DA, Milburn PJ, and Hendry IA. Pertussis toxin inhibits EGF-, phorbol ester- and insulin-stimulated DNA synthesis in BALB/c3T3 cells: evidence for post-receptor activation of Gi{alpha}. Biochem Biophys Res Commun 167: 1369–1376, 1990.[CrossRef][Medline]
  15. Davey MG and Luscher EF. Actions of thrombin and other coagulant and proteolytic enzymes on blood platelets. Nature 216: 857–858, 1967.[CrossRef][Medline]
  16. Erdös EG (Editor). Bradykinin, Kallidin and Kallikrein. Handbook of Experimental Pharmacology. Heidelberg: Springer-Verlag, 1979, p. 1–817.
  17. Gohla A, Offermanns S, Wilkie TM, and Schultz G. Differential involvement of G{alpha}12 and G{alpha}13 in receptor-mediated stress fiber formation. J Biol Chem 274: 17901–17907, 1999.[Abstract/Free Full Text]
  18. Gutowski S, Smrcha A, Nowak L, Wu D, Simon M, and Sternweis P. Antibodies to the {alpha}q subfamily of guanine nucleotide-binding regulatory protein {alpha} subunits attenuate activation of phosphatidylinositol 4,5-bisphosphate hydrolysis by hormones. J Biol Chem 266: 20519–20524, 1991.[Abstract/Free Full Text]
  19. Hecquet C, Becker RP, Tan F, and Erdös EG. Kallikreins when activating bradykinin B2 receptor induce its redistribution on plasma membrane. Int Immunopharmacol 2: 1795–1806, 2002.[CrossRef][Web of Science][Medline]
  20. Hecquet C, Tan F, Marcic BM, and Erdös EG. Human bradykinin B2 receptor is activated by kallikrein and other serine proteases. Mol Pharmacol 58: 828–836, 2000.[Abstract/Free Full Text]
  21. Holinstat M, Mehta D, Kozasa T, Minshall RD, and Malik AB. Protein kinase C{alpha}-induced p115RhoGEF phosphorylation signals endothelial cytoskeletal rearrangement. J Biol Chem 278: 28793–28798, 2003.[Abstract/Free Full Text]
  22. Huntington JA and Baglin TP. Targeting thrombin—rational drug design from natural mechanisms. Trends Pharmacol Sci 24: 589–595, 2003.[Medline]
  23. Hur EM, Park YS, Lee BD, Jang IH, Kim HS, Kim TD, Suh PG, Ryu SH, and Kim KT. Sensitization of epidermal growth factor-induced signaling by bradykinin is mediated by c-Src. Implications for a role of lipid microdomains. J Biol Chem 279: 5852–5860, 2004.[Abstract/Free Full Text]
  24. Kramer RM and Sharp JD. Structure, function and regulation of Ca2+-sensitive cytosolic phospholipase A2 (cPLA2). FEBS Lett 410: 49–53, 1997.[CrossRef][Web of Science][Medline]
  25. LaMorte VJ, Harootunian AT, Spiegel AM, Tsien RY, and Feramisco JR. Mediation of growth factor induced DNA synthesis and calcium mobilization by Gq and Gi2. J Cell Biol 121: 91–99, 1993.[Abstract/Free Full Text]
  26. Liao JK and Homcy CJ. The G proteins of the G{alpha}i and G{alpha}q family couple the bradykinin receptor to the release of endothelium-derived relaxing factor. J Clin Invest 92: 2168–2172, 1993.[Web of Science][Medline]
  27. Mackie EJ, Pagel CN, Smith R, de Niese MR, Song SJ, and Pike RN. Protease-activated receptors: a means of converting extracellular proteolysis into intracellular signals. IUBMB Life 53: 277–281, 2002.[Medline]
  28. Marcic B, Deddish PA, Skidgel RA, Erdös EG, Minshall RD, and Tan F. Replacement of the transmembrane anchor in angiotensin I-converting enzyme (ACE) with a glycosylphosphatidylinositol tail affects activation of the B2 bradykinin receptor by ACE inhibitors. J Biol Chem 275: 16110–16118, 2000.[Abstract/Free Full Text]
  29. Marcic BM and Erdös EG. Protein kinase C and phosphatase inhibitors block the ability of angiotensin I-converting enzyme inhibitors to resensitize the receptor to bradykinin without altering the primary effects of bradykinin. J Pharmacol Exp Ther 294: 605–612, 2000.[Abstract/Free Full Text]
  30. Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug H, Marme D, and Schachtele C. Selective inhibition of protein kinase C isozymes by the indolocarbazole Gö 6976. J Biol Chem 268: 9194–9197, 1993.[Abstract/Free Full Text]
  31. Mehta D, Ahmmed GU, Paria BC, Holinstat M, Voyno-Yasenetskaya T, Tiruppathi C, Minshall RD, and Malik AB. RhoA interaction with inositol 1,4,5-trisphosphate receptor and transient receptor potential channel-1 regulates Ca2+ entry. Role in signaling increased endothelial permeability. J Biol Chem 278: 33492–33500, 2003.[Abstract/Free Full Text]
  32. Oldgren J, Wallentin L, Grip L, Linder R, Norgaard BL, and Siegbahn A. Myocardial damage, inflammation and thrombin inhibition in unstable coronary artery disease. Eur Heart J 24: 86–93, 2003.[Abstract/Free Full Text]
  33. Popov S, Yu K, Kozasa T, and Wilkie TM. The regulators of G protein signaling (RGS) domains of RGS4, RGS10, and GAIP retain GTPase activating protein activity in vitro. Proc Natl Acad Sci USA 94: 7216–7220, 1997.[Abstract/Free Full Text]
  34. Schievella AR, Regier MK, Smith WL, and Lin LL. Calcium-mediated translocation of cytosolic phospholipase A2 to the nuclear envelope and endoplasmic reticulum. J Biol Chem 270: 30749–30754, 1995.[Abstract/Free Full Text]
  35. Schmitz JM, Apprill PG, Buja LM, Willerson JT, and Campbell WB. Vascular prostaglandin and thromboxane production in a canine model of myocardial ischemia. Circ Res 57: 223–231, 1985.[Abstract/Free Full Text]
  36. Takada Y, Skidgel RA, and Erdös EG. Purification of human prokallikrein. Identification of the site of activation by the metalloproteinase thermolysin. Biochem J 232: 851–858, 1985.[Web of Science][Medline]
  37. Vanhauwe JF, Thomas TO, Minshall RD, Tiruppathi C, Li A, Gilchrist A, Yoon EJ, Malik AB, and Hamm HE. Thrombin receptors activate Go proteins in endothelial cells to regulate intracellular calcium and cell shape changes. J Biol Chem 277: 34143–34149, 2002.[Abstract/Free Full Text]
  38. Vu TK, Wheaton VI, Hung DT, Charo I, and Coughlin SR. Domains specifying thrombin-receptor interaction. Nature 353: 674–677, 1991.[CrossRef][Medline]




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