ACE INHIBITORS DIRECTLY ACTIVATE BRADYKININ B1 RECEPTORS TO RELEASE NO Chapter

Chapter 13

ACE INHIBITORS DIRECTLY ACTIVATE BRADYKININ B1 RECEPTORS

TO RELEASE NO

Tatj ana Ignj atovic Sinisa Stanisavljevic Viktor Brovkovych Fulong Tan

Randal A. Skidgel Ervin G . Erdos

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

INTRODUCTION

Angiotensin I-converting (ACE; kininase 11) inhibitors have been successfully used to treat millions of patients world-wide, but their cellular, subcellular and molecular modes of action are still being unraveled. The inhibition of ACE (kininase 11) blocks angiotensin I1 release or bradykinin inactivation [l-31 but these alone do not fully account for the usefulness of ACE inhibitors. They also potentiate the effects of bradykinin and its ACE-resistant analogs on the B2 receptors by inducing an enzymelreceptor interaction (ACE/B2), heterodimer formation [4-7].We showed this by using atrial strips and ileal segments of guinea pigs in bio-assay and in cultured cells of human and animal origin.

Of the two bradykinin receptors B1 and B2, B2 is ubiquitous as a first line, primary mediator of kinin action under physiological conditions [8, 91. Normally, few cell types express B1 receptors, but various pathologic conditions such as ischemia, myocardial infarction, atheromatous disease, or exposure to inflammatory cytokines rapidly induce its expression [9]. The elimination of the B2 receptor gene in knockout mice also up-regulated the B1 receptor [lo]. The peptide agonists of the receptors differ, as plasma carboxypeptidase N or cell

membrane carboxypeptidase M cleave the C-terminal Arg of the B2 receptor agonists kallidin (Lys-bradykinin) and bradykinin to generate B1 agonists deskg-kinins (Fig. 1) [9, 1 1, 121. Both of these kininase I-type human carboxypeptidases have been cloned and the crystal structure of carboxypeptidase M was recently published by R.A. Skidgel and F. Tan with the collaboration of the Max Planck Institute in Munich [13].

Carbcxypeptidase M Carbcxypeptidase N

L 4rg-Pro-Pro-Gly-Phe-Ser-Pro-P e

~ d e s A t $ - b f a d y k i n ~ de~Arg'~-kallidin

Figure 1. Schematic diagram that illustrates how carboxypeptidases generate speciJc BI receptor agonists (desArg9-bradykinin, desArgl0-kallidin)

fvom

the B2 agonists (bradykinin, kallidin).

Multiple contributions of the kinin B2 receptors to the effects of ACE inhibitors have already been published [I, 71. In contrast, little was known about the relationship between the B1 and ACE inhibitors. We hypothesized an unexplored role for the B1 receptors from description of conditions of many patients treated with ACE inhibitors that can also lead to B1 receptor induction. Following this lead, we found that ACE inhibitors in nanomolar concentrations directly activate the bradykinin B1 receptors in cells without an intermediate peptide ligand and in the absence of ACE [14]. This interaction leads to a prolonged NO release from endothelial cells. In subsequent studies we investigated further how the activation of B1 leads to NO release [15]. We also tested whether the two B1 receptor agonists, the peptide desArg10 -kallidin and the ACE inhibitor enalaprilat, acting on different domains on the receptor could initiate transduction through different signaling pathways.

METHODS

Cell Culture.

Effects of ACE inhibitors were tested using several cultured cell types, which included:

A. CHO, HEK293 and COS7 cells that were transfected to express human B1 receptors [14]

B. Bovine pulmonary artery endothelial (BPAE) cells and IMR-90 fibroblasts, which constitutively express B1 receptors [14, 151

ACE Inhibitors directly activate bradykinin BI receptors to release NO 165 C. Human pulmonary artery endothelial (HLMVE) cells, which were routinely treated with interleukin-1

p

(IL-1 p) and interferon-y (IFN- y) for 16 to 18 h prior to the experiment in order to induce B1 receptors [15]

Methods used.

Measurement of [ca2+]i and detection of NO were performed as reported [14, 151. Real time NO was measured using a porphyrinic microsensor. Current generated on the porphyrinic electrode was proportional to the NO released and was quantitated with a known standard NO solution.

Phosphoinositide hydrolysis was assayed as previously described [15]. Briefly, cells were labeled with [ 3 ~ ] inositol for 16-24 h, then washed with 5 mM LiC1. Cells were extracted and inositol phosphates were separated by anion exchange chromatography (Seuwen et al., 1990). Results are expressed as the ratio of inositol phosphates / inositol phosphates

+

inositol counts.

L-arginine transport

^- Transport was assayed in whole cells using L-[2,3,4,53~] arginine monohydrochloride as reported [15].

RESULTS

As a prototype of an active ACE inhibitor we used enalaprilat and initially measured the increase in [ca2+li in IMR-90 and BPAE cells, which express B1 and B2 receptors [14]. Subsequently, we also measured NO release from BPAE cells using a porphyrinic electrode in real-time (Fig. 2). The receptors were activated with either the peptide agonists de~Ar~'~-kallidin and bradykinin or ACE inhibitor enalaprilat.

Bradykinin (10 nM) and de~-Ar~'~-kallidin (10 nM) released NO in distinctly different patterns. Bradykinin, a B2 receptor ligand, released NO in a brief burst, ~hile'desAr~'~-kallidin, agonist of the B1 receptor, brought about a more prolonged and substantially higher liberation of NO than after B2 receptor stimulation (Fig 2A). Enalaprilat (10 nM), in the absence of a peptide agonist, significantly increased NO, similarly to deskg''-kallidin (Fig.2A). The response to enalaprilat was completely inhibited by the B1 antagonist d e ~ A r ~ ' ~ - ~ e u ~ - k a l l i d i n (Fig.2C).

BK IOnM

DAKD IOnM

F Ept 10nM

20 rnin

0 J .

1 10 100 ,om ,moo

Enalaprilat concentrat~on, nM

C

^~7 Preincubation with

100 nM DALKD

DAKD, 10 nM Enalaprilat, 10 nM

-

Figure 2. Stimulation of NO generation in endothelial cells by Bl agonists. NO production was measured in BPAE cells using a porphyrinic microsensor in real time. A, the addition of enalaprilat (Ept) or des-Argl0-kallidin (DAKD) (denoted by the arrows) caused an immediate generation of NO, which continued to increase over 20 min. In contrast, BK stimulated a transient increase in NO, which returned to baseline by about 5 rnin. B, the dose-response curve for enalaprilat is shown. BPAE cells were stimulated with increasing concentrations of enalaprilat and the NO concentration generated at 20 min taken as a measure of the response. Results represent the mean values offive (10 and 100 nM) or two (pM concentration points) independent experiments. C, the BI receptor blocker inhibits enalaprilat stimulation of NO production. Des-ArglO-kallidin and enalaprilat were added to cells pretreated for 2 min with or without the Bl receptor antagonist (DALKD) as indicated, and the NO concentration generated at 20 min was taken as a measure of the response. Shown are the mean values from four or more independent experiments. Reproduced with permission from ref[l4].

ACE Inhibitors directly activate bradykinin B, receptors to release NO 167

To explain the mode of activation of B1 receptor by ACE inhibitors we performed another set of experiments, where we used various cell lines and transfected them to express the human B1 receptor [14]. These experiments fiuther indicated that ACE inhibitors directly activate the B1 receptor, even in the absence of ACE, behaving essentially as receptor agonists. To clarify how they activate the B1 receptors, we compared the amino acid sequence of ACE with the bradykinin B1 and B2 receptors [16, 171. Although there is little overall homology, the human B1 receptor contains in its second extracellular loop (residues 195-199) an HEAWH sequence (Fig. 2), which is absent in the B2 receptor. This is similar to the sequence in the active centers of the two domains of ACE (HEMGH) [ l l ] and matches the HEXXH zinc- binding consensus sequence [I 81 in other zinc metalloproteases. ACE inhibitors combine with the active centers of ACE via the zn2' cofactor with their SH or COO groups. If ACE inhibitors bind in a similar fashion to the B1 receptor, then mutation in the HEXXH motif should eliminate inhibitor binding [19]. We constructed a point mutation (H195A) at the putative Zn-binding site (HEAWH+AEAWH). The [Hl 95A]B1 receptor mutant was transiently expressed in HEK 293 cells.

In HEW[H195A]BI cells, des-ArglO-kallidin activated the receptor, whereas enalaprilat was inactive (Fig.3). Consequently, the HEAWH sequence in the second extracellular loop is essential for the direct activation of the B1 receptors by enalaprilat but not for de~Ar~'~-kallidin, which acts on other epitopes in the third extracellular loop [20,21].

LLLP

H F A

Figure 3. Site of the activation of the BI receptor by enalaprilat. A, a schematic of the structure of the human B l receptor is shown. The HEAWH motif in the second extracellular loop (residues 195-199), the proposed binding site for enalaprilat, is enlarged. This putative zinc-binding sequence (HEAWH) is well conserved in BI receptors across species. B, tracings from single HEK cells expressing wild type Bl (panels I and II) or the [H195A]Bl mutant (panel III) are shown. The cells were stimulated with des-Argl0-kallidin (DAKD) and enalaprilat (Ept). Notice that the H195A mutation of the BI receptor in the Zn-binding region abolished only the effect of enalaprilat, whereas des-Argl0-kallidin remained active. Results are representative of j v e independent experiments. Reproduced with permission from re$ [14].

To confirm our findings obtained with the [H195A]B1 mutant, we used a synthetic undecapeptide (LLPHEAWI-IFAR) corresponding to residues 192-202 of the B1 receptor, which contains the putative ACE inhibitor binding site [14]. This undecapeptide blocked the effect of ACE inhibitor specifically and completely, whereas de~-Ar~'~-kallidin's activity was not affected [14].

We next sought to investigate how the activation of B1 receptors releases NO from endothelial cells. To this end we used two types of cells: BPAE cells, which constitutively express the B1 receptor and HLMVE cells where we induced the receptor with inflammatory cytokines, IL-1

P

and

IFN-

y. To determine the contributions of different NO synthase (NOS) isoforms to NO production, we used inhibitors relatively specific for endothelial NOS (eNOS) or inducible NOS (iNOS). 1400W, iNOS inhibitor, supressed the majority of NO responses elicited both by de~Ar~'~-kallidin or enalaprilat, when tested in cytokine-

ACE Inhibitors directly activate bradykinin BI receptors to release NO 169

stimulated HLMVE cells (Fig. 4A). In these cells, both agonists release NO via iNOS, in a similar, calcium-independent manner [15] , by increasing the uptake of NOS substrate, arginine (Fig. 4B).

Control DAKD E P ~

100nM 1OOnM

Figure 4. Bl agonist peptide and ACE inhibitor release NO mainly via iNOS and increase arginine uptake in cytokine-stimulated HLMVE cells.

A. NO Release. Cells were preincubated (15 min, 37 7 with selective eNOS (2 pM N w nitro-L-arginine (L-NNA)) or iNOS inhibitor (2 pi4 1400W) and NO production in response to B1 agonists was measured with a porphyrinic electrode in real-time.

Shown are mean values obtained with 100 nM desArgl0-kallidin (DAKD) or 100 nM enalaprilat (Ept) in the absence and presence of eNOS and iNOS inhibitors (n=4).

*, significantly different from control (p8.05);

***,

signijkantly different from control (p4.001)

Reproduced with permission (ref: 15, Fig. 6).

B. L-arginine uptake. HLMVE cells were stimulated for I minute with 100 nM desArgl0-kallidin (DAKD) or 100 nM enalaprilat (Ept). The abscissa shows the uptake of arginine relative to control. The results represent the means f l E from 9 independent experiments (done in triplicate). ***, signzjkantly different from the control ('8.001).

Reproduced with permission (ref15, Fig. 7).

In contrast, in BPAE cells, de~Ar~'~-kallidin and enalaprilat activate NO synthesis by different mechanisms. ~esAr~'O-kallidin releases NO via eNOS activation, dependent on intracellular calcium elevation (not shown;[l5]). Enalaprilat activates mainly eNOS but to some degree iNOS to release NO (Fig.5). Consistent with these findings, the intracellular calcium chelator, BAPTA, had less effect on the response to enalaprilat compared to that obtained with de~Ar~'~-kallidin (not shown; [l5]).

Figure 5. NO release from BPAE cells. BPAE cells were preincubated with L- NNA or 1400W and NO production in response to BI agonists was measured with a porphyrinic electrode.

Mean values ( B E ) obtained with 100 nM desArgl0- kallidin (DAKD) or 100 nM enalaprilat (ept) in the absence and presence of eNOS and iNOS inhibitors (n=4-5).

*, signijcantly different from control (p4.05); ***,

signijicantly different from control (p4.001)

Reproduced with permission from re$ [I 51.

In BPAE cells, B1 receptor ligands de~Ar~'~-kallidin and enalaprilat also raised intracellular calcium by different mechanisms [I 51 (Fig.6). Enalaprilat activates the B1 receptor via a Gs-protein dependent pathway and it stimulates the influx of external calcium, independent of inositol 1,4,5-tris-phosphate (P3) generation. In contrast, desArgI0- kallidin acts through Gq-protein to first release internal calcium by IP3 liberation and then subsequently stimulates calcium entry. In addition, protein kinase C inhibitors (calphostin C and chelerythrine) blocked the elevation of [ca2']i triggered only by ACE inhibitors but not that stimulated by des~r~lO-kallidin [15].

ACE Inhibitors directly activate bradykinin BI receptors to release NO 171

DISCUSSION

Numerous studies demonstrate the effectiveness of ACE inhibitors in cardiac and renal patients. For instance, trials involving more than 9000 patients showed that ACE inhibitor substantially lowered the rates of death, heart attack, stroke, heart failure and complications related to diabetes mellitus [22,23].

We investigated the cellular and molecular mode of action of these drugs in order to gain better understanding of their actions. We found, using several types of cultured cells, that ACE inhibitors, in low, therapeutically relevant concentrations, directly activate the bradykinin B1 receptor in the absence of endogenous kinins or ACE [14, 241. This activity differs fkom the potentiation of bradykinin actions on its B2 receptor by ACE inhibitors, previously decribed by our group [6, 71, which is indirect and requires the expression of both the B2 receptor and ACE. Human B1 and ACE, although lacking overall similarities, share a common structural element- a zinc binding motif. This sequence, represented by HEAWH residues in the B1 receptor, is the likely site of attachment of ACE inhibitors. This hypothesis was confirmed in experiments where we found that point mutation of the zinc binding motif of B1 receptor or the pretreatment of cells with the undecapeptide representing this Zn-binding site blocked the effect of ACE inhibitors but not that of the peptide ligand, d e s ~ r ~ ' ~ - k a l l i d i n [14].

Activation of B1 receptors by either the peptide ligand or ACE inhibitor releases NO [12, 14, 15, 25-28]. B1-mediated NO release is sustained and the time course differs from that caused by B2 receptor activation, which produces a shorter, transient response [12, 141. We investigated the mechanism of NO release triggered by the activation of either a constitutively expressed or induced B1 receptor. To this end we used two types of endothelial cells, BPAE and cytokine-stimulated HLMVE, as suitable models for the native, constitutive [14, 291 or inducible expression of Bl receptors [12]. We hypothesized that different NOS isoforms may be involved under native or cytokine-treated conditions, since interleukin-1

p

stimulates the expression of iNOS [30- 321 and B1 receptor ligands, desArglO-kallidin and enalaprilat may stimulate distinct signaling pathways [14].

In BPAE cells we found that the mode of NO release by ACE inhibitor and peptide ligand differs [15]. The synthesis of NO by de~Ar~'~-kallidin largely depends on the elevation of [ca2']i and eNOS activation. In contrast, enalaprilat releases NO largely independent of [ca2+]i via eNOS and iNOS activation. Although both B1 receptor ligands raise [ca2+li , they do so through stimulation of dissimilar

signaling pathways. ACE inhibitor enalaprilat enhances entry of extracellular calcium, while d e s ~ r ~ l ~ - k a l l i d i n first releases internal calcium via IP3 liberation and then subsequently augments calcium influx. This is mediated by differential coupling to G proteins. ~ e s ~ r ~ ' O - kallidin acts through Gq-protein to activate phospholipase CP, generate IP3 and raise cytosolic calcium, whereas enalaprilat stimulates calcium entry through a Gs- and PKC dependent pathway (Fig.6). PKC may phosphorylate and upregulate calcium channels by opposing GPy- induced inhibition [33].

ACE Inhibitors

PKC

8

\

PLCP

a

Rare earth sensitive Ca2' channel

1

I!= 3

t [C a''] i

NO

Figure 6. Scheme to illustrate how the peptide desArgl0-kallidin and ACE inhibitor enalaprilat activate the BI receptor to stimulate elevation of [Ca2+]i and NO release through dzyerentpathways in BPAE cells.

HLMVE cells, exposed to inflammatory cytokines IL- 1

P

and IFN-y, were used to mimic pathological conditions, which induce the B1 receptor expression in vivo [9,24]. In these cells, both d e s ~ r ~ ' ~ - k a l l i d i n and ACE inhibitor release NO mainly via iNOS stimulation, independent of [ca2"]i elevation. Both of the B1 ligands employed enhanced the uptake of extracellular arginine, which is necessary for iNOS, but not

ACE Inhibitors directly activate bradykinin BI receptors to release NO 173 eNOS activity. These results indicate that although the activation of both constitutively expressed and cytokine-induced B1 receptor causes a prolonged NO release, the mechanisms of these processes may be different. The signaling in cells constitutively expressing B1 receptors or in cells stimulated with cytokines to induce B1 receptors may be predominantly linked to eNOS and iNOS activity respectively.

Upregulation and activation of iNOS to produce high levels of NO can be deleterious, but several reports also document the benefits of its expression. For instance, iNOS protects against oxidative damage [34, 351, inhibits platelet adhesion [36] or contributes to prolonged protective effects of ischemic preconditioning [37]. NO can also blunt the inflammatory response by inhibition of NF-KB activation, increasing the expression, nuclear translocation and stabilization of its inhibitory protein IKB [38,39].

Augmenting NO release by direct activation of B1 receptors may play a significant role in the effectiveness of ACE inhibitors, since a deficiency of NO contributes to a variety of cardiovascular diseases [40- 421. B1 receptors, induced during ischemia are cardioprotective [43].

Activation of B1 receptors also contributes to the protective effects of ischemic preconditioning in coronary arteries [44]. Heart failure is accompanied by increased production of cytokines [45, 461, which, in turn, stimulate B1 receptor expression. The B1 receptors are induced after myocardial infarction and they may contribute to wound healing and tissue repair [47].

In summary, ACE inhibitors directly activate B1 receptor, in the absence of ACE and endogenous kinins and this results in a prolonged NO release [14, 151. B1 receptors contribute to the effects of ACE inhibitors when tested in cultured cells [14, 151, laboratory animals [48- 501 or heart failure patients [51]. Some of the beneficial effects of ACE inhibitor therapy can be due to direct activation of the peptide B1 receptor, even in cells lacking ACE expression, especially in cardiac functions.

Acknowledgements

These studies were supported by National Heart, Lung and Blood Institute grants HL36473, HL68580 and HL 60678.

REFERENCES

Linz, W., et al., Contribution of kinins to the cardiovascular actions of angiotensin- converting enzyme inhibitors. Pharmacol Rev, 1995.47(1): p. 25-49.

Yang, H.Y., E.G. Erdos, and Y. Levin, Characterization of a dipeptide hydrolase (kininase 11: angiotensin I converting enzyme). J Pharmacol Exp Ther, 1971.

177(1): p. 291-300.

Yang, H.Y.T. and E.G. Erdos, Second kininase in human blood plasma. Nature, 1967.215: p. 1402-1403.

Marcic, B., et al., 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, 2000.

275(21): p. 16 1 10-8.

Minshall, R.D., et al., Potentiation of the effects of BK on its receptor in the isolated guinea pig ileum. Peptides, 2000. 2 l(8): p. 1257-64.

Minshall, R.D., et al., Potentiation of the actions of bradykinin by angiotensin I- converting enzyme inhibitors. The role of expressed human bradykinin B2 receptors and angiotensin I-converting enzyme in CHO cells. Circ Res, 1997.

81(5): p. 848-56.

Erdos, E.G., P.A. Deddish, and B.M. Marcic, Potentiation of bradykinin actions by ACE inhibitors. Trends Endocrinol Metab, 1999. lO(6): p. 223-229.

Hall, J.M., Bradykinin receptors. Gen Pharmacol, 1997.28(1): p. 1-6.

McLean, P.G., M. Perretti, and A. Ahluwalia, Kinin B(1) receptors and the cardiovascular system: regulation of expression and function. Cardiovasc Res, 2000.48(2): p. 194-21 0.

Duka, I., et al., Vasoactive potential of the B(1) bradykinin receptor in normotension and hypertension. Circ Res, 2001. 88(3): p. 275-8 1.

Erdos, E.G. and R.A. Skidgel, Metabolism of bradykinin by peptidases in health and disease, in The Kinin System, S.G. Farmer, Editor. 1997, Academic Press, Inc.: San Diego. p. 101-141.

Sangsree, S., et al., Kininase I-type carboxypeptidases enhance nitric oxide production in endothelial cells by generating bradykinin BI receptor agonists. Am J Physiol Heart Circ Physiol, 2003.284(6): p. H1959-68.

Reverter, D., et al., Crystal structure of human carboxypeptidase M, a membrane- bound enzyme that regulates peptide hormone activity. J Mol Biol, 2004. 338(2):

p. 257-69.

Ignjatovic, T., et al., Novel mode of action of angiotensin I converting enzyme inhibitors: direct activation of bradykinin B1 receptor. J Biol Chem, 2002.

277(19): p. 16847-52.

Ignjatovic, T., et al., Kinin B1 receptors stimulate nitric oxide production in endothelial cells: signaling pathways activated by angiotensin I-converting enzyme inhibitors and peptide ligands. Mol Pharmacol, 2004.66(5): p. 1310-6.

Menke, J.G., et al., Expression cloning of a human B1 bradykinin receptor. J Biol Chem, 1994.269(34): p. 21583-6.

Soubrier, F., et al., Two putative active centers in human angiotensin I-converting enzyme revealed by molecular cloning. Proc Natl Acad Sci U S A, 1988. 85(24):

p. 9386-90.

Hooper, N.M., Families of zinc metalloproteases. FEBS Lett, 1994. 354(1): p. 1-6.

Wei, L., et al., The two homologous domains of human angiotensin I-converting enzyme interact differently with competitive inhibitors. J Biol Chem, 1992.

267(19): p. 13398-405.

ACE Inhibitors directly activate bradykinin BI receptors to release NO 175

Fathy, D.B., D.J. Kyle, and L.M. Leeb-Lundberg, High-affinity binding of peptide agonists to the human B1 bradykinin receptor depends on interaction between the peptide N-terminal L-lysine and the fourth extracellular domain of the receptor.

Mol Pharmacol, 2000. 57(1): p. 171-9.

Fathy, D.B., et al., A single position in the third transmembrane domains of the human BI and B2 bradykinin receptors is adjacent to and discriminates between the C-terminal residues of subtype-selective ligands. J Biol Chem, 1998. 273(20):

p. 12210-8.

Yusuf, S., et a]., Effects of an angiotensin-converting-enzyme inhibitor, ramipril, on cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators. N Engl J Med, 2000. 342(3): p. 145-53.

Teo, K.K., et al., Effect of ramipril in reducing sudden deaths and nonfatal cardiac arrests in high-risk individuals without heart failure or left ventricular dysfunction.

Circulation, 2004. 1 lO(11): p. 141 3-7.

Ignjatovic, T., et al., Activation of bradykinin B1 receptor by ACE inhibitors. Int Immunopharmacol, 2002.2(13-14): p. 1787-93.

Drummond, G.R. and T.M. Cocks, Endothelium-dependent relaxations mediated by inducible B 1 and constitutive B2 kinin receptors in the bovine isolated coronary artery. Br J Pharmacol, 1995. 116(5): p. 2473-81.

Parenti, A., et al., The bradykinin/Bl receptor promotes angiogenesis by up- regulation of endogenous FGF-2 in endothelium via the nitric oxide synthase pathway. Faseb J, 2001. 15(8): p. 1487-9.

Pruneau, D. and P. Belichard, Induction of bradykinin B1 receptor-mediated relaxation in the isolated rabbit carotid artery. Eur J Pharmacol, 1993. 239(1-3): p.

63-7.

Pruneau, D., et al., Characterisation of bradykinin receptors from juvenile pig coronary artery. Eur J Pharmacol, 1996.297(1-2): p. 53-60.

Smith, J.A., et a]., Signal transduction pathways for B1 and B2 bradykinin receptors in bovine pulmonary artery endothelial cells. Mol Pharmacol, 1995.

47(3): p. 525-34.

Asano, K., et al., Constitutive and inducible nitric oxide synthase gene expression, regulation, and activity in human lung epithelial cells. Proc Natl Acad Sci U S A,

1994.91(21): p. 10089-93.

Kanno, K., et a]., Regulation of inducible nitric oxide synthase gene by interleukin-l beta in rat vascular endothelial cells. Am J Physiol, 1994. 267(6 Pt 2): p. H23 18-24.

Kolyada, A.Y. and N.E. Madias, Transcriptional regulation of the human iNOS gene by IL-1 beta in endothelial cells. Mol Med, 2001.7(5): p. 329-43.

Zamponi, G.W., et al., Crosstalk between G proteins and protein kinase C mediated by the calcium channel alpha1 subunit. Nature, 1997. 385(6615): p. 442- 6.

Suschek, C.V., et al., Critical role of L-arginine in endothelial cell survival during oxidative stress. Circulation, 2003. 107(20): p. 2607-14.

Hemmrich, K., et al., iNOS activity is essential for endothelial stress gene expression protecting against oxidative damage. J Appl Physiol, 2003. 95(5): p.

1937-46.

Yan, Z.Q., et al., Expression of inducible nitric oxide synthase inhibits platelet adhesion and restores blood flow in the injured artery. Circ Res, 1996. 79(1): p.

38-44.

Park, K.M., et al., Inducible nitric-oxide synthase is an important contributor to prolonged protective effects of ischemic preconditioning in the mouse kidney. J Biol Chem, 2003. 278(29): p. 27256-66.

Peng, H.B., P. Libby, and J.K. Liao, Induction and stabilization of I kappa B alpha by nitric oxide mediates inhibition of NF-kappa B. J Biol Chem, 1995. 270(23): p.

14214-9.

Spiecker, M., H.B. Peng, and J.K. Liao, Inhibition of endothelial vascular cell adhesion molecule-1 expression by nitric oxide involves the induction and nuclear translocation of IkappaBalpha. J Biol Chem, 1997.272(49): p. 30969-74.

Davignon, J. and P. Ganz, Role of endothelial dysfunction in atherosclerosis.

Circulation, 2004. 109(23 Suppl 1): p. 11127-32.

Barbato, J.E. and E. Tzeng, Nitric oxide and arterial disease. J Vasc Surg, 2004.

40(1): p. 187-93.

Cooke, J.P., NO and angiogenesis. Atheroscler Suppl, 2003.4(4): p. 53-60.

Chahine, R., et al., Protective effects of bradykinin on the ischaemic heart:

implication of the B1 receptor. Br J Pharmacol, 1993. 108(2): p. 3 18-22.

Bouchard, J.F., J. Chouinard, and D. Lamontagne, Role of kinins in the endothelial protective effect of ischaemic preconditioning. Br J Pharmacol, 1998. 123(3): p.

4 13-20.

Blum, A., et al., Levels of T-lymphocyte subpopulations, interleukin-1 beta, and soluble interleukin-2 receptor in acute myocardial infarction. Am Heart J, 1994.

l27(5): p. 1226-30.

Valen, G., Z.Q. Yan, and G.K. Hansson, Nuclear factor kappa-B and the heart. J Am Coll Cardiol, 2001. 38(2): p. 307-14.

Tschope, C., et al., Upregulation of bradykinin BI-receptor expression after myocardial infarction. Br J Pharmacol, 2000. 129(8): p. 1537-8.

Marin-Castano, M.E., et al., Induction of functional bradykinin B(1)-receptors in normotensive rats and mice under chronic angiotensin-converting enzyme inhibitor treatment. Circulation, 2002. 105(5): p. 627-32.

Hirata, R., T. Nabe, and S. Kohno, Augmentation of spontaneous cough by enalapril through up-regulation of bradykinin B1 receptors in guinea pigs. Eur J Pharmacol, 2003.474(2-3): p. 255-60.

Mage, M., et al., Induction of Bl receptors in streptozotocin diabetic rats: possible involvement in the control of hyperglycemia-induced glomerular Erk 1 and 2 phosphorylation. Can J Physiol Pharmacol, 2002. 80(4): p. 328-33.

Witherow, F.N., et al., Bradykinin contributes to the vasodilator effects of chronic angiotensin-converting enzyme inhibition in patients with heart failure.

Circulation, 2001. 104(18): p. 2 177-8 1.