Published online before print June 30, 2003, doi: 10.1161/01.HYP.0000082812.88953.66
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(Hypertension. 2003;42:e1.)
© 2003 American Heart Association, Inc.
Letters to the Editor |
B2R of Bradykinin Activated by Proteases
Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois
To the Editor:
We wish to correct the misstatements of our results1 by Houle et al,2 who quoted us: "The human B2R has been proposed to be a specific binding site for serine proteases like human and porcine tissue kallikrein and trypsin, with pharmacological activation of these receptors on binding" (p. 611); "Tissue kallikrein exerts its effect on B2R after high-affinity binding and without the need for a catalytically active enzyme" (p. 616). Both statements are attributed to our report, but they are the opposite of what we published1 in abstract, on p. 831, para. 2, and on p. 834, para. 7; using cultured transfected or native cells, catalytically active enzyme is necessary to activate the bradykinin (BK) receptor and involving cleavage of a peptide bond. Just binding to B2 R did not activate it1 since inactive prokallikrein and DFP-treated kallikrein bind to B2 R (Figure 7); conversely, trypsin or cathepsin G did not bind to (Figure 6, p. 832, para. 3, and p. 836, para. 3), but activated the native receptor (p. 830, paras. 6, 7, 11, and p. 831, para. 1).
These authors also exposed HEK 293 cells to undiluted human plasma. After rinsing cells they added rabbit kallikrein, released BK, and, in Western blot, showed kininogen (Figure 5). Elsewhere they found no competition binding with [3H]BK of kallikrein added to B2-green fluorescent protein-expressing cells. They conclude that kallikrein’s effect on B2 was due to kinin release, but elsewhere (p. 616) to limited proteolysis of B2R.
They detected low levels of BK (0.01 nmol/L) in the medium of HEK 293 cells exposed only to 50 nmol/L kallikrein for 10 minutes but not to plasma. It is questionable whether this amount could be found with cells washed with zinc-free medium or containing Ca2+-EDTA as done1 by us (p. 634, para. 5) to remove traces of kininogen bound via Zn2+.3 If kallikrein released 0.01 nmol/L BK in 10 minutes, no significant amount of BK would be released in the seconds needed to elevate [Ca2+]i by kallikrein in CHO/B2 cells (Figure 2).1 Finally, the authors show that 50 nmol/L kallikrein releases arachidonic acid from HEK-B2-GFP cells as does 1 nmol/L BK (Figure 3) as we reported1 in Figure 1, but certainly 0.01 nmol/L BK would not give such a response.
Of course kallikreins liberate kinins, but when investigating a basic mechanism in cultured cells, they are never exposed to undiluted plasma, but grown with diluted serum where presumably most kininogen was catabolized during clotting.
The authors also found that rabbit kallikrein (50 nmol/L) stimulated B2R, released arachidonic acid, and contracted the rabbit jugular vein without cross-desensitization to BK as we stated.1 These experiments and other ones appear to confirm our results, in contrast to misquotations.
We are puzzled how repeated statements, supported by a figure, can be cited the opposite way. Nevertheless, this does not negate the validity of our finding that B2R is protease-activated.
References
1. Hecquet C, Tan F, Marcic BM, Erdös EG. Human bradykinin B2 receptor is activated by kallikrein and other serine proteases. Mol Pharmacol. 2000; 58: 828–836.
2. Houle S, Molinaro G, Adam A, Marceau F. Tissue kallikrein actions at the rabbit natural or recombinant kinin B2 receptors. Hypertension. 2003; 41: 611–617.
3. Hasan AA, Cines DB, Ngaiza JR, Jaffe EA, Schmaier AH. High-molecular-weight kininogen is exclusively membrane bound on endothelial cells to influence activation of vascular endothelium. Blood. 1995; 85: 3134–3143.
Response: Does the Bradykinin B2 Receptor Function as a Protease-Activated Receptor?
Centre Hospitalier Universitaire de Québec, Centre de recherche Pavillon l’Hôtel-Dieu de Québec, Québec, Canada
Faculté de Pharmacie, Université de Montréal, Montréal, Canada
Our recent article1 was misquoted by the above correspondence of Deddish, Hecquet, and Erdös. We used human, not rabbit tissue kallikrein (TK). HEK 293 cells were washed with a zinc-free solution before TK stimulation in experiments based on enzyme immunoassays for kinins. The possibility that B2 receptor (B2R) proteolysis initiates the pharmacologic effects of kallikrein was mentioned only as a nonpreferred hypothesis among others. Beyond misquotations from either side, we wish to summarize why our experiments do not convincingly support a direct B2R activation by TK.
HEK 293 cells expressing B2R-green fluorescent protein (GFP) were exploited to assess receptor hydrolysis by extracellular proteases, as GFP-related fragments are readily detectable reaction products. The reaction was undetectable for TK (
100 nmol/L) or for plasma kallikrein.1 However, the cells were pharmacologically activated by TK (50 nmol/L). A plausible explanation is that the cells have taken up or produced kininogen. Even without plasma pretreatment, a significant production of immunoreactive bradykinin was detected in TK-stimulated cells. The measured concentration (
10 pmol/L) was low in the supernatant, but the kinin level must have been transiently higher at the cell surface and possibly sufficient for uptake by high affinity receptors, as discussed.1 What could be the source of kininogen for such a reaction? Kininogen production by various types of cultured cells has been documented.2 Kininogens also adhere to HEK 293 cells exposed to human plasma.1 Animal sera used in culture media are not a likely source of high molecular weight kininogen, but may provide cells with low molecular weight kininogen.
Our desensitization experiments constitute the strongest evidence against a proteinase-activated receptor (PAR)-related mechanism for the action of kallikrein at B2Rs. The contraction of the rabbit jugular vein induced by TK (50 nmol/L) is completely desensitized after a single application. Bradykinin does not desensitize the preparation to itself or to TK if the stimulations are made at 30-minute intervals. We and others have shown that the B2Rs are completely recycled to the cell surface after bradykinin-induced desensitization and endocytosis.3 Proteolytically active kallikrein obviously consumes a substrate that is present in a limited amount to contract the vein via a mechanism sensitive to a B2R antagonist. This substrate is not likely to be the B2R, as shown by unabated responses to BK in TK-desensitized tissues. B2Rs hypothetically cleaved by kallikrein are not likely to remain at the cell surface to be available for subsequent bradykinin stimulation because receptors cleaved by proteases are submitted to immediate endocytosis and extensive degradation.1 The most reasonable explanation is that the relevant kallikrein substrate is a kininogen in the rabbit jugular vein.
References
1. Houle S, Molinaro G, Adam A, Marceau F. Tissue kallikrein actions at the rabbit natural or recombinant kinin B2 receptors. Hypertension. 2003; 41: 611–617.
2. Okamoto H, Takano M, Yokoyama K, Kondo J, Yayama K. Fibroblasts synthesize kininogen in response to inflammatory mediators. Immunopharmacology. 1996; 32: 28–33.[Medline] [Order article via Infotrieve]
3. Bachvarov DR, Houle S, Bachvarova M, Bouthillier J, Adam A, Marceau F. Agonist-induced rabbit bradykinin B2 receptor endocytosis and recycling assessed using green fluorescent protein conjugates. J Pharmacol Exp Ther. 2001; 297: 19–26.
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