[Des-Arg9]-Bradykinin acetate

The effects of the neuropeptide bradykinin (BK) and its natural proteolytic fragment Des-Arg9 bradykinin (DBK) on DNA synthesis and phospholipase C activation were investigated in cultured mesangial cells. DBK, acting through a distinct bradykinin receptor, induced DNA synthesis in serum-starved cultured mesangial cells. The effect of DBK was dose dependent (ED50 = 0.6 microM) and was strongly potentiated by insulin. Under the same conditions, BK had no effect. Down-regulation of protein kinase C by long term pretreatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly reduced DBK-induced DNA synthesis. In the same way, co-incubation with the protein kinase C inhibitor staurosporine potently attenuated the response to DBK, suggesting a role of protein kinase C in DBK-induced mitogenesis. Analysis of phosphoproteins from 32P-labeled mesangial cells by two-dimensional gel electrophoresis revealed that DBK, like TPA but not BK, induced a net increase in the phosphorylation of an acidic cellular protein migrating with an apparent Mr = 80,000 (termed 80K), identified as a major and specific substrate of protein kinase C. Phosphorylation of the 80K protein by DBK or TPA was completely abolished in cells depleted of protein kinase C. DBK and TPA also induced an increase in phosphorylation of an Mr = 28,000 protein. Moreover, DBK but not TPA stimulated the phosphorylation of an Mr = 18,000 protein in normal as well as in protein kinase C-depleted cells. Analysis of phospholipase C activation revealed that DBK induced a large and sustained increase in diacylglycerol production and inositol phosphate accumulation over a 10-min incubation. BK had only a minor effect on both parameters. These results demonstrate that DBK, but not BK, modulates DNA synthesis through protein kinase C activation in cultured mesangial cells.

1 The mechanisms by which agents modulate the induction of kinin B1-receptors were investigated by studying the effects of kinins in vitro, by use of the rabbit isolated aorta, and in vivo by measuring the blood pressure of anaesthetized rabbits. 2 The contractile response of the rabbit isolated aorta to kinins increased in a time-dependent manner in vitro. This effect was abolished by continuous exposure to the protein synthesis inhibitor cycloheximide (71 microM). 3 Several substances were found to increase specifically the rate of sensitization to des-Arg9-bradykinin (des-Arg9-Bk), when applied continuously in vitro to tissues isolated from normal animals: bacterial lipopolysaccharide (LPS; 1 micrograms ml-1), muramyl-dipeptide (MDP; 2 micrograms ml-1), phorbol myristate acetate (PMA; 320 nM), epidermal growth factor (EGF; 100 ng ml-1) and endothelial cell growth factor (150 micrograms ml-1). 4 The protease inhibitors phenylmethylsulphonyl fluoride and aprotinin, a non-adjuvant isomer of MDP, rabbit purified leukocyte interferon, fibroblast growth factor and the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) did not have this effect. 5. It has been demonstrated that LPS induces B1-receptors in rabbits enabling des-Arg9-Bk to act as a hypotensive agent. In these experiments neutropenia induced by nitrogen mustard, did not prevent the in vivo effect of LPS. MDP (300 micrograms) and PMA (100 micrograms) were also found to induce a state of responsiveness to des-Arg9-Bk in vivo. FMLP (1 mg i.v.) induced a temporary decrease in blood neutrophil counts but had no effect on the induction of responses to des-Arg9-Bk. 6. The development of responses mediated by the B,-receptor in the two experimental systems seems to be unrelated to the activation of neutrophil leukocytes, but may be related to the activation of tissue macrophages. Approximately 3% of cultured adherent cells derived from rabbit aorta strips following protease digestion were stained for non-specific esterase, supporting such a possibility.

In this study we investigated receptor-specific cellular signals elicited by kinin agonists in cultured rabbit superior mesenteric artery smooth muscle cells. Kinins promoted an increase in inositol phosphate formation and arachidonic acid release in these cells. The responses elicited by des-Arg9-bradykinin (des-Arg9-BK), a B1 kinin agonist, were antagonized by des-Arg9[Leu8]-BK, a B1 kinin antagonist, but not by D-Arg0[Hyp3,D-Phe7]-BK, a B2 kinin antagonist. In contrast, the responses elicited by BK, a B2 kinin agonist, were antagonized with the opposite antagonist specificity. Lys-BK or kallidin displayed a biphasic concentration-response relationship and each response phase was selectively antagonized by each of the above antagonists. Des-Arg9-BK, at 1 microM, promoted a sustained increase primarily in the level of inositol monophosphate which was partially dependent on extracellular Ca++, whereas 1 microM BK promoted a transient increase in the levels of inositol trisphosphate, inositol bisphosphate and inositol monophosphate, and the formation of inositol monophosphate was only marginally dependent on extracellular Ca++. Pretreatment with 0.1 microM phorbol 12-myristate-13-acetate resulted in inhibition of both des-Arg9-BK- and BK-promoted inositol phosphate formation. ADP-ribosylation by pertussis toxin (100 ng/ml) had no effect on the inositol phosphate response elicited by either of these agonists. The major finding in this study is that pharmacologically typical B1 and B2 kinin receptors are both coupled to inositol phospholipid metabolism and arachidonic acid release. These cells should provide an excellent system for further studies of the function and regulation of B1 and B2 kinin receptors.