Search

Pharmacological research in mice and human genetic analyses suggest that the kallikrein-kinin system (KKS) may regulate anxiety. We examined the role of the KKS in anxiety and stress in both species. In human genetic association analysis, variants in genes for the bradykinin precursor (KNG1) and the bradykinin receptors (BDKRB1 and BDKRB2) were associated with anxiety disorders (p < 0.05). In mice, however, neither acute nor chronic stress affected B1 receptor gene or protein expression, and B1 receptor antagonists had no effect on anxiety tests measuring approach-avoidance conflict. We thus focused on the B2 receptor and found that mice injected with the B2 antagonist WIN 64338 had lowered levels of a physiological anxiety measure, the stress-induced hyperthermia (SIH), vs controls. In the brown adipose tissue, a major thermoregulator, WIN 64338 increased expression of the mitochondrial regulator Pgc1a and the bradykinin precursor gene Kng2 was upregulated after cold stress. Our data suggests that the bradykinin system modulates a variety of stress responses through B2 receptor-mediated effects, but systemic antagonists of the B2 receptor were not anxiolytic in mice. Genetic variants in the bradykinin receptor genes may predispose to anxiety disorders in humans by affecting their function.

Bradykinin-related peptides (BRPs) are significant components of the defensive skin secretions of many anuran amphibians, and these secretions represent the source of the most diverse spectrum of such peptides so far encountered in nature. Of the many families of bioactive peptides that have been identified from this source, the BRPs uniquely appear to represent homologues of counterparts that have specific distributions and receptor targets within discrete vertebrate taxa, ranging from fishes through mammals. Their broad spectra of actions, including pain and inflammation induction and smooth muscle effects, make these peptides ideal weapons in predator deterrence. Here, we describe a novel 12-mer BRP (RVALPPGFTPLR-RVAL-(L¹, T⁶, L⁸)-bradykinin) from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis). The C-terminal 9 residues of this BRP (-LPPGFTPLR) exhibit three amino acid substitutions (L/R at Position 1, T/S at Position 6 and L/F at Position 8) when compared to canonical mammalian bradykinin (BK), but are identical to the kinin sequence present within the cloned kininogen-2 from the Chinese soft-shelled turtle (Pelodiscus sinensis) and differ from that encoded by kininogen-2 of the Tibetan ground tit (Pseudopodoces humilis) at just a single site (F/L at Position 8). These data would imply that the novel BRP is an amphibian defensive agent against predation by sympatric turtles and also that the primary structure of the avian BK, ornithokinin (RPPGFTPLR), is not invariant within this taxon. Synthetic RVAL-(L¹, T⁶, L⁸)-bradykinin was found to be an antagonist of BK-induced rat tail artery smooth muscle relaxation acting via the B2-receptor.

Kinins, a group of important pro-inflammatory peptides, are abundantly found in tissues and biological fluids of cancer patients. Bradykinin, the major representative of kinins, induces vascular permeability and, in consequence, promotes tumor expansion. Additionally, the kinin-induced inflammatory responses, especially those mediated by kinin metabolites without the C-terminal arginine residue, lead to enhanced tumor growth. The present study aimed at analyzing the ability of the human glioblastoma cell line U-373, derived from a malignant tumor, to produce kinin peptides. The proteins involved in kinin generation, i.e., the kininogens and the kallikreins, were shown to be expressed in these cells. Moreover, tumor necrosis factor α, a proinflammatory cytokine that mediates tumorigenesis, was found to enhance the expression of enzymes associated with kinin production. The strong binding of kininogen to the cell surface and the enzymatic degradation of this protein by cells suggest the activation of kinin-generating systems. Indeed, glioblastoma cells, pre-treated with tumor necrosis factor α, released kinin peptides from exogenous kininogen. The expression of kinin receptors in these cells was also shown to increase under the influence of this cytokine. Our results suggest that the human glioblastoma cell line U-373 constitutes a good cellular model that can be helpful in cancer research focused on kinin-induced inflammation. Furthermore, our findings can contribute to new approaches in cancer treatment with the use of kinin receptor antagonists and inhibitors of kinin production.

Extensive studies on bradykinin-related peptides (BRPs) generated from plasma kininogens in representative species of various vertebrate taxa, have confirmed that many amphibian skin BRPs reflect those present in putative vertebrate predators. For example, the (Val(1), Thr(6))-bradykinin, present in the defensive skin secretions of many ranids and phyllomedusines, can be generated from plasma kininogens in colubrid snakes-common predators of these frogs. Here, we report the presence of (Arg(0), Trp(5), Leu(8))-bradykinin in the skin secretion of the European edible frog, Pelophylax kl. esculentus, and have found it to be encoded in single copy by a kininogen with an open-reading frame of 68 amino acid residues. This peptide is the archetypal bony fish bradykinin that has been generated from plasma kininogens of the bowfin (Amia calva), the long-nosed gar (Lepisosteus oseus) and the rainbow trout (Onchorhynchus mykiss). More recently, this peptide has been shown to be encoded within cloned kininogens of the Atlantic cod (Gadus morhua) spotted wolf-fish (Anarichas minor), zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis) and Northern pike (Esox lucius). The latter species is regarded as a major predator of P. kl. esculentus. Synthetic (Arg(0), Trp(5), Leu(8))-bradykinin was previously reported as having multiphasic effects on arterial blood pressure in conscious trout and here we have demonstrated that it can antagonize the relaxation in rat arterial smooth muscle induced by canonical mammalian bradykinin. The discovery of (Arg(0), Trp(5), Leu(8))-bradykinin in the defensive skin secretion of this amphibian completes the spectrum of vertebrate taxon-specific BRPs identified from this source., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Bradykinins and related peptides (BRPs) occur in the defensive skin secretions of many amphibians. Here we report the structures of BRPs and their corresponding biosynthetic precursor cDNAs from the Chinese brown frog, Rana chensinensis, and the North American leopard frog, Lithobates pipiens. R. chensinensis skin contained four transcripts each encoding a different kininogen whose organizations and spectrum of encoded BRPs were similar to those reported for the pickerel frog, Lithobates palustris. In contrast, from L. pipiens, a single skin kininogen was cloned whose structural organization and spectrum of mature BRPs were similar to those reported for the Chinese piebald odorous frog, Huia schmackeri. These data also implied that the endogenous precursor processing proteases in each species pair have identical site-directed specificities, which in part may be dictated by the primary structures of encoded BRPs. Thus the spectra of skin BRPs and the organization of their biosynthetic precursors are not consistent with recent taxonomy. The natural selective pressures that mould the primary structures of amphibian skin secretion peptides are thought to be related to the spectrum of predators encountered within their habitats. Thus similarities and differences in skin bradykinins may be reflective of predator spectra rather than indicative of species relatedness.

1 To identify the roles of endogenous kinins in prevention of myocardial infarction (MI), we performed the permanent ligation of coronary artery in rats. 2 The size of MI 12, 24, and 48 h after coronary ligation in kininogen-deficient Brown Norway Katholiek (BN-Ka) rats was significantly larger (49.7+/-0.2%, 49.6+/-2%, and 51.1+/-1%, respectively) than that of kinin-replete Brown Norway Kitasato (BN-Ki) rats (42+/-2%, 38.5+/-4%, and 41.5+/-1%). 3 Hoe140, a bradykinin (BK) B(2) receptor antagonist injected (1.0 mg kg(-1), i.v.) half an hour before, and every 8 h after, coronary ligation, significantly increased the size of MI in Sprague-Dawley rats. Aprotinin, a kallikrein inhibitor, which was infused intravenously (10,000 Units kg(-1) h(-1)) with an osmotic mini-pump, significantly increased the size of an MI 24 h after ligation. 4 When evaluated using microspheres, the regional myocardial blood flow around the necrotic lesion in BN-Ka rats 6 h after ligation was reduced more than that in BN-Ki rats with MI by 41-46%. The same was true in Hoe140-treated BN-Ki rats. 5 FR190997, a nonpeptide B(2) agonist, which was infused (10 microg kg(-1) h(-1)) into the vena cava of BN-Ka rats for 24 h with an osmotic mini-pump, caused significant reduction in the size of MI (38+/-3%), in comparison with the size in vehicle solution-treated rats (51+/-3%). The size of MI in FR190997-treated BN-Ka rats was the same as in BN-Ki rats. 6 These results suggested that endogenous kinin has the capacity to reduce the size of MI via B(2) receptor signalling because of the increase in regional myocardial blood flow around the ischaemic lesion.

Physiological roles of angiotensin II type 2 receptor (AT(2)) are not well defined. This study was designed to investigate the mechanisms of AT(2)-dependent vascular relaxation by studying vasodilation in pressurized and perfused rat mesenteric arterial segments. Perfusion of angiotensin II in the presence of AT(1) antagonist elicited vascular relaxation, which was completely dependent on AT(2) receptors on endothelium. FR173657 (>1 microM), a bradykinin (BK) B(2)-specific antagonist, significantly suppressed AT(2)-dependent vasodilation (maximum inhibition: 68.5% at 10 microM). Kininogen-deficient Brown Norway Katholiek rats showed a significant reduction in AT(2)-mediated vasodilatory response compared with normal wild-type Brown Norway rats. Indomethacin (>1 microM), aprotinin (10 microM) and soybean trypsin inhibitor (10 microM) also reduced AT(2)-dependent vasodilation. Our results demonstrated that stimulation of AT(2) receptors caused a significant vasodilation through local production of BK in resistant arteries of rat mesentery in a flow-dependent manner. Such vasodilation counterbalances AT(1)-dependent vasoconstriction to regulate the vascular tone.

Amphibian skin contains rich bradykinin-related peptides, but the mode of biosynthesis of these peptides is unknown. In the present study, a novel bradykinin-related peptide, termed bombinakinin M, was purified from skin secretions of the Chinese red belly toad Bombina maxima. Its primary sequence was established as DLPKINRKGPRPPGFSPFR that comprises bradykinin extended from its N-terminus by a 10-residue segment DLPKINRKGP. The cDNA structure of bombinakinin M was found to contain a coding region of 624 nucleotides. The encoded precursor of bombinakinin M is composed of a signal peptide, an acidic peptide, six 100% identical copies of a 28-amino-acid peptide unit including bombinakinin M plus a spacer peptide. The sequence of bombinakinin M is preceded by a single basic residue (arginine), which represents the site of cleavage for releasing of mature bombinakinin M. This is the first cDNA cloning of bradykinin-related peptides from amphibian skin. The unique cDNA structure encoding bombinakinin M suggests that the generation modes of bradykinin-related peptides in amphibian skin and in mammalian blood system are different., (Copyright 2001 Academic Press.)

1. In this study, the abilities of PC12 cells to synthesize and degrade kinins were investigated. Kinin formation was assessed as kinin and kininogen content of cells and supernatants in serum-free incubations by use of a bradykinin-specific radioimmunoassay. Expression of kininogen mRNA was demonstrated by reverse-transcriptase PCR. Kinin degradation pathways of intact PC12 cells were characterized by identification of the kinin fragments generated from tritiated bradykinin either in the absence or presence of the angiotensin I-converting enzyme inhibitor ramiprilat. 2. Kinin immunoreactivity in the supernatant of PC12 cell cultures accumulated in a time-dependent fashion during incubations in serum-free media. This effect was solely due to de novo synthesis and release of kininogen (35 pg bradykinin h-1 mg-1 protein) since it could be suppressed by cycloheximide. Continuous synthesis of kininogen was a specific property of PC12 cells, as it was not observed in cultured macro- or microvascular endothelial cells. PC12 cells contained only minor amounts of stored kininogen. The rate of kininogen synthesis was not affected by ramiprilat, bacterial lipopolysaccharide, nerve growth factor or dexamethasone, but was stimulated 1.4 fold when cells were pretreated for 1 day with 1 microM desoxycorticosterone. 3. By use of cDNA probes specific for kininogen subtype mRNAs, expression of low-molecular-weight kininogen and T-kininogen in PC12 cells was confirmed. Expression of high molecular weight kininogen mRNA was also shown, though only at the lowest limit of detection of the assay. 4. Degradation of tritiated bradykinin by PC12 cells occurred with a half-life of 48 min resulting in the main fragments [1-7]- and [1-5]-bradykinin. The degradation rate of bradykinin decreased to 15% in the presence of ramiprilat (250 nM). Apart from angiotensin I-converting enzyme direct cleavage of bradykinin to [1-7]- and [1-5]-bradykinin still occurred under this condition as a result of additional kininase activities. 5. Along with previous findings of B2-receptor-mediated catecholamine release, these results now confirm the hypothesis that a cellular kinin system is expressed in PC12 cells. The presence of such a system may reflect a role of kinins as local neuromodulatory mediators in the peripheral sympathetic system.

Three types of cloned cDNA sequences for rat low molecular weight prekininogens were isolated and determined by molecular cloning and sequence analysis. The deduced amino acid sequences indicated that one, termed K-prekininogen, represents the counterpart of the known low molecular weight prekininogen present in other mammals, while the other two, called T-prekininogens, contain a novel T-kinin sequence which was recently identified from rat plasma. Although T- and K-prekininogens are highly homologous with each other, both of the T-prekininogens contain methionine, instead of arginine or lysine, as an amino acid preceding T-kinin and exhibit two consecutive amino acid deletions in the preceding region of T-kinin as compared with K-prekininogen. The former finding accounts for the previous observation of strong resistance of T-kininogens to cleavage with trypsin or kallikreins, while the latter finding has been explained by the structural analysis of genomic clones in which T-kinin-coding exon is contracted at its intron junction. A partial nucleotide sequence reported recently for the rat major acute phase protein (alpha 1-MAP) mRNA was found to be extremely related to the corresponding portion of the rat T-prekininogen mRNA. Furthermore, consistent with the previous report of the structural identity of major acute phase protein and alpha 1-cysteine proteinase inhibitor, kininogen closely resembles not only the former but also the latter in the amino acid compositions. The interrelationship among the triad of these proteins has been discussed.

The complete amino acid sequence of major acute phase alpha 1-protein of the rat (MAP) was derived from the nucleotide sequence of cloned cDNA. Amino acid analysis and partial sequencing supported the predicted sequence. The amino acid compositions of MAP and rat low-Mr kininogen are identical within experimental variation. The sequence is homologous (60%) to that of bovine low-Mr kininogen and both proteins carry the sequence for the vasoactive nonapeptide bradykinin in their C-terminal region. The rate of synthesis of MAP and the levels of MAP mRNA change coordinately during the acute phase response to inflammation.