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Bradykinin (BK) coronary outflow and left ventricular (LV) performance of kininogen-deficient Brown Norway Katholiek (BNK) rats and Brown Norway Hannover (BNH) controls were investigated. We analyzed whether the angiotensin-converting enzyme (ACE) inhibitor ramipril is able to attenuate LV dysfunction after induction of myocardial infarction (MI) in this animal model. Ex vivo, the basal BK content in the coronary outflow of buffer-perfused, isolated hearts was measured by specific radioimmunoassay. In vivo, left ventricular pressure (LVP), the maximal rate of LVP increase, LV end-diastolic pressure, the maximal rate of LVP decrease and heart rate were determined using a tip catheter 3 weeks after induction of MI. Compared to BNK rats, basal BK outflow was increased 30-fold in controls (p<0.01). In vivo, we found no significant differences between sham-ligated BNK and BNH rats in basal LV function. After MI, the impairment of LV function was significantly worse in BNK rats when compared to BNH rats. ACE inhibition significantly attenuated this LV dysfunction in both groups, when compared to untreated animals. Reduced basal BK level resulting from kininogen deficiency has no effect on basal LV function, but remains to be a risk factor for the ischemic heart. However, ACE inhibition is sufficient to improve LV function despite kininogen deficiency.

C/EBP transcription factors are involved in the regulation of the intestinal epithelial cell response to inflammatory stimuli. GATA transcription factors modulate C/EBP-dependent transcriptional activation in various cell types. We thus determined whether GATA-4 whose expression is restricted to epithelial cells, modulate C/EBP transcriptional activity and C/EBP-dependent acute phase protein expression in intestinal epithelial cells. Interaction between C/EBPdelta and GATA-4 required both C/EBPdelta leucine zipper and basic DNA-binding domains, and the GATA-4 C-terminal region. C/EBP isoforms and GATA-4 synergistically activated the alpha-acid glycoprotein gene while GATA-4 repressed thiostatin and haptoglobin C/EBP-dependent transactivation. In GATA-4 expressing intestinal epithelial cells, GATA-4 led to decreased C/EBPbeta and C/EBPdelta protein levels, and decreased thiostatin expression in response to IL-1beta and dexamethasone. This correlated with decreased C/EBPdelta recruitment to the thiostatin promoter, as assessed by chromatin immunoprecipitation assays. In contrast, increased AGP expression in response to dexamethasone correlated with increased basal histone H4 acetylation. Thus, GATA-4 may be involved in the establishment of specific intestinal epithelial cell C/EBP-dependent and C/EBP-independent transcriptional responses.

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.

High-molecular-weight kininogen (HK) plays an important role in the assembly of the plasma kallikrein-kinin system. While the human genome contains a single copy of the kininogen gene, 3 copies exist in the rat (1 encoding K-kininogen and 2 encoding T-kininogen). Here, we confirm that the mouse genome contains 2 homologous kininogen genes, mKng1 and mKng2, and demonstrate that these genes are expressed in a tissue-specific manner. To determine the roles of these genes in murine development and physiology, we disrupted mKng1, which is expressed primarily in the liver. mKng1(-/-) mice were viable, but lacked plasma HK and low-molecular-weight kininogen (LK), as well as DeltamHK-D5, a novel kininogen isoform that lacks kininogen domain 5. Moreover, despite normal tail vein bleeding times, mKng1(-/-) mice displayed a significantly prolonged time to carotid artery occlusion following Rose Bengal administration and laser-induced arterial injury. These results suggest that a single gene, mKng1, is responsible for production of plasma kininogen, and that plasma HK contributes to induced arterial thrombosis in mice.

Kininogens, the precursors of bradykinins, vary extremely in both structure and function among different taxa of animals, in particular between mammals and amphibians. This includes even the most conserved bradykinin domain in terms of biosynthesis mode and structure. To elucidate the evolutionary dynamics of kininogen genes, we have identified 19 novel amino acid sequences from EST and genomic databases (for mammals, birds, and fishes) and explored their phylogenetic relationships using combined amino acid sequence and gene structure as markers. Our results show that there were initially two paralogous kininogen genes in vertebrates. During their evolution, the original gene was saved with frequent multiplication in amphibians, but lost in fishes, birds, and mammals, while the novel gene was saved with multiple functions in fishes, birds, and mammals, but became a pseudogene in amphibians. We also propose that the defense mechanism against specific predators in amphibian skin secretions has been bradykinin receptor dependent. Our findings may provide a foundation for identification and structural, functional, and evolutionary analyses of more kininogen genes and other gene families.

Genetic predisposition likely plays an important role in the development of intracranial aneurysms. We carried out a genome search meta-analysis to identified loci associated with intracranial aneurysm. We identified previous whole-genome linkage analyses by searching PUBMED. Five studies reported by separate investigators where detailed data could be obtained were included in our analysis. We synthesized the available genome-wide scan data by using a heterogeneity-based genome search meta-analyses. We identified two linkage sites on chromosomes 3 and 17 which had P-values <0.01 for association with intracranial aneurysm. Our findings confirm the association of a locus on chromosome 17 and identify a new linkage site on chromosome 3 for intracranial aneurysm. The new locus contains a number of potential gene candidates including kininogen-1 precursor, fibroblast growth factor-12 and endothelin converting enzyme 2.

Previous peptidomic analyses of the defensive skin secretion from the North American pickerel frog, Rana palustris, have established the presence of canonical bradykinin and multiple bradykinin-related peptides (BRPs). As a consequence of the multiplicity of peptides identified and their diverse primary structures, it was speculated that they must represent the products of expression of multiple genes. Here, we present unequivocal evidence that the majority of BRPs (11/13) identified in skin secretion by the peptidomic approach can be generated by differential site-specific protease cleavage from a single common precursor of 321 amino acid residues, named skin kininogen 1, whose primary structure was deduced from cloned skin secretion-derived cDNA. The organization of skin kininogen 1 consists of a hydrophobic signal peptide followed by eight non-identical domains each encoding a single copy of either canonical bradykinin or a BRP. Two additional splice variants, encoding precursors of 233 (skin kininogen 2) or 189 amino acid residues (skin kininogen 3), were also cloned and were found to lack BRP-encoding domains 5 and 6 or 4, 5 and 6, respectively. Thus, generation of peptidome diversity in amphibian defensive skin secretions can be achieved in part by differential protease cleavage of relatively large and multiple-encoding domain precursors reflecting a high degree of transcriptional economy.

The kininogen gene and its coded proteins are slightly different in various species. In human beings, bovine and mouse, for example, there is an alternatively spliced kininogen gene K, encoding two kinds of proteins. By contrast, there is still another constituted spliced kininogen gene T which encodes only one kind of protein in the rat. The kininogen, belonging to the family 3 of cystatin superfamily, is a kind of multifunctional proteins with multiple domains, which maintains the normal physiological condition in human and some other organisms. The antagonism of hemoglutination and antihemoglutination of kininogen can not only recuperate the damaged blood vessels to prevent them from bleeding ceaselessly, but also restrain the formation of thrombus. In this review, we briefly discussed alternative splicing of kininogen gene and the multifunction of kininogen protein, as well as primarily hemoglutination and antihemoglutination, in human beings, bovine, mouse and rat which have been currently studied in detail. Our aims are to provide the beneficial references for further understanding the mechanism of evolution and alternative splicing of kininogen gene, and elucidating the multifunction roles of kininogen protein. Besides, it would be helpful for developing new medicines to regulate the vascular permeability and blood pressure and to restrain tumor.

We isolated PCR, RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE-PCR)-, and RT-PCR-generated clones from mouse kininogen family transcripts. DNA sequencing indicated that the clones were from two distinct genes. One set (K1) is from the previously reported mouse kininogen gene. The second set (K2) has an open reading frame, is 93% identical to K1 in the overlapping nucleotide sequence, and, unlike T-kininogens in the rat, encodes a bradykinin motif identical to K1. We discovered that K2 exists with two different 5' ends. We used RT-PCR to determine the distribution and relative abundance of K1 and K2 mRNA in mouse tissues. K2 is transcribed and K1 and K2 are generally both expressed in the same tissues; however, they differ in their regulation of the alternative splicing event that yields either low-molecular-weight kininogen (LMWK) or high-molecular-weight kininogen (HMWK). For example, in the liver K1 is expressed as both HMWK and LMWK, whereas K2 is only expressed as LMWK. Conversely, in the kidney K2 is strongly expressed as both HMWK and LMWK, whereas K1 is not expressed as HMWK and expressed only very weakly as LMWK.

One of the most replicated loci influencing type 2 diabetes-related quantitative traits (quantitative trait loci [QTL]) is on chromosome 3q27 and modulates both type 2 diabetes-and metabolic syndrome-associated phenotypes. A QTL for type 2 diabetes age of onset (logarithm of odds [LOD] score = 3.01 at D3S3686, P = 0.0001) was identified in a set of French families. To assess genetic variation underlying both age-of-onset QTL and our previous type 2 diabetes linkage in a 3.87-Mb interval, we explored 36 single nucleotide polymorphisms (SNPs) in two biologically relevant candidate genes for glucose homeostasis, kininogen (KNG1), and eukaryotic translation initiation factor 4alpha2 (EIF4A2). Analysis of 148 families showed significant association of a frequent SNP, rs266714, located 2.47 kb upstream of EIF4A2, with familial type 2 diabetes (family-based association test, P = 0.0008) and early age of onset (P = 0.0008). This SNP also contributes to both age-of-onset QTL (1.13 LOD score decrease P = 0.02) and type 2 diabetes linkage (genotype identical-by-descent sharing test, P = 0.02). However, no association was observed in three independent European diabetic cohorts. EIF4A2 controls specific mRNA translation and protein synthesis rate in pancreatic beta-cells, and our data indicates that EIF4A2 is downregulated by high glucose in rat beta-INS832/13 cells. The potential role of EIF4A2 in glucose homeostasis and its putative contribution to type 2 diabetes in the presence of metabolic stress will require further investigation.

The aim of the present study was to investigate whether pharmacological enhancement of the renal kallikrein-kinin system using the vasopeptidase inhibitor omapatrilat plays a direct role in modulating the fibrotic responses of human mesangial cells to injury. Treatment with 40 micromol/L omapatrilat was able to reduce macrophage-conditioned medium (MPCM)-induced fibronectin levels without affecting mRNA expression. MPCM injury also suppressed kallikrein and low molecular weight kininogen mRNA. Omapatrilat was able to attenuate this suppression. Bradykinin levels in contrast were increased by MPCM and treatment with omapatrilat further augmented levels. Co-incubation with the bradykinin B2 receptor antagonist HOE 140 attenuated the omapatrilat-induced lowering of fibronectin. Moreover, inhibition of cGMP release had a similar effect. Paradoxically, RT-PCR and Southern blotting demonstrated that bradykinin B2 receptor mRNA levels were down regulated in response to omapatrilat. Western blotting supported this data. Supernatant levels of tissue plasminogen activator (tPA), a product of bradykinin stimulation, were decreased by omapatrilat while cell associated tPA levels were increased. Matrix metalloproteinase-9 (MMP-9) mRNA expression was up regulated by omapatrilat treatment, although no difference in active zymogen levels was observed. In conclusion enhancement of kallikrein-kinin system appears to play a direct role in promoting anti-fibrotic responses in MPCM-injured human mesangial cells., (Copyright 2006 S. Karger AG, Basel.)

T-kininogen (T-KG) is a precursor of T-kinin, the most abundant kinin in rat serum, and also acts as a strong and specific cysteine proteinase inhibitor. Its expression is strongly induced during aging in rats, and expression of T-KG in Balb/c 3T3 fibroblasts results in inhibition of cell proliferation. However, T-KG is a serum protein produced primarily in the liver, and thus, most cells are only exposed to the protein from the outside. To test the effect of T-KG on fibroblasts exposed to exogenous T-KG, we purified the protein from the serum of K-kininogen-deficient Katholiek rats. In contrast to the results obtained by transfection, exposure of Balb/c 3T3 fibroblasts to exogenously added T-KG leads to a dose-dependent increase in [3H]-thymidine incorporation. This response does not require kinin receptors, but it is clearly mediated by activation of the ERK pathway. As a control, we repeated the transfection experiments, using a different promoter. The results are consistent with our published data showing that, under these circumstances, T-KG inhibits cell proliferation. We conclude that T-KG exerts opposite effects on fibroblast proliferation, depending exclusively on the way that it is administered to the cells (transfection versus exogenous addition).

Studies of proteinaceous cysteine protease inhibitors originated with the discovery of cystatins in the 1960s. Since that time, a rich and fascinating world of proteins that control and regulate a multitude of important physiological processes, ranging from the basics of protein turnover to development and brain function, has been uncovered. Failures in such important and complex systems inevitably lead to pathologies. Many threatening diseases such as cancer or neurological disorders, to mention only some, are attributed to deregulation of protease-inhibitor balance. Moreover, important aspects of infection pathology and host defense rely on proteolysis and protease inhibition. Recent advances in the field of protease inhibitors have drawn attention to the possible use of this collected knowledge to control related pathological processes. This review attempts to familiarize the reader with proteinaceous cysteine protease inhibitors by providing an overview of current knowledge. The work primarily highlights biological processes in which the inhibitors are involved and focuses on pathologies resulting from aberrant protease-inhibitor balance, pointing out emerging possibilities for their correction.

The human leukocyte antigen B27 (HLA-B27) transgenic rat is a model of human inflammatory bowel disease, rheumatoid arthritis and psoriasis. Studies of chronic inflammation in other rat models have demonstrated activation of the kallikrein-kinin system as well as modulation by a plasma kallikrein inhibitor initiated before the onset of clinicopathologic changes or a deficiency in high-molecular-mass kininogen. Here we study the effects of monoclonal antibody C11C1, an antibody against high-molecular-mass kininogen that inhibits the binding of high-molecular-mass kininogen to leukocytes and endothelial cells in the HLA-B27 rat, which was administered after the onset of the inflammatory changes. Thrice-weekly intraperitoneal injections of monoclonal antibody C11C1 or isotype IgG1 were given to male 23-week-old rats for 16 days. Stool character as a measure of intestinal inflammation, and the rear limbs for clinical signs of arthritis (tarsal joint swelling and erythema) were scored daily. The animals were killed and the histology sections were assigned a numerical score for colonic inflammation, synovitis, and cartilage damage. Administration of monoclonal C11C1 rapidly decreased the clinical scores of pre-existing inflammatory bowel disease (P < 0.005) and arthritis (P < 0.001). Histological analyses confirmed significant reductions in colonic lesions (P = 0.004) and synovitis (P = 0.009). Decreased concentrations of plasma prekallikrein and high-molecular-mass kininogen were found, providing evidence of activation of the kallikrein-kinin system. The levels of these biomarkers were reversed by monoclonal antibody C11C1, which may have therapeutic potential in human inflammatory bowel disease and arthritis.

Inflammation is accompanied by activation of the plasma kallikrein-kinin system (KKS). KKS activation has been demonstrated in a variety of inflammatory human diseases. To further explore the participation of KKS in arthritis and inflammatory bowel disease, we used two experimental animal models in arthritis and enterocolitis. We found that activation of KKS is associated with arthritis induced by intraperitoneal injection of peptidoglycan-polysaccharide polymers (PG-PS) as well as the enterocolitis and systemic inflammation induced also by PG-PS when injected into the intestinal wall of genetically susceptible Lewis rats. We postulated that KKS participates in the pathogenesis of inflammatory reactions involved in cellular injury, coagulation, fibrinolysis, kinin formation, complement activation, cytokine secretion, and release of proteases. We demonstrated that therapy with a specific plasma kallikrein inhibitor modulated the experimental enterocolitis, arthritis, and systemic inflammation. The fact that deficiency of plasma high molecular weight kininogen in the genetically susceptible Lewis rat results in decreased chronic enterocolitis and systemic inflammation also supports our hypothesis. We suggest that KKS plays a similar role in idiopathic human intestinal inflammatory disease and arthritis, making kallikrein-kinin system proteins appealing targets for drug therapy in chronic inflammatory diseases such as rheumatoid arthritis and Crohn's disease.

Brown Norway (BN) and BN Katholiek (BN/Ka) rat strains are both susceptible to develop lesions in the internal elastic lamina (IEL) of the aorta. BN/Ka rats are characterized by a single point mutation in the kininogen gene leading to deficiency in high- and low-molecular-weight kininogen. Recently, a suggestive quantitative trait locus for lesions in the IEL of the abdominal aorta was identified in an F2 intercross between Dahl salt-sensitive (SS) and BN rats, implicating kininogen as a positional candidate gene. Therefore, BN and BN/Ka rat strains represent ideal model organisms with which to study the contribution of kininogen to the genetic predisposition to IEL lesion formation and to characterize the early events underlying vascular remodeling. Here we present data demonstrating that genetic kininogen deficiency promotes the formation of aneurysms in the abdominal aorta but not the development of atherosclerosis upon 12-wk treatment with an atherogenic diet. Aneurysm formation was associated with an enhanced elastolysis, increased expression of MMP-2 and MMP-3, downregulation of TIMP-4, and with FasL- and caspase-3-mediated apoptosis. Kininogen-deficient animals also featured changes in plasma cytokines compatible with apoptotic vascular damage, i.e., upregulation of IFN-gamma and downregulation of GM-CSF and IL-1beta. Finally, in response to atherogenic diet, kininogen-deficient animals developed an increase in HDL/total cholesterol index, pronounced fatty liver and heart degeneration, and lipid depositions in aortic media without atherosclerotic plaque formation. These findings suggest that genetic kininogen deficiency renders vascular tissue prone to aneurysmatic but not to atherosclerotic lesions.

We evaluated the significance of the host kallikrein-kinin system in tumor angiogenesis and tumor growth using two rodent models genetically deficient in a kallikrein-kinin system. Inoculation of Walker 256 carcinoma cells into the s.c. tissues of the back of normal Brown Norway Kitasato rats (BN-Ki rats) resulted in the rapid development of solid tumors with marked angiogenesis. By contrast, in kininogen-deficient Brown Norway Katholiek rats (BN-Ka rats), which cannot generate intrinsic bradykinin (BK), the weights of the tumors and the extent of angiogenesis were significantly less than those in BN-Ki rats. Daily administration of B(2) receptor antagonists significantly reduced angiogenesis and tumor weights in BN-Ki rats to levels similar to those in BN-Ka rats but did not do so in BN-Ka rats. Angiogenesis and tumor growth were significantly suppressed in B(2) receptor knockout mice bearing sarcoma 180 compared with their wild-type counterparts. Immunoreactive vascular endothelial growth factor (VEGF) was localized in Walker tumor stroma more extensively in BN-Ki rats than in BN-Ka rats, although immunoreactive B(2) receptor also was detected in the stroma to the same extent in both types of rats. Cultured stromal fibroblasts isolated from BN-Ki rats and BN-Ka rats produced VEGF in response to BK (10(-8)-10(-6) m), and this stimulatory effect of BK was abolished with a B(2) receptor antagonist, Hoe140 (10(-5) m). These results suggest that BK generated from kininogens supplied from the host may facilitate tumor-associated angiogenesis and tumor growth by stimulating stromal B(2) signaling to up-regulate VEGF production mainly in fibroblasts.

Kininogens serve dual functions by forming a scaffold for the assembly of the protein complex initiating the surface-activated blood coagulation cascade and as precursors for the kinin hormones. While rats have three kininogen genes, for mice, cattle, and humans only one gene has been described. Here, we present sequence and expression data of a second mouse kininogen gene. The two genes, kininogen-I and kininogen-II, are located in close proximity on chromosome 16 in a head-to-head arrangement. In liver and kidney, both genes are expressed and for each gene three alternative splice variants are synthesized. Two of them are the expected high and low molecular weight isoforms known from all mammalian kininogens. However, for both genes also a third, hitherto unknown splice variant was detected which lacks part of the high molecular weight mRNA due to splicing from the low molecular weight donor site to alternative splice acceptor sites in exon 10. The physiological functions of the six kininogen isoforms predicted by these findings need to be determined.

Crohn disease is immunologically mediated and characterized by intestinal and systemic chronic inflammation. In a rat model, injection of peptidoglycan-polysaccharide complexes into the intestinal wall induced chronic inflammation in Lewis but neither Fischer nor Buffalo rats, indicating a differential genetic susceptibility. Proteolysis of plasma high molecular weight kininogen (HK) yielding bradykinin and cleaved HK (HKa) was faster in Lewis than in Fischer or Buffalo rat plasma. A single point mutation at nucleotide 1586 was found translating from Ser511 (Buffalo and Fisher) to Asn511 (Lewis). The latter defines an Asn-Xaa-Thr consensus sequence for N-glycosylation. We expressed these domains in Escherichia coli and found no differences in the rate of cleavage by purified kallikrein in the 3 strains in the absence of N-glycosylation. We then expressed these domains in Chinese hamster ovary (CHO) cells, which are capable of glycosylation, and found an increased rate of cleavage of Lewis HK. The Lewis mutation is associated with N-glycosylation as evidenced by a more rapid migration after treatment with N-glycosidase F. When CHO cells were cultured in the presence of tunicamycin, the kallikrein-induced cleavage rate of Lewis HK was not increased. This molecular alteration might be one contributing factor resulting in chronic inflammation in Lewis rats.

Roles for the kallikrein-kinin system in inflammation have been investigated extensively, and many reviews on this topic have been published during the 50 years since the discovery of bradykinin in 1949. Recent progress in the field has been remarkable with the help of experiments using gene-targetted transgenic or knockout mice, which have added further valuable information in addition to previous results obtained from pharmacological and biochemical studies using purified and isolated components of the system. Furthermore, much knowledge has been accumulated as a result of the development of various bradykinin agonists and antagonists. In this review, we focused on the data obtained from the kininogen-deficient rat, which is a natural mutant, and discuss the results in comparison with those from bradykinin receptor knockout mice. These data have clarified that endogenous bradykinin exerts a most important role in inflammatory exudation along with prostanoids, preferentially to histamine, serotonin, or neuropeptides. In inflammatory pain perception also, bradykinin produced in the local perivascular spaces stimulates polymodal pain receptors in conjunction with co-helpers such as prostanoids, vanilloids, and neuropeptides. These important roles are concluded based on consistent results obtained from experiments using several antagonists of bradykinin, kininogen-deficient rats, and bradykinin receptor knockout mice.

Human kininogen belongs to the plasma kallikreinkinin system. High molecular weight kininogen is the precursor for two-chain kinin-free kininogen and bradykinin. It has been shown that the two-chain kinin-free kininogen has the properties of anti-adhesion, anti-platelet aggregation, and anti-thrombosis, whereas bradykinin is a potent vasodilator and mediator of inflammation. In this study we show that the human kininogen gene is strongly up-regulated by agonists of the farnesoid X receptor (FXR), a nuclear receptor for bile acids. In primary human hepatocytes, both the endogenous FXR agonist chenodeoxycholate and synthetic FXR agonist GW4064 increased kininogen mRNA with a maximum induction of 8-10-fold. A more robust induction of kininogen expression was observed in HepG2 cells, where kininogen mRNA was increased by chenodeoxycholate or GW4064 up to 130-140-fold as shown by real time PCR. Northern blot analysis confirmed the up-regulation of kininogen expression by FXR agonists. To determine whether kininogen is a direct target of FXR, we examined the sequence of the kininogen promoter and identified a highly conserved FXR response element (inverted repeat, IR-1) in the proximity of the kininogen promoter (-66/-54). FXR/RXRalpha heterodimers specifically bind to this IR-1. A construct of a minimal promoter with the luciferase reporter containing this IR-1 was transactivated by FXR. Deletion or mutation of this IR-1 abolished FXR-mediated promoter activation, indicating that this IR-1 element is responsible for the promoter transactivation by FXR. We conclude that kininogen is a novel and direct target of FXR, and bile acids may play a role in the vasodilation and anti-coagulation processes.

A 6-year-old male with vertebral-basilar artery thrombosis was recognized to have high-molecular-weight kininogen (HK) deficiency. The propositus had no HK procoagulant activity and antigen (< 1%). Using monoclonal antibodies (Mabs) to kininogen domain 3, the propositus, family members, and Fitzgerald plasma were determined to have detectable low-molecular-weight kininogen. Mabs to HK domains 5 and 6 do not detect HK antigen in the propositus' plasma. The propositus has a single base pair (bp) deletion in cDNA position 1492 of exon 10 affecting amino acid 480 of the mature protein and resulting in a frameshift and a premature stop codon at position 1597 (amino acid 532). Unexpectedly, Mabs to the heavy chain and domain 5 of HK detect a 92-kDa form of HK in Fitzgerald plasma, the first HK-deficient plasma. The 92-kDa Fitzgerald HK has amino acid residues through 502, corresponding to domains 1 through 5, but lacks epitopes of domain 6 (positions 543 to 595). Fitzgerald DNA has a normal exon 10, but a 17-bp mutation in intron 9. These combined results indicate that mutations in the kininogen gene may differentially affect biosynthesis, processing, and/or secretion of HK.

To determine the potential role of the cardiac kallikrein-kinin system in the development of cardiac hypertrophy, we studied the expression patterns of kallikrein, kininogen, and bradykinin receptor mRNA in the heart by polymerase chain reaction during the development of pressure-overload-induced left ventricular hypertrophy (LVH) in rats. The abdominal aortic constriction produced LVH after 7, 14, and 28 days. Neither mRNA levels for high-molecular-weight (H-) or low-molecular-weight (L-) kininogens and T-kininogen, nor those for tissue kallikreins, changed during LVH. B(2)-receptor mRNA levels in the left ventricles decreased 4 and 7 days after aortic constriction, subsequently returning to the levels in sham-operated animals. B(2)-receptor densities in cardiac membrane preparations obtained 4 days after aortic constriction significantly decreased compared to preparations from sham-operated rats, whereas the receptor affinity was unchanged. Down-regulation of B(2)-receptor mRNA levels was abolished by oral administration of an angiotensin II type 1 (AT1) receptor antagonist, candesartan, for 4 days after aortic constriction. Both cardiomyocytes and nonmyocytes obtained from neonatal rat hearts expressed B(2)-receptor mRNA in vitro, and the levels were not changed in either cell type by culture with 1 microM angiotensin II (Ang II). However, when a mixture of cardiomyocytes and nonmyocytes was cultured with 1 microM Ang II, B(2)-receptor mRNA levels decreased within 12 hr; this in vitro effect of Ang II was inhibited by the AT1-receptor antagonist losartan. These results indicate that the mechanical load in the myocardium caused by pressure-overload rapidly produces a down-regulation of B(2)-receptor expression during the initial stage of LVH, probably mediated by activating the AT1-receptor.

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.

Antiphosphatidylethanolamine antibodies (APE) have been described in patients with thrombotic diseases and recurrent pregnancy loss (RPL). It has been reported that certain APE are not specific for phosphatidylethanolamine (PE) per se, but are directed to PE-binding plasma proteins, called kininogens. Our recent in vitro data suggest that APE may recognize the domain 3 (D3) region of kininogens. In this study, we have used synthetic peptides that span the D3 of kininogens in inhibition and direct binding studies to identify epitopes that are sites for binding APE. Our present data demonstrate that among 24 RPL patients who were positive for kininogen-dependent immunoglobulin (IgG) APE, 17 patients (70.8%) recognized the LDC27 peptide. We mapped the APE-binding region on D3 using plasma from a RPL patient (X) who had a high titer of IgG APE that recognized LDC27. APE of patient X recognized a 13-residue segment in LDC27, named CNA13. Leu331-Met357 (LDC27) and Cys333-Lys345 (CNA13) are located on the carboxyl-terminal portion of kininogen D3, which is known as the major kininogen heavy chain cell attachment site where it overlaps its cysteine protease inhibitory region. Because APE interferes with the balance of hemostasis in vitro, APE may therefore induce a similar condition in patients thereby causing thrombosis and RPL.

Various proinflammatory mediators are believed to be involved in the processes and symptoms of ulcerative colitis (UC). To determine whether endogenous kinin enhances the severity of UC, we induced experimental colitis (EC) in kininogen-deficient mutant rats and tested the effect of a non-peptide B2 receptor antagonist. EC was induced in male kininogen-deficient Brown Norway-Katholiek rats (BN-Ka) and normal Brown Norway-Kitasato rats (BN-Ki) with 5% dextran sulfate sodium (DSS). Sprague-Dawley rats (SD) were also used. Colon length, body weight and hematocrit were determined for 7 days. Effects of FR173657, an orally active B2 antagonist, were tested. The colon length was shortened in BN-Ki with DSS treatment, but not in BN-Ka, and the difference between their lengths was significant. The hematocrit value was also reduced in BN-Ki, and the difference in hematocrit between BN-Ki and BN-Ka was significant. In SD, shortening of the colon and reduction in hematocrit were also observable, and both were blunted by FR173657. The survival rate in SD given DSS for 7 days was 68%, but FR173657 treatment restored it significantly to 100%. These results suggest that the endogenous kinins generated from the kallikrein-kinin system have a significant role in the development of EC.

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.

Kininogens are multifunctional proteins found so far mainly in mammals. They carry vasoactive kinins as well as participate in defense, blood coagulation and the acute phase response. In this study, novel kininogens were isolated from Atlantic cod (Gadus morhua L.) and spotted wolffish(Anarhichas minor) by papain-affinity chromatography. The molecular mass of cod kininogen determined by MALDI-TOF mass spectrometry to be 51.0 kDa and it had pI values of 3.6, 3.9 and 4.4. The molecular mass of wolffish kininogen was 45.8 kDa and it had pI values of 4.1, 4.3, 4.35 and 4.4. Partial amino-acid sequences determined from both kininogens showed clear homology with previously determined kininogen sequences. Both kininogens were found to inhibit cysteine proteinases like papain and ficin but they had no effect on trypsin, a serine proteinase. Wolffish kininogen carried alpha2,3-sialylated biantennary and triantennary N-glycans with extensive sialic acid O-acetylation. Cod kininogen carried similar glycan structures but about 1/3 of its glycans carried sulfate at their N-acetylglucosamine units.

Differential mRNA display showed that a cDNA band disappeared after treatment of mice with 3-methylcholanthrene (MC). The cDNA encoded low-molecular-weight (LMW) prekininogen, known to be the precursor of a potent vasodilator, bradykinin. MC is generally known to bind to aryl hydrocarbon receptor (AhR) as an initial event to cause effects in vivo. In accordance with the results, Northern blot analysis for LMW prekininogen mRNA using total RNAs from wild-type and AhR-null mice indicated that the suppression of the mRNA expression by MC was seen in wild-type mice but not in AhR-null mice. The expression of LMW prekininogen mRNA was almost completely lost within 1 h after treatment of mice with MC, while a clear increase of CYP1A2 mRNA, as a positive control, was noted 4 h after the treatment. The plasma concentration of bradykinin released from LMW prekininogen was decreased by MC in wild-type mice, but not in AhR-null mice. Based on these results, we conclude that AhR inhibits bradykinin synthesis in mice via suppression of the expression of LMW prekininogen. Possible mechanism(s) responsible for hypertension caused by treatment of mice with MC is also discussed., (Copyright 2001 Academic Press.)

Objectives: In different vulvar pathologies inflammatory process and pain are often observed. In these processes, kinins, released from kininogen, play an important role. Their effects are mediated by at least two types of bradykinin receptors--B1 and B2. B1 receptor appears in certain pathological states, B2 is widely distributed in normal tissues. The expression of genes coding kininogen, B1 and B2 receptors can be a very sensitive marker of tissue pathology., Design: In the present study, the analysis of expression of genes coding kininogen, B1 and B2 was performed. The relation between the analysed genes expression and the pathology stage was analysed., Materials and Methods: The specimens from condylomata accuminata, vulvar cancer and surgical margin were analysed. The number of DNA and mRNA copies of beta-actine, kininogen, B1 and B2 were examined basing on Q-PCR standard curves for beta-actine by use of Perkin Elmer-kit and the sequence detector ABI PRISM 7700-Taq Man application., Results: In condylomata accuminata the high expression of mRNA of kininogen, B1 and B2 was found, while in vulvar cancer tissue, the expression of analysed genes was low. In the tissue from the tumour center, the lowest kinin genes expression was stated., Conclusions: The absence of kininogen and B2 mRNA expression characterised vulvar cancer tissue. The profile of expression of kininogen and its receptor genes can be a useful marker in the assessment of vulvar cancer surgical margin.

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.)

The mRNAs for acute-phase proteins and kininogens were found to be increased in the submandibular gland (SMG) and extraorbital and intraorbital lacrimal gland (ELG and ILG) in response to experimentally induced inflammation in rats; i.e., 24 hours after subcutaneous injection of turpentine oil, mRNAs for C-reactive protein (CRP), serum amyloid P component (SAP), and H- and T-kininogens were induced in the SMG, ELG, and ILG of rats, whereas these mRNAs were not detected in the same tissues of normal control rats. The induction of mRNAs for these inflammatory proteins by turpentine oil was preceded by a transient increase in the level of mRNA for tumor necrosis factor-alpha (TNF-alpha) at 6 hours after subcutaneous injection of the oil. This was confirmed by injection of another inflammation inducer, lipopolysaccharide (LPS), which induced the TNF-alpha mRNA in the same way at 6 hours as turpentine oil did. The up-regulation of acute-phase proteins including kininogens in the SMG, ELG, and ILG suggest the existence of a strict defense system in the exocrine glands.

Recently RT-PCR studies had demonstrated the expression of plasma prekallikrein (PPK) mRNA in extrahepatic tissues. The questions arose whether that is illegitimate or regular expression, and whether the mRNAs of blood coagulation factors XI and XII, and high molecular weight kininogen, components of the contact activation cascade of blood coagulation are also expressed in non-hepatic tissues. These questions were addressed in the present study by employing quantitative RT-PCR. The relative mRNA levels of the respective proteins determined in 16 human tissues indicate legitimate extrahepatic transcription of at least three of the genes. Transcription of all genes was highest in the liver, but only PPK mRNA was detected in all 16 tissues, especially high levels in pancreas, kidney, testis, spleen and prostate. We conclude from these results that PPK is synthesized in significant amounts in non-hepatic tissues and that this locally synthesized PPK may have special local functions.

Background: The metabolic state effect of liver failure on liver gene regulation was evaluated in a rat model., Methods: Following 70 or 90% hepatectomy and lipopolysaccharide or vehicle treatment at intervals up to 24 h, the liver remnants were analyzed for mRNA levels for acute-phase, liver-specific and growth-related proteins., Results: After 70% hepatectomy mRNA for alpha 1-acid glycoprotein, alpha 2-macroglobulin, thiostatin and fibrinogen, haptoglobin increased three- to sevenfold (P < 0.05), and mRNA for cyclin D and histone 3 increased seven- and 15-fold (P < 0.05), respectively. After lipopolysaccharide injection and 70% hepatectomy were done, mRNA for acute-phase proteins raised significantly (P < 0.05), more to five to 20-fold, while mRNA for growth-related proteins raised significantly (P < 0.05) less to three- to fourfold. After 90% hepatectomy, acute-phase protein mRNA increased five- to ninefold (P < 0.05) more than after 70% hepatectomy, while mRNA for histone 3 and cyclin D did not increase within 24 h, which indicates a delayed growth after 90% hepatectomy. In 90% of hepatectomized rats treated with lipopolysaccharide, acute-phase protein mRNA raised three- to sixfold (P < 0.05) less than after vehicle treatment., Conclusion: In endotoxemia from liver failure, the synthesis of acute-phase proteins is upregulated by gene regulation at the expense of that for regeneration, which may be an appropriate response for immediate survival. In severe liver failure, endotoxin may interfere with the appropriate gene regulation.

Angiotensin-converting enzyme inhibitors prevent cardiac hypertrophy in vivo, and a component of this ameliorative effect has been attributed to accumulation of kinins in cardiac tissues. However, little is known regarding the levels of kallikrein-kinin components in the heart during the development of cardiac hypertrophy. The objectives of the present study were to define the effects of pressure-overload cardiac hypertrophy on cardiac levels of kininogen, kallikrein and bradykinin B2 receptor mRNAs. The pressure-overload induced by aortic constriction produced cardiac hypertrophy in mice after 14 and 28d, assessed from the increased ratios of heart weight to body weight and elevation of brain natriuretic peptide mRNA in the heart. B2 receptor mRNA rapidly decreased in the heart within 7 d after the operation, subsequently returning to those of sham-operated animals. In contrast, levels of both low-molecular-weight kininogen and tissue kallikrein mRNAs were increased 7, 14 and 28 d after aortic constriction. These findings suggest that the mechanical load or stretch in cardiac tissue by pressue-overload rapidly produces the downregulation of B2 receptor expression during the initial stage which may allow the promotion of cardiac hypertrophy induced by a mediation of hypertophic factors such as angiotensin II, while upregulation of kininogen and kallikrein mRNAs during the chronic stage may lead to an enhancement of local kinin generation in the heart, from which further progression of cardiac hypertrophy during later stages may be regulated.

Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit mu- and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and deltaL110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefin B by the corresponding KD2 sequence resulted in a calpain inhibitor of Ki = 188 nM. Deletion of L110, which forms a beta-bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of mu-calpain 4- to 8-fold. All engineered cystatins were temporary inhibitors of calpain due to slow substrate-like cleavage of a single peptide bond corresponding to Gly9-Ala10 in chicken cystatin. Biomolecular interaction analysis revealed that, unlike calpastatin, the cystatin-type inhibitors do not bind to the calmodulin-like domain of the small subunit of calpain, and their interaction with the mu-calpain heterodimer is completely prevented by a synthetic peptide comprising subdomain B of calpastatin domain 1. Based on these results we propose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatin-like mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold.

Cleavage of high molecular weight kininogen (HK) by plasma kallikrein results in a light chain and a heavy chain (HK). The light chain has two domains: D6, which binds (pre)kallikrein, and D5, which binds to anionic surfaces, including heparin as well as zinc. Initially, HK was thought to be important for surface-activated coagulation. HKa or D5 binds to the urokinase receptor on endothelial cells, thereby enhancing the conversion of prourokinase to urokinase by kallikrein, and, thus, cell-associated fibrinolysis. HKa or D5 is antiadhesive by competing with vitronectin binding to the urokinase receptor and/or forming a complex with vitronectin. D5 inhibits endothelial cell migration, proliferation, tube formation and angiogenesis, thus modulating inflammation and neovascularization.

The connective tissue-type mast cells present in the submandibular gland (SMG) and peritoneal cavity of rats were found to express kininogens (KGs), the expression of which was demonstrated by Western blotting, reverse transcription-polymerase chain reaction (RT-PCR), RT-PCR Southern blotting, and light- and electron-microscopic immunocytochemistry. In the SMG, the analysis of cDNA amplified by RT-PCR revealed that the molecular species of mRNAs expressed were high-molecular-weight (HMW)-K KG and T-I KG. Light microscopic immunocytochemistry exclusively localized the KG protein(s) in the mast cells present in the SMG. The signals in the mast cells were very strong, but no positive reaction was observed in the granular convoluted tubular cells, acinar cells or striated duct cells. As determined by using electron microscopy, extremely strong labelling with immunogold was observed in the secretory granules of the mast cells, but no labelling in their nucleus or cytoplasm. Analysis by Western blotting and RT-PCR Southern blotting indicated that both protein and mRNA of KGs were present in the mast cells separated from the peritoneal cavity, indicating de novo synthesis of KG in these cells. Preliminary experiments implied that the connective tissue-type mast cells in other rat tissues also expressed KG.

Using Brown Norway Katholiek (BNK) rats, which are deficient in kininogen (kinin precursor) due to a mutation in the kininogen gene, we examined the role of endogenous kinins in 1) normal cardiac function; 2) myocardial infarction (MI) caused by coronary artery ligation; 3) cardiac remodeling in the development of heart failure (HF) after MI; and 4) the cardioprotective effect of angiotensin-converting enzyme inhibitors (ACEI) on HF after MI. Two months after MI, rats were randomly treated with vehicle or the ACEI ramipril for 2 mo. Brown Norway rats (BN), which have normal kininogen, were used as controls. Left ventricular (LV) end-diastolic volume (EDV), end-systolic volume (ESV), end-diastolic pressure (EDP), and ejection fraction (EF) as well as myocardial infarct size (IS), interstitial collagen fraction (ICF), cardiomyocyte cross-sectional area (MCA), and oxygen diffusion distance (ODD) were measured. We found that 1) cardiac hemodynamics, function, and histology were the same in sham-ligated BN and BNK rats; 2) IS was similar in BN and BNK; 3) in rats with HF treated with vehicle, the decrease in LVEF and the increase in LVEDV, LVESV, LVEDP, ICF, MCA, and ODD did not differ between BNK and BN; and 4) ACEI increased EF, decreased LVEDV and LVESV, and improved cardiac remodeling in BN-HF rats, and these effects were partially blocked by the bradykinin B(2) receptor antagonist icatibant (HOE-140). In BNK-HF rats, ACEI failed to produce these beneficial cardiac effects. We concluded that in rats, lack of kinins does not influence regulation of normal cardiac function, myocardial infarct size, or development of HF; however, kinins appear to play an important role in the cardioprotective effect of ACEI, since 1) this effect was significantly diminished in kininogen-deficient rats and 2) it was blocked by a B(2) kinin receptor antagonist in BN rats.

To ascertain the existence of the kallikrein-kinin system in the heart, we have studied in vivo and in vitro whether rat cardiac tissue expresses kininogen, kallikrein and kinin receptor mRNAs. The reverse transcription-polymerase chain reaction demonstrated that the ventricular myocardium of adult male rats expressed mRNAs for T- and low-molecular-weight (L-) kininogens, tissue kallikreins such as true kallikrein and T-kininogenase, and bradykinin B2 receptor, but not those for high-molecular-weight kininogen and B1 receptor. Lipopolysaccharide (LPS; 0.5 mg/kg, i.v.) increased the levels of mRNA for T-kininogen at 12 h and the bradykinin B1 receptor at 24 h without affecting that for other components. All of these mRNAs for the kallikrein-kinin system were also detected in cultured cardiomyocytes derived from neonatal rat ventricles; dibutyryl cyclic AMP, LPS or inflammatory cytokines such as interleukin-1 and tumor necrosis factor, up-regulated mRNA expression of T-kininogen, T-kininogenase, or B1 receptor in these cells in vitro. These results suggest that there are two kinin-generating systems in rat myocardium comprising T-kininogen/T-kininogenase and L-kininogen/true kallikrein respectively, and that the former may be relatively important in inflammatory diseases or conditions in which cAMP levels increase in cardiomyocytes.

We describe the purification and characterization of two novel cysteine proteinase inhibitors found in Atlantic salmon skin. One of these, salmon kininogen, has a molecular mass of 52 kDa as determined by matrix-assisted laser desorption/ionization time-of-flight MS, is multiply charged with pI values of 4.0, 4.2 and 4.6 and shows homology to kininogens including the bradykinin motif. The other, salarin, has a molecular weight of 43 kDa, a pI of 5.1 and shows weak homology to cysteine proteinases. Both proteins are N- and O-glycosylated and inhibit papain and ficin but not trypsin.

Expression of kininogen mRNAs has been studied in cultures of three different types of cells in rat brain, including neurons and astrocytes from cerebral cortex and meningeal cells from the leptomeninges/choroid plexus. T-kininogen mRNA was expressed by meningeal cells, but not by neurons and astrocytes, and the expression in meningeal cells was enhanced by culture with prostaglandin E2 (PGE2) or dibutyryl cAMP (Bt2cAMP). Low-molecular-weight kininogen mRNA was not detected in these cultures of cells, even after treatment with PGE2. Although expression of high-molecular-weight kininogen mRNA was very low in these cultures of cells, PGE2 or Bt2cAMP markedly stimulated its expression in cultures of meningeal cells and slightly in neurons, but not in astrocytes. We also found that expression of plasma kallikrein mRNA was strong in cultures of meningeal cells and slight in astrocytes, but absent in neurons. These results suggest that cells in the leptomeninges/choroid plexus are major sources of kininogens in rat brain which may function as precursor proteins for kinins and/or potent cysteine proteinase inhibitors during cerebral inflammation.

To determine the existence of the kallikrein-kinin system in the heart, we have studied in vitro and in vivo whether rat heart expresses kininogens (KGNs). The reverse transcription-polymerase chain reaction (RT-PCR) for KGN mRNAs demonstrated that the cardiac tissue of adult male rats expresses T-KGN mRNA but not high-molecular-weight (H-) KGN mRNA. An intravenous injection of lipopolysaccharide (LPS) resulted in a significant increase in T-KGN mRNA levels of rat heart within 12 h. Polyacrylamide gel electrophoresis of cDNA products generated by RT-PCR from heart mRNA using primers specific for either T- or low-molecular-weigh (L-) KGN revealed that rat heart expressed not only T-KGN gene but also L-KGN gene, and that LPS injection exclusively stimulated the expression of T-KGN but not of L-KGN gene. T-KGN mRNA was also detected in cultured myocytes derived from fetal rat heart, and the expression was markedly enhanced by an addition of LPS to cultures. These results demonstrated that rat cardiomyocytes are the source of T- and L-KGNs but not of H-KGN, and that their expression of T-KGN mRNA is stimulated by LPS, probably via LPS-receptor CD14.

Bradykinin and kallidin are released during dermal injury and inflammation as a result of activation of kallikreins which cleave high- and low-molecular weight kininogen (HMW and LMW kininogen, respectively). In the skin, kinins are involved, e.g., as co-mitogens in cellular proliferation or in processes propagating pain and inflammation. The aim of our study was to investigate the specific occurrence of mRNAs for components of the kallikrein-kinin system in normal human skin and in skin biopsies of patients with selected skin diseases (psoriasis, lichenificated atopic eczema, basalioma). In normal skin, reverse transcription polymerase chain reaction (RT-PCR) with specific primer pairs followed by separation of products by polyacrylamide gel electrophoresis (PAGE) revealed the presence of mRNAs for tissue kallikrein, for the B2 and the B1 bradykinin receptors, but not for kininogen. In biopsies of lichenificated atopic eczema and basalioma, additionally, the mRNAs for HMW and LMW kininogen were detected, whereas in psoriatic skin mRNA for HMW kininogen was not expressed. These differences in mRNA expression may reflect the different contribution of kallikrein-kinin system components to the maintenance of chronic skin diseases like psoriasis. In acute dermal reactions occurring in lichenificated atopic eczema or in basalioma, tissue mRNA for HMW kininogen appears to be arisen from sources not pre-existing in normal skin.

Kininogens have recently been shown to possess antiadhesive, anticoagulant, and profibrinolytic properties and can inhibit platelet activation at low thrombin concentrations. To test whether kininogens have antithrombotic properties in vivo, we devised a model of limited arterial injury confined to removal of the endothelium. Brown-Norway Katholiek strain rats with an absence of low- and high-molecular-weight kininogen due to a single point mutation, A163T, were compared in the thrombosis model to the wild-type animals, which were otherwise genetically identical. Despite an equivalent vascular injury, the mean time (+/-SEM) for a 90% decrease in flow measured by laser Doppler was 38.4+/-17 minutes in the kininogen-deficient rats compared with 194+/-29 minutes in the wild-type animals (P<0.002). The degree of vascular injury was the same. No evidence for disseminated intravascular coagulation (decrease in factor V, antithrombin, or fibrinogen) or excessive fibrinolysis (elevation of fibrinogen degradation products) was found in either group of animals. The results suggest that kininogens have antithrombotic properties at low concentrations of thrombin and that inhibitory peptides derived from kininogen may constitute a new antithrombotic strategy.

High potassium intake is known to attenuate hypertension, glomerular lesion, ischemic damage, and stroke-associated death. Our recent studies showed that expression of recombinant kallikrein by somatic gene delivery reduced high blood pressure, cardiac hypertrophy, and renal injury in hypertensive animal models. The aim of this study is to explore the potential role of the tissue kallikrein-kinin system in blood pressure reduction and renal protection in spontaneously hypertensive rats (SHR) on a high-potassium diet. Young SHR were given drinking water with or without 1% potassium chloride for 6 wk. Systolic blood pressure was significantly reduced beginning at 1 wk, and the effect lasted for 6 wk in the potassium-supplemented group compared with that in the control group. Potassium supplement induced 70 and 40% increases in urinary kallikrein levels and renal bradykinin B2 receptor density, respectively (P < 0.05), but did not change serum kininogen levels. Similarly, Northern blot analysis showed that renal kallikrein mRNA levels increased 2.7-fold, whereas hepatic kininogen mRNA levels remained unchanged in rats with high potassium intake. No difference was observed in beta-actin mRNA levels in the kidney or liver of either group. Competitive RT-PCR showed a 1.7-fold increase in renal bradykinin B2 receptor mRNA levels in rats with high potassium intake. Potassium supplement significantly increased water intake, urine excretion, urinary kinin, cAMP, and cGMP levels. This study suggests that upregulation of the tissue kallikrein-kinin system may be attributed, in part, to blood pressure-lowering and diuretic effects of high potassium intake.

Kininogens are multifunctional plasma glycoproteins. There are two forms of human kininogen: low molecular weight kininogen (LK) and high molecular weight kininogen (HK). Both are derived from the same gene by alternative splicing. Some patients with kininogen deficiency have been reported to be deficient only in HK while others are deficient in both HK and LK (total kininogen deficiency). We analyzed three Japanese patients with total kininogen deficiency by the Csp45I digestion study of exon 5 as previously reported in Williams trait and found that two had the same point mutation of C to T at base 22 of exon 5, resulting in a transition of CGA (Arg) codon to TGA (Stop) codon. This is the first report of molecular characterization of total kininogen deficiency in the Japanese population.

We have previously reported on the identification of T-kininogen (T-KG) as a gene whose expression is increased during senescence in male Sprague-Dawley (S-D) rats. Serum T-KG levels increase 2.5-4 months before the time of death for any given animal, irrespective of the actual age of the animal at the time of this event. Furthermore, dietary restriction (DR) delays, but does not prevent, the increase in serum T-KG levels. In the present study, we have assessed whether or not the age-related increase in T-KG is a common feature of senescence in other strains of rat. We have analyzed hepatic T-KG mRNA levels in male Fischer 344 rats (F344), as well as in male and female (Fischer 344 x Brown Norway)F1 rats (F1). In both of these strains, we observed a dramatic increase in hepatic T-KG mRNA levels when male rats approach senescence. The mRNA levels behave similarly in F1 and S-D rats, in that the increase occurs late in life, and it is either repressed or delayed by DR. In contrast, the increase in T-KG mRNA levels in F344 rats occurs earlier in life, and is not significantly affected by DR. Young female F1 rats fed ad libitum (AL) show a statistically significant (P = 0.0009) 2.6-fold higher level of T-KG mRNA, as compared to their male counterparts. Thus, while we still observe an age-related increase in this parameter in both AL and DR female F1 rats, the difference is statistically significant (P = 0.0001) only in DR animals. We conclude that the increase in T-KG gene expression is a common feature of senescence and that, at least in males of these commonly used rat strains, T-KG can be used as a reliable biomarker of aging. Since the increase in T-KG gene expression does not appear to correlate with inflammatory processes, and since different strains of animals succumb to different pathologies, these results further suggest that the increase in T-KG expression might be related to the process of aging per se, rather than to any given age-related pathology.