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Obstructive uropathy impairs nephron growth and function and is a major cause of end-stage renal disease in both adults and children. The major focus of this review article is to examine the evidence implicating a role for the kallikrein-kinin system in the pathophysiology of obstructive uropathy. Recent in vivo studies using specific kinin receptor antagonists and transgenic animals overexpressing or lacking various components of the kallikrein-kinin system have documented that kinins are involved in the regulation of renal function and blood pressure. Multiple roles have been proposed for kinins in obstructive uropathy. Renal kallikrein gene expression is suppressed in the kidney with chronic (>7 days) complete ureteral obstruction. In contrast, ureteral obstruction stimulates renin expression, creating a state of intrarenal angiotensin excess and kinin deficiency, which plays an important role in mediating the increased renal vascular resistance and decreased renal blood flow in the obstructed kidney. In addition to their hemodynamic effects, kallikrein and kinins influence tubular functions. For example, kallikrein influences urinary acidification in the distal nephron, suggesting that dysregulation of kallikrein expression may contribute to the acidification defect in the obstructed kidney. Also, kinins exert direct diuretic and natriuretic effects in the collecting duct and may be important in mediating the post-obstructive diuresis after the relief of urinary obstruction. The kinin substrate, kininogen, is a potent inhibitor of lysosomal cysteine proteases. Unlike kallikrein, kininogen synthesis is upregulated in the kidneys and liver of animals with urinary obstruction. By neutralizing cysteine proteases, kininogen may protect the tubular epithelium of obstructed nephrons from excessive apoptosis. The beneficial actions of kinins and kininogens on renal hemodynamics, tubular function, and cell survival suggest that strategies aimed at increasing intrarenal kinins, eg, ACE-kininase II inhibitors and kallikrein gene therapy, may represent a useful adjunct in the medical treatment of obstructive uropathy.

To identify the presence of a local kallikrein-kinin system in vascular wall, we have studied whether rat vascular smooth muscle cells (VSMC) express kininogen in vitro and in vivo. Western blots using anti-T-kininogen antibody revealed the presence of T-kininogen in conditioned medium of cultured VSMC. T-Kininogen secretion by VSMC was markedly enhanced by the addition of lipopolysaccharide (LPS), angiotensin II (AII) and phorbol 12-myristate 13-acetate (PMA) to the culture. Experiments using specific inhibitors for protein kinases and on the PMA-induced down-regulation of protein kinase C suggested that a protein kinase C-dependent or unidentified pathway is involved in AII or LPS action, respectively. The intravenous injection of LPS (0.5 mg/kg) resulted in an increase in T-kininogen mRNA levels in the vascular smooth muscle of rat aorta, peaking at 16 h. Polyacrylamide gel electrophoresis of cDNA products generated by reverse transcription-polymerase chain reaction (RT-PCR) from aortic mRNA using primers specific for either T- or low-molecular-weight kininogen revealed that rat vascular smooth muscle expressed T-kininogen gene but not low-molecular-weight kininogen gene, and that LPS exclusively stimulated T-kininogen expression. The mRNA for high-molecular-weight kininogen was undetectable in either aortic smooth muscle or cultured VSMC by means of RT-PCR analysis. RT-PCR using specific primers for rat tissue kallikrein genes showed that aortic smooth muscle expressed KLK1 (true kallikrein) mRNA, but not KLK10 (T-kininogenase) mRNA. These results demonstrated that rat VSMC are a source of T-kininogen but not of low-molecular-weight- or high-molecular-weight kininogen, in contrast to the expression of true kallikrein but not of T-kininogenase by these cells.

To determine the existence of the kallikrein-kinin system in vascular wall, the expression of low-molecular-weight (LMW) kininogen, a precursor protein of kinins, was studied in mouse aortic smooth muscle in vivo or in cultured vascular smooth muscle cells (VSMC) derived from mouse aorta in vitro. Although LMW-kininogen mRNA was undetectable in aortic smooth muscle of untreated mice using either Northern blotting or reverse transcription-polymerase chain reactions (RT-PCR) followed by Southern blotting, administration of lipopolysaccharide (LPS; 1 mg/kg, i.v.) induced the expression of LMW-kininogen mRNA at levels detectable by RT-PCR within 12 h. Cultured VSMC not only expressed LMW-kininogen mRNA at levels easily detectable by RT-PCR, but also secreted LMW-kininogen-like protein that was immunoreactive to anti-mouse LMW-kininogen antibody. These results demonstrate that VSMC are a source of LMW-kininogen in the mouse, and suggest the presence of a local kallikrein-kinin system in vascular tissue. LPS-induced up-regulation of LMW-kininogen expression suggests a role for vascular LMW-kininogen in tissue trauma or inflammation.

Low and high molecular weight kininogens (LK and HK), containing 409 and 626 amino acids with masses of approximately 65 and 120 kDa after glycosylation, respectively, are coded by a single gene mapped to the human chromosome 3 by alternative splicing of the transcribed mRNA. The NH2-termini Glu1-Thr383 region, identical in LK and HK, contains bradykinin (BK) moieties Arg363-Arg371. LK, HK and their kinin products Lys-BK and BK are involved in several biologic processes. They are evolutionarily conserved and only 7 patients, all apparently normal, have been reported to lack them. In one of these patients (Williams' trait), a codon mutation (Arg178-->stop) has been blamed for the absence of LK and HK. However, using Western blots with 2 monoclonal anti-HK antibodies, one that recognizes the region common to LK and HK and the other that recognizes only HK, 1 detected approximately 110-kDa bands in the plasma of this LK/HK-deficient patient vs approximately 120-kDa bands in normal human and ape plasmas. With polyclonal anti-Lys-BK antibody, which strongly detects BK cleaved at its COOH-terminus in purified HK, 1 detected approximately 110-kDa bands in the normal and the deficient plasmas. Western blots with a monoclonal anti-prekallikrein (PK) antibody showed that surface activation of PK and distribution of PK activation products, both dependent on HK, were similar in these plasmas. These findings suggest that a mutant gene yielded a kininogen-like species possibly involving aberrant mRNA splicing-structurally different from normal HK, but apparently with the capacity to carry out seemingly vital HK functions.

The nature of all of the peptides critical to the mechanism(s) of the antihypertensive action of neutral endopeptidase (NEP) inhibitors is still unclear, but bradykinin is thought to be one such peptide. This study was designed to assess the effectiveness of an NEP inhibitor in deoxycorticosterone acetate (DOCA)-salt treated kininogen-deficient Brown Norway Katholiek (BN-Ka) rats. Oral administration of BP102 (10-100 mg/kg), an NEP inhibitor, increased urine volume and urinary sodium excretion in a dose-dependent manner in anesthetized Sprague-Dawley rats. DOCA-salt hypertension was induced in both BN-Ka and Brown Norway Kitasato (BN-Ki) rats after left nephrectomy. The development of DOCA-salt hypertension in normal BN-Ki rats was prevented, and that in BN-Ka rats was also significantly reduced, by an 8-day administration of BP102. When BP102 was administered for 5 weeks, the high blood pressure of DOCA-salt treated BN-Ka rats was markedly lowered, and their heart weights were reduced. These results suggest that kinins play no role in the antihypertensive effect of this inhibitor and that other factors may be involved in this effect.

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.

The kallikrein-kinin system (KKS) plays an important role in the regulation of renal function. Endogenous kinins modulate renal microvascular resistance, medullary blood flow, and distal nephron sodium and water reabsorption. All the components of the KKS, including tissue kallikrein, kininogen, kininase II, and kinin receptors are expressed within the kidney, establishing a paracrine system capable of controlling local nephron functions. In this review, data will be presented demonstrating that the developing kidney expresses an endogenous, functionally active KKS. Molecular studies have shown that gene expression of the renal KKS in the rat is activated postnatally, and that the intrarenal distribution of KKS components is subject to developmental control. Furthermore, the developmental expression of KKS appears to be regulated primarily at the transcriptional level. Ontogenetic studies have also revealed that the bradykinin B-2 receptor gene is overexpressed in the developing rat kidney. As kinins are potent vasoactive and growth-promoting factors, it is proposed that endogenous kinins mediate developmental renal growth and differentiation, and modulate the maturational changes which occur in renal hemodynamics.

We isolated cDNAs encoding low-molecular-weight (L-) and high-molecular-weight (H-) prekininogens from a mouse liver cDNA library using rat T-kininogen cDNA and rat H-kininogen cDNA respectively, as probes. The signal peptide, the heavy chain, and the bradykinin moiety, which are common between the two prekininogens, consist of 20, 359, and 9 amino acids, respectively, while the light chains of the L- and H-prekininogens are composed of 44 and 273 amino acids, respectively. All 19 cysteine residues present in both mouse prekininogens are located at the same positions relative to those of human origin. The light chain of H-prekininogen contains a characteristic 15-repeated His-Gly sequence and a conserved sequence for binding prekallikrein or factor XI. Northern blotting or reverse transcription-polymerase chain reaction followed by Southern blotting using mouse L- and H-kininogen cDNAs demonstrated that both L- and H-kininogens are predominantly expressed in the liver and kidney. L-Kininogen mRNA was also expressed in other tissues, such as the adrenal gland, brain, spinal cord, testis, lung, heart, and skin, while levels of H-kininogen mRNA in these tissues were too low to detect, suggesting that L-kininogen is synthesized in various tissues of mouse, while H-kininogen is exclusively synthesized in the liver and kidney. A genomic Southern blot using H-prekininogen cDNA revealed that the L- and H-prekininogen mRNAs in mouse are probably encoded by a single gene, as is the case in both human and bovine.

The effect of chronic treatment with zidovudine (AZT) on the inflammatory response was examined in the rat. AZT was administered orally for 36 days. On day 35, inflammation was induced by hindpaw injection of 1% carrageenan lambda. Paw edema over a 24-hour period was used as a marker of the local inflammatory reaction. On day 36, quantification of immunoreactive T-kininogen and alpha 1-inhibitor-3 in liver and serum was used to assess the systemic inflammatory response. Albumin was selected as a protein whose concentration is modified only slightly or not at all during the acute-phase response. Animals treated with AZT transiently exhibited significantly greater (18%) paw edema 3 hours after carrageenan injection. AZT treatment alone induced a 1.8-fold increase in serum T-kininogen concentration, but it had no effect on albumin and alpha 1-inhibitor-3. In rats with inflamed paws, AZT administration led to a significant increase in liver (3.4-fold) and serum (1.8-fold) immunoreactive T-kininogen content. Dot blot analysis of total RNA isolated from liver correlated with the protein measurements. Our results indicate that chronic treatment with AZT potentiates the nonspecific local and the systemic inflammatory responses in the rat.

Hepatocyte growth factor activator (HGFA) is a serine protease that is responsible for localized activation of hepatocyte growth factor (HGF) in injured tissue. The activated HGF may be involved in regeneration of the injured tissue. HGFA is produced and secreted by the liver and circulates in the plasma as an inactive zymogen. In response to tissue injury, the HGFA zymogen is converted to the active form by limited proteolysis. In this study, we isolated a rat HGFA complementary DNA (cDNA) clone and analyzed the production of HGFA messenger RNA (mRNA) in response to tissue injury using this cDNA clone as a probe. The nucleotide sequence of the cDNA revealed that the amino acid sequences of rat and human HGFA showed a high degree of conservation in the regions of the characteristic domain structures, suggesting that rat and human HGFA are activated by a similar mechanism and have similar enzymatic activities in vivo. Tissue distribution analysis showed that the liver was the major site of rat HGFA mRNA synthesis. Moreover, the cells producing HGFA mRNA were identified as parenchymal liver cells. The level of HGFA mRNA increased in the liver after hepatotoxin or nephrotoxin treatment. This increase was also observed during acute inflammation induced by turpentine. These results suggest that the increase in production of HGFA mRNA in response to tissue injury is the result of an inflammatory response, and that HGFA is an acute phase protein.

The tissue kallikrein-kinin system has been implicated in regulating blood pressure and electrolyte homeostasis. To understand the function of this system, we identified the expression and cellular localization of its components including tissue kallikrein, kallistatin, kininogen, and bradykinin B1 and B2 receptors in human adrenal gland. Reverse transcription-polymerase chain reaction followed by Southern blot analysis showed that these five components of this system were all expressed in human adrenal gland. In situ hybridization histochemistry with respective digoxigenin-labeled antisense riboprobes revealed localization of kallikrein transcript throughout the adrenal cortex and medulla except the zona glomerulosa, whereas kallistatin mRNA was only localized in the zona fasciculata. Low-molecular-weight kininogen and B2 receptor mRNAs were colocalized in the zona glomerulosa and zona fasciculata and also in the zona reticularis and chromaffin cells but to a lesser degree. The B1 receptor mRNA was stained in the zona fasciculata and medulla. These results show the expression and differential colocalization of the components of the tissue kallikrein-kinin system and reveal the potential action sites of this system in the adrenal gland.

Expression of the low-molecular-weight kininogen (L-kininogen) mRNA in the human fibroblast cell line WI38 was examined by means of the reverse-transcription polymerase chain reaction and Southern blotting using human L-kininogen cDNA as a probe. The results demonstrated that WI38 fibroblasts expressed L-kininogen mRNA and that the expression was stimulated by 1 mM dibutyryl cAMP or 10 microM prostaglandin E2. Dexamethasone (1 microM) inhibited the stimulatory effect of prostaglandin E2. These results suggest that human fibroblasts supply L-kininogen, a protein precursor of the inflammatory mediator kinins, to connective tissues in response to inflammatory stimuli and that glucocorticoids may exert the antiinflammatory effect in part by inhibiting the local production of L-kininogen.

Human high (H) and low (L) molecular weight kininogens are encoded by distinct mRNAs derived by alternative splicing from a single kininogen gene. Previous studies have demonstrated the presence of L-kininogen but not of H-kininogen in the distal nephron structures of the kidney. Using the highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) we have been able to demonstrate the expression of both H-kininogen mRNA and L-kininogen mRNA in kidney and liver. The presence of H- and L-kininogen antigen was shown immunohistochemically by applying specific antibodies that discriminate between the two types of kininogens. Immunoreactive kininogens were localized in the cortical and medullary collecting ducts. Our results indicate that both types of kinin-bearing kallikrein substrates are expressed in the human kidney where they might contribute to the suggested roles of the kallikrein-kinin system in the regulation of renal blood flow and electrolyte excretion.

Tissue kallikrein is a serine proteinase which processes kininogens to release bioactive kinins. Kinins mediate a variety of biological processes through the interaction with kinin receptors. Kinins are involved in the regulation of blood pressure and local blood flow, vasodilation, smooth muscle contraction and relaxation, production of pain and inflammation, and stimulation of cell proliferation. The tissue kallikrein-kinin system has been implicated in a number of pathophysiological processes such as hypertension, allergy and diabetes mellitus. In the present study, we have identified the expression and localization of components of the kallikrein-kinin system in the human eye by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analyses, and in situ hybridization histochemistry. RT-PCR and Southern blot analyses have detected mRNAs of the key components of the system including tissue kallikrein, low molecular weight kininogen, and bradykinin B1 and B2 receptors at high levels in human retina, choroid and ciliary body, and relatively low levels in the optic nerve. In situ hybridization has identified cellular localization of these four mRNAs in ocular tissues. They are expressed in retinal neuronal cells including the outer nuclear layer, inner nuclear layer and ganglion cell layer. These mRNAs were also identified in endothelial cells of ocular blood vessels, ciliary muscle and lens epithelial cells. The sense riboprobes showed negative staining, which indicates the specificity of the antisense riboprobes. These results suggest that the tissue kallikrein-kinin system is produced endogenously in human ocular tissues. Similar expression patterns of kallikrein, kininogen and kinin receptors indicate that the kallikrein-kinin system may function in an autocrine or paracrine fashion in the eye.

This study was designed to determine whether the kallikrein-kinin system exerts a protective action in hypertension induced by chronic inhibition of nitric oxide synthase. N omega-nitro-L-arginine methyl ester (L-NAME, 40 mg/100 ml water) was given orally to Sprague-Dawley rats, while controls received regular tap water. Hepatic kininogen mRNA levels in the L-NAME-treated group were 2.9- and 2.5-fold higher at 3 and 4 wk, respectively, compared with control rats, whereas kallikrein-binding protein (KBP) mRNA levels were 82% and 45% of the values found in control rats at 3 and 4 wk, respectively. There was no significant change in hepatic alpha 1-antitrypsin mRNA levels under the same conditions. At 3 and 4 wk post L-NAME treatment, renal kallikrein mRNA levels were 2.5- and 3.4-fold higher than in controls, whereas renal beta-actin mRNA levels were similar between groups. Changes in the transcript levels of renal kallikrein, kininogen, and KBP were consistent with their protein levels. Immunoreactive total kininogen and low-Mr kininogen levels in sera and tissue kallikrein levels in kidney were significantly higher in the L-NAME-treated group, whereas KBP levels in the circulation were lower compared with controls. Systolic blood pressure was increased by 58 +/- 4 mmHg after 4 wk of L-NAME treatment. This effect was enhanced in rats given L-NAME in combination with HOE-140, a bradykinin B2-receptor antagonist, at the dose of 100 micrograms/day ip (79 +/- 5 vs. 58 +/- 4 mmHg, P < 0.05). This difference was confirmed by direct measurement of mean blood pressure (MBP). An intra-arterial bolus injection of 200 ng bradykinin significantly decreased MBP of L-NAME-treated rats, and this effect was blunted in the group treated with the bradykinin antagonist (-29 +/- 3 vs. -9 +/- 2 mmHg, P < 0.01). These results suggest that enhanced kallikrein and kininogen synthesis may have a protective role against the cardiovascular effects induced by chronic inhibition of nitric oxide synthesis.

Kininogen is the precursor of the kinin peptide, which binds to kinin receptors and mediates a broad spectrum of physiological effects. To understand the function of kinin in the kidney, we have identified the cellular localization of the human low-molecular-weight (LMW) kininogen and bradykinin B2 receptor mRNAs in the human kidney by in situ hybridization histochemistry. Kininogen mRNA was found in the juxtaglomerular cells, mesangial areas, epithelium of parietal and visceral (podocytes) layers of Bowman's capsule, proximal and distal tubules, thin and thick segments of Henle's loop, collecting ducts, and the endothelial cells of the blood vessels. B2 receptor mRNA was colocalized with kininogen mRNA in the kidney except the podocytes. The most intense signals were observed in the distal tubules and collecting ducts for both kininogen and B2 receptor mRNAs. No signals were observed in the interstitial cells and macula densa. Control sections did not stain with either the kininogen or B2 receptor sense riboprobe. A Northern blot showed that the expression of LMW kininogen is in the liver and the kidney. Reverse transcription-polymerase chain reaction Southern blot showed expression of B2 receptor mRNA in the endothelial cells, renal proximal tubular cells, and kidney. Our results show the sites of action of kinin in the human kidney and provide further insight into the physiological role of the kallikrein-kinin system on renal function.

RP 67850, a NK1 receptor antagonist, inhibited the sialogogic effect of Substance P (SP). Heat-induced salivation was indirectly measured through the changes in body weight. Thermolytic salivation was reduced by atropine and RP 67580 and thus would be mainly controlled by acetylcholine and tachykinins. This salivation was associated with a large depletion of kallikrein in submaxillary glands. This depletion was not inhibited by atropine and RP 67580. Feeding has been reported to reduce amylase activity in parotid glands but did not modify the kallikrein content in submaxillary glands. Heat exposure did not modify amylase activity. There is a dissociation between the releases of amylase and kallikrein from salivary glands.

Each of 11 exons of the human kininogen gene has the potential to code for different functional activities of the molecule in both bradykinin formation and interactions with platelets, neutrophils and endothelial cells. Our recent studies have localized amino acid sequences in exon 4 product and exon 5 product in domain 2, which bind and inhibit platelet calpain, respectively. Furthermore, we have shown that the exon 7 product expressed in domain 3 contains a decapeptide which interacts with thrombospondin on activated platelets, and a distinct septapeptide which inhibits thrombin-induced activation of platelets. Exon 8 and 9 products cooperate to inhibit cathepsins B and H. Domain 3 also contains a cell binding site for neutrophils, as does domain 5. Fine mapping of both cell binding domains has been performed by several groups of investigators for neutrophils, endothelial cells and platelets. The cell binding domain in D5 overlaps with the anionic surface binding subdomain of D5. The monomeric structure of kininogen assures that it will function as an antiadhesive protein, unlike dimeric fibrinogen. High molecular weight kininogen competes with fibrinogen binding to neutrophils and platelets. We have also fine mapped the domains for binding of kininogen (domain 6) to prekallikrein 'apple' domains 1 and 4. Kininogens can serve as proinflammatory proteins by releasing bradykinin, but cleaved kininogens exhibit antiadhesive and anti-inflammatory properties.

Mouse fibroblasts in vitro secret kininogen (KGN). Rat fibroblasts also synthesized and secreted T-KGN in vitro. KGN production by these fibroblasts is greatly stimulated by dibutyryl cAMP, prostaglandin E2 and tumor necrosis factor. Human fibroblast WI-38 cells also express the L-KGN gene, which was stimulated by dibutyryl cAMP and prostaglandin E2. These results demonstrate that fibroblasts express the KGN gene, and suggest that the expression is regulated by inflammatory mediators. RT-PCR, using specific primers for the T-KGN and L-KGN genes, reveals that the rat hind-paw express both T- and L-KGN mRNAs, and the expression of both KGN mRNAs was increased in the hind-paw 24 h after inducing inflammation by injecting Freund's complete adjuvant into the paw. Thus, it is suggested that local connective tissues express the KGN gene, and that the expression is enhanced under pathological conditions, such as inflammation.

We have previously shown that senescent Sprague-Dawley rats have significantly increased levels of kininogen (KG) mRNA and protein, when compared with younger counterparts. In the rat, five different isoforms of kininogen have been identified: high-molecular-weight K-kininogen, low-molecular-weight K-kininogen, and T1 alpha, T1 beta, and T2 kininogens. Several of these isoforms have been shown to differ in their biological properties. It was therefore considered relevant to establish which of these isoforms are expressed in the liver of old rats. To this end, we have isolated and sequenced nine independent cDNA clones from a library constructed using liver mRNA from healthy senescent rats. Predicted protein sequences indicate the presence of T-kininogens only. The relative lack of induction of K-kininogens during aging was further confirmed by RNA hybridization experiments. The nucleotide sequences reveal a microheterogeneity of silent polymorphisms, suggesting the presence and expression of several different alleles of the genes. From our data we conclude that (i) Several isoforms of T-KG are expressed in the liver of senescent Sprague-Dawley rats and (ii) T1 kininogens appear to be the most highly represented T-KG mRNA species in old rat livers.

Objective: The purpose of this study was to determine the source(s) of the reproductive tract kininogen and to assess whether kininogen transcription is influenced by reproductive conditions., Study Design: Rats in various reproductive states (immature, mature, ovulatory, luteal phase, pregnancy, parturition, postpartum) were used to obtain reproductive tissues (follicles, corpora lutea, oviduct, uterus, testes) and liver. Complementary deoxyribonucleic acid probes for rat prekininogens were used to quantify kininogen messenger ribonucleic acid synthesis., Results: The T-prekininogen complementary deoxyribonucleic acid probe detected a single 1.6 kb message, whereas the k-prekininogen complementary deoxyribonucleic acid probe identified two messages, an abundantly expressed 1.6 kb band and a 2.2 kb band. The source of all the three prekininogen messages appears to be the liver. Naturally occurring reproductive conditions such as ovulation, implantation, and parturition, did not turn on prekininogen message transcription in the rat gonad or genital tract. Only decidualization of the uterus was associated with the induction of kininogen transcription in the liver., Conclusion: There appears to be little, if any, contribution of local gene expression to the kininogen present in the reproductive tissues. Apparently, the reproductive tract increases uptake of kininogen from plasma as needed.

The calpain-binding domain 2 of the kininogens, the major plasma inhibitors of cysteine proteinases, was expressed in Escherichia coli. Expression of soluble protein was optimal at 15 degrees C and was augmented by growing the bacteria in sorbitol and betaine. The recombinant domain showed high affinity (Ki 0.3-1 nM) for cathepsin L and papain, and a somewhat lower affinity (Ki approximately 15 nM) for calpain. The binding to cathepsin H was substantially weaker, and no inhibition of actinidin and cathepsin B was detected. The affinity for cathepsin L was comparable to that reported for the domain isolated from plasma L-kininogen, whereas the affinities for papain and calpain were about tenfold lower. The latter difference may be due to the recombinant domain being nonglycosylated.

While constructing a contig in the human chromosome region 3q27, we identified two YAC clones that were positive for the polymorphic marker D3S1602. One of these clones was also positive for a sequence-tagged site derived from the kininogen (KNG) gene. Because of the known evolutionary and structural relationship of KNG to other members of the cystatin gene superfamily, we tested the physical linkage of the genes encoding alpha-2HS-glycoprotein (AHSG), KNG, and histidine-rich glycoprotein (HRG), all of which were previously mapped to the long arm of chromosome 3. Our results show the colocalization of the three genes in two independent, partially overlapping YAC clones. The genomic inserts of the two clones were 1 Mb and 1.3 Mb in size, indicating that the three genes map within 1 Mb of DNA. The largest YAC was also positive for the polymorphic marker D3S1262, substantiating previously reported data of genetic linkage between this marker and HRG. Fluorescence in situ hybridization localized the two YAC clones to chromosome band 3q27.

1. The Brown Norway (B/N) Katholiek rat is a mutant strain of plasma kininogen deficiency. The plasma of B/N-Katholiek rats was shown to contain only 3-5% of high-molecular-weight and low-molecular-weight kininogens (HK and LK) of the normal level by specific RIA, and 30% of prekallikrein was detected by amidase activity. However, HK antigen in the liver microsomal fraction of B/N-Katholiek rats was about 60% of that of normal rats. 2. In this paper we compare and discuss synthesis and secretion of HK and LK by primary cultures of livers of deficient and normal rats. The deficient hepatocytes could synthesize HK and LK in the same way as normal cells but could not secrete mature forms of HK and LK in the medium. Examination of the subcellular localization of the mutant HK in the hepatocytes showed that a larger amount of mutant HK antigen, compared to normal rats, was found in the 10,000 g fraction, which is rich in lysosomes, suggesting that the mutant HK may be transported to the lysosomes. 3. We also analyzed sequence of the HK cDNA of B/N-Katholiek and B/N-Kitasato rats and found a point mutation of G to A at nucleotide 487, which locates at the heavy chain region of HK and LK. 4. We constructed five expression plasmids to transfect COS-1 cells to examine HK secretion. COS-1 cells transfected with the plasmids containing the G to A transition could not secrete and retained HK, while those cells transfected with the plasmids containing normal G released HK into the medium. 5. These results indicate that a point mutation G to A at nucleotide 487, resulting in an amino acid transition from alanine (163) to threonine, is responsible for the defective secretion of HK and LK by the liver of B/N-Katholiek rats. We also discuss other cases of secretion defect of plasma proteins reported in the literature.

High molecular weight kininogen (HK) binds specifically, saturably, and reversibly to neutrophils and also reciprocally inhibits the binding of fibrinogen to neutrophils. Since fibrinogen binds to the leukocyte integrin CD11b/18 (Mac-1, alpha M beta 2), we investigated whether HK bound to Mac-1 and whether the binding site was similar to that for factor X. We also examined whether one or both chains of cleaved HK (HKa) were involved. Two monoclonal antibodies, 2B5 (0.29 microM) to HK heavy chain domains 2 (D2) and 3 (D3), and C11C1 (0.26 microM) to HK light chain domain 5 (D5), inhibited by 99 and 93% the binding, respectively, of 125I-HK (8.3 nM) to neutrophils. To minimize steric hindrance, we further demonstrated that the Fab' fragments of 2B5 and C11C1 were able to inhibit the binding of this ligand to virtually the same extent as the intact antibody, indicating that, as in binding of HK to platelets and endothelial cells, both chains are involved. To directly demonstrate the involvement of each chain, we showed that the reduced alkylated light chain derived from HK and low molecular weight kininogen, which contains the same heavy chain as HK, each markedly inhibited the binding of HK to neutrophils. We localized the domain responsible for the binding in each chain by showing that recombinant D3 and D5 decreased the binding of HK to neutrophils. To define the receptor for HK, we employed three monoclonal antibodies to Mac-1: OKM1 and OKM10 to epitopes on the alpha M subunit and IB4 to an epitope on the beta 2 chain. OKM1, which can inhibit fibrinogen binding to neutrophils, inhibited HK binding by 79%, whereas the other antibodies inhibited HK binding less than 25%. Coagulation factor X also binds to Mac-1 on monocytes at a similar site to C3bi. Synthetic peptides which define noncontiguous surface loops in factor X that interact with Mac-1, failed to inhibit 125I-HK binding to neutrophils. We conclude that HK binds, via domains on its heavy chain, D3, and light chain, D5, to Mac-1 on the neutrophil surface, and HK occupies a site overlapping with fibrinogen and different from factor X.

The precursors of kinins, K-kininogens and T-kininogens (KG), are encoded by separate genes that display 90% nucleotide sequence homology. Despite their homology, K-KG and T-KG genes are differentially regulated. The K-KG gene is expressed constitutively and encodes high- and low-molecular-weight KG, the precursors of the vasoactive nonapeptide bradykinin. In contrast, the T-KG gene is inducible, and its protein is a potent thiol-protease inhibitor. Given their potential role in the regulation of blood pressure, renal hemodynamics, and the response to inflammation and tissue injury, K-KG and T-KG gene expression in rats subjected to chronic (1 or 5 wk) unilateral ureteral obstruction (UUO), a maneuver that suppresses renal kallikrein synthesis to 25% of controls, has been examined. Northern and slot blots of total liver and kidney RNA were probed with oligonucleotides complementary to either T-KG or K-KG mRNA under high-stringency conditions. Steady-state levels of hepatic T-KG mRNA were increased in the UUO compared with sham-operated rats--2.7-fold at 1 wk and 4.1-fold at 5 wk (P < 0.05). Western blot analysis revealed that the 68-kd T-KG protein was up-regulated 2.5- to 3-fold in the liver of UUO rats (P < 0.05). In marked contrast, the abundance of high (2.3-kb)- and low (1.6-kb)-molecular-weight splicing transcripts of hepatic pre-K-KG mRNA was not altered at either time after UUO.(ABSTRACT TRUNCATED AT 250 WORDS)

The serum concentration of rat T1 kininogen increases 20-30-fold in response to acute inflammation. This increase, induced in the liver, is regulated primarily at the transcriptional level. In contrast, synthesis of a homologous K kininogen is not induced. In this study, we further analyzed a 321-base pair interleukin (IL)-6 response element in the T1 kininogen promoter and showed that it consists of at least three functionally distinct sequences (A, B, and C boxes). All three sequences were required for full promoter activity. The B box, a strong C/EBP-binding site, was crucial for T1 kininogen's basal expression, whereas A and C boxes resembled the type II IL-6 response elements and were critical for the cytokine response. C/EBP alpha, -beta, and -delta interacted with the B box sequence; however, upon IL-6 stimulation, C/EBP delta binding activity was dramatically induced and became the predominant factor binding to this site. Consistent with these binding studies were the cotransfection experiments, revealing that C/EBP delta was the most potent transactivator under induced conditions and that its transactivation on the T1 kininogen promoter required an intact B box. These findings substantiated the importance of the B box in eliciting the full acute-phase response. A sequence comparison showed the K kininogen promoter contained identical A and B boxes but differed from the T1 kininogen promoter by two nucleotides at the C box. This divergence reduced the IL-6 response by approximately 4-fold, thus contributing to the differential inflammatory response. Our studies demonstrate that evolutionary divergence of a few nucleotides at a critical sequence in the promoter regions can profoundly alter the expression patterns of downstream genes.

High and low molecular weight kininogens (HK and LK) modulate inflammatory responses, serve as precursors of kinins, inhibit cysteine proteinases, and modulate thrombin activation of platelets. Differential splicing yields two different mRNAs for HK and LK. We report the first molecular characterization of a kininogen gene mutation in a patient lacking LK and HK. No gross DNA deletion or insertion was detected by Southern blots, and LK and HK mRNAs were normal size on Northern blots. Exon sequences amplified by polymerase chain reaction showed a C-->T transition at nucleotide 587, resulting in a CGA (Arg)--> TGA (Stop) mutation in exon 5 before the splice site in exon 10, thus preventing synthesis of both HK and LK. The mutation eliminated the recognition site of the restriction enzyme Csp45I. Results of Csp45I digestion of the polymerase chain reaction-amplified exon 5 DNA fragment of the patient (lacking kininogens), three daughters (approximately 50% HK), and 1 granddaughter (normal HK) revealed that the patient was homozygous, while the three daughters were heterozygous, and the granddaughter was normal. We conclude that a single base mutation in the kininogen gene exon 5 was responsible for kininogen deficiency in the Williams family.

Serum concentration of rat T1 kininogen increases 20- to 30-fold in response to acute inflammation, an induced hepatic synthesis regulated primarily at the transcriptional level. We have demonstrated by transient transfection analyses that rat T1 kininogen gene/chloramphenicol acetyltransferase (T1K/CAT) constructs are highly responsive to interleukin-6 and dexamethasone. In these studies we examined the regulation of a highly homologous K kininogen gene promoter and showed that it is minimally induced under identical conditions. The basal expression of the KK/CAT construct was, however, five- to sevenfold higher than that of the analogous T1K/CAT construct. Promoter-swapping experiments to examine the molecular basis of this differentially regulated basal expression showed that at least two K kininogen promoter regions are important for conferring its high basal expression: a distal 19-bp region (C box) constituted a binding site for C/EBP family proteins, and a proximal 66-bp region contained two adjacent binding sites for hepatocyte nuclear factor 3 (HNF-3). While the C box in the K kininogen promoter was able to interact with C/EBP transcription factors, the T1 kininogen promoter C box could not. In addition, HNF-3 binding sites of the K kininogen promoter demonstrated stronger affinities than those of the T1 kininogen promoter. Since C/EBP and HNF-3 are highly enriched in the liver and are known to enhance transcription of liver-specific genes, these differences in their binding activities thus accounted for the K kininogen gene's higher basal expression. Our studies demonstrated that evolutionary divergence of a few critical nucleotides may lead to subtle changes in the binding affinities of a transcription factor to its recognition site, profoundly altering expression of the downstream gene.

Kinins are vasoactive paracrine peptides which participate in a wide range of functions, including the regulation of local organ blood flow, systemic blood pressure, transepithelial water and electrolyte transport, cellular growth, capillary permeability and inflammatory response, and pain. The recent introduction of specific bradykinin receptor subtype antagonists has greatly advanced our understanding of the role of the kallikrein-kinin system (KKS) in various physiological and disease states. However, a major gap remains in our knowledge of the role of kinins in early development. In this review, evidence is presented that the developing nephron expresses both tissue kallikrein and kininogen, and that the genes encoding the components of the KKS are subject to considerable developmental regulation. The activity of the intrarenal kinin-generating system is lowest in the developing kidney and increases with age. Completion of nephrogenesis is characterized by a marked surge in intrarenal kallikrein synthesis and gene transcription. Maturation is associated with redistribution of intrarenal kallikrein and its messenger RNA from the inner to outer cortical nephrons following the centrifugal pattern of nephron development. Challenges for the future include delineation of the direct role of kinins in the maturation of renal functions and elucidation of the molecular mechanisms underlying the developmental expression of the KKS.

To clarify the mechanism of the secretion defect of high molecular weight kininogen (HK) and low molecular weight kininogen (LK) by the liver of Brown Norway (B/N) Katholiek rats causing plasma kininogen deficiency, we cloned cDNAs for HK from cDNA libraries of the livers of B/N Katholiek and B/N Kitasato rats. A point mutation of G to A at nucleotide 487 was found in the cDNA of B/N Katholiek rats by sequence analysis of the cDNAs (including the entire HK-coding region) obtained from both strains. Both B/N Katholiek and B/N Kitasato rat cDNA fragments were introduced into a eukaryotic vector, pRc/CMV, to construct their respective expression plasmid, which was used to transfect COS-1 cells. At 24 h of incubation, the culture medium of COS-1 cells transfected with the B/N Katholiek rat cDNA contained only 10% of the HK antigen that was found in COS-1 cells transfected with the B/N Kitasato rat cDNA. More HK antigen was retained in the former cells. Moreover, cells transfected with B/N Katholiek rat cDNA, in which the A at nucleotide 487 was artificially replaced by G, secreted a significant amount of HK into the medium. These results suggest that a point mutation of G to A at nucleotide 487, which causes a substitution of Ala163 to Thr in the heavy chain of HK and LK, is responsible for the defective secretion of HK and LK by the liver of B/N Katholiek rats.

Pyrrolizidine monocrotaline (MCT) from plant seed produces pulmonary endothelial cell injury, pulmonary hypertension, and inflammation in rats, providing a useful animal model for studying progressive pulmonary vascular disease. Kininogen is the precursor of proinflammatory kinins and may also exert anti-inflammatory actions by inhibiting cysteine proteinases. Given the potential roles of kininogen in vascular injury and inflammation, we have investigated the regulation of kininogen gene expression in the MCT-induced pulmonary hypertensive rat model. Sprague-Dawley rats, in groups of six, were given a single subcutaneous injection of monocrotatine (60 mg/kg body wt) and sacrificed 10 and 20 days later. Northern blot hybridization using a kininogen cDNA probe showed kininogen gene expression in the liver, lung, and kidney. MCT treatment induced a time-dependent increase in kininogen mRNA levels, whereas it reduced rat alpha 1-antitrypsin and kallikrein-binding protein mRNA levels in the liver. Similarly, kininogen mRNA levels were low in the normal lung and were increased 7.5- and 13.7-fold, respectively, after MCT injection for 10 and 20 days. Immunoreactive kininogen levels in perfused liver and lung extracts of rats receiving MCT injection increased up to 20-fold, as measured by a T-kininogen radioimmunoassay. Western blot analyses showed that a 68-kilodalton immunoreactive kininogen increased in the serum and lung extracts of MCT-treated rats compared to those in the control rats. In control rats, immunostaining for kininogen in the lung was most marked in venous endothelial cells and alveolar macrophages. After MCT treatment, staining for kininogen increased dramatically throughout the lung tissues, often covering the epithelial surfaces of alveoli and bronchi. The present studies have shown that the toxin MCT altered the synthesis and distribution of pulmonary kininogen and suggest that the kininogen/kinin system may be associated with the pulmonary vascular injury, remodeling, and inflammation seen in this animal model.

The molecular diversity of fibronectin arises from alternative RNA splicing at regions termed ED-A, ED-B, and IIICS. We investigated the splicing patterns of fibronectin pre-mRNA at both ED-B and IIICS regions in various human liver tissues with an emphasis on the expression of the alternative cell adhesive site CS1 within the IIICS region. The relative abundance of the fibronectin mRNA containing the CS1 sequence was significantly increased in both fetal and cancerous liver tissues, although it was not affected in nonmalignant tissues with chronic hepatitis and cirrhosis. Similarly, the relative abundance of the fibronectin mRNA containing the ED-B region was also increased in both fetal liver and liver tumors, showing a close parallelism with the splicing pattern at the ED-A region. Immunohistochemical examination of cancerous liver tissues with monoclonal antibodies directed to the ED-A and ED-B segments revealed that the fibronectin isoforms containing these extra peptide segments were specifically deposited in the tumor nodules. Other genes encoding kininogen, gamma chain of fibrinogen, and beta-amyloid protein precursor, all of which had been shown to be alternatively processed, did not show any significant alteration in the splicing pattern in cancerous liver tissues. These results indicate that the alternative splicing of fibronectin pre-mRNA at the ED-A, ED-B, and IIICS regions is coordinately modulated in both fetal and cancerous liver tissues toward inclusion of the extra peptide segments and that not all but only selected genes are susceptible for "fine tuning" of alternative RNA splicing in cancerous liver tissues.

The internal domain 3 of the heavy chain of human kininogen, a cysteine proteinase inhibitor, was amplified by a polymerase chain reaction from the kininogen cDNA clone phKG36. The DNA fragment was expressed in Escherichia coli using the ompA expression vector pASK40 and the resulting protein was isolated from periplasm, purified by S-carboxymethylpapain affinity- and ion-exchange chromatography. The recombinant human kininogen domain 3 is 92% pure, reacts with anti-kininogen antibodies and is actively inhibitory. The expected amino acid sequence of ANSM-[G253-S377] kininogen was confirmed; the inhibitor has a molecular mass of 14,396 Da and an isoelectric point of 6.0 (pH). The determined Ki values of the complexes with papain and cathepsin L are similar to those measured previously with proteolytically liberated kininogen domain 3, and those of single-domain cystatins, like chicken egg white cystatin. However, recombinant kininogen domain 3 is a weak inhibitor of cathepsin B (Ki = 63 nM) as it has been found for native L-kininogen (Ki = 340 nM).

The light chain (LC) of cleaved high molecular weight kininogen (HK) binds to anionic surfaces as well as the zymogens prekallikrein and factor XI and thus accelerates activation of the kallikrein-kinin, fibrinolytic, and coagulation pathways. The binding sites on HK LC for factor XI (amino acid residues 574-631) and prekallikrein (residues 583-613) have been localized to domain 6. Domain 5 (residues 438-520) has been postulated to contain the anionic surface binding subdomain. In order to define this subdomain we have expressed HK LC (residues Lys438-Ser644) as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli and generated various HK LC deletion mutants. The recombinant HK LC (rHK LC) and various HK LC fragments were purified as GST fusion proteins by glutathione-Sepharose affinity chromatography from bacterial cell extracts. The rHK LC and recombinant fragments His459-Ser644, Glu466-Ser644, Leu483-Ser644, His493-Ser644, Lys438-Asp492, Lys438-Ser531, and His493-Lys520 inhibited 125I-HKa binding to kaolin, a model anionic surface used in the contact system, in a concentration-dependent manner. Deletion mutant proteins lacking domain 5, Thr521-Ser644 and Ser583-Ser644, did not inhibit the radiolabeled HKa binding to kaolin. The rHK LC and recombinant fragments Lys438-Asp492, Lys438-Ser531, His493-Ser644, His493-Lys520, Thr521-Ser644, and Ser583-Ser644 were radiolabeled with 125I and were then tested for their ability to bind to kaolin in the presence of fibrinogen and albumin. Except for the Thr521-Ser644 and Ser583-Ser644 fragments, all other radiolabeled HK LC deletion mutant proteins and rHK LC bound to kaolin in a concentration-dependent manner. This binding to kaolin was specific since it was inhibited by the addition of excess unlabeled HKa. The rHK LC, His493-Ser644 and delta 493-520 HK LC have coagulant activity, while other deletion mutant proteins did not exhibit coagulant properties. We conclude that there are at least two anionic surface binding subdomains, one in the histidine-glycine-rich region (Lys438-Asp492) and the other in the histidine-glycine-lysine-rich region (His493-Lys520), in the domain 5 of HK LC. Either subdomain, in the presence of the zymogen binding domain 6, is sufficient to impart coagulant activity to HK LC, while the presence of both did not increase the coagulant activity of HK LC additively.

The rat liver expresses two low molecular weight kininogens (T-KG and K-KG). Although they share 90% of the nucleotide sequence in their 5' flanking regions, T- and K-KG genes are differentially regulated. The T-KG gene is inducible, and its protein is a potent thiol-protease inhibitor. In contrast, K-KG gene is expressed constitutively and encodes the precursor of the vasoactive nonapeptide bradykinin. To further elucidate the differential regulation of T- and K-KG genes, we examined their developmental expression in the Sprague-Dawley rat. Northern blots of total liver RNA were probed with oligonucleotides complementary to T and K-KG mRNA under high-stringency conditions. A single T-KG mRNA (1.8 kb) and two K-KG mRNA species (1.6 and 2.3 kb) were consistently detected at all ages studied. Steady state T-KG mRNA levels increased 3.5-fold at birth and remained high during the 1st week of postnatal life only to decline thereafter. T-KG immunoreactivity in the liver and plasma determined by Western blot analysis paralleled T-KG mRNA expression. In marked contrast, K-KG mRNA expression was not altered during the transition from fetal to neonatal life, nor was it affected by postnatal maturation. The results demonstrate that the fetal rat liver synthesizes kininogens and that T- and K-KG genes are differentially regulated during development. Up-regulation of T-KG synthesis after birth may serve a protective function in the newborn via its antiprotease activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Kininogens which have multifunctional domains, serve as the precursors of potent vasoactive kinin peptides and also function as cysteine proteinase inhibitors. Given its potential role in blood pressure homeostasis and inflammation, we have examined the regulation of rat kininogen gene expression by sex hormones in vivo. Our studies indicate a differential regulation of kininogen gene expression in rat liver by estrogen and progesterone. Northern and dot blot analysis using a rat low molecular weight kininogen cDNA probe show that kininogen mRNA levels in the liver of female rats are 4-fold higher than those in male rats. Ovariectomy results in a reduction of kininogen transcripts in the liver, while estradiol replacement of the ovariectomized rats increases kininogen mRNA levels. Similarly, Northern blot analysis using a kallikrein cDNA probe shows that estradiol treatment induces an increase of kallikrein gene expression in the kidney of the same animals. In contrast, progesterone treatment of the ovariectomized rats results in an increase in renal kallikrein mRNA levels while it reduces kininogen gene expression as compared to vehicle-treated ovariectomized animals. Immunoreactive kininogen levels in the serum, analyzed by a direct radioimmunoassay and Western blot, are increased by estradiol but slightly decreased by progesterone treatment. Western blot of serum proteins on a two-dimensional polyacrylamide gel reveals that in estradiol-treated ovariectomized rats, the levels of several 68,000 Da kininogens varying in charge are markedly higher than those in ovariectomized rats. The results indicate that estrogen is one of the determinants in regulating low molecular weight kininogen gene expression in vivo. The impact of estrogen-regulated kininogen expression on cardiovascular function awaits further investigation.

Human high molecular weight kininogen (HK), a single chain plasma glycoprotein, serves as a cofactor in the contact system of blood coagulation. After cleavage by human plasma kallikrein, the nonapeptide bradykinin is released. The HK light chain (LC) contains coagulant activity, which requires both the ability to bind the contact system zymogens, prekallikrein and factor XI, and the ability to interact with negatively charged surfaces. Since bacterial expression might not be successful if carbohydrate was required for activity, we evaluated that possibility by incubating plasma HK with endoglycosydase F. Although the procedure removed detectable N-linked carbohydrate, no change in specific activity occurred. We then developed a bacterial expression system to produce recombinant HK LC. The cDNA coding for the HK LC was prepared by polymerase chain reaction (PCR), digested with restriction enzymes EcoRI and PstI, and introduced into the bacterial expression vector pKK223-3. E. coli harboring this recombinant plasmid (pSK1) expressed HK LC upon induction with isopropylthio-galactoside (IPTG). The recombinant protein (27 kDa), when transferred onto a PVDF membrane, was recognized by monospecific polyclonal anti-HK LC-antibodies. The recombinant HK LC was purified by heparin agarose affinity chromatography to homogeneity and found to have a specific activity of 28 coagulant units per mg protein, similar to the specific activity of the LC derived by proteolytic digestion of human plasma HK. We conclude: 1) The HK LC synthesized in bacteria is biologically active, and 2) the 40% carbohydrate content of the HK LC is not required for its cofactor activity.

The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by defining the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.

Brown Norway rat strain has been studied for mode of inheritance of its congenital deficiency in plasma high molecular weight (HMW)-kininogen and low molecular weight (LMW)-kininogen, and low plasma level of prekallikrein. We examined the genetics of the deficiency by performing a mixed breeding experiment between B/N-Katholiek (B/N-Ka, deficient) and B/N-Kitasato (B/N-Ki, normal) strains. Incidence of the deficiency was judged by the plasma level of HMW-kininogen. Plasma level of HMW-kininogen was around 50% of the normal level in all F1 generations of the hybrid between male B/N-Ka and female B/N-Ki (Exp. 1), and between female B/N-Ka and male B/N-Ki (Exp. 2). Incidence of deficiency (plasma HMW-kininogen level less than 5%) in Exp. 1 was 23.8% in male F2 and 20.0% in female F2 generations. By Exp. 2 also the incidence was 25.0% in male and 30.0% in female F2 generations. There was no significant difference of the incidence between the two experiments or sexes. These results indicate the inheritance of the kininogen-deficiency to be Mendelian autosomal recessive, the same as for the reported cases of human kininogen deficiency. Gel filtration study suggests that prekallikrein in the B/N-Ka plasma may be free form, while that in the B/N-Ki plasma may form complex with HMW-kininogen.

The Brown Norway Katholiek (B/N-Ka) strain rat is the only animal strain that demonstrates deficiency in plasma HMW- and LMW-kininogens with a low level of prekallikrein. We developed an RIA for rat HMW-kininogen, LMW-kininogen, and T-kininogen, and using them measured these proteins in B/N-Ka and normal strain (B/N-Ki) rats. Plasma level of immunoreactive as well as kinin-releasing HMW-kininogen and LMW-kininogen in B/N-Ka rats was either around 3% of their levels in the normal B/N-Ki rats. The cause of the plasma deficiency of kininogens in the B/N-Ka strain was examined by 35S-methionine uptake of primary cultures of hepatocytes from the B/N-Ki and B/N-Ka strains. The results indicated that the kininogens were synthesized in the B/N-Ka liver but not secreted into the medium. Northern blot analysis of poly A(+)RNA extracted from the livers of both strains demonstrated that the band corresponding to mRNA of HMW-kininogen was present in the mRNA from B/N-Ka liver as well as in that from the B/N-Ki one. The band was similar in size and intensity in both cases. This result confirmed the data that immunoreactive HMW-kininogen was found in the liver of B/N-Ka rats (12). Thus, the cause of plasma deficiency of HMW-kininogen in the mutant appears to be secretory defect in nature. The B/N-Ka rats showed less reactivity to the inflammatory stimulus, such as carrageenin or kaolin, but the strain expressed almost the same response as normal rats to phorbol ester (PMA) or zymosan for pleurisy induction. These results indicate that kinin may play an important role in exudation in carrageenin- and kaolin-induced edema but not in that induced by PMA or zymosan. The deficient rat strain could be useful for differentiation of the inflammatory model which shows involvement of the kinin system.

The structural gene for human kininogen (KNG) was localized to chromosome 3q26----qter by in situ hybridization. The assignment substantiates the evolutionary relationship of kininogen to two other members of the cystatin superfamily, alpha-2-HS-glycoprotein and histidine-rich glycoprotein, which also map to chromosome 3.

Studies are presented which indicate that T-kininogen, the acute phase kininogen of the rat, could be a healing protein because of its properties as a cysteine protease inhibitor. Evidence is also presented that mRNA of T-kininogen synthesis may be a function of interleukin 6 production. A regulatory mechanism is postulated by which SH cofactors could determine if T-kinin is released or whether the T-kininogen molecule would remain intact. Evidence is also presented that T-kinin acts through kinin B2 receptors. No specific binding of bradykinin or T-kinin could be detected in rat heart preparations.

T-kininogen plays an important role in the acute phase response to trauma in the rat as a possible source of kinins and as a cysteine proteinase inhibitor. Two T-kininogens are expressed by rat liver from two separate genes. T-kininogen expression in liver during the acute phase response is regulated at the level of transcription. The similarity in T- and K-kininogen gene structure suggests divergent evolution from a common gene ancestor.