Kinin Peptides Research Articles

Cellular localization of low-molecular-weight kininogen and bradykinin B2 receptor mRNAs in human kidney.

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.

Tissue kallikreins in evolutionarily diverse vertebrates

A search for tissue kallikreins in lower vertebrates resulted in the discovery of three novel kallikreins. Tissue kallikrein was isolated from the salivary gland of the Eastern Atlantic mole, Scalopus aquaticus, and the pancreas of the Southern frog, Rana berlandieri. A prokallikrein was identified in skeletal muscle of the black sea bass, Centropristis striata. These enzymes range in molecular mass from 27 to 36 kDa and are acidic proteins with pls between 4.2 and 5.3. Bass prokallikrein was activated by trypsin cleavage. These novel kallikreins were compared with human and rat tissue kallikreins in regard to immunoreactivity, molecular weight, isoelectric point, extinction coefficient, susceptibility to serine proteinase inhibitors and their ability to cleave low molecular weight dog kininogen to release kinin peptides.

ANGIOTENSIN CONVERTING ENZYME (ACE) INHIBITORS AND KININ METABOLISM: EVIDENCE THAT ACE INHIBITORS MAY INHIBIT A KININASE OTHER THAN ACE

1. Angiotensin converting enzyme (ACE) converts angiotensin I to angiotensin II, and also metabolizes bradykinin-(1-9) to bradykinin-(1-7) and bradykinin-(1-7) to bradykinin-(1-5). Increases in endogenous kinin levels may contribute to the therapeutic effects of ACE inhibitors. 2. ACE inhibitors increase vascular levels of both bradykinin-(1-9) and its ACE cleavage product bradykinin-(1-7), at doses below the threshold for ACE inhibition, leading to the proposal that ACE inhibitors may also inhibit a non-ACE kininase which cleaves both kinin peptides; this non-ACE kininase may be the major pathway of kinin metabolism in the vasculature and some other tissues. 3. In support of this proposal, ACE inhibitors potentiate bradykinin-(1-9) effects at doses which have little or no effect on ACE activity, as indicated by angiotensin I conversion to angiotensin II. ACE inhibitors also potentiate the actions of ACE-resistant kinin analogues, which may be susceptible to metabolism by a non-ACE kininase. 4. Identification and characterization of the putative non-ACE kininase which is inhibited by ACE inhibitors may reveal novel approaches to the tissue-specific modulation of kinin levels.

Role of Kinins in Inflammatory Responses

Kinins are ubiquitous mediators that produce many of the cardinal manifestations of inflammation, and for that reason are an important target for therapeutic intervention. The cellular effects produced by the vasoactive kinin peptides are mediated by at least 2 different classes of receptors, B1 and B2. The development of specific, competitive antagonists of these receptors has provided a powerful tool for elucidating the physiopathological actions of kinins.

Intramuscular delivery of rat kallikrein-binding protein gene reverses hypotension in transgenic mice expressing human tissue kallikrein.

The tissue kallikrein-kinin system has been postulated to play a role in blood pressure regulation. The activity of tissue kallikrein is controlled by a number of factors in vivo. Rat kallikrein-binding protein (RKBP) is a serine proteinase inhibitor which binds to and inhibits tissue kallikrein's activity in vitro. We have recently developed several hypotensive transgenic mouse lines which express human tissue kallikrein. In order to investigate the role of RKBP in blood pressure regulation, we delivered the RKBP to these transgenic mice by intramuscular injection. Expression of the RKBP was detected in skeletal muscle by reverse transcription-polymerase chain reaction and Southern blot analysis at 10, 20, 30, and 40 days post-injection. Immunoreactive RKBP levels in the muscle and serum of these mice were quantified by a RKBP-specific enzyme-linked immunosorbent assay and Western blot analysis. The levels of RKBP mRNA and immunoreactive protein were detectable at 10 days post-injection and increased significantly at 20 and 30 days. During this period, RKBP delivery significantly increased systemic blood pressure in the kallikrein transgenic mice to a level comparable to that of normotensive control mice. The RKBP and vector DNA delivery had no effect on the blood pressure of normotensive control mice. No serum antibodies to RKBP or its DNA were detected in the mice 40 days post injection. These results suggest that the increase of systemic blood pressure by RKBP delivery in these hypotensive transgenic mice may be mediated by inhibiting tissue kallikrein activity.

Structural dissection of the multidomain kininogens. Fine mapping of the target epitopes of antibodies interfering with their functional properties.

Kininogens, the large precursor molecules of the vasoactive kinin peptides, are prototypic multidomain proteins serving numerous functions. To investigate their structure-function relationships, we have raised a panel of monoclonal antibodies against human H-kininogen and L-kininogen and fragments thereof and characterized them with respect to their target epitopes. Of 35 antibodies, 12 were directed to the amino-terminal domains (D1 to D3) of cystatin-like structure, 3 recognized domain D4 bearing the kinin segment, 17 bound to the carboxyl-terminal domains of H-kininogen (D5H and D6H), and 3 bound to the carboxyl-terminal domain D5L of L-kininogen. At least 14 distinct epitopes spread over the kininogen molecules were identified: 9 epitopes located on L-kininogen and 13 epitopes harbored by H-kininogen. Of these, 8 are shared by the two types of kininogens. Fine mapping of the epitopes by proteolytic fragments, recombinant proteins, and anti-idiotypic antibodies demonstrated that most but not all of the antibodies recognize liner epitopes. Synthesis of 28 peptides covering more than one-third of the entire kininogen sequences allowed us to narrow down seven major epitopes to 7-31 residues. Functional analyses identified 14 antibodies interfering with specific biological roles of the kininogens, i.e. cysteine proteinase inhibition, platelet attachment, cofactor binding, contact activation, and kinin delivery. Cross-reactivity studies indicated that three of the epitopes are present throughout the mammalian kininogens and further identified a unique epitope characteristic for the H/L-type of kininogens not present in their T-type. The panel of mapped antibodies provides powerful tools for the characterization of relevant interaction sites exposed by the pleiotropic kininogens and for the development of molecular surrogates mimicking these functional loci.

The Effect of Propofol on Histamine and Prostaglandine Like Activites at Serum in Rats.

In this experimental study propofol's hypotensive effect has been investigated. 30 rats were anesthetized and divided randomly into 3 groups. One group received propofol 10 mg/kg via the mesenteric vein while the other trasylol 100000 IU/kg intraperitoneally. The third group was for control. Serum PGE2 and histamine-like activities of the three groups were compared while carotid artery blood pressure was bein simultaneously recorded on the chimograph.
 According to our results, histamine levels increased and PGE2 levels deçreased in the trasylol group whereas they both, increased in the propofol, group. Simultaneous b1ood pressure recordings showed a decrease of 30 %. There fore, it could be' stated that the increase of histamine which has a strong vasodilating action could contribute to the hypotensive effect -of propofol. However, it should not be forgotten that in rats, mesenteric vein bed is rich in mast cells. Moreover, the changes of PGE2 levels caused by propofol and trasylol activate the kallikrein-kinin system. These results also bring to mind a possibility of the role of kinin peptides in the hypotension created by propofol.

Purification and biological activity of alligator bradykinin.

Incubation of plasma form the alligator (Alligator mississipiensis) with glass beads in the presence of a kininase inhibitor resulted in the activation of the kallikrein-kinin system and generation of bradykinin-like immunoreactivity. The kinin peptides were purified to homogeneity and were shown to comprise [Thr6]-bradykinin and des-Arg9[Thr6]bradykinin in the molar ratio of approximately 10:1. Bolus injections of synthetic [Thr6]bradykinin into the jugular vein of the anesthetized alligator resulted in a dose-dependent decrease in mean arterial blood pressure. The minimum dose of kinin producing a significant fall in pressure was 0.07 micrograms/kg body wt and the maximum response (25 +/- 6% fall; mean +/- SD, n = 8) was produced by a dose of 0.56 micrograms/kg body wt. The dose producing a half-maximum response was 0.19 +/- 0.08 micrograms/kg. The data indicate that alligator plasma contains all the components of the kallikrein-kinin system found in mammals and suggest that the system may be of physiological importance in the regulation of cardiovascular function in these reptiles.

Differential regulation of kininogen gene expression by estrogen and progesterone in vivo

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.

Receptors for kinins in isolated arterial vessels of dogs

A variety of kinin peptides, agonists and antagonists were tested with dog carotid and renal arteries in order to characterize kinin receptor types and functions. The dog carotid artery responds to bradykinin with concentration-dependent relaxation only when the endothelium is intact but des-Arg 9-bradykinin is practically inactive. The effect of bradykinin is blocked by B 2 receptor antagonists, suggesting that the dog carotid artery has B 2 receptors in the endothelium. These receptors mediate relaxation of the arterial smooth muscles by promoting the release of an endothelium-derived relaxing factor whose action is prevented by methylene blue. Kinins relax the dog renal artery with or without endothelium. Methylene blue prevents the effect of bradykinin only, suggesting that B 2 receptors, promoting the release of endothelium-derived relaxing factor, are present in the endothelium of the dog renal artery. Moreover, the dog renal artery appears to have both B 2 and B 1 receptors mediating relaxation of the arterial smooth muscle. The presence of the two receptor types has been demonstrated by means of specific agonists and antagonists. Indomethacin blocks the effects of both bradykinin and des-Arg 9-bradykinin on the dog renal artery without endothelium, suggesting that muscular B 1 and B 2 receptors act by promoting the release of prostaglandins. Captopril, a kininase II inhibitor, potentiates the effect of bradykinin on the dog carotid artery more than on the dog renal artery.

Kinin and angiotensin metabolism by purified renal post-proline cleaving enzyme

Post-proline cleaving enzyme (PPCE; EC 3.4.21.26) is a proline specific endopeptidase capable of hydrolyzing biologically active peptides. The present studies examined the hydrolysis of kinin- and angiotensin-related peptides by cytosolic PPCE purified from porcine kidney. PPCE hydrolysis of the synthetic substrate Z-Gly-Pro-MCA (30.7 ± 0.3 μmol · min −1 · mg −1) was competitively inhibited by saralasin, bradykinin, des(Arg 9)bradykinin, [Leu 8], des(Arg 9)bradykinin and angiotensin II (IC 50 = 0.5 to 7.0 μM). Qualitative TLC studies demonstrated that each peptide was degraded by hydrolysis on the carboxyl side of proline residues (positions 7 or 8). Quantitative HPLC studies established that peptide degradation was optimal at pH 8.2 to 8.7 and was inhibited by the specific PPCE inhibitor Z-Pro-prolinal (IC 50 = 0.8 ± 0.1 nM). Conversely, degradation was unaffected by inhibitors of aminopeptidases (amastatin), neutral endopeptidase (phosphoramidon), carboxypeptidase N (MERGETPA) or angiotensin I converting enzyme (captopril). Apparent K m values, obtained from Lineweaver-Burk analysis, were comparable for all kinin and angiotensin peptides ( K m = 5.5 to 12.8 μM), whereas V max values ranged from 1.7μmol · min −1 · mg −1 for angiotensin II to 0.44 μmol · min −1 · mg −1 for saralasin. These data are consistent with a role for PPCE in the degradation of kinins and angiotensin in vivo.

Intrarenal Kallikrein-Kinin Activity in Acute Renovascular Hypertension in Dogs

The intrarenal kallikrein-kinin system was studied during the acute phase of renovascular hypertension induced by renal artery constriction and during teprotide inhibition of kininase II in the dog. Kallikrein-like activity measured by both kininogenase and esterolytic assays, was increased during renal artery constriction (p less than 0.5) and (p less than 0.01). The administration of teprotide resulted in a further increase of renal cortical kallikrein-like activity and inhibited kininase II activity (p less than 0.01). Following the inhibition of kininase II, the plasma concentration of kininogen was also significantly decreased (p less than 0.01). These results suggest that kininase II inhibition may increase levels of intrarenal and plasma kinins and that decreased degradation of kinin peptides may contribute significantly to the acute hypertensive effect of teprotide.

Effects of bradykinin on DNA synthesis in resting NIL8 hamster cells and human fibroblasts

Bradykinin stimulates [ 3H]thymidine incorporation and DNA synthesis in resting, serum-deprived NIL8 hamster cells. The ED 50 for this stimulation is 4.52 ± 2.91 nM. Other kinin peptides including lys-bradykinin (kallidin) and met-lys-bradykinin also stimulate [ 3H]thymidine incorporation in the NIL8 cells, whereas desarg 9-bradykinin is without effect, suggesting action of the kinin peptides through type B 2 receptors. Bradykinin also stimulates DNA synthesis in IMR-90 human fibroblasts; however, this effect is observed only in the presence of indomethacin, which blocks prostaglandin synthesis. These results suggest that prostaglandins act as negative modulators of the growth-stimulatory effects of bradykinin in the fibroblasts. This conclusion is supported by the observation that exogenously added PGE 1, PGE 2, PGA 1, PGA 2, PGB 1, and PGB 2 strongly inhibit [ 3H]thymidine incorporation in the human fibroblasts. The direct effect of bradykinin observed in the NIL8 cells may be attributable to the relative resistance of these cells to growth inhibition by prostaglandins.

A radioimmunoassay for the detection of molecular forms of human plasma kininogen

Kininogen is a heterogenous plasma glycoprotein and source of the vasoactive kinin peptides. Two main forms of human kininogen are well known, the high molecular mass (Wr 120 000) and the low molecular mass (IiWr 50 000-78 000) kininogens [l-3]. I-I$ kininogen functions as a cofactor in the contact activation of F XIIa and F XIa [4]. The cascade reactions leading to the formation of fibrin in the intrinsic pathway of blood coagulation also involve activation of kallikrein (EC 3.4.2 1.8) in an amplifying feedback mechanism between prekallikrein and F XII. A small amount of surface-bound F XII has been shown to catalyze the formation of low levels of kallikrein. Whether these reactions shown to occur in vitro when blood plasma comes into contact with glass occur under physiological conditions is not yet known [5,6]. The two molecular forms differ primarily by the structure of their respective light chain, which in the HMr kininogen contains a histidine-rich fragment responsible for the coagulation effect and covalently bound to the heavy chain through the vasoactive peptide segment (bradykinin). However, UM, is present in normal human plasma in a higher concentration while HMr kininogen constitutes an average 15% of the total kininogen content [7,8]. A relationship between the two molecular forms has been suggested

The differentiation of fluorescamine-modified kinins by gel electrophoresis

A technically simple procedure has been found for the identification and differentiation of bradykinin, leukokinin-H, and lysyl-containing analogs of bradykinin. By reacting the kinins with fluorescamine and thus altering the charges, the modified kinins may be separated by polyacrylamide gel electrophoresis. At pH 4.2 fluorescamine-modified (FM) bradykinin may be separated from FM-leukokinin-H and from FM-Lys-bradykinin and FM-Met-Lys-bradykinin. Similarly at pH 9.8, FM-Lys-bradykinin and FM-Met-Lys-bradykinin can be separated from FM-bradykinin and FM-leukokinin-H. FM-Lysyl-bradykinin and FM-Met-Lys-bradykinin are not separable at either pH. This procedure may prove to be extremely useful in the identification and purification of kinin peptides.

Relationship of Contact Activation Cofactor (CAC) Procoagulant Activity to Kininogen

Contact activation cofactor (CAC) facilitates the interaction of factors XI and XII. Patients lacking CAC have a coagulation defect and are deficient in high molecular weight kininogen. The coincidence of these two defects suggests that a single protein may be responsible for both physiologic functions. Immunologic and activity studies have been made on isolated CAC to clarify the relationship between CAC and kininogen. CAC forms a single precipitin line with anti-human kininogen, and antikininogen neutralizes CAC activity. CAC and high molecular weight kininogen show a reaction of identity on immunodiffusion against rabbit anti-CAC. Anti-CAC forms two precipitin lines with normal plasma which can be identified as high and low molecular weight kininogen. Monospecific immunoabsorbed anti-CAC forms a single precipitin line with plasma high molecular weight kininogen and neutralizes CAC activity. Cleavage of kinin fragment from CAC by insoluble trypsin or kalikrein does not proportionally reduce procoagulant activity. CAC neutralized by anti-CAC can release kinins on exposure to trypsin or kallikrein. The results support the conclusions that CAC procoagulant activity is a function of high molecular weight kininogen, that antigenic determinants unique to high molecular weight kininogen are shared by the CAC portion of the molecule, and that the clotting reactions may occur at a site removed from the kinin peptide.

Relationship of contact activation cofactor (CAC) procoagulant activity to kininogen

Contact activation cofactor (CAC) facilitates the interaction of factors XI and XII. Patients lacking CAC have a coagulation defect and are deficient in high molecular weight kininogen. The coincidence of these two defects suggests that a single protein may be responsible for both physiologic functions. Immunologic and activity studies have been made on isolated CAC to clarify the relationship between CAC and kininogen. CAC forms a single precipitin line with anti-human kininogen, and antikininogen neutralizes CAC activity. CAC and high molecular weight kininogen show a reaction of identity on immunodiffusion against rabbit anti-CAC. Anti-CAC forms two precipitin lines with normal plasma which can be identified as high and low molecular weight kininogen. Monospecific immunoabsorbed anti-CAC forms a single precipitin line with plasma high molecular weight kininogen and neutralizes CAC activity. Cleavage of kinin fragment from CAC by insoluble trypsin or kalikrein does not proportionally reduce procoagulant activity. CAC neutralized by anti-CAC can release kinins on exposure to trypsin or kallikrein. The results support the conclusions that CAC procoagulant activity is a function of high molecular weight kininogen, that antigenic determinants unique to high molecular weight kininogen are shared by the CAC portion of the molecule, and that the clotting reactions may occur at a site removed from the kinin peptide.

Assay of Kinins by Their Effects on Canine Femoral Blood Flow

In pentobarbital anesthetized dogs, close arterial injections of bradykinin and kallidin elicit a dose related increase in femoral blood flow. Treatment with the kininase inhibitor BPP9alpha augments the femoral blood flow responses to bradykinin and kallidin by five and threefold respectively. The sensitivity of the preparation permits the detection of 0.5-1 ng of either bradykinin or kallidin in untreated dogs and as little as 0.1 ng of kinin peptides in animals receiving BPP9alpha. This sensitivity and the steepness of the dose response curves make this procedure suitable for the assay of kinins.

Bovine Plasma Kininogens<subtitle>III. Structural Comparison of High Molecular Weight and Low Molecular Weight Kininogens</subtitle>

A method was developed for the purification of low molecular weight (LMW) kininogen from the same batch of bovine fresh plasma used for the isolation of high molecular weight (HMW) kininogen. Purified HMW kininogen reacted with both HMW and LMW kininogen antisera prepared, respectively, from immunized rabbit serum. Purified LMW kininogen also reacted with both antisera. Thus, the immunological properties of HMW and LMW kininogens were very similar, and their antigenic determinant groups seem to be identical. On treatment of purified HMW kininogen with cyanogen bromide, a fragment was obtained containing a kinin peptide sequence, from which kallidin was liberated by trypsin [EC 3.4.21.4]. The amino acid sequence of this fragment was identical to that of a fragment previously obtained from purified LMW kininogen by cyanogen bromide treatment. Moreover, tryptic peptide maps of S-carboxymethylated derivatives of HMW and LMW kininogens were very similar: the former gave 43 peptide spots and the latter 30 peptide spots; 28 peptide spots were found to be common to both kininogens. Thus, HMW kininogen seems to contain all the structural segments constituting LMW kininogen, though more detailed studies on the primary structures of the two are required to confirm this. The physicochemical and chemical properties of HMW and LMW kininogens were compared and differences were discussed.

Hormonal factors stimulating the motility of the intestinal tract in inflammation of its wall.

The tissues of the gastrointestinal tract constitute a rich store of biologically active proteins, peptides and amines. Many of these substances are known to exert actions on the motility, blood supply or secretion of the gastrointestinal tract. Frequently, however, the precise physiologic functions of such substances are unknown and their clinical importance is obscure. The kinin peptides and 5-hydroxytryptam ine (5-HT) are compounds which fall into this category. 5-HT and the inactive precursor and releasing enzyme of the kinins may be found in tissues along the entire length of the gastrointestinal tract. Kinins and 5-HT in low concentration are known to inhibit or stimulate peristalsis and to increase or decrease local blood flow, respectively. Release of these substances from gastrointestinal tissue has been demonstrated in response to various agencies (I, 2). It would be of interest if changes in the tissue content of these substances or in the sensitivity of the tissue to their actions were consistently related to diseases of the gastrointestinal tract. The present paper describes some preliminary findings in tissue taken at operation from patients with colitis and diverticular disease.