The renin-angiotensin system (RAS) has been widely known as a circulating endocrine system involved in the control of blood pressure. However, components of RAS have been found to be localized in rather unexpected sites in the body including the kidneys, brain, bone marrow, immune cells, and reproductive system. These discoveries have led to steady, growing evidence of the existence of independent tissue RAS specific to several parts of the body. It is important to understand how RAS regulates these systems for a variety of reasons: It gives a better overall picture of human physiology, helps to understand and mitigate the unintended consequences of RAS-inhibiting or activating drugs, and sets the stage for potential new therapies for a variety of ailments. This review fulfills the need for an updated overview of knowledge about local tissue RAS in several bodily systems, including their components, functions, and medical implications.

We engineered a monoclonal antibody (mAb) against the human C-terminus of angiotensin-(1-12) [h-Ang-(1-12)] and performed a biochemical characterization in concert with direct in vivo and ex vivo (carotid artery strips) assessments of h-Ang-(1-12) vasoconstrictor activity in 78 (36 females) transgenic rats expressing the human angiotensinogen gene [TGR(hAGT)L1623] and 26 (10 female) Sprague Dawley (SD) controls. The mAb shows high specificity in neutralizing angiotensin II formation from h-Ang-(1-12) and did not cross-react with human and rat angiotensins. Changes in arterial pressure and heart rate in Inactin® hydrate anesthetized rats were measured before and after h-Ang-(1-12) injections [dose range: 75-300 pmol/kg i.v.] prior to and 30-60 minutes after administration of the h-Ang-(1-12) mAb. Neutralization of circulating Ang-(1-12) inhibited the pressor action of h-Ang-(1-12), prevented Ang-(1-12) constrictor responses in carotid artery rings in both SD and TGR(hAGT)L1623 rats, and caused a fall in the arterial pressure of male and female transgenic rats. The Ang-(1-12) mAb did not affect the response of comparable dose-related pressor responses to Ang II, pre-immune IgG, or the rat sequence of Ang-(1-12). This h-Ang-(1-12) mAb can effectively suppress the pressor actions of the substrate in the circulation of hypertensive rats or in carotid artery strips from both SD and transgenic rats. The demonstration that this Ang-(1-12) mAb by itself, induced a fall in arterial pressure in transgenic hypertensive rats supports further exploring the potential abilities of Ang-(1-12) mAb in the treatment of hypertension.

Systemic inflammation correlates with an increased risk of atrial fibrillation (AF) and thrombogenesis. Systemic inflammation alters vessel permeability, allowing inflammatory and immune cell migration toward target organs, including the heart. Among inflammatory cells infiltrating the atria, macrophages and mast cell have recently attracted the interest of basic researchers due to the pathogenic mechanisms triggered by their activation. This chemotactic invasion is likely implicated in short- and long-term changes in cardiac cell-to-cell communication and in triggering fibrous tissue accumulation in the atrial myocardium and electrophysiological re-arrangements of atrial cardiomyocytes, thus favoring the onset and progression of AF. Serine proteases are a large and heterogeneous class of proteases involved in several processes that are important for cardiac function and are involved in cardiac diseases, such as (i) coagulation, (ii) fibrinolysis, (iii) extracellular matrix degradation, (iv) activation of receptors (i.e., protease-activated receptors [PPARs]), and (v) modulation of the activity of endogenous signals. The recognition of serine proteases substrates and their involvement in inflammatory/profibrotic mechanisms allowed the identification of novel cardio-protective mechanisms for commonly used drugs that inhibit serine proteases. The aim of this review is to summarize knowledge on the role of inflammation and fibrosis as determinants of AF. Moreover, we will recapitulate current findings on the role of serine proteases in the pathogenesis of AF and the possible beneficial effects of drugs inhibiting serine proteases in reducing the risk of AF through decrease of cardiac inflammation and fibrosis. These drugs include thrombin and factor Xa inhibitors (used as oral anticoagulants), dipeptidyl-peptidase 4 (DPP4) inhibitors, used for type-2 diabetes, as well as novel experimental inhibitors of mast cell chymases.

In its classical view, the renin angiotensin system (RAS) was defined as an endocrinesystem involved in blood pressure regulation and body electrolyte balance. However, the emergingconcept of tissue RAS, along with the discovery of new RAS components, increased thephysiological and clinical relevance of the system. Indeed, RAS has been shown to be expressed invarious tissues where alterations in its expression were shown to be involved in multiple diseasesincluding atherosclerosis, cardiac hypertrophy, type 2 diabetes (T2D) and renal fibrosis. In thischapter, we describe the new components of RAS, their tissue-specific expression, and theiralterations under pathological conditions, which will help achieve more tissue- and conditionspecifictreatments.

The classical view of biochemical pathways for the formation of biologically active angiotensins continues to undergo significant revision as new data uncovers the existence of important species differences between humans and rodents. The discovery of two novel substrates that, cleaved from angiotensinogen, can lead to direct tissue angiotensin II formation has the potential of radically altering our understanding of how tissues source angiotensin II production and explain the relative lack of efficacy that characterizes the use of angiotensin converting enzyme inhibitors in cardiovascular disease. This review addresses the discovery of angiotensin-(1-12) as an endogenous substrate for the production of biologically active angiotensin peptides by a non-renin dependent mechanism and the revealing role of cardiac chymase as the angiotensin II convertase in the human heart. This new information provides a renewed argument for exploring the role of chymase inhibitors in the correction of cardiac arrhythmias and left ventricular systolic and diastolic dysfunction.

The title of the proposed series of reviews is Translational Success Stories. The definition of "translation" according to Webster is, "an act, process, or instance of translating as a rendering of one language into another." In the context of this inaugural review, it is the translation of Tigerstedt's and Bergman's(1) discovery in 1898 of the vasoconstrictive effects of an extract of rabbit kidney to the treatment of heart failure. As recounted by Marks and Maxwell,(2) their discovery was heavily influenced by the original experiments of the French physiologist Brown-Séquard, who was the author of the doctrine that "many organs dispense substances into the blood which are not ordinary waste products, but have specific functions." They were also influenced by Bright's(3) original observation that linked kidney disease with hypertension with the observation that patients dying with contracted kidneys often exhibited a hard, full pulse and cardiac hypertrophy. However, from Tigerstedt's initial discovery, there was a long and arduous transformation of ideas and paradigms that eventually translated to clinical applications. Although the role of the renin-angiotensin system in the pathophysiology of hypertension and heart failure was suspected through the years, beneficial effects from its blockade were not realized until the early 1970s. Thus, this story starts with a short historical perspective that provides the reader some insight and appreciation into the long delay in translation.

The renin-angiotensin system (RAS) has been implicated in vessel wall remodeling. This investigation tested the hypothesis that the RAS is altered during experimental rodent aneurysm formation.

Rat aortas were perfused with saline (controls, N = 45) or elastase (6 U/ml, N = 45). At 4, 7, and 14 days after aortic perfusion, aortic diameters were measured (n = 15/time point/group) and aortic wall mRNA and protein were extracted. Real time polymerase chain reation (PCR) measured RNA levels of angiotensin, angiotensin converting enzyme (ACE), angiotensin II receptor 1 (AT(1)), and angiotensin II receptor 2 (AT(2)). Western blot analysis measured ACE protein levels. Immunohistochemical studies localized ACE within the aortic wall. Statistical analyses were performed with the unpaired t-test and ANOVA.

Elastase perfusion significantly increased aortic diameter (P < 0.01), with no significant changes in saline control aortic diameters. ACE mRNA did not become elevated in elastase-perfused aortas, yet ACE protein levels were elevated on days 4 and 7 of perfusion (P < 0.01) compared to controls, and ACE staining was noted in these aortas. This difference resolved by 14 days. In neither group were there significant alterations in AT(1), AT(2), or An mRNA levels, although ACE mRNA was elevated in controls after 7 days of perfusion compared to elastase perfused aortas (P < 0.005).

Experimental aortic aneurysm formation may be associated with increased aortic wall ACE protein levels. The mechanisms by which these proteins contribute to, or serve as markers of, aneurysm formation in vivo requires further intervention.

The serine proteases of the trypsin superfamily are versatile enzymes involved in a variety of biological processes. In the cardiovascular system, the importance of these enzymes in blood coagulation, platelet activation, fibrinolysis, and thrombosis has been well established. Recent studies have shown that trypin-like serine proteases are also important in maintaining cardiac function and contribute to heart-related disease processes. In this review, we describe the biological function of corin, tissue kallikrein, chymase and urokinase and discuss their roles in cardiovascular diseases such as hypertension, cardiac hypertrophy, heart failure, and aneurysm.

Roles of each angiotensin II producing enzymes of each of the angiotensin II-producing enzymes were reviewed based on experimental models. In vascular tissues, angiotensin II is potentially cleaved from angiotensin I by angiotensin converting enzyme (ACE) and chymase. It has been confirmed that vascular tissues of humans, monkeys, dogs and hamsters have a chymase-dependent angiotensin II-forming pathway. Much like other hypertensive models, hamster hypertensive models show high levels of vascular ACE activity, but not chymase activity. In hypertensive hamsters, administration of either an ACE inhibitor or an angiotensin II type 1 (AT1) receptor antagonist resulted in similar reductions in blood pressure, suggesting that chymase is not involved in the maintenance of high blood pressure in this model. In monkeys fed a high-cholesterol diet, ACE activity was increased in the atherosclerotic lesions, and an ACE inhibitor and an AT1 receptor antagonist prevented atherosclerosis to a similar degree, suggesting that ACE may be mainly involved in the development of atherosclerosis. After balloon injury in dog vessels, both ACE and chymase activities were locally increased about 3-fold in the injured arteries, and an AT1 receptor antagonist was effective in preventing the intimal formation, but an ACE inhibitor was ineffective. In dog grafted veins, the activities of chymase were increased 15-fold, but those of ACE were increased only 2-fold, and the intimal formation was suppressed by either an AT1 receptor antagonist or a chymase inhibitor. In the normal vascular tissues, ACE plays a crucial role for angiotensin II production, whereas chymase is stored in mast cells in an inactive form. Chymase acquires the ability to form angiotensin II following mast cells activation followed by mast cells activation by a strong stimulus such as occurs in catheter-injury or grafting. Together, these results indicate that chymase plays a major role in the vascular angiotensin II-generating system, particularly in cases of vascular injury.

The renin-angiotensin system has been studied and recognized as one of the major blood pressure-regulating systems for the past century. In the last quarter century, however, many alternative pathways of angiotensin II formation have been found, and among them, chymase has been a focus of interest because of its specificity and potency in the human cardiovascular system. Chymase evidently is not involved in functional regulation of blood pressure at least in the short term, but evidence is accumulating that it may be involved in structural remodeling of the cardiovascular system. We found increased vascular chymase activity in atherosclerotic lesions of the human aorta as well as in cardiac remodeling after myocardial infarction. We found a significant positive correlation between serum total or LDL cholesterol levels and arterial chymase-dependent angiotensin II-forming activity in patients who were undergoing coronary artery bypass operation, suggesting that high serum cholesterol may trigger upregulation of vascular chymase and facilitate the development of atherosclerosis. This hypothesis was tested in Syrian hamsters fed a high cholesterol diet containing 0.5% cholesterol: A marked lipid deposition in the aortic cusp developed and the plasma cholesterol levels were positively correlated with aortic chymase activity. An orally active nonpeptide chymase inhibitor almost canceled this lipid deposition. These clinical and experimental data indicated an association between cholesterol and vascular chymase upregulation that may facilitate the development of atherosclerosis.

In the normal state, vascular ACE regulates local angiotensin II formation and plays a crucial role in the regulation of blood pressure, whereas chymase is stored in secretory granules in mast cells and has no enzymatic effects such as angiotensin II-forming activity. Chymase has a maximal activity immediately upon release into the extracellular matrix in vascular tissues after mast cells have been activated by a strong stimulus such as experienced by catheter-injured and grafted vessels. Therefore, chymase plays an important role in forming local angiotensin II when vascular tissues are injured, and inhibition of chymase may be useful for preventing vascular proliferation in grafted vessels and after PTCA (Figure 6).

We previously purified a kallikrein-like enzyme from the dog heart and demonstrated that it is not only able to form kinins but can also convert angiotensin (Ang) I to Ang II. The aim of the present study was to clarify the structure and tissue localization of this enzyme. Western blot analysis of various canine tissues was performed with antiserum against the purified dog heart enzyme. The purified enzyme was subjected to a determination of its amino acid composition and a sequence analysis. Western blotting indicated that this enzyme was present in the heart, aorta, kidney, pancreas, lung, liver, spleen, small intestine, and skeletal muscle. The amino acid composition of the enzyme was different from that of dog urinary kallikrein. Amino acid sequence analysis indicated that it is likely to be N-terminally blocked. The present study showed that this kallikrein-like enzyme is different from previously reported kallikrein and is distributed not only in the heart, but also in other tissues such as the aorta, kidney, lung, liver, spleen, small intestine, and skeletal muscle. This enzyme may exert local effects by generating kinins and Ang II.

Locally formed angiotensin II (Ang II) and mast cells may participate in the development of atherosclerosis. Chymase, which originates from mast cells, is the major Ang II-forming enzyme in the human heart and aorta in vitro. The aim of the present study was to investigate aortic Ang II-forming activity (AIIFA) and the histochemical localization of each Ang II-forming enzyme in the atheromatous human aorta. Specimens of normal (n=9), atherosclerotic (n=8), and aneurysmal (n=6) human aortas were obtained at autopsy or cardiovascular surgery from 23 subjects (16 men, 7 women). The total, angiotensin-converting enzyme (ACE)-dependent, and chymase-dependent AIIFAs in aortic specimens were determined. The histologic and cellular localization of chymase and ACE were determined by immunocytochemistry. Total AIIFA was significantly higher in atherosclerotic and aneurysmal lesions than in normal aortas. Most of AIIFA in the human aorta in vitro was chymase-dependent in both normal (82%) and atherosclerotic aortas (90%). Immunocytochemical staining of the corresponding aortic sections with antichymase, antitryptase or anti-ACE antibodies showed that chymase-positive mast cells were located in the tunica adventitia of normal and atheromatous aortas, whereas ACE-positive cells were localized in endothelial cells of normal aorta and in macrophages of atheromatous neointima. The density of chymase- and tryptase-positive mast cells in the atherosclerotic lesions was slightly but not significantly higher than that in the normal aortas, and the number of activated mast cells in the aneurysmal lesions (18%) was significantly higher than in atherosclerotic (5%) and normal (1%) aortas. Our results suggest that local Ang II formation is increased in atherosclerotic lesions and that chymase is primarily responsible for this increase. The histologic localization and potential roles of chymase in the development of atherosclerotic lesions appear to be different from those of ACE.

Angiotensin (Ang) II plays an important role in cardiovascular homeostasis, not only in the systemic circulation but also at the tissue level, and is involved in the remodeling of the heart and vasculature under pathological conditions. Although alternative Ang II-forming pathways are known to exist in various tissues, the details of such pathways remain unclear. The aim of this study was to examine tissue Ang II-forming activities and to identify the responsible enzyme in several organs (lung, heart, and aorta) in various species (human, hamster, rat, rabbit, dog, pig, and marmoset). Among the organs examined, the lung contained the highest Ang II-forming activity. The responsible enzyme for pulmonary Ang II formation was angiotensin I-converting enzyme (ACE) in all of the species except the human lung, in which a chymaselike enzyme was dominant. In the heart, the highest total Ang II-forming activity was observed in humans, and a chymaselike enzyme was dominant in all of the species except rabbit and pig. Aorta exhibited a relatively high total Ang II-forming activity, with a predominance of chymaselike activity in all of the species except rabbit and pig, in which ACE was dominant. Our results indicate that there were remarkable differences in Ang II-forming pathways among the species and organs we examined. To study the pathophysiological roles of ACE-independent Ang II formation, one should choose species and/or organs that have Ang II-forming pathways similar to those in humans.

1. To determine whether total interruption of the local cardiac renin-angiotensin system by angiotensin II (AngII) receptor antagonist limits myocardial ischaemia, intracoronary (i.c.) or intravenous (i.v.) infusion of AngII receptor antagonists was compared in ischaemic dogs. 2. Dogs subjected to 90 min coronary artery occlusion and 270 min reperfusion assigned to saline (n = 10) or i.c. low dose (LD, n = 10), i.v. low dose (n = 10) or i.v. high dose (HD, n = 10) of AngII AT1-receptor antagonist, CV-11974. The CV-11974 was infused from 15 min pre-occlusion for 180 min. Cardiac and regional function, area at risk and infarct size were measured. 3. Although i.c. CV-11974 did not cause systemic haemodynamic changes, it abolished reduction in coronary blood flow induced by i.c. AngII injection. Elevation in LV end-diastolic pressure during ischaemia was smaller in both i.c. and i.v.-HD CV-11974 dogs than in i.v.-LD and control dogs. Regional wall thickening was not different among the four groups. With comparable area at risk, i.c. CV-11974 reduced infarct size to the same extent as i.v.-HD CV-11974 (18 vs 21%), which was smaller than i.v.-LD CV or the controls (38 and 42%). 4. The results indicate that AngII receptor antagonist can reduce ischaemia-reperfusion injury in experimental ischaemia. These cardioprotective effects might be mediated through direct inhibition of local angiotensin action in the heart. Local cardiac AngII formation may play a crucial role in cardiac injury during ischaemia and reperfusion.

To purify and characterize a kinin-forming enzyme in the dog heart and to examine the ability of this enzyme to generate angiotensin (Ang) II from Ang I.

The enzyme was isolated from heart homogenate using a diethylaminoethyl-Sepharose column, an aprotinin affinity column and a wheat germ lectin-Sepharose 6MB column. Kininogenase activity was assessed with a kinin radioimmunoassay after samples had been incubated with bovine low-molecular-mass kininogen at 37 degrees C for 1 h. Ang I-converting activity was assessed by the quantitation of Ang II formed by incubation of the sample with Ang I at 37 degrees C for 3 h, using high performance liquid chromatography. The enzyme was subjected to 12.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, stained by Coomassie brilliant blue and transferred electrically to a membrane with glycoprotein staining.

The purified enzyme is a glycoprotein with an apparent relative molecular mass of 65 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Its kininogenase activity was approximately 20 micrograms bradykinin/h per mg protein at an optimal pH of 8.0. The enzyme also converted Ang I to Ang II at an optimal pH of 6.5. Its specific activity was approximately 2 micrograms Ang II/h per mg protein. Both activities were inhibited by aprotinin, a tissue kallikrein inhibitor. Western blot analysis using polyclonal antibody against this enzyme demonstrated that this enzyme exists both in the myocardium and in the coronary artery.

The present study showed that the kinin-forming enzyme in the dog heart is a kallikrein-like enzyme that is different from cathepsin D, cathepsin G and chymase. It is also able to Ang I to Ang II. This enzyme might play a role in regulating myocardial perfusion, mainly by generating kinins and in part by forming Ang II.

To study the oxytocic effect of trypsin, we measured the force of isometric contraction in uteri isolated from estrogenized rats exposed to trypsin (8.8 x 10(-10) to 1.7 x 10(-6) mol/L) either alone or in the presence of receptor antagonists to angiotensin II [saralasin ([Sar1,Ala8]angiotensin II) or DuP 753 (losartan)] or to kinins (D-[Arg0,Hyp3,Thi5,8,D-Phe7]-bradykinin). We found that saralasin or DuP 753, but not the kinin antagonist, displaced the dose-response curve to the right. Exposure to exogenous angiotensin I desensitized the preparation to further doses of either angiotensin I or II or trypsin, without altering the effects of oxytocin or bradykinin. Enalaprilat (an angiotensin I converting enzyme inhibitor) or pepstatin A (a renin inhibitor) also displaced the dose-response curve to trypsin to the right, without altering the effects of oxytocin or angiotensin II. Our results indicate that the response to trypsin is mediated by an agent produced from a substrate present in the uterus and acting on the angiotensin II type 1 receptor and are consistent with both renin and angiotensin I converting enzyme being involved in its mechanism of action, thus supporting the notions that the renin-angiotensin system may be important in the late stages of pregnancy and that serine proteases existing in the uterus may contribute to its activation.

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.

Kallikrein, a serine protease known to generate bradykinin (a vasodilating peptide) in alkaline conditions, also generates angiotensin II (a vasoconstricting peptide) in weak acidic conditions. Based on this previous observation, the present study was performed to determine whether ischemic muscle tissue, in which the regional pH must decrease, produces angiotensin II by kallikrein or a similar enzyme, and whether nafamostat (NAF), a serine protease inhibitor, improves local hemodynamics under ischemic conditions caused by exercise in patients with ischemic peripheral vascular disease. NAF was administered intravenously to 20 patients with peripheral vascular disease. Lower-limb thermograms and blood flow were measured before and after exercise. Femoral venous blood of affected limbs was obtained to measure viscosity and humoral variables (i.e., pH, lactate, angiotensin II and bradykinin). Walking distance and subjective symptoms were also recorded. As a control, the same patients repeated this test with saline infusion on a separate day. NAF significantly increased maximal walking distance, improved subjective symptoms during exercise, and attenuated exercise-induced venous lactate and blood viscosity increases, and pH reduction. The blood viscosity increase correlated with the lactate increase. Pretreatment with NAF also resulted in a higher lower-limb skin temperature, and a greater increase of blood flow in the lower limbs after exercise than did pretreatment with saline. The results suggest that kallikrein-like serine protease may exacerbate ischemic symptoms. Changes in plasma bradykinin and angiotensin II in the femoral vein were not detectable, probably because of the lower levels of these peptides in the peripheral circulation.

An alternative angiotensin II-forming system distinct from the vascular renin-angiotensin system was demonstrated using a rat hindlimb perfusion system and synthetic substrates. This pathway was resistant to captopril and aprotinin, but was highly sensitive to chymostatin. Moreover, angiotensin II formation was substrate-dependent, i.e. angiotensin II formation from tridecapeptide human renin substrate in the presence of captopril was more than twice than that from an equimolar amount of angiotensin I. Both pathways may play a role in regulating the peripheral circulation.

The lack of kinin formation in systemic circulation and in the renal system may lead to the pathogenesis of high blood pressure (hypertension). Angiotensin converting enzyme inhibitors are able to protect the kinin inactivation by kininase II, therefore, causing an accumulation of kinin. Although the concentrations of kinin in plasma after oral administration of ACE inhibitors are conflicting this is mainly due to methodological difficulties. Kinin receptor antagonists are becoming most reliable pharmacological probes for defining the molecular actions of kinin in several physiopathological states, and in the mechanism of actions of drugs which are dependent on the kinin system. The blood pressure lowering effect of ACE inhibitors can be antagonized by the pretreatment with kinin receptor antagonists. I have therefore proposed that the hypotensive action of ACE inhibitors may reflect the activation of kinin receptor. It is suggested that the development of compounds having protective properties on the kallikrein-kinin system might be therapeutically applicable as anti-hypertensive drugs.

Human glandular kallikrein was purified from urine and subjected to detailed structural characterization. The protein was carboxymethylated with iodoacetic acid and digested with TPCK-trypsin, Staphylococcal aureus V-8 protease and endo LysC peptidase. The resulting peptide fragments were separated by reverse-phase HPLC using C-4 columns and acetonitrile-trifluoroacetic acid gradient elution. The complete amino acid sequence of the carboxymethylated derivative was elucidated by sequence analysis and alignment of peptides derived from different proteolytic cleavages. A procedure using in situ CNBr cleavage of a large endo LysC peptidase-derived peptide followed by direct sequencing was carried out to provide overlap for two glycosylation sites at residues 78 and 84. Three Asn-linked glycosylation sites were confirmed by the direct sequence analysis of the isolated glycopeptides. However, the third glycosylation at Asn-144 occurs only in 60% of kallikrein molecules. Reverse-phase HPLC effectively separates two species of HUK which correspond to molecules glycosylated and non-glycosylated at Asn-144, respectively. The human urinary kallikrein contains 238 amino acid residues with Ile and Ser as N- and C-terminal amino acids, respectively. The primary structure is completely identical to that deduced from a human genomic DNA sequence (F.K. Lin et al., manuscript in preparation) and is different in one amino acid (Lys-162 vs. Glu-162) from that deduced from pancreatic or kidney cDNA sequence.

Tonin is a mammalian serine protease that is capable of generating the vasoconstrictive agent, angiotensin II, directly from its precursor protein, angiotensinogen, a process that normally requires two enzymes, renin and angiotensin-converting enzyme. The X-ray crystallographic structure determination and refinement of tonin at 1.8 A resolution and the analysis of the resulting model are reported. The initial phases were obtained by the method of molecular replacement using as the search model the structure of bovine trypsin. The refined model of tonin consists of 227 amino acid residues out of the 235 in the complete molecule, 149 water molecules, and one zinc ion. The R-factor (R = sigma Fo - Fc/sigma Fo) is 0.196 for the 14,997 measured data between 8 and 1.8 A resolution with I greater than or equal to sigma (I). It is estimated that the overall root-mean-square error in the coordinates is about 0.3 A. The structure of tonin that has been determined is not in its active conformation, but one that has been perturbed by the binding of Zn2+ in the active site. Zn2+ was included in the buffer to aid the crystallization. Nevertheless, the structure of tonin that is described is for the most part similar to its native form as indicated by the close tertiary structural homology with kallikrein. The differences in the structures of the two enzymes are concentrated in several loop regions; these structural differences are probably responsible for the differences in their reactivities and specificities.

A method has been developed to measure the relative rate of rat tissue kallikrein synthesis which employs a specific antiserum raised against a purified rat urinary kallikrein. Incorporation of [35S]methionine into kallikrein and protein 20 min after intraperitoneal injection was measured in submaxillary gland, pancreas, kidney and descending colon. Kallikrein content was measured with a direct radioimmunoassay, and kallikrein-specific incorporation of [35S]methionine measured after immunoprecipitation. Kallikrein specific radioactivity (c.p.m./mg of enzyme) was about 100-fold greater than that in total protein in both kidney and colon. In contrast, in pancreas the incorporation into the enzyme was only 5-fold higher than into protein, and in submaxillary gland the incorporation was equivalent. Measured as kallikrein-specific radioactivity relative to total protein radioactivity incorporated in 20 min, kallikrein represents 0.18% of total protein synthesis in the kidney, 0.34% in the pancreas, 0.41% in the colon, but 7.29% in the submaxillary gland. Dietary Na+ restriction increased the relative rate of kallikrein synthesis 1.8-fold in the kidney without a comparable effect in submaxillary gland. In contrast, testosterone increased the relative rate of synthesis 2.3-fold in submaxillary gland, but decreased it in kidney. The data show that endogenous kallikrein synthesis differs markedly in various tissues, and that interventions which are known to change kallikrein content or excretion also change the relative rate of enzyme synthesis.

This study was undertaken to confirm our previous preliminary observation that hog pancreas kallikrein (EC 3.4.21.35) directly liberated an angiotensin-like substance from human plasma protein Cohn fraction IV-4 at an acidic pH of 4.0-5.0. First, the possibility of proangiotensin or des-Asp1-angiotensin being the pressor substance was ruled out by t.l.c. Secondly, the pressor substance was purified by Sephadex G-25 and Bio-Gel P-2 gel filtration, and finally by high-performance liquid chromatography. The amino acid composition of the isolated pressor substance (residues/mol) was: Asp, 1.03; Val, 1.03; Ile, 1.00; Tyr, 0.69; Phe, 1.04; His, 0.91; Arg, 0.86; Pro, 0.86. This composition was identical with that of angiotensin. Since the reaction mixture was not contaminated with common proteolytic enzymes, such as trypsin, chymotrypsin, renin, cathepsin D and proangiotensin-converting enzyme, and other enzymes activated by kallikrein, it is clear that hog kallikrein directly produces angiotensin in vitro.

Responses of smooth muscle to kallikreins (EC 3.4.21.8) are generally considered to result from kinin formation. This premise was reexamined with the isolated rat uterus. Rat urinary kallikrein or bradykinin produced dose-dependent contractions of rat uterus but kallikrein was 5-fold more potent than bradykinin. Kallikrein caused an immediate series of rhythmic contractions which could be increased gradually with subsequent addition of kininogen substrate. Kallikrein-induced contractions were unaffected by carboxypeptidase B or a bradykinin antiserum whereas bradykinin-induced contractions were attenuated or abolished. Other serine proteinases, including trypsin, either did not induce contraction in the absence of added kininogen or did so minimally. Although small amounts of kininogen-like substrate were found in uterine tissue, detectable kinin levels (greater than 4 pg) could not be found in bathing media during maximal kallikrein-induced contractions or after uterine tissue was incubated with high concentrations of the enzyme in the presence of SQ 20881, a kininase II inhibitor. The data suggest that uterine contraction produced by a homologous kallikrein does not involve kinin formation but results from an action of this serine proteinase upon other accessible systems coupled to the contractile response.