Inhibition Of Kininase Research Articles

Genetic Deletion of Carboxypeptidase N Attenuates Angiotensin II-Induced Hypertension and Target Organ Damage

Objectives: Kininase I transforms bradykinin and kallidin, into kinin B1 receptor (B1R) agonists des-Arg9-BK and des-Arg10-kallidin, respectively. Carboxypeptidase M (CPM) is the membrane bound isoform of kininase I and carboxypeptidase N (CPN) is the isoform located in the plasma. Activation of the B1R has been implicated in the development of hypertension, diabetes and atherosclerosis. Pharmacologic inhibition of kininase I is associated with reductions in B1R expression, inflammation, oxidative stress, and improvements in insulin resistance. B1R can interact with aminopeptidase N (CD13) or high-mobility group box 1 (HMGB1) to mediate an inflammatory response. However, the exact role of kininase I in the development of hypertension and the regulation of these B1R-mediated signaling mechanisms remains unknown. In this study, we hypothesize that gene deletion of CPN will prevent B1R activation, reduce inflammation via interfering with CD13 and HMGB1 signaling, and ultimately attenuate the progression of hypertension and target organ damage. Methods and Results: To test our hypothesis we utilized 12-week-old male and female C57BL/6NJ wild-type (WT) and CPN gene knockout (CPNKO) mice. These mice were infused angiotensin II (Ang II, 600 ng/kg/min) or saline (vehicle) via osmotic minipumps for 2 weeks. Vehicle treated CPNKO and WT mice showed no significant difference in mean arterial blood pressure (MAP) (104 ±7 vs 102 ±5 mmHg, n=5, p≤0.05). Ang II-induced elevations in MAP were significantly reduced in CPNKO mice compared to WT mice infused with Ang II (129 ±8 vs 150 ±6 mmHg, n=5, p≤0.05). Interestingly, we did not find any significant sex differences in MAP in CPNKO groups. Additionally, Ang II treatment significantly increased cardiomyocyte hypertrophy in male and female WT mice, but this effect was blunted in CPNKO mice (n=5, p≤0.05; wheat germ agglutinin staining). Ang II infusion promoted collagen deposition in the aorta of WT male and female mice, but not in CPNKO mice (n=5, p≤0.05; picrosirius red/Masson’s trichrome). In male WT mice, Ang II significantly increased the immunoreactivity of CPM in the heart, but this was abrogated in CPNKO mice infused with Ang II (n=4, p≤0.05). Similarly, B1R was also upregulated in male WT mice hearts when compared to CPNKO mice after Ang II infusions (n=3, p≤0.05). We also found that Ang II significantly increased HMGB1 and CD13 in male WT hearts and brains, but not in CPNKO mice (n=4, p≤0.05). In addition, oxidative stress levels in the brain were significantly increased by Ang II in WT mice and was attenuated in CPNKO mice (n=4, p≤0.05; dihydroethidium staining). Conclusions: Genetic deletion of CPN attenuates Ang II-induced hypertension by reducing B1R mediated reductions in CD13 and HMGB1-mediated inflammatory responses and oxidative stress. Our data suggests that targeting kininase I may be a novel therapeutic target for hypertension and target organ damage. Funding: This study was supported by NHLBI/NIH 5R01HL153115 (S. Sriramula). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Kininase I inhibition reduces B1R mediated signaling and attenuates angiotensin II-induced hypertension

Objectives: Kininase I, or carboxypeptidase M and N (CPM/CPN), metabolizes bradykinin and kallidin into endogenous kinin B1 receptor (B1R) agonists. B1R expression in the brain is linked to neuroinflammation and autonomic dysfunction, and the development of hypertension. The metalloprotease aminopeptidase N (CD13) can interact with B1R to enhance proinflammatory cytokine production. However, the exact role of kininase I in the development of hypertension has not been yet investigated. In the present study, we tested the hypothesis that inhibition of kininase I will prevent the activation of the B1R, and reduce CD13 signaling, thus ultimately attenuating the progression of hypertension. Methods and Results: To inhibit the kininase I activity, we have utilized carboxypeptidase N gene deleted knockout (CPNKO) mice. Twelve-week-old male and female C57BL/6NJ wild-type (WT) and CPNKO mice, were administered angiotensin II (Ang II, 600 ng/kg/min; 2 weeks) or saline (vehicle) via osmotic minipumps. The mean arterial blood pressure (MAP) was measured using radio telemetry probes for 2 weeks. CPNKO mice did not show any significant differences in MAP at baseline. Ang II-infusion significantly increased the MAP in WT mice compared to saline infused control mice (147 ±5 vs 102 ±2 mmHg, n=5, p<0.05), which was attenuated in CPNKO mice (125 ±4 mmHg, n=5, p<0.01). Interestingly, there were no significant differences in MAP following Ang II-infusion in male and female CPNKO mice. Ang II treatment resulted in sympathoexcitation in WT mice, as indicated by increased urinary norepinephrine levels (446 ±38 ng/ml, p<0.05 vs. control), but this increase was blunted in CPNKO mice infused with Ang II (246 ±14 ng/ml, p<0.05 vs. WT+Ang II). WT mice treated with Ang II had increased inflammation, as shown by elevated gene expression (measured by qRT-PCR) of TNF, IL-1β and IL-6 (5-, 3.6-, and 4-fold, respectively, n=6, p<0.01 vs. WT) in the hypothalamic paraventricular nucleus (PVN). The expression of these inflammatory genes was attenuated in CPNKO mice with Ang II infusion. Immunofluorescence staining showed that Ang II-infusion also significantly increased the expression of B1R, CPM, and CD13 in the PVN, a central regulatory center for cardiovascular and autonomic control. CPM has been shown to interact directly with B1R. To further determine interactions between B1R and CPM, we used proximity ligation assay (PLA) to elucidate close (<40 nM) protein-protein interactions, as indicated by fluorescent PLA signals. Our data suggest that direct B1R-CPM interactions occurring within the PVN as the PLA signals are significantly increased by Ang II treatment in WT mice (p<0.05, n=3), and blunted in CPNKO mice. Conclusions: Kininase I inhibition by CPN gene deletion attenuates Ang II-induced hypertension by reducing B1R and CD13 acti vation, sympathoexcitation, and inflammation. Our data suggest that targeting kininase I may be a novel therapeutic target for hypertension. Funding: This study was supported by NHLBI/NIH 5R01HL153115 (S. Sriramula). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The effect of bradykinin on the pro-inflammatory response of human adipocytes.

The proper functioning of adipose tissue is one of the factors in maintaining energy homeostasis. Adipocytes not only store lipids but also produce active molecules such as adipokines and adipocytokines, which are involved in many functions of adipose tissue, including the secretion of hormones that regulate energy and lipid metabolism. Inflammation has been shown to underlie the deregulation of adipose tissue function. Bradykinin belongs to a family of pro-inflammatory kinin peptides that are abundant in most tissues and biological fluids. This study aimed to determine the ability to produce kinin peptides and characterize the effect of bradykinin on pro-inflammatory responses in adipocytes. The Chub-S7 human preadipocyte line was differentiated to show specific properties for adipose tissue cells. The differentiated cells expressed genes that encode proteins such as kininogen, kallikrein, and prolylcarboxypeptidase that are involved in the production of kinins and also showed the expression of kinin receptors. The response of adipocytes to bradykinin was examined in relation to kinin concentration and the presence of kininase inhibitors. The high concentration of bradykinin induced a moderate increase in lipid accumulation, increased release of pro-inflammatory cytokines, and altered gene expression of molecules involved in adipocyte function, such as adiponectin, lipoprotein lipase, and other transcription factors. This study suggests an important role for kinin peptides in inducing inflammatory responses in adipocytes, which can modify the function of adipose tissue and ultimately lead to diseases related to disturbance of energy homeostasis. The results obtained may enrich our understanding of the mechanisms underlying obesity-related disorders.

Kininase I Inhibition Prevents Angiotensin II‐Induced Hypertension and Target Organ Damage

Activation of the kinin B1 receptor (B1R), a major component of the kallikrein kinin system (KKS), is associated with the pathogenesis of hypertension. Kininase I (or carboxypeptidase N/M; CPN/CPM), an enzyme primarily responsible for the synthesis of endogenous B1R agonist, has been implicated in the progression of cardiovascular diseases. However, the exact role of kininase I in hypertension has not been investigated. Our preliminary data shows that CPN activity is significantly increased in the brain of angiotensin (Ang) II‐induced hypertensive mice compared to vehicle treated mice (Ang II: 7.1 +0.4 AU/min vs. vehicle: 2.8 +0.3 AU/min, n=5, p<0.05). In this study, our objective was to clarify the role of kininase I in the pathogenesis of hypertension, particularly the effects of this enzyme on blood pressure, cardiac and renal hypertrophy. We hypothesized that inhibition of kininase I will attenuate the development of Ang II‐induced hypertension and target organ damage. To test this hypothesis, 12‐week old male and female C57BL/6NJ wild‐type (WT) and CPN gene deleted (CPNKO) mice, were administered Ang II (600 ng/kg/min; 2 weeks) or saline (vehicle) via osmotic minipumps. Blood pressure was measured using radio telemetry probes and hypertrophy was assessed in the heart and kidney after 14 days of Ang II‐infusion, using weight/tibial length. Baseline blood pressure was similar between WT and CPNKO mice (102 +2 mmHg vs. 104 +2 mmHg). Ang II‐infusion significantly increased the blood pressure in WT mice compared to saline infused mice (147 +5 mmHg, n=5, p<0.05). This Ang II‐increased blood pressure response was attenuated in CPNKO mice (125 +4 mmHg, n=5, p<0.01). Both male and female CPNKO mice did not show any significant differences in blood pressure following Ang II‐infusion. In addition, Ang II‐infusion also induced significant cardiac (heart weight/tibia length: 8.5 +0.9 vs. 6.8 +0.6 mg/mm) and renal (kidney weight/tibia length: 11.8 +1.5 vs. 8.7 +0.9 mg/mm) hypertrophy in WT mice compared to vehicle treated mice. However, CPNKO mice exhibited an attenuation in cardiac and renal hypertrophy following Ang II administration. Furthermore, Ang II‐infusion increased collagen deposition in the heart, measured by picrosirius red staining, which was attenuated in CPNKO mice (percent area 9.8 +1.4 vs. 7.2 +1.4). Ang II‐infusion also significantly increased B1R gene and protein expression in the hypothalamus, heart, kidney, and aorta of WT mice, but this expression was abrogated in CPNKO mice. In conclusion, our data suggest that CPN gene deletion protects from Ang II‐induced hypertension and target organ damage, and indirectly prevents the B1R activation. This is the first evidence to show that inhibition of kininase I prevents the development of Ang II‐induced hypertension. Targeting kininase I might be a novel therapeutic approach for the treatment of hypertension.

Kininase 1 As a Preclinical Therapeutic Target for Kinin B1 Receptor in Insulin Resistance.

Kinin B1 receptor (B1R) contributes to insulin resistance, an early event in type 2 diabetes, through the upregulation and activation of the inducible form of nitric oxide synthase (iNOS), pro-inflammatory cytokines and the oxidative stress. This study addresses the hypothesis that inhibition of kininase 1 (carboxypeptidase M, CPM), the key enzyme involved in the biosynthesis of B1R agonists, could exert the same beneficial effects to B1R antagonism in insulin resistance. Male Sprague-Dawley rats were made insulin resistant with a drinking solution containing 10% D-glucose for a period of 9 weeks. Control rats received tap water. During the last week, kininase 1 was blocked with Mergetpa (1 mg kg−1 twice daily, s.c.) and the impact was determined on insulin resistance (HOMA index), metabolic hormone levels, oxidative stress and the expression of several markers of inflammation by western blot and qRT-PCR. Glucose-fed rats displayed hyperglycemia, hyperinsulinemia, hyperleptinemia, insulin resistance, hypertension, positive body weight gain, and enhanced expression of B1R, CPM, iNOS, and IL-1β in renal cortex, aorta and liver. Markers of oxidative stress (superoxide anion and nitrotyrosine expression) were also enhanced in aorta and renal cortex. Mergetpa reversed and normalized most of those alterations, but failed to affect leptin levels and hypertension. Pharmacological blockade of kininase 1 (CPM) exerted similar beneficial effects to a 1-week treatment with a B1R antagonist (SSR240612) or an iNOS inhibitor (1,400 W). These data reinforce the detrimental role of B1R in insulin resistance and recommend CPM as a new therapeutic target.

Renal (tissue) kallikrein-kinin system in the kidney and novel potential drugs for salt-sensitive hypertension.

A large variety of antihypertensive drugs, such as angiotensin converting enzyme inhibitors, diuretics, and others, are prescribed to hypertensive patients, with good control of the condition. In addition, all individuals are generally believed to be salt sensitive and, thus, severe restriction of salt intake is recommended to all. Nevertheless, the physiological defense mechanisms in the kidney against excess salt intake have not been well clarified. The present review article demonstrated that the renal (tissue) kallikrein-kinin system (KKS) is ideally situated within the nephrons of the kidney, where it functions to inhibit the reabsorption of NaCl through the activation of bradykinin (BK)-B2 receptors localized along the epithelial cells of the collecting ducts (CD). Kinins generated in the CD are immediately inactivated by two kidney-specific kinin-inactivating enzymes (kininases), carboxypeptidase Y-like exopeptidase (CPY), and neutral endopeptidase (NEP). Our work demonstrated that ebelactone B and poststatin are selective inhibitors of these kininases. The reduced secretion of the urinary kallikrein is linked to the development of salt-sensitive hypertension, whereas potassium ions and ATP-sensitive potassium channel blockers ameliorate salt-sensitive hypertension by accelerating the release of renal kallikrein. On the other hand, ebelactone B and poststatin prolong the life of kinins in the CD after excess salt intake, thereby leading to the augmentation of natriuresis and diuresis, and the ensuing suppression of salt-sensitive hypertension. In conclusion, accelerators of the renal kallikrein release and selective renal kininase inhibitors are both novel types of antihypertensive agents that may be useful for treatment of salt-sensitive hypertension.

A Proposal----The Renal Kallikrein-Kinin System as a Compensating System for Salt Accumulation after Excess Salt Intake

Terrestrial mammals, like human, must reserve water and NaCl to counter the effects of dry-environments. Their kidneys need to reabsorb as much water and sodium as possible along the proximal tubules and collecting duct. Thus, excess sodium intake tends to sodium accumulation in the body. The renal (tissue) kallikrein-kinin system (KKS) exists to prevent excessive sodium reabsorption. Renal kallikrein is secreted from the distal connecting tubule cells, which are located just distal to the major reabsorbing system. Kallikrein releases kinins, which inhibit sodium reabsorption along the collecting ducts. The kinins in the tubules are quickly destroyed by kidney-specific destroying enzymes (kininases), carboxypeptidase Y-like exopeptidase and neutral endopeptidase, which differ completely from those in plasma. Ebelactone B and poststatin were discovered as selective inhibitors of urinary kininases. Excess sodium intake causes hypertension, since sodium accumulation in the blood vessel walls increases sensitivity to hypertensive agents. Potassium, and ATP-sensitive potassium channel blockers cause renal kallikrein secretion. In bradykinin B2 receptor gene knockout mice, tissue kallikrein gene knockout mice, and kininogen-deficient rats, the knockout or deficiency does not itself induce hypertension, but these animals are salt-sensitive. Mutant kininogen-deficient (kinin-free) rats on 2% sodium intake quickly develop hypertension. Urinary kininase inhibitors reduce high blood pressure due to sodium accumulation on high sodium intake. Our ample supporting data suggest that the renal KKS prevents sodium accumulation, and that reduced levels of renal kallikrein may cause salt-sensitive hypertension. Keywords: Excess salt intake, The renal kallikrein-kinin system, Potassium, ATP-sensitive potassium channel blockers, Ebelactone B, Poststatin, Neutral endopeptidase, Renal Kallikrein-Kinin System, Renal kallikrein, Y-like exopeptidase, urinary kininases, Mutant kininogen-deficient, kallikrein-kinin system, renin-angiotensin system, systemic blood pressure, angiotensin-converting enzymes, lysyl-bradykinin, bradykinin, kallikrein-secreting cells, Henle's loop, BK-B2 antagonist, carboxypeptidase Y-like exopeptidase, human urinary kininases, epithelial Na channels, Rattus norvegicus, LMW kininogens, cerebrospinal fluid, deoxycorticosterone acetate, Weinberger's group

Potentiation of Bradykinin Effect by Angiotensin-Converting Enzyme Inhibition Does Not Correlate With Angiotensin-Converting Enzyme Activity in the Rat Mesenteric Arteries

Angiotensin-converting enzyme (kininase II [ACE]) inhibitors are capable of potentiating bradykinin (BK) effects by enhancing the actions of bradykinin on B(2) receptors independent of blocking its inactivation. To investigate further the importance of ACE kininase activity on BK-induced vasodilation, we investigated the effect of inhibiting ACE, as well as other kininases, on both BK metabolism and vasodilator effect in preparations that exhibit increased ACE activity. Mesenteric arterial beds obtained from 1-kidney, 1-clip hypertensive rats presented augmented ACE and angiotensin I converting activities compared with normotensive rats. The isolated and perfused mesenteric beds were exposed to BK for 15 minutes in the absence or in the presence of kininase inhibitors; then, the perfusate was collected for analysis of the products of BK metabolism by high-performance liquid chromatography. BK was metabolized to the fragments BK(1-8), BK(1-7), and BK(1-5), and the recovery of intact BK was reduced by 47% in the hypertensive group. Recovery of BK was increased in both groups in the presence of a kininase I inhibitor and in the hypertensive group by neutral endopeptidase 24.11 inhibitor; however, ACE inhibition did not affect BK metabolism in both groups. In contrast, only the ACE inhibitor potentiated the vasodilator effect of BK in a mesenteric bed preconstricted with phenylephrine; the increase in BK effect, nevertheless, was not greater in arteries from hypertensive rats that presented an increased ACE activity when compared with those in the normotensive group. These data demonstrated that ACE inhibitor-induced potentiation of BK vasodilator effects is not related to their actions on BK degradation.

Transvascular dissemination of Porphyromonas gingivalis from a sequestered site is dependent upon activation of the kallikrein/kinin pathway

Epidemiological evidence implicates a connection between human periodontitis and systemic diseases. One possible mechanism involves the direct dissemination of periodontopathogens to the target organs through the circulation. The aim of this work was to define the mechanism used by Porphyromonas gingivalis for dissemination from a sequestered infection site. BALB/c mice were subcutaneously infected with P. gingivalis via use of a mouse chamber model. Tissue fluids from various sites were collected and cultured to determine the presence of P. gingivalis. Evans Blue dye was used to measure the dissemination ability of P. gingivalis. Kinin-associated molecules were introduced into mice, and their effects on bacterial dissemination and mouse pathology were monitored. P. gingivalis strain A7436 caused remote lesions and septicemia with severe cachexia, resulting in animal death. Intrachamber challenge with A7436 resulted in vascular permeability enhancement (VPE), as measured by the systemic infiltration of Evans Blue dye into chamber fluids. VPE was blocked by kininase and kinin receptor antagonist and enhanced by exogenous bradykinin and kininase inhibitor. Live bacteria were recovered from the subcutaneous perichamber and abdominal spaces (spreading), and from the blood (disseminating) of infected mice. Both kininase and kinin receptor antagonist reduced animal mortality as a result of infection with strain A7436 and decreased the number of bacteria recoverable from the blood, but they were not associated with bacterial spreading. The results suggest that activation of the kinin system is involved in the breach of the vascular barrier that permits dissemination of P. gingivalis.

A Missing Link Between a High Salt Intake and Blood Pressure Increase

It is widely accepted that a high sodium intake triggers blood pressure rise. However, only one-third of the normotensive subjects were reported to show salt-sensitivity in their blood pressure. Many factors have been proposed as causes of salt-sensitive hypertension, but none of them provides a satisfactory explanation. We propose, on the basis of accumulated data, that the reduced activity of the kallikrein-kinin system in the kidney may provide this link. Renal kallikrein is secreted by the distal connecting tubular cells and all kallikrein-kinin system components are distributed along the collecting ducts in the distal nephron. Bradykinin generated is immediately destroyed by carboxypeptidase Y-like exopeptidase and neutral endopeptidase, both quite independent from the kininases in plasma, such as angiotensin converting enzyme. The salt-sensitivity of the blood pressure depends largely upon ethnicity and potassium intake. Interestingly, potassium and ATP-sensitive potassium (K(ATP)) channel blockers accelerate renal kallikrein secretion and suppress blood pressure rises in animal hypertension models. Measurement of urinary kallikrein may become necessary in salt-sensitive normotensive and hypertensive subjects. Furthermore, pharmaceutical development of renal kallikrein releasers, such as K(ATP) channel blockers, and renal kininase inhibitors, such as ebelactone B, may lead to the development of novel antihypertensive drugs.

The Plasma and Tissue Kininogen-kallikrein-kinin System: Role in the Cardiovascular System

Bradykinin and Lys-bradykinin are potent peptide mediators implicated in several physiopathological effects in mammals. They act through activation of G-protein-coupled constitutive B(2) or inducible kinin B(1) receptors linked to signaling pathways involving increased intracellular Ca(++) concentrations and/or release of mediators including arachidonic acid metabolites, NO and EDHF. In the cardiovascular system, the kallikrein-kinin system exerts a fine control of vascular smooth muscle tone and arterial blood pressure, and plays a significant cardioprotective effect. This has been lately confirmed in experimental studies employing transgenic mice overexpressing human tissue kallikrein and animals with knockout of kinin B(1) and B(2) receptor gene. Disturbances in this system are associated with arterial hypertension, myocardial ischaemia and other clinical complications. Inhibitors of kininase II (angiotensin-converting enzyme) have been prescribed successfully to patients with cardiovascular diseases, but there is still a great interest in developing drugs or pharmacological strategies that augment the activity of kininogen-kallikrein-kinin system in pathological conditions. Delivery of adenovirus vector containing the human tissue kallikrein gene (gene kallikrein therapy) has emerged as a great potential to satisfy these conditions. This review provides a summary of plasma and tissue kallikrein-kinin system, focusing on the pharmacological properties, kinin receptors and drugs reported to interfere with their actions. The modulatory effects of the kallikrein-kinin system on cardiovascular system, particularly in regulating smooth muscle tone and arterial blood pressure and in preventing myocardium ischaemia have also been explored in the review.

Prostaglandin receptors EP2, EP3, and IP mediate exudate formation in carrageenin-induced mouse pleurisy.

The roles of prostaglandins (PGs) as mediators of inflammation have been extensively studied, and production of PGI2 and PGE2 at inflammatory sites has been reported. However, it has not yet been clarified which type of PG receptors has a major role in inflammatory exudation. To examine in vivo role of PG receptors in inflammatory exudation, we induced pleurisy in PG receptors (IP, EP1, EP2, EP3, or EP4) knockout mice by intrapleural injection of carrageenin. Pleural exudate accumulation in wild-type (WT) mice at 1 to 5 h, but not at 24 h, was significantly attenuated by the pretreatment with indomethacin, indicating that PGs are responsible for exudate formation at the early phase of pleurisy. Pleural exudation at 1 to 5 h in IP, EP2, or EP3 knockout mice, but not in EP1 and EP4 knockout, was significantly reduced compared with in WT mice. In the exudates, 6-keto-PGF1alpha and PGE2 were detected as the major PGs, each with its peak concentration at 3 h. In addition, involvement of bradykinin in the phenomenon was suggested by the fact that captopril, a kininase inhibitor, enhanced the exudate formation and increased the amount of 6-keto-PGF1alpha and PGE2 and that a bradykinin B2-receptor antagonist inhibited the exudate formation. In contrast, leukocyte migration into pleural cavity was not influenced by indomethacin-treatment nor by these receptor deficiencies. These results demonstrate participation of EP2 and EP3 along with IP in pleural exudate formation but not in leukocyte migration in carrageenin-induced mouse pleurisy.

Cardiac kinin level in experimental diabetes mellitus: role of kininases.

Diabetes mellitus impairs the cardiac kallikrein-kinin system by reducing cardiac kallikrein (KLK) and kininogen levels, a mechanism that may contribute to the deleterious outcome of cardiac ischemia in this disease. We studied left ventricular (LV) function and bradykinin (BK) coronary outflow in buffer-perfused, isolated working hearts (n = 7) of controls and streptozotocin (STZ)-induced diabetic rats before and after global ischemia. With the use of selective kininase inhibitors, the activities of angiotensin I-converting enzyme, aminopeptidase P, and neutral endopeptidase were determined by analyzing the degradation kinetics of exogenously administered BK during sequential coronary passages. Basal LV function and coronary flow were impaired in STZ-induced diabetic rats. Neither basal nor postischemic coronary BK outflow differed between control and diabetic hearts. Reperfusion after 15 min of ischemia induced a peak in coronary BK outflow that was of the same extent and duration in both groups. In diabetic hearts, total cardiac kininase activity was reduced by 41.4% with an unchanged relative kininase contribution compared with controls. In conclusion, despite reduced cardiac KLK synthesis, STZ-induced diabetic hearts are able to maintain kinin liberation under basal and ischemic conditions because of a primary impairment or a secondary downregulation of kinin-degrading enzymes.

Evidence for a direct effect of captopril on early steps of insulin action in BC3H-1 myocytes

Captopril, an angiotensin-converting enzyme (ACE) inhibitor, has been reported to improve insulin sensitivity. However, despite extensive investigation, the mechanisms responsible for this effect are not fully understood. Reduction of plasma angiotensin II and inhibition of kininase II have been suggested to contribute to improve insulin sensitivity. Insulin binding was measured at tracer insulin concentration in intact cells with or without captopril treatment. Specific binding, expressed as percent of total insulin added, was not different in control and captopril-treated cells. However, captopril treatment caused an increase in insulin-induced insulin receptor substrate-1 (IRS-1) phosphorylation accompanied by an increased association of IRS-1 with phosphoinositide-3 kinase (PI-3 kinase), despite no change on insulin receptor (IR) autophosphorylation. There was also an increased threonine kinase B (AKT) phosphorylation in captopril-treated cells followed by enhanced basal and insulin-stimulated glucose uptake. These results indicate that captopril treatment has a direct effect on early phosphorylation events induced by insulin in BC3H-1 myocytes. Copyright 2003, Elsevier Science (USA). All rights reserved.

Inhibition of kinin breakdown prolongs retention and action of bradykinin in a myocardial B2 receptor compartment.

1. The high efficacy of ACE inhibitors to potentiate the actions of kinins might be explained by a hypothetical compartment in which B(2)-receptors are colocalized with kinin degrading enzymes. To demonstrate the functional consequence of such a compartment we compared the myocardial uptake and the persistence of action of bradykinin under the influence of kininase inhibitors. 2. Bradykinin-induced vasodilation and uptake of tritiated bradykinin were studied in perfused rat hearts during inhibition of ACE and aminopeptidase P. B(2)-receptors were localized by immuno-gold labelling and electron-microscopy. 3. The EC(50) of bradykinin-induced vasodilation (5.1+/-0.8 nM) was shifted to 14 fold lower concentrations during inhibition of both kininases. The maximum persistence of vasodilation after termination of bradykinin application (half-life 112+/-20 s) was increased by kininase inhibitors to 398+/-130 s. This prolongation was reversed when B(2)-receptors were blocked simultaneously with the termination of bradykinin infusion. 4. Tritiated bradykinin (perfused for 1 min) was partially (1.7+/-0.24%) retained by the myocardium and consecutively released with a half-life of 70+/-9 s. Kinin uptake was increased during kininase inhibition (7.7+/-2.6%), and was normalized by HOE 140 (2.0+/-0.34%), or when a tritiated B(2)-receptor antagonist (NPC 17731) was used as label. 5. B(2)-receptors were localized in plasmalemmal and cytosolic vesicles of capillary endothelium. 6. Bradykinin is locally incorporated and can associate with B(2)-receptors repeatedly when kinin breakdown is inhibited. This is the kinetic and functional consequence of a colocalization of kininases and B(2)-receptors in a compartment constituted by endothelial membrane vesicles.

Trypanosoma cruzi induces edematogenic responses in mice and invades cardiomyocytes and endothelial cells in vitro by activating distinct kinin receptor (B1/B2) subtypes.

Trypanosoma cruzi, the protozoan that causes Chagas' heart disease, invades endothelial cells in vitro by activating the B2 kinin receptor (B2R). Here, we demonstrate that mice infected with trypomastigotes develop potent edema after treatment with the angiotensin-converting enzyme (ACE) (or kininase II) inhibitor captopril. Experiments performed with specific kinin receptor (B2R/B1R) antagonists and knockout mice revealed that the early-phase (3-h) edema is mediated by the constitutive B2R, whereas the late-phase (24-h) response depends on stimulation of the up-regulated B1R. Given previous evidence that parasite invasion of cells expressing B2R is potentiated by captopril, we investigated the prerequisites for in vitro infection of Chinese hamster ovary cells overexpressing either B1R or B2R, human umbilical vein endothelial cells activated by lipopolysaccharide, and neonatal rat cardiomyocytes. Our results indicate that captopril potentiates parasite invasion regardless of the kinin (B2/B1) activation pathways, whereas DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid (MGTA), an inhibitor of kininase I (carboxypeptidase M/N), selectively decreases parasite infectivity for B1R-expressing cells. These data suggest that formation of the B1R agonist, i.e., [des-Arg] kinins, critically depends on the processing action of kininase I, here proposed as a potential pathogenesis cofactor. Collectively, our data suggest that fluctuations in the levels of kininases may modulate parasite infectivity and pathological outcome in Chagas' disease.

Determination of Bradykinin in Rat Urine by Coupled-Column High Pressure Liquid Chromatography with Precolumn Derivatization with a Water-Soluble Fluorogenic Reagent

Bradykinin (BK) in rat urine was determined by coupled-column HPLC with precolumn fluorogenic derivatization with a water-soluble reagent, 3-(7-fluoro-2,1,3-benzoxadiazole-4-sulfonamido)benzenesulfonic acid (m-BS-ABD-F). The derivatization of BK with m-BS-ABD-F was completed at 70°C for 100 min and gave only a single peak of BK derivative in addition to the peaks of the blank. The hydrophilicity of the derivatization reagent effectively prevented the adsorption of BK during the sample pretreatment and improved the recovery of BK. Good linearity was shown between the amount of BK spiked in urine (0–10 pmol) and the peak area of the BK derivatives (correlation coefficients >0.999), and the detection limits of the BK derivative were 35 fmol (S/N = 3). The precisions (cv, %) of intra- and interday assay were not more than 5.5% and the accuracies were in the range of 95.3–111% (1 and 5 pmol of BK in urine, n = 3). Although the peak regarded as that of the BK derivative rapidly decreased after incubation at 37°C, addition of urinary kininase inhibitors to the urine samples drastically suppressed the decrease of this peak, confirming that the identified peak was that of the BK derivative. The urinary kinin excretion in male SD rats (9–11 weeks old) determined by the present method was 56.0 ± 22.1 pg/min/kg (mean ± SE, n = 5).

Anti-ischemic effects of angiotensin-converting enzyme inhibitors: a future therapeutic perspective.

The renin-angiotensin system and angiotensin-converting enzyme (ACE) are increasingly being implicated in the pathogenesis of coronary artery disease and its sequelae and the potential of ACE inhibitors to protect the heart is a topic that has emerged recently as a matter for scientific discussion. Experimental and clinical studies have shown the beneficial effects of ACE inhibitors on the metabolism, function and structure of healthy and damaged hearts and these data support the concept of both primary and secondary cardioprotection with this class of drugs. Animal studies have demonstrated the potential beneficial effects of ACE inhibition at a variety of sites, including improvement of endothelial function, inhibition of platelet aggregation, prevention of atherosclerotic lesions and inhibition of myointimal proliferation, extending the concept to a more general definition of cardiovascular protection with ACE inhibitors involving both the heart and the vessels. ACE inhibitors prevent stimulation of smooth muscle cell angiotensin-II (A-II) receptors, thereby blocking both contractile and proliferative actions of A-II. In addition, ACE inhibition of kininase inhibits the breakdown of bradykinin, a direct stimulant of nitric oxide release from the intact endothelial cell. Thus, at the cellular level ACE inhibition shifts the balance of ongoing mechanisms in favour of those promoting vasodilatory, anti-aggregatory, antithrombotic and antiproliferative effects. Although these data have not all been validated in human studies, the reduction of ischemic events in studies of ACE inhibition in left ventricular dysfunction (LVD) and, more recently, also in patients without LVD, cannot be explained solely by improved hemodynamics, and it is possible that actions on the endothelium, the atherosclerotic process and platelets are at least in part responsible. So, the available data underlie the potential benefits of ACE inhibition in the field of ischemic heart disease and atherosclerosis; the results of ongoing studies in humans looking more directly at the influence of ACE inhibitors in this setting are awaited with interest.

Losartan, an angiotensin type 1 receptor antagonist, improves endothelial function in non-insulin-dependent diabetes

OBJECTIVESThe present study examined the effect on forearm endothelial function of an angiotensin II type 1 receptor antagonist, losartan, in subjects with non-insulin-dependent diabetes mellitus (NIDDM).BACKGROUNDAngiotensin-converting enzyme (ACE) inhibition with enalapril improves acetylcholine (ACh)-dependent endothelial function in patients with NIDDM. This could be mediated through angiotensin II and the type 1 receptor or could be due to inhibition of kininase II and a bradykinin preserving effect. It is therefore relevant to determine whether a type 1 receptor antagonist improves endothelial function.METHODSThe influence of losartan (50 mg daily for four weeks) on endothelium-dependent and independent vasodilator function was determined in 9 NIDDM subjects using a double-blinded placebo-controlled crossover protocol. Forearm blood flow was measured using strain-gauge plethysmography.RESULTSLosartan significantly decreased infused arm vascular resistance in response to three incremental doses of intrabrachial acetylcholine (p < 0.05, ANOVA). The forearm blood flow ratio (flow in infused to noninfused arm) was also increased (p < 0.01). Responses to sodium nitroprusside and monomethyl arginine were not significantly changed.CONCLUSIONSLosartan administration at 50 mg per day improved endothelium-dependent dilation of resistance vessels in patients with NIDDM. That is, blockade of the angiotensin II type 1 receptors improves endothelial function in NIDDM. At least some of the similarly beneficial effect of ACE inhibition is probably mediated also through the angiotensin II-type 1 receptor pathway. The use of a type 1 receptor antagonist seems a reasonable alternative to an ACE inhibitor to maintain endothelial function in NIDDM subjects.

Inhibition of kinin degradation on the luminal side of renal tubules reduces high blood pressure in deoxycorticosterone acetate salt-treated rats.

1. To determine whether the antihypertensive response in deoxycorticosterone acetate (DOCA) salt-treated rats was mediated by kinins on the luminal side of renal tubules or in the circulation, selective urinary kininase inhibitors were administered to normal Brown Norway Kitasato (BN-Ki) rats and kininogen-deficient Brown Norway Katholiek (BN-Ka) rats. 2. Kinins were degraded by neutral endopeptidase (NEP) and carboxypeptidase Y-like kininase (CPY) in urine, but were inactivated mainly by angiotensin-converting enzyme (ACE) in the plasma. 3. Ebelactone B inhibited CPY, while poststatin inhibited CPY and NEP. 4. Daily administration of poststatin (5 mg/kg per day, s.c.) for 3 days reduced blood pressure (BP) in DOCA salt-treated BN-Ki rats, but not in BN-Ka rats. 5. Ebelactone B (5 mg/kg per day, s.c.) also reduced BP in BN-Ki rats, which was accompanied by increased urinary sodium excretion, but had no effect on BP in BN-Ka rats. 6. Lisinopril (5 mg/kg per day, s.c.) had no effect on BP in either rat strain. 7. Arterial kinin levels in BN-Ki rats increased significantly (2.2-4.6 pg/mL) with captopril (10 mg/kg, s.c.). However, arterial kinin levels that induced hypotension following the infusion of bradykinin (1000 ng/kg per min, i.v.) were 110-fold higher than endogenous arterial kinin levels attained following captopril. 8. These results suggest that inhibition of kinin degradation on the luminal side of the renal tubules may effectively attenuate hypertension.