Kinetic and thermodynamic characterization of the interactions between the components of human plasma kinin-forming system and isolated and purified cell wall proteins of Candida albicans.

Cell wall proteins of Candida albicans, besides their best known role in the adhesion of this fungal pathogen to host's tissues, also bind some soluble proteins, present in body fluids and involved in maintaining the biochemical homeostasis of the human organism. In particular, three plasma factors - high-molecular-mass kininogen (HK), factor XII (FXII) and prekallikrein (PPK) - have been shown to adhere to candidal cells. These proteins are involved in the surface-contact-catalyzed production of bradykinin-related peptides (kinins) that contribute to inflammatory states associated with microbial infections. We recently identified several proteins, associated with the candidal cell walls, and probably involved in the binding of HK. In our present study, a list of potential FXII- and PPK-binding proteins was proposed, using an affinity selection (on agarose-coupled FXII or PPK) from a whole mixture of ß-1,3-glucanase-extrated cell wall-associated proteins and the mass-spectrometry protein identification. Five of these fungal proteins, including agglutinin-like sequence protein 3 (Als3), triosephosphate isomerase 1 (Tpi1), enolase 1 (Eno1), phosphoglycerate mutase 1 (Gpm1) and glucose-6-phosphate isomerase 1 (Gpi1), were purified and characterized in terms of affinities to the human contact factors, using the surface plasmon resonance measurements. Except Gpm1 that bound only PPK, and Als3 that exhibited an affinity to HK and FXII, the other isolated proteins interacted with all three contact factors. The determined dissociation constants for the identified protein complexes were of 10(-7) M order, and the association rate constants were in a range of 10(4)-10(5) M(-1)s(-1). The identified fungal pathogen-host protein interactions are potential targets for novel anticandidal therapeutic approaches.

Inhibition of kinin B1 receptors attenuates pulmonary hypertension and vascular remodeling.

This study examined whether the kinin B1 receptor is involved in the pathogenesis of pulmonary hypertension, and whether its inhibition could reduce inflammation, pulmonary hypertension, vascular remodeling, and right heart dysfunction. Male Wistar rats underwent left pneumonectomy. Seven days later, the rats were injected subcutaneously with monocrotaline (60 mg/kg). The rats were then randomly assigned to receive treatment with vehicle or with BI113823 (a selective B1 receptor antagonist, 30 mg/kg, twice per day) via oral gavage from the day of monocrotaline injection to day 28. By day 28, BI113823-treated rats had significantly lower mean pulmonary artery pressure, less right ventricular hypertrophy, and pulmonary arterial neointimal formation than that of the vehicle-treated rats. Real-time polymerase chain reaction revealed that there was a significant increase in mRNA expression of B1 receptors in the lungs of monocrotaline-challenged pneumonectomized rats. Treatment with BI113823 significantly reduced macrophage recruitment, as measured via bronchoalveolar lavage. It also markedly reduced CD-68 positive macrophages and proliferating cell nuclear antigen positive cells in the perivascular areas, reduced expression of inducible nitric oxide synthase, matrix metalloproteinase 2 and 9, and B1 receptors compared with measurements in vehicle-treated rats. These findings demonstrate that kinin B1 receptors represent a novel therapeutic target for pulmonary arterial hypertension.

Binding and activation of the human plasma kinin-forming system on the cell walls of Candida albicans and Candida tropicalis.

Bacterial infections often upregulate the plasma kinin-forming cascade of the host (the 'contact system') which is triggered by adsorption of high molecular mass kininogen (HK), coagulation factor XII (FXII) and prekallikrein (pHPK) on the host or pathogen cell surfaces. A possible activation of the contact system upon infection of the human host by major fungal pathogens of Candida species has not been extensively explored until a recent report of tight binding of HK to the cell walls of these fungi. In the current study, the adsorption of the other contact system components to the cell surfaces of Candida albicans and Candida tropicalis was characterized. FXII was found to be tightly bound by Candida germ tube forms, to a level 5-fold higher than that for HK. In contrast, pHPK bound poorly but its additional amounts could dock to the cell wall through the surface-bound HK. It was also shown that within the complex of these proteins assembled on the cell walls of fungal hyphae, pHPK could be activated by FXIIa and the active HPK effectively produced kinins from HK. It is suggested that kinins, released at the Candida cell wall, can promote host colonization by the pathogen and the development of infection.

Kallikreins, kininogens and kinin receptors on circulating and synovial fluid neutrophils: role in kinin generation in rheumatoid arthritis.

OBJECTIVES: Neutrophils traffic into and have the capacity to generate kinins in SF of RA patients. The aim of this study was to assess the expression of kallikreins, kininogens and kinin receptors in circulating and SF neutrophils, as well as synovial tissue of RA patients, and to assess kinin generation in SF. METHODS: Neutrophils were isolated from blood and SF of RA patients and blood of healthy volunteers. Expression of kallikreins, kininogens and kinin receptors in neutrophils and synovial tissue was assessed by immunocytochemistry using specific antibodies, with visualization by brightfield and confocal microscopy. Levels of basal and generated kinins in SF of RA patients were measured by ELISA. RESULTS: Kinin labelling was significantly reduced, indicating the loss of the kinin moiety from kininogen on circulating (P < 0.001) and SF neutrophils (P < 0.05) of RA patients. Immunolabelling of tissue kallikrein was also decreased, whereas kinin B(1) and B(2) receptor expression was increased in circulating and SF neutrophils of RA patients. Immunolabelling of kallikreins and kinin receptor proteins was similar in RA and normal synovial tissues. The basal kinin level in SF of RA patients was 5.7 +/- 6.1 ng/ml and the mean concentration of kinins generated in vitro was 80.6 +/- 56.3 ng/ml. The capacity for kinin generation was positively correlated with measures of disease activity. CONCLUSIONS: Kallikrein-kinin proteins on neutrophils play an important role in kinin generation and the pathophysiology of RA. Specific kallikrein and kinin receptor antagonists may be useful as IA therapies for inflamed joints.

Generation of kinins during preparation and storage of whole blood-derived platelet concentrates.

BACKGROUND: Leukoreduction of platelet (PLT) concentrates (PCs) may be associated with hypotension in recipients, and a role for bradykinin (BK)-related peptides has been proposed for this side effect. STUDY DESIGN AND METHODS: The concentration of BK and one of its vasoactive metabolites, des-arginine(9)-BK (des-Arg(9)-BK), was measured in a large number of PCs as a function of leukoreduction and storage duration with specific enzyme immunoassays and complementary techniques. RESULTS: On Day 0 of storage, kinins were detected in leukoreduced and unfiltered PCs at a concentration lower than 100 pg per mL. During storage, both kinin levels peaked on Day 5 of storage, with a concentration higher than 1 ng per mL in 22 percent of PCs whether filtered on Day 0 or not. Physicochemical and pharmacologic characterizations of immunoreactive kinins confirm their nature. In vitro activation of the contact system of the corresponding PLT-poor plasma showed that a high kinin concentration on Day 5 of the storage corresponded with a low kinin-forming capacity of plasma. On Day 7, BK was no longer elevated presumably due to its degradation and the depletion of kinin-forming capacity of the plasma in stored PCs. The activities of metallopeptidases that metabolize BK-related peptides in plasma from PCs were at levels similar to those recorded in the plasma of a normal reference population and were unaffected by storage. CONCLUSION: Storage of PCs contributes to the hydrolysis of high-molecular-weight kininogen and generation of pharmacologically relevant BK levels that might pose a hazard in susceptible patients.

Neurotransmitter receptor expression and activity during neuronal differentiation of embryonal carcinoma and stem cells: from basic research towards clinical applications.

Embryonal carcinoma and embryonic stem cells have served as models to understand basic aspects of neuronal differentiation and are promising candidates for regenerative medicine. Besides being well characterized regarding the capability of embryonal carcinoma and embryonic stem cells to be precursors of different tissues, the molecular mechanisms controlling neuronal differentiation are hardly understood. Neuropeptide and neurotransmitter receptors are expressed at early stages of differentiation prior to synaptogenesis, triggering transient changes in calcium concentration and inducing neurone-specific gene expression. In vitro neuronal differentiation of embryonal carcinoma and embryonic stem cells closely resembles early neuronal development in vivo. Murine P19 EC cells are a well-characterized model for in vitro differentiation, which upon treatment with retinoic acid differentiate into neurones. Expression and activity of various receptor proteins is regulated during their differentiation. Stimulation of kinin-B2, endothelin-B, muscarinic acetylcholine, and N-methyl-D-aspartate receptors results in transient increases of intracellular free calcium concentration [Ca(2+)](i) in P19 cells undergoing neuronal differentiation, whereas embryonal cells do not respond or show a smaller change in [Ca(2+)](i) than differentiating cells. Receptor inhibition, as studied with the example of the kinin-B2 receptor, aborts neuronal maturation of P19 cells, demonstrating the crucial importance of B2 receptors during the differentiation process. Future success in obtaining desired neuronal phenotypes from pluripotent cells in vitro may offer new therapeutic perspectives for curing genetic and acquired dysfunctions of the developing and adult nervous system.

Modulation of kinin B1 receptor expression by endogenous angiotensin II in hypertensive rats.

We investigated the expression and localization of B1 receptor in tissues of rats submitted to a renin-dependent model of hypertension (2K-1C), and analyzed the influence of endogenous Ang II in modulating the in vivo expression of these receptors. B1 mRNA levels in the heart, kidney and thoracic aorta were quantified by real time PCR, B1 receptor protein expression was assessed by immunohistochemistry, plasma Ang II levels were analyzed by radioimmunoassay and the effects of AT1 receptor blockade were determined after losartan treatment. 2K-1C rats presented a marked increase in Ang II levels when compared to sham-operated rats. In parallel, cardiac- (but not renal and aortic) B1 mRNA levels were 15-fold higher in 2K-1C than in sham rats. In 2K-1C, B1 expression was detected in the endothelium of small cardiac arteries and in cardiomyocytes. Losartan completely reverted the increased B1 mRNA levels and significantly decreased the protein expression observed in 2K-1C rats, despite reducing, but not normalizing blood pressure. We conclude that in the 2K-1C rat, induction of cardiac B1 receptor might be tightly linked to AT1 receptor activation. These data suggest the existence of a new site of interaction between kinins and angiotensins, and might provide important contributions for a better understanding of the pathophysiology of hypertension.

Assembly, activation, and signaling by kinin-forming proteins on human vascular smooth muscle cells.

Cardiovascular disease is the number one cause of death in the United States. Vascular smooth muscle cells (VSMC) are an important constituent of the vessel wall that can bring about pathological changes leading to vascular disease. Depending on the environment, the function of VSMC can deviate profoundly from its normal contractile role. Despite advances in research, the underlying mechanisms that activate VSMC toward vascular disease are poorly understood. For the first time, we have observed that factor XII and high-molecular-weight kininogen, constituents of the blood plasma, can bind to VSMC in a Zn2+-dependent manner. In the presence of prekallikrein, this assembly of factor XII and high-molecular-weight kininogen on VSMC leads to the activation of prekallikrein to kallikrein with a rapid formation of bradykinin. The amount of bradykinin in the culture medium then decreases, presumably because of the presence of a kininase activity. p44/42 mitogen-activated protein kinase is rapidly phosphorylated in response to in situ-generated or in vitro-added bradykinin and is inhibited by bradykinin antagonist HOE-140. Binding of factor XII to VSMC also results in a concentration-dependent phosphorylation of p44/42 mitogen-activated protein kinase. This early mitogenic signal, which is also implicated in atherogenesis, may change the metabolic and proliferative activity of VSMC, which are key steps in the progression of atherosclerosis.

Release of a new vascular permeability enhancing peptide from kininogens by human neutrophil elastase.

Stimulated neutrophils produced vascular permeability enhancing (VPE) activity in the presence of high molecular weight kininogen (HMWK), which was inhibited mainly by a neutrophil elastase (NE) inhibitor or a bradykinin (BK) B(2)-receptor antagonist. NE (>3 nM) generated VPE activity from kininogens at normal plasma concentrations with the smaller protein being several fold more responsive than the larger protein, through releasing a new VPE peptide (E-kinin), SLMKRPPGFSPFRSSRI. Synthetic E-kinin, SLMKRPPGFSPFRSS and SLMKRPPGFSPFR had VPE and blood pressure lowering activities, which were comparable to the activities of BK and completely inhibited by B(2)-receptor antagonists. Interestingly, E-kinin and SLMKRPPGFSPFRSS did not induce smooth muscle contraction. These results suggest that E-kinin formed in vivo may be processed at the carboxy-terminus to give a peptide that can bind to the B(2)-receptor. The molecular mechanism for neutrophil-associated VPE may be explained by excision of E-kinin from kininogens by NE, followed by further processing of the peptide.

Expression of the tissue kallikrein-kinin cascade in granulosa cells of the ovary.

The serine protease, tissue kininogenase (kallikrein), belongs to a unique family of enzymes that cleaves the decapeptide, kallidin, from the endogenous substrate kininogen. By analysis of genealogy patterns rat KLK gene family members have been detected in ovarian luteinizing granulosa cells of both gonadotrophin-treated and non-treated control rats. Recently, we demonstrated that tissue kininogenase showed intense immunolabeling in angiogenic endothelial cells isolated from bovine mature and regressing corpora lutea. Therefore, the question to answer was whether granulosa cells associated with ovarian vascularization possess the same capacity to express the kallikrein-kinin cascade as do microvascular endothelial cells. As a first step, experiments were designed to determine the expression and visualization of tissue kininogenase (both active and pro forms) as well as kininogen and kinin receptors in granulosa cells of different developmental stages and segments of the ovarian follicle by immunoperoxidase assay, confocal fluorescent microscopy and in situ hybridization.

Activation of the kinin-forming cascade on the surface of endothelial cells.

Activation of the plasma kallikrein-kinin forming cascade takes place upon incubation with human umbilical vein endothelial cells. The mechanism by which initiation occurs is uncertain. Zinc-dependent binding of plasma proteins to gC1qR, cytokeratin 1, and perhaps u-PAR is requisite for activation to take place. We demonstrate here that during a 2 hour incubation time plasma deficient in either factor XII or high molecular weight kininogen (HK) fails to activate, as compared to normal plasma, but with more prolonged incubation, factor XII-deficient plasma gradually activates while HK-deficient plasma does not. Our data support both factor XII-dependent (rapid) and factor XII-independent (slow) mechanisms; the latter may require a cell-derived protease to activate prekallikrein and the presence of zinc ions and HK.

C1 inhibitor deficiency: hereditary and acquired forms.

C1 inhibitor deficiency can be hereditary or acquired. The hereditary disorder has two types, each of which is inherited as a dominant disorder, with genetic mechanisms leading either to low levels of normal C1 INH and little or no mutant problem as a result of mRNA or protein synthetic defects or degradative mechanisms (Type I) or with point mutations and synthesis of a functionless protein product with transinhibition of the normal allele (Type II). The acquired disorder with low C1q is due to C1 INH consumption associated with lymphoma or connective tissue disease (Type I) and/or autoimmune mechanisms (Type II). The swelling of all types is due to absence of inhibition of the plasma kinin forming cascade with liberation of bradykinin while complement activation, a critical marker of the disorder, is not responsible for the swelling. Treatment employs androgenic compounds, antifibrinolytic agents, or replacement therapy.

Evidence for activation of the tissue kallikrein-kinin system in nociceptive transmission and inflammatory responses of mice using a specific enzyme inhibitor.

The pharmacological activity of phenylacetyl-Phe-Ser-Arg-N-(2, 4-dinitrophenyl)-ethylenediamine (TKI), a tissue kallikrein specific inhibitor, was assessed using models of nociception and inflammation in mice. Injection of TKI (13.6 - 136 micromol kg(-1), i.p. or 41 - 410 micromol kg(-1), s.c.) produced a dose-related inhibition of the acetic acid-induced writhes (by 37 to 85% or 34 to 80%, respectively). The antinociceptive activity of TKI (41 micromol kg(-1), i.p.) was maximal after 30 min injection and lasted for 120 min. The effect was unaltered by pretreatment with naloxone (8.2 micromol kg(-1), s.c.) or bilateral adrenalectomy. TKI (41 and 136 micromol kg(-1), i.p.) produced a dose-related decrease of the late phase of formalin-induced nociception by 79 and 98%, respectively. At 136 micromol kg(-1), i.p., TKI also shortened the duration of paw licking in the early phase by 69%. TKI (41 and 136 micromol kg(-1), i.p.) also reduced the capsaicin-induced nociceptive response (by 51 to 79%). TKI (41 micromol kg(-1), i.p. or 410 micromol kg(-1), s.c.) reduced the oedematogenic response, from the second to the fifth hour after carrageenin injection by 36 to 30% or by 47 to 39%, respectively. Pretreatment with TKI (41 micromol kg(-1), i.p.) reduced the capsaicin-induced neurogenic inflammation in the mouse ear by 54%. It is concluded that TKI presents antinociceptive and antiinflammatory activities mediated by inhibition of kinin formation by tissue kallikrein in mice. The results also indicate that the tissue kallikrein-dependent pathway contributes to kinin generation in nociceptive and inflammatory processes in mice.

[Local cerebral blood flow, cortical impedance, cerebral intravascular kinin production during forebrain ischemia and reperfusion in rats]. / Lokal'nyi mozgovoi krovotok, kortikal'nyi impedans, tserebral'noe vnutrisosudistoe kininoobrazovanie pri perednemozgovoi ishemii i reperfuzii u krys.

In the rat model of forebrain ischemia with subsequent reperfusion, an obvious formation of intravascular kinin (IVKF) occurred. The IVKF preceded and coincided in time with the maximum hyperemia and a vasogenic oedema. Local cerebral blood flow (LCBF) increased up to about 187%, at that. In three groups of experimental rats, a correlation was revealed among the ischemia obviousness, IVKF, and development of the brain oedema.

Interactions of angiotensin-converting enzyme, kinins and nitric oxide in circulation and the beneficial effects of ACE inhibitors in cardiovascular diseases.

Renin-angiotensin-aldosterone systems play a critical role in the development and progression of cardiovascular diseases, and inhibitors of angiotensin-converting enzyme have proven effective for the treatment of these diseases. Since angiotensin II receptor antagonists can inhibit the effects of angiotensin II via ACE-independent pathways, e.g., chymase, they were considered to be more effective than ACEIs. On the other hand, ACE inhibitors can increase bradykinin, and thus, nitric oxide, which may cause potent cardioprotection, inhibition of smooth muscle proliferation and attenuation of inflammation mechanisms. It appears that angiotensin II receptor antagonists and ACEIs may mediate cardioprotection in different ways. This is the rationale to explore the possibility of a combined administration of both drugs for the treatment of chronic heart failure and other cardiovascular pathology. In this review we try to analyze the role of ACE, kinins and chymase inhibition in the pathophysiology and treatment of cardiovascular diseases.

Correlation of kinin generating activity with Helicobacter pylori-associated gastric infection.

The kallikrein-kinin system involves a biologically complex set of interactive proteases that signal the first-line onset of inflammation and associated cellular processes. The basic enzymatic cleavage of kininogen substrate by the serine protease tissue kallikrein to liberate kinins is regulated by a number of factors. These may include the recently discovered bacterial involvement in the causation of gastritis. The gram-negative Helicobacter pylori organism, colonises the human gastric epithelium and initiates ulcerogenesis and may induce, in the longer term, tumour formation. The aim of this study was to investigate the role of kinins in H. pylori-induced gastric dyspepsia. During endoscopic examination, lavage aspirates of 23 patients were collected, and the tissue kallikrein content measured by a kinin-generating assay and an enzyme-linked immunosorbent assay. Gastric antral and pyloric biopsy tissue was histologically examined for degrees of inflammation and H. pylori infection, and then immunolabelled for tissue kallikrein and kinin receptors. Results show that labelled tissue kallikrein in the fundic glands and parietal cells of the diseased antrum was elevated with increasing severity of gastritis. Further, kinin-generating potential of the lavage fluid appeared to be greater with increasing evidence of infection. Tissue kallikrein immunosorbent assay levels were significantly raised in patients showing mild to moderate H. pylori infection. One outcome of this study may be the inclusion of kinin antagonists in management of gastric dyspepsia.

[Kinin generation in acute and chronic airway inflammation].

OBJECTIVE: To investigate the kinin generation pathways in acute and chronic airway inflammation. METHODS: BALF from patients with acute, chronic airway inflammation and healthy controls were collected. Kinins, Plasma kallikrein, alpha 2-macroglobulin and toluenesulphonyl-arginine methyl ester esterase activity (TAME-ea) in BALF were studied. RESULTS: Kinins and TAME-ea values were significantly higher in the BALF of patients with acute and chronic airway inflammation than those in the controls, but there was no significant difference between acute and chronic groups; PK and alpha 2-M values were significantly higher in the acute group than the in chronic one. Gel filtration revealed the highest TAME-ea peak at about 800,000 in the acute group, corresponding with the first alpha 2-M peak, whereas at about 40,000 in chronic bronchitis. The inhibition test of the TAME-ea showed that the TAME-ea peak at 800,000 was mainly due to PK and the TAME-ea peak at 40,000 was mainly due to TK. CONCLUSIONS: The results indicated that in acute airway inflammation kinins seem to be mainly generated by PK, whereas in chronic inflammation kininogenases other than PK--such as TK--seem to be more important.

Heparin, derived from the mast cells of human lungs is responsible for the generation of kinins in allergic reactions due to the activation of the contact system.

BACKGROUND: In a recent study mast cell heparin proteoglycan (HepPG) of a cell line derived from a mouse mastocytoma was isolated. Glycosaminoglycans proved to be an initiating surface for starting contact activation and could explain kinin generation present in allergic reactions. It is the aim of the present study to prove that HepPG or glycosaminoglycan derived from human mast cells is also capable of acting as a physiologic macromolecule and to induce contact activation. METHODS: HepPG molecules were isolated by anionic column chromatography. Their ability to accelerate reciprocal activation of factor XII was investigated by spectrophotometry. The anticoagulant effect was demonstrated by an increase in partial thromboplastin time. HPLC was performed to correlate these effects with molecular weight (MW). RESULTS: The isolated heparin showed high contact-activating and anticoagulant potency. Both actions were suppressed by incubation with heparinase I. The maximum contact activation peak appeared at a lower MW than the anticoagulant effect. CONCLUSION: These in vitro results explain the results of in vivo allergen challenge studies where a high degree of kinin generation occurs. Heparin derived from human mast cells therefore seems to represent the physiological macromolecule capable of activating the contact system and could be a missing link between cellular and humoral responses in allergic reactions.

Cardiovascular kinin-generating capability in hypertensive fructose-fed rats.

OBJECTIVE: The hypertensive state is often associated with metabolic abnormalities, including glucose intolerance. Tissue kallikrein, a potent kinin-generating enzyme, is present in the vascular wall and heart tissue. High dietary fructose consumption is reported to induce hyperinsulinemia, hypertriglyceridemia and hypertension. The objective of the present study was to examine the status of kallikrein in vascular and cardiac tissue from highly fructose-fed rats and to delineate the effect of kinins and the angiotensin converting enzyme inhibitor ramipril in this animal model of glucose intolerance. DESIGN AND METHODS: Male Wistar rats (350 g body weight) were divided into four groups of 10 rats each: (1) controls; (2) oral ramipril at 500 microg/kg per day for the last 2 study weeks; (3) fructose in drinking water as a 10% (w/v) solution for 4 weeks; and (4) fructose + ramipril, with fructose administered as in group 3 plus the administration of ramipril for the last 2 study weeks. Systolic blood pressure (tail-cuff method), glucose tolerance (2 g/kg body weight intraperitoneally) and metabolic parameters were recorded. Kallikrein activity in tail artery and heart tissue homogenates was estimated at the end of the 4th study week from measurements of kininogenase activity and kinins generated by a radioimmunoassay. RESULTS: The area under the curve for the glucose tolerance test increased from 1265 +/- 103 mmol/l after 120 min in the control and 1152 +/- 36 mmol/l in the ramipril group (NS) to 2628 +/- 143 mmol/l in the fructose group (P<0.01). The administration of ramipril to fructose-treated rats in group 4 improved glucose tolerance (2160 +/- 100 mmol/l; P<0.05 versus group 3). Blood pressure increased significantly in fructose-fed rats but fell markedly in fructose-fed rats treated with ramipril (P<0.01). Kallikrein activity measured in the heart and vessels increased as a consequence of fructose administration (P<0.05), but the administration of ramipril increased this parameter to a much greater extent (P<0.01 versus control group), which correlated closely with the decrease in blood pressure and the improvement in glucose tolerance observed in the fructose + ramipril group. CONCLUSIONS: The administration of fructose as a solution in the drinking water induced glucose intolerance and increased blood pressure. Treatment with the angiotensin converting enzyme inhibitor ramipril improved glucose tolerance and significantly diminished blood pressure. Cardiovascular kinin-generating capability increased in treated animals and this increase was even higher when rats were treated with ramipril, suggesting that kinins, acting as a paracrine hormonal system, can exert cardiovascular protection and contribute to the beneficial effects of angiotensin converting enzyme inhibitor.