Tissue kallikrein is required for the cardioprotective effect of cyclosporin A in myocardial ischemia in the mouse.

Clinical and experimental studies suggest that pharmacological postconditioning with Cyclosporin A (CsA) reduces infarct size in cardiac ischemia and reperfusion. CsA interacts with Cyclophilin D (CypD) preventing opening of the mitochondrial permeability transition pore (mPTP). Tissue kallikrein (TK) and its products kinins are involved in cardioprotection in ischemia. CypD knockout mice are resistant to the cardioprotective effects of both CsA and kinins suggesting common mechanisms of action. Using TK gene knockout mice, we investigated whether the kallikrein-kinin system is involved in the cardioprotective effect of CsA. Homozygote and heterozygote TK deficient mice (TK(-/-), TK(+/-)) and wild type littermates (TK(+/+)) were subjected to cardiac ischemia-reperfusion with and without CsA postconditioning. CsA reduced infarct size in TK(+/+) mice but had no effect in TK(+/-) and TK(-/-) mice. Cardiac mitochondria isolated from TK(-/-) mice had indistinguishable basal oxidative phosphorylation and calcium retention capacity compared to TK(+/+) mice but were resistant to CsA inhibition of mPTP opening. TK activity was documented in mouse heart and rat cardiomyoblasts mitochondria. By proximity ligation assay TK was found in close proximity to the mitochondrial membrane proteins VDAC and Tom22, and CypD. Thus, partial or total deficiency in TK induces resistance to the infarct size reducing effect of CsA in cardiac ischemia in mice, suggesting that TK level is a critical factor for cardioprotection by CsA. TK is required for the mitochondrial action of CsA and may interact with CypD. Genetic variability in TK activity has been documented in man and may influence the cardioprotective effect of CsA.

Pathophysiology of genetic deficiency in tissue kallikrein activity in mouse and man.

Study of mice rendered deficient in tissue kallikrein (TK) by gene inactivation and human subjects partially deficient in TK activity as consequence of an active site mutation has allowed recognising the physiological role of TK and its peptide products kinins in arterial function and in vasodilatation, in both species. TK appears as the major kinin forming enzyme in arteries, heart and kidney. Non-kinin mediated actions of TK may occur in epithelial cells in the renal tubule. In basal condition, TK deficiency induces mild defective phenotypes in the cardiovascular system and the kidney. However, in pathological situations where TK synthesis is typically increased and kinins are produced, TK deficiency has major, deleterious consequences. This has been well documented experimentally for cardiac ischaemia, diabetes renal disease, peripheral ischaemia and aldosterone-salt induced hypertension. These conditions are all aggravated by TK deficiency. The beneficial effect of ACE/kininase II inhibitors or angiotensin II AT1 receptor antagonists in cardiac ischaemia is abolished in TK-deficient mice, suggesting a prominent role for TK and kinins in the cardioprotective action of these drugs. Based on findings made in TK-deficient mice and additional evidence obtained by pharmacological or genetic inactivation of kinin receptors, development of novel therapeutic approaches relying on kinin receptor agonism may be warranted.

Cardioprotection and kallikrein-kinin system in acute myocardial ischaemia in mice.

1. Acute myocardial ischaemia and reperfusion trigger cardioprotective mechanisms that tend to limit myocardial injury. These cardioprotective mechanisms remain for a large part unknown, but can be potentiated by performing ischaemic preconditioning or by administering drugs such as angiotensin-I-converting enzyme (kininase II) inhibitors (ACEI). 2. This brief review summarizes the findings concerning the role of tissue kallikrein (TK), a major kinin-forming enzyme, kinins and kinin receptors in the cardioprotection afforded by ischaemic preconditioning (IPC) or by pharmacological postconditioning by drugs originally targeted at the renin-angiotensin system, ACEI and type 1 angiotensin-II receptor blockers (ARB) in acute myocardial ischaemia. Myocardial ischaemia was induced by left coronary occlusion and was followed after 30 min by a 3 h reperfusion period (IR), performed in vivo in mice. The role of the kallikrein-kinin system (KKS) was studied by using genetically engineered mice deficient in TK gene and their wild-type littermates, or by blocking B1 or B2 bradykinin receptors in wild-type mice using selective pharmacological antagonists. 3. Ischaemic preconditioning (three cycles: 3 min occlusion/5 min reperfusion) enhances the ability of the heart of wild-type mice to tolerate IR. Tissue kallikrein plays a major role in the cardioprotective effect afforded by IPC, which is largely reduced in TK-deficient mice. The B2 receptor is the main kinin receptor involved in the cardioprotective effect of IPC. 4. Tissue kallikrein is also required for the cardioprotective effects of pharmacological postconditioning with ACEI (ramiprilat) or ARB (losartan), which are abolished for both classes of drugs in TK-deficient mice. The B2 receptor mediates the cardioprotective effects of these drugs. Activation of angiotensin-II type 2 (AT2) receptor is involved in the cardioprotective effects of losartan, suggesting a functional coupling between AT2 receptor and TK during angiotensin-II type 1 (AT1) receptor blockade. 5. The demonstration of a cardioprotective effect of the KKS in acute myocardial ischaemia involving TK and the B2 receptor and playing a major role in IPC or pharmacological postconditioning by ACEI or ARB, suggests a potential therapeutic approach based on pharmacological activation of the B2 receptor.

Murine models of myocardial and limb ischemia: diagnostic end-points and relevance to clinical problems.

Ischemic disease represents the new epidemic worldwide. Animal models of ischemic disease are useful because they can help us to understand the underlying pathogenetic mechanisms and develop new therapies. The present review article summarizes the results of a consensus conference on the status and future development of experimentation in the field of cardiovascular medicine using murine models of peripheral and myocardial ischemia. The starting point was to recognize the limits of the approach, which mainly derive from species- and disease-related differences in cardiovascular physiology. For instance, the mouse heart beats at a rate 10 times faster than the human heart. Furthermore, healing processes are more rapid in animals, as they rely on mechanisms that may have lost relevance in man. The main objective of the authors was to propose general guidelines, diagnostic end points and relevance to clinical problems.

[Gene transfers of kallikrein, bradykinin receptor B2 and a mutated B2 receptor stimulate neoangiogenesis in peripheral ischemia]. / La surexpression de la kallicréine tissulaire, du récepteur B2 de la bradykinine et d'une forme mutée du récepteur B2, stimule la néoangiogenèse dans un modèle d'ischémie périphérique.

In this work, we evaluated the angiogenic effect of the gene transfer of human tissue kallikrein (TK), bradykinin B2 receptor (B2R) and a mutated form (RB2m) in a rabbit peripheral model of ischaemia. We studied the effects of the transfection of each of these factors and the effects of their co-transfection. In New Zealand anesthetised rabbits we first induced an ischaemia of the left posterior leg by ligation-excision of the superficial femoral artery and its collaterals. Seven days later, we performed i.m. injections in the ischemic tight with transfection solutions containing either the control (pcDNA3 empty backbone) or the pcDNA3-TK, the pcDNA3-TK and the pcDNA3-B2R, the pcDNA3-TK and the pcDNA3-B2Rm. Twenty eight days later, the therapeutic effect was evaluated using ultrasonographic debitmetry of the common iliac artery, perfusion index (PI) = ischemic leg blood flow /non ischemic leg blood flow (%) and capillaries measurements i.e. capillary density: number of vessels/mm2 and the ratio of vessels/muscular fiber, in the adductors and gastrocnemian muscles. The PI was increased in each treated group vs control (32.61 +/- 5.2%), pcDNA3-TK: 59.72 +/- 2.33%; p = 0.001; pcDNA3-RB2: 55.25 +/- 2.29%; p = 0.008; pcDNA3-TK + pcDNA3-RB2: 84.77 +/- 3.15%; p < 0.001; pcDNA3-TK + pcDNA3-RB2m: 103.25 +/- 4.9%; p < 0.001. The capillary density and the vessel/muscular fiber ratio increased in a parallel with the hemodynamic in the ischemic adductors (pcDNA3-TK + pcDNA3-B2Rm > pcDNA3-TK + pcDNA3-RB2 > pcDNA3-TK = pcDNA3-B2R; p < 0.001). There was no angiogenic effect measurable neither in the non ischemic adductors (right) nor in the gastrocnemian muscles. In rabbit peripheral ischaemia, the cotransfection of TK and B2R increases the arterial flow in the treated leg and potentiates the neoangiogenesis. Cotransfection of the B2Rm cDNA enhanced the synergic effect of this therapeutic strategy.

Role of tissue kallikrein in the cardioprotective effects of ischemic and pharmacological preconditioning in myocardial ischemia.

Tissue kallikrein (TK), a major kinin-forming enzyme, is synthesized in the heart and arteries. We tested the hypothesis that TK plays a protective role in myocardial ischemia by performing ischemia-reperfusion (IR) injury, with and without ischemic preconditioning (IPC) or ACE inhibitor (ramiprilat) pretreatment, in vivo in littermate wild-type (WT) or TK-deficient (TK-/-) mice. IR induced similar infarcts in WT and TK-/-. IPC reduced infarct size by 65% in WT, and by 40% in TK-/- (P<0.05, TK-/- vs WT). Ramiprilat also reduced infarct size by 29% in WT, but in TK-/- its effect was completely suppressed. Pretreatment of WT with a B2, but not a B1, kinin receptor antagonist reproduced the effects of TK deficiency. However, B2 receptor-deficient mice (B2-/-) unexpectedly responded to IPC or ramiprilat like WT mice. But pretreatment of the B2-/- mice with a B1 antagonist suppressed the cardioprotective effects of IPC and ramiprilat. In B2-/-, B1 receptor gene expression was constitutively high. In WT and TK-/- mice, both B2 and B1 mRNA levels increased several fold during IR, and even more during IPC+IR. Thus TK and the B2 receptor play a critical role in the cardioprotection afforded by two experimental maneuvers of potential clinical relevance, IPC and ACE inhibition, during ischemia.

Angiotensin-I-converting enzyme insertion/deletion polymorphism and high urinary albumin concentration in French Type 2 diabetes patients.

AIMS: Family-based studies suggest a genetic basis for nephropathy in Type 2 diabetes. The angiotensin-I-converting enzyme (ACE) gene is a candidate gene for Type 1 diabetes nephropathy. We assessed the association between high urinary albumin concentration and ACE insertion/deletion (I/D) polymorphism, in French Type 2 diabetes patients. METHODS: We studied 3139 micro/macroalbuminuric French patients recruited in the DIABHYCAR Study, an ACE inhibition trial in Type 2 diabetes patients with renal and cardiovascular outcomes. The main inclusion criteria were age >/= 50 years, urinary albumin concentration >/= 20 mg/l assessed centrally during two consecutive screening visits, and plasma creatinine concentration

Palmitoylation of the human bradykinin B2 receptor influences ligand efficacy.

To investigate the palmitoylation of the human bradykinin B2 receptor, we have mutated individually or simultaneously into glycine two potential acylation sites (cysteines 324 and 329) located in the carboxyl terminus of the receptor and evaluated the effects of these mutations by transfection in COS-7, CHO-K1, and HEK 293T. The wild-type receptor and the single mutants, but not the double mutant, incorporated [3H]palmitate, indicating that the receptor carboxyl tail can be palmitoylated at both sites. The mutants did not differ from the wild-type receptor for the kinetics of [3H]bradykinin binding, the basal and bradykinin-stimulated coupling to phospholipases C and A2, and agonist-induced phosphorylation. The nonpalmitoylated receptor had a 30% reduced capacity to internalize [3H]bradykinin. This indicates that palmitoylation does not influence the basal activity of the receptor and its agonist-driven activation. However, the mutants triggered phospholipid metabolism and MAP kinase activation in response to B2 receptor antagonists. Pseudopeptide and nonpeptide compounds that behaved as antagonists on the wild-type receptor became agonists on the nonpalmitoylated receptor and produced phospholipases C and A2 responses of 25-50% as compared to that of bradykinin. These results suggest that palmitoylation is required for the stabilization of the receptor-ligand complex in an uncoupled conformation.

Genetically increased angiotensin I-converting enzyme level and renal complications in the diabetic mouse.

Diabetic nephropathy is a major risk factor for end-stage renal disease and cardiovascular diseases and has a marked genetic component. A common variant (D allele) of the angiotensin I-converting enzyme (ACE) gene, determining higher enzyme levels, has been associated with diabetic nephropathy. To address causality underlying this association, we induced diabetes in mice having one, two, or three copies of the gene, normal blood pressure, and an enzyme level range (65-162% of wild type) comparable to that seen in humans. Twelve weeks later, the three-copy diabetic mice had increased blood pressures and overt proteinuria. Proteinuria was correlated to plasma ACE level in the three-copy diabetic mice. Thus, a modest genetic increase in ACE levels is sufficient to cause nephropathy in diabetic mice.

Determination of bradykinin B2 receptor in vivo phosphorylation sites and their role in receptor function.

Reversible phosphorylation plays important roles in G protein-coupled receptor signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. Using metabolic 32P labeling and phosphopeptide sequencing we provide a complete phosphorylation map of the human bradykinin B2 receptor in its native cellular environment. We identified three serine residues, Ser(339), Ser(346), and Ser(348), at the C-terminal tail as principal phosphorylation sites. Constitutive phosphorylation occurs at Ser(348), while ligand-induced phosphorylation is found at Ser(339) and Ser(346)/Ser(348) that could be executed by several G protein-coupled receptor kinases. In addition, we found a protein kinase C-dependent phosphorylation of Ser(346) that was mutually exclusive with the basal phosphorylation at Ser(348) and therefore may be implicated in differential regulation of B2 receptor activation. Functional analysis of receptor mutants revealed that a low phosphorylation stoichiometry is sufficient to initiate receptor sequestration while a clustered phosphorylation around Ser(346) is necessary for desensitization of the B2 receptor-induced phospholipase C activation. This was further supported by the specifically reduced Ser(346)/Ser(348) phosphorylation observed upon stimulation with a nondesensitizing B2 receptor agonist. The differential usage of clustered phosphoacceptor sites points to distinct roles of multiple kinases in controlling G protein-coupled receptor function.

Decreased flow-dependent dilation in carotid arteries of tissue kallikrein-knockout mice.

- Flow-dependent dilation is a fundamental mechanism by which large arteries ensure appropriate blood supply to tissues. We investigated whether or not the vascular kallikrein-kinin system, especially tissue kallikrein (TK), contributes to flow-dependent dilation by comparing wild-type and TK-knockout mice in which the presence or absence of TK expression was verified. We examined in vitro changes in the outer diameter of perfused carotid arteries from TK(+/+) and TK(-/-) mice. In both groups, exogenous bradykinin caused a similar dilation that was abolished by the B(2) receptor antagonist HOE-140, as well as by the NO synthase inhibitor N:(omega)-nitro-L-arginine methyl ester. However, purified kininogen dilated only TK(+/+) arteries, demonstrating the essential role of TK in the vascular formation of kinins. In TK(+/+) arteries, increasing intraluminal flow caused a larger endothelium-dependent dilation than that seen in TK(-/-). In both strains the flow response was mediated by NO and by endothelium-derived hyperpolarizing factor, whereas in TK(-/-) vasoconstrictor prostanoids participated as well. HOE-140 impaired flow-dependent dilation in TK(+/+) arteries while showing no effect in TK(-/-). This compound reduced the flow response in TK(+/+) arteries to a level similar to that in TK(-/-). After NO synthase inhibition, HOE-140 no longer affected the response of TK(+/+). Impaired flow-dependent dilation was also observed in arteries from knockout mice lacking bradykinin B(2) receptors as compared with wild-type animals. This study demonstrates the active contribution of the vascular kallikrein-kinin system to one-third of the flow-dependent dilation response via activation of B(2) receptors coupled to endothelial NO release.

Bradykinin attenuates the [Ca(2+)](i) response to angiotensin II of renal juxtamedullary efferent arterioles via an EDHF.

1. Bradykinin (BK) effect on the [Ca(2+)](i) response to 1 nM angiotensin II was examined in muscular juxtamedullary efferent arterioles (EA) of rat kidney. 2. BK (10 nM) applied during the angiotensin II-stimulated [Ca(2+)](i) increase, induced a [Ca(2+)](i) drop (73+/-2%). This drop was prevented by de-endothelialization and suppressed by HOE 140, a B2 receptor antagonist. It was neither affected by L-NAME or indomethacin, nor mimicked by sodium nitroprusside, 8-bromo-cyclic GMP or PGI(2). The BK effect did not occur when the [Ca(2+)](i) increase was caused by 100 mM KCl-induced membrane depolarization and was abolished by 0.1 microM charybdotoxin, a K(+) channel blocker. 3. Although proadifen prevented the BK-caused [Ca(2+)](i) fall, more selective cytochrome P450 inhibitors, 17-octadecynoic acid (50 microM) and 7-ethoxyresorufin (10 microM) were without effect. 4. Increasing extracellular potassium from 5 to 15 mM during angiotensin II stimulation caused a [Ca(2+)](i) decrease (26+/-4%) smaller than BK which was charybdotoxin-insensitive. Inhibition of inward rectifying K(+) channels by 30 microM BaCl(2) and/or of Na(+)/K(+) ATPase by 1 mM ouabain abolished the [Ca(2+)](i) decrease elicited by potassium but not by BK. 5. A voltage-operated calcium channel blocker, nifedipine (1 microM) did not prevent the BK effect but reduced the [Ca(2+)](i) drop. 6. These results indicate that the BK-induced [Ca(2+)](i) decrease in angiotensin II-stimulated muscular EA is mediated by an EDHF which activates charybdotoxin-sensitive K(+) channels. In these vessels, EDHF seems to be neither a cytochrome P450-derived arachidonic acid metabolite nor K(+) itself. The closure of voltage-operated calcium channels is not the only cellular mechanism involved in this EDHF-mediated [Ca(2+)](i) decrease.

Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice.

Tissue kallikrein is a serine protease thought to be involved in the generation of bioactive peptide kinins in many organs like the kidneys, colon, salivary glands, pancreas, and blood vessels. Low renal synthesis and urinary excretion of tissue kallikrein have been repeatedly linked to hypertension in animals and humans, but the exact role of the protease in cardiovascular function has not been established largely because of the lack of specific inhibitors. This study demonstrates that mice lacking tissue kallikrein are unable to generate significant levels of kinins in most tissues and develop cardiovascular abnormalities early in adulthood despite normal blood pressure. The heart exhibits septum and posterior wall thinning and a tendency to dilatation resulting in reduced left ventricular mass. Cardiac function estimated in vivo and in vitro is decreased both under basal conditions and in response to beta-adrenergic stimulation. Furthermore, flow-induced vasodilatation is impaired in isolated perfused carotid arteries, which express, like the heart, low levels of the protease. These data show that tissue kallikrein is the main kinin-generating enzyme in vivo and that a functional kallikrein-kinin system is necessary for normal cardiac and arterial function in the mouse. They suggest that the kallikrein-kinin system could be involved in the development or progression of cardiovascular diseases.

Microalbuminuria and markers of the atherosclerotic process: the D.E. S.I.R. study.

The relationship between microalbuminuria and tissue-type plasminogen activator antigen (tPA-ag) and fibrinogen was evaluated in non-diabetic subjects. Subjects were participants of the D.E.S.I. R. (Data from an Epidemiological Study on the Insulin Resistance syndrome) Study. Analyses were carried out on 2248 women and 2402 men for fibrinogen and on 272 women and 284 men for tPA-ag. Microalbuminuria was defined as urinary albumin concentration greater than 20 mg/l. Men with microalbuminuria had a 6% higher fibrinogen concentration than those without (3.07 g/l (95% confidence interval: 2.99,3.15) vs. 2.89 g/l (2.87,2.91), adjusted for age and smoking). This relationship existed in hypertensive as well as non-hypertensive subjects. The association between microalbuminuria and tPA-ag existed only in hypertensive men, those with microalbuminuria having a 21% higher tPA-ag than those without (4.39 ng/ml (3.70,5.08) vs. 3.63 ng/ml (3.32,3.94), adjusted for age and smoking). Adjustment for other risk markers for cardiovascular disease did not change the results. There was no relationship between microalbuminuria and these haemostatic factors in women. The results of this study suggest that in non-diabetic men, microalbuminuria is associated with fibrinogen, but with tPA-ag only when concomitant with hypertension.

[The renin-angiotensin system and progression of kidney disease]. / Système rénine-angiotensine et progression des maladies rénales.

CONSTRICTIVE ACTIVITY OF THE RAS AND DEVELOPMENT OF RENAL DISEASES: Several studies have suggested that the constitutive level of activity of the renin-angiotensin system (RAS), and especially of angiotensin converting enzyme (ACE) which plays an important role in the kidney in determining intrarenal angiotensin and kinin concentrations, is genetically determined and linked to the risk of developing several vascular diseases, including diabetic nephropathy, and to the risk of renal function deterioration in glomerular diseases of several origins. INHIBITION OF THE RAS AND PROGRESSION OF RENAL DISEASES: Large controlled clinical trials have shown over the past years that inhibition of ACE has a beneficial effect and protects against degradation of renal function in type I diabetes with microalbuminuria and also in renal diseases of several origins. PATHOPHYSIOLOGY: All these observations taken together suggest that activation of the RAS, which is necessary in certain circumstances to maintain glomerular filtration and tissue perfusion, can have a long-term deleterious effect on the heart, vessels, and kidneys, especially through glomerular hypertension which can lead to glomerulosclerosis. They support the use of ACE inhibitors, within the indications deduced from analyses of the large clinical trials, for protecting renal function in kidney diseases.

Somatic isoform of angiotensin I-converting enzyme in the pathology of testicular germ cell tumors.

Retained fetal expression of angiotensin I-converting enzyme (ACE, CD143) has recently been shown in intratubular germ cell neoplasms (IGCN) and invasive germ cell tumors (GCT), suggesting the somatic isoform (sACE) as a characteristic component of neoplastic germ cells. We analyzed the distribution of sACE in 159 testicular GCT, including 87 IGCN. sACE protein was determined by immunohistochemistry (MAb CG2) on routinely formalin-fixed and paraffin-embedded tissue sections, supplemented by mRNA expression analysis using in situ hybridization. These data were compared with those obtained by germ cell/placental alkaline phosphatases (PIAP; MAbs PL8-F6 and 8A9) employing an uniform score system for the evaluation of immunoreactivity (IRS; possible values from 0 to 12). Expression of sACE and PIAP was found in all 87 analyzed IGCN (IRS > 4, median IRS of 12). Heterogeneous staining patterns were not related to the type of adjacent GCT but correlated with low expression in adjacent seminomas (P =.032 for sACE; P =.005 for PIAP). Both sACE and PIAP often showed a decreased and more heterogeneous but still moderate expression in 91 classic seminomas (median IRS of 8) and were completely absent in tumor cells of spermatocytic seminomas. Despite all similarities, we found sACE and PIAP differently regulated during GCT progression. This was documented by a well-preserved expression of either sACE or PIAP or both in all classic seminomas, low PIAP immunoreactivity in metastasis of seminomas, and completely diverging expression patterns in nonseminomatous GCT. Our findings underline the close molecular relationship between IGCN and seminoma, and suggest sACE as an appropriate marker for seminomatous differentiated tumors. HUM PATHOL 31:1466-1476.

Dissimilar mechanisms of Ca(2+) response to bradykinin in different types of juxtamedullary glomerular arterioles.

Bradykinin (BK)-induced changes in intracellular calcium level ([Ca(2+)](i)) were studied on fura 2-loaded afferent (AA) and efferent glomerular arterioles (EA) microdissected from juxtamedullary renal cortex. A distinction was made between thin and muscular EA. In AA and both types of EA, BK increased [Ca(2+)](i) through activation of B(2) receptors located only on the endothelium. The responses were not affected by nifedipine (10(-6) M) and were smaller in a Ca(2+)-free medium, providing evidence that BK opens voltage-independent Ca(2+) channels and mobilizes intracellular Ca(2+). Thin EA differed from AA and muscular EA by a lower sensitivity to BK (EC(50) = 6.95 +/- 3.81 vs. 0.21 +/- 0.08 and 0.18 +/- 0.13 nM, respectively; P < 0.05), a higher maximal response (89 +/- 5 vs. 57 +/- 5 and 44 +/- 7 nM; P < 0.001), and a spontaneous return to basal Ca(2+) level, even in the presence of BK. Genistein (10(-4) M) and herbimycin A (25 x 10(-6) M), specific inhibitors of tyrosine kinases, inhibited the [Ca(2+)](i) responses exclusively in AA. Genistein reduced the peak and plateau phases of responses by 69 +/- 9 and 82 +/- 6%, respectively, in a medium with Ca(2+) and the peak by 48 +/- 9% in a Ca(2+)-free medium. Similar reductions were observed with herbimycin A. These results show that dissimilar signal transduction pathways are involved in BK effects on juxtamedullary arterioles and that a tyrosine kinase activity could participate in the regulation of BK effect on AA but not on EA.

Angiotensin-converting enzyme in non-neoplastic kidney diseases.

BACKGROUND: The angiotensin I-converting enzyme (ACE, CD143, kininase II) plays a critical role in controlling the level of vasoactive peptides such as angiotensins and kinins in the local circulations and tissue interstitium. Because recent work has documented a vessel-, organ-, and species-specific pattern of endothelial ACE expression in the vascular system, we have analyzed whether or not changes of this pattern occur in vessels, tubules, and interstitium of the human kidney that is affected by different non-neoplastic diseases. METHODS: Using a set of well-characterized monoclonal antibodies (mAbs), ACE was assessed on renal tissue of 135 patients by immunohistochemistry, including an additional analysis at the ultrastructural level. A semiquantitative evaluation allowed the estimation and comparison of ACE content in different renal compartments. These data were compared with several clinical findings, diagnosis, therapeutic modalities, and histological features. RESULTS: In contrast to the normal human kidney, where ACE is abundant in the brush border of the proximal tubule but is usually absent in endothelial cells of any vessel type, an endothelial neoexpression of ACE was observed in different diseases. In general, this neoexpression was associated with histological sites of interstitial fibrosis and showed some selectivity for glomerular endothelial cells in diabetes mellitus and chronic arterial hypertension. There was also a loss of epithelial ACE in the proximal tubule in certain pathological conditions, for example, in chronic fibroplastic processes, acute pyelonephritis, and different stages of acute renal failure. CONCLUSIONS: Neoexpression of ACE by renal endothelial cells, as well as changes of the tubular ACE content, is a common finding in diseased human kidneys. As associated with certain tissue sites, clinical and/or morphological features, these changes may be involved in parenchymal remodeling and renal pathophysiology.

Decreased expression of angiotensin-converting enzyme in the airway epithelium of asthmatic subjects is associated with eosinophil inflammation.

BACKGROUND: Angiotensin-converting enzyme (ACE) is a peptidase involved in the metabolism of several bioactive peptides. It may be involved in the airway inflammation and hyperresponsiveness that occur in asthma. OBJECTIVE: We studied the expression of ACE in the airway mucosa of normal and asthmatic subjects and assessed the relationship between ACE expression and airway inflammation and bronchial hyperresponsiveness in asthma. METHODS: We used immunohistochemistry to study the ACE expression and airway inflammation in bronchial biopsy samples obtained by fiberoptic bronchoscopy from 20 asthmatic subjects randomly assigned to groups treated with (n = 10) or without inhaled corticosteroids (n = 10) and from normal subjects (n = 10). Airway response to methacholine and bradykinin was also determined for all subjects. RESULTS: In normal subjects ACE was present in the surface epithelium, the endothelial cells of the lamina propria, and the submucosal glands, in which ACE was found in seromucous cells and in secreted mucus. ACE was not detected in smooth muscle cells and in most of the endothelial cells of the vascular network surrounding the glands. ACE was absent or present at lower levels in the surface epithelium of asthmatic subjects not treated with corticosteroids compared with those treated with corticosteroids and the control group. In asthmatic subjects low levels of ACE in the epithelium were associated with larger numbers of eosinophils in the epithelium and lamina propria. There was no relationship between ACE levels in the airway mucosa and airway responsiveness to methacholine and bradykinin. CONCLUSION: ACE expression is decreased in the epithelium of asthmatic patients and is associated with increased eosinophil inflammation.

Activation of mitogen-activated protein kinase by the bradykinin B2 receptor is independent of receptor phosphorylation and phosphorylation-triggered internalization.

Recent evidence suggests that serine/threonine phosphorylation and internalization of beta2-adrenergic receptors play critical roles in signalling to the mitogen-activated protein kinase cascade. To investigate whether this represents a general mechanism employed by G protein-coupled receptors, we studied the requirement of these processes in the activation of mitogen-activated protein kinase by G alpha(q)-coupled bradykinin B2 receptors. Mutant B2 receptors impaired in receptor phosphorylation and internalization are fully capable to activate mitogen-activated protein kinase. Bradykinin-induced long-term effects on mitogenic signalling monitored by measuring the transcriptional activity of Elk1 were identical in cells expressing the wild-type or mutant B2 receptors. Therefore, G protein-coupled bradykinin receptors activate the mitogen-activated protein kinase pathway independently of receptor phosphorylation and internalization.