Bradykinin Signaling Research Articles

Bradykinin acutely inhibits activity of the epithelial Na+channel in mammalian aldosterone-sensitive distal nephron

Activation of the renal kallikrein-kinin system results in natriuresis and diuresis, suggesting its possible role in renal tubular sodium transport regulation. Here, we used patch-clamp electrophysiology to directly assess the effects of bradykinin (BK) on the epithelial Na(+) channel (ENaC) activity in freshly isolated split-opened murine aldosterone-sensitive distal nephrons (ASDNs). BK acutely inhibits ENaC activity by reducing channel open probability (P(o)) in a dose-dependent and reversible manner. Inhibition of B2 receptors with icatibant (HOE-140) abolished BK actions on ENaC. In contrast, activation of B1 receptors with the selective agonist Lys-des-Arg(9)-BK failed to reproduce BK actions on ENaC. This is consistent with B2 receptors playing a critical role in mediating BK signaling to ENaC. BK has little effect on ENaC P(o) when G(q/11) was inhibited with Gp antagonist 2A. Moreover, inhibition of phospholipase C (PLC) with U73122, but not saturation of cellular cAMP levels with the membrane-permeable nonhydrolysable cAMP analog 8-cpt-cAMP, prevents BK actions on ENaC activity. This argues that BK stimulates B2 receptors with subsequent activation of G(q/11)-PLC signaling cascade to acutely inhibit ENaC activity. Activation of BK signaling acutely depletes apical PI(4,5)P(2) levels. However, inhibition of Ca(2+) pump SERCA of the endoplasmic reticulum with thapsigargin does not prevent BK signaling to ENaC. Furthermore, caffeine, while producing a similar rise in [Ca(2+)](i) as in response to BK stimulation, fails to recapitulate BK actions on ENaC. Therefore, we concluded that BK acutely inhibits ENaC P(o) in mammalian ASDN via stimulation of B2 receptors and following depletion of PI(4,5)P(2), but not increases in [Ca(2+)](i).

Mast Cells Increase Vascular Permeability by Heparin-Initiated Bradykinin Formation In Vivo

Activated mast cells trigger edema in allergic and inflammatory disease. We report a paracrine mechanism by which mast cell-released heparin increases vascular permeability invivo. Heparin activated the protease factor XII, which initiates bradykinin formation in plasma. Targeting factor XII or kinin B2 receptors abolished heparin-triggered leukocyte-endothelium adhesion and interfered with a mast cell-driven drop in blood pressure in rodents. Intravital laser scanning microscopy and tracer measurements showed heparin-driven fluid extravasation in mouse skin microvessels. Ablation of factor XII or kinin B2 receptors abolished heparin-induced skin edema and protected mice from allergen-activated mast cell-driven leakage. In contrast, heparin and activated mast cells induced excessive edema in mice deficient in the major inhibitor of factor XII, C1 esterase inhibitor. Allergen exposure triggered edema attacks in hereditary angioedema patients, lacking C1 esterase inhibitor. The data indicate that heparin-initiated bradykinin formation plays a fundamental role in mast cell-mediated diseases.

17β-Estradiol Rapidly Enhances Bradykinin Signaling in Primary Sensory Neurons In Vitro and In Vivo

Many studies have demonstrated that premenopausal women are at increased risk for various pain disorders. Pain-sensing neurons, termed "nociceptors," in the trigeminal ganglia (TG) and dorsal root ganglia (DRG) express receptors for inflammatory mediators and noxious physical stimuli and transmit signals for central processing of pain sensation. Estrogen receptors (ERs) are also expressed on nociceptors in the TG and DRG, and there is ample literature to suggest that activation of ERs can influence pain mechanisms. However, the mechanism for ER modulation of nociceptor activity is incompletely understood. The aim of this study was to characterize the effect of 17β-estradiol (17β-E(2)) on signaling of the inflammatory mediator bradykinin (BK) in primary cultures of rat sensory neurons and a behavioral model of thermal allodynia in rats. Here, we show that exposure to 17β-E(2) rapidly (within 15 min) enhanced responses to BK in vitro and in vivo. The 17β-E(2)-mediated enhancement of BK signaling was not blocked by the transcription inhibitor anisomycin and was mediated by a membrane-associated ER. The effect of 17β-E(2) to enhance BK responses required activation of β1-containing, RGD-binding integrins. These data show that 17β-E(2) rapidly enhances inflammatory mediator responses both in vitro and in vivo and suggest that 17β-E(2) acting at primary sensory pain neurons may participate in regulating the sensitivity of women to painful stimuli.

Loss of Bradykinin Signaling Does Not Accelerate the Development of Cardiac Dysfunction in Type 1 Diabetic Akita Mice

Bradykinin signaling has been proposed to play either protective or deleterious roles in the development of cardiac dysfunction in response to various pathological stimuli. To further define the role of bradykinin signaling in the diabetic heart, we examined cardiac function in mice with genetic ablation of both bradykinin B1 and B2 receptors (B1RB2R(-/-)) in the context of the Akita model of insulin-deficient type 1 diabetes (Ins2(Akita/+)). In 5-month-old diabetic and nondiabetic, wild-type and B1RB2R(-/-) mice, in vivo cardiac contractile function was determined by left-ventricular (LV) catheterization and echocardiography. Reactive oxygen species levels were measured by 2'-7'-dichlorofluorescein diacetate fluorescence. Mitochondrial function and ATP synthesis were determined in saponin-permeabilized cardiac fibers. LV systolic pressure and the peak rate of LV pressure rise and decline were decreased with diabetes but did not deteriorate further with loss of bradykinin signaling. Wall thinning and reduced ejection fractions in Akita mouse hearts were partially attenuated by B1RB2R deficiency, although other parameters of LV function were unaffected. Loss of bradykinin signaling did not increase fibrosis in Ins2(Akita/+) diabetic mouse hearts. Mitochondrial dysfunction was not exacerbated by B1RB2R deficiency, nor was there any additional increase in tissue levels of reactive oxygen species. Thus, loss of bradykinin B2 receptor signaling does not abrogate the previously reported beneficial effect of inhibition of B1 receptor signaling. In conclusion, complete loss of bradykinin expression does not worsen cardiac function or increase myocardial fibrosis in diabetes.

Multiple Cross Talk between Angiotensin II, Bradykinin, and Insulin Signaling in the Cortical Thick Ascending Limb of Rat Kidney

Cortical thick ascending limb (CTAL) naturally expresses the angiotensin II (AngII) receptor type 1A (AT(1)-R), the bradykinin (BK) receptor type 2 (B(2)-R), and the insulin receptor. This segment is made of a single morphologically distinct cell type. AngII and BK are involved in same transduction pathways but differ markedly in their physiological actions on Na(+) transport. Besides, the insulin signaling intersects with those of AngII and BK at multiple levels and especially by stimulation on Na(+) reabsorption. Thus, the CTAL is a biologically suitable model to study the cross talk between G protein-coupled receptors or G protein-coupled receptors and receptor tyrosine kinase. In this work, the cross talks between AngII, BK, and insulin signaling are studied in rat CTAL by measuring changes in [Ca(2+)](i). We show that BK exerts negative modulatory effects on AngII-induced [Ca(2+)](i) responses dependent on tyrosine kinase and MAPK pathways. Moreover, in the presence of BK, AngII-induced Na(+) transport is suppressed. These effects suggest an interaction between AT(1)-R and B(2)-R. We show a positive interaction between the insulin receptor and the AT(1)-R through a protein kinase A-dependent mechanism that involves MAPK cascade, leading to the stimulation of the Ca(2+) influx induced by AngII. The presence of such interactions brings additional arguments for a complex and fine regulation of CTAL functions and puts forward the potentially beneficial effect of BK across this segment, in case of hyperinsulinemia or insulin resistance, by its negative feedback on AngII actions.

Differential effects of a bradykinin receptor 2 (B2R) and bradykinin receptor 1 (B1R) in vasculogenesis of human endothelial progenitor cells (EPCs) compared to human umbilical vein endothelial cells (HUVECs)

Bradykinin (BK), a potent activator of endothelial cells, is known to induce vasodilation and vascular permeability. BK signaling is initiated by binding to G protein‐coupled receptors B2R and/or B1R. B2R is constitutively expressed in endothelial cells while B1R is upregulated by inflammatory stimuli.We now report that EPCs undergo differentiation (tube formation) in a 3D collagen gel. Pre‐incubation of EPCs with a B2R selective antagonist (HOE‐140) reduced differentiation (p<0.001). We found that EPCs stimulated with the selective B2R agonist FR190997 (50 nM) enhanced migration, a response abrogated by HOE‐140 (p<0.05). We conclude that EPCs utilize B2R for vasculogenesis and migration.In contrast, HUVECs did not display a significant reduction in vasculogenesis upon addition of exogenous BK. After pre‐incubation with [des‐Arg10]‐HOE 140, (B1R selective antagonist), BK‐stimulated HUVECs displayed a reduction in tube formation (p<0.001), suggesting that HUVECs utilize B1R for vasculogenesis. In the presence of the B1R antagonist, BK‐stimulated HUVEC migration was inhibited (p<0.01). In addition, proliferation of HUVECs remained unchanged upon stimulation with BK. Upon exposure to the B1R antagonist, proliferation was unchanged.In conclusion, in BK‐stimulated EPCs, B2R mediates differentiation and migration. In contrast, in BK‐stimulated HUVECs, the B1R antagonist selectively inhibits vasculogenesis and migration but not proliferation.

Characterization of estradiol‐mediated, rapid enhancement of bradykinin signaling in sensory neurons

Premenopausal women are at increased risk for numerous pain disorders. Although this sex difference is far from understood, many studies have shown the importance of estrogen in the enhancement of nociceptive signaling. Estrogen receptors (ERs) are present on sensory neurons, and we have previously shown that 17β‐estradiol (E2) rapidly enhances signaling of the inflammatory mediator, bradykinin (BK), in vitro and in vivo. The aim of this study was to further characterize and delineate mechanisms involved in the enhancement of BK signaling by E2. Pretreatment (15 min) with the ERα‐selective agonist, PPT, significantly enhanced BK‐induced PLC activity in cultures of primary sensory neurons and thermal allodynia. The ERβ‐selective agonist, DPN, and the GPR30‐selective agonist, G‐1, had no effect. Enhancement of BK signaling by E2 was seen with pretreatment times ranging from 0 (co‐injection) to 60 min, and persisted for up to 60 min following washout of E2. The effect of E2 was not blocked by the transcription inhibitor anisomycin and was mimicked by E2 conjugated to BSA (membrane impermeable). These data suggest that ERα mediates the rapid onset effect of E2 to enhance BK signaling and that the effect of E2 is non‐genomic.

Physiological regulation of ENaC activity in the distal nephron by local bradykinin signaling

ENaC activity in the aldosterone‐sensitive distal nephron sets sodium plasma levels to control blood pressure in response to negative feedback regulation by the renin‐angiotensin‐aldosterone system. In complement to this, paracrine factors intrinsic to the collecting duct, including bradykinin (BK), may also have an important role in controlling ENaC‐mediated sodium reabsorption. Patch clamp electrophysiology on freshly isolated murine collecting ducts (CD) was used to test whether ENaC is a proximal target for bradykinin signaling. Bradykinin acutely and reversibly decreases ENaC open probability via B2 receptors: Inhibition of B2 receptors with HOE‐140 prevents BK‐mediated decreases in ENaC Po. Disruption of this paracrine signaling in B2 −/− mice abolishes BK actions on ENaC and results in elevated resting ENaC Po. We propose that sodium retention due to increased basal ENaC activity contributes, in part, to the salt‐sensitive hypertension in B2 −/− mice. B2 receptors couple to both Gq/11‐PLC, and Gαi‐mediating decreases in adenylyl cyclase activity. Inhibition of PLC with U73122 but not saturation of intracellular [cAMP] with 8‐cpt‐cAMP disrupts regulation of ENaC by BK. Rapid decreases in apical PI(4,5)P2 levels in response to BK stimulation parallel decreases in ENaC activity. Inhibition of Ca2+ release from the ER does not prevent decreases in ENaC activity in response to bradykinin. This is consistent with decreases in PI(4,5)P2 levels but not subsequent increases in intracellular Ca2+ account for BK‐mediated decreases in ENaC activity in response to stimulation of B2‐Gq/11‐PLC.

Bradykinin B2 receptor activates extracellular signal regulated kinase in renal cells from a mouse model of polycystic kidney disease

The vasoactive peptide bradykinin (BK) is a mitogen for normal renal cells. We explored the effects of BK in orpk cilia (+) and orpk cilia (−) collecting duct cell lines, which were derived from a mouse model of polycystic kidney disease (PKD). mRNAs for BK B1 and BK B2 receptors were demonstrated by RT‐PCR in both cell lines. To study BK signaling pathways, we employed fluorescent measurements of intracellular Ca2+, a Cytosensor microphysiometer to measure changes in extracellular pH as a reflection of activation of the Na+/H+ exchanger (NHE), and Western blotting to assess extracellular signal regulated kinase (ERK) activation.Exposure to 100 nM BK resulted in rapid elevation of intracellular Ca2+, caused a ≥ 20% increase in NHE activity, and a ≥ 200% increase in ERK phosphorylation. While BK‐induced Ca2+ signals and NHE activation were similar in orpk cilia (+) and orpk cilia (−) cell lines, BK‐induced ERK phosphorylation was less pronounced in orpk cilia (−) cells. The BK B2 receptor antagonist and an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor blocked BK‐induced ERK phosphorylation suggesting a role for the B2 receptor‐induced EGFR transactivation in the mitogenic ERK pathway. We speculate that cilium is required for effective transactivation of EGFR and ERK activation by BK B2 receptors in PKD cells. These studies are important because of proposed role of ERK pathway in pathophysiology of PKD.

Novel bradykinin signaling in adult rat cardiac myocytes through activation of p21-activated kinase

Although bradykinin (BK) is known to exert effects on the myocardium, its intracellular signaling pathways remain poorly understood. Experiments in other cell types indicated that p21-activated kinase-1 (Pak1), a Ser/Thr kinase downstream of small monomeric G proteins, is activated by BK. We previously reported that the expression of active Pak1 in adult cardiac myocytes induced activation of protein phosphatase 2A and dephosphorylation of myofilament proteins (Ke et al. Circ Res 94: 194–200, 2004). In experiments reported here, we tested the hypothesis that BK signals altered protein phosphorylation in adult rat cardiac myocytes through the activation and translocation of Pak1. Treatment of myocytes with BK resulted in the activation of Pak1 as demonstrated by increased autophosphorylation at Thr423 and a diminished striated localization, which is present in the basal state. BK induced dephosphorylation of both cardiac troponin I and phospholamban. Treatment of isolated myocytes with BK also blunted the effect of isoproterenol to enhance peak Ca2+ and relaxation of Ca2+ transients. Protein phosphatase 2A was demonstrated to associate with both Pak 1 and phospholamban. Our studies indicate a novel signaling mechanism for BK in adult rat cardiac myocytes and support our hypothesis that Pak 1 is a significant regulator of phosphatase activity in the heart.

Surface Assisted Laser Desorption/Ionization on Two-Layered Amorphous Silicon Coated Hybrid Nanostructures

Matrix-free laser desorption/ionization was studied on two-layered sample plates consisting of a substrate and a thin film coating. The effect of the substrate material was studied by depositing thin films of amorphous silicon on top of silicon, silica, polymeric photoresist SU-8, and an inorganic-organic hybrid. Des-arg(9)-bradykinin signal intensity was used to evaluate the sample plates. Silica and hybrid substrates were found to give superior signals compared with silicon and SU-8 because of thermal insulation and compatibility with amorphous silicon deposition process. The effect of surface topography was studied by growing amorphous silicon on hybrid micro- and nanostructures, as well as planar hybrid. Compared with planar sample plates, micro- and nanostructures gave weaker and stronger signals, respectively. Different coating materials were tested by growing different thin film coatings on the same substrate. Good signals were obtained from titania and amorphous silicon coated sample plates, but not from alumina coated, silicon nitride coated, or uncoated sample plates. Overall, the strongest signals were obtained from oxygen plasma treated and amorphous silicon coated inorganic-organic hybrid, which was tested for peptide-, protein-, and drug molecule analysis. Peptides and drugs were analyzed with little interference at low masses, subfemtomole detection levels were achieved for des-arg(9)-bradykinin, and the sample plates were also suitable for ionization of small proteins.

Abnormal bradykinin signalling in fibroblasts deficient in the PIP2 5‐phosphatase, ocrl1

The oculocerebrorenal syndrome of Lowe (Lowe syndrome) is an X-linked disorder of phosphatidylinositol metabolism characterized by congenital cataracts, renal proximal tubulopathy and neurological deficits. The disorder is due to the deficiency of the phosphatidylinositol 4,5-bisphosphate (PIP(2)) 5-phosphatase, ocrl1. PIP(2) is critical for numerous cellular processes, including cell signalling, actin reorganization and protein trafficking, and is chronically elevated in patients with Lowe syndrome. The elevation of PIP(2) cells of patients with Lowe syndrome provides the unique opportunity to investigate the roles of this phospholipid in fundamental cellular processes. We previously demonstrated that ocrl1 deficiency causes alterations in the actin cytoskeleton. Since actin remodelling is strongly activated by [Ca(+2)], which increases in response to IP(3) production, we hypothesized that altered calcium signalling might contribute to the observed abnormalities in actin organization. Here we report a specific increase in bradykinin-induced Ca(+2) mobilization in Lowe fibroblasts. We show that the abnormal bradykinin signalling occurs in spite of normal total cellular receptor content. These data point to a novel role for ocrl1 in agonist-induced calcium release.

Regulation of the novel Mg2+ transporter transient receptor potential melastatin 7 (TRPM7) cation channel by bradykinin in vascular smooth muscle cells

Transient receptor potential melastatin 7 (TRPM7) channels have been identified in the vasculature. However, their regulation and function remain unclear. Here, we tested the hypothesis that bradykinin and its second messenger cAMP upregulate TRPM7, which stimulates activation of annexin-1 (TRPM7 substrate) and increases transmembrane Mg2+ transport and vascular smooth muscle cell (VSMC) migration. Human and rat VSMCs were studied. TRPM7 phosphorylation was assessed by immunoprecipitation:immunoblotting using antiphospho-serine/threonine and anti-TRPM7 antibodies. [Mg2+]i was measured by mag-fura-2. TRPM7 was downregulated by small interfering RNA and 2-aminoethoxydiphenyl borate. Annexin-1 activity was assessed by cytosol-to-membrane translocation. Cell migration and invasion, functional responses to bradykinin, were assessed in transwell chambers. Bradykinin increased expression of TRPM7 and annexin-1. TRPM7 was rapidly (5 min) phosphorylated on serine/threonine but not on tyrosine residues by bradykinin. [Mg2+]i was increased in bradykinin-stimulated cells (0.53 versus 0.68 mmol/l, basal versus bradykinin, P < 0.01). Annexin-1 activation was increased by bradykinin and inhibited by 2-aminoethoxydiphenyl borate. Although Hoe 140 (B2 receptor antagonist), U-73122 (phospholipase C inhibitor), 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (c-Src inhibitor) and chelerythrine (protein kinase C inhibitor) blocked bradykinin actions, dibutyryl-c-AMP was without effect. In small interfering RNA-transfected and in 2-aminoethoxydiphenyl borate-treated cells, bradykinin-induced Mg2+ influx and VSMC migration were reduced. Our results demonstrate that bradykinin regulates TRPM7 and its downstream target annexin-1 through phospholipase C-dependent, protein kinase C-dependent and c-Src-dependent and cAMP-independent pathways; effects are mediated through bradykinin type 2 receptor; and bradykinin regulates VSMC [Mg2+]i and migration through TRPM7. These data identify TRPM7/annexin-1/Mg2+ as a novel pathway in bradykinin signaling.

Damaging effects of intense repetitive treadmill running on murine intestinal musculature

Several gastrointestinal symptoms associated with prolonged intense exercise (IE) have been reported, although the mechanisms underlying its effects on the intestine remain poorly understood. The aim of the present study was to investigate whether IE may induce oxidative stress in the intestine, as well as its possible relationship with intestinal signaling impairments, leading to contractile disturbances. C57BL/6 mice were submitted to 4 days (EX.4D) and 10 days (EX.10D) of IE. The daily exercise session consisted of a running session until exhaustion, with the treadmill speed set at 85% of each animal's maximum velocity. The decrease in exhaustion time was exponential, and the reduction in the maximum velocity, as assessed by an incremental test, was higher in EX.4D than in EX.10D animals. The ileum mucosa layer was partially destroyed after 4 days of IE, where 37% and 11% muscle layer atrophies were observed in EX.4D and EX.10D animals, respectively. Ileum contractility was significantly impaired in the EX.4D animal group, with reduced efficacy for carbachol, bradykinin, and KCl signaling associated with a decrease in lipid peroxidation and with no alteration of protein oxidation. Intestinal myocytes from EX.10D animals displayed areas containing structurally disorganized mitochondria, which were associated with increased levels of protein oxidation, without alteration of contractility, except for a reduction in the potency of bradykinin signaling. Finally, no clear relationship between ileum contractility and oxidative stress was shown. Together, these results argue in favor of significant functional, biochemical, and morphological disturbances caused by exercise, thus demonstrating that intestinal tissue is very sensitive to exercise.

Expression of endogenous nuclear bradykinin B2 receptors mediating signaling in immediate early gene activation

Bradykinin (BK) represents a pro-inflammatory mediator that partakes in many inflammatory diseases. The mechanism of action of BK is thought to be primarily mediated by specific cell surface membrane B2 receptors (B2Rs). Some evidence has suggested, however, the existence of an intracellular/nuclear B2R population. Whether these receptors are functional and contribute to BK signaling remains to be determined. In this study, by mean of Western blotting, 3D-confocal microscopy, receptor autoradiography and radioligand binding analysis, we showed that plasma membrane and highly purified nuclei from isolated rat hepatocytes contain specific B2R that bind BK. The results depicting B2R nuclear expression in isolated nuclear organelles were reproduced in situ on hepatic sections by immunogold labeling and transmission electron microscopy. Functional tests on single nuclei, by means of confocal microscopy and the calcium-sensitive probe fluo-4AM, showed that BK induces concentration-dependent transitory mobilization of nucleoplasmic calcium; these responses were blocked by B2R antagonist HOE 140, not by the B1R antagonist R954 and, were also found in wild-type C57/Bl6 mice, but not in B2R-KO mice. In isolated nuclei, BK elicited activation/phosphorylation of Akt, acetylation of histone H3 and ensuing pro-inflammatory iNOS gene induction as determined by Western blot and RT-PCR. ChIP assay confirmed binding of acetylated-histone H3 complexes, but not B2R, to promoter region of iNOS gene suggesting that B2R-mediated gene expression is bridged with accessory downstream effectors. This study discloses a previously undescribed mechanism in BK-induced transcriptional events, via intracrine B2R-mediated signaling, occurring in rat autologous hepatic cells.

Acute angiotensin-converting enzyme inhibition evokes bradykinin-induced sympathetic activation in diabetic rats

We have previously shown that acute intravenous injection of the angiotensin-converting enzyme (ACE) inhibitor enalapril in diabetic rats evokes a baroreflex-independent sympathoexcitatory effect that does not occur with angiotensin receptor blockade alone. As ACE inhibition also blocks bradykinin degradation, we sought to determine whether bradykinin mediated this effect. Experiments were performed in conscious male Sprague-Dawley rats, chronically instrumented to measure mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA), 2 wk after streptozotocin (55 mg/kg iv, diabetic, n = 11) or citrate vehicle (normal, n = 10). Enalapril (2.5 mg/kg iv) decreased MAP in normal rats (-15 +/- 3 mmHg), while a smaller response (-4 +/- 1 mmHg) occurred in diabetic rats. Despite these different depressor responses to enalapril, HR (+44 +/- 8 vs. +26 +/- 7 bpm) and RSNA (+90 +/- 21 vs +71 +/- 8% baseline) increased similarly between the groups (P > or = 0.22 for both). Pretreatment with the bradykinin B2 receptor antagonist Hoe 140 (10 microg/kg bolus followed by 0.8.mug(-1)kg.min(-1) infusion) attenuated the decrease in MAP observed with enalapril in normal rats but had no effect in diabetic rats. Moreover, the normal group had smaller HR and RSNA responses (HR: +13 +/- 8 bpm; RSNA: +32 +/- 13% baseline) that were abolished in the diabetic group (HR: -4 +/- 5 bpm; RSNA: -5 +/- 9% baseline; P < 0.05 vs. preenalapril values). Additionally, bradykinin (20 microg/kg iv) evoked a larger, more prolonged sympathoexcitatory effect in diabetic compared with normal rats that was further potentiated after treatment with enalapril. We conclude that enhanced bradykinin signaling mediates the baroreflex-independent sympathoexcitatory effect of enalapril in diabetic rats.

Comparison of angiotensin converting enzyme inhibition and angiotensin II receptor blockade for the prevention of experimental autoimmune myocarditis

The angiotensin converting enzyme inhibitor captopril prevents myosin-induced experimental autoimmune myocarditis. Captopril inhibits production of angiotensin II and increases bradykinin signaling, among other actions. To test whether captopril inhibits disease through blockade of angiotensin signaling, we tested the ability of losartan, an angiotensin II receptor blocker, to prevent myosin-induced myocarditis. A/J mice immunized with the heavy chain of cardiac myosin in complete Freund's adjuvant develop acute myocarditis by day 21 post-immunization, consisting of severe focal inflammation, necrosis and fibrosis. Administration of losartan (250 mg/L in the drinking water) or captopril (75 mg/L in the drinking water) significantly reduced inflammation, necrosis and fibrosis in myosin-immunized mice. The heart weights and the heart weight-to-body weight ratios were also significantly reduced in both treatment groups. However, whereas captopril reduced myosin-specific delayed-type hypersensitivity, losartan did not. Both captopril-treated mice and losartan-treated mice showed a decrease in myosin-specific autoantibody production. Because losartan treatment significantly reduced myocarditis, fibrosis and autoantibody production in EAM, it is likely that prevention of angiotensin II receptor stimulation is a major mechanism underlying the inhibition of myosin-induced myocarditis by captopril.

The Renin Angiotensin System in the Regulation of Angiogenesis

Decades of experimentation on angiotensin and bradykinin have focused on macrovascular systemic effects. However, angiotensin II and bradykinin are both angiogenic agents, highlighting their ability to also effect the microvascular circulation. Not surprisingly, inhibition of angiotensin converting enzyme, which inhibits angiotensin II synthesis and bradykinin degradation, would have different impacts on angiogenesis in vivo dependent upon what factors were present in the system. Several pathological states in which angiogenesis is important, including peripheral ischemia, stroke, retinopathy, and cancer are examined in this review with respect to activity of angiotensin II and bradykinin and the impact of angiotensin converting enzyme inhibition. Although generalizations are not without legitimate criticism, one can think about peripheral ischemia and stroke as being more dependent upon bradykinin signaling and retinopathy and cancer as more dependent upon angiotensin II signaling to drive angiogenesis. Many exceptions are found that are specific to individual animal model systems. Furthermore, cancer systems that have been examined at any depth are few. However, published data on in vitro cultures and animal models present interesting predictions about how the renin angiotensin and bradykinin systems may function in humans. Since angiotensin converting enzyme inhibitors have been widely utilized pharmaceuticals for many years, we are now accumulating epidemiological data that test our predictions. The importance of understanding which agent, angiotensin and/or bradykinin, appears to be the more important regulator of angiogenesis in a given pathology will become increasing evident as more specific angiotensin II and bradykinin receptor blocking drugs make their way into clinical use.

Increased Activity of Coagulation Factor XII (Hageman Factor) Causes Hereditary Angioedema Type III

Hereditary angioedema (HAE) is characterized clinically by recurrent acute skin swelling, abdominal pain, and potentially life-threatening laryngeal edema. Three forms of HAE have been described. The classic forms, HAE types I and II, occur as a consequence of mutations in the C1-inhibitor gene. In contrast to HAE types I and II, HAE type III has been observed exclusively in women, where it appears to be correlated with conditions of high estrogen levels--for example, pregnancy or the use of oral contraceptives. A recent report proposed two missense mutations (c.1032C-->A and c.1032C-->G) in F12, the gene encoding human coagulation factor XII (FXII, or Hageman factor) as a possible cause of HAE type III. Here, we report the occurrence of the c.1032C-->A (p.Thr328Lys) mutation in an HAE type III-affected family of French origin. Investigation of the F12 gene in a large German family did not reveal a coding mutation. Haplotype analysis with use of microsatellite markers is compatible with locus heterogeneity in HAE type III. To shed more light on the pathogenic relevance of the HAE type III-associated p.Thr328Lys mutation, we compared FXII activity and plasma levels in patients carrying the mutation with that of healthy control individuals. Our data strongly suggest that p.Thr328Lys is a gain-of-function mutation that markedly increases FXII amidolytic activity but that does not alter FXII plasma levels. We conclude that enhanced FXII enzymatic plasma activity in female mutation carriers leads to enhanced kinin production, which results in angioedema. Transcription of F12 is positively regulated by estrogens, which may explain why only women are affected with HAE type III. The results of our study represent an important step toward an understanding of the molecular processes involved in HAE type III and provide diagnostic and possibly new therapeutic opportunities.

Resonant Waveguide Grating Biosensor for Living Cell Sensing

This article presents theoretical analysis and experimental data for the use of resonant waveguide grating (RWG) biosensors to characterize stimulation-mediated cell responses including signaling. The biosensor is capable of detecting redistribution of cellular contents in both directions that are perpendicular and parallel to the sensor surface. This capability relies on online monitoring cell responses with multiple optical output parameters, including the changes in incident angle and the shape of the resonant peaks. Although the changes in peak shape are mainly contributed to stimulation-modulated inhomogeneous redistribution of cellular contents parallel to the sensor surface, the shift in incident angle primarily reflects the stimulation-triggered dynamic mass redistribution (DMR) perpendicular to the sensor surface. The optical signatures are obtained and used to characterize several cellular processes including cell adhesion and spreading, detachment and signaling by trypsinization, and signaling through either epidermal growth factor receptor or bradykinin B2 receptor. A mathematical model is developed to link the bradykinin-mediated DMR signals to the dynamic relocation of intracellular proteins and the receptor internalization during B2 receptor signaling cycle. This model takes the form of a set of nonlinear, ordinary differential equations that describe the changes in four different states of B2 receptors, diffusion of proteins and receptor-protein complexes, and the DMR responses. Classical analysis shows that the system converges to a unique optical signature, whose dynamics (amplitudes, transition time, and kinetics) is dependent on the bradykinin signal input, and consistent with those observed using the RWG biosensors. This study provides fundamentals for probing living cells with the RWG biosensors, in general, optical biosensors.