Distinct metabolic and vascular effects of dietary triglycerides and cholesterol in atherosclerotic and diabetic mouse models.

Cholesterol and triglyceride-rich Western diets are typically associated with an increased occurrence of type 2 diabetes and vascular diseases. This study aimed to assess the relative impact of dietary cholesterol and triglycerides on glucose tolerance, insulin sensitivity, atherosclerotic plaque formation, and endothelial function. C57BL6 wild-type (C57) mice were compared with atherosclerotic LDLr(-/-) ApoB(100/100) (LRKOB100) and atherosclerotic/diabetic IGF-II × LDLr(-/-) ApoB(100/100) (LRKOB100/IGF) mice. Each group was fed either a standard chow diet, a 0.2% cholesterol diet, a high-fat diet (HFD), or a high-fat 0.2% cholesterol diet for 6 mo. The triglyceride-rich HFD increased body weight, glucose intolerance, and insulin resistance but did not alter endothelial function or atherosclerotic plaque formation. Dietary cholesterol, however, increased plaque formation in LRKOB100 and LRKOB100/IGF animals and decreased endothelial function regardless of genotype. However, cholesterol was not associated with an increase of insulin resistance in LRKOB100 and LRKOB100/IGF mice and, unexpectedly, was even found to reduce the insulin-resistant effect of dietary triglycerides in these animals. Our data indicate that dietary triglycerides and cholesterol have distinct metabolic and vascular effects in obese atherogenic mouse models resulting in dissociation between the impairment of glucose homeostasis and the development of atherosclerosis.

Effects of tempol on endothelial and vascular dysfunctions and insulin resistance induced by a high-fat high-sucrose diet in the rat.

We investigated the effects of treatment with tempol (an antioxidant) on vascular and metabolic dysfunction induced by a high-fat high-sucrose (HFHS) diet. Rats were randomized to receive an HFHS or chow diet with or without tempol treatment (1.5 mmol·(kg body mass)(-1)·day(-1)) for 4 weeks. Blood pressure, heart rate, and blood flow were measured in the rats by using intravascular catheters and Doppler flow probes. Insulin sensitivity and vascular responses to insulin were assessed during a euglycemic-hyperinsulinemic clamp. In-vitro studies were performed to evaluate vascular reactivity and endothelial and inducible nitric oxide synthase (eNOS; iNOS) expression in vascular and muscle tissues. Endothelin, nitrotyrosine, and NAD(P)H oxidase expressions were determined in vascular tissues, and glucose transport activity and glucose transporter 4 (GLUT4) expression were examined in muscles. Tempol treatment was found to prevent alterations in insulin sensitivity, glucose transport activity, GLUT4 expression, and vascular reactivity, and to prevent increases in plasma insulin, blood pressure, and heart rate noted in the untreated HFHS-fed rats. These were associated with increased levels of eNOS expression in vascular and muscle tissues, but reductions in nitrotyrosine, endothelin, NAD(P)H oxidase, and iNOS expressions. Therefore, oxidative stress induced by a relatively short-term HFHS diet could contribute to the early development of vascular and metabolic abnormalities in rats.

Are noscapine and raloxifene ligands of the bradykinin B2 receptor? An assessment based on the human umbilical vein contractility assay.

The centrally acting antitussive opiate derivative, noscapine, has been claimed to be a non-competitive bradykinin B2 receptor antagonist. Raloxifene, a selective estrogen receptor modulator, was predicted to bind the bradykinin B2 receptor and to exert a partial agonist activity. These intriguing claims suggest that new molecular scaffolds ("chemotypes") may be identified for small molecule ligands of kinin receptors and that some off-target effects of noscapine or raloxifene may be mediated by bradykinin B2 receptors. An established contractile bioassay for ligands of the bradykinin B2 receptor, the isolated human umbilical vein, was exploited to characterize the inhibitory effect of noscapine and raloxifene on the B2 receptor-mediated contractile response to bradykinin. Observed effects were compared with those of the peptide antagonist icatibant, a potent, selective and competitive B2 receptor antagonist. Our results indicate that neither noscapine (2.5 µM) nor raloxifene (20 µM) behave as B2 receptor antagonists in concentrations that vastly exceeded an effective concentration of the control antagonist, icatibant; further, none of these drugs had direct contractile effects. It is suggested that the previously reported B2 receptor inhibitory effect of noscapine, a putative sigma-receptor agonist, might result from an indirect physiological antagonism, while raloxifene did not appear to have any significant affinity for the B2 receptors.

Picomolar Sensitivity Analysis of Multiple Bradykinin-Related Peptides in the Blood Plasma of Patients With Hereditary Angioedema in Remission: A Pilot Study.

Background: Hereditary angioedema (HAE) is a rare autosomal dominant disease; the most well understood forms concern the haplodeficiency of C1 esterase inhibitor (C1INH) and a gain of function mutation of factor XII (FXII). The acute forms of these conditions are mediated by an excessive bradykinin (BK) formation by plasma kallikrein. Methods: A validated LC-MS/MS platform of picomolar sensitivity developed for the analysis of eleven bradykinin-related peptides was applied to the plasma of HAE-C1INH and HAE-FXII sampled during remission. Results: In HAE-C1INH plasma, the concentrations of the relatively stable BK1-5 fragment (mean ± S.E.M.: 12.0 ± 4.2 pmol/L), of BK2-9 (0.7 ± 0.2 pmol/L) and of the sums of BK and its tested fragments (18.0 ± 6.4 pmol/L) are significantly greater than those recorded in the plasma of healthy volunteers (1.9 ± 0.6, 0.03 ± 0.03 and 4.3 ± 0.8 pmol/L, respectively), consistent with the previous evidence of permanent plasma kallikrein activity in this disease. Kinin levels in the plasma of HAE-FXII patients did not differ from controls, suggesting that triggering factors for contact system activation are not active during remission. Conclusion: BK1-5, BK2-9 and the sum of BK and its fragments determined by the sensitive LC-MS/MS technique are proposed as potential biomarkers of HAE-C1INH in remission while this was not applicable to HAE-FXII patients.

A Robust Bioassay of the Human Bradykinin B2 Receptor that Extends Molecular and Cellular Studies: The Isolated Umbilical Vein.

Bradykinin (BK) has various physiological and pathological roles. Medicinal chemistry efforts targeted toward the widely expressed BK B2 receptor (B2R), a G-protein-coupled receptor, were primarily aimed at developing antagonists. The only B2R antagonist in clinical use is the peptide icatibant, approved to abort attacks of hereditary angioedema. However, the anti-inflammatory applications of B2R antagonists are potentially wider. Furthermore, the B2R antagonists notoriously exhibit species-specific pharmacological profiles. Classical smooth muscle contractility assays are exploited over a time scale of several hours and support determining potency, competitiveness, residual agonist activity, specificity, and reversibility of pharmacological agents. The contractility assay based on the isolated human umbilical vein, expressing B2R at physiological density, was introduced when investigating the first non-peptide B2R antagonist (WIN 64338). Small ligand molecules characterized using the assay include the exquisitely potent competitive antagonist, Pharvaris Compound 3 or the partial agonist Fujisawa Compound 47a. The umbilical vein assay is also useful to verify pharmacologic properties of special peptide B2R ligands, such as the carboxypeptidase-activated latent agonists and fluorescent probes. Furthermore, the proposed agonist effect of tissue kallikrein on the B2R has been disproved using the vein. This assay stands in between cellular and molecular pharmacology and in vivo studies.

In Vitro Modeling of Bradykinin-Mediated Angioedema States.

Kinins (peptides related to bradykinin, BK) are formed from circulating substrates, the kininogens, by the action of two proteases, the kallikreins. The only clinical application of a BK receptor ligand, the B2 receptor antagonist icatibant, is the treatment of the rare hereditary angioedema (HAE) caused by the deficiency of C1-esterase inhibitor (C1-INH). Less common forms of HAE (genetic variants of factor XII, plasminogen, kininogen) are presumably mediated by increased BK formation. Acquired forms of BK-mediated angioedema, such as that associated with angiotensin-I converting enzyme (ACE) inhibition, are also known. Antibody-based analytical techniques are briefly reviewed, and support that kinins are extremely short-lived, prominently cleared by ACE. Despite evidence of continuous activation of the kallikrein-kinin system in HAE, patients are not symptomatic most of the time and their blood or plasma obtained during remission does not generate excessive immunoreactive BK (iBK), suggesting effective homeostatic mechanisms. HAE-C1-INH and HAE-FXII plasmas are both hyperresponsive to fibrinolysis activation. On another hand, we suggested a role for the alternate tissue kallikrein-kinin system in patients with a plasminogen mutation. The role of the BK B1 receptor is still uncertain in angioedema states. iBK profiles under in vitro stimulation provide fresh insight into the physiopathology of angioedema.

Bradykinin receptors: Agonists, antagonists, expression, signaling, and adaptation to sustained stimulation.

Bradykinin-related peptides, the kinins, are blood-derived peptides that stimulate 2 G protein-coupled receptors, the B1 and B2 receptors (B1R, B2R). The pharmacologic and molecular identities of these 2 receptor subtypes will be succinctly reviewed herein, with emphasis on drug development, receptor expression, signaling, and adaptation to persistent stimulation. Peptide and non-peptide antagonists and fluorescent ligands have been produced for each receptor. The B2R is widely and constitutively expressed in mammalian tissues, whereas the B1R is mostly inducible under the effect of cytokines during infection and immunopathology. The B2R is temporarily desensitized by a cycle of phosphorylation/endocytosis followed by recycling, whereas the nonphosphorylable B1R is relatively resistant to desensitization and translocated to caveolae on activation. Both receptor subtypes, mainly coupled to protein G Gq, phospholipase C and calcium signaling, mediate the vascular aspects of inflammation (vasodilation, edema formation). On this basis, icatibant, a peptide antagonist of the B2R, is approved in the management of hereditary angioedema attacks. This disease is the therapeutic showcase of the kallikrein-kinin system, with an orally bioavailable B2R antagonist under development, as well as other agents that inhibit the kinin forming protease, plasma kallikrein. Other clinical applications are still elusive despite the maturity of the medicinal chemistry efforts applied to kinin receptors.

The development of a targeted and more potent, anti-Inflammatory derivative of colchicine: Implications for gout.

BACKGROUND: Colchicine is routinely used for its anti-inflammatory properties to treat gout and Familial Mediterranean fever. More recently, it was also shown to be of therapeutic benefit for another group of diseases in which inflammation is a key component, namely, cardiovascular disease. Whilst there is considerable interest in repurposing this alkaloid, it has a narrow therapeutic index and is associated with undesirable side effects and drug interactions. We, therefore, developed a derivatives of colchicine that preferentially target leukocytes to increase their potency and diminish their side effects. The anti-inflammatory activity of the colchicine derivatives was tested in experimental models of neutrophil activation by the etiological agent of gout, monosodium urate crystals (MSU). METHODS: Using a rational drug design approach, the structure of colchicine was modified to increase its affinity for ßVI-tubulin, a colchicine ligand preferentially expressed by immune cells. The ability of the colchicine analogues with the predicted highest affinity for ßVI-tubulin to dampen neutrophil responses to MSU was determined with in vitro assays that measure MSU-induced production of ROS, release of IL-1 and CXCL8/IL-8, and the increase in the concentration of cytoplasmic calcium. The anti-inflammatory property of the derivatives was assessed in the air pouch model of MSU-induced inflammation in mice. RESULTS: The most effective compound generated, CCI, is more potent than colchicine in all the in vitro assays. It inhibits neutrophil responses to MSU in vitro at concentrations 10-100-fold lower than colchicine. Similarly, in vivo, CCI inhibits the MSU-induced recruitment of leukocytes at a 10-fold lower concentration than colchicine when administered prior to or after MSU. CONCLUSIONS: We provide evidence that colchicine can be rendered more potent atinhibiting MSU-induced neutrophil activation and inflammation using a rational drug design approach. The development of compounds such as CCI will provide more efficacious drugs that will not only alleviate gout patients of their painful inflammatory episodes at significantly lower doses than colchicine, but also be of potential therapeutic benefit for patients with other diseases treated with colchicine.

Measurement of Bradykinin Formation and Degradation in Blood Plasma: Relevance for Acquired Angioedema Associated With Angiotensin Converting Enzyme Inhibition and for Hereditary Angioedema Due to Factor XII or Plasminogen Gene Variants.

Bradykinin (BK)-mediated angioedema (AE) states are rare acquired or hereditary conditions involving localized edema of the subcutaneous and submucosal tissues. Citrated plasma from healthy volunteers or patients with hereditary angioedema (HAE) with normal level of C1-inhibitor (C1-INH) was used to investigate pathways of BK formation and breakdown relevant to AE physiopathology. The half-life of BK (100 nM) added to normal plasma was 34 s, a value that was increased ~12-fold when the angiotensin converting enzyme (ACE) inhibitor enalaprilat (130 nM) was added (enzyme immunoassay measurements). The BK half-life was similarly increased ~5-fold following 2 daily oral doses of enalapril maleate in healthy volunteers, finding of possible relevance for the most common form of drug-associated AE. We also addressed the kinetics of immunoreactive BK (iBK) formation and decline, spontaneous or under three standardized stimuli: tissue kallikrein (KLK-1), the particulate material Kontact-APTT™ and tissue plasminogen activator (tPA). Relative to controls, iBK production was rapid (10-20 min) and very intense in response to tPA in plasma of female heterozygotes for variants in gene F12 coding for factor XII (FXII) (p.Thr328Lys, 9 patients; p.Thr328Arg, one). An increased response to Kontact-APTT™ and an early tPA-induced cleavage of anomalous FXII (immunoblots) were also observed. Biotechnological inhibitors showed that the early response to tPA was dependent on plasmin, FXIIa and plasma kallikrein. Results from post-menopausal and pre-menopausal women with HAE-FXII were indistinguishable. The iBK production profiles in seven patients with the plasminogen p.Lys330Glu variant (HAE-PLG) did not significantly differ from those of controls, except for an unexpected, rapid and lanadelumab-resistant potentiation of KLK-1 effect. This enzyme did not cleave plasminogen or factor XII, suggesting a possible idiosyncratic interaction of the plasminogen pathogenic variant with KLK-1 activity. KLK-1 abounds in salivary glands and human saliva, hypothetically correlating with the clinical presentation of HAE-PLG that includes the swelling of the tongue, lips and contiguous throat tissues. Samples from HAE patients with normal C1-INH levels and F12 gene did not produce excessive iBK in response to stimuli. The ex vivo approach provides physiopathological insight into AE states and supports the heterogeneous physiopathology of HAE with normal C1-INH.

Increased fibrinolysis-induced bradykinin formation in hereditary angioedema confirmed using stored plasma and biotechnological inhibitors.

OBJECTIVE: We recently investigated the pathways of immunoreactive bradykinin (iBK) formation in fresh blood of normal volunteers and of patients with hereditary angioedema due to C1-esterase inhibitor deficiency (HAE-1/-2). Herein, we adapted the techniques to small volumes (200 µl) of previously frozen citrated plasma and further analyzed the mechanisms of iBK formation with additional biotechnological inhibitors. RESULTS: Measurable iBK formation was observed under stimulation with tissue kallikrein (KLK-1, 10 nM), the particulate material Kontact-APTT (concentration reduced to 2% v/v) or recombinant tissue plasminogen activator (tPA, 169 nM), with little background in unstimulated plasma incubated for up to 2 h. Plasma samples from HAE-1/-2 patients responded earlier to tPA than those from controls, as previously reported with whole blood. Lanadelumab inhibited iBK formation induced by Kontact-APTT and tPA. A highly specific plasmin inhibitor, DX-1000, abolished tPA-induced iBK formation in plasma but had no effect against Kontact-APTT, confirming the role of fibrinolysis in tPA-induced kinin formation. The anti-lanadelumab neutralizing antibody M293-D02 reversed the inhibitory effects of lanadelumab. Frozen plasma is a suitable material for measuring iBK formation kinetics, with possible applications such as investigating the effect of rare disease states on the kallikrein-kinin system and monitoring the effect of HAE prophylactic treatments.

D-Arg0-Bradykinin-Arg-Arg, a Latent Vasoactive Bradykinin B2 Receptor Agonist Metabolically Activated by Carboxypeptidases.

We previously reported hypotensive and vasodilator effects from C-terminally extended bradykinin (BK) sequences that behave as B2 receptor (B2R) agonists activated by vascular or plasma peptidases. D-Arg0-BK-Arg-Arg (r-BK-RR) is a novel prodrug peptide hypothetically activated by two catalytic cycles of Arg-carboxypeptidases (CPs) to release the direct agonist D-Arg0-BK. N-terminally extending the BK sequence with D-Arg0 in the latter peptide was meant to block the second kinin inactivation pathway in importance, aminopeptidase P. The affinity of r-BK and r-BK-RR for recombinant B2R was assessed using a [3H]BK binding displacement assay. Their pharmacology was evaluated in human isolated umbilical vein, a contractile bioassay for the B2R, in a morphological assay involving the endocytosis of B2R-green fusion protein (GFP) and in anesthetized rats instrumented to record hemodynamic responses to bolus intravenous injection of both peptides. r-BK exhibited an affinity equal to that of BK for the rat B2R, while r-BK-RR was 61-fold less potent. In the vein and the B2R-GFP internalization assay, r-BK was a direct agonist unaffected by the blockade of angiotensin converting enzyme (ACE) with enalaprilat, or Arg-CPs with Plummer's inhibitor. However, the in vitro effects of r-BK-RR were reduced by these inhibitors, more so by enalaprilat. In anesthetized rats, r-BK and r-BK-RR were equipotent hypotensive agents and their effects were inhibited by icatibant (a B2R antagonist). The hypotensive effects of r-BK were potentiated by enalaprilat, but not influenced by the Arg-CPs inhibitor, which is consistent with a minor role of Arg-CPs in the metabolism of r-BK. However, in rats pretreated with both enalaprilat and Plummer's inhibitor, the hypotensive responses and the duration of the hypotensive episode to r-BK were significantly potentiated. The hypotensive responses to r-BK-RR were not affected by enalaprilat, but were reduced by pre-treatment with the Arg-CPs inhibitor alone or combined with enalaprilat. Therefore, in vivo, Arg-CPs activity is dominant over ACE to regenerate the B2R agonist r-BK from r-BK-RR, a prodrug activator of the B2R. A B2R agonist activated only at the level of the microcirculation by resident peptidases could be developed as an intravenously infused drug for ischemic diseases.

Bifunctional ligands of the bradykinin B2 and B1 receptors: An exercise in peptide hormone plasticity.

Kinins are the small and fragile hydrophilic peptides related to bradykinin (BK) and derived from circulating kininogens via the action of kallikreins. Kinins bind to the preformed and widely distributed B2 receptor (B2R) and to the inducible B1 receptor (B1R). B2Rs and B1Rs are related G protein coupled receptors that possess natural agonist ligands of nanomolar affinity (BK and Lys BK for B2Rs, Lys-des-Arg9-BK for B1R). Decades of structure-activity exploration have resulted in the production of peptide analogs that are antagonists, one of which is clinically used (the B2R antagonist icatibant), and also non-peptide ligands for both receptor subtypes. The modification of kinin receptor ligands has made them resistant to extracellular or endosomal peptidases and/or produced bifunctional ligands, defined as agonist or antagonist peptide ligands conjugated with a chemical fluorophore (emitting in the whole spectrum, from the infrared to the ultraviolet), a drug-like moiety, an epitope, an isotope chelator/carrier, a cleavable sequence (thus forming a pro-drug) and even a fused protein. Dual molecular targets for specific modified peptides may be a source of side effects or of medically exploitable benefits. Biotechnological protein ligands for either receptor subtype have been produced: they are enhanced green fluorescent protein or the engineered peroxidase APEX2 fused to an agonist kinin sequence at their C-terminal terminus. Antibodies endowed with pharmacological actions (agonist, antagonist) at B2R have been reported, though not monoclonal antibodies. These findings define classes of alternative ligands of the kinin receptor of potential therapeutic and diagnostic value.

Cholinergic involvement in vascular and glucoregulatory actions of insulin in rats.

This study was designed to test the glucose metabolic and vasodilator actions of insulin in rats and its relation to cholinergic system-dependent mechanisms. The first group of rats had pulsed Doppler flow probes and intravascular catheters implanted to determine blood pressure, heart rate, and regional blood flows. Insulin sensitivity was assessed by the euglycemic-hyperinsulinemic clamp technique carried out in the absence or presence of atropine. The second group of rats was used to determine the cholinergic contribution to in vivo insulin-mediated glucose utilization in individual muscles. Glucose uptake was examined by using [(3)H]2-deoxy-D-glucose. Muscarinic cholinergic blockade was found to significantly (P = 0.002) reduce insulin sensitivity and to completely abrogate the renal (P = 0.008) and hindquarter (P = 0.02) vasodilator responses to euglycemic infusion of insulin. A significant reduction in insulin-stimulated in vivo glucose uptake was also noted in soleus (P = 0.006), quadriceps (P = 0.03), gastrocnemius (P = 0.02), and extensor digitorum longus (EDL) (P = 0.001) muscles, when insulin was infused at a rate of 4 mU . kg(-1) . min(-1), whereas at the rate of 16 mU . kg(-1) . min(-1), a significant reduction in glucose uptake was only observed in EDL (P = 0.03) and quadriceps (P = 0.01) muscles. Together, these results demonstrate a potential role for cholinergic involvement with physiological insulin actions in glucose clearance and blood flow regulation in rats.

Species-specific pharmacology of maximakinin, an amphibian homologue of bradykinin: putative prodrug activity at the human B2 receptor and peptidase resistance in rats.

Maximakinin (MK), an amphibian peptide possessing the C-terminal sequence of bradykinin (BK), is a BK B2 receptor (B2R) agonist eliciting prolonged signaling. We reinvestigated this 19-mer for species-specific pharmacologic profile, in vivo confirmation of resistance to inactivation by angiotensin converting enzyme (ACE), value as a module for the design of fusion proteins that bind to the B2R in mammalian species and potential activity as a histamine releaser. Competition of the binding of [3H]BK to recombinant human myc-B2Rs in cells that express these receptors revealed that MK possessed a tenuous fraction (<0.1%) of the affinity of BK, despite being only ∼20-fold less potent than BK in a contractility assay based on the human isolated umbilical vein. These findings are reconciled by the generation of C-terminal fragments, like Lys-Gly-Pro-BK and Gly-Pro-BK, when the latent MK is incubated with human venous tissue (LC-MS), supporting activation via hydrolysis upstream of the BK sequence. At the rat recombinant myc-B2R, MK had a lesser affinity than that of BK, but with a narrower margin (6.2-fold, radioligand binding competition). Accordingly, MK (10 nM) stimulated calcium transients in cells that expressed the rat receptors, but not the human B2R. Recombinant MRGPRX2, a receptor that mediates cationic peptide-induced mast cell secretion, minimally responded by increased [Ca+2]i to MK at 10 µM. Enhanced green fluorescent protein fused to MK (EGFP-MK) labeled cells that expressed rat, but not human B2Rs. Intravenous MK induced dose-dependent hypotensive, vasodilator and tachycardic responses in anesthetized rats and the effects were antagonized by pretreatment with icatibant but not modified by pyrilamine or enalaprilat. Strong species-specific responses to the toxin-derived peptide MK and its prodrug status in the isolated human vein were evidenced. Accordingly, MK in the EGFP-MK fusion protein is a pharmacophore module that confers affinity for the rat B2R, but not for the human form of the B2R. MK is unlikely to be an efficient mast cell activator, but its resistance to inactivation by ACE was confirmed in vivo.

Infrared-emitting, peptidase-resistant fluorescent ligands of the bradykinin B2 receptor: application to cytofluorometry and imaging.

BACKGROUND: We have previously reported the design, pharmacological properties and imaging application of bradykinin (BK) B2 receptor (B2R) ligands conjugated with fluorophores such as fluorescein derivatives at their N-terminus. To take advantage of the high penetration of infrared light into living tissues and their low autofluorescence in this region of the spectrum, additional probes conjugated with cyanine dye 7 (Cy7) were synthesized and characterized. RESULTS: The antagonist B-9430 (D-Arg-[Hyp3,Igl5,D-Igl7,Oic8]-BK) and the agonist B-9972 (D-Arg-[Hyp3,Igl5,Oic7,Igl8]-BK) were N-terminally extended with the infrared fluorophore Cy7, producing the peptides B-10665 and B-10666, respectively. Pharmacological studies indicated that the agonist B-10666 lost much affinity for the B2R vs. the parent peptide, whereas the antagonist B-10665 better retained its potency vs. B-9430 (competition of [3H]BK binding to human B2R, contractility of the human isolated umbilical vein for which potency losses were more important in each case). Both probes stained HEK 293 cells that expressed the B2R-green fluorescent protein (GFP) construction in a specific manner (confocal microscopy) and with very extensive co-localization of the green and infrared fluorescence in either case. The agonist B-10666 at 100 nM promoted the endocytosis of B2R-GFP in live cells, but not the antagonist version at 10-25 nM. The Cy7-labeled peptides did not label cells expressing the ß2-adrenoceptor-GFP construction. B-10665 at low nanomolar concentrations was an effective probe for the recombinant B2Rs in cytofluorometry and macroscopic imaging of cell wells (IVIS imaging system operated for infrared fluorescence detection). CONCLUSIONS: Despite a propensity for non-specific binding when used at high concentrations and limited sensitivity, Cy7-conjugated peptidase-resistant B2R ligands support original imaging and cytofluorometric applications.

Fatty acid-induced changes in vascular reactivity in healthy adult rats.

Dietary fatty acids (FAs) are known to modulate endothelial dysfunction, which is the first stage of atherosclerosis. However, their exact role in this initial phase is still unclear. The effects of isolated or combined (by 2) purified FAs from the main FA families were studied on the vascular response of isolated thoracic aorta in healthy rats to get a better understanding of the mechanisms of action of dietary FAs in regulating vascular endothelial function. Cumulative contraction curves to phenylephrine and relaxation curves to carbachol and then to sodium nitroprusside were obtained in the absence or presence of the FAs studied allowing endothelium-dependent and endothelium-independent ability of the smooth muscle to relax to be assessed in each experimental group. The endothelium-dependent vasodilator response to carbachol was lowered by eicosapentaenoic acid, whereas it was not altered either by docosahexaenoic acid alone or by combined eicosapentaenoic acid-docosahexaenoic acid, oleic acid, or stearic acid, and it was increased by linoleic acid (LA). A decreased phenylephrine-induced contraction was observed after incubation with arachidonic acid and with stearic acid. On the other hand, the endothelium-dependent relaxation was reduced by the addition of combined LA-arachidonic acid and LA-oleic acid. In conclusion, these data point out the differential effects of different types of FAs and of FAs alone vs combined on vascular reactivity. The complex nature of these effects could be partially linked to metabolic specificities of endothelial cells and to interactions between some FAs.

In Vivo Effects of Bradykinin B2 Receptor Agonists with Varying Susceptibility to Peptidases.

We reported evidence of bradykinin (BK) regeneration from C-terminal extended BK sequences that behave as peptidase-activated B2 receptor (B2R) agonists. Further to these in vitro studies, we carried out in vivo experiments to verify hemodynamic effects of BK analogs exhibiting variable susceptibility toward vascular and blood plasma peptidases. Rats were anesthetized and instrumented to record blood pressure and heart rate responses to bolus intravenous (i.v.) injection of increasing doses of BK, B-9972 (D-Arg-[Hyp(3),Igl(5),Oic(7),Igl(8)]-BK), BK-Arg, BK-His-Leu or BK-Ala-Pro, in the absence or presence of specific inhibitors. In some experiments, pulsed Doppler flow probes measured hindquarter Doppler shift in response to i.v. injections of kinins. BK caused rapid, transient and dose-related hypotensive effects. These effects were potentiated ∼15-fold by the angiotensin converting enzyme (ACE) inhibitor, enalaprilat, but extensively inhibited by icatibant (a B2R antagonist) and not influenced by the Arg-carboxypeptidase (CP) inhibitor (Plummer's inhibitor). The hypotensive responses elicited by the peptidase-resistant B2R agonist, B-9972, were not affected by enalaprilat, but were inhibited by icatibant. The hypotensive responses to BK-Arg were abolished by pre-treatment with either the Arg-CP inhibitor or icatibant, pharmacologically evidencing BK regeneration. The hypotensive effects of BK-His-Leu and BK-Ala-Pro, previously reported as ACE-activated substrates, were abolished by icatibant, but not by enalaprilat. In vivo regeneration of BK from these two C-terminally extended analogs with no affinity for the B2R must follow alternative cleavage rules involving unidentified carboxypeptidase(s) when ACE is blocked. The transient hypotensive responses to BK and three tested analogs coincided with concomitant vasodilation (increased Doppler shift signal). Together, these results provide in vivo evidence that interesting hypotensive and vasodilator effects can be extracted from prodrug peptides that behave as peptidase-activated B2R agonists.

Tonic inhibitory control exerted by opioid peptides in the paraventricular nuclei of the hypothalamus on regional hemodynamic activity in rats.

1. Systemic and regional cardiovascular changes were measured following bilateral microinjection of specific and selective opioid-receptor antagonists into the paraventricular nuclei of the hypothalamus (PVN) of awake, freely moving rats. 2. PVN microinjection of increasing doses of the specific opioid antagonist naloxone - methiodide (1 - 5.0 nmol), or a selective mu-opioid receptor antagonist, beta-funaltrexamine (0.05 - 0.5 nmol), evoked important cardiovascular changes characterized by small and transient increases in heart rate (HR) and mean arterial pressure (MAP), vasoconstriction in renal and superior mesenteric vascular beds and vasodilation in the hindquarter vascular bed. 3. No significant cardiovascular changes were observed following PVN administration of the highly selective delta-opioid-receptor antagonist, ICI 174864 (0.1 - 1 nmol), or the selective kappa-opioid-receptor antagonist, nor-binaltorphine (0.1 - 1 nmol). 4. Most of the cardiovascular responses to naloxone (3 nmol) and beta-funaltrexamine (0.5 nmol) were attenuated or abolished by an i.v. treatment with a specific vasopressin V(1) receptor antagonist. 5. These results suggest that endogenous opioid peptides and mu-type PVN opioid receptors modulate a tonically-active central depressor pathway acting on systemic and regional haemodynamic systems. Part of this influence could involve a tonic inhibition of vasopressin release.

Induction of insulin resistance by high-sucrose feeding does not raise mean arterial blood pressure but impairs haemodynamic responses to insulin in rats.

1. This study was undertaken to further investigate the effects of a sucrose-enriched diet on vascular function and insulin sensitivity in rats. 2. Male Sprague-Dawley rats were randomized to receive a sucrose- or regular rat chow-diet for 4 weeks. A first group of sucrose- and chow-fed rats was instrumented with pulsed Doppler flow probes and intravascular catheters to determine blood pressure, heart rate, regional blood flows and insulin sensitivity in conscious rats. Insulin sensitivity was assessed by the euglycemic hyperinsulinemic clamp technique. Glucose transport activity was examined in isolated muscles by using the glucose analogue [(3)H]-2-deoxy-D-glucose. A second group of sucrose- and chow-fed rats was used to obtain information regarding nitric oxide synthase (NOS) isozymes protein expression in muscles, and determine endothelin content in vascular tissues isolated from both dietary groups. 3. Sucrose feeding was found to induce insulin resistance, but had no effect on resting blood pressure, heart rate, or regional haemodynamics. This insulin resistance was accompanied by alteration in the vascular responses to insulin. Insulin-mediated skeletal muscle vasodilation was impaired, whereas the mesenteric vasoconstrictor response was potentiated in sucrose-fed rats. A reduction in eNOS protein content in muscle and an increase in vascular endothelin peptide were noted in these animals. Moreover, a reduction in insulin-simulated glucose transport activity was also noted in muscles isolated from sucrose-fed rats. 4. Together these data suggest that a cluster of metabolic and haemodynamic abnormalities occur in response to the intake of simple sugars in rats.

Effect of troglitazone on vascular and glucose metabolic actions of insulin in high-sucrose-fed rats.

In rats, diets high in simple sugar induce insulin resistance and alter vascular reactivity. The present study was designed to evaluate the effects of 5 weeks treatment with troglitazone on insulin sensitivity, regional hemodynamics, and vascular responses to insulin in chow-fed and high-sucrose-fed rats. Male rats were randomly divided in 4 groups to receive a regular chow diet in the absence (group 1) or presence of troglitazone (0.2% in food; group 2), or a sucrose-enriched diet in the absence (group 3) or presence of troglitazone (group 4) for 5 weeks. The rats were instrumented with Doppler flow probes and intravascular catheters to determine blood pressure, heart rate, and regional blood flows. Insulin sensitivity was assessed by the euglycemic hyperinsulinemic clamp technique. Glucose transport activity was examined in isolated muscles. Sucrose feeding was found to induce insulin resistance and to impair the insulin-mediated skeletal muscle vasodilation. Treatment with troglitazone was found to increase whole-body insulin sensitivity in sucrose- and chow-fed rats, but had no effect on skeletal muscle glucose transport activity measured in isolated muscles from both dietary groups. Changes in regional hemodynamics were observed in both dietary cohorts treated with troglitazone, and the hindquarter vasoconstrictor response to insulin noted in sucrose-fed rats was abolished by the treatment. The vascular effects of troglitazone, and its insulin-related attenuating effects on contractile tone, could have contributed, in part, to improve insulin action on peripheral glucose disposal, presumably by improving blood flow distribution and glucose delivery.