Hyperosmolar glucose induces vasoconstriction through Rho/Rho‐kinase pathway in the rat aorta

Rho/Rho‐kinase signalling pathway plays a substantial role in vascular contractions. In this study, we investigated any roles of Rho/Rho‐kinase pathway in the vasoconstriction of the rat conductance and capacitance vessels by hyperosmolar glucose solution. Isolated aortic, mesenteric and renal rings were suspended and exposed to hyperosmolar glucose, sucrose and NaCl in the organ chambers filled with Krebs solution gassed with 95% O₂ and 5% CO₂ and maintained at 37 °C. The effect of a Rho‐kinase inhibitor, (+)‐(R)‐trans‐4‐(1‐aminoethyl)‐N‐(4‐pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y‐27632, 10^?5 m ), was tested on the contraction induced by hypertonic solutions. Endothelial integrity was also assessed after hyperosmolar glucose exposure. Moreover, the activity and expression of Rho‐kinase (ROCK‐2) as well as RhoA translocation were detected by Western blotting and enzyme‐linked immunosorbent assay‐based RhoA activity detection method detection kit. The vessels produced substantial contractions in response to hyperosmolar solutions. Y‐27632 significantly reduced hyperosmolarity‐induced vasoconstrictions (P < 0.05). Phosphorylation of myosin‐phosphatase target 1 increased after hyperosmolar glucose exposure, and this phosphorylation was significantly decreased by Y‐27632 (P < 0.05) in the aorta. Furthermore, RhoA translocation but not ROCK‐2 expression markedly increased by hyperosmolar glucose solution. These results may indicate that hyperosmolarity could induce vasoconstriction through Rho/Rho‐kinase signalling.     

In vitro effects of the cyclooxygenase inhibitor indomethacin and of the phospholipase‐C inhibitor U‐73122 on carbachol‐induced contractions of porcine detrusor muscle

Prostaglandin synthetase inhibitors belong to one substance class additionally used in the treatment of bladder dysfunctions associated with involuntary bladder contractions. However, the mechanism of action of non‐steroidal anti‐inflammatory drugs (NSAIDs) on the detrusor muscle is not clear. In this study, it was examined in vitro whether the NSAID indomethacin exhibited an inhibitory effect on carbachol‐induced contractions of the porcine detrusor muscle. Additionally, the inhibitory effect of the phospholipase‐C inhibitor U‐73122 on carbachol‐induced contractions of the porcine detrusor muscle was investigated. Experiments were performed on the muscle strips of the porcine detrusor muscle suspended in a tissue bath. Effects of indomethacin at 10^?6 and 10^?5 m on the maximum carbachol‐induced contraction and on the carbachol–response curve were investigated. Additionally, the inhibitory influence of U‐73122 at a concentration of 10^?5.5 m on the carbachol–response curve was investigated. Pretreatment with indomethacin at both concentrations did not result in a significant reduction in the maximum contraction compared with the control. In the experiments in which carbachol concentration‐response curves were generated, indomethacin exhibited at both concentrations a very small but significant change at carbachol concentrations of 10^?8 and 10^?7.5 m . In the experiments with U‐73122, a significant change was found in the concentration–response curve of carbachol at all concentrations of carbachol from 10^?6.5 to 10^?4 m . The mean maximum carbachol‐induced contraction was 141.8 ± 6.8% after incubation with U‐73122 and 166.0 ± 6.4% in the control group (P < 0.05). Indomethacin did not inhibit the carbachol‐induced contractions of the porcine detrusor muscle. The cyclooxygenase does not play a significant role in the carbachol‐induced bladder contraction of the porcine detrusor muscle. The inhibitory action of the phospholipase‐C inhibitor U‐73122 on the carbachol‐induced contraction was significant, but small. The results point to an inferior role of this pathway.     

Effects induced by inhibitors of the phosphatidylinositol 3‐kinase/Akt and nitric oxide synthase/guanylyl cyclase pathways on the isometric contraction in rat aorta: a comparative study

This work was aimed to determine whether isometric contraction in Wistar rat aorta is related to the phosphatidylinositol 3‐kinase (PI3K)/Akt‐dependent activation of endothelial nitric oxide synthase (eNOS). Basically, we hypothesized that additional increases in active tone occur after the pharmacological inhibition of a transduction pathway involved in NO synthesis or action. In intact aortic rings contracted with phenylephrine or high K⁺, the cumulative administration of the PI3K inhibitor, LY294002, elicited significant decreases – but not supplementary increases – in tone. In endothelium‐intact tissues, on the other hand, the Akt1/2 kinase inhibitor did not alter phenylephrine‐ and K⁺‐induced isometric contractions. The PI3K inhibitor wortmannin (1 × 10^?7 m ) produced a significant supplementary contraction only in endothelium‐intact aortic rings precontracted with phenylephrine. Higher concentrations of this inhibitor produced relaxations of phenylephrine and high K⁺‐constricted endothelium‐intact and endothelium‐denuded aortic rings. LY294002 and wortmannin did not cause any potentiating effect on phenylephrine‐ and angiotensin II‐induced concentration‐dependent contractile responses in endothelium‐intact tissues. In intact aortic rings contracted with phenylephrine or high K⁺, the addition of the NOS inhibitor, L‐NAME, or the guanylyl cyclase inhibitor, ODQ, further augmented tone in a concentration‐dependent manner, and these supplementary contractions were significantly reduced by endothelium removal. Taken together, our data suggest that the PI3K/Akt pathway is not counteracting aortic isometric contractions by activation of the eNOS. It appears, on the other hand, that the smooth muscle PI3K can stimulate contraction without activation of the protein kinase Akt in response to GPCR agonists and high K⁺.     

The role of Rho kinase and extracellular regulated kinase-mitogen-activated protein kinase in alpha2-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein

Alpha2-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon activation of the extracellular signal-regulated kinase-mitogen-activated protein (ERK-MAP) kinase signal transduction pathway. Recent studies have shown that alpha2-adrenoceptor-mediated vasoconstriction in the rat aorta is also dependent upon activation of Rho kinase. The aim of this study was to determine whether Rho kinase and ERK-MAP kinase are part of the same signaling pathway. The Rho kinase inhibitor Y27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride) (10 microM) almost completely inhibited the contractile response to the alpha2-adrenoceptor agonist UK14304 (5-bromo-6-[2-imidazolin-2-ylamine]-quinoxaline bitartrate) in segments of porcine palmar lateral vein [maximum response 2.9 +/- 2.3% of 60 mM KCl response (mean +/- S.E.M.) in the presence of Y27632, compared with 64.9 +/- 7.1% in control tissues, n = 4]. However, Y27632 had no effect on alpha2-adrenoceptor-mediated ERK activation, as measured by Western blotting. Alpha2-adrenoceptor-mediated vasoconstriction was associated with an increase in phosphorylation of the myosin phosphatase-targeting subunit (MYPT) at Thr696 (the Rho kinase phosphorylation site). This phosphorylation was inhibited by 10 microM Y27632. In contrast, inhibition of ERK activation with the MAP kinase kinase inhibitor PD98059 (2-amino-3-methoxyflavone) (50 microM) had no effect on MYPT phosphorylation. Both Y27632 and PD98059 inhibited myosin light chain phosphorylation. These data indicate that alpha2-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon both Rho kinase and ERK activation, although these are separate pathways. Rho kinase causes vasoconstriction through inhibition of myosin phosphatase and an increase in myosin light chain phosphorylation, whereas ERK causes vasoconstriction through a myosin phosphatase-independent pathway.     

Vasorelaxant effects of the monoterpenic phenol isomers,carvacrol and thymol,on rat isolated aorta

Various essential oils are rich in carvacrol, a monoterpenic phenol isomeric with thymol. This study was undertaken to assess the vasorelaxant effects of thymol and carvacrol in rat isolated aorta and the putative mechanisms underlying these effects. Thymol and carvacrol produced a concentration‐dependent relaxation on the aortic ring preparations pre‐contracted using KCl (IC₅₀ value of 64.40 ± 4.41 and 78.80 ± 11.91 μm , respectively) or using phenylephrine (PHE, 0.1 μm ) (IC₅₀ value of 106.40 ± 11.37 and 145.40 ± 6.07 μm , respectively) and inhibited the concentration‐response curves of aortic rings to PHE or KCl. In Ca²⁺‐free medium with ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid (2 mm ), thymol and carvacrol both at 1000 μm completely abolished the phasic component of PHE‐induced endothelium‐containing ring contractions. At 400 μm , thymol and carvacrol significantly reduced the CaCl₂‐induced contractions in Ca²⁺‐free medium. Furthermore, both thymol and carvacrol (300 and 1000 μm ) significantly reduced the contraction evoked by phorbol dibutyrate (1 μm ), an activator of protein kinase C. Magnitude of this inhibitory effect was enhanced in the presence of the Ca²⁺ pump inhibitor, thapsigargin (1 μm ). At 1000 μm , neither thymol nor carvacrol altered the resting potential of vascular smooth muscle cells. In conclusion, thymol and carvacrol induced an endothelium‐independent relaxation in rat isolated aorta, an effect that seems mediated through some mechanisms probably involving a transduction pathway between Ca²⁺ release from sarcoplasmic reticulum and/or regulation of the Ca²⁺ sensitivity of the contractile system. Moreover, it’s conceivable that thymol and carvacrol, at low concentrations, block the Ca²⁺ influx through the membrane.     

Effects of SCA40 on human bronchi and on guinea pig main bronchi in vitro. Comparison with cromakalim

Summary— The aim of this study was to examine the activity of SCA40, a novel charybdotoxin-sensitive potassium channel opener, against a variety of spasmogens or against electrical field stimulation in guinea pig isolated main bronchi and in human isolated bronchi; the effects of SCA40 were compared with those of cromakalim. Like cromakalim, SCA40 reduced the contractility of guinea pig and human isolated bronchi precontracted with acetylcholine 10^?6 M or neurokinin A 10^?6 M, SCA40 being more efficient and more potent than cromakalim. Moreover, on guinea pig isolated main bronchi, SCA40 can exert a preventive effect on contractions induced by acetylcholine, neurokinin A or capsaicin, that is, it shifts to the right the concentration-effect curves of these substances, whereas cromakalim has no such effect. The effects of cromakalim were antagonized by glibenclamide 10^?5 M, whereas the effects of SCA40 were inhibited by tetraethylammonium (TEA 10^?2 M) and charybdotoxin (3 × 10^?8 M), but this inhibitory effect of TEA was reversed by nifedipine (10^?6 M). Electrical field stimulation of guinea pig isolated main bronchi induced two successive contractile responses. Both contractions were significantly reduced by SCA40 (10^?6 and 10^?5 M) and cromakalim (10^?5 M). Since cromakalim was unable to inhibit the effects of acetylcholine or neurokinin A, it might be suggested that for this latter compound the inhibition seems to take place prejunctionally and to affect the release of neuromediators produced by electrical field stimulation. In contrast, in the case of SCA40, a postjunctional effect seems to be likely, owing to its preventive effects, although a prejunctional effect cannot be excluded. Finally, on guinea pig isolated main bronchi, SCA40 (10^?8-10^?6 M) did not potentiate the relaxant effect of isoprenaline or sodium nitroprusside, suggesting a lack of functional manifestation of inhibition of phosphodiesterase for these concentrations. In conclusion, these results demonstrate that SCA40 is a potent and efficient relaxant of guinea pig and human airway smooth muscle, and is able to inhibit, in the guinea pig isolated main bronchi, the contractions induced by electrical field stimulation. It has an effect on TEA-sensitive K+ channels, but this effect is probably not involved in its relaxant effect which does not also rest on an inhibitory effect of phosphodiesterase.     

Vasodilatory activity and antihypertensive profile mediated by inhibition of phosphodiesterase type 1 induced by a novel sulfonamide compound

LASSBio‐985 is a sulfonamide compound designed as a simplified structure of a nonselective phosphodiesterase type 4 (PDE‐4) inhibitor that promotes vasodilatory activity in vitro. PDE are enzymes responsible for the hydrolysis of cyclic adenosine 3′,5′‐ monophosphate and cyclic guanosine 3′,5′‐monophosphate. Five different isozymes of PDE are found in vascular smooth muscle (PDE1–PDE5). Aortic rings, with or without endothelium, from male normotensive and spontaneously hypertensive rats (SHR) were prepared for isometric tension recording. Blood pressure was measured in Wistar Kyoto (WKY) rats and SHR during intravenous infusion of LASSBio‐985 (10 mg/kg/min) during 15 min. LASSBio‐985 induced a concentration‐dependent vasodilation in aortic rings from normotensive and SHR, which was almost completely inhibited in endothelium‐denuded vessels. Vasodilatory activity was also reduced in endothelium‐intact aortic rings that had been pretreated with N_ω‐nitro‐l ‐arginine methyl ester hydrochloride (l ‐NAME), a nitric oxide synthase inhibitor and 1H‐[1,2,4]oxadiazolod[4,3‐a]quinoxalin‐1‐one (ODQ), a guanylate cyclase inhibitor. LASSBio‐985‐induced vasodilation was also inhibited by sildenafil (100 μm ) and SQ 22536, a PDE5 inhibitor and adenylate cyclase inhibitor, respectively. To evaluate the involvement of some endothelial receptors, atropine, diphenhydramine, HOE 140, naloxone, propranolol, indomethacin, and wortmannin were tested, but none inhibited the effects of LASSBio‐985. The residual effect observed on endothelium‐denuded aortic rings was abolished by nicardipine, a voltage‐sensitive‐Ca²⁺‐channel blocker. Intravenous infusion of LASSBio‐985 (10 mg/kg/min) significantly reduced systolic and diastolic pressures in both WKY and SHR. LASSBio‐985 is a compound with vasodilatory activity, which could be consequent to PDE1 inhibition and voltage‐sensitive‐Ca²⁺‐channel blockade.     

Effects of levosimendan on isolated human internal mammary artery and saphenous vein: concurrent use with conventional vasodilators

Graft spasm is a common problem in coronary artery bypass grafting (CABG). In this study, we aimed to investigate the interaction of levosimendan, a novel inodilator, with vasodilator agents that are clinically used for the treatment of graft spasm and with endogenous vasoconstrictors that are thought to play a role in graft vasospasm, in human internal mammary artery (IMA) and saphenous vein (SV). Isolated human IMA and SV segments derived from patients undergoing CABG were suspended in an organ bath. Responses to cumulative concentrations of noradrenaline (NA), serotonin (5‐HT), papaverine, nitroglycerin (NG), and diltiazem were recorded before and after 10^?5 m levosimendan incubation (30 min). In addition, cumulative levosimendan responses were taken in vessels precontracted with NA or 5‐HT. 10^?5 m levosimendan reduced NA E_max and sensitivity in IMA and SV, and 5‐HT E_max responses in IMA. Moreover, levosimendan caused concentration‐dependent relaxation in both grafts. Papaverine E_max or sensitivity was not altered by levosimendan neither in IMA nor in SV. Levosimendan diminished NG sensitivity in IMA and E_max responses in SV and decreased diltiazem E_max responses both in IMA and SV. Our results suggest that levosimendan may be used alone for prevention or treatment of graft spasm in IMA or in combination with papaverine in IMA and SV grafts. However, as concurrent administration with diltiazem or NG causes a reduction in relaxation in vitro, we suggest caution should be exercised when using levosimendan in combination with these agents.     

Paroxetine inhibited the relaxations induced by EFS in mice corpus cavernosum: is it a NOS inhibition?

Selective serotonin reuptake inhibitors are used in the treatment of psychiatric disorders but are associated with high incidence of sexual dysfunction such as ejaculation disorders by sertraline and fluoxetine, erection disorders by paroxetine. The aim of this study is to evaluate the effects of paroxetine, sertraline and fluoxetine on relaxation of smooth muscle of corpus cavernosum on the basis of nitric oxide (NO). Male mice were killed by cervical dislocation and their penile tissues were immediately removed. The tissues were incubated in organ baths containing Krebs solution at 37°C and bubbled with 95% O₂ and 5% CO₂. The corpus cavernosum strips were contracted with 10^?5 m phenylephrine (PHE) and relaxed with either paroxetine, sertraline, fluoxetine (10^?8–10^?4 m ) or electrical field stimulation (EFS). The effects of paroxetine, sertraline and fluoxetine were examined on EFS‐induced relaxations. While paroxetine did not show any effect on the corpus cavernosum strips precontracted with PHE, sertraline and fluoxetine caused a relaxation at concentrations of 3 × 10^?5–10^?4 m . The relaxations induced by sertraline and fluoxetine were completely abolished by l ‐NAME, but not d ‐NAME. The relaxations induced by EFS could be inhibited by l ‐NAME but not d ‐NAME. Paroxetine inhibited the relaxations at high concentrations. l ‐Arginine potentiated the relaxations induced by EFS; however in the presence of paroxetine these relaxations were not observed. In contrast, sertraline (10^?8–10^?5 m ) and fluoxetine (10^?8–10^?5m ) increased the relaxations induced by EFS. Sertraline and fluoxetine seem to be releasing some relaxing factor(s) and this factor may be NO. Paroxetine probably has a NOS inhibitory activity either on nNOS or eNOS, in contrast to sertraline and fluoxetine.     

Cloning and paratope analysis of an antibody fragment, a rational approach for the design of a PAI-1 inhibitor

Summary. This study reports the cloning, characterization and paratope analysis of the plasminogen activator inhibitor‐1 (PAI‐1) neutralizing single‐chain variable fragment 56A7C10 (scFv‐56A7C10). ScFv‐56A7C10‐wt exhibits a similar affinity (K_A = 1.01 ± 0.3 × 10⁹ m ^?1) and PAI‐1 inhibitory capacity (90 ± 6% PAI‐1 inhibition at a 16‐fold molar excess and IC₅₀ = 44 ± 14 ng mL^?1) as MA‐56A7C10 (K_A = 1.43 ± 0.4 × 10⁹ m ^?1, 90 ± 2% PAI‐1 inhibition at a 16‐fold molar excess and IC₅₀ = 122 ± 26 ng mL^?1). Subsequently, alanine scanning of the six complementarity determining regions (CDRs) was performed and the scFv‐56A7C10‐mutants (n = 26) were analyzed for their PAI‐1 binding and PAI‐1 inhibitory properties. Mutation of the residues Y32 and V33 in the CDR1 of the heavy chain (HCDR1) and the residues R98, H99, W100 or F100a (HCDR3) resulted in reduced PAI‐1 inhibitory capacities (IC₅₀ ≥ 418 ng mL^?1), confirmed by reduced affinities (14‐, 17‐, 7‐, 9‐ and 16‐fold reduced, respectively, vs. scFv‐56A7C10‐wt). In the light chain, mutation of the residues W50 (LCDR2), H91, Y92, D93, or W96 (LCDR3) resulted in reduced PAI‐1 inhibitory properties (IC₅₀ ≥ 160 ng mL^?1) and decreased affinities (i.e. 4‐, 9‐, 3‐, 3‐ and 2‐fold reduced affinity, respectively, vs. scFv‐56A7C10‐wt). Furthermore, an overlapping peptide scan confirmed the importance of the HCDR3 region. These data, combined with a three‐dimensional model of scFv‐56A7C10, reveal the molecular and structural properties of the paratope and contribute to the rational design of PAI‐1 neutralizing compounds.     

β‐Cell subcellular localization of glucose‐stimulated Mn uptake by X‐ray fluorescence microscopy: implications for pancreatic MRI

Manganese (Mn) is a calcium (Ca) analog that has long been used as a magnetic resonance imaging (MRI) contrast agent for investigating cardiac tissue functionality, for brain mapping and for neuronal tract tracing studies. Recently, we have extended its use to investigate pancreatic β‐cells and showed that, in the presence of MnCl₂, glucose‐activated pancreatic islets yield significant signal enhancement in T₁‐weigheted MR images. In this study, we exploited for the first time the unique capabilities of X‐ray fluorescence microscopy (XFM) to both visualize and quantify the metal in pancreatic β‐cells at cellular and subcellular levels. MIN‐6 insulinoma cells grown in standard tissue culture conditions had only a trace amount of Mn, 1.14 ± 0.03 × 10^?11 µg/µm², homogenously distributed across the cell. Exposure to 2 m m glucose and 50 µ m MnCl₂ for 20 min resulted in nonglucose‐dependent Mn uptake and the overall cell concentration increased to 8.99 ± 2.69 × 10^?11 µg/µm². When cells were activated by incubation in 16 m m glucose in the presence of 50 µ m MnCl₂, a significant increase in cytoplasmic Mn was measured, reaching 2.57 ± 1.34 × 10^?10 µg/µm². A further rise in intracellular concentration was measured following KCl‐induced depolarization, with concentrations totaling 1.25 ± 0.33 × 10^?9 and 4.02 ± 0.71 × 10^?10 µg/µm² in the cytoplasm and nuclei, respectively. In both activated conditions Mn was prevalent in the cytoplasm and localized primarily in a perinuclear region, possibly corresponding to the Golgi apparatus and involving the secretory pathway. These data are consistent with our previous MRI findings, confirming that Mn can be used as a functional imaging reporter of pancreatic β‐cell activation and also provide a basis for understanding how subcellular localization of Mn will impact MRI contrast. Copyright © 2011 John Wiley & Sons, Ltd.     

Lenograstim with or without dexamethasone for neutrophil mobilization in healthy donors: short-term kinetics of white blood cells and effects of granulocyte apheresis

Objectives : To determine the optimal time schedule for neutrophil collection after single mobilization with glycosylated recombinant granulocyte colony‐stimulating factor (G‐CSF, lenograstim) with or without dexamethasone (DXM). Donors and Methods : In this prospective randomized trial, 26 healthy volunteers were randomly assigned to a single subcutaneous dose of lenograstim 6 μg/kg plus 8‐mg DXM (G‐CSF/DXM, n = 13) or placebo (G‐CSF/placebo, n = 13). Hematological and biochemical parameters were analyzed before and 12, 15, 18, 21, 24, 27, 29, 36, 48, 60, 72, and 84 h and 7 and 30 days after mobilization. Six G‐CSF/DXM subjects underwent standard neutrophil apheresis (NA) 12 and 36 h after mobilization. Results : Polymorphonuclear neutrophil (PMN) counts 12 and 21 h after mobilization were 22.7 (16.6?32.8) × 10⁹/L and 22.4 (18.6?30.6) × 10⁹/L for G‐CSF/placebo versus 33.1 (24.2–44.9) × 10⁹/L and 32.5 (17.4–39.6) × 10⁹/L for G‐CSF/DXM. This mobilization plateau was followed by slow normalization at 72–84 h. The six NA subjects had median PMN yields of 62 (47–101) × 10⁹ and 39 (23–42) × 10⁹ per therapeutic unit. After the first apheresis, PMN counts sharply decreased to 21.1 (14.8–26.3) × 10⁹/L and then temporarily recovered to 25.9 (18.9–36.5) × 10⁹/L (P ≤ 0.001) over the next 8 h. Conclusions : Single doses of lenograstim with or without DXM induced a PMN plateau that lasted 9 h (12–21 h after mobilization), with PMN counts suitable for neutrophil collection. Lenograstim plus DXM made it possible to perform NA twice, 12 and 36 h after mobilization. © 2011 Wiley Periodicals, Inc.     

Intestinal permeability and P‐glycoprotein‐mediated efflux transport of ticagrelor in Caco‐2 monolayer cells

Ticagrelor is the unique reversible oral antiplatelet drug commercialized today. During this study, the intestinal permeability of ticagrelor and its potential P‐glycoprotein (P‐gp)‐mediated active transport were assessed. To this end, bidirectional transport of ticagrelor was performed across Caco‐2 (human epithelial colorectal adenocarcinoma) monolayer model in the presence and absence of potent P‐gp inhibitor valspodar. Ticagrelor presented an apical–basolateral apparent permeability coefficient (P_app) of 6.0 × 10^?6 cm/s. On the other hand, mean efflux ratio (ER) of 2.71 was observed for ticagrelor describing a higher efflux permeability compared to the influx component. Valspodar showed a significant inhibitory effect on the efflux of ticagrelor suggesting involvement of P‐gp in its oral disposition. Co‐incubation of the P‐gp inhibitor decreased the efflux P_app of ticagrelor from 1.60 × 10^?5 to 1.13 × 10^?5 cm/s and decreased its ER by 70%. Results suggest a modest active transport of ticagrelor by P‐gp across the Caco‐2 cell monolayer. The co‐administration of ticagrelor with a P‐gp inhibitor seems altogether unlikely to have an extended impact on pharmacokinetics of ticagrelor and cause bleeding events in patients.     

Antispasmodic effects of eugenol on rat airway smooth muscle

This study was undertaken to assess the effects of eugenol (EUG) on tracheal muscle (TM) and the putative mechanisms underlying these effects. Cumulatively increasing concentrations (1–1000 μm ) of EUG did not affect the resting tonus of TM. However, EUG (1–2000 μm ) reduced the contractions induced by electrical field stimulation (IC₅₀ = 842.3 ± 52.7 μm ), an effect that was unaltered by either 10 μm montelukast (IC₅₀ = 816.1 ± 70.1 μm ) or 2 μm indomethacin (IC₅₀ = 693.1 ± 170.8 μm ). EUG also completely relaxed the sustained contractile responses to 80 mM K⁺ (IC₅₀ = 597.3 ± 60.6 μm ) and 1 μm carbamoylcholine (IC₅₀ = 571.3 ± 148.8 μm ), an effect that was unaltered by indomethacin (2 μm ). Under Ca²⁺‐free conditions, EUG reduced the ACh‐induced contractions (IC₅₀ = 703.4 ± 256.1 μm ), the CaCl₂‐induced contractions in preparations pretreated with 60 μm ACh in the presence of nifedipine, and the Ba²⁺‐induced contractions in preparations depolarized with K⁺. In tracheal preparations maintained in Ca²⁺‐containing solution, EUG (300–2000 μm ) relaxed the contractile response to phorbol dibutyrate (1 μm ), an activator of protein kinase C. It is concluded that in TM, EUG induces a myogenic antispasmodic effect (not modulated by arachidonic acid derivatives) either through various mechanisms almost with the same pharmacological potency or via an action on a step common to all of them. These mechanisms seem to include blockade of voltage‐ and receptor‐operated Ca²⁺ channels, IP₃‐induced Ca²⁺ release from sarcoplasmic reticulum and reduction of the sensitivity of contractile proteins to Ca²⁺.     

New insights on relaxant effects of (—)‐borneol monoterpene in rat aortic rings

The monoterpene alcohol (?)‐borneol has many biological effects such as sedative, anti‐inflammatory, analgesic, anti‐nociceptive, antithrombotic and vasorelaxant effects. Our objective in this study was to investigate the mechanism of action of (?)‐borneol and determine its vasorelaxant effect. (?)‐Borneol was tested on isolated aortic rings contracted with PE (10^?6 m ). This study was performed in the absence or in the presence of endothelium, L‐NAME (100 μm ), indomethacin (10 μm ), TEA (1 and 10 mm ), 4‐AP (1 mm ) or glibenclamide (1 mm ) to assess the participation of EDRF, nitric oxide, prostanoids and potassium channels on the relaxing effect of (?)‐borneol. In this work, (?)‐borneol induced a relaxant effect in aortic rings, with and without endothelium, in a concentration‐dependent manner. The pharmacological characterization obtained using L‐NAME, indomethacin, TEA, 4‐AP and glibenclamide demonstrates that the effect of (?)‐borneol was modified in the presence of L‐NAME, indomethacin and glibenclamide showing that these signal transduction pathways are involved in the relaxing effect of the monoterpene. (?)‐Borneol has a vasorelaxant effect that depends on the presence of vascular endothelium, with the participation of nitric oxide and prostanoids. Also, (?)‐borneol displayed a direct action on the vascular smooth muscle, greatly dependent on K_ATP channels.     

Effect of long‐chain triglyceride lipid emulsion on bupivacaine‐induced changes in electrophysiological parameters of rabbit Purkinje cells

Lipid emulsions are used in the reversal of local anesthetic toxicity. The aim of this study was to investigate the cellular electrophysiological effects of long‐chain triglyceride lipid emulsion (LCTE) on cardiac action potential characteristics and conduction disturbances induced by bupivacaine. Purkinje fibers were dissected from the left ventricle of New Zealand white rabbit hearts and superfused with either Tyrode's solution during 30 min (control group), with bupivacaine 10^?6 m , 10^?5 m , and 5.10^?5 m alone, or in the presence of LCTE 0.5%, in addition, LCTE at 0.1%, 0.5%, and 1% was perfused alone. Electrophysiological parameters were recorded using the conventional microelectrode technique (37 °C, 1 Hz frequency). Bupivacaine 5.10^?5 m ‐induced conduction blocks (8/8 preparations): LCTE 0.5% suppressed the bupivacaine 5.10^?5 m ‐induced conduction blocks (1/8 preparations). Exposure to bupivacaine 10^?6 m , 10^?5 m , and 5.10^?5 m resulted in a significant decrease in the maximal rate of depolarization (V_max) (respectively, 25%, 55%, 75%; P < 0.002 vs. control group). In the presence of LCTE 0.5%, bupivacaine 10^?6 m did not significantly decreased V_max (13%; P = 0.10 vs. control group). The decrease in V_max resulting from bupivacaine 10^?5 m alone was significantly less in the presence of LCTE 0.5% (P < 0.01 vs. bupivacaine 10^?5 m alone). Exposure to bupivacaine 10^?6 m , 10^?5 m , and 5.10^?5 m alone or in the presence of LCTE 0.5% resulted in a significant decrease in action potential duration measured at 50% and 90% repolarization (APD₅₀ and APD₉₀; P < 0.01 vs. control group). LCTE inhibited the Purkinje fibers conduction blocks induced by bupivacaine. Moreover, LCTE 0.5% attenuates the decrease in V_max induced by bupivacaine 10^?6 m and 10^?5 m .     

Mannitol clearance for the determination of glomerular filtration rate–a validation against clearance of 51Cr‐EDTA

We studied the agreement between plasma clearance of mannitol and the reference method, plasma clearance of ⁵¹Cr‐EDTA in outpatients with normal to moderately impaired renal function. Forty‐one patients with a serum creatinine <200 μmol l^?1 entered the study. ⁵¹Cr‐EDTA clearance was measured with the standard bolus injection technique and glomerular filtration rate (GFR) was calculated by the single‐sample method described by Jacobsson. Mannitol, 0·25 g kg^?1 body weight (150 mg ml^?1), was infused for 4–14 min and blood samples taken at 1‐, 2‐, 3‐ and 4‐h (n = 24) or 2‐, 3‐, 3·5‐ and 4‐h after infusion (n = 17). Mannitol in serum was measured by an enzymatic method. Plasma clearance for mannitol and its apparent volume of distribution (Vd) were calculated according to Brøchner‐Mortensen. Mean plasma clearance (±SD) for ⁵¹Cr‐EDTA was 59·7 ± 18·8 ml min^?1. The mean plasma clearance for mannitol ranged between 57·0 ± 20·1 and 61·1 ± 16·7 ml min^?1 and Vd was 21·3 ± 6·2% per kg b.w. The between‐method bias ranged between ?0·23 and 2·73 ml min^?1, the percentage error between 26·7 and 39·5% and the limits of agreement between ?14·3/17·2 and ?25·3/19·9 ml min^?1. The best agreement was seen when three‐ or four‐sample measurements of plasma mannitol were obtained and when sampling started 60 min after injection. Furthermore, accuracy of plasma clearance determinations was 88–96% (P30) and 41–63% (P10) and was highest when three‐ or four‐sample measurements of plasma mannitol were obtained, including the first hour after the bolus dose. We conclude that there is a good agreement between plasma clearances of mannitol and ⁵¹Cr‐EDTA for the assessment of GFR.     

Is the effect of acute hyperglycaemia on interdigestive antroduodenal motility and small-bowel transit mediated by insulin?

Acute hyperglycaemia inhibits antroduodenal motility. In non-diabetic subjects this inhibitory effect may result from reactive endogenous hyperinsulinaemia. Therefore, we investigated the effects of hyperinsulinaemia during both hyperglycaemia and euglycaemia on interdigestive antroduodenal motility (perfusion manometry) and duodenocaecal transit time (DCTT; lactulose breath-H₂ test). Six healthy volunteers (age 20–26 years) were studied for 240 min on three separate occasions in random order during: (a) i.v. saline (control); (b) acute hyperglycaemic hyperinsulinaemia (HG) with plasma glucose at 15 mmol L^?1; and (c) euglycaemic hyperinsulinaemia (HI) with plasma insulin at 80 mU L^?1 and glucose at 4–5 mmol L^?1. Results: DCTT was significantly (P < 0.05) prolonged during HG (158 ± 23 min) compared with control (95 ± 25 min), whereas HI had no effect (100 ± 17 min). Mean duration of complete migrating motor complex (MMC) cycles was significantly (P < 0.05) reduced during HG (63 ± 9 min) compared with control (103 ± 15 min) and HI (105 ± 16 min), which resulted from a significantly (P < 0.05) shorter duration of phase II. Antral motility was significantly (P < 0.05) reduced during both HI (20 ± 8 contractions 240 min^?1) and HG (9 ± 5) compared with control (43 ± 7). It is concluded that in healthy subjects hyperglycaemia prolongs DCTT, increases duodenal MMC cycle frequency and inhibits antral motility. Hyperinsulinaemia reduces antral motor activity but has no effect on interdigestive duodenal motility or DCTT. Thus, other factors, apart from insulin, mediate the inhibitory effect of hyperglycaemia on interdigestive intestinal motility and transit.     

Effect of the selective serotonin reuptake inhibitor paroxetine on platelet function is modified by a SLC6A4 serotonin transporter polymorphism

Summary. Background: Selective serotonin reuptake inhibitors (SSRIs) have been associated with an increased bleeding tendency. Objectives: To prospectively quantify the dose‐response effects of paroxetine and the influence of the serotonin transporter gene (SLC6A4) promoter polymorphism (5‐HTTLPR) on platelet function. Methods: Nineteen drug‐free psychiatric outpatients (44.5 ± 10.8 years) were tested before and after 6 weeks of paroxetine treatment (20 mg day^?1). Based on clinical symptoms, paroxetine dosages were increased (40–50 mg day^?1) for 6 more weeks in 11 patients. Parameters related to platelet function were assessed by bleeding time, platelet function analyzer (PFA), platelet serotonin, platelet factor 4 (PF4), β‐thromboglobulin (β‐TG), and aggregation tests. Results: Paroxetine 20 mg day^?1 increased mean bleeding time by 1.2 min (95% confidence interval (95% CI) ?0.2–2.7) and reduced median platelet serotonin level (463 ng 10^?9 platelets; inter quartile range (IQR) 361–666), and platelet ß‐TG concentration (3.1 IU 10^?6 platelets; IQR 0.3–6.0). Other platelet parameters did not change significantly. Serial platelet aggregation tests did not become abnormal. Paroxetine dose‐escalation did not further influence platelet function. However, 5‐HTTLPR polymorphisms modified these effects: in L_A/L_A‐carriers, bleeding times did not change (?0.2 min; 95% CI ?0.6 to 0.9), while bleeding times significantly increased in <2L_A‐allele carriers (2.3 min; 95% CI 0.5 to 4.07; P = 0.032). Platelet serotonin decreases were larger in patients without L_A‐alleles (868 ng 10^?9 platelets; IQR 585 to 1213) than in ≥1 L_A‐allele carriers (457 ng 10^?9 platelets; IQR 392 to 598; P = 0.035). PFA closure time and PF4 increased significantly in patients without L_A‐alleles. Conclusions: Paroxetine 20 mg day^?1 does not increase overall bleeding time, but impairs platelet function by decreasing the levels of platelet serotonin and platelet ß‐TG. These paroxetine effects appear to be mediated by 5‐HTTLPR, with most pronounced effects in patients without L_A‐alleles.     

Influence of methoctramine on the acetylcholine-isoprenaline functional antagonism in the guinea pig isolated trachea

Summary— It has been suggested that activation of muscarinic M₂ receptors is one of the components of the functional antagonism between muscarinic and β-adrenoceptor agonists in canine and guinea pig tracheal smooth muscle. The aim of the present study was to determine in the guinea pig trachea the importance of this component according to the magnitude of the acetylcholine-induced contraction. Cumulative concentration-response curves for isoprenaline were obtained in the absence or presence of the muscarinic M₂ receptor antagonist methoctramine (3 × 10^?7 M) in tracheal rings under basal tension or precontracted by acetylcholine 2 × 10^?7, 3 × 10^?6 and 10^?4 M, giving contractions of 25, 50 or 75%, respectively, of the maximal tension induced by acetylcholine 3 × 10^?3 M. In the absence of methoctramine, acetylcholine induced a concentration-dependent shift of the concentration-response curves of isoprenaline (-log EC₅₀ of isoprenaline are 8.09 ± 0.07, 7.85 ± 0.08, 7.38 ± 0.12 and 6.49 ± 0.12, n = 6 for basal tension and for acetylcholine concentrations of 2 × 10^?7, 3 × 10^?6 and 10^?4 M, respectively). In the presence of methoctramine, the basal -log EC₅₀ of isoprenaline was unmodified, whereas the acetylcholine-induced shifts of concentration-response curves of isoprenaline were abolished for low levels of contraction (25%) and significantly reduced to 50 and 75% levels of contraction. Under similar conditions, acetylcholine-induced shifts of concentration-response curves of isoprenaline were unmodified by the muscarinic M₁ receptor antagonist pirenzepine (10^?7 M). These results suggest that the inhibitory effect of M₂ receptors on β-adrenoceptor agonists effects is important for low contraction levels induced by acetylcholine, and that this effect becomes less important for higher concentrations of acetylcholine.