Effects of Extracts and Flavonoids from Drosera rotundifolia L. on Ciliary Beat Frequency and Murine Airway Smooth Muscle.

Extracts from Drosera rotundifolia are traditionally used to treat cough symptoms during a common cold. The present study aimed to investigate the impact of extracts from D. rotundifolia and active compounds on the respiratory tract. Tracheal slices of C57BL/6N mice were used ex vivo to examine effects on airway smooth muscle (ASM) and ciliary beat frequency (CBF). Phosphodiesterase (PDE) inhibition assays were carried out to test whether PDE1 or PDE4 are targeted by the active compounds. An ethanol-water extract, as well as an aqueous fraction of this extract, exerted antispasmodic properties against acetylcholine-induced contractions. In addition, contractions induced by 60 mM K+ were abrogated by the aqueous fraction. Effects on ASM could be attributed to the flavonoids quercetin, 2″-O-galloylhyperoside and hyperoside. Moreover, the Drosera extract and the aqueous fraction increased the CBF of murine tracheal slices. Quercetin and 2″-O-galloylhyperoside were identified as active compounds involved in the elevation of CBF. Both compounds inhibited PDE1A and PDE4D. The elevation of CBF was mimicked by the subtype-selective PDE inhibitor rolipram (PDE4) and by 8-methoxymethyl-IBMX. In summary, our study shows, for the first time, that a Drosera extract and its flavonoid compounds increase the CBF of murine airways while antispasmodic effects were transferred to ASM.

In vitro and in vivo biotransformation of WMS-1410, a potent GluN2B selective NMDA receptor antagonist.

Structural modification of the GluN2B selective NMDA receptor antagonist ifenprodil led to the 3-benzazepine WMS-1410 with similar GluN2B affinity but higher receptor selectivity. Herein the in vitro and in vivo biotransformation of WMS-1410 is reported. Incubation of WMS-1410 with rat liver microsomes and different cofactors resulted in four hydroxylated phase I metabolites, two phase II metabolites and five combined phase I/II metabolites. With exception of catechol 4, these metabolites were also identified in the urine of a rat treated with WMS-1410. However the metabolites 7, 8 and 12 clearly show that the catechol metabolite 4 was also formed in vivo. As shown for ifenprodil the phenol of WMS-1410 represents the metabolically most reactive structural element. The biotransformation of WMS-1410 is considerably slower than the biotransformation of ifenprodil indicating a higher metabolic stability. From the viewpoint of metabolic stability the bioisosteric replacement of the phenol of WMS-1410 by a metabolically more stable moiety should be favourable.

Metabolism studies of ifenprodil, a potent GluN2B receptor antagonist.

The NMDA receptor antagonist ifenprodil is an important lead structure for developing new GluN2B selective NMDA receptor antagonists. Ifenprodil itself has a high affinity to the GluN2B subunit but a poor selectivity for the NMDA receptor. This aspect and the fast biotransformation are the major drawbacks of ifenprodil. In order to optimize the development of new and more selective GluN2B (NMDA) receptor antagonists, the identification of the main metabolic pathways of ifenprodil is necessary. Herein the in vitro and in vivo phase I and phase II metabolites of ifenprodil were generated and analyzed via LC-MS(n) experiments. In vitro experiments were carried out with rat liver microsomes and various co-factors to generate phase I and phase II metabolites. The application of ifenprodil to a rat and the analysis of its urine led to the identification of diverse formed in vivo metabolites. The phenol represents the metabolically most labile structural element since glucuronide 7 and 8 appeared as main metabolites.

Impact and benefit of A(2B)-adenosine receptor agonists for the respiratory tract: mucociliary clearance, ciliary beat frequency, trachea muscle tonus and cytokine release.

OBJECTIVES: Adenosine is known to induce a bronchospasm in asthma- and COPD patients. The role of A(2B) receptors was investigated with respect to several parameters of the respiratory tract: tonus of smooth muscle, ciliary beat frequency as measured by high-speed video camera connected to a microscope (both in rats) and mucociliary clearance (MCC; transport of a fluorescent dye using a microdialysis procedure) in mice. KEY FINDINGS: NECA (5'-N-ethylcarboxamidoadenosine) (a non-selective adenosine receptor agonist) was able to acutely induce a contraction, which was reversed to a relaxation after repeated dosing. This relaxation was completely abolished by PSB-1115, an A(2B) receptor antagonist. IL-13 (cytokine) was not involved mediating acute contractility effects. MCC was increased by BAY 60-6583 (A(2B) receptor agonist) and NECA (counteracted by the A(2B) receptor antagonist PSB-1115). Activation of A(2B) adenosine receptors by BAY 60-6583 induced an increase of the ciliary beat frequency, which could be reduced by administration of PSB-1115. Several cytokines were increased by NECA although only some are relevant because they are not blocked by A(2B) receptor antagonism. CONCLUSIONS: The A(2B) receptors are involved in airway relaxation, MCC improvement and ciliary beat frequency. A(2B) receptor agonists may be of therapeutic value and should be developed.

Effect of myrtol standardized and other substances on the respiratory tract: ciliary beat frequency and mucociliary clearance as parameters.

INTRODUCTION: Myrtol standardized is a phytomedicine obtained by distillation, consisting of many constituents. In vitro and in vivo, the major monterpenes, d-limonene, 1,8-cineole, and alpha-pinene, are used as biological marker substances. Myrtol standardized has secretolytic, secretomotor, and mucolytic effects in addition to anti-inflammatory and antioxidative actions. The aim of the study was to investigate the effects of different concentrations of Myrtol standardized on in vivo mucociliary clearance in mice and the ciliary beat frequency (CBF) in rat tracheal rings. METHODS: Data regarding the effects of 1,8-cineole and N-acetylcysteine (NAC) were compared. Salbutamol was used as a positive control. CBF was measured using rat tracheal explants and a high-speed video camera linked to a microscope with specific software equipment. Mucociliary clearance was determined using the microdialysis technique, which measured the acceleration of a fluorescent sample in the trachea in vivo. RESULTS: Myrtol standardized accelerated both CBF and mucociliary transport in a concentration-dependent manner. Significant effects were seen at a concentration of 0.01% Myrtol regarding CBF (P<0.01) and 17.1 mg/kg body weight regarding mucociliary clearance experiments (P<0.05) according to doses relevant to humans. The 1,8-cineole dosage relative to humans only accelerated the mucociliary clearance in vivo without having an effect on the CBF. Extremely high doses of Myrtol were not able to additionally increase the CBF effect in comparison to salbutamol. Compared to NAC, also used in a dosage relative to humans, Myrtol standardized showed a tendency to be more effective. CONCLUSION: In summary, the present data suggest that Myrtol standardized is a pharmacologically important drug which, when used at a dose relative to humans, shows positive effects on both measured parameters, CBF and mucociliary clearance, in vivo.

Effect of silymarin and harpagoside on inflammation reaction of BEAS-2B cells, on ciliary beat frequency (CBF) of trachea explants and on mucociliary clearance (MCC).

Silymarin and harpagoside are derived from drugs which are used for their protective effects against hepatotoxicity and inflammatory processes. Both are now investigated with respect to the respiratory tract. They were able to reduce the release of the inflammatory cytokine RANTES (regulated on activation, normal T cells expressed and secreted) from BEAS-2B cells in a concentration-dependent manner when stimulated by a cytokine mix (10 ng/mL of TNF- α and IFN- γ). This effect was not due to a possible toxic effect (control experiments using LDH release as a marker). Silymarin but not harpagoside was able to increase ciliary beat frequency. Effects were comparable to positive controls (isoprenaline and salbutamol). Silymarin also increases mucociliary clearance. In conclusion, silymarin should be further investigated for its clinical use in distinct respiratory diseases.

Impact of thymol in thyme extracts on their antispasmodic action and ciliary clearance.

Thyme is a herb with broncholytic und secretomotoric effects. Its activity on beta (2) receptors as a possible mechanism of action was demonstrated. Major components are thymol and carvacrol which are claimed to be responsible for its effects and, therefore, used for standardization in the German pharmacopoeia (0.03 % phenols calculated as thymol). Our aim was to investigate the impact of thymol by using thyme extracts with either normal or extremely low thymol concentrations (< 0.005 % or > 0.038 %). The antispasmodic effect on smooth muscles of the trachea and the ileum and the effect on ciliary activity (respiratory clearance) were investigated. In addition, pure thymol and carvacrol were investigated separately and in spiking experiments. Thymol and carvacrol had a concentration-dependent antispasmodic effect in the rat trachea either being stimulated by acetylcholine, K (+) or Ba (++). The same result was observed with respect to the increase of mucociliary transport in mice. Extracts with very low thymol contents are effective in all models used except acetylcholine-induced rat ileum contraction. When thyme extracts with normal thymol contents or with very low thymol contents were compared, the extract with normal thymol contents was more effective, both as a relaxant (rat ileum) and as an antispasmodic compound (rat trachea contraction induced by either acetylcholine, Ba (++) or K (+)) and in ciliary transport experiments. Thyme extracts with very low thymol contents (practically free of volatile oil) were equally effective with respect to endothelin effects. When an extract with very low thymol contents is spiked with increasing concentrations of thymol, a concentration-dependent increase concerning the antispasmodic effect (Ba (++)-induced trachea contraction) is observed. In conclusion, the data show that in various models of antispasmodic effect (ileum and trachea) and by measuring ciliary activity, thymol (and carvacrol) is (are) active, although other not identified components of thyme extract appear to be very important as well, since extracts with very low thymol contents are active. On the basis of these results the standardization on thymol alone appears not to be justified.

Ventricular arrhythmias, increased cardiac calmodulin kinase II expression, and altered repolarization kinetics in ANP receptor deficient mice.

Cardiac hypertrophy is associated with ventricular arrhythmias and sudden death. The molecular mechanisms that predispose the hypertrophied heart to arrhythmias are not well understood. In mice, deletion of the gene coding for the atrial natriuretic peptide receptor, guanylyl cyclase A (GC-A-/-), causes arterial hypertension, cardiac hypertrophy and sudden death. We used this mouse model to study molecular mechanisms of arrhythmias in the hypertrophied heart. Right and left ventricular monophasic action potential durations (APD) were recorded in isolated, Langendorff-perfused hearts during pacing from the right atrium and ventricle. The atrioventricular (AV) node was ablated to provoke bradycardia. Intracellular Ca(2+) transients were measured in isolated INDO-1 loaded ventricular myocytes. Cardiac expression of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) was analyzed by western blotting. Polymorphic ventricular arrhythmias (pVT) occurred spontaneously after mechanical AV block in 20/45 hearts from 12-month-old GC-A-/- mice (P < 0.05), but neither in age-matched GC-A+/+ hearts nor in hearts from 3-month-old mice of either genotype. Triggered activity preceded pVT. APD were prolonged and systolic Ca(i)(2+) levels were increased in GC-A-/- hearts independently of age. In 12-month-old GC-A-/- hearts only, dispersion of APD and expression levels of CaMKII were increased. CaMKII expression was particularly increased in hearts with pVT. Direct inhibition of CaMKII activation by KN93 (0.5 or 2 microM) or inhibition of Ca(2+)/calmodulin-dependent activation of CaMKII by W-7 (25 microM) suppressed pVT in GC-A-/- hearts (P < 0.05) while prolonging APD. The combination of increased CaMKII activity and altered action potential characteristics facilitates ventricular arrhythmias in hypertrophic GC-A-/- hearts.

Transgenic triadin 1 overexpression alters SR Ca2+ handling and leads to a blunted contractile response to beta-adrenergic agonists.

OBJECTIVE: Ca2+ release from the cardiac junctional sarcoplasmic reticulum (SR) is regulated by a complex of proteins, including the ryanodine receptor (RyR), calsequestrin (CSQ), junctin (JCN), and triadin 1 (TRD). Moreover, triadin 1 appears to anchor calsequestrin to the ryanodine receptor. METHODS: To determine whether triadin 1 overexpression alters excitation-contraction coupling, we examined the effects of cardiac-specific overexpression of triadin 1 on SR Ca2+ handling and contractility in transgenic (TG) compared to wild-type (WT) mice. RESULTS: The overexpression of triadin 1 was associated with an enhanced SR Ca2+ load, reflected by a 22% higher amplitude of caffeine-induced Ca2+ transients. The decline of Ca2+ transients during caffeine exposure was prolonged by 57%. The detection of resting spontaneous SR Ca2+ release events (Ca2+ sparks) revealed an increased amplitude (by 16%), decline (by 47%), and width (by 47%) in TG. This was associated with a redistribution of Ca2+ spark amplitudes from one population to two populations. Measurement of cardiac function by echocardiography and left ventricular (LV) catheterization revealed a decreased cardiac contractility in vivo. The impaired response to beta-adrenergic receptor (beta-AR) stimulation in TG hearts was associated with an increased protein expression of beta-AR kinase 1. In addition, the increase of the L-type Ca2+ peak current and the increase of phospholamban (PLB) phosphorylation at Thr17 were reduced under beta-AR stimulation. CONCLUSION: Taken together, our data suggest that triadin 1 overexpression results in a complex modulation of SR Ca2+ handling, which may contribute, at least in part, to the depressed basal contractility and the blunted response to beta-adrenergic agonists in TG mice.

Altered signal transduction in cardiac ventricle overexpressing A(1)-adenosine receptors.

OBJECTIVE: The aim of the present study was to assess the effects of A(1)-adenosine receptor (A1-AR) stimulation in ventricle of A(1)-adenosine receptor overexpressing mice (transgenic mice, TG). METHODS: Effects of the A(1)-adenosine receptor agonist R-PIA ((-)-N(6)-phenylisopropyladenosine) on phosphorylation of phospholamban (PLB), Ca(2+) transients, Ca(2+) currents and cell shortening were studied in isolated ventricular cardiomyocytes. RESULTS: R-PIA alone did not affect contractility in isolated electrically stimulated cardiomyocytes from wild-type mice (WT) or TG. However, after pre-stimulation of beta-adrenoceptors by isoproterenol, R-PIA reduced contractility in cardiomyocytes from WT but increased contractility in TG. Under the same conditions, R-PIA reduced isoproterenol-stimulated currents through L-type Ca(2+) channels, Ca(2+) transients and phosphorylation of PLB in cardiomyocytes from WT. In contrast, R-PIA diminished phospholamban phosphorylation induced by isoproterenol but augmented isoproterenol-elevated currents through L-type Ca(2+) channels, and isoproterenol-heightened Ca(2+) transients in cardiomyocytes from TG. CONCLUSIONS: We suggest that A(1)-adenosine receptor overexpression reverses the interaction of beta-adrenergic and A(1)-adenosine receptor stimulation, at least in part. Hence, the receptor/effector coupling is dependent on receptor density in this model.

Increased effects of C-type natriuretic peptide on contractility and calcium regulation in murine hearts overexpressing cyclic GMP-dependent protein kinase I.

1. C-type natriuretic peptide (CNP) and its receptor guanylyl cyclase (GC-B) are expressed in the heart and modulate cardiac contractility in a cGMP-dependent manner. Since the distal cellular signalling pathways remain unclear, we evaluated the peptide effects on cardiac function and calcium regulation in wild-type (WT) and transgenic mice with cardiac overexpression of cGMP-dependent protein kinase I (PKG ITG). 2. In isolated, perfused working WT hearts, CNP (10 nm) provoked an immediate increase in the maximal rates of contraction and relaxation, a small increase in the left ventricular systolic pressure and a decrease in the time of relaxation. These changes in cardiac function were accompanied by a marked increase in the levels of Ser16-phosphorylated phospholamban (PLB). 3. In PKG ITG hearts, the effects of CNP on cardiac contractility and relaxation as well as on PLB phosphorylation were markedly enhanced. 4. CNP increased cell shortening and systolic Cai2+ levels, and accelerated Cai2+ decay in isolated, Indo-1/AM-loaded WT cardiomyocytes, and these effects were enhanced in PKG I-overexpressing cardiomyocytes. 5. 8-pCPT-cGMP, a membrane-permeable PKG activator, mimicked the contractile and molecular actions of CNP, the effects again being more pronounced in PKG ITG hearts. In contrast, the cardiac responses to beta-adrenergic stimulation were not different between genotypes. 6. Taken together, our data indicate that PKG I is a downstream target activated by the CNP/GC-B/cGMP-signalling pathway in cardiac myocytes. cGMP/PKG I-stimulated phosphorylation of PLB and subsequent activation of the sarcoplasmic reticulum Ca2+ pump appear to mediate the positive inotropic and lusitropic responses to CNP.

Pressure-independent cardiac hypertrophy in mice with cardiomyocyte-restricted inactivation of the atrial natriuretic peptide receptor guanylyl cyclase-A.

Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Atrial natriuretic peptide (ANP) has been postulated to exert local antihypertrophic effects in the heart. Thus, a loss of function of the ANP receptor guanylyl cyclase-A (GC-A) might contribute to the increased propensity to cardiac hypertrophy, although a causative role in vivo has not been definitively demonstrated. To test whether local ANP modulates cardiomyocyte growth, we inactivated the GC-A gene selectively in cardiomyocytes by homologous loxP/Cre-mediated recombination. Thereby we have circumvented the systemic, hypertensive phenotype associated with germline inactivation of GC-A. Mice with cardiomyocyte-restricted GC-A deletion exhibited mild cardiac hypertrophy, markedly increased mRNA expression of cardiac hypertrophy markers such as ANP (fivefold), alpha-skeletal actin (1.7-fold), and beta-myosin heavy chain (twofold), and increased systemic circulating ANP levels. Their blood pressure was 7-10 mmHg below normal, probably because of the elevated systemic levels and endocrine actions of ANP. Furthermore, cardiac hypertrophic responses to aortic constriction were enhanced and accompanied by marked deterioration of cardiac function. This phenotype is consistent with a local function of the ANP/GC-A system to moderate the molecular program of cardiac hypertrophy.

Junctional sarcoplasmic reticulum transmembrane proteins in the heart.

The uptake of calcium into the sarcoplasmic reticulum (SR) and its subsequent release from the SR play a key role in the regulation of the cytosolic calcium concentration and the excitation-contraction coupling in cardiac muscle. While calcium uptake, catalyzed by the calcium-dependent ATPase, is thought to occur throughout the SR, the release of calcium is controlled by a complex of proteins localized to a distinct region, the junctional SR. This complex consists of the calcium release channel or ryanodine receptor (RyR), the high capacity calcium-binding protein calsequestrin located in the lumen of the junctional SR, and the junctional SR transmembrane proteins triadin 1 and junctin which are hypothesized to anchor calsequestrin to the RyR. Transgenic mice with cardiac-specific overexpression of triadin 1 or junctin show distinct cardiac phenotypes with altered cellular and subcellular morphology, changes in contractile properties and/or in the expression of junctional SR proteins suggesting that these junctional sarcoplasmic reticulum transmembrane proteins are of functional relevance for the regulation of calcium release in the heart.