Frank Begrow

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.

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

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). 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). In PKG ITG hearts, the effects of CNP on cardiac contractility and relaxation as well as on PLB phosphorylation were markedly enhanced. 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. 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 reponses to β‐adrenergic stimulation were not different between genotypes. 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.

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.

The Effect of Nigellone and Thymoquinone on Inhibiting Trachea Contraction and Mucociliary Clearance

NIGELLA SATIVA L. has many effects including those on the gastrointestinal tract and trachea and is, therefore, used in the Mediteranean area and in India/Pakistan. Our aim was to investigate the effect of two main constituents, nigellone and thymoquinone, on trachea (antispasmodic effect) and their influence on respiratory clearance. The effects on Ba (2+)-, carbachol- and leukotriene-induced trachea contractions and the transport of the fluorescence dye rhodamin B concerning ciliary action in the tracheal area were investigated using a microdialysis technique. Nigellone and high concentrations of thymoquinone had a concentration-dependent inhibitory effect on the trachea when being contracted by the depolarizing effect of Ba (2+). The trachea contractions induced by leukotriene-d (4) were inhibited by nigellone and by thymoquinone. The cholinergic system (stimulation by carbachol) was hardly involved. The rate of ciliary clearance (mucociliary transport) was slightly modified by a high thymoquinone concentration (153.0 vs. 505.0 sec/12 mm distance, respectively), and was highly increased by nigellone (217.5 vs. 505.0 sec/12 mm distance). In conclusion, this study provides evidence for an antispasmodic effect and an increase in mucociliary clearance for nigellone but not for thymoquinone. Altogether the data indicate that nigellone but not thymoquinone may be useful in treatment of different respiratory diseases.

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.

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

IntroductionMyrtol 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.MethodsData 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.ResultsMyrtol 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.ConclusionIn 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.

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.

Impact and benefit of A2B-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 A2B 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.