Marc Brede

Publications of Marc Brede

• α(2)-adrenoceptors do not mediate neuroprotection in acute ischemic stroke in mice.

  Authors: Marc Brede, Stefan Braeuninger, Friederike Langhauser, Lutz Hein, Norbert Roewer, Guido Stoll, Christoph Kleinschnitz

  Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 07/2011; 31(10):e1-7.

  We assessed the neuroprotective potential of α(2)-adrenoceptors in ischemic stroke using mice with targeted deletions of individual α(2)-adrenoceptor subtypes (α(2A)(-/-), α(2B)(-/-), α(2C)(-/-),We assessed the neuroprotective potential of α(2)-adrenoceptors in ischemic stroke using mice with targeted deletions of individual α(2)-adrenoceptor subtypes (α(2A)(-/-), α(2B)(-/-), α(2C)(-/-), α(2A/C)(-/-)). The effects of the α(2)-adrenoceptor agonist clonidine were studied in parallel. Focal cerebral ischemia was induced with or without clonidine pretreatment by transient middle cerebral artery occlusion. Neurologic outcome and infarct volumes were evaluated on day 1. Cerebral blood flow (CBF) and mean arterial pressure were determined. α(2)-Adrenoceptor null mice did not display larger infarct volumes compared with wild-type (WT) mice under basal conditions (P>0.05). In line with this finding, pretreatment with clonidine did not protect from ischemic brain damage in WT mice or α(2A)(-/-), α(2B)(-/-), and α(2C)(-/-) mice. Clonidine induced smaller infarct volumes only in α(2A/C)(-/-) mice (P<0.05), but this did not translate into improved neurologic function (P>0.05). Importantly, while clonidine caused a significant decrease in arterial blood pressure in all groups, it had no blood pressure lowering effect in α(2A/C)(-/-) mice, and this correlated with higher CBF and smaller infarct volumes in this group. In summary, we could not demonstrate a neuroprotective function of α(2)-adrenoceptors in focal cerebral ischemia. Careful controlling of physiological parameters relevant for stroke outcome is recommended in experimental stroke studies.

• Phosducin influences sympathetic activity and prevents stress-induced hypertension in humans and mice.

  Authors: Nadine Beetz, Michael D Harrison, Marc Brede, Xiangang Zong, Michal J Urbanski, Anika Sietmann, Jennifer Kaufling, Michel Barrot, Mathias W Seeliger, Maria Augusta Vieira-Coelho [......] Mary Kaldunski, Rolf Nüsing, Bela Szabo, Howard J Jacob, Allen W Cowley, Martin Biel, Monika Stoll, Martin J Lohse, Ulrich Broeckel, Lutz Hein

  The Journal of clinical investigation. 12/2009; 119(12):3597-3612.

  Hypertension and its complications represent leading causes of morbidity and mortality. Although the cause of hypertension is unknown in most patients, genetic factors are recognized as contributingHypertension and its complications represent leading causes of morbidity and mortality. Although the cause of hypertension is unknown in most patients, genetic factors are recognized as contributing significantly to an individual's lifetime risk of developing the condition. Here, we investigated the role of the G protein regulator phosducin (Pdc) in hypertension. Mice with a targeted deletion of the gene encoding Pdc (Pdc-/- mice) had increased blood pressure despite normal cardiac function and vascular reactivity, and displayed elevated catecholamine turnover in the peripheral sympathetic system. Isolated postganglionic sympathetic neurons from Pdc-/- mice showed prolonged action potential firing after stimulation with acetylcholine and increased firing frequencies during membrane depolarization. Furthermore, Pdc-/- mice displayed exaggerated increases in blood pressure in response to post-operative stress. Candidate gene-based association studies in 2 different human populations revealed several SNPs in the PDC gene to be associated with stress-dependent blood pressure phenotypes. Individuals homozygous for the G allele of an intronic PDC SNP (rs12402521) had 12-15 mmHg higher blood pressure than those carrying the A allele. These findings demonstrate that PDC is an important modulator of sympathetic activity and blood pressure and may thus represent a promising target for treatment of stress-dependent hypertension.

• Genetic dissection of {alpha}2-adrenoceptor functions in adrenergic versus non-adrenergic cells.

  Authors: Ralf Gilsbach, Christoph Roeser, Nadine Beetz, Marc Brede, Kerstin Hadamek, Miriam Haubold, Jost Leemhuis, Melanie Philipp, Johanna Schneider, Michal Urbanski, Bela Szabo, David Weinshenker, Lutz Hein

  Molecular pharmacology. 03/2009;

  alpha2-adrenoceptors mediate diverse functions of the sympathetic system and are targets for the treatment of cardiovascular disease, depression, pain, glaucoma, and sympathetic activation duringalpha2-adrenoceptors mediate diverse functions of the sympathetic system and are targets for the treatment of cardiovascular disease, depression, pain, glaucoma, and sympathetic activation during opioid withdrawal. To determine whether alpha2-adrenoceptors on adrenergic neurons or alpha2-adrenoceptors on non-adrenergic neurons mediate the physiological and pharmacological responses of alpha2-agonists, we used the dopamine beta-hydroxylase (Dbh) promoter to drive expression of alpha2A-adrenoceptors exclusively in noradrenergic and adrenergic cells of transgenic mice. Dbh-alpha2A transgenic mice were crossed with double knockout mice lacking both alpha2A- and alpha2C-receptors to generate lines with selective expression of alpha2A-autoreceptors in adrenergic cells. These mice were subjected to a comprehensive phenotype analysis and compared to wild-type mice, which express alpha2A- and alpha2C-receptors in both adrenergic and non-adrenergic cells, and alpha2A/alpha2C double knockout mice, which do not express these receptors in any cell type. Surprisingly, only a few functions previously ascribed to alpha2-adrenoceptors were mediated by receptors on adrenergic neurons, including feedback inhibition of norepinephrine release from sympathetic nerves and spontaneous locomotor activity. Other agonist effects including analgesia, hypothermia, sedation and anesthetic-sparing were mediated by alpha2-receptors in non-adrenergic cells. In dopamine beta-hydroxylase knockout mice lacking norepinephrine, the alpha2-agonist medetomidine still induced a loss of the righting reflex, confirming that the sedative effect of alpha2-adrenoceptor stimulation is not mediated via autoreceptor-mediated inhibition of norepinephrine release. The present study paves the way for a revision of the current view of the alpha2-adrenergic receptors and it provides important new considerations for future drug development.

• Blockade of Bradykinin Receptor B1 but Not Bradykinin Receptor B2 Provides Protection From Cerebral Infarction and Brain Edema.

  Authors: Madeleine Austinat, Stefan Braeuninger, João B Pesquero, Marc Brede, Michael Bader, Guido Stoll, Thomas Renné, Christoph Kleinschnitz

  Stroke; a journal of cerebral circulation. 12/2008;

  BACKGROUND AND PURPOSE: Brain edema is detrimental in ischemic stroke and its treatment options are limited. Kinins are proinflammatory peptides that are released during tissue injury. The effects ofBACKGROUND AND PURPOSE: Brain edema is detrimental in ischemic stroke and its treatment options are limited. Kinins are proinflammatory peptides that are released during tissue injury. The effects of kinins are mediated by 2 different receptors (B1 and B2 receptor [B1R and B2R]) and comprise induction of edema formation and release of proinflammatory mediators. METHODS: Focal cerebral ischemia was induced in B1R knockout, B2R knockout, and wild-type mice by transient middle cerebral artery occlusion. Infarct volumes were measured by planimetry. Evan's blue tracer was applied to determine the extent of brain edema. Postischemic inflammation was assessed by real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry. To analyze the effect of a pharmacological kinin receptor blockade, B1R and B2R inhibitors were injected. RESULTS: B1R knockout mice developed significantly smaller brain infarctions and less neurological deficits compared to wild-type controls (16.8+/-4.7 mm(3) vs 50.1+/-9.1 mm(3), respectively; P<0.0001). This was accompanied by a dramatic reduction of brain edema and endothelin-1 expression, as well as less postischemic inflammation. Pharmacological blockade of B1R likewise salvaged ischemic tissue (15.0+/-9.5 mm(3) vs 50.1+/-9.1 mm(3), respectively; P<0.01) in a dose-dependent manner, even when B1R inhibitor was applied 1 hour after transient middle cerebral artery occlusion. In contrast, B2R deficiency did not confer neuroprotection and had no effect on the development of tissue edema. CONCLUSIONS: These data demonstrate that blocking of B1R can diminish brain infarction and edema formation in mice and may open new avenues for acute stroke treatment in humans.

• The anesthetic effects of etomidate: species-specific interaction with alpha 2-adrenoceptors.

  Authors: Andrea Paris, Lutz Hein, Marc Brede, Philipp-Alexander Brand, Jens Scholz, Peter H Tonner

  Anesthesia and analgesia. 01/2008; 105(6):1644-9, table of contents.

  BACKGROUND: The IV anesthetic, etomidate, has structural and clinical similarities to specific alpha2-adrenoceptor agonists such as dexmedetomidine. We investigated whether the sedative effects ofBACKGROUND: The IV anesthetic, etomidate, has structural and clinical similarities to specific alpha2-adrenoceptor agonists such as dexmedetomidine. We investigated whether the sedative effects of etomidate may be mediated by alpha2-adrenoceptors. METHODS: The anesthetic potency of etomidate (1-20 microM) was determined in Xenopus laevis tadpoles in the absence and presence of the specific alpha2-adrenoceptor antagonist atipamezole (10 microM). Anesthesia was defined as loss of righting reflex. Nonlinear logistic regression curves were fitted to the data and half-maximal effective concentrations and the slopes of the curves were calculated. Additionally, sedative/ hypnotic effects of etomidate (8 mg/kg IP) were studied by rotarod test in wild-type (WT) mice and mice carrying targeted deletions of the alpha2A-adrenoceptor gene (alpha2A-KO). Data are presented as mean +/- sem. RESULTS: The fraction of anesthetized tadpoles increased with increasing concentrations of etomidate. Atipamezole significantly increased the half-maximal effective concentration of etomidate (4.5 +/- 0.2 microM; slope: 2.6 +/- 0.3) to 8.4 +/- 0.4 microM (slope: 2.3 +/- 0.3). Etomidate resulted in time-dependent sedative effects in all mice, as assessed by rotarod performance. In WT mice, the sedative effects of etomidate were not decreased by atipamezole (2 mg/kg). Consistently, etomidate-induced sedation was not reduced in alpha2A-KO animals compared with WT mice. CONCLUSIONS: The sedative effects of etomidate exhibit a species-specific interaction with alpha2-adrenoceptors. Although the decrease in potency of etomidate by atipamezole may be caused by an interaction with alpha2-adrenoceptors in X. laevis tadpoles, results in mice indicate that the hypnotic effect of etomidate does not require alpha2-adrenoceptors.

• Heterozygous alpha 2C-adrenoceptor-deficient mice develop heart failure after transverse aortic constriction.

  Authors: Ralf Gilsbach, Marc Brede, Nadine Beetz, Eduardo Moura, Verena Muthig, Carolin Gerstner, Frederico Barreto, Stefan Neubauer, Maria Augusta Vieira-Coelho, Lutz Hein

  Cardiovascular research. 10/2007; 75(4):728-37.

  OBJECTIVE: Feedback regulation of norepinephrine release from sympathetic nerves is essential to control blood pressure, heart rate and contractility. Recent experiments in gene-targeted mice haveOBJECTIVE: Feedback regulation of norepinephrine release from sympathetic nerves is essential to control blood pressure, heart rate and contractility. Recent experiments in gene-targeted mice have suggested that alpha(2C)-adrenoceptors may operate in a similar feedback mechanism to control the release of epinephrine from the adrenal medulla. As heterozygous polymorphisms in the human alpha(2C)-adrenoceptor gene have been associated with cardiovascular disease including hypertension and chronic heart failure, we have sought to characterize the relevance of alpha(2C)-gene copy number for feedback control of epinephrine release in gene-targeted mice. METHODS: Adrenal catecholamine release, basal hemodynamics and susceptibility to develop heart failure after transverse aortic constriction were tested in mice with two copies (+/+), one copy (+/-) or no functional alpha(2C)-adrenoceptor gene (alpha(2C)-/-). RESULTS: Heterozygous alpha(2C)-receptor deletion (alpha(2C)+/-) resulted in a 43% reduction of adrenal alpha(2C) mRNA copy number and in a similar decrease in alpha(2)-receptor-mediated inhibition of catecholamine release from isolated adrenal glands in vitro. Urinary excretion of epinephrine was increased by 74+/-15% in alpha(2C)+/- and by 142+/-23% in alpha(2C)-/- mice as compared with wild-type control mice. Telemetric determination of cardiovascular function revealed significant tachycardia but no hypertension in alpha(2C)-adrenoceptor-deficient mice. alpha(2C)+/- mice were more susceptible to develop cardiac hypertrophy, failure and mortality after left-ventricular pressure overload than alpha(2C)+/+ mice. CONCLUSION: Adrenal alpha(2)-mediated feedback regulation of epinephrine secretion differs fundamentally from sympathetic feedback control. A single adrenoceptor subtype, alpha(2C), operates without a significant receptor reserve to prevent elevation of circulating epinephrine levels. This genetic model may provide an experimental basis to study the pathophysiology of alpha(2C)-adrenoceptor dysfunction in humans.

• The effects of dexmedetomidine on perinatal excitotoxic brain injury are mediated by the alpha2A-adrenoceptor subtype.

  Authors: Andrea Paris, Jean Mantz, Peter H Tonner, Lutz Hein, Marc Brede, Pierre Gressens

  Anesthesia and analgesia. 03/2006; 102(2):456-61.

  We performed the current study in mice lacking individual alpha2-adrenoceptor subtypes to elucidate the contribution of alpha(2)-adrenoceptor subtypes to the neuroprotective properties ofWe performed the current study in mice lacking individual alpha2-adrenoceptor subtypes to elucidate the contribution of alpha(2)-adrenoceptor subtypes to the neuroprotective properties of dexmedetomidine in a model of perinatal excitotoxic brain injury. On postnatal Day 5, wild-type mice and mice lacking alpha2A-adrenoceptor (alpha2A-KO) or alpha2C-adrenoceptor subtypes (alpha2C-KO) were randomly assigned to receive dexmedetomidine (3 microg/kg) or phosphate-buffered saline intraperitoneally. Thirty minutes after the intraperitoneal injection, the glutamatergic agonist ibotenate (10 microg) was intracerebrally injected, producing transcortical necrosis and white matter lesions that mimic perinatal human hypoxic-like lesions. Quantification of the lesions was performed on postnatal Day 10 by histopathologic examination. Dexmedetomidine reduced mean lesion size in the cortex of wild-type mice and alpha2C-KO mice by 44% and 49%, respectively. Ibotenate-induced white matter lesions were reduced by 71% (wild-type mice) and 75% (alpha2C-KO mice) after pretreatment with dexmedetomidine. In contrast, in alpha2A-KO mice, dexmedetomidine did not protect against the cortical excitotoxic insult, and white matter lesions were even more pronounced (82% increase of mean lesion size). Dexmedetomidine provides potent neuroprotection in a model of perinatal excitotoxic brain damage. This effect was completely abolished in alpha2A-KO mice, suggesting that the neuroprotective effect is mediated via the alpha2A-adrenoceptor subtype.

• Alpha2C-adrenoceptor polymorphism is associated with improved event-free survival in patients with dilated cardiomyopathy.

  Authors: Vera Regitz-Zagrosek, Berthold Hocher, Martin Bettmann, Marc Brede, Kerstin Hadamek, Carolin Gerstner, Hans Brendan Lehmkuhl, Roland Hetzer, Lutz Hein

  European heart journal. 03/2006; 27(4):454-9.

  AIMS: The sympathetic nervous system plays a central role in cardiac growth but its overstimulation is associated with increased mortality in patients with chronic heart failure. Pre-synapticAIMS: The sympathetic nervous system plays a central role in cardiac growth but its overstimulation is associated with increased mortality in patients with chronic heart failure. Pre-synaptic alpha2-adrenoceptors are essential feedback regulators to control the release of norepinephrine from sympathetic nerves. In this study we tested whether a deletion polymorphism in the human alpha2C-adrenoceptor gene (alpha2CDel322-325) affects progression of heart failure in patients with dilated cardiomyopathy (DCM). METHODS AND RESULTS: We genotyped and phenotyped 345 patients presenting with DCM in the heart transplant unit of the German Heart Institute, starting in 1994. Patients were treated according to guidelines (99% ACEI, 76% beta-blockers) and were followed until December 2002 or until a first event [death, heart transplantation, or implantation of a left ventricular assist device (LVAD) for a life-threatening condition] occurred. Mean follow-up time was 249 weeks (4.9 years) in event-free patients and 104 weeks (2 years) in patients with events. During follow-up, 51% of the patients exhibited an event: death (18%), implantation of LVAD as bridging for transplantation (7%), or heart transplantation (25%). By Kaplan-Meier analysis, DCM patients with the deletion variant Del322-325 in the alpha2C-adrenoceptor showed significantly decreased event rates (P=0.0043). Cox regression analysis revealed that the presence of the deletion was associated with reduced death rate (relative risk: 0.129, 95% CI: 0.18-0.9441, P=0.044) and event rates (relative risk: 0.167, 95% CI: 0.041-0.685, P=0.012). CONCLUSION: Alpha2C-adrenoceptor deletion may be a novel, strong, and independent predictor of reduced event rates in DCM patients treated according to guidelines.

• Pulmonary hypertension and right heart failure in pituitary adenylate cyclase-activating polypeptide type I receptor-deficient mice.

  Authors: Christiane Otto, Lutz Hein, Marc Brede, Roland Jahns, Stefan Engelhardt, Hermann-Josef Gröne, Günther Schütz

  Circulation. 12/2004; 110(20):3245-51.

  BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP), acting via 3 different G protein-coupled receptors, has been implicated in the regulation of several homeostatic systems in theBACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP), acting via 3 different G protein-coupled receptors, has been implicated in the regulation of several homeostatic systems in the body, including cardiopulmonary control. To define the physiologic role of the PACAP-preferring type I receptor, PAC1, in cardiopulmonary function, we developed a mutant mouse strain lacking functional PAC1 receptors. METHODS AND RESULTS: When PAC1-deficient mice were crossed onto a C57BL/6 background, almost all mutants died during the second postnatal week. Whereas mutant mice were indistinguishable from their wild-type littermates at birth, they showed progressive weakness and died from rapidly developing heart failure. Right ventricles of PAC1 mutants were massively dilated and showed cardiac myocyte hypertrophy, whereas left ventricular structure was unaltered. On direct cardiac catheterization, right ventricular pressure was elevated by 45% in PAC1-deficient mice, indicating increased pulmonary artery pressure, as no malformations were detected in the valves or outflow tract of the right ventricle. Consistent with elevated pulmonary pressure, lung capillary density was decreased by 30% and small pulmonary arteries of mutant mice had significant vascular smooth muscle cell hypertrophy compared with wild-type mice. CONCLUSIONS: Whereas PACAP induces vasodilation in isolated pulmonary vessels in wild-type mice, the absence of its specific receptor PAC1 causes pulmonary hypertension and right heart failure after birth. These in vivo findings demonstrate the crucial importance of PAC1-mediated signaling for the maintenance of normal pulmonary vascular tone during early postnatal life.

• alpha2-adrenergic receptor subtypes - novel functions uncovered in gene-targeted mouse models.

  Authors: Marc Brede, Melanie Philipp, Anne Knaus, Verena Muthig, Lutz Hein

  Biology of the cell / under the auspices of the European Cell Biology Organization. 07/2004; 96(5):343-8.

  The family of adrenergic receptors contains nine different subtypes of G protein-coupled receptors which mediate the biological effects of adrenaline and noradrenaline. With few exceptions, the fullThe family of adrenergic receptors contains nine different subtypes of G protein-coupled receptors which mediate the biological effects of adrenaline and noradrenaline. With few exceptions, the full therapeutic potential of subtype-selective therapy has not yet been explored for the group of adrenergic receptors. In the absence of sufficiently subtype-selective ligands which can distinguish between individual receptor subtypes of the adrenergic family, gene-targeted mouse models with deletions in these receptor genes have recently been generated and characterized. These genetic mouse models have helped to assign specific pharmacological effects of alpha(2)-receptor agonists or antagonists to individual receptor subtypes. However, some unexpected and novel functions of alpha(2)-adrenergic receptors were also uncovered in these mouse models: Presynaptic control of catecholamine release from adrenergic nerves in the central and sympathetic nervous system may be regulated by three different alpha(2)-receptor subtypes, alpha(2A), alpha(2B), and alpha(2C). A similar feedback loop also controls the release of catecholamines from the adrenal gland. alpha(2B)-receptors are not only involved in regulating vascular tone in the adult organism, but they are essential for the development of the vascular system of the placenta during prenatal development. The challenge will now be to generate strategies to identify whether the findings obtained in gene-targeted mice may predict the action of receptor subtype-selective drugs in humans.

• Cardiac hypertrophy is associated with decreased eNOS expression in angiotensin AT2 receptor-deficient mice.

  Authors: Marc Brede, Wilhelm Roell, Oliver Ritter, Frank Wiesmann, Roland Jahns, Axel Haase, Bernd K Fleischmann, Lutz Hein

  Hypertension. 01/2004; 42(6):1177-82.

  Angiotensin II receptors play an essential role in cardiovascular physiology and disease. The significance of angiotensin type II (AT2) receptors in cardiac disease still remains elusive. Thus, weAngiotensin II receptors play an essential role in cardiovascular physiology and disease. The significance of angiotensin type II (AT2) receptors in cardiac disease still remains elusive. Thus, we tested in gene-targeted mice whether AT2 receptors modulate cardiac function and remodeling after experimental myocardial injury. To generate myocardial infarcts of reproducible size, a cryolesion was generated at the free wall of the left ventricle of wild-type mice (Agtr2+/Y) and mice carrying a deletion of the AT2 receptor gene (Agtr2-/Y). Postinjury remodeling was followed up for 4 weeks after cryoinjury. The cryoprocedure led to an increased heart weight/body weight ratio and heart weight/tibia length ratio in AT2-deficient mice compared with control mice. Morphometric analysis revealed a significant increase in myocyte cross-sectional area after cardiac injury (infarct vs sham Agtr2+/Y, +53%; vs Agtr2-/Y, +95%). Expression of endothelial nitric oxide synthase (eNOS) was significantly lower in hearts from Agtr2-/Y than from Agtr2+/Y mice. eNOS downregulation was accompanied by a decrease in cardiac cGMP levels in Agtr2-/Y mice. In isolated murine cardiomyocytes, angiotensin II induced eNOS expression through AT2 receptors, and inhibition of NO production by NG-nitro-l-arginine methyl ester abolished the antihypertrophic effect of AT2 on cardiac myocytes. Our results demonstrate in a genetic mouse model that angiotensin II AT2 receptors exert an antihypertrophic effect in cardiac remodeling after myocardial cryoinjury and link the expression of cardiac eNOS to AT2 receptor activation.

• Inhibition of insulin secretion via distinct signaling pathways in alpha2-adrenoceptor knockout mice.

  Authors: Melanie Peterhoff, Andrea Sieg, Marc Brede, Cho-Ming Chao, Lutz Hein, Susanne Ullrich

  European journal of endocrinology / European Federation of Endocrine Societies. 11/2003; 149(4):343-50.

  OBJECTIVE: Adrenaline inhibits insulin secretion through activation of alpha(2)-adrenoceptors (ARs). These receptors are linked to pertussis toxin-sensitive G proteins. Agonist binding leads toOBJECTIVE: Adrenaline inhibits insulin secretion through activation of alpha(2)-adrenoceptors (ARs). These receptors are linked to pertussis toxin-sensitive G proteins. Agonist binding leads to inhibition of adenylyl cyclase, inhibition of Ca(2+) channels and activation of K(+) channels. Recently, three distinct subtypes of alpha(2)-AR were described, alpha(2A)-AR, alpha(2B)-AR and alpha(2C)-AR. At present, it is unknown which of these alpha(2)-AR subtype(s) may regulate insulin secretion. We used mice deficient in alpha(2)-ARs to analyze the coupling and role of individual alpha(2)-AR subtypes in insulin-secreting beta cells. METHODS: The inhibitory effect of adrenaline on insulin secretion was measured in freshly isolated and cultured wild type (wt) and alpha(2)-AR knockout (KO) mouse islets in order to examine the receptor subtypes which mediate adrenaline-induced inhibition of insulin secretion. Adenylyl cyclase activity was measured in isolated cultured islets. Membrane potential was measured using the amphotericin B permeabilized patch clamp method in isolated and cultured single islet cells. RESULTS: In wt, alpha(2A)- and alpha(2C)-AR KO mouse islets, adrenaline, 1 microM/L, inhibited secretion by 83, 80 and 100% respectively. In contrast, in alpha(2A/2C)-AR double KO mouse islets, adrenaline had no effect on stimulated secretion indicating that both alpha(2A)-AR and alpha(2C)-AR, but not alpha(2B)-AR, are functionally expressed in mouse islets. Surprisingly, glucose (16.7 mM/L)-induced secretion in the presence of 1 microM/L forskolin was greatly impaired in alpha(2A)-AR KO islets. However, when cAMP levels were increased further by the combination of forskolin (5 microM/L) and 3-isobutyl-1-methylxanthine (100 microM/L), secretion was stimulated 2.7-fold (8.5-fold in wt islets). Adrenaline lowered the concentration of cAMP in wt and alpha(2C)-AR KO mouse islets by 74%. Adrenaline also hyperpolarized wt and alpha(2C)-AR KO beta cells. In contrast, adrenaline did not inhibit adenylyl cyclase in islets of alpha(2A)-AR KO mice, nor did it hyperpolarize alpha(2A)-AR KO beta cells. CONCLUSION: Adrenaline inhibits insulin release through alpha(2A)- and alpha(2C)-ARs via distinct intracellular signaling pathways.

• Differential control of adrenal and sympathetic catecholamine release by alpha 2-adrenoceptor subtypes.

  Authors: Marc Brede, Gabor Nagy, Melanie Philipp, Jakob B Sorensen, Martin J Lohse, Lutz Hein

  Molecular endocrinology (Baltimore, Md.). 09/2003; 17(8):1640-6.

  In the adrenergic system, release of the neurotransmitter norepinephrine from sympathetic nerves is regulated by presynaptic inhibitory alpha2-adrenoceptors, but it is unknown whether release ofIn the adrenergic system, release of the neurotransmitter norepinephrine from sympathetic nerves is regulated by presynaptic inhibitory alpha2-adrenoceptors, but it is unknown whether release of epinephrine from the adrenal gland is controlled by a similar short feedback loop. Using gene-targeted mice we demonstrate that two distinct subtypes of alpha2-adrenoceptors control release of catecholamines from sympathetic nerves (alpha 2A) and from the adrenal medulla (alpha 2C). In isolated mouse chromaffin cells, alpha2-receptor activation inhibited the electrically stimulated increase in cell capacitance (a correlate of exocytosis), voltage-activated Ca2+ current, as well as secretion of epinephrine and norepinephrine. The inhibitory effects of alpha2-agonists on cell capacitance, voltage-activated Ca2+ currents, and on catecholamine secretion were completely abolished in chromaffin cells isolated from alpha 2C-receptor-deficient mice. In vivo, deletion of sympathetic or adrenal feedback control led to increased plasma and urine norepinephrine (alpha 2A-knockout) and epinephrine levels (alpha 2C-knockout), respectively. Loss of feedback inhibition was compensated by increased tyrosine hydroxylase activity, as detected by elevated tissue dihydroxyphenylalanine levels. Thus, receptor subtype diversity in the adrenergic system has emerged to selectively control sympathetic and adrenal catecholamine secretion via distinct alpha2-adrenoceptor subtypes. Short-loop feedback inhibition of epinephrine release from the adrenal gland may represent a novel therapeutic target for diseases that arise from enhanced adrenergic stimulation.

• AT2 receptor activation regulates myocardial eNOS expression via the calcineurin-NF-AT pathway.

  Authors: Oliver Ritter, Kai Schuh, Marc Brede, Nicola Röthlein, Natalie Burkard, Lutz Hein, Ludwig Neyses

  The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 03/2003; 17(2):283-5.

  The role of AT2-receptors has recently been subject of considerable debate. We investigated the influence of AT2-stimulation/inhibition on myocardial endothelial NO-synthase (eNOS, NOS-III) promoterThe role of AT2-receptors has recently been subject of considerable debate. We investigated the influence of AT2-stimulation/inhibition on myocardial endothelial NO-synthase (eNOS, NOS-III) promoter activity and eNOS protein expression. Stimulation of rat cardiomyocytes with angiotensin II (AngII) increased eNOS protein expression 3.3-fold. This was blocked by Cyclosporin A (CsA). Inhibition of the AT1-receptor did not reduce AngII-mediated eNOS protein expression, whereas AT2 stimulation increased it 2.4-fold and AT2 inhibition suppressed it. The modulatory effects of the AT2-receptor on eNOS expression was confirmed in mice with a genetic deletion of the AT2-receptor (AT2-KO). In gel shift assays two putative NF-AT sites in a 1.6 kb eNOS promoter fragment showed NF-AT binding and a supershift by NF-AT2(-c1)-specific antibodies. Stimulation of transfected cells with AngII or specific AT2-receptor agonists resulted in a significant increase in eNOS promoter activity, which was blocked by CsA, MCIP1, and mutation of an upstream NF-AT site. CONCLUSION: 1) AngII-stimulation of the myocardium, both in vivo and in vitro, is accompanied by increased expression of eNOS. 2) This effect is mediated by the calcineurin pathway and is induced by the AT2-receptor. 3) These results define a calcineurin/NF-AT/eNOS pathway as downstream effector of AT2-receptor activation in the myocardium.

• Feedback inhibition of catecholamine release by two different alpha2-adrenoceptor subtypes prevents progression of heart failure.

  Authors: Marc Brede, Frank Wiesmann, Roland Jahns, Kerstin Hadamek, Carsten Arnolt, Stefan Neubauer, Martin J Lohse, Lutz Hein

  Circulation. 12/2002; 106(19):2491-6.

  BACKGROUND: Elevated plasma norepinephrine levels are associated with increased mortality in patients and in animal models with chronic heart failure. To test which alpha2-adrenoceptor subtypesBACKGROUND: Elevated plasma norepinephrine levels are associated with increased mortality in patients and in animal models with chronic heart failure. To test which alpha2-adrenoceptor subtypes operate as presynaptic inhibitory receptors to control norepinephrine release in heart failure, we investigated the response of gene-targeted mice lacking alpha2-adrenoceptor subtypes (alpha2-KO) to chronic left ventricular pressure overload. In addition, we determined the functional consequences of genetic variants of alpha2-adrenoceptors in human patients with chronic heart failure. METHODS AND RESULTS: Cardiac pressure overload was induced by transverse aortic constriction. Three months after aortic banding, survival was dramatically reduced in alpha2A-KO (52%) and alpha2C-KO (47%) mice compared with wild-type and alpha2B-deficient (86%) animals. Excess mortality in alpha2A- and alpha2C-KO strains was attributable to heart failure with enhanced left ventricular hypertrophy and fibrosis and elevated circulating catecholamines. The clinical importance of this finding is emphasized by the fact that heart failure patients with a dysfunctional variant of the alpha2C-adrenoceptor had a worse clinical status and decreased cardiac function as determined by invasive catheterization and by echocardiography. CONCLUSIONS: Our results indicate an essential function of alpha2A- and alpha2C-adrenoceptors in the prevention of heart failure progression in mice and human patients. Identification of heart failure patients with genetic alpha2-adrenoceptor variants as well as new alpha2-receptor subtype-selective drugs may represent novel therapeutic strategies in chronic heart failure and other diseases with enhanced sympathetic activation.

• Physiological significance of alpha(2)-adrenergic receptor subtype diversity: one receptor is not enough.

  Authors: Melanie Philipp, Marc Brede, Lutz Hein

  American journal of physiology. Regulatory, integrative and comparative physiology. 09/2002; 283(2):R287-95.

  Alpha(2)-adrenergic receptors mediate part of the diverse biological effects of the endogenous catecholamines epinephrine and norepinephrine. Three distinct subtypes of alpha(2)-adrenergic receptors,Alpha(2)-adrenergic receptors mediate part of the diverse biological effects of the endogenous catecholamines epinephrine and norepinephrine. Three distinct subtypes of alpha(2)-adrenergic receptors, alpha(2A), alpha(2B), alpha(2C), have been identified from multiple species. Because of the lack of sufficiently subtype-selective ligands, the specific biological functions of these receptor subtypes were largely unknown until recently. Gene-targeted mice carrying deletions in the genes encoding for individual alpha(2)-receptor subtypes have added important new insight into the physiological significance of adrenergic receptor diversity. Two different strategies have emerged to regulate adrenergic signal transduction. Some biological functions are controlled by two counteracting alpha(2)-receptor subtypes, e.g., alpha(2A)-receptors decrease sympathetic outflow and blood pressure, whereas the alpha(2B)-subtype increases blood pressure. Other biological functions are regulated by synergistic alpha(2)-receptor subtypes. The inhibitory presynaptic feedback loop that tightly regulates neurotransmitter release from adrenergic nerves also requires two receptor subtypes, alpha(2A) and alpha(2C). Similarly, nociception is controlled at several levels by one of the three alpha(2)-receptor subtypes. Further investigation of the specific function of alpha(2)-subtypes will greatly enhance our understanding of the relevance of closely related receptor proteins and point out novel therapeutic strategies for subtype-selective drug development.

• Transgenic mouse models of angiotensin receptor subtype function in the cardiovascular system

  Authors: Marc Brede, Lutz Hein

  Regulatory Peptides.

  Angiotensin II mediates is biological actions via different subtypes of G protein-coupled receptors, termed AT1 and AT2 receptors. In rodents, two AT1 receptors have been identified, AT1A and AT1B,Angiotensin II mediates is biological actions via different subtypes of G protein-coupled receptors, termed AT1 and AT2 receptors. In rodents, two AT1 receptors have been identified, AT1A and AT1B, whereas in humans a single AT1 receptor exists. Recently, a number of transgenic animal models have been generated which overexpress or lack functional angiotensin II receptor subtypes. This review focuses on the physiological significance of angiotensin II receptor subtype diversity in the cardiovascular system. In the mouse, AT1A receptors are the major regulators of cardiovascular homeostasis by determining vascular tone and natriuresis. In addition, AT1A receptors mediate growth-stimulating signals in vascular and cardiac myocytes. AT1B receptors participate in blood pressure regulation, and their functions become apparent when the AT1A receptor gene is deleted. Deletion of the mouse gene for the AT2 receptor subtype led to hypersensitivity to pressor and antinatriuretic effects of angiotensin II in vivo, suggesting that the AT2 receptor subtype counteracts some of the biological effects of AT1 receptor signalling.