Crosstalk between ß2- and α2-Adrenergic Receptors in the Regulation of B16F10 Melanoma Cell Proliferation.

Adrenergic receptors (AR) belong to the G protein-coupled receptor superfamily and regulate migration and proliferation in various cell types. The objective of this study was to evaluate whether ß-AR stimulation affects the antiproliferative action of α2-AR agonists on B16F10 cells and, if so, to determine the relative contribution of ß-AR subtypes. Using pharmacological approaches, evaluation of Ki-67 expression by flow cytometry and luciferase-based cAMP assay, we found that treatment with isoproterenol, a ß-AR agonist, increased cAMP levels in B16F10 melanoma cells without affecting cell proliferation. Propranolol inhibited the cAMP response to isoproterenol. In addition, stimulation of α2-ARs with agonists such as clonidine, a well-known antihypertensive drug, decreased cancer cell proliferation. This effect on cell proliferation was suppressed by treatment with isoproterenol. In turn, the suppressive effects of isoproterenol were abolished by the treatment with either ICI 118,551, a ß2-AR antagonist, or propranolol, suggesting that isoproterenol effects are mainly mediated by the ß2-AR stimulation. We conclude that the crosstalk between the ß2-AR and α2-AR signaling pathways regulates the proliferative activity of B16F10 cells and may therefore represent a therapeutic target for melanoma therapy.

Role of ß-Adrenergic Receptors and Estrogen in Cardiac Repair after Myocardial Infarction: An Overview.

Acute myocardial infarction (MI) is associated with an intense inflammatory response that is critical for cardiac repair but is also involved in the pathogenesis of adverse cardiac remodeling, i.e., the set of size, geometry, and structure changes that represent the structural substrate for the development of post-MI heart failure. Deciphering the pathophysiological mechanisms underlying cardiac repair after MI is, therefore, critical to favorably regulate cardiac wound repair and to prevent development of heart failure. Catecholamines and estrogen play an active role in regulating the inflammatory response in the infarcted area. For example, stress-induced catecholamines alter recruitment and trafficking of leukocytes to the heart. Additionally, estrogen affects rate of cardiac rupture during the acute phase of MI, as well as infarct size and survival in animal models of MI. In this review, we will summarize the role of ß-adrenergic receptors and estrogen in cardiac repair after infarction in preclinical studies.

ß-blockers Reverse Agonist-Induced ß2-AR Downregulation Regardless of Their Signaling Profile.

Altered ß-adrenergic receptor (ß-AR) density has been reported in cells, animals, and humans receiving ß-blocker treatment. In some cases, ß-AR density is upregulated, but in others, it is unaffected or even reduced. Collectively, these results would imply that changes in ß-AR density and ß-blockade are not related. However, it has still not been clarified whether the effects of ß-blockers on receptor density are related to their ability to activate different ß-AR signaling pathways. To this aim, five clinically relevant ß-blockers endowed with inverse, partial or biased agonism at the ß2-AR were evaluated for their effects on ß2-AR density in both human embryonic kidney 293 (HEK293) cells expressing exogenous FLAG-tagged human ß2-ARs and human lymphocytes expressing endogenous ß2-ARs. Cell surface ß2-AR density was measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Treatment with propranolol, carvedilol, pindolol, sotalol, or timolol did not induce any significant change in surface ß2-AR density in both HEK293 cells and human lymphocytes. On the contrary, treatment with the ß-AR agonist isoproterenol reduced the number of cell surface ß2-ARs in the tested cell types without affecting ß2-AR-mRNA levels. Isoproterenol-induced effects on receptor density were completely antagonized by ß-blocker treatment. In conclusion, the agonistic activity of ß-blockers does not exert an important effect on short-term regulation of ß2-AR density.

Inflammatory cytokines associated with cancer growth induce mitochondria and cytoskeleton alterations in cardiomyocytes.

Cardiac dysfunction is often observed in patients with cancer also representing a serious problem limiting chemotherapeutic intervention and even patient survival. In view of the recently established role of the immune system in the control of cancer growth, the present work has been undertaken to investigate the effects of a panel of the most important inflammatory cytokines on the integrity and function of mitochondria, as well as of the cytoskeleton, two key elements in the functioning of cardiomyocytes. Either mitochondria features or actomyosin cytoskeleton organization of in vitro-cultured cardiomyocytes treated with different inflammatory cytokines were analyzed. In addition, to investigate the interplay between tumor growth and cardiac function in an in vivo system, immunocompetent female mice were inoculated with cancer cells and treated with the chemotherapeutic drug doxorubicin at a dosing schedule able to suppress tumor growth without inducing cardiac alterations. Analyses carried out in cardiomyocytes treated with the inflammatory cytokines, such as tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), interleukin 6 (IL-6), IL-8, and IL-1ß revealed severe phenotypic changes, for example, of contractile cytoskeletal elements, mitochondrial membrane potential, mitochondrial reactive oxygen species production and mitochondria network organization. Accordingly, in immunocompetent mice, the tumor growth was accompanied by increased levels of the inflammatory cytokines TNF-α, IFN-γ, IL-6, and IL-8, either in serum or in the heart tissue, together with a significant reduction of ventricular systolic function. The alterations of mitochondria and of microfilament system of cardiomyocytes, due to the systemic inflammation associated with cancer growth, could be responsible for remote cardiac injury and impairment of systolic function observed in vivo.

ß-Blockers promote angiogenesis in the mouse aortic ring assay.

Recent results indicate that the reduction of ß-adrenergic signaling impairs angiogenesis under ischemic conditions. Because angiogenesis may occur in the absence of ischemia, it remains to be determined whether and how ß-adrenergic signaling regulates angiogenesis, which develops under normoxic conditions. The effect of ß-adrenergic ligands on angiogenesis was investigated using 3-dimensional cultures of mouse aortic rings embedded in collagen type I, in which luminized microvessels develop in response to vascular endothelial growth factor (VEGF). Under normoxic conditions, both isoproterenol, a ß-adrenergic receptor (ß-AR) agonist, and forskolin, an adenylate cyclase activator, were unable to influence aortic microvessel sprouting. On the contrary, treatment with propranolol, a ß-AR antagonist, caused an approximately 70% increase in VEGF-mediated microvessel sprouting. This effect was abolished in rings from both double ß-AR and ß1-AR knockout mice, but not in rings from ß2-AR knockout mice. Significant increases in microvessel sprouting were also observed when mouse aortic rings from C57BL/6 mice were treated with the ß1-AR-selective antagonists metoprolol and bisoprolol or with the ß2-AR-selective antagonist ICI 118,551. Conversely, carvedilol, a nonselective ß-AR antagonist, was unable to affect aortic sprouting. These findings suggest that some ß-blockers display proangiogenic activity through a mechanism that is independent of their ability to antagonize catecholamine action. The present results also identify a new function for ß-AR signaling as a facilitator for VEGF-mediated angiogenesis and have implications for understanding the mechanisms that regulate angiogenic responses under normoxic conditions.

Benzodiazepine diazepam regulates cell surface ß1-adrenergic receptor density in human monocytes.

Downregulation of cell surface ß-adrenergic receptors (ß-AR) is an important adaptive response that prevents deleterious effects of receptor overstimulation. Various factors including reactive oxygen species cause ß-AR downregulation. In this study, we evaluated the effects of ligands of the peripheral benzodiazepine receptor (PBR), a key protein in regulating oxidative stress, on surface density of endogenous ß1-and ß2-ARs in highly differentiated cells such as human monocytes, which express both ß-AR subtypes. ß-AR expression in human monocytes was evaluated by flow cytometry, qPCR and western blotting. Monocyte treatment with ß-AR agonist isoproterenol did not change surface ß1-AR density while downregulating surface ß2-AR density. This effect was antagonized by the ß-blocker propranolol. An opposite response was observed with benzodiazepine diazepam that led to a time-dependent reduction in ß1-AR density. In particular, while no significant downregulation was observed after 3 h of treatment, only 63% of ß1-ARs were still present on the cell surface after 48 h of treatment with diazepam at 1 µM. Treatment with the PBR antagonist PK11195, but not with propranolol, antagonized the effects of diazepam. No change in ß1-AR-mRNA or protein levels was observed at any time after diazepam treatment. We also found that diazepam did not affect Gs-protein or ß-arrestin-2 recruitment for both ß-ARs in engineered fibroblasts, further suggesting that diazepam activity on ß1-AR density is mediated by PBR. Finally, no sex-related differences were found. Collectively, these results indicate that monocyte ß1-ARs are resistant to catecholamine-mediated downregulation and suggest that PBR plays an important role in regulating ß1-AR density.

α-Adrenoceptor stimulation attenuates melanoma growth in mice.

BACKGROUND AND PURPOSE: Recently, ß-adrenoceptor blockade has emerged as a potential strategy to inhibit melanoma growth. It remains to be ascertained whether ß-adrenoceptor stimulation by circulating catecholamines increases melanoma growth in mice. EXPERIMENTAL APPROACH: B16F10 melanoma-bearing mice were used to evaluate effects of adrenaline and specific adrenoceptor (AR) ligands on tumour volume. AR expression and effects of AR ligands on cell viability, production of mitochondrial reactive oxygen species (mROS), and proliferation activity in B16F10 cells, were determined by biochemical analyses. KEY RESULTS: Real-time polymerase chain reaction (qPCR) analyses revealed that B16F10 cells express α1B-, α2A-, α2B- and ß2-ARs. We found that treatment with the α- and ß-AR agonist adrenaline or with the synthetic catecholamine isoprenaline, which selectively stimulates ß-ARs, did not affect melanoma growth. Conversely, adrenaline reduced tumour growth in mice cotreated with propranolol, a ß1ß2-AR antagonist. Adrenaline had no effect in tumour-bearing ß1ß2-AR knockout mice, in which ß1- and ß2-ARs are lacking, but it reduced tumour growth when co-administered with propranolol suggesting that tumour ß2-ARs negatively regulate adrenaline antitumour activity. Additionally, we found that α1-AR stimulation with cirazoline yielded a decrease in B16F10 melanoma size. These effects on melanoma growth were paralleled by reduced cell viability and proliferation activity as well as increased mROS production in α1-AR-stimulated B16F10 cells. Decreased viability, proliferation and mitochondrial function in B16F10 cells also occurred after α2-AR stimulation by α2-AR agonist ST91. CONCLUSIONS AND IMPLICATIONS: In the B16F10 melanoma model, stimulation of α-AR subtypes yields in vivo and in vitro anticancer activity.

Intermittent ß-adrenergic blockade downregulates the gene expression of ß-myosin heavy chain in the mouse heart.

Expression of the ß-myosin heavy chain (ß-MHC), a major component of the cardiac contractile apparatus, is tightly regulated as even modest increases can be detrimental to heart under stress. In healthy hearts, continuous inhibition of ß-adrenergic tone upregulates ß-MHC expression. However, it is unknown whether the duration of the ß-adrenergic inhibition and ß-MHC expression are related. Here, we evaluated the effects of intermittent ß-blockade on cardiac ß-MHC expression. To this end, the ß-blocker propranolol, at the dose of 15mg/kg, was administered once a day in mice for 14 days. This dosing schedule caused daily drug-free periods of at least 6 h as evidenced by propranolol plasma concentrations and cardiac ß-adrenergic responsiveness. Under these conditions, ß-MHC expression decreased by about 75% compared to controls. This effect was abolished in mice lacking ß1- but not ß2-adrenergic receptors (ß-AR) indicating that ß-MHC expression is regulated in a ß1-AR-dependent manner. In ß1-AR knockout mice, the baseline ß-MHC expression was fourfold higher than in wild-type mice. Also, we evaluated the impact of intermittent ß-blockade on ß-MHC expression in mice with systolic dysfunction, in which an increased ß-MHC expression occurs. At 3 weeks after myocardial infarction, mice showed systolic dysfunction and upregulation of ß-MHC expression. Intermittent ß-blockade decreased ß-MHC expression while attenuating cardiac dysfunction. In vitro studies showed that propranolol does not affect ß-MHC expression on its own but antagonizes catecholamine effects on ß-MHC expression. In conclusion, a direct relationship occurs between the duration of the ß-adrenergic inhibition and ß-MHC expression through the ß1-AR.

cAMP-mediated beta-adrenergic signaling negatively regulates Gq-coupled receptor-mediated fetal gene response in cardiomyocytes.

The treatment with beta-blockers causes an enhancement of the norepinephrine-induced fetal gene response in cultured cardiomyocytes. Here, we tested whether the activation of cAMP-mediated beta-adrenergic signaling antagonizes alpha(1)-adrenergic receptor (AR)-mediated fetal gene response. To address this question, the fetal gene program, of which atrial natriuretic peptide (ANP) and the beta-isoform of myosin heavy chain are classical members, was induced by phenylephrine (PE), an alpha(1)-AR agonist. In cultured neonatal rat cardiomyocytes, we found that stimulation of beta-ARs with isoproterenol, a beta-AR agonist, inhibited the fetal gene expression induced by PE. Similar results were also observed when cardiomyocytes were treated with forskolin (FSK), a direct activator of adenylyl cyclase, or 8-CPT-6-Phe-cAMP, a selective activator of protein kinase A (PKA). Conversely, the PE-induced fetal gene expression was further upregulated by H89, a selective PKA inhibitor. To evaluate whether these results could be generalized to Gq-mediated signaling and not specifically to alpha(1)-ARs, cardiomyocytes were treated with prostaglandin F(2)alpha, another Gq-coupled receptor agonist, which is able to promote fetal gene expression. This treatment caused an increase of both ANP mRNA and protein levels, which was almost completely abolished by FSK treatment. The capability of beta-adrenergic signaling to regulate the fetal gene expression was also evaluated in vivo conditions by using beta1- and beta2-AR double knockout mice, in which the predominant cardiac beta-AR subtypes are lacking, or by administering isoproterenol (ISO), a beta-AR agonist, at a subpressor dose. A significant increase of the fetal gene expression was found in beta(1)- and beta(2)-AR gene deficient mice. Conversely, we found that ANP, beta-MHC and skACT mRNA levels were significantly decreased in ISO-treated hearts. Collectively, these data indicate that cAMP-mediated beta-adrenergic signaling negatively regulates Gq cascade activation-induced fetal gene expression in cultured cardiomyocytes and that this inhibitory regulation is already operative in the mouse heart under physiological conditions.

Propranolol promotes Egr1 gene expression in cardiomyocytes via beta-adrenoceptors.

Recent research has revealed that propranolol, a beta-adrenoceptor antagonist, causes extracellular signal-regulated kinase (ERK) cascade activation, nuclear translocation of phospho-ERK and increased transcriptional activity in cultured cell lines. Given the importance of beta-adrenoceptor antagonists in the treatment of heart failure, we evaluated the capability of propranolol of promoting the ERK-dependent gene expression at the cardiomyocyte level. To this end, the gene expression of the early growth response factor 1 (Egr1), a well-recognized indicator of nuclear extracellular signal-regulated kinase 1/2 (ERK1/2) activation, was assessed by quantitative real-time RT-PCR in vivo as well as in vitro experiments. Propranolol, administered at the dose of 10 mg/kg/day in C57BL/6 mice, caused a approximately 19-fold increase of Egr1 mRNA expression in left ventricular myocardium along with a approximately 2.1-fold increase of Egr1 protein expression. Isoproterenol, a nonselective beta-adrenoceptor agonist, also increased Egr1 mRNA and protein expression but to a lesser degree. Remarkably, isoproterenol administration was associated with the development of cardiac hypertrophy, whereas propranolol-treated mice showed a completely normal cardiac morphology. The effect of propranolol on Egr1 mRNA expression was abrogated in mice lacking beta(1)- and beta(2)-adrenoceptors indicating that propranolol increases Egr1 mRNA expression in a beta-adrenoceptor-dependent manner. The role of beta-adrenoceptors was further confirmed by showing that propranolol was able to increase Egr1 mRNA and protein levels in cultured neonatal cardiomyocytes. Collectively, these results indicate that propranolol promotes Egr1 gene expression in cardiomyocytes via beta-adrenoceptors with a mechanism which is independent of its ability to antagonize the effects of catecholamines. It is also suggested that cardiomyocyte growth and Egr1 gene overexpression are not obligate processes.

Givinostat reduces adverse cardiac remodeling through regulating fibroblasts activation.

Cardiovascular diseases (CVDs) are a major burden on the healthcare system: indeed, over two million new cases are diagnosed every year worldwide. Unfortunately, important drawbacks for the treatment of these patients derive from our current inability to stop the structural alterations that lead to heart failure, the common endpoint of many CVDs. In this scenario, a better understanding of the role of epigenetics - hereditable changes of chromatin that do not alter the DNA sequence itself - is warranted. To date, hyperacetylation of histones has been reported in hypertension and myocardial infarction, but the use of inhibitors for treating CVDs remains limited. Here, we studied the effect of the histone deacetylase inhibitor Givinostat on a mouse model of acute myocardial infarction. We found that it contributes to decrease endothelial-to-mesenchymal transition and inflammation, reducing cardiac fibrosis and improving heart performance and protecting the blood vessels from apoptosis through the modulatory effect of cardiac fibroblasts on endothelial cells. Therefore, Givinostat may have potential for the treatment of CVDs.

Biphasic effects of propranolol on tumour growth in B16F10 melanoma-bearing mice.

BACKGROUND AND PURPOSE: Propranolol is a vasoactive drug that shows antiangiogenic and antitumour activities in melanoma. However, it is unknown whether these activities are dose-dependent and whether there is a relationship between systemic vascular effects of propranolol and anti-melanoma activity. EXPERIMENTAL APPROACH: Effects of increasing doses of propranolol (10, 20, 30 and 40 mg·kg-1 ·day-1 ) on tumour growth were studied in B16F10 melanoma-bearing mice. Histological and biochemical analyses were used to assess propranolol effects on angiogenesis and cancer cell proliferation. Systemic vascular resistance (SVR) was evaluated by measuring cardiac output and arterial BP. KEY RESULTS: In vitro analyses revealed that B16F10 cells expressed ß-adrenoceptors, but neither isoprenaline, a ß-adrenoceptor agonist, nor the ß-blocker propranolol affected cancer cell proliferation. In vivo studies showed that the antitumour efficacy of propranolol follows a U-shaped biphasic dose-response curve. Low doses (10 and 20 mg·kg-1 ·day-1 ) significantly inhibit tumour growth, whereas higher doses are progressively less effective. We also found that high-dose propranolol stimulates tumour arteriogenesis whereas no effect on angiogenesis was observed at any dose. Based on these data and considering that propranolol is a vasoactive drug, we hypothesized that it causes systemic vasoconstriction or vasodilation depending on the dose and thus alters tumour perfusion and growth. Consistent with this hypothesis, we found that propranolol has a biphasic effect on SVR with low and high doses producing vasoconstriction and vasodilation respectively. CONCLUSIONS AND IMPLICATIONS: Propranolol inhibits melanoma growth in a U-shaped biphasic manner. A direct relationship exists between SVR and anti-melanoma activity.

Pressure overload causes cardiac hypertrophy in beta1-adrenergic and beta2-adrenergic receptor double knockout mice.

OBJECTIVE: Cardiac hypertrophy arises as an adaptive response to increased afterload. Studies in knockout mice have shown that catecholamines, but not alpha1-adrenergic receptors, are necessary for such an adaptation to occur. However, whether beta-adrenergic receptors are critical for the development of cardiac hypertrophy in response to pressure overload is not known at this time. METHODS AND RESULTS: Pressure overload was induced by transverse aortic banding in beta1-adrenergic and beta2-adrenergic receptor double knockout (DbetaKO) mice, in which the predominant cardiac beta-adrenergic receptor subtypes are lacking. Chronic pressure overload for 4 weeks induced cardiac hypertrophy in both DbetaKO and wild-type mice. There were no significant differences between banded mice in left ventricular weight to body weight ratio, in the left ventricular wall thickness, in the cardiomyocyte size or in the expression levels of the load-sensitive cardiac genes such as ANF and beta-MHC. Additionally, the left ventricular systolic pressure, an index of afterload, and cardiac contractility, evaluated as dp/dtmax, the maximal slope of systolic pressure increment, and Ees, end-systolic elastance, were increased at a similar level in both wild-type and DbetaKO banded mice, and were significantly greater than in sham controls. CONCLUSION: Despite chronic activation of the cardiac beta-adrenergic system being sufficient to induce a pathological hypertrophy, we show that beta1-adrenergic and beta2-adrenergic receptors are not an obligatory component of the signaling pathway that links the increased afterload to the development of cardiac hypertrophy.

Na+/H+ exchange inhibition attenuates left ventricular remodeling and preserves systolic function in pressure-overloaded hearts.

Cardiac hypertrophy is a homeostatic response to elevated afterload. Na+/H+ exchanger (NHE) inhibition reduces the hypertrophic response in animal models of left ventricular hypertrophy (LVH) and myocardial infarction. We examined the effect of chronic treatment with cariporide, a selective inhibitor of Na+/H+ exchanger isoform 1 (NHE-1), on left ventricular (LV) systolic and diastolic function under pressure overload conditions. Male CD-1 mice were randomized to receive either a control diet or an identical diet supplemented with 6000 p.p.m. of cariporide. Cardiac pressure overload was induced by thoracic aortic banding. LV dimension and systolic and diastolic function were assessed in sham and banded mice by echocardiography and cardiac catheterization 2 and 5 weeks after surgery. Histological analysis was also performed. After 2 weeks of pressure overload, the vehicle-treated banded mice (Veh-Bd) had enhanced normalized LV weight (about +50%) and normal chamber size and function, whereas cariporide-treated banded mice (Car-Bd) showed a preserved contractility and systolic function despite a marked attenuation of LVH. Diastolic function did not differ significantly among groups. After 5 weeks, the Veh-Bd developed LV chamber enlargement and systolic dysfunction as evidenced by a 16% increase in LV end-diastolic diameter, a 36% decrease in myocardial contractility, and a 26% reduction in percent fractional shortening. In contrast, Car-Bd showed an attenuated increase in LV mass, normal chamber size, and a maintained systolic function. A distinct histological feature was that in banded mice, cariporide attenuated the development of cardiomyocyte hypertrophy but not the attendant myocardial fibrosis. In conclusion, the results of the present study indicate that (i) the hypertrophic response to pressure overload is dependent on NHE-1 activity, and (ii) at the 5-week stage, banding-induced deterioration of LV performance is prevented by NHE-1 inhibition.British Journal of Pharmacology (2004) 141, 526-532. doi:10.1038/sj.bjp.0705631

Iron excretion in iron dextran-overloaded mice.

BACKGROUND: Iron homeostasis in humans is tightly regulated by mechanisms aimed to conserve iron for reutilisation, with a negligible role played by excretory mechanisms. In a previous study we found that mice have an astonishing ability to tolerate very high doses of parenterally administered iron dextran. Whether this ability is linked to the existence of an excretory pathway remains to be ascertained. MATERIALS AND METHODS: Iron overload was generated by intraperitoneal injections of iron dextran (1 g/kg) administered once a week for 8 weeks in two different mouse strains (C57bl/6 and B6D2F1). Urinary and faecal iron excretion was assessed by inductively coupling plasma-mass spectrometry, whereas cardiac and liver architecture was evaluated by echocardiography and histological methods. For both strains, 24-hour faeces and urine samples were collected and iron concentration was determined on days 0, 1 and 2 after iron administration. RESULTS: In iron-overloaded C57bl/6 mice, the faecal iron concentration increased by 218% and 157% on days 1 and 2, respectively (p<0.01). The iron excreted represented a loss of 14% of total iron administered. Similar but smaller changes was also found in B6D2F1 mice. Conversely, we found no significant changes in the concentration of iron in the urine in either of the strains of mice. In both strains, histological examination showed accumulation of iron in the liver and heart which tended to decrease over time. CONCLUSIONS: This study indicates that mice have a mechanism for removal of excess body iron and provides insights into the possible mechanisms of excretion.

Signaling pathway-focused gene expression profiling in pressure overloaded hearts.

The ß-blocker propranolol displays antihypertrophic and antifibrotic properties in the heart subjected to pressure overload. Yet the underlying mechanisms responsible for these important effects remain to be completely understood. The purpose of this study was to determine signaling pathway-focused gene expression profile associated with the antihypertrophic action of propranolol in pressure overloaded hearts. To address this question, a focused real-time PCR array was used to screen left ventricular RNA expression of 84 gene transcripts representative of 18 different signaling pathways in C57BL/6 mice subjected to transverse aortic constriction (TAC) or sham surgery. On the surgery day, mice received either propranolol (80 mg/kg/day) or vehicle for 14 days. TAC caused a 49% increase in the left ventricular weight-to-body weight (LVW/BW) ratio without changing gene expression. Propranolol blunted LVW/BW ratio increase by approximately 50% while causing about a 3-fold increase in the expression of two genes, namely Brca1 and Cdkn2a, belonging to the TGF-beta and estrogen pathways, respectively. In conclusion, after 2 weeks of pressure overload, TAC hearts show a gene expression profile superimposable to that of sham hearts. Conversely, propranolol treatment is associated with an increased expression of genes which negatively regulate cell cycle progression. It remains to be established whether a mechanistic link between gene expression changes and the antihypertrophic action of propranolol occurs.

Propranolol enhances cell cycle-related gene expression in pressure overloaded hearts.

BACKGROUND AND PURPOSE: Cell cycle regulators are regarded as essential for cardiomyocyte hypertrophic growth. Given that the ß-adrenoceptor antagonist propranolol blunts cardiomyocyte hypertrophic growth, we determined whether propranolol alters the expression of cell cycle-related genes in mouse hearts subjected to pressure overload. EXPERIMENTAL APPROACH: Pressure overload was induced by transverse aortic constriction (TAC), whereas the expression levels of 84 cell cycle-related genes were assayed by real-time PCR. Propranolol (80 mg·kg(-1) ·day(-1) ) was administered in drinking water for 14 days. KEY RESULTS: Two weeks after surgery, TAC caused a 46% increase in the left ventricular weight-to-body weight (LVW/BW) ratio but no significant changes in cell cycle gene expression. Propranolol, at plasma concentrations ranging from 10 to 140 ng·mL(-1) , blunted the LVW/BW ratio increase in TAC mice, while significantly increasing expression of 10 cell cycle genes including mitotic cyclins and proliferative markers such as Ki67. This increase in cell cycle gene expression was paralleled by a significant increase in the number of Ki67-positive non-cardiomyocyte cells as revealed by immunohistochemistry and confocal microscopy. ß-Adrenoceptor signalling was critical for cell cycle gene expression changes, as genetic deletion of ß-adrenoceptors also caused a significant increase in cyclins and Ki67 in pressure overloaded hearts. Finally, we found that metoprolol, a ß(1) -adrenoceptor antagonist, failed to enhance cell cycle gene expression in TAC mice. CONCLUSIONS AND IMPLICATIONS: Propranolol treatment enhances cell cycle-related gene expression in pressure overloaded hearts by increasing the number of cycling non-cardiomyocyte cells. These changes seem to occur via ß(2) -adrenoceptor-mediated mechanisms.

Bile acid depletion and repletion regulate cholangiocyte growth and secretion by a phosphatidylinositol 3-kinase-dependent pathway in rats.

BACKGROUND & AIMS: We tested the hypothesis that during bile duct obstruction, increased biliary bile acids trigger cholangiocyte proliferation and secretion by a phosphatidylinositol 3-kinase (PI3-K)-dependent pathway. METHODS: In bile duct-incannulated (BDI) rats, bile duct obstruction present for 7 days was relieved for 24 hours by external bile drainage. During the 24-hour drainage period, animals received either Krebs Ringer Henseleit (the bile-depleted group), or sodium taurocholate (the bile-depleted, taurocholate-infused group). We evaluated cholangiocyte proliferation and secretin-stimulated ductal secretion. Apical bile acid transporter (ABAT) expression and bile acid transport activity was determined. In pure preparations of cholangiocytes, we examined the effect of taurocholate (in the absence or presence of wortmannin or PI 3,4-bisphosphate the lipid product of PI3-K) on cholangiocyte proliferation and secretin-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) levels. RESULTS: Bile depletion reduced cholangiocyte proliferation and secretin-stimulated ductal secretion and ABAT expression and bile acid transport activity compared with 1-week BDI control rats. In bile-depleted, taurocholate-infused rats, cholangiocyte proliferation and secretion and ABAT expression and bile acid transport activity were maintained at levels similar to those seen in BDI control rats. In vitro, taurocholate stimulation of DNA replication and secretin-stimulated cAMP levels was blocked by wortmannin. The inhibitory effect of wortmannin on taurocholate stimulation of cholangiocyte proliferation and secretion was prevented by PI 3,4-bisphosphate. CONCLUSIONS: Bile acid uptake by ABAT and the PI3-K pathway are important for bile acids to signal cholangiocyte proliferation. In bile duct obstruction, increased biliary bile acid concentration and ABAT expression initiate increased cholangiocyte proliferation and secretion.