Phosphodiesterase PDE3, but not PDE4, reduces β1- and β2-adrenoceptor-mediated inotropic and lusitropic effects in failing ventricle from metoprolol-treated patients

Background and purpose Phosphodiesterases PDE3 and/or PDE4 control ventricular effects of catecholamines in several species but their relative effects in failing human ventricle are unknown. We investigated whether the PDE3-selective inhibitor cilostamide (0.3-1μM) or PDE4 inhibitor rolipram (1-10μM) modified the positive inotropic and lusitropic effects of catecholamines in human failing myocardium. Experimental approach Right and left ventricular trabeculae from freshly explanted hearts of 5 non-β-blocker-treated and 15 metoprolol-treated patients with terminal heart failure were paced to contract at 1Hz. The effects of (-)-noradrenaline, mediated through β1-adrenoceptors (β2-adrenoceptors blocked with ICI118551), and (-)-adrenaline, mediated through β2-adrenoceptors (β1-adrenoceptors blocked with CGP20712A), were assessed in the absence and presence of PDE inhibitors. Catecholamine potencies were estimated from –logEC50s. Key results Cilostamide did not significantly potentiate the inotropic effects of the catecholamines in non-β-blocker-treated patients. Cilostamide caused greater potentiation (P=0.037) of the positive inotropic effects of (-)-adrenaline (0.78±0.12 log units) than (-)-noradrenaline (0.47±0.12 log units) in metoprolol-treated patients. Lusitropic effects of the catecholamines were also potentiated by cilostamide. Rolipram did not affect the inotropic and lusitropic potencies of (-)-noradrenaline or (-)-adrenaline on right and left ventricular trabeculae from metoprolol-treated patients. Conclusions and implications Metoprolol induces a control by PDE3 of ventricular effects mediated through both β1- and β2-adrenoceptors, thereby further reducing sympathetic cardiostimulation in patients with terminal heart failure. Concurrent therapy with a PDE3 blocker and metoprolol could conceivably facilitate cardiostimulation evoked by adrenaline through β2-adrenoceptors. PDE4 does not appear to reduce inotropic and lusitropic effects of catecholamines in failing human ventricle.

Oncogenic BRAF induces melanoma cell invasion by downregulating the cGMP-specific phosphodiesterase PDE5A

We show that in melanoma cells oncogenic BRAF, acting through MEK and the transcription factor BRN2, downregulates the cGMP-specific phosphodiesterase PDE5A. Although PDE5A downregulation causes a small decrease in proliferation, its major impact is to stimulate a dramatic increase in melanoma cell invasion. This is because PDE5A downregulation leads to an increase in cGMP, which induces an increase in cytosolic Ca2+, stimulating increased contractility and inducing invasion. PDE5A downregulation also this leads to an increase in short-term and long-term colonization of the lungs by melanoma cells. We do not observe this pathway in NRAS mutant melanoma or BRAF mutant colorectal cells. Thus, we show that in melanoma cells oncogenic BRAF induces invasion through downregulation of PDE5A.

B-adrenoceptor determinants of contractility in the human heart: The role of phosphodiesterase enzymes

This thesis investigated how enzymes called phosphodiesterases control changes in contractility mediated by noradrenaline and adrenaline through activation of β1- and β2-adrenoceptors in live human heart tissue from patients with advanced heart failure undergoing transplantation. The study compared patients who had been administered β-blocker medicines metoprolol or carvedilol or no β-blocker treatment. This work helped to further elucidate the complex roles of target receptors and enzymes that are integral to the progression of heart failure, to compare the mechanisms of action of β-blockers currently used to manage heart failure and to identify new drug targets for heart failure treatment.

(-)-CGP12177 increases contractile force through beta1l-adrenoceptors but not through beta3-adrenoceptors in human right atrial myocardium

(-)-CGP12177 is a non-conventional partial agonist that causes modest and transient increases of contractile force in human atrial trabeculae (Kaumann and Molenaar, 2008). These effects are markedly increased and maintained by inhibition of phosphodiesterase PDE3. As verified with recombinant receptors, the cardiostimulant effect of (-)-CGP12177 is mediated through a site at the beta1-adrenoceptor with lower affinity (beta1LAR) compared to the site through which (-)-CGP12177 antagonizes the effects of catecholamines (beta1HAR). However, in a recent report it was proposed that the positive inotropic effects of CGP12177 are mediated through beta3-adrenoceptors (Skeberdis et al 2008). We therefore investigated whether the effects of (-)-CGP12177 on human atrial trabeculae are antagonized by the beta3-adrenoceptor-selective antagonist L-748,337 (1 microM). (-)-CGP12177 (200 nM) caused a stable increase in force which was significantly reduced by the addition of (-)-bupranolol (1 microM), P = 0.002, (basal 4.45 ± 0.78 mN, IBMX (PDE inhibitor) 5.47 ± 1.01 mN, (-)-CGP12177 9.34 ± 1.33 mN, (-)-bupranolol 5.79 ± 1.08 mN, n = 6) but not affected by the addition of L-748,337 (1 microM), P = 0.12, (basal 4.48 ± 1.32 mN, IBMX 7.15 ± 2.28 mN, (-)-CGP12177 12.51 ± 3.71 mN, L-748,337 10.90 ± 3.49 mN, n = 6). Cumulative concentration-effect curves for (-)-CGP12177 were not shifted to the right by L-748,337 (1 microM). The –logEC50M values of (-)-CGP12177 in the absence and presence of L-748,337 were 7.21±0.09 and 7.41±0.13, respectively (data from 25 trabeculae from 8 patients, P=0.2) The positive inotropic effects of (-)-CGP12177 (IBMX present) were not antagonized by L-748,337 but were blunted by (-)-bupranolol (1 microM). The results rule out an involvement of beta3-adrenoceptors in the positive inotropic effects (-)-CGP12177 in human right atrial myocardium and are consistent with mediation through beta1LAR. Kaumann A and Molenaar P (2008) Pharmacol Ther 118, 303-336 Skeberdis VA et al (2008) J Clin Invest, 118, 3219-3227

The highs (B1H) and lows (B1L) of human heart B1-adrenoceptors (AR)

The 1AR has two binding sites which can be activated to cause cardiostimulation. The first, termed, 1HAR (high affinity site of 1AR) is activated by noradrenaline and adrenaline and is blocked by relatively low concentrations of β-blockers including carvedilol (Kaumann and Molenaar, 2008). The other, termed, 1LAR (low affinity site of 1AR) has lower affinity for noradrenaline and adrenaline and is activated by some β-blockers including CGP12177 and pindolol, at higher concentrations than those required to block the receptor (Kaumann and Molenaar, 2008). (-)-CGP12177 is a non-conventional partial agonist that causes modest and transient increases of contractile force in human atrial trabeculae (Kaumann and Molenaar, 2008). These effects are markedly increased and maintained by inhibition of phosphodiesterase PDE3. The stimulant effects of (-)-CGP12177 at human β1ARs was verified with recombinant receptors (Kaumann and Molenaar, 2008). However, in a recent report it was proposed that the positive inotropic effects of CGP12177 are mediated through 3ARs in human right atrium (Skeberdis et al 2008). This proposal was not consistent with the lack of blockade of (-)-CGP12177 inotropic effects or increases in L-type Ca2+ current (ICa-L ) by the β3AR blocker 1 μM LY748,337 (Christ et al, 2010). On the otherhand, (-)-CGP12177 increases in inotropic effects and ICa-L were blocked by (-)-bupranolol 1-10 μM (Christ et al, 2010). Chronic infusion of (-)-CGP 12177 (10 mg/Kg/24 hours) for four weeks in an aortic constriction mouse model of heart failure caused an increase in left ventricular wall thickness, fibrosis and inflammation-related left ventricular gene expression levels. Christ T et al (2010) Br J Pharmacol, In press Kaumann A and Molenaar P (2008) Pharmacol Ther 118, 303-336 Skeberdis VA et al (2008) J Clin Invest, 118, 3219-3227

Unswitch-ABL drugs overcome resistance in chronic myeloid leukemia

ABL inhibitors have revolutionized the clinical management of chronic myeloid leukemia, but the BCR-ABLT315I mutation confers resistance to currently approved drugs. Chan et al. show, in this issue of Cancer Cell, that " switch-control" inhibitors block BCR-ABLT315I activity by preventing ABL from switching from the inactive to active conformation.

Antidepressants, but not antipsychotics, modulate GR function in human whole blood: An insight into molecular mechanisms

Clinical studies have demonstrated an impairment of glucocorticoid receptor (GR)-mediated negative feedback on the hypothalamic-pituitary-adrenal (HPA) axis in patients with major depression (GR resistance), and its resolution by antidepressant treatment. Recently, we showed that this impairment is indeed due to a dysfunction of GR in depressed patients (Carvalho et al., 2009), and that the ability of the antidepressant clomipramine to decrease GR function in peripheral blood cells is impaired in patients with major depression who are clinically resistant to treatment (Carvalho et al. 2008). To further investigate the effect of antidepressants on GR function in humans, we have compared the effect of the antidepressants clomipramine, amytriptiline, sertraline, paroxetine and venlafaxine, and of the antipsychotics, haloperidol and risperidone, on GR function in peripheral blood cells from healthy volunteers (n=33). GR function was measured by glucocorticoid inhibition of lypopolysaccharide (LPS)-stimulated interleukin-6 (IL-6) levels. Compared to vehicle-treated cells, all antidepressants inhibited dexamethasone (DEX, 10-100nM) inhibition of LPS-stimulated IL-6 levels (p values ranging from 0.007 to 0.1). This effect was specific to antidepressants, as antipsychotics had no effect on DEX-inhibition of LPS-stimulated IL-6 levels. The phosphodiesterase (PDE) type 4 inhibitor, rolipram, potentiated the effect of antidepressants on GR function, while the GR antagonist, RU-486, inhibited the effect of antidepressants on GR function. These findings indicate that the effect of antidepressants on GR function are specific for this class of psychotropic drugs, and involve second messenger pathways relevant to GR function and inflammation. Furthermore, it also points towards a possible mechanism by which one maybe able to overcome treatment-resistant depression. Research in this field will lead to new insights into the pathophysiology and treatment of affective disorders.

Genetic factors in the pathogenesis of CPPD crystal deposition disease

Crystal deposition is a very complex process ruled by numerous factors. A small but important proportion of cases of chondrocalcinosis are monogenic, and many of the genes involved have been identified. These genetic findings strongly point to control of the level of extracellular inorganic pyrophosphate as the primary mechanism for their association with either calcium pyrophosphate dihydrate or hydroxyapatite deposition. However, effects on extracellular inorganic pyrophosphate levels do not explain the mechanism of association in all of these monogenic diseases. Further, there are likely to be several as yet unidentified genes that are important in this common condition. This review highlights what genetic studies have demonstrated about the processes involved in these diverse but related disorders.

Carvedilol induces greater control of β2- than β1-adrenoceptor-mediated inotropic and lusitropic effects by PDE3, while PDE4 has no effect in human failing myocardium

The beta-blockers carvedilol and metoprolol provide important therapeutic strategies for heart failure treatment. Therapy with metoprolol facilitates the control by phosphodiesterase PDE3, but not PDE4, of inotropic effects of catecholamines in human failing ventricle. However, it is not known whether carvedilol has the same effect. We investigated whether the PDE3-selective inhibitor cilostamide (0.3 mu M) or PDE4-selective inhibitor rolipram (1 mu M) modified the positive inotropic and lusitropic effects of catecholamines in ventricular myocardium of heart failure patients treated with carvedilol. Right ventricular trabeculae from explanted hearts of nine carvedilol-treated patients with terminal heart failure were paced to contract at 1 Hz. The effects of (-)-noradrenaline, mediated through beta(1)-adrenoceptors (beta(2)-adrenoceptors blocked with ICI118551), and (-)-adrenaline, mediated through beta(2)-adrenoceptors (beta(1)-adrenoceptors blocked with CGP20712A), were assessed in the absence and presence of the PDE inhibitors. The inotropic potency, estimated from -logEC(50)s, was unchanged for (-)-noradrenaline but decreased 16-fold for (-)-adrenaline in carvedilol-treated compared to non-beta-blocker-treated patients, consistent with the previously reported beta(2)-adrenoceptor-selectivity of carvedilol. Cilostamide caused 2- to 3-fold and 10- to 35-fold potentiations of the inotropic and lusitropic effects of (-)-noradrenaline and (-)-adrenaline, respectively, in trabeculae from carvedilol-treated patients. Rolipram did not affect the inotropic and lusitropic potencies of (-)-noradrenaline or (-)-adrenaline. Treatment of heart failure patients with carvedilol induces PDE3 to selectively control the positive inotropic and lusitropic effects mediated through ventricular beta(2)-adrenoceptors compared to beta(1)-adrenoceptors. The beta(2)-adrenoceptor-selectivity of carvedilol may provide protection against beta(2)-adrenoceptor-mediated ventricular overstimulation in PDE3 inhibitor-treated patients. PDE4 does not control beta(1)- and beta(2)-adrenoceptor-mediated inotropic and lusitropic effects in carvedilol-treated patients.

Strong evidence for a novel schizophrenia risk locus on chromosome 1p31.1 in homogeneous pedigrees from Tamil Nadu, India

OBJECTIVE: The study of ethnically homogeneous populations may help to identify schizophrenia risk loci. The authors conducted a genomewide linkage scan for schizophrenia in an Indian population. METHOD: Participants were 441 individuals (262 affected probands and siblings) who were recruited primarily from one ethnically homogeneous group, the Tamil Brahmin caste, although individuals from other geographically proximal castes also participated. Genotyping of 124 affected sibling pair pedigrees was performed with 402 short tandem repeat polymorphisms. Linkage analyses were conducted using nonparametric exponential LOD (logarithm of the odds ratio for linkage) scores and parametric heterogeneity LOD scores. Parametric heterogeneity scores were calculated using simple dominant and recessive models, correcting for multiple statistics. The data were examined for evidence of consanguinity. Genomewide significance levels were determined using 10,000 gene dropping simulations. RESULTS: These findings revealed genomewide significant linkage to chromosome 1p31.1, through the use of both exponential and heterogeneity LOD scores, incorporating correction for multiple statistics and mild consanguinity. The estimated sibling recurrence risk associated with this putative locus was 1.95. Analysis for heterogeneity LOD scores also detected suggestive linkage to chromosomes 13q22.1 and 16q12.2. Using 117 tag single nucleotide polymorphisms (SNPs), family-based association analyses of phosphodiesterase 4B (PDE4B), the closest schizophrenia candidate gene, detected no convincing evidence of association, suggesting that the chromosome 1 peak represents a novel risk locus. CONCLUSIONS: This is the first study-to the authors' knowledge-to report significant linkage of schizophrenia to chromosome 1p31.1. Further investigation of this chromosome region in diverse populations is warranted to identify underlying sequence variants.