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ABSTRACT: The establishment of set points for cellular activities is essential in regulating homeostasis. Here we demonstrate key determinants of the fibrogenic set point of cardiac fibroblasts (CFs) by focusing on the pro-fibrotic activity of ATP, which is released by CFs. We tested the hypothesis that the hydrolysis of extracellular ATP by ectonucleoside triphosphate diphosphohydrolases (ENTPDs) regulates pro-fibrotic nucleotide signaling. We detected two ENTPD isoforms, ENTPD-1 and -2, in adult rat ventricular CFs. Partial knockdown of ENTPD-1 and -2 with siRNA increased basal extracellular ATP concentration and enhanced the pro-fibrotic effect of ATP stimulation. Sodium polyoxotungstate (POM)-1, an ENTPD inhibitor, not only enhanced the pro-fibrotic effects of exogenously added ATP but also increased basal expression of α-smooth muscle actin (α-SMA), plasminogen activator inhibitor (PAI)-1 and transforming growth factor (TGF)-β, collagen synthesis and gel contraction. Furthermore, we found that adenosine, a product of ATP hydrolysis by ENTPD, acts via A2B receptors to counterbalance the pro-fibrotic response to ATP. Removal of extracellular adenosine or inhibition of A2B receptors enhanced pro-fibrotic ATP signaling. Together, these results demonstrate the contribution of basally released ATP in establishing the set point for fibrotic activity in adult rat CFs and identify a key role for the modulation of this activity by hydrolysis of released ATP by ENTPDs. These findings also imply that cellular homeostasis and fibrotic response involves the integration of signaling that is pro-fibrotic by ATP and anti-fibrotic by adenosine and which is regulated by ENTPDs.
Journal of Biological Chemistry 05/2013; · 4.77 Impact Factor
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ABSTRACT: Introduction: Cyclic AMP (cAMP) promotes growth arrest and/or apoptosis of various types of lymphoma, in particular chronic lymphocytic leukemia (CLL). These responses have spurred the interest in developing agents that increase cAMP to treat such malignancies and to identify mechanisms of the responses. Areas covered: The murine T-lymphoma cell line S49, has provided an important, pioneering model to define mechanisms of cAMP-mediated lymphoid cell death. Studies with S49 cells demonstrated that cAMP, acting via protein kinase A (PKA), is pro-apoptotic through a mitochondria-dependent pathway and identified cAMP/PKA-regulated targets involved in apoptosis. Akin to such findings, cAMP promotes apoptosis via PKA of cells from patients with CLL. Analysis of mediators of cAMP accumulation and cAMP-promoted apoptosis in CLL cells has revealed approaches to increase cAMP and engage its pro-apoptotic action. Expert opinion: This 'pathway approach' targeted to cAMP has identified GPCR agonists/antagonists, AC activators (e.g., AC7), PDE inhibitors (e.g., PDE7B) and/or activators or inhibitors of downstream mediators (PKA and Epac, respectively), which might be utilized therapeutically in CLL. Therapy directed at such targets may prove to be clinically useful and may also provide a proof-of-principle of the utility of targeting cAMP signaling in other types of cancer.
Expert opinion on therapeutic targets 05/2013; · 3.72 Impact Factor
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ABSTRACT: In order to define the proteins whose expression is regulated by cAMP and protein kinase A (PKA), we used a quantitative proteomic approach in studies of wild-type (WT) and kin- (PKA-null) S49 murine T lymphoma cells. We also compared the impact of endogenous increases in cAMP (Forskolin [Fsk] plus the phosphodiesterase inhibitor isobutylmethylxanthine [IBMX]) or by a cAMP analog (8-CPT-cAMP). We identified 1056 proteins in WT and kin- S49 cells and found that 8-CPT-cAMP and Fsk/IBMX produced differences in protein expression. WT S49 cells had a correlation coefficient of 0.41 between DNA microarray data and the proteomic analysis in cells incubated with 8-CPT-cAMP for 24 h and a correlation coefficient of 0.42 between the DNA microarray data obtained at 6 h and the changes in protein expression after 24 h incubation with 8-CPT-cAMP. Glutathione reductase (Gsr) had higher basal expression in kin- S49 cells than in WT cells. Consistent with this finding, kin- cells are less sensitive to cell killing and generation of malondialdehyde than are WT cells incubated with H2O2. Cyclic AMP acting via PKA thus has a broad impact on protein expression in mammalian cells, including in the regulation of Gsr and oxidative stress.
Biochemistry 10/2012; · 3.42 Impact Factor
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Heidi N Fridolfsson,
Yoshitaka Kawaraguchi,
Sameh S Ali,
Mathivadhani Panneerselvam,
Ingrid R Niesman,
J Cameron Finley,
Sarah E Kellerhals,
Michael Y Migita,
Hideshi Okada,
Ana L Moreno, [......],
Ravi C Balijepalli,
Robert S Ross,
Piyush M Patel,
Atsushi Miyanohara,
Qun Chen,
Edward J Lesnefsky,
Brian P Head,
David M Roth, Paul A Insel,
Hemal H Patel
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ABSTRACT: We show here that the apposition of plasma membrane caveolae and mitochondria (first noted in electron micrographs >50 yr ago) and caveolae-mitochondria interaction regulates adaptation to cellular stress by modulating the structure and function of mitochondria. In C57Bl/6 mice engineered to overexpress caveolin specifically in cardiac myocytes (Cav-3 OE), localization of caveolin to mitochondria increases membrane rigidity (4.2%; P<0.05), tolerance to calcium, and respiratory function (72% increase in state 3 and 23% increase in complex IV activity; P<0.05), while reducing stress-induced generation of reactive oxygen species (by 20% in cellular superoxide and 41 and 28% in mitochondrial superoxide under states 4 and 3, respectively; P<0.05) in Cav-3 OE vs. TGneg. By contrast, mitochondrial function is abnormal in caveolin-knockout mice and Caenorhabditis elegans with null mutations in caveolin (60% increase free radical in Cav-2 C. elegans mutants; P<0.05). In human colon cancer cells, mitochondria with increased caveolin have a 30% decrease in apoptotic stress (P<0.05), but cells with disrupted mitochondria-caveolin interaction have a 30% increase in stress response (P<0.05). Targeted gene transfer of caveolin to mitochondria in C57Bl/6 mice increases cardiac mitochondria tolerance to calcium, enhances respiratory function (increases of 90% state 4, 220% state 3, 88% complex IV activity; P<0.05), and decreases (by 33%) cardiac damage (P<0.05). Physical association and apparently the transfer of caveolin between caveolae and mitochondria is thus a conserved cellular response that confers protection from cellular damage in a variety of tissues and settings.-Fridolfsson, H. N., Kawaraguchi, Y., Ali, S. S., Panneerselvam, M., Niesman, I. R., Finley, J. C., Kellerhals, S. E., Migita, M. Y., Okada, H., Moreno, A. L., Jennings, M., Kidd, M. W., Bonds, J. A., Balijepalli, R. C., Ross, R. S., Patel, P. M., Miyanohara, A., Chen, Q., Lesnefsky, E. J., Head, B. P., Roth, D. M., Insel, P. A., Patel, H. H. Mitochondria-localized caveolin in adaptation to cellular stress and injury.
The FASEB Journal 08/2012; · 5.71 Impact Factor
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AJP Cell Physiology 07/2012; 303(5):C488-9. · 3.54 Impact Factor
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ABSTRACT: Because the complex phenotype of human hypertension is at least in part genetically determined, how individual genes ultimately
contribute to the disease is not well understood. By contrast, intermediate phenotypes are traits associated with complex
disease, but which may display simpler genetic properties such as greater heritability, more consistent and earlier penetrance
and bimodality, and may suggest particular candidate susceptibility genes. Because autonomic nervous system activity is altered
in hypertension, we examined biochemical, physiologic, and pharmacologic autonomic traits that fulfill at least some of these
properties. Such biochemical, physiologic, or pharmacologic autonomic traits may be especially valuable as phenotypic anchor
points in linkage or association studies probing the genetic basis of human hypertension.
Current Hypertension Reports 04/2012; 2(1):16-22. · 2.50 Impact Factor
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ABSTRACT: The directional movement of cells can be regulated by ATP, certain other nucleotides (e.g., ADP, UTP), and adenosine. Such regulation occurs for cells that are "professional phagocytes" (e.g., neutrophils, macrophages, certain lymphocytes, and microglia) and that undergo directional migration and subsequent phagocytosis. Numerous other cell types (e.g., fibroblasts, endothelial cells, neurons, and keratinocytes) also change motility and migration in response to ATP, other nucleotides, and adenosine. In this article, we review how nucleotides and adenosine modulate chemotaxis and motility and highlight the importance of nucleotide- and adenosine-regulated cell migration in several cell types: neutrophils, microglia, endothelial cells, and cancer cells. We also discuss difficulties in conducting experiments and drawing conclusions regarding the ability of nucleotides and adenosine to modulate the migration of professional and non-professional phagocytes.
Purinergic Signalling 04/2012; 8(3):587-98. · 3.16 Impact Factor
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ABSTRACT: Cardiac fibroblasts (CFs) play an essential role in remodeling of the cardiac extracellular matrix. Extracellular nucleotide signaling may provoke a profibrotic response in CFs. We tested the hypothesis that physical perturbations release ATP from CFs and that ATP participates in profibrotic signaling. ATP release was abolished by the channel inhibitor carbenoxolone and inhibited by knockdown of either connexin (Cx)43 or Cx45 (47 and 35%, respectively), implying that hypotonic stimulation induces ATP release via Cx43 and Cx45 hemichannels, although pannexin 1 may also play a role. ATP released by hypotonic stimulation rapidly (<10 min) increased phosphorylated ERK by 5-8 fold, an effect largely eliminated by P2Y(2) receptor knockdown or ATP hydrolysis with apyrase. ATP stimulation of P2Y(2) receptors increased α-smooth muscle actin (α-SMA) production, and in an ERK-dependent manner, ATP increased collagen accumulation by 60% and mRNA expression of profibrotic markers: plasminogen activator inhibitor-1 and monocyte chemotactic protein-1 by 4.5- and 4.0-fold, respectively. Apyrase treatment substantially reduced the basal profibrotic phenotype, decreasing collagen and α-SMA content and increasing matrix metalloproteinase expression. Thus, ATP release activates P2Y(2) receptors to mediate profibrotic responses in CFs, implying that nucleotide release under both basal and activated states is likely an important mechanism for fibroblast homeostasis.
The FASEB Journal 03/2012; 26(6):2580-91. · 5.71 Impact Factor
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ABSTRACT: Fibrosis, the result of excess deposition of extracellular matrix (ECM), in particular collagen, leads to scarring and loss of function in tissues that include the heart, lung, kidney and liver. The second messenger cAMP can inhibit the formation and extent of ECM during this late phase of inflammation, but the mechanisms for these actions of cAMP and of agents that elevate tissue cAMP levels are not well understood. In this article, we review the fibrotic process and focus on two recently recognized aspects of actions of cAMP and its effector Epac (Exchange protein activated by cAMP): (a) blunting of epithelial-mesenchymal transformation (EMT) and (b) down-regulation of Epac expression by profibrotic agents (e.g. TGF-β, angiotensin II), which may promote tissue fibrosis by decreasing Epac-mediated antifibrotic actions. Pharmacological approaches that raise cAMP or blunt the decrease in Epac expression by profibrotic agents may thus be strategies to block or perhaps reverse tissue fibrosis. LINKED ARTICLES This article is part of a themed section on Novel cAMP Signalling Paradigms. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-2.
British Journal of Pharmacology 01/2012; 166(2):447-56. · 4.41 Impact Factor
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ABSTRACT: Ligand binding to receptors is a key step in the regulation of cellular function by neurotransmitters, hormones, and many drugs. Not surprisingly then, genome projects have found that families of receptor genes form the largest groups of functional genes in mammalian genomes. A large body of experimental data have thus been generated on receptor-ligand interactions, and in turn, numerous computational tools for the in silico prediction of receptor-ligand interactions have been developed. Websites containing ligand binding data and tools to assess and manipulate such data are available in the public domain. Such Websites provide a resource for experimentalists studying receptor binding and for scientists interested in utilizing large data sets for other purposes, which include modeling structure-function relationships, defining patterns of interactions of drugs with different receptors, and computational comparisons among receptors. The Websites include databases of receptor protein and nucleotide sequences for particular classes of receptors (such as G-protein-coupled receptors and nuclear receptors) and of experimental results from receptor-ligand binding assays, as well as computational tools for modeling the interactions between ligands and receptors and predicting the function of orphan receptors. In this chapter, we provide information and Uniform Resource Locators (URLs) for Websites that facilitate computational and experimental studies of receptor-ligand interactions. This list will be periodically updated at https://sites.google.com/site/receptorligandbinding/.
Methods in molecular biology (Clifton, N.J.) 01/2012; 897:1-13.
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ABSTRACT: The mechanism of cAMP-promoted apoptosis is not well defined. In wild-type (WT) murine S49 lymphoma cells, cAMP promotes apoptosis in a protein kinase A (PKA)-dependent manner. We find that treatment of WT S49 cells with 8-CPT-cAMP prominently increases the expression (as determined by DNA microarray analysis, real-time PCR and immunblotting) of cytotoxic T lymphocyte antigen-2α (CTLA-2α), a cathepsin L-like cysteine protease inhibitor. By contrast, CTLA-2α expression is only slightly increased by 8-CPT-cAMP treatment of D-S49 cells, which lack cAMP/PKA-promoted apoptosis. Raising endogenous cAMP (by use of forskolin or inhibition of phosphodiesterase [PDE] 4) or a PKA-selective, but not an Epac-selective, cAMP analogue, increases CTLA-2α mRNA expression; PKA, and not Epac, thus mediates the increase in CTLA-2α expression. An adenoviral CLTA-2α (Ad-CTLA-2α) construct induces apoptosis and enhances cAMP-promoted apoptosis in WT S49 cells but such cells do not have an increase in cathepsin L activity nor does a cathepsin L inhibitor alter cAMP-promoted apoptosis. 8-CPT-cAMP also increases CTLA-2α expression and induces apoptosis in murine cardiac fibroblasts; knockdown of CTLA-2α expression by siRNA blocks 8-CPT-cAMP-promoted apoptosis. Thus, cAMP increases CTLA-2α expression in murine lymphoma and cardiac fibroblasts and this increase in CTLA-2α contributes to cAMP/PKA-promoted apoptosis by mechanisms that are independent of the ability of CTLA-2α to inhibit cathepsin L.
Cellular signalling 10/2011; 23(10):1611-6. · 4.09 Impact Factor
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Brian P. Head,
Yue Hu,
J. Cameron Finley,
Michelle D. Saldana,
Jacqueline A. Bonds,
Atsushi Miyanohara,
Ingrid R. Niesman,
Sameh S. Ali,
Fiona Murray, Paul A. Insel,
David M. Roth,
Hemal H. Patel,
Piyush M. Patel
[show abstract]
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ABSTRACT: Decreased expression of prosurvival and progrowth-stimulatory pathways, in addition to an environment that inhibits neuronal
growth, contribute to the limited regenerative capacity in the central nervous system following injury or neurodegeneration.
Membrane/lipid rafts, plasmalemmal microdomains enriched in cholesterol, sphingolipids, and the protein caveolin (Cav) are
essential for synaptic development/stabilization and neuronal signaling. Cav-1 concentrates glutamate and neurotrophin receptors
and prosurvival kinases and regulates cAMP formation. Here, we show that primary neurons that express a synapsin-driven Cav-1
vector (SynCav1) have increased raft formation, neurotransmitter and neurotrophin receptor expression, NMDA- and BDNF-mediated prosurvival
kinase activation, agonist-stimulated cAMP formation, and dendritic growth. Moreover, expression of SynCav1 in Cav-1 KO neurons restores NMDA- and BDNF-mediated signaling and enhances dendritic growth. The enhanced dendritic growth
occurred even in the presence of inhibitory cytokines (TNFα, IL-1β) and myelin-associated glycoproteins (MAG, Nogo). Targeting
of Cav-1 to neurons thus enhances prosurvival and progrowth signaling and may be a novel means to repair the injured and neurodegenerative
brain.
Journal of Biological Chemistry 09/2011; 286(38):33310-33321. · 4.77 Impact Factor
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Paul A Insel
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ABSTRACT: Nonsynonymous, coding sequence single-nucleotide polymorphisms in β(2)-adrenergic receptors were first recognized almost 20 years ago, but a full understanding of their impact on signal transduction-especially on receptor abundance in native cells and their clinical importance-remains unclear. New evidence has revealed a feature of the Arg(16)Gly variant of β(2)-adrenergic receptors that has not been previously noted: a difference in the rate of response upon repeated stimulation of the receptors, such that the Arg(16) variant shows slower activation and the Gly(16) variant faster activation of cyclic adenosine monophosphate (cAMP) formation-a feature that the authors term "receptor memory." This is an intriguing idea but will require confirmation and demonstration of its functional importance in vivo and its possible contribution to clinical responses, especially in terms of the administration of β(2)-adrenergic agonists.
Science Signaling 08/2011; 4(185):pe37. · 7.50 Impact Factor
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ABSTRACT: The second messenger cAMP is proapoptotic for numerous cell types, but the mechanism for this proapoptotic action is not defined. Here, we use murine CD4(+)/CD8(+) S49 lymphoma cells and isolated thymocytes to assess this mechanism. In WT S49 cells, cAMP acts via protein kinase A (PKA) to induce G(1) phase cell cycle arrest and apoptosis. Treatment of WT and cAMP-Deathless (D-) S49 cells, which lack cAMP-promoted apoptosis, with the PKA agonist 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) differentially regulates transcripts for numerous proapoptotic and antiapoptotic proteins. In contrast, kin-S49 cells (which lack PKA) show no cAMP-promoted changes in transcript expression. In this study, we use knockdown and overexpression approaches to define the role in cAMP/PKA-promoted apoptosis of the proapoptotic factor BIM (Bcl-2 interacting mediator of cell death), whose expression prominently increases in response to CPT-cAMP treatment of WT but not D- or kin- S49 cells. Conditional expression of BimL, one of the three major forms of Bim, increases apoptosis of WT, D-, and kin-S49 cells, whereas inhibition of cAMP-mediated induction of Bim isoforms by shRNAi attenuates CPT-cAMP-mediated apoptosis of WT S49 cells. Bim protein levels increase in subpopulations of CPT-cAMP-treated cells that undergo apoptosis. Thymic CD4(+)/CD8(+) cells isolated from Bim(-/-) mice corroborated the requirement of Bim expression for cAMP-promoted apoptosis. Thus, up-regulation of Bim appears to be an important determinant of cAMP/PKA-mediated apoptosis in immature T cells and may be a mechanism for such apoptosis in other cell types as well.
Journal of Biological Chemistry 08/2011; 286(38):33260-7. · 4.77 Impact Factor
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ABSTRACT: Expression of cyclic adenosine monophosphate-specific phosphodiesterase 7B (PDE7B) mRNA is increased in patients with chronic lymphocytic leukemia (CLL), thus suggesting that variation may occur in the PDE7B gene in CLL. As genetic variation in other PDE family members has been shown to associate with numerous clinical disorders (reviewed in this manuscript), we sought to identify single-nucleotide polymorphisms (SNPs) in the PDE7B gene promoter and coding region of 93 control subjects and 154 CLL patients. We found that the PDE7B gene has a 5' non-coding region SNP -347C>T that occurs with similar frequency in CLL patients (1.9%) and controls (2.7%). Tested in vitro, -347C>T has less promoter activity than a wild-type construct. The low frequency of this 5' untranslated region variant indicates that it does not explain the higher PDE7B expression in patients with CLL but it has the potential to influence other settings that involve a role for PDE7B.
Journal of Human Genetics 07/2011; 56(9):676-81. · 2.57 Impact Factor
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Brian P Head,
Yue Hu,
J Cameron Finley,
Michelle D Saldana,
Jacqueline A Bonds,
Atsushi Miyanohara,
Ingrid R Niesman,
Sameh S Ali,
Fiona Murray, Paul A Insel,
David M Roth,
Hemal H Patel,
Piyush M Patel
[show abstract]
[hide abstract]
ABSTRACT: Decreased expression of prosurvival and progrowth-stimulatory pathways, in addition to an environment that inhibits neuronal growth, contribute to the limited regenerative capacity in the central nervous system following injury or neurodegeneration. Membrane/lipid rafts, plasmalemmal microdomains enriched in cholesterol, sphingolipids, and the protein caveolin (Cav) are essential for synaptic development/stabilization and neuronal signaling. Cav-1 concentrates glutamate and neurotrophin receptors and prosurvival kinases and regulates cAMP formation. Here, we show that primary neurons that express a synapsin-driven Cav-1 vector (SynCav1) have increased raft formation, neurotransmitter and neurotrophin receptor expression, NMDA- and BDNF-mediated prosurvival kinase activation, agonist-stimulated cAMP formation, and dendritic growth. Moreover, expression of SynCav1 in Cav-1 KO neurons restores NMDA- and BDNF-mediated signaling and enhances dendritic growth. The enhanced dendritic growth occurred even in the presence of inhibitory cytokines (TNFα, IL-1β) and myelin-associated glycoproteins (MAG, Nogo). Targeting of Cav-1 to neurons thus enhances prosurvival and progrowth signaling and may be a novel means to repair the injured and neurodegenerative brain.
Journal of Biological Chemistry 07/2011; 286(38):33310-21. · 4.77 Impact Factor
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Yousuke T Horikawa,
Mathivadhani Panneerselvam,
Yoshitaka Kawaraguchi,
Yasuo M Tsutsumi,
Sameh S Ali,
Ravi C Balijepalli,
Fiona Murray,
Brian P Head,
Ingrid R Niesman,
Timo Rieg,
Volker Vallon, Paul A Insel,
Hemal H Patel,
David M Roth
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ABSTRACT: We hypothesized that cardiac myocyte-specific overexpression of caveolin-3 (Cav-3), a muscle-specific caveolin, would alter natriuretic peptide signaling and attenuate cardiac hypertrophy.
Natriuretic peptides modulate cardiac hypertrophy and are potential therapeutic options for patients with heart failure. Caveolae, microdomains in the plasma membrane that contain caveolin proteins and natriuretic peptide receptors, have been implicated in cardiac hypertrophy and natriuretic peptide localization.
We generated transgenic mice with cardiac myocyte-specific overexpression of caveolin-3 (Cav-3 OE) and also used an adenoviral construct to increase Cav-3 in cardiac myocytes.
The Cav-3 OE mice subjected to transverse aortic constriction had increased survival, reduced cardiac hypertrophy, and maintenance of cardiac function compared with control mice. In left ventricle at baseline, messenger ribonucleic acid for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were increased 7- and 3-fold, respectively, in Cav-3 OE mice compared with control subjects and were accompanied by increased protein expression for ANP and BNP. In addition, ventricles from Cav-3 OE mice had greater cyclic guanosine monophosphate levels, less nuclear factor of activated T-cell nuclear translocation, and more nuclear Akt phosphorylation than ventricles from control subjects. Cardiac myocytes incubated with Cav-3 adenovirus showed increased expression of Cav-3, ANP, and Akt phosphorylation. Incubation with methyl-β-cyclodextrin, which disrupts caveolae, or with wortmannin, a PI3K inhibitor, blocked the increase in ANP expression.
These results imply that cardiac myocyte-specific Cav-3 OE is a novel strategy to enhance natriuretic peptide expression, attenuate hypertrophy, and possibly exploit the therapeutic benefits of natriuretic peptides in cardiac hypertrophy and heart failure.
Journal of the American College of Cardiology 05/2011; 57(22):2273-83. · 14.16 Impact Factor
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ABSTRACT: ATP and UTP are endogenous agonists of P2Y(2/4) receptors. To define the in vivo effects of P2Y(2) receptor activation on blood pressure and urinary excretion, we compared the response to INS45973, a P2Y(2/4) receptor agonist and UTP analog, in wild-type (WT) and P2Y(2) receptor knockout (P2Y(2)-/-) mice. INS45973 was administered intravenously as a bolus injection or continuous infusion to determine effects on blood pressure and renal function, respectively. Within seconds, bolus application of INS45973 (0.1 to 3 mg/kg body wt) dose-dependently decreased blood pressure in WT (maximum response -35 ± 2 mmHg) and to a similar extent in endothelial nitric oxide synthase knockout mice. By contrast, blood pressure increased in P2Y(2)-/- (maximum response +18 ± 1 mmHg) but returned to basal levels within 60 s. Continuous infusion of INS45973 (25 to 750 μg·min(-1)·kg(-1) body wt) dose-dependently increased urinary excretion of Na(+) in WT (maximum response +46 ± 15%) but reduced Na(+) excretion in P2Y(2)-/- (maximum responses of -45 ± 15%) mice. In renal clearance experiments, INS45973 did not affect glomerular filtration rate but lowered blood pressure and increased fractional excretion of fluid, Na(+), and K(+) in WT relative to P2Y(2)-/- mice. The blood pressure responses to INS45973 are consistent with P2Y(2) receptor-mediated NO-independent vasodilation and implicate responses to endothelium-derived hyperpolarizing factor, and P2Y(2) receptor-independent vasoconstriction, probably via activation of P2Y(4) receptors on smooth muscle. Systemic activation of P2Y(2) receptors thus lowers blood pressure and inhibits renal Na(+) reabsorption, effects suggesting the potential utility of P2Y(2) agonism in the treatment of hypertension.
AJP Regulatory Integrative and Comparative Physiology 05/2011; 301(2):R510-8. · 3.34 Impact Factor
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ABSTRACT: Caveolin (Cav) proteins in the plasma membrane have numerous binding partners, but the determinants of these interactions are poorly understood. We show here that Cav-3 has a small ubiquitin-like modifier (SUMO) consensus motif (ΨKX(D/E, where Ψ is a hydrophobic residue)) near the scaffolding domain and that Cav-3 is SUMOylated in a manner that is enhanced by the SUMO E3 ligase PIASy (protein inhibitor of activated STAT-y). Site-directed mutagenesis revealed that the consensus site lysine is the preferred SUMOylation site but that mutation of all lysines is required to abolish SUMOylation. Co-expression of a SUMOylation-deficient mutant of Cav-3 with β-adrenergic receptors (βARs) alters the expression level of β(2)ARs but not β(1)ARs following agonist stimulation, thus implicating Cav-3 SUMOylation in the mechanisms for β(2)AR but not β(1)AR desensitization. Expression of endothelial nitric-oxide synthase (NOS3) was not altered by the SUMOylation-deficient mutant. Thus, SUMOylation is a covalent modification of caveolins that influence the regulation of certain signaling partners.
Journal of Biological Chemistry 03/2011; 286(17):14830-41. · 4.77 Impact Factor
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ABSTRACT: The second messenger cyclic AMP (cAMP) can either stimulate or inhibit programmed cell death (apoptosis). Here, we review examples of cell types that show pro-apoptotic or anti-apoptotic responses to increases in cAMP. We also show that cells can have both such responses, although predominantly having one or the other. Protein kinase A (PKA)-promoted changes in phosphorylation and gene expression can mediate pro-apoptotic responses, such as in murine S49 lymphoma cells, based on evidence that mutants lacking PKA fail to undergo cAMP-promoted, mitochondria-dependent apoptosis. Mechanisms for the anti-apoptotic response to cAMP likely involve Epac (Exchange protein activated by cAMP), a cAMP-regulated effector that is a guanine nucleotide exchange factor (GEF) for the low molecular weight G-protein, Rap1. Therapeutic approaches that activate PKA-mediated pro-apoptosis or block Epac-mediated anti-apoptotisis may provide a means to enhance cell killing, such as in certain cancers. In contrast, efforts to block PKA or stimulate Epac have the potential to be useful in diseases settings (such as heart failure) associated with cAMP-promoted apoptosis.
Acta Physiologica 03/2011; 204(2):277-87. · 3.09 Impact Factor
