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See detailPlasma membrane calcium pump (PMCA4)-neuronal nitric-oxide synthase complex regulates cardiac contractility through modulation of a compartmentalized cyclic nucleotide microdomain.
Mohamed, Tamer M. A.; Oceandy, Delvac; Zi, Min et al

in The Journal of biological chemistry (2011), 286(48), 41520-9

Identification of the signaling pathways that regulate cyclic nucleotide microdomains is essential to our understanding of cardiac physiology and pathophysiology. Although there is growing evidence that ... [more ▼]

Identification of the signaling pathways that regulate cyclic nucleotide microdomains is essential to our understanding of cardiac physiology and pathophysiology. Although there is growing evidence that the plasma membrane Ca(2+)/calmodulin-dependent ATPase 4 (PMCA4) is a regulator of neuronal nitric-oxide synthase, the physiological consequence of this regulation is unclear. We therefore tested the hypothesis that PMCA4 has a key structural role in tethering neuronal nitric-oxide synthase to a highly compartmentalized domain in the cardiac cell membrane. This structural role has functional consequences on cAMP and cGMP signaling in a PMCA4-governed microdomain, which ultimately regulates cardiac contractility. In vivo contractility and calcium amplitude were increased in PMCA4 knock-out animals (PMCA4(-/-)) with no change in diastolic relaxation or the rate of calcium decay, showing that PMCA4 has a function distinct from beat-to-beat calcium transport. Surprisingly, in PMCA4(-/-), over 36% of membrane-associated neuronal nitric-oxide synthase (nNOS) protein and activity was delocalized to the cytosol with no change in total nNOS protein, resulting in a significant decrease in microdomain cGMP, which in turn led to a significant elevation in local cAMP levels through a decrease in PDE2 activity (measured by FRET-based sensors). This resulted in increased L-type calcium channel activity and ryanodine receptor phosphorylation and hence increased contractility. In the heart, in addition to subsarcolemmal calcium transport, PMCA4 acts as a structural molecule that maintains the spatial and functional integrity of the nNOS signaling complex in a defined microdomain. This has profound consequences for the regulation of local cyclic nucleotide and hence cardiac beta-adrenergic signaling. [less ▲]

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See detailEndothelial nitric oxide synthase activity is inhibited by the plasma membrane calcium ATPase in human endothelial cells.
Holton, Marylouisa; Mohamed, Tamer M. A.; Oceandy, Delvac et al

in Cardiovascular research (2010), 87(3), 440-8

AIMS: Nitric oxide (NO) plays a pivotal role in the regulation of cardiovascular physiology. Endothelial NO is mainly produced by the endothelial nitric oxide synthase (eNOS) enzyme. eNOS enzymatic ... [more ▼]

AIMS: Nitric oxide (NO) plays a pivotal role in the regulation of cardiovascular physiology. Endothelial NO is mainly produced by the endothelial nitric oxide synthase (eNOS) enzyme. eNOS enzymatic activity is regulated at several levels, including Ca(2+)/calmodulin binding and the interaction of eNOS with associated proteins. There is emerging evidence indicating a role for the plasma membrane calcium ATPase (PMCA) as a negative regulator of Ca(2+)/calmodulin-dependent signal transduction pathways via its interaction with partner proteins. The aim of our study was to investigate the possibility that the activity of eNOS is regulated through its association with endothelial PMCA. METHODS AND RESULTS: We show here a novel interaction between endogenous eNOS and PMCA in human primary endothelial cells. The interaction domains were located to the region 735-934 of eNOS and the catalytic domain of PMCA. Ectopic expression of PMCA in endothelial cells resulted in an increase in phosphorylation of the residue Thr-495 of endogenous eNOS. However, disruption of the PMCA-eNOS interaction by expression of the PMCA interaction domain significantly reversed the PMCA-mediated effect on eNOS phosphorylation. These results suggest that eNOS activity is negatively regulated via interaction with PMCA. Moreover, NO production by endothelial cells was significantly reduced by ectopic expression of PMCA. CONCLUSION: Our results show strong evidence for a novel functional interaction between endogenous PMCA and eNOS in endothelial cells, suggesting a role for endothelial PMCA as a negative modulator of eNOS activity, and, therefore, NO-dependent signal transduction pathways. [less ▲]

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See detailPlasma membrane calcium ATPase proteins as novel regulators of signal transduction pathways.
Holton, Mary Louisa; Wang, Weiguang; Emerson, Michael et al

in World Journal of Biological Chemistry (2010), 1(6), 201-8

Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs ... [more ▼]

Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathways by targeting specific proteins to cellular sub-domains where the levels of intracellular free calcium are kept low by the calcium ejection properties of PMCAs. According to this model, PMCAs have been shown to interact functionally with the calcium-sensitive proteins neuronal nitric oxide synthase, calmodulin-dependent serine protein kinase, calcineurin and endothelial nitric oxidase synthase. Transgenic animals with altered expression of PMCAs are being used to evaluate the physiological significance of these interactions. To date, PMCA interactions with calcium-dependent partner proteins have been demonstrated to play a crucial role in the pathophysiology of the cardiovascular system via regulation of the nitric oxide and calcineurin/nuclear factor of activated T cells pathways. This new evidence suggests that PMCAs play a more sophisticated role than the mere ejection of calcium from the cells, by acting as modulators of signaling transduction pathways. [less ▲]

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See detailThe cardiovascular phenotype of a mouse model of acromegaly.
Izzard, Ashley S.; Emerson, Michael; Prehar, Sukhpal et al

in Growth hormone & IGF research : official journal of the Growth Hormone Research Society and the International IGF Research Society (2009), 19(5), 413-9

BACKGROUND: Although, it is accepted that there is an excess of cardiovascular mortality in acromegaly, it is uncertain whether this is due to the direct effects of growth hormone-induced-cardiomyopathy ... [more ▼]

BACKGROUND: Although, it is accepted that there is an excess of cardiovascular mortality in acromegaly, it is uncertain whether this is due to the direct effects of growth hormone-induced-cardiomyopathy or is a consequence of atherosclerosis secondary to the metabolic syndrome often observed in this condition. Direct comparison of a mouse model of acromegaly to a mouse model of Laron's syndrome allowed us to carry out detailed phenotyping and better understand the role GH plays in the circulatory system. METHODS AND RESULTS: Transgenic mice that overexpress the growth hormone gene (GH) developed gigantism, including insulin resistance and higher blood pressures commensurate with increased body mass. In these giant mice, the hearts were hypertrophied but haemodynamic studies suggested contractile function was normal. Segments of small arteries mounted in a pressure myograph showed vascular wall hypertrophy but a preserved lumen diameter. Vascular contractile function was normal. Mice in which the GH receptor gene was disrupted or 'knocked out' were dwarf and had low blood pressure, small hearts and blood vessels but a normally functioning circulation. Correlations of body mass with cardiovascular parameters suggested that blood pressure and structural characteristics develop in line with body size. CONCLUSION: In this transgenic mouse model of acromegaly, there is cardiac and vascular hypertrophy commensurate with GH excess but normal function. Our findings support the contention that the excess mortality in this condition may be due to the development of hypertrophic cardiomyopathy rather than increased rates of atherosclerotic coronary artery disease. [less ▲]

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See detailNeuronal nitric oxide synthase signaling in the heart is regulated by the sarcolemmal calcium pump 4b.
Oceandy, Delvac; Cartwright, Elizabeth J.; Emerson, Michael et al

in Circulation (2007), 115(4), 483-92

BACKGROUND: Neuronal nitric oxide synthase (nNOS) has recently been shown to be a major regulator of cardiac contractility. In a cellular system, we have previously shown that nNOS is regulated by the ... [more ▼]

BACKGROUND: Neuronal nitric oxide synthase (nNOS) has recently been shown to be a major regulator of cardiac contractility. In a cellular system, we have previously shown that nNOS is regulated by the isoform 4b of plasma membrane calcium/calmodulin-dependent ATPase (PMCA4b) through direct interaction mediated by a PDZ domain (PSD 95, Drosophilia Discs large protein and Zona occludens-1) on nNOS and a cognate ligand on PMCA4b. It remains unknown, however, whether this interaction has physiological relevance in the heart in vivo. METHODS AND RESULTS: We generated 2 strains of transgenic mice overexpressing either human PMCA4b or PMCA ct120 in the heart. PMCA ct120 is a highly active mutant form of the pump that does not interact with or modulate nNOS function. Calcium was extruded normally from PMCA4b-overexpressing cardiomyocytes, but in vivo, overexpression of PMCA4b reduced the beta-adrenergic contractile response. This attenuated response was not observed in ct120 transgenic mice. Treatment with a specific nNOS inhibitor (N omega-propyl-L-arginine) reduced the beta-adrenergic response in wild-type and ct120 transgenic mice to levels comparable to those of PMCA4b transgenic animals. No differences in lusitropic response were observed in either transgenic strain compared with wild-type littermates. CONCLUSIONS: These data demonstrate the physiological relevance of the interaction between PMCA4b and nNOS and suggests its signaling role in the heart. [less ▲]

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See detailAbsence of alpha 7 integrin in dystrophin-deficient mice causes a myopathy similar to Duchenne muscular dystrophy.
Guo, Chun; Willem, Michael; Werner, Alexander et al

in Human molecular genetics (2006), 15(6), 989-98

Both the dystrophin-glycoprotein complex and alpha7beta1 integrin have critical roles in the maintenance of muscle integrity via the provision of mechanical links between muscle fibres and the basement ... [more ▼]

Both the dystrophin-glycoprotein complex and alpha7beta1 integrin have critical roles in the maintenance of muscle integrity via the provision of mechanical links between muscle fibres and the basement membrane. Absence of either dystrophin or alpha7 integrin results in a muscular dystrophy. To clarify the role of alpha7 integrin and dystrophin in muscle development and function, we generated integrin alpha7/dystrophin double-mutant knockout (DKO) mice. Surprisingly, DKO mice survived post-natally and were indistinguishable from wild-type, integrin alpha7-deficient and mdx mice at birth, but died within 24-28 days. Histological analysis revealed a severe muscular dystrophy in DKO mice with endomysial fibrosis and ectopic calcification. Weight loss was correlated with the loss of muscle fibres, indicating that progressive muscle wasting in the double mutant was most likely due to inadequate muscle regeneration. The data further support that premature death of DKO mice is due to cardiac and/or respiratory failure. The integrin alpha7/dystrophin-deficient mouse model, therefore, resembles the pathological changes seen in Duchenne muscular dystrophy and suggests that the different clinical severity of dystrophin deficiency in human and mouse may be due to a fine-tuned difference in expression of dystrophin and integrin alpha7 in both species. Together, these findings indicate an essential role for integrin alpha7 in the maintenance of dystrophin-deficient muscles. [less ▲]

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See detailThe sarcolemmal calcium pump, alpha-1 syntrophin, and neuronal nitric-oxide synthase are parts of a macromolecular protein complex.
Williams, Judith C.; Armesilla, Angel L.; Mohamed, Tamer M. A. et al

in The Journal of biological chemistry (2006), 281(33), 23341-8

The main role of the plasma membrane Ca2+/calmodulin-dependent ATPase (PMCA) is in the removal of Ca2+ from the cytosol. Recently, we and others have suggested a new function for PMCA as a modulator of ... [more ▼]

The main role of the plasma membrane Ca2+/calmodulin-dependent ATPase (PMCA) is in the removal of Ca2+ from the cytosol. Recently, we and others have suggested a new function for PMCA as a modulator of signal transduction pathways. This paper shows the physical interaction between PMCA (isoforms 1 and 4) and alpha-1 syntrophin and proposes a ternary complex of interaction between endogenous PMCA, alpha-1 syntrophin, and NOS-1 in cardiac cells. We have identified that the linker region between the pleckstrin homology 2 (PH2) and the syntrophin unique (SU) domains, corresponding to amino acids 399-447 of alpha-1 syntrophin, is crucial for interaction with PMCA1 and -4. The PH2 and the SU domains alone failed to interact with PMCA. The functionality of the interaction was demonstrated by investigating the inhibition of neuronal nitric-oxide synthase-1 (NOS-1); PMCA is a negative regulator of NOS-1-dependent NO production, and overexpression of alpha-1 syntrophin and PMCA4 resulted in strongly increased inhibition of NO production. Analysis of the expression levels of alpha-1 syntrophin protein in the heart, skeletal muscle, brain, uterus, kidney, or liver of PMCA4-/- mice, did not reveal any differences when compared with those found in the same tissues of wild-type mice. These results suggest that PMCA4 is tethered to the syntrophin complex as a regulator of NOS-1, but its absence does not cause collapse of the complex, contrary to what has been reported for other proteins within the complex, such as dystrophin. In conclusion, the present data demonstrate for the first time the localization of PMCA1b and -4b to the syntrophin.dystrophin complex in the heart and provide a specific molecular mechanism of interaction as well as functionality. [less ▲]

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See detailThe sarcolemmal calcium pump inhibits the calcineurin/nuclear factor of activated T-cell pathway via interaction with the calcineurin A catalytic subunit.
Buch, Mamta H.; Pickard, Adam; Rodriguez, Antonio et al

in The Journal of biological chemistry (2005), 280(33), 29479-87

The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma ... [more ▼]

The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active mutant calcineurin A suggesting a calcium/calmodulin-dependent mechanism. The modulatory function of PMCA4b is further supported by the observation that endogenous calcineurin moves from the cytoplasm to the plasma membrane when PMCA4b is overexpressed. We suggest recruitment by PMCA4b of calcineurin to a low calcium environment as a possible explanation for these findings. In summary, our results offer strong evidence for a novel functional interaction between PMCA and calcineurin, suggesting a role for PMCA as a negative modulator of calcineurin-mediated signaling pathways in mammalian cells. This study reinforces the emerging role of PMCA as a molecular organizer and regulator of signaling transduction pathways. [less ▲]

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See detailNovel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1).
Armesilla, Angel L.; Williams, Judith C.; Buch, Mamta H. et al

in The Journal of biological chemistry (2004), 279(30), 31318-28

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions ... [more ▼]

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling. [less ▲]

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See detailPlasma membrane Ca2+ ATPase 4 is required for sperm motility and male fertility.
Schuh, Kai; Cartwright, Elizabeth J.; Jankevics, Eriks et al

in The Journal of biological chemistry (2004), 279(27), 28220-6

Calcium and Ca(2+)-dependent signals play a crucial role in sperm motility and mammalian fertilization, but the molecules and mechanisms underlying these Ca(2+)-dependent pathways are incompletely ... [more ▼]

Calcium and Ca(2+)-dependent signals play a crucial role in sperm motility and mammalian fertilization, but the molecules and mechanisms underlying these Ca(2+)-dependent pathways are incompletely understood. Here we show that homozygous male mice with a targeted gene deletion of isoform 4 of the plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA), which is highly enriched in the sperm tail, are infertile due to severely impaired sperm motility. Furthermore, the PMCA inhibitor 5-(and-6)-carboxyeosin diacetate succinimidyl ester reduced sperm motility in wild-type animals, thus mimicking the effects of PMCA4 deficiency on sperm motility and supporting the hypothesis of a pivotal role of the PMCA4 on the regulation of sperm function and intracellular Ca(2+) levels. [less ▲]

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