Calcium Extrusion Pump PMCA4: A New Player in Renal Calcium Handling?

Calcium (Ca²⁺) is vital for multiple processes in the body, and maintenance of the electrolyte concentration is required for everyday physiological function. In the kidney, and more specifically, in the late distal convoluted tubule and connecting tubule, the fine-tuning of Ca²⁺ reabsorption from the pro-urine takes place. Here, Ca²⁺ enters the epithelial cell via the transient receptor potential vanilloid receptor type 5 (TRPV5) channel, diffuses to the basolateral side bound to calbindin-D_28k and is extruded to the blood compartment via the Na⁺/Ca²⁺ exchanger 1 (NCX1) and the plasma membrane Ca²⁺ ATPase (PMCA). Traditionally, PMCA1 was considered to be the primary Ca²⁺ pump in this process. However, in recent studies TRPV5-expressing tubules were shown to highly express PMCA4. Therefore, PMCA4 may have a predominant role in renal Ca²⁺ handling. This study aimed to elucidate the role of PMCA4 in Ca²⁺ homeostasis by characterizing the Ca²⁺ balance, and renal and duodenal Ca²⁺-related gene expression in PMCA4 knockout mice. The daily water intake of PMCA4 knockout mice was significantly lower compared to wild type littermates. There was no significant difference in serum Ca²⁺ level or urinary Ca²⁺ excretion between groups. In addition, renal and duodenal mRNA expression levels of Ca²⁺-related genes, including TRPV5, TRPV6, calbindin-D_28k, calbindin-D_9k, NCX1 and PMCA1 were similar in wild type and knockout mice. Serum FGF23 levels were significantly increased in PMCA4 knockout mice. In conclusion, PMCA4 has no discernible role in normal renal Ca²⁺ handling as no urinary Ca²⁺ wasting was observed. Further investigation of the exact role of PMCA4 in the distal convoluted tubule and connecting tubule is required.     

SOCCER: Self-Optimization of Energy-efficient Cloud Resources

Cloud data centers often schedule heterogeneous workloads without considering energy consumption and carbon emission aspects. Tremendous amount of energy consumption leads to high operational costs and reduces return on investment and contributes towards carbon footprints to the environment. Therefore, there is need of energy-aware cloud based system which schedules computing resources automatically by considering energy consumption as an important parameter. In this paper, energy efficient autonomic cloud system [Self-Optimization of Cloud Computing Energy-efficient Resources (SOCCER)] is proposed for energy efficient scheduling of cloud resources in data centers. The proposed work considers energy as a Quality of Service (QoS) parameter and automatically optimizes the efficiency of cloud resources by reducing energy consumption. The performance of the proposed system has been evaluated in real cloud environment and the experimental results show that the proposed system performs better in terms of energy consumption of cloud resources and utilizes these resources optimally.     

Specific Role of Neuronal Nitric-oxide Synthase when Tethered to the Plasma Membrane Calcium Pump in Regulating the ��-Adrenergic Signal in the Myocardium

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates β-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser¹⁶ phospholamban (PLB) phosphorylation as well as Ser²² and Ser²³ cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the β-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.Neuronal nitric-oxide synthase (nNOS)⁵ is involved in a number of key processes in cardiomyocytes including calcium cycling (1), the β-adrenergic contractile response (2, 3), post-infarct left ventricular remodeling (4), and the regulation of redox equilibrium (5). Moreover, a polymorphism in an nNOS-interacting protein, CAPON, has been found to form a quantitative trait for the determination of the QT interval in humans (6), whereas a mutation in α1-syntrophin (SNTA1), another interacting partner of nNOS, has been associated with long QT syndrome (7). The signaling events downstream of the nNOS-CAPON (8) and nNOS-SNTA1 (7) complexes, which are responsible for mediating cardiac repolarization and sodium current respectively, have been elucidated. The nNOS-containing protein complex is therefore of immediate relevance to human pathology.In recent years, we have shown that the sarcolemmal calcium pump, which ejects calcium to the extracellular compartment (reviewed in Refs. 9 and 10), is an important molecule involved in signal regulation and transmission in the heart (11). We have demonstrated that isoform 4b of the sarcolemmal calcium pump (also known as PMCA4b for plasma membrane calcium/calmodulin-dependent ATPase 4b) modulates signaling through a tight molecular interaction with nNOS, leading to the modulation of β-adrenergic responsiveness in the heart (12). However, the events following signaling through the PMCA4b-nNOS complex remain unknown.In myocardial cells, nNOS has been localized to the sarcolemma (13), sarcoplasmic reticulum (2), and mitochondria (14), and translocation between compartments has been demonstrated (15). It has been speculated that these various localizations provide specificity to NO signaling, but the exact mechanisms have yet to be elucidated. In this study, we show a mechanism by which one fraction of nNOS serves highly specific functions through binding to PMCA4b. As PMCA4b is confined to the sarcolemma and is a calcium pump, it is the first identified protein to fulfill these aggregate functions. 1) It acts as an anchoring protein; 2) it regulates nNOS activity; and 3) it modulates a process at the plasma membrane, i.e. β-adrenergic signaling.     

Acute inhibition of PMCA4, but not global ablation,reduces blood pressure and arterial contractility via a nNOS‐dependent mechanism

Cardiovascular disease is the world's leading cause of morbidity and mortality, with high blood pressure (BP) contributing to increased severity and number of adverse outcomes. Plasma membrane calcium ATPase 4 (PMCA4) has been previously shown to modulate systemic BP. However, published data are conflicting, with both overexpression and inhibition of PMCA4 in vivo shown to increase arterial contractility. Hence, our objective was to determine the role of PMCA4 in the regulation of BP and to further understand how PMCA4 functionally regulates BP using a novel specific inhibitor to PMCA4, aurintricarboxylic acid (ATA). Our approach assessed conscious BP and contractility of resistance arteries from PMCA4 global knockout (PMCA4KO) mice compared to wild‐type animals. Global ablation of PMCA4 had no significant effect on BP, arterial structure or isolated arterial contractility. ATA treatment significantly reduced BP and arterial contractility in wild‐type mice but had no significant effect in PMCA4KO mice. The effect of ATAin vivo and ex vivo was abolished by the neuronal nitric oxide synthase (nNOS) inhibitor Vinyl‐l ‐NIO. Thus, this highlights differences in the effects of PMCA4 ablation and acute inhibition on the vasculature. Importantly, for doses here used, we show the vascular effects of ATA to be specific for PMCA4 and that ATA may be a further experimental tool for elucidating the role of PMCA4.     

Plasma membrane calcium pump (PMCA4)-neuronal nitric-oxide synthase complex regulates cardiac contractility through modulation of a compartmentalized cyclic nucleotide microdomain

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 β-adrenergic signaling.     

Reduced expression of PMCA1 is associated with increased blood pressure with age which is preceded by remodelling of resistance arteries

Hypertension is a well‐established risk factor for adverse cardiovascular events, and older age is a risk factor for the development of hypertension. Genomewide association studies have linked ATP2B1, the gene for the plasma membrane calcium ATPase 1 (PMCA1), to blood pressure (BP) and hypertension. Here, we present the effects of reduction in the expression of PMCA1 on BP and small artery structure and function when combined with advancing age. Heterozygous PMCA1 null mice (PMCA1^Ht) were generated and conscious BP was measured at 6 to 18 months of age. Passive and active properties of isolated small mesenteric arteries were examined by pressure myography. PMCA1^Ht mice exhibited normal BP at 6 and 9 months of age but developed significantly elevated BP when compared to age‐matched wild‐type controls at ≥12 months of age. Decreased lumen diameter, increased wall thickness and increased wall:lumen ratio were observed in small mesenteric arteries from animals 9 months of age and older, indicative of eutrophic remodelling. Increases in mesenteric artery intrinsic tone and global intracellular calcium were evident in animals at both 6 and 18 months of age. Thus, decreased expression of PMCA1 is associated with increased BP when combined with advancing age. Changes in arterial structure precede the elevation of BP. Pathways involving PMCA1 may be a novel target for BP regulation in the elderly.     

The Mammalian Ste20-like Kinase 2 (Mst2) Modulates Stress-induced Cardiac Hypertrophy

The Hippo signaling pathway has recently moved to center stage in cardiac research because of its key role in cardiomyocyte proliferation and regeneration of the embryonic and newborn heart. However, its role in the adult heart is incompletely understood. We investigate here the role of mammalian Ste20-like kinase 2 (Mst2), one of the central regulators of this pathway. Mst2^−/− mice showed no alteration in cardiomyocyte proliferation. However, Mst2^−/− mice exhibited a significant reduction of hypertrophy and fibrosis in response to pressure overload. Consistently, overexpression of MST2 in neonatal rat cardiomyocytes significantly enhanced phenylephrine-induced cellular hypertrophy. Mechanistically, Mst2 positively modulated the prohypertrophic Raf1-ERK1/2 pathway. However, activation of the downstream effectors of the Hippo pathway (Yes-associated protein) was not affected by Mst2 ablation. An initial genetic study in mitral valve prolapse patients revealed an association between a polymorphism in the human MST2 gene and adverse cardiac remodeling. These results reveal a novel role of Mst2 in stress-dependent cardiac hypertrophy and remodeling in the adult mouse and likely human heart.     

Identification of [I]endothelin binding sites in human coronary tissue

In vitro receptor autoradiography has been used to study the distribution of [¹²⁵I]endothelin binding sites in human coronary tissue from patients undergoing cardiac transplantation. Dense binding of [¹²⁵I]endothelin was associated with the smooth muscle of epicardial coronary arteries as well as to perivascular regions of these vessels. Binding was also associated with the ventricular myocardium. There was an increased binding of [¹²⁵I]endothelin to atheromatous tissue, both coronary arteries and vein graft.

The [¹²⁵I]endothelin binding sites identified using in vitro autoradiography are likely to be functionally relevant since endothelin causes a concentration-dependent contraction of segments of human epicardial coronary arteries in vitro and also has positive inotropic activity on isolated human cardiomyocytes.

The presence of specific binding sites for [¹²⁵I]endothelin on coronary tissue and the increased binding in atheromatous tissue suggest that endothelin is a peptide which may play a role in the maintenance of vascular tone and/or the pathogenesis of ischaemic heart disease.     

The formation of polymeric model biomembranes from diacetylenic fatty acids and phospholipids

Diacetylenic fatty acid monolayers at the air/water interface and multilayers on suitable supports polymerise when exposed to ultraviolet radiation. It has been found that polymerisation still occurs when monolayers are diluted with cholesterol or gramicidin. The rigid, crystalline nature of the films formed makes them useful biomembrane models. Phospholipids made from the fatty acids were less reactive. Multilayers deposited on hydrophobic supports would polymerise but not monolayers on water.