Echocardiographic parameters for the assessment of congestive heart failure in dogs with myxomatous mitral valve disease and moderate to severe mitral regurgitation.

The objective of this study was to evaluate the influence of congestive heart failure (CHF) on echocardiographic variables in dogs with moderate or severe mitral valve regurgitation (MR) secondary to myxomatous mitral valve disease (MMVD). The secondary objective was to investigate the preliminary use of left ventricular early inflow-outflow index (LVEIO) and L-waves as indicators of CHF. Forty-five dogs with moderate or severe MR without (n = 23) or with (n = 22) CHF were retrospectively included. Echocardiographic variables of elevated left ventricular (LV) filling pressures and cardiac function, LVEIO and the presence of L-waves were obtained and statistically compared between the two groups. On general linear model analysis, mitral inflow peak E-wave velocity, E:A ratio, ratio of E to isovolumic relaxation time (IVRT) and LVEIO were increased, while left atrium (LA) fractional shortening was reduced, in dogs with CHF. Peak E-wave velocity, peak A-wave velocity, E:A ratio, LA minimum diameter, LA diameter before the P-wave, LA fractional shortening and LVEIO were found to be predictive of CHF. The absence of L-waves indicated a reduced risk of CHF. Several echocardiographic variables may be useful in identifying elevated LV filling pressures, consistent with CHF, in dogs with moderate and severe MR. Left ventricular early inflow-outflow index and L-waves might be of interest as clinical and prognostic markers in dogs with MMVD and require further investigation.

Short communication: Characterizing ovine serum stress biomarkers during endotoxemia.

Breeding stress-resilient livestock is a potential strategy to help mitigate the negative effect of environmental and pathogenic stressors. The hypothalamic-pituitary-adrenal axis and immune system are activated during stress events and release mediators into the circulation that help restore physiological homeostasis. The purpose of this study was to assess a comprehensive set of circulatory mediators released in response to an acute immune stress challenge to identify candidate biomarkers that can be used for the selection of stress-resilient animals. Fifteen female lambs were stress challenged with an intravenous bolus of lipopolysaccharide (LPS; 400 ng/kg), and blood was collected from the jugular vein at 0, 2, 4, and 6 h after LPS challenge to identify and monitor candidate stress biomarkers; temperature was also recorded over time. Biomarker responses were evaluated with a repeated-measures model to compare time points with baseline values. As expected, all sheep had a monophasic febrile response to LPS challenge, and cortisol increased and returned to baseline by 6 h. The cytokines tumor necrosis factor-α, IL-6, IFN-γ (proinflammatory), and IL-10 (anti-inflammatory) increased, but only tumor necrosis factor-α returned to baseline during the monitoring period. The cytokines IL-1α, IL-1ß, IL-17α (proinflammatory), and IL-4 (anti-inflammatory) did not respond to LPS challenge. All chemokines (CCL2, CCL3, CCL4, CXCL10, and IL-8) responded to LPS challenge; however, only CCL2, CCL3, CCL4, and CXCL10 increased over time, and only CCL3, CCL4, and CXCL10 returned to baseline during the monitoring period. MicroRNA (miR-145, miR-233, and miR-1246) also increased and remained elevated during the study. In summary, the LPS challenge induced a strong stress response in Rideau-Dorset sheep that could be monitored with a distinct profile of circulatory biomarkers.

Assessment of CXC ligand 12-mediated calcium signalling and its regulators in basal-like breast cancer cells.

CXC ligand (L)12 is a chemokine implicated in the migration, invasion and metastasis of cancer cells via interaction with its receptors CXC chemokine receptor (CXCR)4 and CXCR7. In the present study, CXCL12-mediated Ca2+ signalling was compared with two basal-like breast cancer cell lines, MDA-MB-231 and MDA-MB-468, which demonstrate distinct metastatic potential. CXCL12 treatment induced Ca2+ responses in the more metastatic MDA-MB-231 cells but not in the less metastatic MDA-MB-468 cells. Assessment of mRNA levels of CXCL12 receptors and their potential modulators in both cell lines revealed that CXCR4 and CXCR7 levels were increased in MDA-MB-231 cells compared with MDA-MB-468 cells. Cluster of differentiation (CD)24, the negative regulator of CXCL12 responses, demonstrated increased expression in MDA-MB-468 cells compared with MDA-MB-231 cells, and the two cell lines expressed comparable levels of hypoxia-inducible factor (HIF)2α, a CXCR4 regulator. Induction of epithelial-mesenchymal transition (EMT) by epidermal growth factor exhibited opposite effects on CXCR4 mRNA levels compared with hypoxia-induced EMT. Neither EMT inducer exhibited an effect on CXCR7 expression, however hypoxia increased HIF2α expression levels in MDA-MB-468 cells. Analysis of the gene expression profiles of breast tumours revealed that the highest expression levels of CXCR4 and CXCR7 were in the Claudin-Low molecular subtype, which is markedly associated with EMT features.

Oncosis and apoptosis induction by activation of an overexpressed ion channel in breast cancer cells.

The critical role of calcium signalling in processes related to cancer cell proliferation and invasion has seen a focus on pharmacological inhibition of overexpressed ion channels in specific cancer subtypes as a potential therapeutic approach. However, despite the critical role of calcium in cell death pathways, pharmacological activation of overexpressed ion channels has not been extensively evaluated in breast cancer. Here we define the overexpression of transient receptor potential vanilloid 4 (TRPV4) in a subgroup of breast cancers of the basal molecular subtype. We also report that pharmacological activation of TRPV4 with GSK1016790A reduced viability of two basal breast cancer cell lines with pronounced endogenous overexpression of TRPV4, MDA-MB-468 and HCC1569. Pharmacological activation of TRPV4 produced pronounced cell death through two mechanisms: apoptosis and oncosis in MDA-MB-468 cells. Apoptosis was associated with PARP-1 cleavage and oncosis was associated with a rapid decline in intracellular ATP levels, which was a consequence of, rather than the cause of, the intracellular ion increase. TRPV4 activation also resulted in reduced tumour growth in vivo. These studies define a novel therapeutic strategy for breast cancers that overexpress specific calcium permeable plasmalemmal ion channels with available selective pharmacological activators.

Calcium influx pathways in breast cancer: opportunities for pharmacological intervention.

Ca(2+) influx through Ca(2+) permeable ion channels is a key trigger and regulator of a diverse set of cellular events, such as neurotransmitter release and muscle contraction. Ca(2+) influx is also a regulator of processes relevant to cancer, including cellular proliferation and migration. This review focuses on calcium influx in breast cancer cells as well as the potential for pharmacological modulators of specific Ca(2+) influx channels to represent future agents for breast cancer therapy. Altered expression of specific calcium permeable ion channels is present in some breast cancers. In some cases, such changes can be related to breast cancer subtype and even prognosis. In vitro and in vivo models have now helped identify specific Ca(2+) channels that play important roles in the proliferation and invasiveness of breast cancer cells. However, some aspects of our understanding of Ca(2+) influx in breast cancer still require further study. These include identifying the mechanisms responsible for altered expression and the most effective therapeutic strategy to target breast cancer cells through specific Ca(2+) channels. The role of Ca(2+) influx in processes beyond breast cancer cell proliferation and migration should become the focus of studies in the next decade.

Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent.

Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of calcium-signaling pathways controlling EMT induction in cancer cells may therefore be an important therapeutic strategy for preventing metastases.

Evaluating Australia's National Medicines Policy using geographical mapping.

BACKGROUND: There has been a proliferation of quality use of medicines activities in Australia since the 1990s. However, knowledge of the nature and extent of these activities was lacking. A mechanism was required to map the activities to enable their coordination. AIMS: To develop a geographical mapping facility as an evaluative tool to assist the planning and implementation of Australia's policy on the quality use of medicines. METHODS: A web-based database incorporating geographical mapping software was developed. Quality use of medicines projects implemented across the country was identified from project listings funded by the Quality Use of Medicines Evaluation Program, the National Health and Medical Research Council, Mental Health Strategy, Rural Health Support, Education and Training Program, the Healthy Seniors Initiative, the General Practice Evaluation Program and the Drug Utilisation Evaluation Network. In addition, projects were identified through direct mail to persons working in the field. RESULTS: The Quality Use of Medicines Mapping Project (QUMMP) was developed, providing a Web-based database that can be continuously updated. This database showed the distribution of quality use of medicines activities by: (i) geographical region, (ii) project type, (iii) target group, (iv) stakeholder involvement, (v) funding body and (vi) evaluation method. At September 2001, the database included 901 projects. Sixty-two per cent of projects had been conducted in Australian capital cities, where approximately 63% of the population reside. Distribution of projects varied between States. In Western Australia and Queensland, 36 and 73 projects had been conducted, respectively, representing approximately two projects per 100,000 people. By comparison, in South Australia and Tasmania approximately seven projects per 100,000 people were recorded, with six per 100,000 people in Victoria and three per 100,000 people in New South Wales. Rural and remote areas of the country had more limited project activity. CONCLUSIONS: The mapping of projects by geographical location enabled easy identification of high and low activity areas. Analysis of the types of projects undertaken in each region enabled identification of target groups that had not been involved or services that had not yet been developed. This served as a powerful tool for policy planning and implementation and will be used to support the continued implementation of Australia's policy on the quality use of medicines.

PMCA1 mRNA expression in rat aortic myocytes: a real-time RT-PCR study.

The plasmalemmal Ca(2+) adenosine triphosphatase (PMCA) is a key regulator of Ca(2+) efflux in vascular smooth muscle. In these studies we developed a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay for assessing PMCA1 mRNA levels in rat primary cultured aortic myocytes. This assay detected fetal bovine serum-induced increases in PMCA1 mRNA (relative to 18S rRNA) 4, 8, and 24 h after stimulation. Early fetal bovine serum-induced increases in PMCA1 mRNA were insensitive to the Ca(2+) channel blockers nifedipine, flunarizine, and SKF-96365. These studies demonstrate the feasibility of real-time RT-PCR to assess mRNA levels of PMCA1 and illustrate dynamic regulation of this Ca(2+) pump isoform in rat primary cultured aortic myocytes.

Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line.

The plasma membrane Ca(2+) pump is a key regulator of cytosolic free Ca(2+). Recent studies have demonstrated the dynamic expression of the plasma membrane Ca(2+) pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca(2+) ATPase (PMCA1) isoform of the plasma membrane Ca(2+) pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously.

Heterogeneity of mitochondrial matrix free ca2+: resolution of Ca2+ dynamics in individual mitochondria in situ.

The role of mitochondria in Ca2+ homeostasis is controversial. We employed the Ca2+-sensitive dye rhod 2 with novel, high temporal and spatial resolution imaging to evaluate changes in the matrix free Ca2+ concentration of individual mitochondria ([Ca2+]m) in agonist-stimulated, primary cultured aortic myocytes. Stimulation with 10 microM serotonin (5-HT) evoked modest cytosolic Ca2+ transients [cytosolic free Ca2+ concentration ([Ca2+]cyt) <500 nM; measured with fura 2] and triggered contractions in short-term cultured myocytes. However, 5-HT triggered a large mitochondrial rhod 2 signal (indicating pronounced elevation of [Ca2+]m) in only 4% of cells. This revealed heterogeneity in the responses of individual mitochondria, all of which stained with MitoTracker Green FM. In contrast, stimulation with 100 microM ATP evoked large cytosolic Ca2+ transients (>1,000 nM) and induced pronounced, reversible elevation of [Ca2+]m (measured as rhod 2 fluorescence) in 60% of cells. This mitochondrial Ca2+ uptake usually lagged behind the cytosolic Ca2+ transient peak by 3-5 s, and [Ca2+]m declined more slowly than did bulk [Ca2+]cyt. The uptake delay may prevent mitochondria from interfering with rapid signaling events while enhancing the mitochondrial response to large, long-duration elevations of [Ca2+]cyt. The responses of arterial myocytes to modest physiological stimulation do not, however, depend on such marked changes in [Ca2+]m.

Different effects of low and high dose cardiotonic steroids on cytosolic calcium in spontaneously active hippocampal neurons and in co-cultured glia.

The Na+ pump is crucial for the regulation of [Na+]i (the intracellular Na+ concentration) in all cells. Three Na+ pump alpha subunit isoforms, alpha1, alpha2 and alpha3, are expressed in rat hippocampal neurons, and alpha1 and alpha2 are expressed in glia, but the significance of these isoforms is not understood. We exploited the different ouabain affinities of the Na+ pump alpha subunit isoforms in rat (alpha1, low ouabain affinity; alpha2 and alpha3, high ouabain affinity) to probe their possible physiological roles. Low and intermediate doses (1-10 microM) of ouabain and its readily reversible analog, dihydroouabain, altered the spontaneous elevations of [Ca2+]i (the intracellular Ca2+ concentration) in neurons and induced [Ca2+]i transients in glia. Complete inhibition of all Na+ pump isoforms (>/=100 microM ouabain) caused sustained increases in global neuronal [Ca2+]i in rat neuronal/glial hippocampal co-cultures and transient [Ca2+]i increases in surrounding glia. High dose ouabain was also associated with increased [Na+]i and [H+]i in neurons and glia. In contrast, 1 microM ouabain (a concentration that completely inhibits only alpha2 and alpha3) was not associated with sustained increases in global neuronal [Ca2+]i or the sustained derangements in [Na+]i and [H+]i observed with high dose ouabain. Reduction of [K+]o to 1 mM suppressed the spontaneous [Ca2+]i oscillations in neurons and induced Ca2+ transients in some glia; removal of external K+ induced sustained elevation of neuronal [Ca2+]i. These studies indicate that the alpha1 isoform is the 'housekeeper' required for maintenance of the global Na+ gradient. As suggested by their restricted plasmalemmal distribution, the high ouabain-affinity Na+ pump isoforms may have more specific roles in neurons and glia.

The plasma membrane calcium pump, its role and regulation: new complexities and possibilities.

Significant progress has been achieved in elucidating the role of the plasma membrane Ca2(+)-ATPase in cellular Ca2+ homeostasis and physiology since the enzyme was first purified and physiology since the enzyme was first purified and cloned a number of years ago. The simple notion that the PM Ca2(+)-ATPase controls resting levels of [Ca2+]CYT has been challenged by the complexity arising from the finding of four major isoforms and splice variants of the Ca2+ pump, and the finding that these are differentially localized in various organs and subcellular regions. Furthermore, the isoforms exhibit differential sensitivities to Ca2+, calmodulin, ATP, and kinase-mediated phosphorylation. The latter pathways of regulation can give rise to activation or inhibition of the Ca2+ pump activity, depending on the kinase and the particular Ca2+ pump isoform. Significant progress is being made in elucidating subtle and more profound roles of the PM Ca2(+)-ATPase in the control of cellular function. Further understanding of these roles awaits new studies in both transfected cells and intact organelles, a process that will be greatly aided by the development of new and selective Ca2+ pump inhibitors.

Elevated plasma membrane and sarcoplasmic reticulum Ca2+ pump mRNA levels in cultured aortic smooth muscle cells from spontaneously hypertensive rats.

We have observed previously that Ca2+ pump-mediated Ca2+ efflux is elevated in cultured aortic smooth muscle cells from spontaneously hypertensive rats compared to those from Wistar-Kyoto rat controls. The objective of this work was to determine if these strains differ in mRNA levels for the PMCA1 isoform of the plasma membrane Ca2+-ATPase and the SERCA2 isoform of the sarcoplasmic reticulum Ca2+-ATPase. mRNA levels were compared in cultured aortic smooth muscle cells from 10-week-old male rats. PMCA1 and SERCA2 mRNA levels were elevated in SHR compared to WKY. Angiotensin II increased the level of PMCA1 and SERCA2 mRNA in both strains. These studies provide further evidence for altered Ca2+ homeostasis in hypertension at the level of Ca2+ transporting ATPases in the spontaneously hypertensive rat model. These data are also consistent with the hypothesis that the expression of these two Ca2+ pumps may be linked.

Plasma membrane calcium pump-mediated calcium efflux and bulk cytosolic free calcium in cultured aortic smooth muscle cells from spontaneously hypertensive and Wistar-Kyoto normotensives rats.

OBJECTIVE: To compare the efficacy of the calcium pump-mediated calcium efflux pathway in spontaneously hypertensive rats (SHR) with that in Wistar-Kyoto normotensive rats (WKY), at rest and after angiotensin II stimulation. DESIGN: The intracellular free calcium concentration and calcium-45 efflux were measured in parallel, in cultured aortic smooth muscle cells isolated from 10-week-old male SHR and WKY rats. METHODS: The intracellular free calcium concentration and calcium-45 efflux were studied in confluent vascular smooth muscle cells in culture. Experiments were performed in the absence of added extracellular calcium and sodium. Fura-2 was used to measure basal and angiotensin II-stimulated intracellular free calcium concentration. Effluxed calcium-45 was measured over 5s intervals to determine basal and angiotensin II-stimulated calcium efflux rates in SHR and in WKY rats. RESULTS: No significant difference between SHR and WKY rats was observed in basal intracellular free calcium concentration or 100nmol/l angiotensin II-stimulated peak intracellular free calcium concentration. However, significantly elevated basal and 100 nmol/l angiotensin II-stimulated calcium-45 efflux rates were found in SHR. The calcium-45 efflux rates in SHR were elevated when the efflux was normalized with respect to the bulk intracellular free calcium concentration. The time taken to reach the maximum calcium-45 efflux rate after angiotensin II stimulation was reduced in SHR compared with that in WKY rats and was dose-dependent in both rat strains. CONCLUSION: The calcium-pump mediated calcium efflux pathway appears to be more efficient in SHR. This may be the result of post-translational modification, enhanced calcium pump sites in a critical region of the membrane, or the presence of a pool of calcium near the plasma membrane that is not readily detected by cytosolic Fura-2 but is higher in SHR both before and after angiotensin II stimulation.

The plasma membrane calcium pump--a physiological perspective on its regulation.

This review focuses on the physiological role of the plasma membrane Ca(2+)+ Mg(2+)-dependent adenosine triphosphatase (PM Ca(2+)-ATPase) in cellular signalling. Particular attention has been paid to the regulation of the PM Ca(2+)-ATPase (PM Ca2+ pump) by calmodulin, proteases, protein kinases, acidic phospholipids and oligomerization in intact cells. We also review recent work investigating the possible regulation of the PM Ca2+ pump by G proteins and agonists. The source of adenosine triphosphate (ATP) and Ca2+ in fueling and activating the Ca2+ pump is discussed, as well as the possible role of the PM Ca(2+)-ATPase in subplasma membrane Ca2+ regulation. The physiological implication of the localisation of the PM Ca2+ pump in caveolae is also considered.

Measurement of Ca2+ pump-mediated efflux in hypertension.

Ca2+ homeostasis has been a prominent research area in the study of hypertension. There is convincing evidence that hypertension in spontaneously hypertensive rats is characterized by enhanced Ca2+ influx in various cell types. It is, however, still unclear whether hypertension is associated with reduced or enhanced Ca2+ efflux. Reduced Ca2+ efflux would augment the effects of enhanced Ca2+ influx. However, enhanced Ca2+ extrusion may occur as an adaptive process to minimize the effects of Ca2+ overload. This question remains unanswered because of inconsistent results obtained using a variety of experimental techniques. In this article we have reviewed the research findings and discuss existing and possible new techniques to assess Ca2+ efflux in hypertension, with particular attention to vascular smooth muscle. We have focused mainly on studies using the spontaneously hypertensive rat and discuss its appropriateness as a model for essential hypertension.

The inhibition of ATP-dependent shape change of human erythrocyte ghosts correlates with an inhibition of Mg(2+)-ATPase activity by fluoride and aluminofluoride complexes.

The vanadate-sensitive Mg(2+)-dependent ATPase activity of the human erythrocyte ghost is believed to be involved in the shape change events that convert echinocytic ghosts to smoothed forms (biconcave discs and stomatocytes). At physiological salt concentration, pH 7.4, 2 mM ATP, 5 mM Mg2+ and 1 mM EGTA, the Mg(2+)-ATPase activity of ghosts was inhibited strongly by millimolar concentrations of sodium fluoride: I50 = 1.31 +/- 0.23 mM (mean +/- S.D.; n = 12). The addition of aluminium chloride to 15 microM reduced the concentration of NaF required for 50% inhibition to 0.76 +/- 0.21 mM (n = 10). Aluminium alone had only a small inhibitory effect on the ATPase activity (13 +/- 9%; n = 10). Desferrioxamine, a strong chelator of tervalent aluminium ion, failed to reverse the inhibition by fluoride and reversed the inhibition in the presence of aluminium and fluoride back to those values obtained with fluoride alone. Of several metal salts tested only beryllium sulfate was able to replace aluminium as an effective inhibitor in the presence of fluoride. Inhibition of the Mg(2+)-ATPase activity by fluoride and the aluminofluoride complexes correlated with an inhibition of the rate of MgATP-dependent change in red cell ghost shape from echinocytes to smoothed forms. All gross morphological changes of the smoothing process were affected, including the production of discocytes, stomatocytes and endocyctic vesicles.