Ion channels as effectors in carbon monoxide signaling

A wealth of recent studies has highlighted the diverse and important influences of carbon monoxide (CO) on cellular signaling pathways. Such studies have implicated CO, and the enzymes from which it is derived (heme oxygenases) as potential therapeutic targets, particularly (although not exclusively) in inflammation, immunity and cardiovascular disease.1 In a recent study,2 we demonstrated that CO inhibited cardiac L-type Ca(2+) channels. This effect arose due to the ability of CO to bind to mitochondria (presumably at complex IV of the electron transport chain) and so cause electron leak, which resulted in increased production of reactive oxygen species. These modulated the channel's activity through interactions with three cysteine residues in the cytosolic C-terminus of the channel's major, pore-forming subunit. Our study provided a potential mechanism for the cardioprotective effects of CO and also highlighted ion channels as a major potential target group for this gasotransmitter.

Mapping the platelet profile for functional genomic studies and demonstration of the effect size of the GP6 locus

BACKGROUND: Evidence suggests the wide variation in platelet response within the population is genetically controlled. Unraveling the complex relationship between sequence variation and platelet phenotype requires accurate and reproducible measurement of platelet response. OBJECTIVE: To develop a methodology suitable for measuring signaling pathway-specific platelet phenotype, to use this to measure platelet response in a large cohort, and to demonstrate the effect size of sequence variation in a relevant model gene. METHODS: Three established platelet assays were evaluated: mobilization of [Ca(2+)](i), aggregometry and flow cytometry, each in response to adenosine 5'-diphosphate (ADP) or the glycoprotein (GP) VI-specific crosslinked collagen-related peptide (CRP). Flow cytometric measurement of fibrinogen binding and P-selectin expression in response to a single, intermediate dose of each agonist gave the best combination of reproducibility and inter-individual variability and was used to measure the platelet response in 506 healthy volunteers. Pathway specificity was ensured by blocking the main subsidiary signaling pathways. RESULTS: Individuals were identified who were hypo- or hyper-responders for both pathways, or who had differential responses to the two agonists, or between outcomes. 89 individuals, retested three months later using the same methodology, showed high concordance between the two visits in all four assays (r(2) = 0.872, 0.868, 0.766 and 0.549); all subjects retaining their phenotype at recall. The effect of sequence variation at the GP6 locus accounted for approximately 35% of the variation in the CRP-XL response. CONCLUSION: Genotyping-phenotype association studies in a well-characterized, large cohort provides a powerful strategy to measure the effect of sequence variation in genes regulating the platelet response.

Transient receptor potential ion channels V4 and A1 contribute to pancreatitis pain in mice.

The mechanisms of pancreatic pain, a cardinal symptom of pancreatitis, are unknown. Proinflammatory agents that activate transient receptor potential (TRP) channels in nociceptive neurons can cause neurogenic inflammation and pain. We report a major role for TRPV4, which detects osmotic pressure and arachidonic acid metabolites, and TRPA1, which responds to 4-hydroxynonenal and cyclopentenone prostaglandins, in pancreatic inflammation and pain in mice. Immunoreactive TRPV4 and TRPA1 were detected in pancreatic nerve fibers and in dorsal root ganglia neurons innervating the pancreas, which were identified by retrograde tracing. Agonists of TRPV4 and TRPA1 increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in these neurons in culture, and neurons also responded to the TRPV1 agonist capsaicin and are thus nociceptors. Intraductal injection of TRPV4 and TRPA1 agonists increased c-Fos expression in spinal neurons, indicative of nociceptor activation, and intraductal TRPA1 agonists also caused pancreatic inflammation. The effects of TRPV4 and TRPA1 agonists on [Ca(2+)](i), pain and inflammation were markedly diminished or abolished in trpv4 and trpa1 knockout mice. The secretagogue cerulein induced pancreatitis, c-Fos expression in spinal neurons, and pain behavior in wild-type mice. Deletion of trpv4 or trpa1 suppressed c-Fos expression and pain behavior, and deletion of trpa1 attenuated pancreatitis. Thus TRPV4 and TRPA1 contribute to pancreatic pain, and TRPA1 also mediates pancreatic inflammation. Our results provide new information about the contributions of TRPV4 and TRPA1 to inflammatory pain and suggest that channel antagonists are an effective therapy for pancreatitis, when multiple proinflammatory agents are generated that can activate and sensitize these channels.

Probabilistic downscaling of remote sensing data with applications for multi-scale biogeochemical flux modeling

Upscaling ecological information to larger scales in space and downscaling remote sensing observations or model simulations to finer scales remain grand challenges in Earth system science. Downscaling often involves inferring subgrid information from coarse-scale data, and such ill-posed problems are classically addressed using regularization. Here, we apply two-dimensional Tikhonov Regularization (2DTR) to simulate subgrid surface patterns for ecological applications. Specifically, we test the ability of 2DTR to simulate the spatial statistics of high-resolution (4 m) remote sensing observations of the normalized difference vegetation index (NDVI) in a tundra landscape. We find that the 2DTR approach as applied here can capture the major mode of spatial variability of the high-resolution information, but not multiple modes of spatial variability, and that the Lagrange multiplier (γ) used to impose the condition of smoothness across space is related to the range of the experimental semivariogram. We used observed and 2DTR-simulated maps of NDVI to estimate landscape-level leaf area index (LAI) and gross primary productivity (GPP). NDVI maps simulated using a γ value that approximates the range of observed NDVI result in a landscape-level GPP estimate that differs by ca 2% from those created using observed NDVI. Following findings that GPP per unit LAI is lower near vegetation patch edges, we simulated vegetation patch edges using multiple approaches and found that simulated GPP declined by up to 12% as a result. 2DTR can generate random landscapes rapidly and can be applied to disaggregate ecological information and compare of spatial observations against simulated landscapes.

Contrasting behaviour of cadmium and zinc in a soil–plant–arthropod system

This study investigates the transfer of Cd and Zn from a soil amended with sewage sludge at rates up to 100 t ha(-1) through a multi-trophic system consisting of barley, the aphid Sitobion avenae and the larvae of the lacewing Chrysoperla carnae. Results show marked differences in the transfer of the two metals. Cadmium was freely accumulated in barley roots, but accumulation in the shoot was restricted to a concentration of around 0.22 mg kg(-1) (dry weight). This limited the transfer of Cd to higher trophic levels and resulted in no significant accumulation of Cd in S. avenae or in C. carnae. Zinc transfer in the system was largely unrestricted, resulting in significant accumulation in roots and shoots, in S. avenae and in C. carnae. Cadmium biomagnification occurred in lacewing pupae, with concentrations up to 3.6 times greater than in aphids. S. avenae biomagnified Zn by a factor of ca. 2.5 at low sludge amendment rates, but biomagnification decreased to a factor of 1.4 at the highest amendment rate. Biomagnification of Zn did not occur in C. carnae, but concentrations were up to 3.5 time higher than in soil. Results are discussed in light of the mechanisms regulating transfer of the two metals in the system.

Targeting myocardial remodelling to develop novel therapies for heart failure: a position paper from the Working Group on Myocardial Function of the European Society of Cardiology

The failing heart is characterized by complex tissue remodelling involving increased cardiomyocyte death, and impairment of sarcomere function, metabolic activity, endothelial and vascular function, together with increased inflammation and interstitial fibrosis. For years, therapeutic approaches for heart failure (HF) relied on vasodilators and diuretics which relieve cardiac workload and HF symptoms. The introduction in the clinic of drugs interfering with beta-adrenergic and angiotensin signalling have ameliorated survival by interfering with the intimate mechanism of cardiac compensation. Current therapy, though, still has a limited capacity to restore muscle function fully, and the development of novel therapeutic targets is still an important medical need. Recent progress in understanding the molecular basis of myocardial dysfunction in HF is paving the way for development of new treatments capable of restoring muscle function and targeting specific pathological subsets of LV dysfunction. These include potentiating cardiomyocyte contractility, increasing cardiomyocyte survival and adaptive hypertrophy, increasing oxygen and nutrition supply by sustaining vessel formation, and reducing ventricular stiffness by favourable extracellular matrix remodelling. Here, we consider drugs such as omecamtiv mecarbil, nitroxyl donors, cyclosporin A, SERCA2a (sarcoplasmic/endoplasmic Ca(2 +) ATPase 2a), neuregulin, and bromocriptine, all of which are currently in clinical trials as potential HF therapies, and discuss novel molecular targets with potential therapeutic impact that are in the pre-clinical phases of investigation. Finally, we consider conceptual changes in basic science approaches to improve their translation into successful clinical applications.

Genomic and nongenomic stimulatory effect of aldosterone on H(+)-ATPase in proximal S3 segments

Leite-Dellova DC, Malnic G, Mello-Aires M. Genomic and non-genomic stimulatory effect of aldosterone on H(+)-ATPase in proximal S3 segments. Am J Physiol Renal Physiol 300: F682-F691, 2011. First published December 29, 2010; doi:10.1152/ajprenal.00172.2010.-The genomic and nongenomic effects of aldosterone on the intracellular pH recovery rate (pHirr) via H(-)(+)ATPase and on cytosolic free calcium concentration ([Ca(2+)](i)) were investigated in isolated proximal S3 segments of rats during superfusion with an Na(+)-free solution, by using the fluorescent probes BCECF-AM and FLUO-4-AM, respectively. The pHirr, after cellular acidification with a NH(4)Cl pulse, was 0.064 +/- 0.003 pH units/min (n = 17/74) and was abolished with concanamycin. Aldosterone (10(-12), 10(-10),10(-8), or 10(-6) M with 1-h or 15- or 2-min preincubation) increased the pHirr. The baseline [Ca(2+)](i) was 103 +/- 2 nM (n = 58). After 1 min of aldosterone preincubation, there was a transient and dose-dependent increase in [Ca(2+)](i) and after 6-min preincubation there was a new increase in [Ca(2+)](i) that persisted after 1 h. Spironolactone [mineralocorticoid (MR) antagonist], actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min preincubation) on pHirr and on [Ca(2+)](i) but inhibited the effects of aldosterone (1-h preincubation) on these parameters. RU 486 [glucocorticoid (GR) antagonist] and dimethyl-BAPTA (Ca(2+) chelator) prevented the effect of aldosterone on both parameters. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on the H(+)-ATPase and on [Ca(2+)](i). The results are compatible with stimulation of the H(+)-ATPase by increases in [Ca(2+)](i) (at 10(-12)-10(-6) M aldosterone) and inhibition of the H(+)-ATPase by decreases in [Ca(2+)](i) (at 10(-12) or 10(-6) M aldosterone plus RU 486).

Cramoll 1,4 lectin increases ROS production, calcium levels, and cytokine expression in treated spleen cells of rats

This study reports the in vivo stimulatory effects of Cramoll 1,4 on rat spleen lymphocytes as evidenced by an increase in intracellular reactive oxygen species (ROS) production, Ca(2+) levels, and interleukin (IL)-1 beta expression. Cramoll 1,4 extracted from seeds of the Leguminosae Cratylia mollis Mart., is a lectin with antitumor and lymphocyte mitogenic activities. Animals (Nine-week-old male albino Wistar rats, Rattus norvegicus) were treated with intraperitoneal injection of Cramoll 1,4 (235 mu g ml(-1) single dose) and, 7 days later, spleen lymphocytes were isolated and analyzed for intracellular ROS, cytosolic Ca(2+), and IL-6, IL-10, and IL-1 mRNAs. Cell viability was investigated by annexin V-FITC and 7-amino-actinomycin D staining. The data showed that in lymphocytes activated by Cramoll 1,4 the increase in cytosolic and mitochondrial ROS was related to higher cytosolic Ca(2+) levels. Apoptosis and necrosis were not detected in statistically significant values and thus the lectin effector activities did not induce lymphocyte death. In vivo Cramoll 1,4 treatment led to a significant increase in IL-1 beta but IL-6 and -10 levels did not change. Cramoll 1,4 had modulator activities on spleen lymphocytes and stimulated the Th2 response.

Purinergic signalling is involved in the malaria parasite Plasmodium falciparum invasion to red blood cells

Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for its successful replication within the host. A number of evidence suggest that changes in parasite Ca(2+) levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca(2+) increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca(2+) levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an increase of cytosolic Ca(2+) in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise in [Ca(2+)](c). Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca(2+)](c) in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells.

Role of alpha 7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrP(C)) is a conserved glycosylphosphatidyl-inositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP(C) extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrPC-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP(C) engagement induces an increase in intracellular Ca(2+) levels. This effect was not detected in PrP(C)-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP(C). Using a best candidate approach to test for potential channels involved in Ca(2+) influx evoked by STI1-PrP(C), we found that alpha-bungarotoxin, a specific inhibitor for alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR), was able to block PrP(C)-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when alpha 7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP(C) and allowed reconstitution of signaling by PrP(C)-STI1 interaction. These results indicate that STI1 can interact with the PrP(C).alpha 7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.

Light modulates the melanophore response to alpha-MSH in Xenopus laevis: An analysis of the signal transduction crosstalk mechanisms involved

Melanin granule (melanosome) dispersion within Xenopus laevis melanophores is evoked either by light or alpha-MSH. We have previously demonstrated that the initial biochemical steps of light and alpha-MSH signaling are distinct, since the increase in cAMP observed in response to alpha-MSH was not seen after light exposure. cAMP concentrations in response to alpha-MSH were significantly lower in cells pre-exposed to light as compared to the levels in dark-adapted melanophores. Here we demonstrate the presence of an adenylyl cyclase (AC) in the Xenopus melanophore, similar to the mammalian type IX which is inhibited by Ca(2+)-calmodulin-activated phosphatase. This finding supports the hypothesis that the cyclase could be negatively modulated by a light-promoted Ca(2+) increase. In fact, the activity of calcineurin PP2B phosphatase was increased by light, which could result in AC IX inhibition, thus decreasing the response to alpha-MSH. St-Ht31, a disrupting agent of protein kinase A (PKA)-anchoring kinase A protein (AKAP) complex totally blocked the melanosome dispersing response to alpha-MSH, but did not impair the photo-response in Xenopus melanophores. Sequence comparison of a melanophore AKAP partial clone with GenBank sequences showed that the anchoring protein was a gravin-like adaptor previously sequenced from Xenopus non-pigmentary tissues. Co-immunoprecipitation of Xenopus AKAP and the catalytic subunit of PKA demonstrated that PKA is associated with AKAP and it is released in the presence of alpha-MSH. We conclude that in X laevis melanophores, AKAP12 (gravin-like) contains a site for binding the inactive PKA thus compartmentalizing PKA signaling and also possesses binding sites for PKC. Light diminishes alpha-MSH-induced increase of cAMP by increasing calcineurin (PP2B) activity, which in turn inhibits adenylyl cyclase type IX, and/or by activating PKC, which phosphorylates the gravin-like molecule, thus destabilizing its binding to the cell membrane. (C) 2009 Elsevier Inc. All rights reserved.

Adenosine modulates alpha2-adrenergic receptors through a phospholipase C pathway in brainstem cell culture of rats

Adenosine acts in the nucleus tractus solitarii (NTS), one of the main brain sites related to cardiovascular control. In the present study we show that A(1) adenosine receptor (A(1R)) activation promotes an increase on alpha(2)-adrenoceptor (Alpha(2R)) binding in brainstem cell culture from newborn rats. We investigated the intracellular cascade involved in such modulatory process using different intracellular signaling molecule inhibitors as well as calcium chelators. Phospholipase C, protein kinase Ca(2+)-dependent, IP(3) receptor and intracellular calcium were shown to participate in A(1R)/Alpha(2R) interaction. In conclusion, this result might be important to understand the role of adenosine within the NTS regarding autonomic cardiovascular control. (C) 2009 Elsevier B.V. All rights reserved.

FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely. E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

ATP pulse and calcium homeostasis in cells from hepatopancreas of Dilocarcinus pagei, a freshwater crab

Calcium (Ca) is critical for crustaceans due to their molting cycle and its presence in the carapace as calcium carbonate, apart from the usual functions of Ca, such as cell signalling. Ca transport in Dilocarcinus pagei, a freshwater crab, was studied in isolated cells from hepatopancreas to further characterize Ca transport mechanisms in these crabs. Cells were isolated and loaded with Fluo-3, a calcium fluorescent dye. Three different cell treatments were performed: Group 1 cells were Ca free during cell dissociation, and calcium was present (at 1mM) for fluorescence cell loading and transport experiments (FC); Group 2 cells were calcium free during cell dissociation and for transport experiments, but not during cell loading (LC); and Group 3 cells were Ca free during cell dissociation, cell loading and transport experiments (WC). Intracellular Ca was recorded through time after ATP was added to the cells and ATP caused an increase in Ca efflux within 30s in all cells. WC cells showed the smallest Ca efflux compared to the other cells, probably because it was intracellularly Ca ""depleted"". Vanadate and amiloride decreased the Ca efflux when ATP was added to the cells, while verapamil did not cause any effect in Ca efflux, confirming the presence of a Ca(2+)-ATPase sensitive to vanadate in hepatopancreas of D. pagei. In a different set of experiments, cells were also exposed to a Ca pulse of 1 and 10mM during 180s. 10mM Ca increased intracellular Ca compared to 1mM, and the increase was not recovered during the experimental time. Additionally, Ca influx was reduced by verapamil and amiloride, but not completely. The results suggest that Ca influx probably occurs through an undefined exchanger, apart from Ca channels (verapamil sensitive) and electrogenic 1Na(+)(1H(+))/1 Ca(2+) exchanger (amiloride-sensitive). Similarities between freshwater and seawater crabs, lobsters and crayfish in relation to plasma membrane Ca transporters, although the environment where they live is quite diverse, suggest that universal mechanisms for Ca homeostasis are widespread among crustaceans. (C) 2010 Elsevier Inc. All rights reserved.

Direct action of aldosterone on bicarbonate reabsorption in in vivo cortical proximal tubule

Pergher PS, Leite-Dellova D, de Mello-Aires M. Direct action of aldosterone on bicarbonate reabsorption in in vivo cortical proximal tubule. Am J Physiol Renal Physiol 296: F1185-F1193, 2009. First published February 18, 2009; doi:10.1152/ajprenal.90217.2008.-The direct action of aldosterone (10(-12) M) on net bicarbonate reabsorption (J(HCO3)(-)) was evaluated by stationary microperfusion of an in vivo middle proximal tubule (S2) of rat kidney, using H ion-sensitive microelectrodes. Aldosterone in luminally perfused tubules caused a significant increase in J(HCO3)(-) from a mean control value of 2.84 +/- 0.08 [49/19 (n degrees of measurements/n degrees of tubules)] to 4.20 +/- 0.15 nmol.cm(-2).s(-1) (58/10). Aldosterone perfused into peritubular capillaries also increased J(HCO3)(-), compared with basal levels during intact capillary perfusion with blood. In addition, in isolated perfused tubules aldosterone causes a transient increase of cytosolic free calcium ([Ca(2+)](i)), monitored fluorometrically. In the presence of ethanol ( in similar concentration used to prepare the hormonal solution), spironolactone (10(-6) M, a mineralocorticoid receptor antagonist), actinomycin D (10(-6) M, an inhibitor of gene transcription), or cycloheximide (40 mM, an inhibitor of protein synthesis), the J(HCO3)(-) and the [Ca(2+)](i) were not different from the control value; these drugs also did not prevent the stimulatory effect of aldosterone on J(HCO3)(-) and on [Ca(2+)](i). However, in the presence of RU 486 alone [10(-6) M, a classic glucocorticoid receptor (GR) antagonist], a significant decrease on J(HCO3)(-) and on [Ca(2+)](i) was observed; this antagonist also inhibited the stimulatory effect of aldosterone on J(HCO3)(-) and on [Ca(2+)](i). These studies indicate that luminal or peritubular aldosterone (10(-12) M) has a direct nongenomic stimulatory effect on J(HCO3)(-) and on [Ca(2+)](i) in proximal tubule and that probably GR participates in this process. The data also indicate that endogenous aldosterone stimulates J(HCO3)(-) in middle proximal tubule.