H(2)O(2) modulates purinergic-dependent calcium signalling in osteoblast-like cells.

Reactive oxygen species (ROS) have long been considered as toxic by-products of aerobic metabolism and appear involved in the pathogenesis of degenerative diseases. The physiological role of ROS as second messengers in cell signal transduction is, on the other hand, increasingly recognized. Here we investigated the effects of H(2)O(2) and extracellular nucleotides on calcium signalling in four osteoblastic cell lines. In the highly differentiated HOBIT cells, sensitive to nanomolar concentrations of ADP and UTP, millimolar H(2)O(2) induced oscillatory increases of the cytosolic calcium concentration followed by a steady and sustained calcium increase. Long lasting rhythmic calcium activity was induced by micromolar H(2)O(2) doses. The H(2)O(2)-induced calcium signals, due to both release from intracellular stores and influx from the extracellular milieu, were totally prevented by incubating the cells with the P2 receptor antagonist suramin or with the ATP/ADP hydrolyzing enzyme apyrase. In the osteosarcoma SaOS-2 cells micromolar H(2)O(2) failed to evoke calcium signals and millimolar H(2)O(2) induced a slowly developing calcium influx which was unaffected by suramin and apyrase. These cells responded to micromolar concentrations of ATP and ADP, but were largely insensitive to UTP. ROS 17/2.8 osteosarcoma cells were totally insensitive to ATP, ADP and UTP in keeping with the evidence that these cells lack functional purinergic receptors. In these cells, H(2)O(2) up to 1mM did not increase the cytosolic calcium concentration. In ROS/P2Y(2) cells, stably expressing the P2Y(2) receptor, spontaneous calcium oscillations were observed in 38% of the population and nanomolar concentration of extracellular ATP or UTP activated oscillations in quiescent cells. Spontaneous calcium signals were inhibited by suramin and apyrase. In these cells H(2)O(2) induced oscillatory calcium activity that was blocked by suramin and apyrase. The sensitivity of ROS/P2Y(2) cells to UTP decreased significantly in the presence of DTT, which was effective also in inhibiting spontaneous calcium oscillations. On the other hand, the membrane-impermeant thiol oxidant DTNB induced calcium oscillations that were inhibited by incubating the cells with suramin or apyrase. Since peroxide did not increase extracellular ATP in these cell lines, we propose that, in osteoblasts, mild oxidative conditions could activate purinergic signalling through the sensitization of P2Y(2) receptor.

Hypertension and prolonged vasoconstrictor signaling in RGS2-deficient mice.

Signaling by hormones and neurotransmitters that activate G protein-coupled receptors (GPCRs) maintains blood pressure within the normal range despite large changes in cardiac output that can occur within seconds. This implies that blood pressure regulation requires precise kinetic control of GPCR signaling. To test this hypothesis, we analyzed mice deficient in RGS2, a GTPase-activating protein that greatly accelerates the deactivation rate of heterotrimeric G proteins in vitro. Both rgs2+/- and rgs2-/- mice exhibited a strong hypertensive phenotype, renovascular abnormalities, persistent constriction of the resistance vasculature, and prolonged response of the vasculature to vasoconstrictors in vivo. Analysis of P2Y receptor-mediated Ca2+ signaling in vascular smooth muscle cells in vitro indicated that loss of RGS2 increased agonist potency and efficacy and slowed the kinetics of signal termination. These results establish that abnormally prolonged signaling by G protein-coupled vasoconstrictor receptors can contribute to the onset of hypertension, and they suggest that genetic defects affecting the function or expression of RGS2 may be novel risk factors for development of hypertension in humans.

Diminished zonula occludens-1 expression in the failing human heart.

BACKGROUND: Reduced expression of the major gap junction protein connexin 43 (Cx43) in the failing human heart may lead to arrhythmias and sudden cardiac death. Cx43 interacts with the actin binding protein, zonula occludens-1 (ZO-1), and it has recently been demonstrated that ZO-1 regulates the formation and function of Cx43 gap junctions. We hypothesize that normal expression of ZO-1 and its interaction with Cx43 are required for appropriate assembly and function of Cx43 gap junctions in the heart. Here, we determined whether expression of ZO-1 is altered in patients with heart failure. METHODS: We examined ventricular myocardium from hearts of patients in end-stage heart failure, obtained at transplant, for ZO-1 expression by immunohistochemistry. We also subjected lysates made from this tissue to immunoblotting to determine the level of ZO-1 expression. RESULTS AND CONCLUSIONS: ZO-1 was found at 96% of the intercalated discs in nonfailing control human hearts, where it colocalized with Cx43. In contrast, there was ZO-1 immunostaining at 5% of intercalated discs in failing hearts, coincident with a reduction in Cx43 staining in intercalated discs. Immunoblotting analysis showed that there was a 95% reduction in ZO-1 expression in human heart failure. Loss of ZO-1 at intercalated discs in heart failure may play a critical role in remodeling of Cx43 gap junctions, which may contribute to abnormal impulse propagation and arrhythmogenesis, thereby predisposing patients in heart failure to sudden cardiac death.

P2X7Rs are involved in cell death, growth and cellular signaling in primary human osteoblasts.

The ionotropic ATP-gated P2X7 receptor (P2X7R) is involved in the regulation of many physiological functions including bone metabolism. Several studies on osteoblasts from rodents and human osteoblast-like cell lines have addressed the expression and function of P2X7R on these bone-forming cells however; its role in human primary osteoblasts has not yet been reported. The aim of this study was to assess the expression of the P2X7R in bone marrow-derived stromal cells and in primary human trabecular osteoblasts and to determine the function in bone formation and cell signaling. We report that osteoblasts derived from human trabecular explants express a functional P2X7R capable of agonist-induced increase in intracellular calcium concentration and a positive permeability to fluorescent dyes. These osteoblasts are fully differentiated cells with alkaline phosphatase activity and the ability to form mineralized nodules. We show that the transcriptional regulation of osteoblastic markers can be modulated by P2X7R activity or blockade thereby influencing the differentiation, proliferation and bone matrix formation by these primary human osteoblasts. Finally, we demonstrate that the P2X7R is involved in propagation of mechanically-induced intercellular signaling in addition to the known mechanisms involving calcium signaling via P2Y2 receptors and gap junction.

The predominant mechanism of intercellular calcium wave propagation changes during long-term culture of human osteoblast-like cells.

Intercellular calcium waves (ICW) are calcium transients that spread from cell to cell in response to different stimuli. We previously demonstrated that human osteoblast-like cells in culture propagate ICW in response to mechanical stimulation by two mechanisms. One mechanism involves autocrine activation of P2Y receptors, and the other requires gap junctional communication. In the current work we ask whether long-term culture of osteoblast-like cells affects the propagation of ICW by these two mechanisms. Human osteoblast-like cells were isolated from bone marrow. Mechanically induced ICW were assessed by video imaging of Fura-2 loaded cells after 1, 2 and 4 months culture. The P2Y2 receptor and the gap junction protein Cx43 were assessed by Western blot and real-time PCR. In resting conditions, P2Y mediated ICW prevailed and spread rapidly to about 13 cells. P2Y receptor desensitization by ATP disclosed gap junction-mediated ICW which diffused more slowly and involved not more than five to six cells. After 2 months in culture, ICW appeared slower and wave propagation was much less inhibited by P2Y desensitization, suggesting an increase in gap junction-mediated ICW. After 4 months in culture cells still responded to addition of ATP, but P2Y desensitization did not inhibit ICW propagation. Our data indicate that the relative role of P2Y-mediated and gap junction-mediated ICW changes during osteoblast differentiation in vitro. In less differentiated cells, P2Y-mediated ICW predominate, but as cells differentiate in culture, gap-junction-mediated ICW become more prominent. These results suggest that P2Y receptor-mediated and gap junction-mediated mechanisms of intercellular calcium signaling may play different roles during differentiation of bone-forming cells.

Two-dimensional gel electrophoresis; better than a poke in the ICAT?

To date, the most widely used technology for conducting proteomic studies has been two-dimensional gel electrophoresis (2DGE), but this approach does have drawbacks. Isotope-coded affinity tagging (ICAT) is starting to challenge 2DGE as a new proteomic tool for the analysis of proteins in complex biological specimens. An appraisal of these two methodologies reveals that neither ICAT nor 2DGE provide comprehensive coverage on a proteome-wide scale.

Redistribution of connexin45 in gap junctions of connexin43-deficient hearts.

OBJECTIVE: Adult ventricular myocytes express two gap junction channel proteins: connexin43 (Cx43) and connexin45 (Cx45). Cx43-deficient mice exhibit slow ventricular epicardial conduction, suggesting that Cx43 plays an important role in intercellular coupling in the ventricle. Cx45 is much less abundant than Cx43 in working ventricular myocytes. Its role in ventricular conduction has not been defined, nor is it known whether expression or distribution of Cx45 is altered in Cx43-deficient mice. The present study was undertaken to determine (1) whether expression of Cx45 is upregulated and (2) whether gap junction structure and distribution are altered in Cx43-deficient mice. METHODS: Ventricular tissue from neonatal Cx43(+/+), Cx43(+/-) and Cx43(-/-) and adult Cx43(+/+) and Cx43(+/-) mice was analyzed by immunoblotting and confocal immunofluorescence microscopy. RESULTS: Total Cx45 protein abundance measured by immunoblotting was not different in Cx43-deficient or null hearts compared to wild-type control hearts. However, the amount and distribution of Cx45 immunoreactive signal measured by quantitative confocal analysis were markedly reduced in both Cx43(+/-) and Cx43(-/-) hearts. CONCLUSION: Although the total content of Cx45 is not upregulated in Cx43-deficient hearts, the localization of Cx45 to cardiac gap junctions depends on the expression level of Cx43 and is dramatically altered in mice that express no Cx43.

Sequence-Dependent Conformational Heterogeneity and Proton-Transfer Reactivity of the Fluorescent Guanine Analogue 6-Methyl Isoxanthopterin (6-MI) in DNA.

The local conformations of individual nucleic acid bases in DNA are important components in processes fundamental to gene regulation. Fluorescent nucleic acid base analogues, which can be substituted for natural bases in DNA, can serve as useful spectroscopic probes of average local base conformation and conformational heterogeneity. Here we report excitation-emission peak shift (EES) measurements of the fluorescent guanine (G) analogue 6-methyl isoxanthoptherin (6-MI), both as a ribonucleotide monophosphate (NMP) in solution and as a site-specific substituent for G in various DNA constructs. Changes in the peak positions of the fluorescence spectra as a function of excitation energy indicate that distinct subpopulations of conformational states exist in these samples on time scales longer than the fluorescence lifetime. Our pH-dependent measurements of the 6-MI NMP in solution show that these states can be identified as protonated and deprotonated forms of the 6-MI fluorescent probe. We implement a simple two-state model, which includes four vibrationally coupled electronic levels to estimate the free energy change, the free energy of activation, and the equilibrium constant for the proton transfer reaction. These parameters vary in single-stranded and duplex DNA constructs, and also depend on the sequence context of flanking bases. Our results suggest that proton transfer in 6-MI-substituted DNA constructs is coupled to conformational heterogeneity of the probe base, and can be interpreted to suggest that Watson-Crick base pairing between 6-MI and its complementary cytosine in duplex DNA involves a "low-barrier-hydrogen-bond". These findings may be important in using the 6-MI probe to understand local base conformational fluctuations, which likely play a central role in protein-DNA and ligand-DNA interactions.

Strategies and solid-phase formats for the analysis of protein and peptide phosphorylation employing a novel fluorescent phosphorylation sensor dye.

Protein kinases represent one of the largest families of regulatory enzymes, with more than 2,000 of them being encoded for by the human genome. Many cellular processes are regulated by the reversible phosphorylation of proteins and upwards of 30% of the proteins comprising the eukaryotic proteome are likely to be phosphorylated at some point during their existence. In the past, analysis of global protein phosphorylation has been accomplished through radiolabelling of samples with inorganic (32P or [gamma-32)P] ATP. The approach is limited to specimens amenable to radiolabelling and poses certain safety and disposal problems. Alternatively, immunodetection with antibodies to the common phosphoamino acids may be employed, but the antibodies are relatively expensive and exhibit limited specificity and a certain degree of cross-reactivity. Pro-Q Diamond dye is a new fluorescent phosphosensor technology suitable for the detection of phosphoserine-, phosphothreonine- and phosphotyrosine-containing proteins directly in isoelectric focusing gels, SDS-polyacrylamide gels and two-dimensional gels. Additionally, the technology is appropriate for the detection of phosphoproteins or phosphopeptides arrayed on protein chips or affixed to beads. Dye-stained proteins and peptides can be excited with a laser-based light source of 532 or 543 nm or with a xenon-arc lamp-based system equipped with appropriate band pass filters. Alternatively, ultraviolet light of about 302 nm may be employed, providing that sufficiently long exposure times are used to collect the fluorescence signal. Pro-Q Diamond dye emits maximally at approximately 580 nm. The fluorescence-based detection technology is easy to conduct, cost effective and allows rapid large-scale screening of protein and peptide phosphorylation in a variety of solid-phase assay formats.

Use of the oral platelet inhibitors dipyridamole and acetylsalicylic acid is associated with increased risk of fracture.

BACKGROUND: Platelet inhibitors are widely used in the treatment and prevention of coronary artery disease. In addition to acetylsalicylic acid, two major groups of platelet inhibitors are used; phosphodiesterase inhibitors including dipyridamole, and thienopyridines (ticlopidine and clopidogrel). Clopidogrel is the most widely used, and in combination with acetylsalicylic acid it is the standard of care for acute coronary syndromes and percutaneous coronary interventions. However, the modes of action involve pathways that are involved in the metabolic activity in bone cells and pharmacologic modulation of these pathways may therefore have effects on bone. METHODS: In the current study, we assessed the association between platelet inhibitor use and fracture incidence in a population-based epidemiological study performed within the Danish population consisting of approximately 5.3 million individuals, where all patients sustaining a fracture during the year of 2000 were included (124,655 cases). The hypotheses were to investigate if use of thienopyridines or phosphodiesterase inhibitors were associated with increased risk of fractures after adjustment for potential confounders. RESULTS: We found that treatment with dipyridamole is associated with increased overall fracture risk, but not to the risk of osteoporotic fractures. In contrast, low-dose acetylsalicylic acid is associated to increased risk of overall fractures and fractures of the hip. Finally, in the current study clopidogrel is not associated with increased fracture risk. CONCLUSIONS: Use of some oral platelet inhibitors is associated with increased risk of fractures, and more studies are warranted to determine the potential effect of platelet inhibitors on bone metabolism in vivo.

An unusual cause of membranous glomerulonephritis in a patient with HIV.

A 68-year old Caucasian male with a past medical history of human immunodeficiency virus (HIV) infection presented with acute oliguric renal failure and maculopapular rash. Renal biopsy demonstrated extensive foot process effacement as well as confluent small subepithelial electron-dense deposits, which is diagnostic of membranous glomerulonephritis. Subsequent serological tests showed venereal disease research laboratory test was positive in both serum and cerebral spinal fluid. Following penicillin treatment, the patient's creatinine returned to baseline 4 weeks later. Secondary membranous glomerulonephritis caused by syphilis in patients with HIV is discussed.

Genetic Background Strongly Influences the Bone Phenotype of P2X7 Receptor Knockout Mice.

The purinergic P2X7 receptor is expressed by bone cells and has been shown to be important in both bone formation and bone resorption. In this study we investigated the importance of the genetic background of the mouse strains on which the P2X7 knock-out models were based by comparing bone status of a new BALB/cJ P2X7(-/-) strain with a previous one based on the C57BL/6 strain. Female four-month-old mice from both strains were DXA scanned on a PIXImus densitometer; femurs were collected for bone strength measurements and serum for bone marker analysis. Bone-related parameters that were altered only slightly in the B6 P2X7(-/-) became significantly altered in the BALB/cJ P2X7(-/-) when compared to their wild type littermates. The BALB/cJ P2X7(-/-) showed reduced levels of serum C-telopeptide fragment (s-CTX), higher bone mineral density, and increased bone strength compared to the wild type littermates. In conclusion, we have shown that the genetic background of P2X7(-/-) mice strongly influences the bone phenotype of the P2X7(-/-) mice and that P2X7 has a more significant regulatory role in bone remodeling than found in previous studies.

Association between P2X7 Receptor Polymorphisms and Bone Status in Mice.

Macrophages from mouse strains with the naturally occurring mutation P451L in the purinergic receptor P2X7 have impaired responses to agonists (1). Because P2X7 receptors are expressed in bone cells and are implicated in bone physiology, we asked whether strains with the P451L mutation have a different bone phenotype. By sequencing the most common strains of inbred mice, we found that only a few strains (BALB, NOD, NZW, and 129) were harboring the wild allelic version of the mutation (P451) in the gene for the purinergic receptor P2X7. The strains were compared by means of dual energy X-ray absorptiometry (DXA), bone markers, and three-point bending. Cultured osteoclasts were used in the ATP-induced pore formation assay. We found that strains with the P451 allele (BALB/cJ and 129X1/SvJ) had stronger femurs and higher levels of the bone resorption marker C-telopeptide collagen (CTX) compared to C57Bl/6 (B6) and DBA/2J mice. In strains with the 451L allele, pore-formation activity in osteoclasts in vitro was lower after application of ATP. In conclusion, two strains with the 451L allele of the naturally occurring mutation P451L, have weaker bones and lower levels of CTX, suggesting lower resorption levels in these animals, which could be related to the decreased ATP-induced pore formation observed in vitro. The importance of these findings for the interpretation of the earlier reported effects of P2X7 in mice is discussed, along with strategies in developing a murine model for testing the therapeutic effects of P2X7 agonists and antagonists upon postmenopausal osteoporosis.

The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling.

The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12-/- OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12-/-, OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent αIIbß3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin ß3 gene knockout (Itgb3-/-) OCs, but its effects were substantially blunted in P2ry12-/- OCs. In vivo, P2ry12-/- mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss.

Fever in a soldier returned from Afghanistan.

We present a case of Plasmodium vivax infection in a soldier, 4 months after returning from Afghanistan. Primary care physicians should be reminded of the possible delay in presentation of P. vivax when evaluating fever and the importance of terminal prophylaxis with primaquine to prevent relapse following return from malarious regions.

Protein gel staining methods: an introduction and overview.

Laboratory scientists who encounter protein biochemistry in many of its myriad forms must often ask: is my protein pure? The most frequent response: run a denaturing SDS polyacrylamide gel. Running this gel raises another series of considerations regarding detection, quantitation, and characterization and so the next questions invariably center on suitable protein gel staining and detection methods. A total protein profile can be determined with the colorimetric methods embodied in Coomassie Blue and silver staining methods, or increasingly, with fluorescent stains. Protein quantitation can be done following staining, with fluorescence- and instrumentation-based methods offering the greatest sensitivity and linear dynamic range. Protein posttranslational modifications such as phosphorylation and glycosylation can be reliably determined with several fluorescence-based protocols. Staining and detection with two or more different stains can be done in series to establish relative profiles of modified versus total protein or to assess purity at two levels of quantitative sensitivity. The choice of staining method and protocol depends on the required rigor of detection and quantitation combined with available instrumentation and documentation capabilities. Other considerations for staining methods include intended downstream analytical procedures such as mass spectrometry or peptide sequencing, which preclude some methods. Nonfixative staining methods allow western blotting after gel staining. Laboratory custom and budget or intellectual curiosity may be the ultimate determinate of the chosen gel staining protocol.

Sphingosine-1-phosphate receptors stimulate macrophage plasma-membrane actin assembly via ADP release, ATP synthesis and P2X7R activation.

Eukaryotic plasma membranes assemble actin filaments within seconds of activation of many receptors, especially during chemotaxis. Here, serum or sphingosine-1-phosphate stimulation of J774 and RAW macrophages released ADP within seconds into the extracellular medium, along with an adenylate kinase activity that converted ADP to ATP. ATP then activated the P2X7 receptor (P2X7R) that was necessary for a peak of plasma-membrane actin assembly within 5 to 10 seconds in P2X7R-expressing J774, RAW and primary macrophages. Neither actin assembly nor characteristic P2X7R channel activity was seen in response to ATP in P2X7R-knockout macrophages, as detected by patch-clamp analysis. Since P2X7R has been shown previously to form a macromolecular complex with actin we propose that it is involved in the membrane assembly of actin. Our data reveal a surprisingly rapid and complex relay of signaling and externalization events that precede and control actin assembly induced by sphingosine-1-phosphate. The overall model we present is strongly supported by the data presented in the accompanying paper that focuses on latex bead phagosomes.

P2 receptors in macrophage fusion and osteoclast formation.

Cells of the mononuclear phagocyte lineage fuse to form multinucleated giant cells and osteoclasts. Several lines of evidence suggest that P2 receptors, in particular P2X7, are involved in this process, although P2X7 is not absolutely required for fusion because P2X7-null mice form multinucleated osteoclasts. Extracellular ATP may be an important regulator of macrophage fusion.

Regulation of RGS2 and second messenger signaling in vascular smooth muscle cells by cGMP-dependent protein kinase.

RGS2, a GTPase-activating protein (GAP) for G(q)alpha, regulates vascular relaxation and blood pressure. RGS2 can be phosphorylated by type Ialpha cGMP-dependent protein kinase (cGKIalpha), increasing its GAP activity. To understand how RGS2 and cGKIalpha regulate vascular smooth muscle signaling and function, we identified signaling pathways that are controlled by cGMP in an RGS2-dependent manner and discovered new mechanisms whereby cGK activity regulates RGS2. We show that RGS2 regulates vasoconstrictor-stimulated Ca(2+) store release, capacitative Ca(2+) entry, and noncapacitative Ca(2+) entry and that RGS2 is required for cGMP-mediated inhibition of vasoconstrictor-elicited phospholipase Cbeta activation, Ca(2+) store release, and capacitative Ca(2+) entry. RGS2 is degraded in vascular smooth muscle cells via the proteasome. Inhibition of cGK activity blunts RGS2 degradation. However, inactivation of the cGKIalpha phosphorylation sites in RGS2 does not stabilize the protein, suggesting that cGK activity regulates RGS2 degradation by other mechanisms. cGK activation promotes association of RGS2 with the plasma membrane by a mechanism requiring its cGKIalpha phosphorylation sites. By regulating GAP activity, plasma membrane association, and degradation, cGKIalpha therefore may control a cycle of RGS2 activation and inactivation. By diminishing cGK activity, endothelial dysfunction may impair RGS2 activation, thereby blunting vascular relaxation and contributing to hypertension.