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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

ZO-1 alters the plasma membrane localization and function of Cx43 in osteoblastic cells.

ZO-1 is the major connexin-interacting protein in ROS 17/2.8 (ROS) osteoblastic cells. We examined the role of ZO-1 in Cx43-mediated gap junction formation and function in ROS cells that expressed the connexin-interacting fragment of ZO-1 (ROS/ZO-1dn) cells. Expression of this ZO-1(7-444) fusion protein in ROS cells disrupted the Cx43/ZO-1 interaction and decreased dye transfer by 85%, although Cx43 was retained on the plasma membrane as assessed by surface biotinylation. Fractionation of lysates derived from ROS/ZO-1dn cells on a 5-30% sucrose flotation gradient showed that 40% of the Cx43 floated into these sucrose gradients, whereas none of the Cx43 in ROS cell lysates entered the gradients, suggesting that more Cx43 is associated with lipid rafts in the transfected ROS cells than in lysates derived from untransfected ROS cells. In contrast to the ROS/ZO-1dn cells, ROS cells that over-expressed ZO-1 protein (ROS/ZO-1myc cells) exhibited increased gap junctional permeability and appositional membrane staining for Cx43. These data demonstrate that ZO-1 regulates Cx43-mediated gap junctional communication in osteoblastic cells and alters the membrane localization of Cx43. They suggest that ZO-1-mediated delivery of Cx43 from a lipid raft domain to gap junctional plaques may be an important regulatory step in gap junction formation.

RGS2 is a mediator of nitric oxide action on blood pressure and vasoconstrictor signaling.

The nitric oxide (NO)-cGMP pathway regulates vascular tone and blood pressure by mechanisms that are incompletely understood. RGS2, a GTPase-activating protein for Gqalpha that is critical for blood pressure homeostasis, has been suggested to serve as an effector of the NO-cGMP pathway that promotes vascular relaxation based on studies of aortic rings in vitro. To test this hypothesis and its relevance to blood pressure control, we determined whether RGS2 functions as an NO effector in smooth muscle of the resistance vasculature. We report that 1) the ability of the NO donor sodium nitroprusside to reduce blood pressure is impaired in RGS2-/- mice, 2) vasopressin-triggered Ca2+ transients are augmented in smooth muscle cells from resistance arteries of RGS2-/- mice, and 3) cGMP analogs fail to inhibit vasopressin-triggered Ca2+ transients in smooth muscle cells from resistance arteries of RGS2-/- mice even though cGMP-dependent protein kinase (PKG)1alpha and PKG1beta are expressed and activated normally. These results indicated that the NO-cGMP pathway uses RGS2 as a novel downstream effector to promote vascular relaxation by attenuating vasoconstrictor-triggered Ca2+ signaling in vascular smooth muscle cells. Genetic or epigenetic impairment of this mechanism may contribute to the development of hypertension, and augmenting it pharmacologically may provide a novel means of treating this disease.

Detection of phosphoproteins on electroblot membranes using a small-molecule organic fluorophore.

A new formulation of the small-molecule organic fluorophore, Pro-Q Diamond dye, has been developed that permits rapid and simple detection of phosphoproteins directly on polyvinylidene difluoride (PVDF) or nitrocellulose membranes (electroblots). Protein samples are first separated by electrophoresis and then electroblotted to membranes, stained and destained, in an analogous manner as typically performed with Amido Black or Ponceau S dye staining of total protein profiles. After staining, blots are imaged using any of a variety of laser-based gel scanners, xenon-arc lamp-based gel scanners or charge-coupled device (CCD) camera-based imaging devices equipped with UV trans- or epi-illumination. The uncomplicated and reliable staining protocol delivers results in as little as 1 h and the limit of detection for the stain is typically 2-4 ng of phosphoprotein with a linear dynamic range of approximately 15-fold. Compared with traditional radiolabeling and antibody-based approaches, the new method offers significant advantages, including avoidance of radioactivity, no need for expensive antibodies, no requirement for blocking unoccupied sites on the membrane with protein or detergent solutions, no sequence context-specific binding to phosphorylated amino acid residues and the ability to analyze the native, steady-state phosphorylation of proteins obtained directly from tissue specimens or body fluids. Pro-Q Diamond dye binds directly and exclusively to the phosphate moiety, allowing it to detect the broadest spectrum of phosphorylated proteins possible. The stain binds noncovalently to phosphoproteins and is thus fully compatible with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) or Edman sequencing. The blot stain is also compatible with standard colorimetric, fluorogenic, and chemiluminescent detection techniques employed in immunoblotting.

ATP downregulates P2X7 and inhibits osteoclast formation in RAW cells.

Multinucleated giant cells derive from fusion of precursor cells of the macrophage lineage. It has been proposed that the purinoreceptor P2X(7) is involved in this fusion process. Prolonged exposure of macrophages to ATP, the ligand for P2X(7), induces the formation of plasma membrane pores and eventual cell death. We took advantage of this cytolytic property to select RAW 264.7 (RAW) cells that lacked P2X(7) function by maintaining them in ATP (RAW ATP-R cells). RAW ATP-R cells failed to fuse to form multinucleated osteoclasts in response to receptor activator nuclear factor-kappaB ligand, although they did become positive for the osteoclast marker enzyme tartrate-resistant acid phosphatase, and upregulated expression of other osteoclast marker genes. RAW ATP-R cells and wild-type RAW cells expressed similar amounts of P2X(7) protein, but little P2X(7) was present on the surface of RAW ATP-R cells. After ATP was removed from the medium of RAW ATP-R cells, the cells reexpressed P2X(7) on the cell surface, regained sensitivity to ATP, and formed multinucleated osteoclasts. These results suggest that P2X(7) or another protein that is downregulated in concert with P2X(7) is involved either in the mechanics of cell fusion to form osteoclasts or in a signaling pathway proximal to this event. These results also suggest that P2X(7) may be regulated by ligand-mediated internalization and that extracellular ATP may regulate the formation of osteoclasts and other multinucleated giant cells.

Gap junctional communication modulates agonist-induced calcium oscillations in transfected HeLa cells.

Gap junctional communication modulates intercellular calcium signaling in many cell types. We have investigated whether gap junctional communication modulates calcium oscillatory behavior of cells responding to an agonist. Extracellular UTP induced calcium oscillations in 70% of HeLa cells cultured in monolayer, and neighboring cells oscillated independently of each other. In HeLa cell transfectants expressing connexin43 (HeLa/Cx43), extracellular UTP induced calcium transients, but calcium oscillations occurred in only 10% of cells. Inhibition of gap junctional communication with anandamide in HeLa/Cx43 transfectants substantially restored oscillations (55% of cells). In HeLa/Cx45 transfectants, UTP initiated calcium oscillations similar to those seen in HeLa cells (63% of cells), but HeLa/Cx46 transfectants demonstrated calcium oscillations that were dampened compared to those of the parental HeLa cells, and occurred in only 40% of cells. These experiments demonstrate that gap junctional communication modulates calcium oscillatory behavior in cell monolayers, presumably by allowing cells to share a small molecule such as inositol trisphosphate. These studies suggest that gap junctional communication may alter the nature of signals induced by calcium mobilizing agonists in a connexin-dependent fashion by modulating calcium oscillatory behavior.