Intercellular Ca2+ waves in mechanically stimulated articular chondrocytes

Articular cartilage is a tissue designed to withstand compression during joint movement and, in vivo, is subjected to a wide range of mechanical loading forces. Mechanosensitivity has been demonstrated to influence chondrocyte metabolism and cartilage homeostasis, but the mechanisms underlying mechanotransduction in these cells are poorly understood. In many cell types mechanical stimulation induces increases of the cytosolic Ca2+ concentration that propagates from cell to cell as an intercellular Ca2+ wave. Cell-to-cell communication through gap junctions underlies tissue co-ordination of metabolism and sensitivity to extracellular stimuli: gap junctional permeability to intracellular second messengers allows signal transduction pathways to be shared among several cells, ultimately resulting in co-ordinated tissue responses. Mechanically-induced Ca2+ signalling was investigated with digital fluorescence video imaging in primary cultures of rabbit articular chondrocytes. Mechanical stimulation of a single cell, obtained by briefly distorting the plasmamembrane with a micropipette, induced a wave of increased Ca2+ that was communicated to surrounding cells. Intercellular Ca2+ spreading was inhibited by 18 alpha-glycyrrhetinic acid, suggesting the involvement of gap junctions in signal propagation. The functional expression of gap junctions was assessed, in confluent chondrocyte cultures, by the intercellular transfer of Lucifer yellow dye in microinjection experiments while the expression of connexin 43 could be detected in Western blots. A series of pharmacological tools known to interfere with the cell calcium handling capacity were employed to investigate the mechanism of mechanically-induced Ca2+ signalling. In the absence of extracellular Ca2+ mechanical stimulation induced communicated Ca2+ waves similar to controls. Mechanical stress induced Ca2+ influx both in the stimulated chondrocyte but not in the adjacent cells, as assessed by the Mn2+ quenching technique. Cells treatment with thapsigargin and with the phospholipase C inhibitor U73122 blocked mechanically-induced signal propagation. These results provide evidence that in chondrocytes mechanical stimulation activates phospholipase C, thus leading to an increase of intracellular inositol 1,4,5-trisphosphate. The second messenger, by permeating gap junctions, stimulates intracellular Ca2+ release in neighbouring cells. Intercellular Ca2+ waves may provide a mechanism to co-ordinate tissue responses in cartilage physiology.     

Extracellular NAD+: a novel autocrine/paracrine signal in osteoblast physiology

Intercellular communication allows co-ordination of cell metabolism and sensitivity to extracellular stimuli. In bone cells, paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex intercellular networks, which favours the intercellular exchange of nutrients and second messengers, ultimately controlling the process of bone remodelling. The importance of local factors in bone remodelling is known since many years. Bone cells secrete and respond to a variety signals, among which include prostaglandins, cytokines, growth factors, and ATP. We here report evidence that extracellular NAD(+) is a novel extracellular signal stimulating osteoblast differentiation. We found that HOBIT human osteoblastic cells, which are known to express ADP-ribosyl cyclase/CD38 activity, respond to micromolar concentrations of extracellular NAD(+) with oscillatory increases of the cytosolic Ca(2+) concentration. The initial Ca(2+) response was followed by a time-dependent inhibition of cell growth, the appearance of an epithelial morphology, and by an increase of alkaline phosphatase and osteocalcin expression. Under resting condition HOBIT cells release NAD(+) in the extracellular medium and the release is significantly potentiated by mechanical stimulation. Taken together these results point to NAD(+) as a novel autocrine/paracrine factor involved in stimulation and maintenance of the osteoblast differentiated phenotype.     

Coordinated intercellular calcium waves induced by noradrenaline in rat hepatocytes: dual control by gap junction permeability and agonist.

Calcium-mobilizing agonists induce intracellular Ca2+ concentration ([Ca2+]i) changes thought to trigger cellular responses. In connected cells, rises in [Ca2+]i can propagate from cell to cell as intercellular Ca2+ waves, the mechanisms of which are not elucidated. Using fura2-loaded rat hepatocytes, we studied the mechanisms controlling coordination and intercellular propagation of noradrenaline-induced Ca2+ signals. Gap junction blockade with 18 alpha-glycyrrhetinic acid resulted in a loss of coordination between connected cells. We found that second messengers and [Ca2+]i rises in one hepatocyte cannot trigger Ca2+ responses in connected cells, suggesting that diffusion across gap junctions, while required for coordination, is not sufficient by itself for the propagation of intercellular Ca2+ waves. In addition, our experiments revealed functional differences between noradrenaline-induced Ca2+ signals in connected hepatocytes. These results demonstrate that intercellular Ca2+ signals in multicellular systems of rat hepatocytes are propagated and highly organized through complex mechanisms involving at least three factors. First, gap junction coupling ensures coordination of [Ca2+]i oscillations between the different cells; second, the presence of hormone at each hepatocyte is required for cell-cell Ca2+ signal propagation; and third, functional differences between adjacent connected hepatocytes could allow a 'pacemaker-like' intercellular spread of Ca2+ waves.     

Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels.

Astrocytes are capable of widespread intercellular communication via propagated increases in intracellular Ca(2+) concentration. We have used patch clamp, dye flux, ATP assay, and Ca(2+) imaging techniques to show that one mechanism for this intercellular Ca(2+) signaling in astrocytes is the release of ATP through connexin channels ("hemichannels") in individual cells. Astrocytes showed low Ca(2+)-activated whole-cell currents consistent with connexin hemichannel currents that were inhibited by the connexin channel inhibitor flufenamic acid (FFA). Astrocytes also showed molecular weight-specific influx and release of dyes, consistent with flux through connexin hemichannels. Transmembrane dye flux evoked by mechanical stimulation was potentiated by low Ca(2+) and was inhibited by FFA and Gd(3+). Mechanical stimulation also evoked release of ATP that was potentiated by low Ca(2+) and inhibited by FFA and Gd(3+). Similar whole-cell currents, transmembrane dye flux, and ATP release were observed in C6 glioma cells expressing connexin43 but were not observed in parent C6 cells. The connexin hemichannel activator quinine evoked ATP release and Ca(2+) signaling in astrocytes and in C6 cells expressing connexin43. The propagation of intercellular Ca(2+) waves in astrocytes was also potentiated by quinine and inhibited by FFA and Gd(3+). Release of ATP through connexin hemichannels represents a novel signaling pathway for intercellular communication in astrocytes and other non-excitable cells.     

Autocrine/paracrine stimulation of purinergic receptors in osteoblasts: contribution of vesicular ATP release

Extracellular nucleotides such as ATP and UTP are released in response to mechanical stimulation in different cell systems. It is becoming increasingly evident that ATP release plays a role in autocrine and paracrine stimulation of osteoblasts. Mechanical stimulation, as shear stress, membrane stretch or hypo-osmotic swelling, as well as oscillatory fluid flow, stimulates ATP release from different osteoblastic cell lines. Human osteoblast-like initial transfectant (HOBIT) cells release ATP in response to mechanical stimulation. In the present study, we show that HOBIT cells are activated by nanomolar levels of extracellular ATP, concentrations that can be detected under resting conditions and increase following hypotonic shock. Cell activation by hypotonic medium induced intracellular Ca2+ oscillations, and Egr-1 synthesis and DNA-binding activity. Quinacrine staining of living, resting cells revealed a granular fluorescence, typical of ATP-storing vesicles. Monensin prevented quinacrine staining and considerably inhibited hypotonic-induced ATP release. Finally, elevated levels of cytosolic Ca2+ activated massive ATP release and a dose-dependent loss of quinacrine granules. The contribution of a vesicular mechanism for ATP release is proposed to sustain paracrine osteoblast activation.     

Photoliberating inositol-1,4,5-trisphosphate triggers ATP release that is blocked by the connexin mimetic peptide gap 26

Calcium signals can be communicated between cells by the diffusion of a second messenger through gap junction channels or by the release of an extracellular purinergic messenger. We investigated the contribution of these two pathways in endothelial cell lines by photoliberating InsP(3) or calcium from intracellular caged precursors, and recording either the resulting intercellular calcium wave or else the released ATP with a luciferin/luciferase assay. Photoliberating InsP(3) in a single cell within a confluent culture triggered an intercellular calcium wave, which was inhibited by the gap junction blocker alpha-glycyrrhetinic acid (alpha-GA), the connexin mimetic peptide gap 26, the purinergic inhibitors suramin, PPADS and apyrase and by purinergic receptor desensitisation. InsP(3)-triggered calcium waves were able to cross 20 microm wide cell-free zones. Photoliberating InsP(3) triggered ATP release that was blocked by buffering intracellular calcium with BAPTA and by applying gap 26. Gap 26, however, did not inhibit the gap junctional coupling between the cells as measured by fluorescence recovery after photobleaching. Photoliberating calcium did not trigger intercellular calcium waves or ATP release. We conclude that InsP(3)-triggered ATP release through connexin hemichannels contributes to the intercellular propagation of calcium signals.     

Connexin 40 and ATP-dependent intercellular calcium wave in renal glomerular endothelial cells

Endothelial intracellular calcium ([Ca(2+)](i)) plays an important role in the function of the juxtaglomerular vasculature. The present studies aimed to identify the existence and molecular elements of an endothelial calcium wave in cultured glomerular endothelial cells (GENC). GENCs on glass coverslips were loaded with Fluo-4/Fura red, and ratiometric [Ca(2+)](i) imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single GENC caused a nine-fold increase in [Ca(2+)](i), which propagated from cell to cell throughout the monolayer (7.9 +/- 0.3 microm/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >400 microm. Inhibition of voltage-dependent calcium channels with nifedipine had no effect on the above parameters, but the removal of extracellular calcium reduced Delta[Ca(2+)](i) by 50%. Importantly, the gap junction uncoupler alpha-glycyrrhetinic acid or knockdown of connexin 40 (Cx40) by transfecting GENCs with Cx40 short interfering RNA (siRNA) almost completely eliminated Delta[Ca(2+)](i) and the calcium wave. Breakdown of extracellular ATP using a scavenger cocktail (apyrase and hexokinase) or nonselective inhibition of purinergic P2 receptors with suramin, had similar blocking effects. Scraping cells off along a line eliminated physical contact between cells but did not effect calcium wave propagation. Using an ATP biosensor technique, we detected a significant elevation in extracellular ATP (Delta = 76 +/- 2 microM) during calcium wave propagation, which was abolished by Cx40 siRNA treatment (Delta = 6 +/- 1 microM). These studies suggest that connexin 40 hemichannels and extracellular ATP are key molecular elements of the glomerular endothelial calcium wave, which may serve important juxtaglomerular functions.     

Intracellular Ca2+ stores are essential for injury induced Ca2+ signaling and re-endothelialization

Endothelialization repairs the lining of damaged vasculature and is a key process in preventing thrombosis and restenosis. It has been demonstrated that extracellular calcium ([Ca2+](o)) influx is important for subsequent endothelialization. The role of intracellular Ca2+ stores in mechanical denudation induced intracellular calcium ([Ca2+](i)) rise and endothelialization remains to be demonstrated. Using monolayer culture of a human endothelial cell line (human umbilical vein endothelial cell, HUVEC), we investigated [Ca2+](i) wave propagation and re-endothelialization following mechanical denudation. Consistent with previous reports for other types of cells, mechanical denudation induces calcium influx, which is essential for [Ca2+](i) rise and endothelialization. Moreover, we found that intracellular Ca(2+) stores are also essential for denudation induced [Ca2+](i) wave initiation and propagation, and the subsequent endothelialization. Thapsigargin which depletes intracellular Ca2+ stores completely abolished [Ca2+](i) wave generation and endothelialization. Xestospongin C (XeC), which prevents Ca2+ release from intracellular Ca2+ stores by inhibition of inositol 1,4,5-trisphosphate (IP(3)) receptor, inhibited intercellular Ca2+ wave generation and endothelialization following denudation. Purinergic signaling through a suramin sensitive mechanism and gap junction communication also contribute to in intercellular Ca(2+) wave propagation and re-endothelialization. We conclude that intracellular Ca2+ stores, in addition to extracellular Ca2+, are essential for intracellular Ca2+ signaling and subsequent endothelialization following mechanical denudation.     

A mechanism for sensing noise damage in the inner ear

Our sense of hearing requires functional sensory hair cells. Throughout life those hair cells are subjected to various traumas, the most common being loud sound. The primary effect of acoustic trauma is manifested as damage to the delicate mechanosensory apparatus of the hair cell stereocilia. This may eventually lead to hair cell death and irreversible deafness. Little is known about the way in which noxious sound stimuli affect individual cellular components of the auditory sensory epithelium. However, studies in different types of cell cultures have shown that damage and mechanical stimulation can activate changes in intracellular free calcium concentration ([Ca(2+)](i)) and elicit intercellular Ca(2+) waves. Thus an attractive hypothesis is that changes in [Ca(2+)](i), propagating as a wave through support cells in the organ of Corti, may constitute a fundamental mechanism to signal the occurrence of hair cell damage. The mechanism we describe here exhibits nanomolar sensitivity to extracellular ATP, involves regenerative propagation of intercellular calcium waves due to ATP originating from hair cells, and depends on functional IP(3)-sensitive intracellular stores in support cells.     

The gap junction cellular internet: connexin hemichannels enter the signalling limelight

Cxs (connexins), the protein subunits forming gap junction intercellular communication channels, are transported to the plasma membrane after oligomerizing into hexameric assemblies called connexin hemichannels (CxHcs) or connexons, which dock head-to-head with partner hexameric channels positioned on neighbouring cells. The double membrane channel or gap junction generated directly couples the cytoplasms of interacting cells and underpins the integration and co-ordination of cellular metabolism, signalling and functions, such as secretion or contraction in cell assemblies. In contrast, CxHcs prior to forming gap junctions provide a pathway for the release from cells of ATP, glutamate, NAD+ and prostaglandin E2, which act as paracrine messengers. ATP activates purinergic receptors on neighbouring cells and forms the basis of intercellular Ca2+ signal propagation, complementing that occuring more directly via gap junctions. CxHcs open in response to various types of external changes, including mechanical, shear, ionic and ischaemic stress. In addition, CxHcs are influenced by intracellular signals, such as membrane potential, phosphorylation and redox status, which translate external stresses to CxHc responses. Also, recent studies demonstrate that cytoplasmic Ca2+ changes in the physiological range act to trigger CxHc opening, indicating their involvement under normal non-pathological conditions. CxHcs not only respond to cytoplasmic Ca2+, but also determine cytoplasmic Ca2+, as they are large conductance channels, suggesting a prominent role in cellular Ca2+ homoeostasis and signalling. The functions of gap-junction channels and CxHcs have been difficult to separate, but synthetic peptides that mimic short sequences in the Cx subunit are emerging as promising tools to determine the role of CxHcs in physiology and pathology.     

Calcium waves in astrocytes-filling in the gaps.

Stimulus-evoked cellular responses are sometimes organized in the form of propagating waves of cytoplasmic Ca2+ increase. Ca2+ waves can be elicited in cultured astrocytes by the neurotransmitter glutamate; however, the propagation mechanism is unknown. Here, qualitative and quantitative features of propagation suggest that astrocytic Ca2+ waves are mediated by an intracellular signal that crosses intercellular junctions. The role of gap junctions in cell-cell Ca2+ wave propagation was specifically tested. Functional gap junctions were demonstrated using a noninvasive fluorescence recovery method and the gap junction blockers halothane and octanol. Gap junction closure prevented intracellular waves from propagating between cells without affecting the velocity of the intracellular wave itself. The pivotal role played by the gap junction creates the potential for dynamic changes in glial connectivity and long-range glial signaling.     

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.     

ATP-induced ATP release from astrocytes

Propagation of interastrocyte Ca2+ waves is mediated by diffusion of extracellular adenosine triphosphate (ATP), and may require regenerative release of ATP. The ability of ATP to initiate release of intracellular ATP was assessed by labeling adenine nucleotide pools in astrocyte cultures with 14C-adenine. The 14C-purines released during exposure to ATP were then identified by thin-layer chromatography. ATP treatment caused a five-fold increase in release of 14C-ATP but not 14C-ADP or 14C-AMP, indicating selectivity for release of ATP. Other P2 receptor agonists also caused significant 14C-ATP release, and the P2 receptor antagonists suramin, reactive blue-2 and pyridoxalphosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) inhibited ATP-induced 14C-ATP release to varying degrees, suggesting the involvement of a P2 receptor. ATP-induced 14C-ATP release was not affected by chelation of intracellular Ca2+ with BAPTA-AM, or by blockers of Ca2+ release from intracellular stores or of extracellular Ca2+ influx, suggesting a Ca2+-independent response. ATP-induced 14C-ATP release was significantly inhibited by non-selective anion channel blockers but not by blockers of ATP-binding cassette proteins, gap junction hemichannels, or vesicular exocytosis. Release of adenine nucleotides induced by 0 Ca2+ was, in contrast, not selective for ATP, and was susceptible to inhibition by gap junction blockers. These findings indicate that astrocytes are capable of ATP-induced ATP release and support a role for regenerative ATP release in glial Ca2+ wave propagation.     

The functional expression of connexin 43 in articular chondrocytes is increased by interleukin 1beta: evidence for a Ca2+-dependent mechanism

Cell-to-cell interactions and gap junctions-dependent communication are crucially involved in chondrogenic differentiation, while in adult articular cartilage direct intercellular communication occurs mainly among chondrocytes facing the outer cartilage layer. Chondrocytes extracted from adult articular cartilage and grown in primary culture express connexin 43 and form functional gap junctions capable of sustaining the propagation of intercellular Ca2+ waves. Degradation of articular cartilage is a characteristic feature of arthritic diseases and is associated to increased levels of interleukin-1 (IL-1) in the synovial fluid. We have examined the effects of IL-1 on gap junctional communication in cultured rabbit articular chondrocytes. Incubation with IL-1 potentiated the transmission of intercellular Ca2+ waves and the intercellular transfer of Lucifer yellow. The stimulatory effect was accompanied by a dose-dependent increase in the expression of connexin 43 and by an enhanced connexin 43 immunostaining at sites of cell-to-cell contact. IL-1 stimulation induced a dose-dependent increase of cytosolic Ca2+ and activates protein tyrosine phosphorylation. IL-1-dependent up-regulation of connexin 43 could be prevented by intracellular Ca2+ chelation, but not by inhibitors of protein tyrosine kinases, suggesting a crucial role of cytosolic Ca2+ in regulating the expression of connexin 43. IL-1 is one of the most potent cytokines that promotes cartilage catabolism: its modulation of intercellular communication represents a novel mechanism by which proinflammatory mediators regulate the activity of cartilage cells.     

Spatio-temporal propagation of Ca2+ signals by cyclic ADP-ribose in 3T3 cells stimulated via purinergic P2Y receptors

The role of cyclic ADP-ribose in the amplification of subcellular and global Ca2+ signaling upon stimulation of P2Y purinergic receptors was studied in 3T3 fibroblasts. Either (1) 3T3 fibroblasts (CD38- cells), (2) 3T3 fibroblasts preloaded by incubation with extracellular cyclic ADP-ribose (cADPR), (3) 3T3 fibroblasts microinjected with ryanodine, or (4) 3T3 fibroblasts transfected to express the ADP-ribosyl cyclase CD38 (CD38+ cells) were used. Both preincubation with cADPR and CD38 expression resulted in comparable intracellular amounts of cyclic ADP-ribose (42.3 +/- 5.2 and 50.5 +/- 8.0 pmol/mg protein). P2Y receptor stimulation of CD38- cells yielded a small increase of intracellular Ca2+ concentration and a much higher Ca2+ signal in CD38-transfected cells, in cADPR-preloaded cells, or in cells microinjected with ryanodine. Confocal Ca2+ imaging revealed that stimulation of ryanodine receptors by cADPR or ryanodine amplified localized pacemaker Ca2+ signals with properties resembling Ca2+ quarks and triggered the propagation of such localized signals from the plasma membrane toward the internal environment, thereby initiating a global Ca2+ wave.     

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.     

Intercellular communication upon mechanical stimulation of CPAE- endothelial cells is mediated by nucleotides

Intercellular Ca(2+)-signaling, after mechanical stimulation of calf pulmonary artery endothelial cells (CPAE), was investigated with fluorescence video imaging. Mechanical stimulation evoked an intracellular Ca(2+)-response in the mechanically stimulated (MS) cell, proceeding to the neighboring (NB) cells as a Ca(2+)-wave. The intercellular propagation of the Ca(2+)-wave was unaffected by the gap junction blockers halothane or heptanol. Therefore the intercellular communication (IC) pathway of the Ca(2+)-wave in CPAE cells does not depend on gap junctional communication but is most likely mediated by release of an extracellular mediator. Continuous unilateral flow experiments confirmed the presence of a diffusible mediator: the Ca(2+)-rise in upstream NB cells is significantly lower than in control experiments. After desensitization of purinergic receptors by pretreatment of CPAE cells with ATP (100mM), UTP (100 microM), 2MeSATP (100microM) or ADPbS (100 microM), the propagation of the intercellular Ca(2+)-wave upon mechanical stimulation was significantly inhibited. Also suramin (200 and 400 microM), a non-specific purinergic receptor blocker, reduced the IC. Application of the nucleotidase apyrase VI (10U/ml), which has a high ATPase/ADPase ratio, enhanced Ca(2+)-signaling and IC. In contrast, apyrase VII (10U/ml), which has a high ADPase/ATPase ratio, significantly depressed the propagation of the intercellular Ca(2+)-wave upon mechanical stimulation. Our experiments therefore demonstrate that the IC, evoked by a mechanical stimulus of CPAE cells, is mediated via release of nucleotides in the extracellular space. The data indicate that the diffusible messenger, responsible for the propagation of a Ca(2+)-wave, is mainly ADP or a combination of ADP/ATP.     

Intercellular signaling in glial cells: calcium waves and oscillations in response to mechanical stimulation and glutamate.

Intercellular Ca2+ signaling in primary cultures of glial cells was investigated with digital fluorescence video imaging. Mechanical stimulation of a single cell induced a wave of increased [Ca2+]i that was communicated to surrounding cells. This was followed by asynchronous Ca2+ oscillations in some cells. Similar communicated Ca2+ responses occurred in the absence of extracellular Ca2+, despite an initial decrease in [Ca2+]i in the stimulated cell. Mechanical stimulation in the presence of glutamate induced a typical communicated Ca2+ wave through cells undergoing asynchronous Ca2+ oscillations in response to glutamate. The coexistence of communicated Ca2+ waves and asynchronous Ca2+ oscillations suggests distinct mechanisms for intra- and intercellular Ca2+ signaling. This intercellular signaling may coordinate cooperative glial function.     

Effects of cAMP on intercellular coupling and osteoblast differentiation

Bone-forming cells are organized in a multicellular network interconnected by gap junctions. Direct intercellular communication via gap junctions is an important component of bone homeostasis, coordinating cellular responses to external signals and promoting osteoblast differentiation. The cAMP pathway, a major intercellular signal transduction mechanism, regulates osteoblastic function and metabolism. We investigated the effects of this second messenger on junctional communication and on the expression of differentiation markers in human HOBIT osteoblastic cells. Increased levels of cAMP induce posttranslational modifications (i.e., phosphorylations) of connexin43 and enhancement of gap junction assembly, resulting in an increased junctional permeance to Lucifer yellow and to a positive modulation of intercellular Ca(2+) waves. Increased intercellular communication, however, was accompanied by a parallel decrease of alkaline phosphatase activity and by an increase of osteocalcin expression. cAMP-dependent stimulation of cell-to-cell coupling induces a complex modulation of bone differentiation markers.