Search PubMed⌕ Search

Biomedical subjects

P D'Andrea

Publications and source records attributed to P D'Andrea.

At least 19 recordsLinked to original sources
PubMed2003

Loss-of-function and residual channel activity of connexin26 mutations associated with non-syndromic deafness.

Connexins are the protein subunits of gap junction channels that allow a direct signaling pathway between networks of cells. The specific role of connexin channels in the homeostasis of different organs has been validated by the association of mutations in several human connexins with a variety of genetic diseases. Several connexins are present in the mammalian cochlea and at least four of them have been proposed as genes causing sensorineural hearing loss. We have started our functional analysis by selecting nine mutations in Cx26 that are associated with non-syndromic recessive deafness (DFNB1). We have observed that both human Cx26 wild-type (HCx26wt) and the F83L polymorphism, found in unaffected controls, generated electrical conductance between paired Xenopus oocytes, which was several orders of magnitude greater than that measured in water-injected controls. In contrast, most recessive Cx26 mutations (identified in DFNB1 patients) resulted in a simple loss of channel activity. In addition, the V37I mutation, originally identified as a polymorphism in heterozygous unaffected individuals, was devoid of function and thus may be pathologically significant. Unexpectedly, we have found that the recessive mutation V84L retained functional activity in both paired Xenopus oocytes and transfected HeLa cells. Furthermore, both the magnitude of macroscopic junctional conductance and its voltage-gating properties were indistinguishable from those of HCx26wt. The identification of functional differences of disease causing mutations may lead to define which permeation or gating properties of Cx26 are necessary for normal auditory function in humans and will be instrumental in identifying the molecular steps leading to DFNB1.

Animals↗
PubMed2001

Mechanically induced ATP release from human osteoblastic cells.

Extracellular ATP is a widespread autocrine/paracrine signal since many animal cells release ATP in the extracellular medium; often this release is mechanosensitive, but the molecular mechanism is still unclear. The involvement of vesicular release, conductive channels, or ABC transporters has been suggested in different cell types. We investigated the mechanism of ATP release in human HOBIT osteoblastic cells, in which mechanical stimulation induced intercellular calcium waves sustained by both cell-to-cell coupling through gap junctions and ATP release. In this study we employed a luciferin-luciferase bioluminescence assay to measure the amount of ATP released under different stimulatory conditions. Given the role of connexin hemichannels in favoring passive NAD(+) transport [Bruzzone, S., et al. (2001) FASEB J. 15, 10-12], the involvement of connexin hemichannels as putative ATP transporters was initially investigated. In HOBIT cells overexpressing connexin43 the amount of nucleotide released under basal and stimulated conditions was similar to non-transfected cells, ruling out a major involvement of connexin hemichannels in ATP transport. In nontransfected HOBIT cells mechanical stimulations induced by medium displacement and hypotonic stress consistently enhanced ATP efflux. Cytochalsin D treatment did not alter basal and stimulated ATP release, while elevated cAMP levels consistently reduced efflux in both cases. ATP released by hypotonic stress and medium displacement evoked intracellular Ca(2+) transients in fura2-loaded HOBIT cells, indicating that different mechanical stimuli activate physiological cell responses.

Adenosine Triphosphate↗
PubMed2001

Extracellular NAD(+) induces calcium signaling and apoptosis in human osteoblastic cells.

ADP-ribosyl cyclase/CD38 is a bifunctional enzyme that catalyzes at its ectocellular domain the synthesis from NAD(+) (cyclase) and the hydrolysis (hydrolase) of the calcium-mobilizing second messenger cyclic ADP ribose (cADPR). Furthermore, CD38 mediates cADPR influx inside a number of cells, thereby inducing Ca(2+) mobilization. Intracellularly, cADPR releases Ca(2+) from ryanodine-sensitive pools, thus activating several Ca(2+)-dependent functions. Among these, the inhibition of osteoclastic-mediated bone resorption has been demonstrated. We found that HOBIT human osteoblastic cells display ADP-ribosyl cyclase activity and we examined the effects of CD38 stimulation on osteoblasts function. Extracellular NAD(+) induced elevation of cytosolic calcium due to both Ca(2+) influx from the extracellular medium and Ca(2+) release from ryanodine-sensitive intracellular stores. Culturing these cells in the presence of NAD(+) caused a complete growth arrest with a time-dependent decrease of cell number and the appearance of apoptotic nuclei. The first changes could be observed after 24 h of treatment and became fully evident after 72-96 h. We propose a role of extracellular NAD(+) in bone homeostatic control.

ADP-ribosyl Cyclase↗
PubMed2001

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.

Alkaline Phosphatase↗
PubMed2001

Dual mechanism of intercellular communication in HOBIT osteoblastic cells: a role for gap-junctional hemichannels.

Intercellular communication allows tissue coordination of cell metabolism and sensitivity to extracellular stimuli. Paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex cellular networks, which favors the intercellular exchange of nutrients and second messengers. Intercellular Ca2+ signaling was investigated in human osteoblast-like initial transfectant (HOBIT) cells, a human osteoblastic cell line in which cells retain most of the osteoblastic differentiation markers. HOBIT cells express connexin43 (Cx43) clustered at the cell-to-cell boundary and display functional intercellular coupling as assessed by the intercellular transfer of Lucifer yellow. Mechanical stimulation of a single cell induced a wave of increased Ca2+ that was radially propagated to surrounding cells. Treatment of cells with thapsigargin blocked mechanically induced signal propagation. Intercellular Ca2+ spreading and dye transfer were inhibited by 18alpha-glycyrrhetinic acid (18-GA), showing the involvement of gap junctions in signal propagation. Pretreatment of cells with suramin or with apyrase decreased the extent of wave propagation, suggesting that ATP-mediated paracrine stimulation contribute to cell-to-cell signaling. The functional expression of gap-junctional hemichannels was evidenced in experiments of Mn2+ quenching, extracellular dye uptake, and intracellular Ca2+ release, activated by uptake of inositol 1,4,5-trisphosphate (InsP3) from the external medium. Gap-junctional hemichannels were activated by low extracellular Ca2+ concentrations and inhibited by 18-GA. A role for Cx hemichannels in adenosine triphosphate (ATP) release and paracrine stimulation is suggested.

Adenosine Triphosphate↗
PubMed2001

Functional analysis of a dominant mutation of human connexin26 associated with nonsyndromic deafness.

Cx26 has been implicated in dominant (DFNA3) and recessive (DFNB1) forms of nonsyndromic sensorineural deafness. While most homozygous DFNB1 Cx26 mutations result in a simple loss of channel activity, it is less clear how heterozygous mutations in Cx26 linked to DFNA3 cause hearing loss. We have tested the ability of one dominant mutation (W44C) to interfere with wild-type human Cx26 (HCx26wt). HCx26wt induced robust electrical conductance between paired oocytes, and facilitated dye transfer between transfected HeLa cells. In contrast, oocyte pairs injected with only W44C were not electrically coupled above background levels, and W44C failed to dye couple transfected HeLa cells. Moreover, W44C dramatically inhibited intercellular conductance of HCx26wt when co-expressed in an equal ratio, and the low levels of residual conductance displayed altered gating properties. A nonfunctional recessive mutation (W77R) did not inhibit the ability of HCx26wt to form functional channels when co-injected in the same oocyte pairs, nor did it alter HCx26wt gating. These results provide evidence for a functional dominant negative effect of the W44C mutant on HCx26wt and explain how heterozygous Cx26 mutations could contribute to autosomal dominant deafness, by resulting in a net loss, and/or alteration, of Cx26 function.

Animals↗
PubMed2000

Interleukin-1beta increases the functional expression of connexin 43 in articular chondrocytes: evidence for a Ca2+-dependent mechanism.

Cell-to-cell interactions and gap junctions-dependent communication are crucially involved in chondrogenic differentiation, whereas 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 (Cx43) 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 Cx43 and by an enhanced Cx43 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 Cx43 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 Cx43. 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.

Animals↗
PubMed2000

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.

Animals↗
PubMed1998

Mechanism of mechanically induced intercellular calcium waves in rabbit articular chondrocytes and in HIG-82 synovial cells.

Intercellular communication through gap junctions allows tissue coordination of cell metabolism and sensitivity to extracellular stimuli. Intercellular Ca2+ signaling was investigated with digital fluorescence video imaging in primary cultures of articular chondrocytes and in HIG-82 synovial cells. In both cell types, mechanical stimulation of a single cell induced a wave of increased Ca2+ that was communicated to surrounding cells. Intercellular Ca2+ spreading was inhibited by 18alpha-glycyrrhetinic acid, demonstrating the involvement of gap junctions in signal propagation. In the absence of extracellular Ca2+, mechanical stimulation induced communicated Ca2+ waves similar to controls; however, the number of HIG-82 cells recruited decreased significantly. Mechanical stress induced Ca2+ influx both in the stimulated chondrocyte and HIG-82 cell, but not in the adjacent cells, as assessed by the Mn2+ quenching technique. Treatment of cells with thapsigargin and with the phospholipase C (PLC) inhibitor U73122 blocked mechanically induced signal propagation. These results provide evidence that in chondrocytes and in HIG-82 synovial cells, mechanical stimulation activates PLC, thus leading to an increase of intracellular inositol 1,4,5-trisphosphate. The second messenger, by permeating gap junctions, stimulates intracellular Ca2+ release in neighboring cells. It is concluded that intercellular Ca2+ waves may provide a mechanism to coordinate tissue responses in joint physiology.

Animals↗
PubMed1997

Propagation of intercellular Ca2+ waves in mechanically stimulated articular chondrocytes.

Intercellular Ca2+ signalling in primary cultures of articular chondrocytes was investigated with digital fluorescence video imaging. Mechanical stimulation of a single cell induced a wave of increased Ca2+ that was communicated to surrounding cells. Intercellular Ca2+ spreading was inhibited by 18alpha-glycyrrhetinic acid, demonstrating the involvement of gap junctions in signal propagation. In the absence of extracellular Ca2+ mechanical stimulation failed to induce Ca2+ responses and communicated Ca2+ waves. Under these conditions Ca2+ microinjection induced intercellular waves involving the cells immediately surrounding the stimulated one. Mechanical stress induced Ca2+ influx in the stimulated, but not in the adjacent cells, as assessed by the Mn2+ quenching technique. Cell treatment with thapsigargin failed to block mechanically induced signal propagation, but significantly reduced the number of cells involved in the communicated Ca2+ wave. Similar results were obtained with the phospholipase C inhibitor U73122, which is known to prevent InsP3 generation. These results provide evidence that mechanical stimulation induces a cytosolic Ca2+ increase that may permeate gap junctions, thus acting as an intercellular messenger mediating cell-to-cell communication in articular chondrocytes.

Animals↗
PubMed1997

Relationship of CD34+ cell dose to early and late hematopoiesis following autologous peripheral blood stem cell transplantation.

We evaluated early and late hematopoietic reconstitution in 27 patients with advanced lymphoma, Hodgkin's disease, and breast or ovarian cancer after treatment using high-dose/myeloablative conditioning regimens and autologous peripheral blood stem cell PBSC) transplantation. Eighteen patients (67%) received G-CSF 5 micrograms/kg/day following chemotherapy and nine (33%) were mobilized using G-CSF alone. Each patient had 7 x 10(8) mononuclear cells (MNC) per kg collected. G-CSF was administered post-PBSC infusion. While all patients showed prompt granulocyte recovery by day 14, platelet recovery failed to occur in our (15%) heavily pretreated patients with non-Hodgkin's lymphoma. Retrospective analysis in 17 patients revealed that the infused number of CD34 surface antigen-positive cells correlated with time to granulocyte (r = 0.59, P = 0.012) and platelet (r = 0.58, P = 0.021) recovery. Patients receiving the higher numbers of CD34+ cells had consistently better hematologic parameters at 11 times examined. At 180 days post-transplant, the median Hb level was 124 g/l vs 88 g/l (P = 0.004); platelet count was 202 x 10(9)/l vs 25 x 10(9)/l (P = 0.004); and neutrophil count was 3100 x 10(6)/l vs 1400 x 10(6)/l (P = 0.15). Hemoglobin strongly correlated with the CD34+ cell dose at 360 days (r = 0.90, P = 0.01). We conclude that graft CD34+ cell content appears to be an indicator of the quality of late as well as early hematopoietic function.

Adult↗
PubMed1996

Dual mechanism for cAMP-dependent modulation of Ca2+ signalling in articular chondrocytes.

The ability of cAMP to modulate the actions of Ca(2+)-mobilizing agonists was studied in single Fura-2-loaded pig articular chondrocytes in primary culture. Forskolin and 8-Br-cAMP increased both the frequency and amplitude of Ca2+ oscillations induced by ATP, and, in unstimulated cells, induced single Ca2+ transients or even Ca2+ oscillations. The cAMP-dependent protein kinase inhibitor H89 totally prevented the effect of cAMP-elevating agents on Ca2+ signalling. Forskolin and 8-Br-cAMP promptly increased the rate of Mn2+ quenching, when administered in the presence of ATP, suggesting a potentiation of receptor-mediated Ca2+ influx. In Ca(2+)-free medium, ATP-induced Ca2+ oscillations decreased and stopped after a few cycles: subsequent ATP additions temporarily resumed the activity, an effect that could be mimicked by forskolin. The same agent induced single Ca2+ transients in 42% of the cell population maintained in Ca(2+)-free medium. Thapsigargin prevented Ca2+ responses to both ATP and forskolin. The results indicate a dual mechanism for cAMP-induced potentiation of Ca2+ signalling in articular chondrocytes: an increase of receptor-mediated Ca2+ influx and a positive modulation of intracellular Ca2+ release.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Ca2+ oscillations and intercellular Ca2+ waves in ATP-stimulated articular chondrocytes.

Cytosolic Ca2+ oscillations are known to occur in many cell types stimulated with agonists linked to the phosphoinositide signaling pathway. Trains of repetitive short-lasting Ca2+ spikes could be induced in articular chondrocytes by extracellular ATP, an agonist potently effective in stimulating cartilage resorption. The mechanism of these Ca2+ oscillations was studied by computerized video imaging on primary cultures of articular chondrocytes. Few cycles of oscillatory activity could be evoked in the absence of extracellular Ca2+, while, for oscillations to be sustained, Ca2+ influx was required. Thapsigargin irreversibly blocked Ca2+ oscillations, thus demonstrating the crucial involvement of intracellular stores in triggering the rhythmic activity. Apart from activating intracellular Ca2+ release, extracellular ATP also induced a noncapacitive Ca2+ influx in these cells. This ATP-mediated influx modulates both the oscillation frequency and intracellular stores refilling. In monolayers of confluent cells, Ca2+ oscillations spread from cell to cell in the form of intercellular waves. Propagating waves could also be observed in the absence of extracellular Ca2+, demonstrating that Ca2+ itself is not required for signal coordination. These results demonstrate that complex spatiotemporal pathways of Ca2+ oscillations and intercellular Ca2+ waves could be activated in articular chondrocytes during degenerative diseases.

Adenosine Triphosphate↗
PubMed1996

Gap junctions mediate intercellular calcium signalling in cultured articular chondrocytes.

Gap junction-mediated intercellular communication has been implicated in a variety of cellular functions. Among these, signal transduction can be coordinated among several cells due to gap junctional permeability to intracellular second messengers. Chondrocytes from articular cartilage in primary culture respond to extracellular ATP by rhythmically increasing their cytosolic Ca2+ concentration. Digital imaging fluorescence microscopy of Fura-2 loaded cells was used to monitor Ca2+ in confluent and semi-confluent cell layers. Under these conditions, Ca2+ spikes propagate from cell to cell giving rise to intercellular Ca2+ waves. The functional expression of gap junctions was assessed, in confluent chondrocyte cultures, by the intercellular transfer of Lucifer yellow dye in scrape-loading experiments. Intercellular dye transfer was blocked by the gap junction inhibitor 18 alpha-glycyrrhetinic acid. In imaging experiments, the inhibitor caused the loss of synchrony of ATP-induced Ca2+ oscillations, and blocked the intercellular Ca2+ propagation induced by mechanical stimulation of a single cell in a monolayer. It is concluded that gap junctions mediate intercellular signal transduction in cartilage cells and may provide a mechanism for co-ordinating their metabolic activity.

Adenosine Triphosphate↗