22 research outputs found
Effect of adenosine on Na+ and Cl- currents in A6 monolayers. Receptor localization and messenger involvement.
No full textThe effect of adenosine regulation on sodium and chloride transport was examined in cultured A6 renal epithelial cells. Adenosine and its analogue N6-cyclopentyladenosine (CPA) had different effects on short-circuit current (Isc) depending on the side of addition. Basolateral CPA addition induced an approximately threefold increase of the Isc that reached a maximum effect 20 min after addition and was completely inhibited by preincubation with either an A2 selective antagonist, CSC, or the sodium channel blocker, amiloride. Apical CPA addition induced a biphasic Isc response characterized by a rapid fourfold transient increase over its baseline followed by a decline and a plateau phase that were amiloride insensitive. The A1 adenosine antagonist, CPX, completely prevented this response. This Isc response to apical CPA was also strongly reduced in Cl--free media and was significantly inhibited either by basolateral bumetanide or apical DPC preincubation. Only basolateral CPA addition was able to induce an increase in cAMP level. CPA, added to cells in suspension, caused a rapid rise in [Ca2+]i that was antagonized by CPX, not affected by CSC and prevented by thapsigargin preincubation. These data suggest that basolateral CPA regulates active sodium transport via A2 adenosine receptors stimulating adenylate cyclase while apical CPA regulates Cl- secretion via A1 receptor-mediated changes in [Ca2+]i
Effects of calcium and calcimimetic nps r-467 on growth/proliferation of size-sieved stem cells from equine umbilical cord matrix. Possible involvement of the extracellular calcium sensing receptor (CaSR)
No full textLa matrice del cordone ombelicale (gelatina di Wharton) è considerata una fonte molto ricca di cellule staminali mesenchimali (MSCs). Obiettivo del presente studio è stato quello di valutare gli effetti del calcio e del calciomimetico NPS R-467 sulla crescita e proliferazione di MSCs isolate dalla matrice di cordone ombelicale equino. Un possibile coinvolgimento del recettore del calcio extracellulare (CaSR), sulla risposta cellulare osservata, è stato approfondito attraverso studi molecolari di Real tTime PCR e studi di localizzazione cellulare mediante analisi di immunofluorescenza con microscopia confocale
CFTR regulation in human airway epithelial cells requires integrity of the actin cytoskeleton and compartmentalized cAMP and PKA activity
No full textThe cystic fibrosis transmembrane conductance regulator (CFTR) mutation ΔF508CFTR still causes regulatory defects when rescued to the apical membrane, suggesting that the intracellular milieu might affect its ability to respond to cAMP regulation. We recently reported that overexpression of the Na+/H+ exchanger regulatory factor NHERF1 in the cystic fibrosis (CF) airway cell line CFBE41o-rescues the functional expression of ΔF508CFTR by promoting F-actin organization and formation of the NHERF1–ezrin–actin complex. Here, using real-time FRET reporters of both PKA activity and cAMP levels, we find that lack of an organized subcortical cytoskeleton in CFBE41o-cells causes both defective accumulation of cAMP in the subcortical compartment and excessive cytosolic accumulation of cAMP. This results in reduced subcortical levels and increased cytosolic levels of PKA activity. NHERF1 overexpression in CFBE41o-cells restores chloride secretion, subcortical cAMP compartmentalization and local PKA activity, indicating that regulation of ΔF508CFTR function requires not only stable expression of the mutant CFTR at the cell surface but also depends on both generation of local cAMP signals of adequate amplitude and activation of PKA in proximity of its target. Moreover, we found that the knockdown of wild-type CFTR in the non-CF 16HBE14o-cells results in both altered cytoskeletal organization and loss of cAMP compartmentalization, whereas stable overexpression of wt CFTR in CF cells restores cytoskeleton organization and re-establishes the compartmentalization of cAMP at the plasma membrane. This suggests that the presence of CFTR on the plasma membrane influences the cytoskeletal organizational state and, consequently, cAMP distribution. Our data show that a sufficiently high concentration of cAMP in the subcortical compartment is required to achieve PKA-mediated regulation of CFTR activity
The metabolic microenvironment finely regulates invadopodia ECM proteolysis through an ezrin-PKA-RhoA-NHE1 signaling axis
No full textInvolvement of the calcium sensing receptor (CaSR) in growth/proliferation of stem cells from equine umbilical cord matrix
No full textInvolvement of the calcium sensing receptor in growth and proliferation of stem cells from equine umbilical cord matrix
No full textThe calcium sensing receptor (CaSR) plays a key role in cells involved in calcium (Ca2+) homeostasis by directly sensing changes in extracellular Ca2+ concentration ([Ca2+]o), and external Ca2+ is a potent mediator of cell proliferation. The present study investigated the effects of high [Ca2+]o and of the CaSR agonist NPS R-467 on growth and proliferation of equine size-sieved umbilical cord matrix mesenchymal stem cells (UCM-MSC). The involvement of CaSR on observed cell response was analysed at the mRNA and protein level. Two subpopulations of UCM-MSC, isolated using multi-dishes with transwell inserts of 8-μm pores and expressing MSC markers (CD105, CD44, CD29; Corradetti et al. 2010 Reprod. Fertil. Dev. 22, 347–348), were analysed. Cells were cultured in medium containing: (A) low [Ca2+]o (0.37 mM), (B) high [Ca2+]o (2.87 mM), (C) NPS R-467 (3 μm) in the presence of high [Ca2+]o, and (D) the CaSR antagonist NPS 2390 (10 μm for 30′) followed by NPS R-467 in the presence of high [Ca2+]o. Growth and proliferation rates were compared among treatments (Student’s t-test). The CaSR expression and subcellular localization were investigated by real-time quantitative RT-PCR, immunofluorescence, and confocal microscopy. In the >8-μm cell line, the addition of NPS R-467, in the presence of [Ca2+]o, significantly increased cell growth after day 7 of culture (C v. A and B; P < 0.001). Increasing [Ca2+]o was not effective in this cell line (B v. A; not significant). In the <8-μm cell line, NPS R-467 increased cell growth, even at a lower extent (C v. A; P < 0.05), as observed on day 9 of culture. In this cell line, an increased proliferation rate was observed upon [Ca2+]o increase (B v. A; P < 0.05). In both cell lines, preincubation with NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, a stimulatory effect of additional calcium and NPS R-467 on cell proliferation, in terms of reduced DT values, was observed. In the 2 cell lines, CaSR expression was down-regulated in the presence of high calcium and in NPS R-467-treated cells compared with controls (B and C v. A cells; P < 0.001). Treatment with high calcium or NPS R-467 reduced CaSR labelling in the cytosol and increased it at the cortical level. We found that CaSR is expressed at mRNA and protein levels in equine UCM-MSC, and it is functionally active because the selective CaSR agonist NPS R-467 induced a stimulatory effect on cell growth and proliferation, which was reversed by the CaSR antagonist NPS 2390. The different responses to treatments between the 2 UCM-MSC subpopulations suggest that CaSR could be differentially activated in these cell lines. The calcimimetic NPS R-467 might be useful as an adjunctive component of media for UCM-MSC culture to obtain enough cells for down-stream purposes
Differential responsiveness of proliferation and cytokeratin release to stripped serum and oestrogen in the human breast cancer cell line, MCF-7
No full textFunctional expression of the extracellular calcium sensing receptor (CaSR) in equine umbilical cord matrix size-sieved stem cells.
No full textBackgroundThe present study investigates the effects of high external calcium concentration ([Ca(2+)](o)) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level.Methodology/principal findingsA large (>8 µm in diameter) and a small (Conclusions/significanceIn conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes
31 Rescue of deltaF508 CFTR in CFBE41o- cells is dependent on actin cytoskeleton interaction with ezrin and NHERF1
No full textEZRIN PHOSPHORYLATION AND ACTIVATION OF RHOA PLAY A ROLE IN THE RESCUE OF ΔF508CFTR IN CFBE41O– CELLS BY NHERF1
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