A novel form of cellular communication among thymic epithelial cells: intercellular calcium wave propagation.

We here describe intercellular calcium waves as a novel form of cellular communication among thymic epithelial cells. We first characterized the mechanical induction of intercellular calcium waves in different thymic epithelial cell preparations: cortical 1-4C18 and medullary 3-10 thymic epithelial cell lines and primary cultures of thymic "nurse" cells. All thymic epithelial preparations responded with intercellular calcium wave propagation after mechanical stimulation. In general, the propagation efficacy of intercellular calcium waves in these cells was high, reaching 80-100% of the cells within a given confocal microscopic field, with a mean velocity of 6-10 microm/s and mean amplitude of 1.4- to 1.7-fold the basal calcium level. As evaluated by heptanol and suramin treatment, our results suggest the participation of both gap junctions and P2 receptors in the propagation of intercellular calcium waves in thymic nurse cells and the more prominent participation of gap junctions in thymic epithelial cell lines. Finally, in cocultures, the transmission of intercellular calcium wave was not observed between the mechanically stimulated thymic epithelial cell and adherent thymocytes, suggesting that intercellular calcium wave propagation is limited to thymic epithelial cells and does not affect the neighboring thymocytes. In conclusion, these data describe for the first time intercellular calcium waves in thymic epithelial cells and the participation of both gap junctions and P2 receptors in their propagation.

Characterization of P2 receptors in thymic epithelial cells.

The presence of P2 receptors was investigated in three distinct preparations of murine thymic epithelial cells (TEC): 2BH4 murine cell line, IT45-R1 rat cell line, and a primary murine cell derived from the Nurse cell lympho-epithelial complex. In all preparations, application of ATP to the extracellular milieu triggered intracellular calcium signals indicating the presence of P2 receptor(s) in these cells. After an initial peak of calcium concentration, a plateau phase that could last more than 10 min was frequently observed. Ion replacement and channel blockage experiments indicated that the initial peak was associated with the release of calcium from intracellular stores, while the plateau phase was associated with an influx from the extracellular medium. ATP and UTP induced similar calcium signals, suggesting the presence of P2Y2 receptors in all three cell types. The murine 2BH4 cells also expressed P2X7/P2Z receptor, since under exposure to millimolar concentrations of ATP, a continuous rise in intracellular calcium concentration was observed and their plasma membranes became permeabilized to the fluorescent dyes Lucifer yellow and ethidium bromide. In addition, this permeabilization phenomenon was blocked by the P2Z-specific antagonist, oxidized ATP. RT-PCR assays confirmed the presence of mRNAs for the P2Y2 molecule in all TEC, while mRNA for the P2X7 molecule was detected only in 2BH4 cells. Our data indicate that P2Y2 purinergic receptors are widely expressed by thymic epithelial cells, whereas the expression of the P2X7 receptor appears to be more restricted, raising the possibility that its expression is related only to a particular epithelial microenvironment within/the thymus.

Pharmacologic properties of P(2Z)/P2X(7 )receptor characterized in murine dendritic cells: role on the induction of apoptosis.

In the immune system, extracellular adenosine 5'-triphosphate (ATP) mediates a variety of effects mainly through activation of a particular receptor subtype, the pore-forming P(2Z)/P2X(7) purinoceptor. This purinergic receptor has been described chiefly in cells of hemopoietic origin such as T cells, thymocytes, monocytes, macrophages, and phagocytic cells of thymic reticulum. In this study, we characterized the P(2Z)/P2X(7) purinoceptor and the ATP-mediated apoptosis in murine spleen-derived dendritic cells (DCs). Dye uptake and apoptosis were evaluated by flow cytometry. ATP-treated DCs were permeable to different low-molecular-weight fluorescent probes such as ethidium bromide, YO-PRO 1, and lucifer yellow. Such an effect was dose-dependent (EC(50): 721 micromol/L); mediated by the fully anionic agonist (ATP(4-)); and specifically stimulated by ATP, BzATP, and ATPgammaS. Additionally, an ATP-induced increase in intracellular calcium was detected by microfluorometry. Furthermore, ATP treatment induced a significant increase in apoptotic DCs (64. 46% +/- 3.8%) when compared with untreated control cells (34% +/- 5. 8%), as ascertained by the TdT-mediated dUTP nick end labeling technique. Both ATP-induced DC permeabilization and apoptosis were inhibited by oxidized ATP, a P(2Z)/P2X(7)-specific antagonist. In conclusion, we characterized the expression of the P(2Z)/P2X(7) purinoceptor in murine spleen-derived DCs and described its role on the induction of apoptosis.

Procedures to characterize and study P2Z/P2X7 purinoceptor: flow cytometry as a promising practical, reliable tool.

The expression of P2Z/P2X7 purinoceptor in different cell types is well established. This receptor is a member of the ionotropic P2X receptor family, which is composed by seven cloned receptor subtypes (P2X1 - P2X7). Interestingly, the P2Z/P2X7 has a unique feature of being linked to a non-selective pore which allows the passage of molecules up to 900 Da depending on the cell type. Early studies of P2Z/P2X7 purinoceptor were exclusively based on classical pharmacological studies but the recent tools of molecular biology have enriched the analysis of the receptor expression. The majority of assays and techniques chosen so far to study the expression of P2Z/P2X7 receptor explore directly or indirectly the effects of the opening of P2Z/P2X7 linked pore. In this review we describe the main techniques used to study the expression and functionality of P2Z/P2X7 receptor. Additionally, the increasing need and importance of a multifunctional analysis of P2Z/P2X7 expression based on flow cytometry technology is discussed, as well as the adoption of a more complete analysis of P2Z/P2X7 expression involving different techniques.

Gap junction modulation by extracellular signaling molecules: the thymus model.

Gap junctions are intercellular channels which connect adjacent cells and allow direct exchange of molecules of low molecular weight between them. Such a communication has been described as fundamental in many systems due to its importance in coordination, proliferation and differentiation. Recently, it has been shown that gap junctional intercellular communication (GJIC) can be modulated by several extracellular soluble factors such as classical hormones, neurotransmitters, interleukins, growth factors and some paracrine substances. Herein, we discuss some aspects of the general modulation of GJIC by extracellular messenger molecules and more particularly the regulation of such communication in the thymus gland. Additionally, we discuss recent data concerning the study of different neuropeptides and hormones in the modulation of GJIC in thymic epithelial cells. We also suggest that the thymus may be viewed as a model to study the modulation of gap junction communication by different extracellular messengers involved in non-classical circuits, since this organ is under bidirectional neuroimmunoendocrine control.

Gap junction modulation by extracellular signaling molecules: the thymus model

Gap junctions are intercellular channels which connect adjacent cells and allow direct exchange of molecules of low molecular weight between them. Such a communication has been described as fundamental in many systems due to its importance in coordination, proliferation and differentiation. Recently, it has been shown that gap junctional intercellular communication (GJIC) can be modulated by several extracellular soluble factors such as classical hormones, neurotransmitters, interleukins, growth factors and some paracrine substances. Herein, we discuss some aspects of the general modulation of GJIC by extracellular messenger molecules and more particularly the regulation of such communication in the thymus gland. Additionally, we discuss recent data concerning the study of different neuropeptides and hormones in the modulation of GJIC in thymic epithelial cells. We also suggest that the thymus may be viewed as a model to study the modulation of gap junction communication by different extracellular messengers involved in non-classical circuits, since this organ is under bidirectional neuroimmunoendocrine control