BACKGROUND
intercellular communication mediated by gap junctions has been considered ubiquitous during the development, maturation, homeostasis and death of diverse cell types and tissues in metazoa  <cit> .
these junctions are membrane specializations located in cell-cell contact regions, where intercellular hydrophilic conduits, assembled as dodecameric protein complexes, directly connect the cytosols of adjacent cells  <cit> .
each complex is composed by two hexameric hemichannels, the connexons, one in each cell  <cit> .
in vertebrates, members of the connexin protein family form these channels, which in rodents has at least  <dig> isoforms  <cit> .
topologically, the connexin protein contains four hydrophobic transmembrane domains , two conserved extracellular loops , one intracellular loop and intracellular c- and n-terminal domains  <cit> .
with an estimated permeability limited to molecules below approximately  <dig> kda, these intercellular channels allow cells to share metabolites such as glucose and nucleotides, buffer ions such as k+ and h+, and convey important intracellular second messengers such as calcium, cyclic 5'-adenosine monophosphate  and  <dig> ,5-inositol-trisphosphate   <cit> .
physiologically, gap junctions have been associated with diverse phenomena such as transmission of electrical signals  and intercellular calcium waves, metabolic and ionic coupling, and cellular synchronization  <cit> .
in this respect, loss or dysfunction of gap junctions have been related to distinct diseases  <cit> .
gap junction channels may be modulated at different levels.
gap junction channel gating, i.e., shifting between open and closed states, is regulated by voltage, intracellular ph  and ca2+ , and phosphorylation  <cit> .
it has been suggested that the connexin c-terminal and the intracellular loop of the protein are associated with gap junction channel sensitivity to phi and i, while the m <dig> domain, the n-terminal and the e <dig> domain have been associated with the voltage sensor  <cit> .
the c-terminal domain exhibits diverse kinase recognition motifs, which allow channel regulation by threonine/serine and tyrosine kinases.
functional gjic has been shown in a variety of cell types of the immune system, such as t and b lymphocytes, dendritic cells, microglia, monocytes, macrophages, neutrophils and mast cells  <cit> .
in vitro experiments have demonstrated cx <dig> mediated functional gjic between thymic epithelial cells  <cit> .
in addition, data obtained from cx43-/- mice revealed that this protein is important to normal t cell lymphopoiesis  <cit> .
despite the multiple possibilities of regulation of thymic physiology by diverse neuroendocrine products  <cit> , few previous studies have evaluated gjic modulation in thymic epithelial cells.
head et al.
<cit>  demonstrated, by dye injection, that treatment of thymic epithelial cells with soluble factors such as interleukin- <dig> , growth hormone , adrenocorticotrophic hormone , steroid hormones and neuropeptides induced a partial inhibition of coupling and in some cases it diminished thymulin secretion.
the modulation of gjic may also be evaluated through the activation of different intracellular signaling pathways by specific second messenger analogs, as well as agonists or antagonists of relevant signaling molecules.
the importance of camp and pkc in mediating intracellular signaling of diverse extracellular messengers is widely recognized  <cit> .
camp activates camp-dependent protein kinase   <cit> ; and pkc is activated by diacylglycerol and/or calcium , as a result of phospholipid signaling pathways  <cit> .
the effects of camp on gjic have been investigated in systems such as hepatocyte primary cultures, cardiac myocytes, ovarian follicles, myometrium, carotid body and retina  <cit> ; and in cell lines derived from ovarian granulosa cells, endometrial and colonic epithelium, endothelium, osteoblasts, and mammary tumor cells  <cit> .
the effects of pkc activation on gjic have also been investigated  <cit> .
in general, camp acts by enhancing gjic while pkc inhibits it  <cit> .
however, contrasting results have been reported for both signaling pathways  <cit> .
nevertheless, to our knowledge the effects of camp elevation or pkc activation on gjic have not been studied in the immune system.
herein we investigated the putative modulatory effects of camp and pkc on gjic between thymic epithelial cells .
our results demonstrate that inter-tec gjic is upregulated by camp and downregulated by pkc.
RESULTS
evaluation of functional gjic-mediated dye transfer by flow cytometry
confirming previous results from our laboratory  <cit>  flow cytometry experiments revealed that after  <dig> hours of co-culture more than 65% of the initial single positive diic18+ tec acquired calcein .
to ascertain that gap junctions mediated this dye transfer we treated the cells for  <dig> hrs with  <dig> μm of 18-β-glycyrrhetinic acid , a gap junction inhibitor.
such procedure inhibited inter-tec gjic by >85% .
similar results were also obtained when the epithelial were treated with carbenoxolone, another gap junction inhibitor .
camp elevation enhances inter-tec gjic
to analyze the putative modulatory effects of camp on gjic in tec cultures, we stimulated the mouse tec line with 8-br-camp, a membrane permeable camp analog; or forskolin, which stimulates camp production through activation of adenylate cyclase isoforms  <cit> .
for that, tec co-cultures were either treated or not with 8-br-camp  or forskolin  for  <dig> hrs and subsequently analyzed by flow cytometry.
two distinct quantitative parameters were calculated comparing the treated versus control double positive tec: the extent of cell coupling  and the cell coupling efficiency .
under these conditions, the extent of coupling was not significantly modified, ranging from  <dig>  ±  <dig> %  at control level to up to  <dig>  ±  <dig> % and  <dig>  ±  <dig> % with 8-br-camp and forskolin, respectively, indicating that the tec monolayer was functionally well coupled under control confluent culture conditions .
nevertheless, treatment with 8-br-camp or forskolin increased transfer efficiency, inducing respectively,  <dig>  ±  <dig>  and  <dig>  ±  <dig>  fold increase in geometric mean fluorescence of calcein labeling in double positive cells .
the half maximal effective concentration  for 8-br-camp and forskolin was  <dig> μm and  <dig>  μm, respectively .
importantly, upregulation of inter-tec gjic triggered by 8-br-camp was not restricted to the mouse tec line, since similar results were seen when tnc-derived primary cultures of human tec  and it <dig> m <dig> cells  were treated with 8-br-camp, as ascertained after lucifer yellow microinjection and blind evaluation of the numbers of coupled cells.
we observed the same effect when the intracellular dye microinjection assay was performed on it76m <dig> cells.
we also tested if inter-tec gjic could be modulated by distinct extracellular messenger molecules, which also signal through cyclic nucleotides.
for that we applied the vasoactive intestinal peptide , adenosine and epinephrine.
vip+ nerve terminals and vip receptors have been characterized in thymic parenchyma  <cit> , whereas adenosine, which activates p <dig> receptors, has been implicated in thymocyte death  <cit>  and epinephrine, a systemic catecholamine, whose actions include control of tec cytokine secretion  <cit> .
we treated mouse tec line cultures with increasing concentrations of vip , adenosine  and epinephrine .
neither vip  nor adenosine  changed in the extent of inter-tec gjic.
however, treatment with the adrenoreceptor agonist epinephrine induced an increase on dye coupling in a dose-dependent manner .
camp elevation upregulates cx <dig> at different levels
we also investigated the possible mechanisms underlying the gjic stimulatory effects of camp elevation.
when mouse tecs were evaluated by immunofluorescence, the labeling pattern generated with the anti-cx <dig> antibody revealed that both 8-br-camp and forskolin induced an accumulation of cx <dig> protein at regions of intercellular contacts, presenting a punctate pattern of distribution .
the northern blot analysis revealed an increase in cx <dig> mrna as early as  <dig> hr after the treatment with 8-br-camp, which continued to be observed after  <dig> and  <dig> hours of treatment .
phorbol ester induced pkc activation and down-regulates inter-tec gjic
pkc is a serine/threonine protein kinase that phosphorylates gap junctions and these events have been correlated with the reduction of gap junction communication  <cit> .
consistent with this finding, and in contrast to camp, when mouse tecs were treated with pma , a decrease of dye coupling was observed, reducing from  <dig>  ±  <dig> % at control conditions to  <dig>  ±  <dig> % and  <dig>  ±  <dig> % with pma at  <dig> and  <dig> ng/ml, respectively , which represents a dye coupling inhibition of up to 60%.
the remaining coupled tec was not significantly affected after pma treatment .
a similar down regulation of inter-tec gjic was seen in tnc-derived human tec primary cultures .
herein we applied microinjection of lucifer yellow, followed by blind counting of coupled cells.
confirming the participation of gap junctions in this process,  <dig> mm heptanol completely inhibited dye coupling among human tec .
since the phospholipid signaling frequently induces both pkc activation and i elevation, we also treated the mouse tec line with both pma and the calcium ionophore ionomycin .
in these conditions, a partial but statistically significant inhibition of dye coupling was still seen .
inter-tec gjic is not modulated by the presence of thymocytes
since thymocytes play an important role in tec differentiation and organization during ontogeny of the thymus  <cit> , we investigated if the contact with thymocytes could modulate gjic in the thymic epithelium.
after the adhesion and establishment of mouse tec co-cultures , the thymocytes were added at  <dig> fold or  <dig> fold excess over tecs.
after an additional  <dig> hours, the thymocytes were discarded and the tec co-cultures were dissociated and analyzed by flow cytometry.
in these conditions, the dye coupling between adjacent tec was not significantly modulated by thymocytes, neither at 1: <dig> nor at 1: <dig> tec:thymocytes proportions .
the calcein fluorescence intensity of calcein+diic18+ was not significantly modified as well .
discussion
in the present study we demonstrated, by various experimental approaches, that camp and pkc are involved in the modulation of inter-tec gjic: the camp agonist 8-br-camp enhanced inter-tec coupling whereas pma-induced pkc activation triggered an opposite effect.
using flow cytometry we first detected in a mouse tec line, that up to 90% of diic18+ cells co-cultured in 1: <dig> ratio with calcein+ cells became double positive, a phenomenon which was readily inhibited by the gap junction inhibitors 18-β-glycyrrhetinic acid and carbenoxolone.
this result clearly demonstrates that the thymic epithelium spontaneously forms a gjic-dependent functional syncytium in vitro.
under these conditions, 8-br-camp and forskolin did not significantly modify this spontaneous percentage of coupled cells.
however, both compounds enhanced up to  <dig> fold the calcein fluorescence intensity of the double positive cells, indicating an increase in the rate of dye transfer among coupled cells.
importantly, 8-br-camp also induced an increment in gjic in tnc-derived primary cultures of human tec.
these data clearly show that elevation of camp upregulates inter-tec gjic, similar to what has been reported for effects on cell types in other systems  <cit> .
experiments performed with the mouse tec line revealed that 8-br-camp and forskolin treatments induced an accumulation of cx <dig> protein at cell-to-cell contact regions, seen as punctate clusters.
furthermore, 8-br-camp induced an increase in cx <dig> gene transcription, suggesting that the protein accumulation may be a consequence of altered cx <dig> protein translation.
such an alteration in connexin synthesis and accumulation in the cell membrane is in accordance with previous description of modulation of gap junctions by camp elevating agonists after long-term evaluations   <cit> .
however, short-term modulation also has been demonstrated, with description of changes in gap junctional conductance within minutes of treatment with 8-br-camp  <cit> ; in some cell types, changes in dye coupling were also detected within 2- <dig> minutes after this treatment  <cit> .
in cases of both short- and long-term evaluations alteration in the degree of connexin phosphorylation has also been demonstrated  <cit> .
interestingly, some of these reports have demonstrated that connexin isoforms other than cx <dig> also may be regulated by camp analogs, such as cx <dig>  cx <dig> and possibly cx <dig>  <cit> .
thus, we cannot exclude the possibility that other connexin isoforms, not yet characterized in tec, might also be regulated by 8-br-camp and forskolin in it-76m <dig> cells.
we also investigated whether physiological stimuli, could signal through cyclic nucleotides, might be involved in modulation of inter-tec gjic.
when we evaluated vip and adenosine, we did not observe any change in the degree of cell coupling in the mouse tec preparation in any concentration of each molecule used.
similar results were seen despite the various concentrations applied for each molecule.
we should mention however, that our finding on vip is at variance with the data reported by head and co-workers  <cit> .
these authors reported an inhibition of gjic in a rat tec.
although by now we cannot explain such a difference, it may be related to cell line variations or presumably due to receptor signaling dynamics that cannot be merely mimicked by a single agonist  <cit> , or may be related to differences in methodological approaches given that the authors treated the cells before gjic formation.
in any case, adenosine, which may also signal through cyclic nucleotides depending on its concentration and the activated p <dig> receptor  <cit> , did not modify the basal level of inter-tec gjic as well.
these results demonstrate that vip and adenosine possibly are not physiological modulators of gjic among tecs.
a recent review indicates that adrenoreceptor agonists mediate thymus homeostasis and local t cell development  <cit> .
in addition, reports have suggested a role for catecholamines on thymic epithelial cells controlling proliferation and cytokine secretion  <cit> .
our data demonstrate that epinephrine, and activation of camp, lead to an increasing dye coupling in tec cells suggesting that this mediator could endogenously control communication between thymic epithelial cells and contribute to thymus physiology.
we are currently investigating other endogenous molecules which also signal through camp, in order to evaluate its potential to modulate inter-tec gjic.
in a second set of experiments, we demonstrated that pkc activation  significantly inhibits the dye coupling in both mouse and human tec in vitro models.
the fact that the same inhibitory effect was seen in a mouse tec line as well as in primary cultures of human tec deserve further discussion.
the study by chanson et al.
<cit>  demonstrated that gjic inhibition was induced by phorbol esters in a liver-derived cell line but not in differentiated primarily cultures of pancreatic exocrine cells.
similarly, the inhibitory effect of phorbol esters on gjic was observed in primary cultured developing lens cells, which express cx <dig> and cx <dig>  but not in lentoid cells , which express cx <dig> and cx <dig>  demonstrating that the effects of these compounds might be dependent on the cellular differentiation stage and its pattern of cx expression  <cit> .
by contrast, the presence of thymocytes apparently is not involved in the control of inter-tec gjic, at least in the co-culture experimental conditions that we used.
nevertheless, further studies are still necessary in order to completely discard a role for thymocytes in the control of inter-tec communication mediated by gap junctions.
CONCLUSIONS
in summary, our data strongly indicate that camp and pkc intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling.
importantly, this control is phylogenetically conserved in the thymus, since it was seen in both mouse and human tec preparations.
in a second vein, we showed that two other extracellular messenger molecules, which also signal through cyclic nucleotides, vip and adenosine, did not mimic the positive action of 8-br-camp, but epinephrine was able to reproduce this effect, suggesting a specificity control of camp in the mechanism of inter-tec gjic.
overall, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium.

 intercellular communication mediated by gap junctions has been considered ubiquitous during the development, maturation, homeostasis and death of diverse cell types and tissues in metazoa. these junctions are membrane specializations located in cell-cell contact regions, where intercellular hydrophilic conduits, assembled as dodecameric protein complexes, directly connect the cytosols of adjacent cells. each complex is composed by two hexameric hemichannels, the connexons, one in each cell. in vertebrates, members of the connexin protein family form these channels, which in rodents has at least isoforms. topologically, the connexin protein contains four hydrophobic transmembrane domains, two conserved extracellular loops, one intracellular loop and intracellular c- and n-terminal domains. with an estimated permeability limited to molecules below approximately kda, these intercellular channels allow cells to share metabolites such as glucose and nucleotides, buffer ions such as k+ and h+, and convey important intracellular second messengers such as calcium, cyclic 5'-adenosine monophosphate and,5-inositol-trisphosphate. physiologically, gap junctions have been associated with diverse phenomena such as transmission of electrical signals and intercellular calcium waves, metabolic and ionic coupling, and cellular synchronization. in this respect, loss or dysfunction of gap junctions have been related to distinct diseases. gap junction channels may be modulated at different levels. gap junction channel gating, i.e., shifting between open and closed states, is regulated by voltage, intracellular ph and ca2+, and phosphorylation. it has been suggested that the connexin c-terminal and the intracellular loop of the protein are associated with gap junction channel sensitivity to phi and i, while the m domain, the n-terminal and the e domain have been associated with the voltage sensor. the c-terminal domain exhibits diverse kinase recognition motifs, which allow channel regulation by threonine/serine and tyrosine kinases. functional gjic has been shown in a variety of cell types of the immune system, such as t and b lymphocytes, dendritic cells, microglia, monocytes, macrophages, neutrophils and mast cells. in vitro experiments have demonstrated cx mediated functional gjic between thymic epithelial cells. in addition, data obtained from cx43-/- mice revealed that this protein is important to normal t cell lymphopoiesis. despite the multiple possibilities of regulation of thymic physiology by diverse neuroendocrine products, few previous studies have evaluated gjic modulation in thymic epithelial cells. head et al. demonstrated, by dye injection, that treatment of thymic epithelial cells with soluble factors such as interleukin-, growth hormone, adrenocorticotrophic hormone, steroid hormones and neuropeptides induced a partial inhibition of coupling and in some cases it diminished thymulin secretion. the modulation of gjic may also be evaluated through the activation of different intracellular signaling pathways by specific second messenger analogs, as well as agonists or antagonists of relevant signaling molecules. the importance of camp and pkc in mediating intracellular signaling of diverse extracellular messengers is widely recognized. camp activates camp-dependent protein kinase; and pkc is activated by diacylglycerol and/or calcium, as a result of phospholipid signaling pathways. the effects of camp on gjic have been investigated in systems such as hepatocyte primary cultures, cardiac myocytes, ovarian follicles, myometrium, carotid body and retina; and in cell lines derived from ovarian granulosa cells, endometrial and colonic epithelium, endothelium, osteoblasts, and mammary tumor cells. the effects of pkc activation on gjic have also been investigated. in general, camp acts by enhancing gjic while pkc inhibits it. however, contrasting results have been reported for both signaling pathways. nevertheless, to our knowledge the effects of camp elevation or pkc activation on gjic have not been studied in the immune system. herein we investigated the putative modulatory effects of camp and pkc on gjic between thymic epithelial cells. our results demonstrate that inter-tec gjic is upregulated by camp and downregulated by pkc.  evaluation of functional gjic-mediated dye transfer by flow cytometry confirming previous results from our laboratory flow cytometry experiments revealed that after hours of co-culture more than 65% of the initial single positive diic18+ tec acquired calcein. to ascertain that gap junctions mediated this dye transfer we treated the cells for hrs with μm of 18-β-glycyrrhetinic acid, a gap junction inhibitor. such procedure inhibited inter-tec gjic by >85%. similar results were also obtained when the epithelial were treated with carbenoxolone, another gap junction inhibitor. camp elevation enhances inter-tec gjic to analyze the putative modulatory effects of camp on gjic in tec cultures, we stimulated the mouse tec line with 8-br-camp, a membrane permeable camp analog; or forskolin, which stimulates camp production through activation of adenylate cyclase isoforms. for that, tec co-cultures were either treated or not with 8-br-camp or forskolin for hrs and subsequently analyzed by flow cytometry. two distinct quantitative parameters were calculated comparing the treated versus control double positive tec: the extent of cell coupling and the cell coupling efficiency. under these conditions, the extent of coupling was not significantly modified, ranging from ± % at control level to up to ± % and ± % with 8-br-camp and forskolin, respectively, indicating that the tec monolayer was functionally well coupled under control confluent culture conditions. nevertheless, treatment with 8-br-camp or forskolin increased transfer efficiency, inducing respectively, ± and ± fold increase in geometric mean fluorescence of calcein labeling in double positive cells. the half maximal effective concentration for 8-br-camp and forskolin was μm and μm, respectively. importantly, upregulation of inter-tec gjic triggered by 8-br-camp was not restricted to the mouse tec line, since similar results were seen when tnc-derived primary cultures of human tec and it m cells were treated with 8-br-camp, as ascertained after lucifer yellow microinjection and blind evaluation of the numbers of coupled cells. we observed the same effect when the intracellular dye microinjection assay was performed on it76m cells. we also tested if inter-tec gjic could be modulated by distinct extracellular messenger molecules, which also signal through cyclic nucleotides. for that we applied the vasoactive intestinal peptide, adenosine and epinephrine. vip+ nerve terminals and vip receptors have been characterized in thymic parenchyma, whereas adenosine, which activates p receptors, has been implicated in thymocyte death and epinephrine, a systemic catecholamine, whose actions include control of tec cytokine secretion. we treated mouse tec line cultures with increasing concentrations of vip, adenosine and epinephrine. neither vip nor adenosine changed in the extent of inter-tec gjic. however, treatment with the adrenoreceptor agonist epinephrine induced an increase on dye coupling in a dose-dependent manner. camp elevation upregulates cx at different levels we also investigated the possible mechanisms underlying the gjic stimulatory effects of camp elevation. when mouse tecs were evaluated by immunofluorescence, the labeling pattern generated with the anti-cx antibody revealed that both 8-br-camp and forskolin induced an accumulation of cx protein at regions of intercellular contacts, presenting a punctate pattern of distribution. the northern blot analysis revealed an increase in cx mrna as early as hr after the treatment with 8-br-camp, which continued to be observed after and hours of treatment. phorbol ester induced pkc activation and down-regulates inter-tec gjic pkc is a serine/threonine protein kinase that phosphorylates gap junctions and these events have been correlated with the reduction of gap junction communication. consistent with this finding, and in contrast to camp, when mouse tecs were treated with pma, a decrease of dye coupling was observed, reducing from ± % at control conditions to ± % and ± % with pma at and ng/ml, respectively, which represents a dye coupling inhibition of up to 60%. the remaining coupled tec was not significantly affected after pma treatment. a similar down regulation of inter-tec gjic was seen in tnc-derived human tec primary cultures. herein we applied microinjection of lucifer yellow, followed by blind counting of coupled cells. confirming the participation of gap junctions in this process, mm heptanol completely inhibited dye coupling among human tec. since the phospholipid signaling frequently induces both pkc activation and i elevation, we also treated the mouse tec line with both pma and the calcium ionophore ionomycin. in these conditions, a partial but statistically significant inhibition of dye coupling was still seen. inter-tec gjic is not modulated by the presence of thymocytes since thymocytes play an important role in tec differentiation and organization during ontogeny of the thymus, we investigated if the contact with thymocytes could modulate gjic in the thymic epithelium. after the adhesion and establishment of mouse tec co-cultures, the thymocytes were added at fold or fold excess over tecs. after an additional hours, the thymocytes were discarded and the tec co-cultures were dissociated and analyzed by flow cytometry. in these conditions, the dye coupling between adjacent tec was not significantly modulated by thymocytes, neither at 1: nor at 1: tec:thymocytes proportions. the calcein fluorescence intensity of calcein+diic18+ was not significantly modified as well. discussion in the present study we demonstrated, by various experimental approaches, that camp and pkc are involved in the modulation of inter-tec gjic: the camp agonist 8-br-camp enhanced inter-tec coupling whereas pma-induced pkc activation triggered an opposite effect. using flow cytometry we first detected in a mouse tec line, that up to 90% of diic18+ cells co-cultured in 1: ratio with calcein+ cells became double positive, a phenomenon which was readily inhibited by the gap junction inhibitors 18-β-glycyrrhetinic acid and carbenoxolone. this result clearly demonstrates that the thymic epithelium spontaneously forms a gjic-dependent functional syncytium in vitro. under these conditions, 8-br-camp and forskolin did not significantly modify this spontaneous percentage of coupled cells. however, both compounds enhanced up to fold the calcein fluorescence intensity of the double positive cells, indicating an increase in the rate of dye transfer among coupled cells. importantly, 8-br-camp also induced an increment in gjic in tnc-derived primary cultures of human tec. these data clearly show that elevation of camp upregulates inter-tec gjic, similar to what has been reported for effects on cell types in other systems. experiments performed with the mouse tec line revealed that 8-br-camp and forskolin treatments induced an accumulation of cx protein at cell-to-cell contact regions, seen as punctate clusters. furthermore, 8-br-camp induced an increase in cx gene transcription, suggesting that the protein accumulation may be a consequence of altered cx protein translation. such an alteration in connexin synthesis and accumulation in the cell membrane is in accordance with previous description of modulation of gap junctions by camp elevating agonists after long-term evaluations. however, short-term modulation also has been demonstrated, with description of changes in gap junctional conductance within minutes of treatment with 8-br-camp; in some cell types, changes in dye coupling were also detected within 2- minutes after this treatment. in cases of both short- and long-term evaluations alteration in the degree of connexin phosphorylation has also been demonstrated. interestingly, some of these reports have demonstrated that connexin isoforms other than cx also may be regulated by camp analogs, such as cx cx and possibly cx. thus, we cannot exclude the possibility that other connexin isoforms, not yet characterized in tec, might also be regulated by 8-br-camp and forskolin in it-76m cells. we also investigated whether physiological stimuli, could signal through cyclic nucleotides, might be involved in modulation of inter-tec gjic. when we evaluated vip and adenosine, we did not observe any change in the degree of cell coupling in the mouse tec preparation in any concentration of each molecule used. similar results were seen despite the various concentrations applied for each molecule. we should mention however, that our finding on vip is at variance with the data reported by head and co-workers. these authors reported an inhibition of gjic in a rat tec. although by now we cannot explain such a difference, it may be related to cell line variations or presumably due to receptor signaling dynamics that cannot be merely mimicked by a single agonist, or may be related to differences in methodological approaches given that the authors treated the cells before gjic formation. in any case, adenosine, which may also signal through cyclic nucleotides depending on its concentration and the activated p receptor, did not modify the basal level of inter-tec gjic as well. these results demonstrate that vip and adenosine possibly are not physiological modulators of gjic among tecs. a recent review indicates that adrenoreceptor agonists mediate thymus homeostasis and local t cell development. in addition, reports have suggested a role for catecholamines on thymic epithelial cells controlling proliferation and cytokine secretion. our data demonstrate that epinephrine, and activation of camp, lead to an increasing dye coupling in tec cells suggesting that this mediator could endogenously control communication between thymic epithelial cells and contribute to thymus physiology. we are currently investigating other endogenous molecules which also signal through camp, in order to evaluate its potential to modulate inter-tec gjic. in a second set of experiments, we demonstrated that pkc activation significantly inhibits the dye coupling in both mouse and human tec in vitro models. the fact that the same inhibitory effect was seen in a mouse tec line as well as in primary cultures of human tec deserve further discussion. the study by chanson et al. demonstrated that gjic inhibition was induced by phorbol esters in a liver-derived cell line but not in differentiated primarily cultures of pancreatic exocrine cells. similarly, the inhibitory effect of phorbol esters on gjic was observed in primary cultured developing lens cells, which express cx and cx but not in lentoid cells, which express cx and cx demonstrating that the effects of these compounds might be dependent on the cellular differentiation stage and its pattern of cx expression. by contrast, the presence of thymocytes apparently is not involved in the control of inter-tec gjic, at least in the co-culture experimental conditions that we used. nevertheless, further studies are still necessary in order to completely discard a role for thymocytes in the control of inter-tec communication mediated by gap junctions.  in summary, our data strongly indicate that camp and pkc intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. importantly, this control is phylogenetically conserved in the thymus, since it was seen in both mouse and human tec preparations. in a second vein, we showed that two other extracellular messenger molecules, which also signal through cyclic nucleotides, vip and adenosine, did not mimic the positive action of 8-br-camp, but epinephrine was able to reproduce this effect, suggesting a specificity control of camp in the mechanism of inter-tec gjic. overall, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium. 
treatment with 8-br-camp, a camp analog; or forskolin, which stimulates camp production, resulted in an increase in dye transfer between adjacent tec, inducing a three-fold enhancement in the mean fluorescence of coupled cells, ascertained by flow cytometry after calcein transfer. lastly, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium. overall, our data indicate that camp and pkc intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. these treatments also increased cx mrna expression, and stimulated cx protein accumulation in regions of intercellular contacts. the first two molecules did not mimic the effects of 8-br-camp, however epinephrine was able to increase gjic suggesting that this molecule functions as an endogenous inter-tec gjic modulators. stimulation of pkc by phorbol-myristate-acetate inhibited inter-tec gjic. lastly, experiments using mouse thymocyte/tec heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-tec gjic. we investigated the effects of the signaling molecules, cyclic amp and protein-kinase c, on gap junctional intercellular communication between thymic epithelial cells. importantly, both the enhancing and the decreasing effects, respectively induced by camp and pkc, were observed in both mouse and human tec preparations. this control is phylogenetically conserved in the thymus, since it was seen in both mouse and human tec preparations. vip, adenosine, and epinephrine which may also signal through cyclic nucleotides were tested.
 we investigated the effects of the signaling molecules, cyclic amp and protein-kinase c, on gap junctional intercellular communication between thymic epithelial cells.  treatment with 8-br-camp, a camp analog; or forskolin, which stimulates camp production, resulted in an increase in dye transfer between adjacent tec, inducing a three-fold enhancement in the mean fluorescence of coupled cells, ascertained by flow cytometry after calcein transfer. these treatments also increased cx mrna expression, and stimulated cx protein accumulation in regions of intercellular contacts. vip, adenosine, and epinephrine which may also signal through cyclic nucleotides were tested. the first two molecules did not mimic the effects of 8-br-camp, however epinephrine was able to increase gjic suggesting that this molecule functions as an endogenous inter-tec gjic modulators. stimulation of pkc by phorbol-myristate-acetate inhibited inter-tec gjic. importantly, both the enhancing and the decreasing effects, respectively induced by camp and pkc, were observed in both mouse and human tec preparations. lastly, experiments using mouse thymocyte/tec heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-tec gjic.  overall, our data indicate that camp and pkc intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. this control is phylogenetically conserved in the thymus, since it was seen in both mouse and human tec preparations. lastly, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium. 