ANÁLISIS DE LA CALIDAD DE LA LECHE

Immunoreactivity for P2X receptors was detected in secretory epithelia o f all three organs studied. The Sertoli cells o f the testis showed immunostaining for P2X2, P2X] and P2X7 receptors (chapter III). The follicular cells o f the thyroid displayed

immunolabelling for P2X3, P2X^ and P2X$ receptors (chapter IV) and medullary cells o f

the thymus showed immunolabelling for P2X% and P2Xg receptors. Surprisingly, only the P2X3 receptor subtype was consistently expressed in secretory epithelia o f all three endocrine organs. P2X3 receptors were initially associated (alone or together with P2X] receptors forming a heterodimeric P2X2/3 receptor) to mediate pain in nociceptive nerve fibers (Kennedy and Leff 1995). However, more recently the expression o f P2X3

receptors is also recognised in epithelial cells and was associated with the mediation o f secretion (Taylor et al. 1999).

It is not likely that P2X receptors are essential for the mediation o f secretion in epithelial cells. The Leydig cells o f the testis, the C-cells o f the thyroid and the cortical epithelial cells o f the thymus are all secretory epithelia, but did not show immunolabelling for P2X receptors.

Hormone secretion from Leydig cells or C-cells may be controlled by PI and P2Y receptors. P2Y receptors were suggested to induce steroidogenesis and testosterone secretion in cultured Leydig cells (Foresta et al. 1996a). PI receptors were also reported to have effects on steroidogenesis in Leydig cells, but only in cell culture and presumably not in situ (Rommerts et al. 1984). ATP and its analogues were shown to inhibit parathyroid hormone secretion from C-cells after mobilising Ca^^ from

intracellular stores (Nemeth and Kosz 1989).

PI and P2Y receptors were shown to stimulate and inhibit secretion from many different epithelial cells. Epithelial receptors (Rugolo et al. 1993), A2 receptors

(Furukawa et al. 1998) and A3 receptors (Mitchell et al. 1999) were all demonstrated to

activate Cl’ conductance in ciliary epithelial cells. Cl’ transport is known to be the driving force for secretion (see: chapter I). A2B receptors however, were shown to inhibit

endothelin synthesis and secretion from tracheal epithelial cells (Pelletier et al. 1998;Pelletier et al. 2000).

P2Y2 receptors were reported to mediate secretion from murine gallbladder (Clarke

et al. 1999). Several P2Y receptors were demonstrated to control Cl’ transport in epithelial cells together with P2X receptors (McCoy et al. 1999;Hede et al. 1999). P2Y% and P2Y2 receptors together with P2Xg and P2X4 receptors were shown to activate Cl’ transport in cultured renal epithelia (McCoy et al. 1999). In native pancreatic ducts P2Y2 and possibly P2Y4 receptors were shown to inhibit secretion whereas P2X7 and possibly P2X4 receptors stimulated secretion (Hede et al. 1999). Whereas P2Y2

receptors were reported to inhibit secretion in nati\e pancreatic ducts in this study it was shown that in cultured canine pancreatic duct cells P2Y2 receptors stimulate secretion (Nguyen et al. 1998).

It was demonstrated that PI receptor expression can be altered in cell culture and that P2Y receptors have opposing effects on secretion from epithelia in cell culture and in situ. PI and P2Y receptor mediated secretion from epithelial cells o f the testis and the thyroid were previously investigated in cell culture studies. PI receptors and G protein coupled ATP receptors were shown to control second messenger pathways in the rat

thyroid cell line FRTL-5 (Sato et al. 1992). P2Y receptors on cultured Sertoli cells were shown to mediate altered hormone responsiveness and hormone synthesis (Filippini et al. 1994;Meroni et al. 1998b). However, if these signalling pathways are present in situ was not described, yet.

Co-labelling for P2X and P2Y receptors was studied in the rat thymus in situ (chapter V). Medullary epithelial cells o f the thymus are known to be the principal hormone secreting cell o f the thymus (Kendall 1991). Only P2 % 2 and P2X] receptors

could be detected on these cells, whereas P2Yi and P2Y2 receptors were shown to be absent from thymic epithelia (chapter V). P2X receptors may however be coexpressed with P2Y4 receptors. The P2Y2 and the P2Y4 receptor subtype were shown to be the principal receptors mediating secretion from airway epithelial cells (see Abbracchio and Bum stock 1998 for a review).

4, Purinergic signalling in endocrine epithelial cells; implications in epithelial

turnover, P2X receptors were previously associated with controlling epithelial cell

proliferation differentiation and apoptosis. P2X receptor mediated cell proliferation was observed in epithelia o f the rat kidney (Palier et al. 1998). Several immunohistochemical studies on epithelia with high turnover rates showed the expression o f P2X$ receptors in differentiating epithelial cells and labelling for P2Xy receptors in apoptotic epithelial cells (Groschel-Stewart et al. 1999a;Grôschel-Stewart et al. 1999b;Bardini et al. 2000).

Epithelial cells o f the adult rat testis, thyroid and thym us are all well differentiated and show only low turnover rates under normal conditions (Kendall 1991;Fritz 1994;Werner and Ingbar 1996). P2X$ receptors, which may indicate differentiating epithelial cells were detected on thyroid follicular cells, but may here participate in

the control o f secretion (chapter IV; Taylor et al. 1999). Epithelial P2Xy receptors were only observed on Sertoli cells o f the testis (chapter 111). These appeared to be expressed in the perinuclear area o f the Sertoli cells and not on the plasma membrane. If P2X? receptors have a functional role in Sertoli cells (although they could not be detected on plasma membranes) they may mediate secretion rather than apoptosis. P2X7 receptors were commonly found in Sertoli cells, although only a small fraction o f these cells is normally apoptotic (Russel and Griswald 1993). P2Xy receptor mediated secretion was described in immune cells (Ferrari et al. 1997b). These data may indicate, that the striking localisation o f P2X$ and P2X7 receptors to differentiating and apoptotic

epithelial cells is only abundant in epithelia with high cellular turnover. P2Xg and P2X7

receptors may have different roles in secretory epithelia with low cellular turnover. Cell culture studies showed that PI and P2Y receptors can modulate epithelial cell differentiation, but do not seem to induce proliferation or apoptosis. Ai and A2

receptors have inhibitory effects on the differentiation o f an epithelial cell line (Lelievre et al. 2 0 0 0). P2Yô receptor were suggested to mediate epithelial-like trophoblastic

differentiation in the placenta (Somers et al. 1999).

5. Purinergic signalling in endocrine epithelial cells; epithelial cells can control

the turnover o f other cells. Some epithelial cells like the cortical and medullary epithelial

cells o f the thymus and the Sertoli cells o f the testis control proliferation, differentiation and apoptosis in other cell types. The epithelial cells o f the thymus have a central function in the process o f T-cell sorting (see chapter 1). The Sertoli cells have a fundamental role in the control o f gamete development (Russel and Griswald 1993). It was shown that Sertoli cells (chapter 111) and thymic epithelial cells (chapter IV) may

fulfil their regulatory role in gamete and T-cell development (at least partly) via communication through P2X receptors.

Development o f gametes takes place in the epithelium o f the testis in periodic cycles. The expression o f P2X2 and P2X] receptor subtypes in Sertoli cells was

dependent on the stage o f the cycle o f this maturation process. P2X receptors could however, only be detected in the perinuclear area o f Sertoli cells and were not detected on Sertoli plasma membranes.

Immature and mature T-cells are found in morphologically different areas o f the thymus. The expression o f P2X2 and P2X] receptors in thymic epithelial cells was

restricted to areas o f T-cell maturation. This may indicate some role for P2X2 and P2Xg

receptors or possibly the heteromeric P2X2/3 receptor in the control o f maturation processes by epithelial cells.

The control o f gamete or immune cell maturation is not dependent on purinergic signalling alone, but may be controlled by purinergic signalling together with e.g. adhesion molecules and growth factors or hormones. Examples for cell-cell communication combining adhesion mediated effects, growth factor secretion and purinergic signalling were previously described. It was shown that P2X7 receptor

expression in macrophages is upregulated after growth factor stimulated cellular adhesion and that P2Xy receptor stimulation induced secretion o f other growth factors (Laliberte et al. I994;Ferrari et al. 1997b). Combined actions by growth factors and by purinergic signalling were also reported in PC -12 cells. PC -12 cells underwent apoptosis after growth factor starvation, but could be rescued from apoptosis after stimulation via P2X2

demonstrated to upregulate P2X2 receptor expression and excitation o f P2X2 receptors

together w ith growth factor stimulation induced neurite outgrowth. Undifferentiated PC- 12 cells were shown to be void o f P2Y2, but these were expressed after growth factor stimulated differentiation (Arslan et al. 2000).

These data show that P2X and P2Y receptor expression can be altered after growth factor treatment or through cellular adhesion and signalling via growth factors together. P2X receptor expression in Sertoli cells or thymic epithelial cells may be influenced by similar mechanisms. Sertoli cells make contact with developing germ cells through adhesion molecules (Orth et al. 2000) and the number o f developing germ cells is limited by interaction with proteins on Sertoli plasma membranes (Lee et al. 1997). Growth factor secretion in seminiferous tubules and expression o f growth factor receptors in germ cells vary over the cycle o f the seminiferous epithelium (Olaso et al. 1998). The differential expression o f growth factor receptors and cell adhesion molecules and the varying levels o f growth factors during the cycle o f the seminiferous epithelium may set the tim e-point for P2X receptor expression. Stimulation o f P2X receptors on Sertoli cells could then induce the secretion o f chemical messengers, which are necessary for germ cell development at a certain time. Alteration o f FSH responsiveness o f cultured Sertoli cells by extracellular ATP was already demonstrated (Filippini et al. 1994).

Similar mechanisms may determine the expression o f P2X receptors in cortical or medullary epithelial cells o f the thymus. T-cells and thymic epithelial cells are both dependent on interactions through cell adhesion molecules for their cellular differentiation and for their survival (Boyd et al. 1993). T-cells communicate through different adhesion molecules w ith subcortical, cortical and medullary epithelial

cells and different growth factors are found in cortical and medullary areas o f the thym us (Kendall 1991). Different combinations o f growth factors and cell adhesion molecules in cortex and medulla may stimulate or inhibit P2X receptor expression in epithelial cells. Stimulation o f P2 receptors on subcortical or medullary epithelial cells may mediate the secretion o f chemical factors, which are important for T-cell development. Secretion o f chemical messengers after the combined application o f ATP derivatives and growth factors on cultured thymic epithelial cells was already observed (Liu et al. 1998).

6. Proliferatioriy differentiation a n d apoptosis in gametes a n d T-cells, Expression

o f P2X receptors changes throughout the cycle o f the seminiferous epithelium and between thymic compartments. These differences in P2X receptor expression are not restricted to the respective epithelial cells, but were also abundant in the developing germ cells (chapter III) and T-cells (chapter IV). In the thym us it was shown that not only P2X receptor but also P2Y receptor expression was dependent on the developmental stage o f the T-cells.

The effects o f P2X-, P2Y- and PI-receptors on T-cell development were investigated in a number o f studies (Apasov et al. 1995;Chow et al. 1997;Dubyak 2000). Only sparse information is available on the expression and role o f PI and P2 receptors in developing gametes. The expression o f P2X^ receptors in spermatids was reported (Tanaka et al. 1996), but could not be observed in the present study (chapter III). P2Y2

and P2Y4 receptors were reported to be absent from the testis after molecular cloning o f

P2Y receptors from several organs o f the rat (Tokuyama et al. 1995).

Signalling via PI receptors, P2Xi and P2X7 receptors and P2Y2 receptors was often suggested to mediate apoptosis in T-cells (Chvatchko et al. 1996;Resta et al.

1997;Koshiba et al. 1997b;Femando et al. 1999). Other reports however denied a role for P2X receptors in the mediation o f apoptosis (Jiang et al. 1996) or proposed a mitogenic role for PI receptors and P2Xy receptors in T-cells (Gregory and K em 1978;Baricordi et al. 1996;Baricordi et al. 1999). Recent reports indicate that P2X7 receptors on lymphocytes may not always be expressed functionally (Gu et al. 2000) and that P2Xy receptors may have a dual role in inducing both, proliferation and apoptosis (Di Virgilio 2000). These contradictive roles for PI and P2 receptors may be explained by the combined function o f several purinergic signalling mechanisms (e.g. combination o f signalling via P I, P2X and P2Y receptors) or by the combined function o f purinergic signalling together with other cell communication systems (e.g. growth factors and/ or adhesion molecules). One combination o f signalling pathways may assign a mitogenic role for PI or P2 receptors, whereas different combinations o f signalling pathways may assign a role for PI and P2 receptors in the induction o f apoptosis or a role outside o f developmental mechanisms.

P2X receptors were found at all stages o f germ cell development in the testis and P2X2, P2Xg and P2X$ receptors were associated w ith the mediation o f developmental

processes (at step 1-17; chapter III). However, P2X2, P2Xg and P2X7 receptor expression in late (step 18-19) spermatids were suggested to induce sperm release and volume reduction o f germ cells.

P2Xi , P2X4 and P2Y2 receptors in T-cells were associated with mediating

proliferation or apoptosis (chapter V). However, T-cells are not only developing in the thymus they also have to migrate from the cortex to the medulla. Purinergic signalling may participate in regulating this cell movement process. Implication o f purinergic

signalling in cellular movement is discussed in the next paragraph.

7. Purinergic signalling and the cytoskeleton.

P2Xô receptors (possibly forming a P2X4/6 heterodimeric channel) in HUVEC were

shown to be associated with the vascular endothelial cell specific adhesion molecule VE- cadherin (chapter VI). It was suggested that stimulation o f P2X receptors may activate protein tyrosine kinases (possibly c-src kinases), which can modulate the binding strength o f cell-cell adhesion molecules.

It was not investigated if P2X receptors in endothelial cells o f the rat show the same colocalisation with VE-cadherin. However, an electron-microscopical study o f endothelial cells in the rat brain demonstrated P2X2 receptor expression at cell-cell

junctions (Loesch and Bumstock 2000). This suggests that P2X receptors have a role in modulating endothelial adherens junctions in vivo and that not only P2X4/6 receptors but

also other P2X receptor subtypes can be expressed in endothelial adherens junctions. Comparing the immunohistochemical labelling for P2X receptors in endothelial cells o f the rat testis, thyroid and thymus immunolabelling was most abundant in endothelial cells o f the thyroid. Immunostaining for P2X receptors in the thymus or the testis was very weak (the thymus, chapter V) or could not be detected (the testis, chapter IV). The relatively high expression o f P2X receptors in endothelial cells o f the thyroid was suggested to participate in controlling the high endothelial turnover rate in the thyroid (chapter VI, Purinergic signalling in blood vessels; implication in vascular turnover) in contrast to low vascular turnover in the testis and the thymus. High vascular turnover and constant changes in vascular shape (due to increase or decrease in follicular lumen; chapter V) imply constant remodelling o f cell-cell junctions. Alterations in cell-cell

adhesion during vascular turnover and during changes in vascular shape are largely mediated by altering the binding strength o f VE-cadherin (Esser et al. 1998;Herren et al. 1998). These changes in cadherin binding strength can be mediated by Ca^^ influx and metalloprotease induction (Ito et al. 1999) or by tyrosine phosphorylation (Schlessinger 2000), which may both be mediated by P2X receptors (Schindelholz and Reber 2000).

Suggesting a role for purinergic signalling in altering cytoskeletal organisation and cellular adhesion may add a new perspective to the 'comparative approach on purinergic receptor-expression in endocrine organs'. It appears that P2X receptors and P2Y receptors are expressed most abundantly in morphologically dynamic structures and least abundantly in morphologically static structures.

P2X receptors in the testis (chapter IV) were shown in Sertoli cells, which undergo profound changes in their cyto-archtitecture throughout the cycle o f the seminiferous epithelium and which constantly alter cell-cell contacts with the surrounding germ cells (Russel and Griswald 1993). P2X receptors were also expressed on germ cells, which undergo fundamental changes in their cellular shape, which migrate throughout the seminiferous epithelium and which constantly renew cell-cell contacts during this migration. Cadherins were shown to be o f central importance to mediate germ cell and Sertoli cell contacts (Johnson et al. 2000). P2X receptors were absent from cells like Leydig cells, which are known to express cadherins, but do not undergo constant changes in cellular form or constantly remodel cell-cell junctions (Payne et al. 1996).

Similar findings were shown for P2X receptor expression in the thyroid. P2X receptors were most abundant in morphologically dynamic structures like the follicles, the capillaries surrounding the follicles and blood vessels. P2X receptors were