ANÁLISIS DE LA CALIDAD DE LA LECHE 1 Contenido de grasa

Cell types

P2X receptors

cortical T-cells medullary T-cells septal epithelial cells subcapsular/ perivascular epithelial cells cortical epithelial cells medullary epithelial cells Hassal's corpuscles vascular smooth muscle endothelial cells

Table 5.1. Summary o f P2X-immunopositive ceils in the adult rat thymus. Cell types studied for P2X receptor expression in adult rat thymi are indicated on the left; subtypes o f P2 receptors are abbreviated as: (X j) P2Xi receptors; (X2) P2X2

receptors; (X3) P2Xg receptors; (X4) P2X^ receptor; (X5) P2X$ receptor; (X^) P2Xô

receptor; (X7) P2X7 receptor; (Y2) P2Y2 receptor; (Y4) P2Y4 receptor; immunopositivity

Discussion.

W estern blot analysis revealed specific detection o f P2X receptor subunit proteins in crude thymic extracts. The detection o f 70 kDa and 140 kDa P2X-proteins is consistent with reports in the literature, which describe the detection o f P2X-proteins o f similar size. Sun et al. (1998) report a single band o f 70 kDa; Scase et al. (1998) tw o bands o f 60 kDa and 45 kDa. Berry et al. (1998) detected three bands o f 160 kDa, 70 kDa and 50 kDa for P2X] in human heart. In HEK293 cells transfected with rat P2X receptors molecular weights between 57 kDa and 64 kDa were found for P2X],3 and 4

(Vulchanova et al. 1997;Lê et al. 1998). The bigger P2X? receptor was described to have a molecular weight o f 70 kDa in transfected HEK293, CHO and N tW8 cells (Collo et al.

1997). W ith our antibodies for P2Xg and 7 a single band o f 70 kDa was observed in western blots o f rat stratified squamous epithelia (Groschel-Stewart et al, 1999a). P2X proteins smaller than 70 kDa in size were not detected.

The molecular weights o f 70 kDa and 140 kDa for each receptor subunit correspond with data from the literature, describing P2X receptors as highly glycosylated proteins that can form dimers (Vulchanova et al. 1997). These receptor proteins also undergo alternative splicing (Le et al. 1997;Townsend-Nicholson et al. 1999)and the presence o f alternative spliced P2X receptor isoforms together with post-translational modifications such as glycosylation (Newbolt et al. 1998)give rise to the potential for great variation in the size o f the detected proteins.

P2X and P2Yreceptors on thymocytes.

Immunoreactivity for P2Xi and P2X^ receptors and expression o f P2Y2 receptor mRNA was shown in thymocytes in this study.

Two lines o f evidence were brought forward, describing functional involvement o f thymocyte P2X and P2Y receptors in mitogenic stimulation and induction o f apoptosis (Chow et al. 1997).

One line comes from the observation that extracellular ATP can cause cell death, probably through the opening o f the membrane channel/pore P2Xy (Di Virgilio et al. 1989). Other authors, however, argued for the participation o f the P2Xi receptor in mediating programmed cell death, since Valera et al. (1994) described extensive sequence homology between P2Xi receptors and RP-2, a gene expressed during thym ocyte apoptosis. Some authors denied a role for P2X receptors in thymocyte activation (Jiang et al. 1996;Koshiba et al. 1997a) while Ross et al. (1997) rejected involvement o f G protein-coupled purinoceptors in thymocyte apoptosis. However, Koshiba et al. (1997b) found P2Y2 mRNA upregulation after steroid hormone treatment and T-cell

receptor crosslinking o f mouse thymocytes. This situation became even more complicated after species differences between mouse and rat in P2Xi mRNA upregulation after glucocorticoid administration were revealed (Koshiba et al. 1997b).

The in vitro stimulation o f thymocytes with cortisol used in many o f these studies gives

no general indication for the onset o f apoptosis, as pointed out by Nakamura et al. (1997). Cortisol-treated cells in vivo die by neglect and become pyknotic, apoptosis being a secondary event. The vast majority o f apoptotic lymphocytes become phagocytosed directly by macrophages, so that there are no free necrotic lymphocytes.

The existence o f P2X4 mRNA in the thymus was described by (Bo et al. 1995). The

finding o f P2Xi in cortical and a minor portion in medullary thym ocytes is consistent

the inhibition o f dexamethasone-induced cell death after application o f suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) in cultured thymocytes. These are antagonists o f P2X% and P2Xs, but they can potentiate agonist responses o f P2X^ (Bo et al. 1995;Townsend-Nicholson et al. 1999). Whether there is collaboration between these purinoceptors resulting in the induction o f cell death remains to be shown. In this model P2Y2 did not seem to be involved in the prevention o f thymocyte apoptosis. The application o f suramin would have antagonised the actions o f P2Y2, whereas PPADS would have had little effect (Harden et al. 1998). However, since no difference was detected between these two P2 receptor antagonists in preventing cell death, P2Y2 receptors do not appear to mediate apoptosis. The localisation o f P2X% and P2Y2 receptors mainly in the cortex and the finding o f P2X^ staining in cortex and medulla may reflect contrasting functions in the medulla and synergistic or reverse effects in the cortex. Some authors found biphasic rises o f intracellular calcium after administration o f ATP to T-cells (Zheng et al. 1991;Ross et al. 1997) and heterogeneous response to ATP in broader populations (Chused et al. 1996). This is consistent with the differential expression o f purinoceptors on lymphocytes, which was found using immunohistochemistry and in situ hybridisation.

Presence o f P2Xy receptors was not detected on T-cells, mast cells and macrophages, although this was demonstrated pharmacologically (Chused et al. 1996). This may be due to a different expression in vivo and in cell culture. In addition, alternative gene splicing could be the cause for this discrepancy. Our P2X antibody is directed against a 15 amino acid portion at the C terminus o f this purinoceptor, which may not be present in some alternatively spliced P2X7 isoforms.

P2X receptors on the vasculature.

On vascular smooth muscle P2X receptors were shown to mediate vasoconstriction. The receptors in the vasculature (a-adrenergic and P2X) can be excited by sympathetic innervation with noradrenaline and ATP as cotransmitters, leading to constriction o f the vessels (Bumstock 1990b).

The vessels o f murine thymus are richly innervated by sympathetic, parasympathetic and sensory nerves (Williams et al. 1981;Weihe et al. 1991). Peptidergic immunomarkers show tyrosine hydroxylase and neuropeptide Y coexistence in sympathetic nerves, vasoactive intestinal polypeptide and peptide histidine isoleucine in parasympathetic neurons and calcitonin gene-related peptide and tachykinins (substance P and neurokinin A) in sensory nerves (Weihe et al. 1991). The P2X2

receptor was mainly expressed in vascular smooth muscle o f the larger septal vessels, the P2Xi receptor was seen in small and big blood vessels o f the septae and in the cortico- medullary junction, whereas the P2X4 receptor occurred predominantly on vascular smooth muscle o f small vessels in cortical and medullary areas and in some septae. The vasoconstrictor tone o f these vessels may be controlled by the sympathetic innervation that, in other sites, was shown to contain both noradrenaline and ATP as well as neuropeptide Y (Bumstock 1990b). P2Xi, P2X2 and P2X4 receptors differ in their pharmacology to extracellular ATP (Ralevic and Bum stock 1998). The differential expression o f P2X receptor subtypes in vascular smooth muscle o f big or small vessels, in the septae, cortical, or medullary areas may reflect physiological needs to differentially control the vascular tone at these sites. Although it was first thought that only P2Xi receptors were prominent in smooth muscle (Collo et al. 1996), in a more

recent studies P2Xi 2 and 4 receptors were found in different regions o f the vasculature (see Nori et al. 1998).

In the present study P2X$ receptors were found on vascular smooth muscle or fibroblasts and it was suggested that P2Xg receptor occur on endothelial cells. So far, P2X5 was related to cell proliferation or differentiation (Groschel-Stewart et al. 1999a)and P2Xg were shown to be expressed largely in sensory neurons (Lewis et al. 1995).

P2X receptors on epithelial cells,

Subcapsular and perivascular epithelial cells secrete chemoattractants recruiting lymphocyte precursors from the blood for intrathymic T-cell development (Kendall

1991). In the present experiments it was shown that such reticular-shaped cells express P2X2 receptors. P2X2-positive epithelial cells were present in cells along the septae.

P2X^ and P2Xy receptors were also found on septal epithelial cells but were seen less frequently than P2X2-positive cells.

The majority o f nerves (sympathetic, parasympathetic and sensory) invade the thymus through the septae travelling along with the vessels (Kendall 1991). Noradrenergic fibres course into the thymus at these sites, branching from the septae into the cortex (Felten and Felten 1988). Nerve fibres can make contacts with reticular shaped cells (like epithelial cells) as demonstrated on the light microscopical and ultrastructural level (Novotny et al. 1990), offering the possibility o f a noradrenergic and purinergic stimulation o f subcapsular, perivascular and septal epithelial cells.

Another area o f extensive noradrenergic innervation and with broad vascular branching exists at the cortico-medullary junction (Felten and Felten 1988). Tyrosine

hydroxylase-positive and noradrenergic fibres were demonstrated to make close contacts to murine macrophages and mast cells (Williams et al. 1981;Weihe et al. 1991), whereas medullary epithelial cells seem to be innervated mainly by sensory nerves (Novotny et al. 1990). However, in cell culture studies p-adrenoceptors could be detected on perivascular, septal and medullary epithelial cells, indicating a role for sympathetic innervation o f medullary epithelial cells (Kurz et al. 1997). In the present study P2X2,3

and 6 receptors were shown on medullary epithelial cells. These cells may be stimulated

via sympathetic innervation co-storing noradrenaline and ATP or by other cells o f the thymic microenvironment, which have yet to be shown to release A l ’P.

Physiologically medullary epithelial cells are important for the synthesis o f neurophysin, oxytocin, arginine vasopressin (AVP), somatostatin, interleukin 1 and 4, colony stimulating factor for macrophages and thymic hormones (Kendall 1991). A VP, somatostatin, cytokines and thymic hormones were shown to effect thym ocyte proliferation in several ways and AVP and oxytocin potently act on blood vessels.

The P2X3 receptors on medullary epithelial cells may form dimers with P2X2

receptors. Dimérisation o f P2X2 and P2X3 receptors was claimed previously in sensory

neurons (Lewis et al. 1995). No clear evidence could be found for P2X3 receptors on

nerve fibres.

The function o f Hassal’s corpuscles, which are formed from specialised epithelial cells in the medulla, where P2X^ staining was detected, is not elucidated yet. However, secretion o f thymic hormones and opioids was associated with them (Ritter and Crispe 1992).

occur at three sites that play key roles in the system o f lymphocyte travel, maturation and sorting. Abundance o f F l Y j receptors was shown in developing thymocytes o f the cortex (whereas P2Y4 was found to be absent from the thymus). Combined in situ

hybridisation and immunohistochemistry showed colocalisation o f the several purinoceptor classes on thymocytes.

CHAPTER VI.

An ATP-gated cation channel (P2X6 receptor) is linked to the