The purinoceptor agonist ATP has been shown to increase [Ca2+]i levels in a sweat gland cell line derived from humans (Wilson et al. 1994) and in cells derived from primary sweat gland cultures (Bovell et al. 2000). ATP can also induce sweating in isolated glands (Sato et al. 1991); however, the mechanism and physiological relevance is not entirely understood. These studies suggest the presence of a P2Y receptor, which we have recently localised in normal human glands (Lindsay et al. 2002). Extracellular nucleotides have also been proposed as a means of alleviating some of the symptoms of the autosomal recessive illness, cystic fibrosis (CF), although the localisation of such a purinoceptor in CF sweat glands has never been investigated. Therefore, immunohistochemistry was employed to investigate the localisation of the P2Y2 receptor in human CF eccrine sweat glands and compare this with the expression in normal glands.
Skin biopsies were obtained with informed consent and local medical ethical committee approval from patients suffering from CF (n = 4) and from patients with no apparent skin disease (n = 11). Samples were fixed, processed and sectioned using standard techniques. Immunohistochemical staining was performed using rabbit antibodies raised against the P2Y2 receptor (Alomone Labs), employing the avidin-biotin complex (ABC) procedure (Vector Labs). Sections were haematoxylin counterstained, dehydrated, cleared, mounted and viewed using light microscopy.
The eccrine reabsorptive duct of CF glands contained dark P2Y2-like immunoreactivity localised to the apical membrane, which was similar to the staining found in normal glands. However, CF glands exhibited staining on the basolateral membranes, which was not seen in normal glands. The secretory coil of CF glands exhibited no staining using both citrate buffer antigen retrieval and no antigen retrieval methods. However, normal glands showed P2Y2-like immunoreactivity localised to the myoepithelial cells of the secretory coil, with some staining seen in the coil itself. Preabsorption of the antibody with the appropriate control peptide abolished all specific staining.
The presence of apical P2Y2-like immunoreactivity in the eccrine sweat gland duct of both normal and CF glands suggests that apical regulation of absorption is important, which has been shown to be the case in colonic epithelia (Cliff & Frizzell, 1990). The reabsorptive duct of CF sweat glands elicited a wider distribution of the P2Y2 receptor compared with normal glands. Those receptors present on the duct may be involved in salt reabsorbtion, as CF glands cannot reabsorb any salt through CFTR. Although the myoepithelial cells of the sweat gland are not regarded as cells involved in either secretion or reabsorption of sweat, the presence of P2Y2 receptor in normal glands would suggest that they do play a role. The absence of any staining in the secretory coil of CF glands may be a result of the condition.
S.L.L. would like to thank GCU studentship.
All procedures accord with current local guidelines.