Purinergic Signalling (2008) 4:331–338 it is convenient to think of stem and transit-amplifying cells as discrete populations from fully differentiated keratino- cytes, there might be gradients of cell behaviour, ranging from a cell that has maximum self-renewal and zero dif- ferentiation capacity to one that has completed terminal differentiation and cannot divide again. Transit-amplifying cells would lie in the middle of each gradient [1]. In anagen hair follicles, P2Y2 receptors are expressed on early dif- ferentiated hair keratinocytes that contribute to the forma- tion of the hair shaft. Cells positive for P2Y2 receptors in hair follicles could still have some proliferative capacity given the right stimulus. In anagen hair follicles, P2X5 receptors were found on cells in the IRS, ORS and medulla. P2X5 receptors are expressed in the basal layer of the interfollicular epidermis but are expressed more strongly in the stratum spinosum and variably into the stratum granulosum [12]. The expression of P2X5 receptors in the ORS was similar to that seen in the epidermis, with basal cells labelling less strongly than suprabasal cells. It has been proposed that P2X5 receptors are involved in human epidermal keratinocyte differentia- tion [10, 12]. There is evidence from other tissues regarding the role of P2X5 receptors. In foetal rat skeletal muscle, P2X5 receptors are sequentially expressed during develop- ment [24] and associated with differentiating cells [25]. P2X5 receptors have also been implicated in the regulation of osteoblastic differentiation and proliferation [26]. Invo- lucrin is a marker of keratinocyte differentiation [27], which is found in the epidermis in the upper stratum spinosum and in the stratum granulosum. In the hair follicle, involucrin is expressed in the hair cortex, medulla, IRS and the innermost cells of the lower ORS [28]. Involucrin colo- calised with P2X5 receptor staining in the IRS and in a few cell layers at the edge of the medulla. This suggests a role for P2X5 receptors in the differentiation of cells of the medulla and IRS. There was no staining with antibodies to P2X7 receptors or active caspase-3 in the anagen hair follicle. Further work on the expression of P2X7 receptors in different stages of the hair cycle would be of interest. Periods of hair growth (anagen) are followed by a regression phase (catagen), when the lower part of the hair follicle undergoes programmed cell death [2], and a resting phase (telogen), before onset of a new growth phase. In this study, only hair follicles in the anagen phase were studied, as hair follicles in catagen or telogen phases of the hair cycle were not found in the sections analysed. At any one time, 84% of scalp hairs are in anagen, 2% in catagen and 14% in telogen [29]. The P2X7 receptor is unlike other P2X receptors because it is a bifunctional molecule that, in addition to forming a channel permeable to small cations, can be triggered to form a cytolytic pore permeable to large hydrophilic molecules up to 900 daltons [30]. The opening of this pore results in the 337 increase in intracellular cytosolic free calcium ions and the induction of cell death [31, 32]. P2X7 receptors are also found on dendritic cells, macrophages and microglial cells, where extracellular ATP can trigger apoptosis via these receptors. There is increasing evidence that this process is dependent on the caspase signalling cascade [33, 34]. It would be of interest to see whether P2X7 receptors are involved in the process of programmed cell death that occurs during the catagen phase of the hair cycle. In summary, P2 purinergic receptors are likely to be involved in ORS cell proliferation via P2Y1 receptors found in the bulb and ORS; IRS and medulla differentiation via activation of P2X5 receptors; and early differentiation of cells of the cortex and medulla of the hair shaft via activation of P2Y2 receptors. Expression of P2 purinergic receptors in the anagen hair follicle may have therapeutic implications for treating hair loss. The expression of P2 receptors in different cell lineages of the anagen hair follicle could provide the prospect of engineering hair follicles in those with hair loss or excess growth by using purinergic receptor agonists and antagonists. Lack of growth reflects follicular dynamics and represents the central mechanisms of most common causes of alopecia. In androgenetic alopecia, the hair-shaft diameter is reduced, and there is a progressive decrease in anagen hair percentage, as well as a significant reduction in proliferating cells [35]. The anagen phase is also shorter [36]. This results in the formation of progres- sively thinner and shorter hair. In some men, there is also prolongation of the interval between shedding of the club hair in telogen and the emergence of a replacement hair in the anagen phase, giving rise to an increase in the number of “empty” follicles [37]. Functional work on hair-follicle cycling, cell proliferation and differentiation using puriner- gic receptor agonists and antagonists may lead to new approaches in the treatment of hair loss. Acknowledgements The support of Roche Bioscience, Palo Alto, CA, USA, who provided P2X5 and P2X7 receptor antibodies, is gratefully acknowledged. Aina Greig is the recipient of a Research Fellowship from The Wellcome Trust and The Simpson Surgical Research Fellowship from The Royal College of Surgeons of England. The authors are grateful to Dr. Gillian E. Knight for editorial assistance. References 1. Niemann C, Watt FM (2002) Designer skin: lineage commitment in postnatal epidermis. Trends Cell Biol 12:185–192 2. Cotsarelis G (1997) The hair follicle: dying for attention. Am J Pathol 151:1505–1509 3. Abbracchio MP, Burnstock G (1998) Purinergic signalling: pathophysiological roles. Jpn J Pharmacol 78:113–145 4. Burnstock G (2002) Purinergic signalling and vascular cell proliferation and death. Arterioscler Thromb Vasc Biol 22:364–373 5. Burnstock G (2007) Physiology and pathophysiology of purinergic neurotransmission. Physiol Rev 87:659–797