Extracellular nucleotides interact with purinergic receptors, which regulate ion transport in a variety of epithelia. With the use of two different human epithelial carcinoma cell lines (HCT8 and Caco-2), we have shown by RT-PCR that the cells express mRNA for P2X₁, P2X₃, P2X₄, P2X₅, P2X₆, P2X₇, P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, and P2Y₁₂receptors. Protein expression for P2Y₁and P2Y₂receptors was also demonstrated immunohistochemically, and P2X receptor subtype protein was present in the following decreasing order: P2X₄> P2X₇> P2X₁> P2X₃> P2X₆> P2X₅>> P2X₂. The functional presence of P2X₇, P2Y₁, P2Y₂, and P2Y₄receptors was shown based on the effect of extracellular nucleotides on apoptosis or cell proliferation, and measurement of nucleotide-dependent calcium fluxes using a fluorometric imaging plate reader in the presence of different selective agonists and antagonists. ATP, at high concentrations, induced apoptosis through ligation of P2X₇and P2Y₁receptors; conversely, ATP, at lower concentrations, and UTP stimulated proliferation, probably acting via P2Y₂receptors. We therefore propose that stimulation or dysfunction of purinergic receptors may contribute at least partially to modulation of epithelial carcinoma cell proliferation and apoptosis.

Muscle atrophy caused by disuse is accompanied by adverse physiological and functional consequences. Satellite cells are the primary source of skeletal muscle regeneration. Satellite cell dysfunction, as a result of impaired proliferative potential and/or increased apoptosis, is thought to be one of the causes contributing to the decreased muscle regeneration capacity in atrophy. We have previously shown that electrical stimulation improved satellite cell dysfunction. Here we test whether electrical stimulation can also enhance satellite cell proliferative potential as well as suppress apoptotic cell death in disuse-induced muscle atrophy. Eight-week-old male BALB/c mice were subjected to a 14-day hindlimb unloading procedure. During that period, one limb (HU-ES) received electrical stimulation (frequency: 20 Hz; duration: 3 h, twice daily) while the contralateral limb served as control (HU). Immunohistochemistry and western blotting techniques were used to characterize specific proteins in cell proliferation and apoptosis. The HU-ES soleus muscles showed significant improvement in muscle mass, cross-sectional area, and peak tetanic force relative to the HU limb (p<0.05). The satellite cell proliferative activity as detected within the BrdU⁺/Pax7⁺ population was significantly higher (p<0.05). The apoptotic myonuclei (detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and the apoptotic satellite cells (detected by cleaved Poly [ADP-ribose] polymerase co-labeled with Pax7) were reduced (p<0.05) in the HU-ES limb. Furthermore the apoptosis-inducing factor and cleaved caspase-3 were down-regulated while the anti-apoptotic Bcl-2 protein was up-regulated (p<0.05), in the HU-ES limb. These findings suggest that the electrical stimulation paradigm provides an effective stimulus to rescue the loss of myonuclei and satellite cells in disuse muscle atrophy, thus maintaining a viable satellite cell pool for subsequent muscle regeneration. Optimization of stimulation parameters may enhance the outcome of the intervention.

Extracellular ATP mediates diverse biological effects by activating two families of receptors, the P2X and P2Y receptors. There is growing evidence to show that activation of G protein‐coupled P2Y receptors can produce trophic effects in many cell types. Yet the expression and function of the P2Y receptors in development has rarely been studied and has never been investigated in mammalian development. This study used the reverse transcription‐polymerase chain reaction and immunohistochemistry to demonstrate the abundant and dynamic expression of P2Y receptors in rat development. These receptors were expressed in a wide range of embryonic structures, notably somites, skeletal muscle, the central and peripheral nervous system, the heart, lung, and liver. All the P2Y receptors studied were expressed as early as embryonic day 11, when most embryonic organs were far from being functional and still in the process of being formed. P2Y receptor proteins were strongly expressed in temporary, developmental structures that do not have a correlate in the adult animal, including the somites (P2Y₁, P2Y₂, and P2Y₄) and the floor plate of the neural tube (P2Y₁). P2Y receptors were also dynamically expressed, with receptor mRNA and protein being both up‐ and down‐regulated at different developmental stages. The down‐regulation of the P2Y₁, ₂, and ₄ receptor proteins in skeletal muscle and heart, and the disappearance of the P2Y₄ receptor from the brainstem and ventral white matter of the spinal cord postnatally, demonstrated that many P2Y receptors were likely to be involved in functions specific to embryonic life. Thus, these findings strongly suggest that P2Y receptors play an important role in the development of many tissues, and pioneer further studies into the role of purinergic signalling in development. Developmental Dynamics 228:254–266, 2003.