Identificazione dei recettori specifici per S1P nel muscolo soleo di ratto. Ruolo dei derivati della sfingomielina sul trofismo del muscolo scheletrico.

S1P is a bioactive metabolite of Sphingomyelin implicated in many biological processes, including growth and proliferation. This molecule can function intracellularly, as a second messenger, or extracellularly, activating S1P receptors present on the cell surface. Skeletal muscle express all the enzymes that compose the Sphingomyelin pathway, that create sphingolipids as demonstrated by the high levels in the T tubular system of Sphyngomyielin and Sphingosine (Sph, precursor of S1P) and by a great activity of sphingomyelinase, that catalyses the first step in the pathway. The sphingolipid derivates are also present at high concentration on blood, and thus skeletal muscle is steadily controlled by these factors. The first part of this research aimed to study the expression and the localization of S1P receptors in rat skeletal muscle. RT-PCR and Western Blot analyses demonstrated the expression of S1P1 and S1P3 receptors in adult soleus muscle. Immunofluorescence revealed that both S1P1 and S1P3 receptors are localized at the cell membrane of muscle fibers and in the T-tubule membranes, but only S1P1 receptor is present at higher expression at the neuromuscular junction. Both the receptors have been found also to decorate the nuclear membrane and to be expressed in the satellite cells. As the presence of S1P1 and S1P3 on skeletal muscle seemed to suggest a possible physiological action of S1P, thus the second part of the research aimed to investigate the possibility that S1P acts as trophic factor. It was studied the action of S1P and Sph by means of the model of atrophy induced by the denervation and the model of in vivo regeneration. During soleus muscle denervation, S1P was continuously delivered through a mini osmotic pump. S1P and Sph, significantly attenuated the progress of denervation-induced muscle atrophy. In other experiments the trophic effect of Sph was prevented by N, N-dimethylsphingosine, an inhibitor of Sph kinase, the enzyme that phosphorylates Sph to S1P, implying that Sph acts by previous transformation in S1P. Neutralization of circulating S1P by a specific antibody, worsen the atrophy, corroborating the trophic effect of S1P during denervation. It has also been demonstrated that S1P and Sph incremented the Myogenin and MyoD expression, and this transcriptional factors, as supposed by others, contrasted the atrophy progression. S1P and Sph also attenuated the slow-to-fast MyHC transformation due to inactivity. Denervation of rat soleus muscle, analyzed 7 and 14 days after motor nerve cut, produced the down regulation of S1P1 and S1P3 receptors, while treatment with S1P and Sph had no effect on their expression. The presence of S1P1 and S1P3 on satellite cells, probably implies function of S1P during regeneration. First, it was studied the time course expression of both receptors during regeneration of rat soleus muscle, induced by bupivacaine. During the first week of regeneration S1P1 expression, initially low, gradually increased, while S1P3, initially high, gradually decreased. These data suggest that S1P1 and S1P3 have distinct roles during regeneration. The direct injection into the regenerating muscle of S1P and Sph determinated an increase in the growth rate of regenerating fibers. In fact, in the presence of S1P and Sph, the mean cross sectional area of the 3-days regenerating fibers was significantly higher compared to the controlateral not injected regenerating muscle. The treatments also increased Akt phosphorylation level, suggesting an augment in the protein synthesis. In conclusion, the results indicate that S1P plays a significant role in the trophism and development of muscle fiber.