Contraction induced secretion of VEGF from 3 skeletal muscle cells is mediated by adenosine 4

25 The role of adenosine and contraction for secretion of VEGF in skeletal muscle was 26 investigated in human subjects and rat primary skeletal muscle cells. Microdialysis probes 27 were inserted into the thigh muscle of seven male subjects and dialysate was collected at 28 rest, during infusion of adenosine and during knee extensor exercise. The dialysate was 29 analyzed for content of VEGF protein and adenosine. The mechanism of VEGF secretion 30 from muscle cells in culture was examined in resting and electro stimulated cells, and in 31 response to the adenosine analogue NECA, and the adenosine A2A receptor specific 32 analog CGS21680. Adenosine receptors A1, A2A and A2B were blocked with DPCPX, 33 ZM241385 and enprofylline, respectively. cAMP dependent protein kinase A (PKA) and 34 Mitogen activated protein kinase (MAPK) were inhibited by H-89 and PD 98509, 35 respectively. The human experiment showed that adenosine infusion enhanced (P<0.05) 36 the interstitial concentration of VEGF protein ~ 4-fold above baseline. Exercise increased 37 (P<0.05) the interstitial VEGF concentration ~6-fold above rest in parallel with a ~3-fold 38 increase in adenosine concentration. In accordance, in cultured muscle cells, NECA and 39 contraction caused secretion of VEGF (p<0.05). The contraction induced secretion of 40 VEGF was abolished by the A2B antagonist enprofylline and by inhibition of PKA or MAPK. 41 The results demonstrate that adenosine causes secretion of VEGF from human skeletal 42 muscle cells and that the contraction induced secretion of VEGF protein is partially 43 mediated via adenosine acting on A2B adenosine receptors. Moreover, the contraction 44 induced secretion of VEGF protein from muscle is dependent on both PKA and MAPK 45 activation, but only the MAPK pathway appears to be adenosine dependent, revealing 46 involvement of additional pathways in VEGF secretion. 47