Intracellular Signalling in Experimental Hypertension

1. The effects of noradrenaline and endothelin-1 on the phosphatidylinositol cycle were investigated in 2 models of experimental hypertension, the spontaneously hypertensive rat (SHR) and the perinephritis hypertensive rabbit, at various stages during the development of hypertension. In both models freshly isolated aorta and cultured aortic smooth muscle cells were examined to ascertain the suitability of cultured aortic smooth muscle cells in this aspect of hypertension research. The coupling of endothelin-1 and noradrenaline to inositol phosphate formation via GTP-binding proteins was also investigated in cultured aortic smooth muscle cells from SHR. 2. In aortic rings from 6, 14, and 40 weeks old SHR basal inositol phosphate formation was unchanged compared to Wistar Kyoto (WKY) rats. Both noradrenaline and endothelin-1 stimulated inositol phosphate formation was unchanged in aortic rings from 6 week SHR. However at 14 and 40 weeks noradrenaline and endothelin-1 stimulated inositol phosphate formation was decreased in aortic rings from SHR compared to WKY. 3. Basal inositol phosphate formation was increased in cultured aortic smooth muscle cells from 6 and 14 week SHR compared to cells from WKY. Both noradrenaline and endothelin-1 stimulated inositol phosphate formation was unchanged in cultured aortic smooth muscle cells from 6 week SHR. In cultured aortic smooth muscle cells from 14 week SHR noradrenaline stimulated inositol phosphate formation was increased while endothelin-1 stimulated inositol phosphate was decreased. 4. In aortic rings from perinephritis hypertensive rabbits 1, 2 and 6 weeks after surgery basal inositol phosphate formation was unchanged compared to uninephrectomized control rabbits. At 1 week after surgery noradrenaline stimulated inositol phosphate formation was unchanged in aortic rings from perinephritis hypertensive rabbits. However, in aortic rings from 2 week perinephritis hypertensive rabbits noradrenaline stimulated inositol phosphate formation was increased but endothelin-1 stimulated inositol phosphate formation was similar to controls. Noradrenaline stimulated inositol phosphate formation was unchanged in aortic rings from 6 week perinephritis hypertensive rabbits. Cultured aortic smooth muscle cells were prepared from the aorta of perinephritis hypertensive rabbits 2 weeks after surgery. Both noradrenaline and endothelin-1 stimulated inositol phosphate formation was unchanged in these cells compared to smooth muscle cells from control rabbits. 5. The effects of pertussis toxin on noradrenaline and endothelin-1 stimulated inositol phosphate formation in cultured aortic smooth muscle cells from 14 week SHR and WKY rats was investigated. Noradrenaline stimulated inositol phosphate formation was unaffected by pertussis toxin preincubation. However, endothelin-1 stimulated inositol phosphate formation was decreased by 50% in both SHR and WKY cells after preincubation with pertussis toxin. Therefore noradrenaline and endothelin-1 appear to be regulated independently at the level of the GTP-binding protein. Confirmation of endothelin-1's ability to activate a pertussis toxin sensitive GTP-binding protein was obtained by measuring endothelin-1's interaction with pertussis toxin dependent ADP-ribosylation in membranes prepared from cultured aortic smooth muscle cells. In the presence of endothelin-1 the pertussis toxin dependent ADP-ribosylation was decreased in both SHR and WKY membranes. 6. The free intracellular calcium concentration in cultured aortic smooth muscle cells from 14 week SHR grown on coverslips was unchanged compared to WKY cells as measured using fura 2.7. Alterations in inositol phosphate formation occur in these 2 models of hypertension. The changes are agonist specific possibly due to independent regulation by GTP-binding proteins and are likely to depend on the system (i. e. fresh tissue or cultured cells), the model and the species studied