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Intracellur signalling pathways and binding of endothelin in vascular smooth muscle cells
Parameter
Maximum Potential (mV) AK efflux (%) ARb efflux (8) APK (%) APRb (55) Relaxation (X)
-19.Qi3.4 233f32 156f13 340 232 97.Oi6.9
Diazoxide 100 FM
Cromakalim 10 pM
Cromakalim 1 pM blax 17.0+2.7 16-20 > 24 16-20 > 24 8.7f1.9
Maximum
t max
Maximum
t marr
- 14.5 + 1.1 337 &43 235 f 18 524 331 97.0f4.0
6.2fO.S 8-12 > 24 8-12 12-16 11.2f3.1
- 10.7 + 3.0 173fi24 1.54%20 233 202 97.9 f 12.5
14.5 f 2.8 ) 24 20-24 > 24 20-24 12.7f0.8
Mean&s-e. mean (n = 3-7); tmX denotes time taken (min) to reach maximum change; % changes are expressed relative to control Values.
J. Longmore was supported by a grant from RhZine Poulenc.
Edwards. G. and Weston, AH. (1989) Br. J. Pharmol. (Proc. Suppl., Manchester meeting), in press.
Quast, U. and Baumlin, Y. (1988) Nauyn-Schmiedeberg’sArch. Phannacol., 238, 319-326.
El
o.tu.09.7
Chabrier,
P-E., Lonchampt,
M-O., Roubert, P., Pinelis, V., Auguet, M., Guillon, Pirotzky, E., Hosford, I). and Bracluet, P.
J-M., V&sat,
I.,
Institut Henri Beaufour, 1, auenue des Tropiques, 9195.2 Les Uiis, France
Endothelin (ET) is a group of 21 aminoacid vasoconstricting peptide. ET-I, the first isoform isolated and characterized from the supematant of cultured porcine aortic endothelial cells, exerts its vas~onst~ctive and pressor action via specific binding sites. In this study, we have examined the binding of ET-1 and its effect on intracellular signalling pathways in vascular smooth muscle cells. Rat aortic smooth muscle cells were used at confluence up to the 6th passage. f125]ET-1 bound to the cells at 37 o C with an apparent single class of high affinity receptors with a maximum binding (Bmax) of 58.6 It 7.5 fmol/106 cells and a dissociation constant (K.d) of 2.6 + 1,6 IO-” M. In competitive experiments, [125]ET-1 was only displaced by structurally related compounds such as ET-1 and ET-2 with a similar inhibition constant (Ki = 5.8 f 0.3 lo-” M); ET-3 showing a biphasic effect (Ki = 4.4 + 0.5 lo-” M and 5.5 f 1.5 lo-* M). Moreover the binding of [125I] ET-I was very tight and almost impossible to dissociate. ET-l did not affect the basal or stimulated production of cyclic GMP or cyclic AMP. It dose-dependently stimulated a rapid and transient increase of 45Ca2+ influx (measured during 105 set) or “Ca’+ -efflux in preloaded cells (18 h) whereas the phosphoinositide IP, was not significantly elevated. In contrast, ET-1 potently stimulated Na+/H+ exchange by measuring the intracellular pH (pHi) with the fluorescent probe (BCECF). ET-1 changed pHi in a biphasic manner, it induced a small acidification (pHi = 6.95 -t 0.04) which was followed by a long and sustained alka?inization (pHi = 7.14 f 0.06, t = 20 min; resting pHi = 7.02 f 0.05). Pretreatment of the cells with amiloride 01 inhibitor of protein kinase C (H7 or staurosporin) inhibited this effect. These results suggest that ET-1 stimulates some intracellular signalling pathways which could be involved in its sustained contractile effect.
Maximum Potential (mV) AK efflux (%) ARb efflux (8) APK (%) APRb (55) Relaxation (X)
-19.Qi3.4 233f32 156f13 340 232 97.Oi6.9
Diazoxide 100 FM
Cromakalim 10 pM
Cromakalim 1 pM blax 17.0+2.7 16-20 > 24 16-20 > 24 8.7f1.9
Maximum
t max
Maximum
t marr
- 14.5 + 1.1 337 &43 235 f 18 524 331 97.0f4.0
6.2fO.S 8-12 > 24 8-12 12-16 11.2f3.1
- 10.7 + 3.0 173fi24 1.54%20 233 202 97.9 f 12.5
14.5 f 2.8 ) 24 20-24 > 24 20-24 12.7f0.8
Mean&s-e. mean (n = 3-7); tmX denotes time taken (min) to reach maximum change; % changes are expressed relative to control Values.
J. Longmore was supported by a grant from RhZine Poulenc.
Edwards. G. and Weston, AH. (1989) Br. J. Pharmol. (Proc. Suppl., Manchester meeting), in press.
Quast, U. and Baumlin, Y. (1988) Nauyn-Schmiedeberg’sArch. Phannacol., 238, 319-326.
El
o.tu.09.7
Chabrier,
P-E., Lonchampt,
M-O., Roubert, P., Pinelis, V., Auguet, M., Guillon, Pirotzky, E., Hosford, I). and Bracluet, P.
J-M., V&sat,
I.,
Institut Henri Beaufour, 1, auenue des Tropiques, 9195.2 Les Uiis, France
Endothelin (ET) is a group of 21 aminoacid vasoconstricting peptide. ET-I, the first isoform isolated and characterized from the supematant of cultured porcine aortic endothelial cells, exerts its vas~onst~ctive and pressor action via specific binding sites. In this study, we have examined the binding of ET-1 and its effect on intracellular signalling pathways in vascular smooth muscle cells. Rat aortic smooth muscle cells were used at confluence up to the 6th passage. f125]ET-1 bound to the cells at 37 o C with an apparent single class of high affinity receptors with a maximum binding (Bmax) of 58.6 It 7.5 fmol/106 cells and a dissociation constant (K.d) of 2.6 + 1,6 IO-” M. In competitive experiments, [125]ET-1 was only displaced by structurally related compounds such as ET-1 and ET-2 with a similar inhibition constant (Ki = 5.8 f 0.3 lo-” M); ET-3 showing a biphasic effect (Ki = 4.4 + 0.5 lo-” M and 5.5 f 1.5 lo-* M). Moreover the binding of [125I] ET-I was very tight and almost impossible to dissociate. ET-l did not affect the basal or stimulated production of cyclic GMP or cyclic AMP. It dose-dependently stimulated a rapid and transient increase of 45Ca2+ influx (measured during 105 set) or “Ca’+ -efflux in preloaded cells (18 h) whereas the phosphoinositide IP, was not significantly elevated. In contrast, ET-1 potently stimulated Na+/H+ exchange by measuring the intracellular pH (pHi) with the fluorescent probe (BCECF). ET-1 changed pHi in a biphasic manner, it induced a small acidification (pHi = 6.95 -t 0.04) which was followed by a long and sustained alka?inization (pHi = 7.14 f 0.06, t = 20 min; resting pHi = 7.02 f 0.05). Pretreatment of the cells with amiloride 01 inhibitor of protein kinase C (H7 or staurosporin) inhibited this effect. These results suggest that ET-1 stimulates some intracellular signalling pathways which could be involved in its sustained contractile effect.