Comparison of signalling mechanisms involved in rat mesenteric microvessel contraction by noradrenaline and sphingosylphosphorylcholine

Abstract

1 We have compared the signalling mechanisms involved in the pertussis toxin-sensitive and -insensitive contraction of rat isolated mesenteric microvessels elicited by sphingosylphosphorylcholine (SPC) and noradrenaline (NA), respectively. 2 The phospholipase D inhibitor butan-1-ol (0.3%), the store-operated Ca(2+) channel inhibitor SK>F 96,365 (10 microM), the tyrosine kinase inhibitor genistein (10 microM), and the src inhibitor PP2 (10 microM) as well as the negative controls (0.3% butan-2-ol and 10 microM diadzein and PP3) had only little effect against either agonist. 3 Inhibitors of phosphatidylinositol-3-kinase (wortmannin and LY 294,002, 10 microM each) or of mitogen-activated protein kinase kinase (PD 98,059 and U 126, 10 microM each) did not consistently attenuate NA- and SPC-induced contraction as compared to their vehicles or negative controls (LY 303,511 or U 124). 4 The phospholipase C inhibitor U 73,122 (10 microM) markedly inhibited the SPC- and NA-induced contraction (70% and 88% inhibition of the response to the highest NA and SPC concentration, respectively), whereas its negative control U 73,343 (10 microM) caused only less than 30% inhibition. 5 The rho-kinase inhibitors Y 27,632 (10 microM) and fasudil (30 microM) caused a rightward-shift of the NA concentration-response curve by 0.7-0.8 log units and reduced the response to 10 microM SPC by 88% and 83%, respectively. 6 These data suggest that SPC and NA, while acting on different receptors coupling to different G-protein classes, elicit contraction of rat mesenteric microvessels by similar signalling pathways including phospholipase C and rho-kinase.

Figure 1

Maximum contraction response to NA and SPC relative to the first NA effect within the same preparation (n=83 vessels each). The linear regression slopes were 1.02±0.04 and 0.61±0.10, respectively, and the r² was 0.87 and 0.31, respectively, yielding P-values of <0.0001 in both cases.

Figure 2

Effect of the phospholipase C inhibitor U 73,122 and its negative control U 73,343 (10 μM) each on NA- and SPC-induced vasoconstriction. Data are means±s.e.mean of eight experiments. P=0.0151 for overall treatment effect of U 73,343 against SPC in a two-way ANOVA, and P<0.0001 for all other overall treatment effects; ^*P<0.05 for individual agonist concentrations vs vehicle in Bonferroni post-tests.

Figure 3

Effects of the phospholipase D inhibitor butan-1-ol and its negative control butan-2-ol (0.3% each) on NA- and SPC-induced vasoconstriction. Data are means±s.e.mean of six experiments. P<0.0001 for overall treatment effects of butan 1-ol-vs butan-2-ol against NA but not SPC effects in a two-way ANOVA; ^*P<0.05 for individual agonist concentrations vs butan-2-ol in Bonferroni post-tests.

Figure 4

Effects of the SK&F 96,365 (10 μM), an inhibitor of store-operated Ca²⁺ channels, on NA- and SPC-induced vasoconstriction. Data are means±s.e.mean of six experiments. P=0.0006 for overall treatment effect of SK&F 96,365 against NA but not SPC effects in a two-way ANOVA.

Figure 5

Effects of the tyrosine kinase inhibitor genistein and the src kinase inhibitor PP2 and their negative controls daidzein and PP3, respectively (10 μM each), on NA- and SPC-induced vasoconstriction. Data are means±s.e.mean of 5–6 experiments. The following P-values for overall treatment effect were obtained for NA (P<0.0001 genistein vs vehicle, P=0.0017 genistein vs daidzein) and SPC (P=0.0248 genistein vs vehicle, P=0.0030 PP2 vs vehicle) in a two-way ANOVA, ^*P<0.05 for individual agonist concentrations vs vehicle in Bonferroni post-tests.

Figure 6

Effects of phosphatidylinositol-3-kinase inhibitors wortmannin and LY 294,002 and the negative control LY 303,511 (10 μM) each on NA- and SPC-induced vasoconstriction. Data are means±s.e.mean of 5–7 experiments. The following P-values for overall treatment effect were obtained for NA (P<0.0001 for LY 294,002 and LY 303,511 vs vehicle and LY 294,002 vs LY 303,511) and SPC (P=0.0018 for wortmannin vs vehicle and P=0.0060 for LY 303,511 vs vehicle) in a two-way ANOVA; ^*P<0.05 for individual agonist concentrations in the presence of LY 294,002 vs LY 303,511 and presence of LY 303,511 vehicle in Bonferroni post-tests.

Figure 7

Effects of the ERK-type mitogen-activated protein kinase inhibitor PD 98,059, U 126 and its negative control U 124 (10 μM each) and their vehicle on NA- and SPC-induced vasoconstriction. Data are means±s.e.mean of 6–11 experiments. In a two-way ANOVA the overall treatment effects were significant for inhibition of NA and SPC by PD 98,059 vs vehicle (P<0.0001) and for inhibition of SPC by U 126 vs vehicle (P=0.0042; ^*P<0.05 for individual agonist concentrations vs vehicle in Bonferroni post-tests.

Figure 8

Effects of the rho-kinase inhibitors Y 27,632 (10 μM) and fasudil (30 μM) and their vehicles on NA- and SPC-induced vasoconstriction. Data are means±s.e.mean of 6–8 experiments. P<0.0001 for overall treatment effect of both Y 27,632 and fasudil vs vehicle in a two-way ANOVA; ^*P<0.05 for individual agonist concentrations vs vehicle in Bonferroni post-tests.