An indirect influence of phenylephrine on the release of endothelium-derived vasodilators in rat small mesenteric artery

Abstract

1. The possibility that stimulation of smooth muscle alpha(1)-adrenoceptors modulates contraction via the endothelium was examined in rat small mesenteric arteries. 2. N(omega)-nitro-L-arginine methyl ester, (L-NAME, 100 microM to inhibit NO synthase) increased contraction to single concentrations of phenylephrine (1 - 3 microM) by approximately 2 fold (from a control level of 14.2+/-3.0 to 34. 1+/-4.2% of the maximum contraction of the artery, n=20). The action of L-NAME was abolished by disrupting the endothelium. 3. The subsequent addition of apamin (to inhibit small conductance Ca(2+)-activated K(+) channels, 50 nM) further augmented phenylephrine contractions, in an endothelium-dependent manner, to more than 3 fold above control (50.4+/-5.3% of the maximum contraction, n=11). 4.Charybdotoxin (non-selective inhibitor of large conductance Ca(2+)-activated K(+) channels, BK(Ca), 50 nM) plus L-NAME augmented the level of phenylephrine contraction to 4 - 5-fold above control (64.1+/-3.1%, n=5), but this effect was independent of the endothelium. The potentiation of contraction by charybdotoxin could be mimicked with the selective BK(Ca) inhibitor, iberiotoxin,. 5. Apamin together with L-NAME and charybdotoxin further significantly increased the phenylephrine contraction by 5 - 6-fold, to 79.9+/-3.5% of the maximum contraction of the artery (n=13). 6. Phenylephrine failed directly to increase the intracellular Ca(2+) concentration in endothelial cells freshly isolated from the small mesenteric artery. 7. Stimulation of smooth muscle alpha(1)-adrenoceptors in the mesenteric artery induces contraction that is markedly suppressed by the endothelium. The attenuation of contraction appears to reflect both the release of NO from the endothelium and the efflux of K(+) from both endothelial and smooth muscle cells. This suggests that the release of NO and endothelium-derived hyperpolarizing factor can be evoked indirectly by agents which act only on the smooth muscle cells.

Figure 1

Typical record demonstrating the augmentation of phenylephrine (3 μM) contraction by a blocker of NO synthesis and inhibitors of Ca²⁺ activated K⁺ channels in wire mounted rat mesenteric arteries with intact endothelium. Addition of L-NAME, followed by charybdotoxin (ChTX) and apamin each augmented the level of contraction. Note the oscillation of contraction under control conditions, but not after treatment with toxins.

Figure 2

Time course of contraction evoked by phenylephrine in arteries with intact endothelium. The level of phenylephrine contraction did not vary after repeated exposure under control conditions (n=4, left). Addition of L-NAME (100 μM, n=20) alone or in combination with apamin (50 nM, n=11) and/or charybdotoxin (ChTX, 50 nM, n=5 alone, n=13 with apamin) augmented the level of contraction compared to control conditions (n=20). For each treatment, values are means±s.e.mean of 5 s averages. Phenylephrine was added for the period indicated by the bar (5 min).

Figure 3

Time course of contraction evoked by phenylephrine in endothelium denuded arteries. The level of phenylephrine contraction did not vary after repeated exposure under control conditions (n=4, left). Addition of L-NAME (100 μM) alone (n=10) or in combination with apamin (50 nM, n=4) did not affect the level of contraction, whereas charybdotoxin (ChTX, 50 nM, n=3 both alone and with apamin) did. For each treatment, values are means±s.e.mean of 5 s averages. Phenylephrine was added for the period indicated by the bar (5 min).

Figure 4

Summary of the effect of blockers on contraction evoked by phenylephrine. Original values from data shown in Figures 2 and 3 were averaged over the final 30 s of phenylephrine application. Iberiotxoin (IbTX, 100 nM) caused the same magnitude of blockade as charybdotoxin (ChTX) when added alone (n=3,4 endothelium intact (+E) and denuded (−E), respectively) or in combination with apamin (n=3,5 endothelium intact and denuded, respectively). Mean values (±s.e.mean) were analysed non-parametrically with the Mann-Whitney Test. The asterisks indicate statistically significant differences from treatment with L-NAME, P<0.05; and the cross indicates a statistically significant difference between endothelium intact and denuded arteries, P<0.05. For each treatment, the maximum contraction for the arteries are given in Table 1.

Figure 5

Typical record demonstrating the change in fluo-3 fluorescence intensity in freshly isolated rat mesenteric artery endothelial cells. Phenylephrine (PE, 10 μM bolus application) was unable to cause a change in fluorescence intensity, whereas acetylcholine (ACh, 10 μM bolus application) caused a rapid rise indicating an increase in intracellular Ca²⁺ concentration.

Figure 6

Time course of changes in fluo-3 fluorescence intensity in freshly isolated endothelial cells. Bolus doses of phenylephrine (PE, 10 μM) and acetylcholine (ACh, 10 μM) were added to each cell. Values are means±s.e.mean of 20 cells from four animals.