Alterations in EDHF-mediated hyperpolarization and relaxation in mesenteric arteries of female rats in long-term deficiency of oestrogen and during oestrus cycle

Abstract

This study was undertaken to determine whether endothelium-dependent relaxations are altered in mesenteric arteries from young female rats during oestrus cycle and after castration. The contractile response to phenylephrine (Phe) was significantly enhanced in arteries from rats subjected to ovariectomy than in those from sham-operated (control) rats. Treatment of ovariectomized rats with 17beta-oestradiol returned the Phe response to the control level. Arteries from rats at the diestrus stage also exhibited greater contraction in response to Phe. In the presence of 100 microM N(G)-nitro-L-arginine (L-NOARG), the enhancement of the Phe contractile response associated with oestrogen deficiency was not observed. Endothelium-dependent relaxations elicited by acetylcholine (ACh) in arteries precontracted with Phe were significantly reduced in ovariectomized and diestrus rats regardless of whether endothelium-derived nitric oxide (NO) was blocked with L-NOARG. Treatment with 17beta-oestradiol prevented the reduced vascular relaxant response to ACh in ovariectomized rats. The reduction in the ACh responses observed in ovariectomized and diestrus rats was eliminated when 500 nM apamin and 100 nM charybdotoxin were present. ACh-induced endothelium-dependent hyperpolarizations were depressed in arteries from ovariectomized and diestrus rats. The hyperpolarizing response to ACh was significantly improved when ovariectomized rats were treated with 17beta-oestradiol. The resting membrane potentials and pinacidil-induced hyperpolarizations were unaffected by ovariectomy or the diestrus stage. These results suggest that oestrogen-deficient states of both short and long duration reduce the basal release of NO from the endothelium and specifically attenuate endothelium-dependent hyperpolarization and relaxation transduced by endothelium-derived hyperpolarizing factor.

Figure 1

Influences of ovariectomy and oestrus cycle on Phe-induced contraction in rat mesenteric arteries. (a) Concentration-response curves for Phe-induced contraction in mesenteric arteries from sham-operated and ovariectomized rats. Phe was added to the bath cumulatively. (b) Effects of treatment with 100 μM L-NOARG on contractions induced by 1 and 10 μM Phe in mesenteric arteries from sham-operated, ovariectomized, ovariectomized and 17β-oestradiol-treated, and diestrus rats. The results are shown as means±s.e.mean of 6–10 experiments. ^*P<0.05 and ^**P<0.01 vs the corresponding value obtained in the control (sham-operated) group.

Figure 2

Influences of ovariectomy and oestrus cycle on endothelium-dependent relaxation induced by ACh in rat mesenteric arteries. (a) Typical traces illustrating the relaxant responses to ACh during 10 μM Phe-induced contraction in mesenteric arteries from sham-operated and ovariectomized rats. (b) Concentration-response curves for ACh-induced relaxation in mesenteric arteries from rats that were sham-operated, ovariectomized, and ovariectomized and 17β-oestradiol-treated. (c) Concentration-response curves for ACh-induced relaxation in mesenteric arteries from oestrus and diestrus rats. ACh was added to the bath cumulatively. The points are means of 6–8 experiments; vertical lines show s.e.mean. Responses are expressed as per cent relaxation of Phe-induced contraction.

Figure 3

Influences of ovariectomy and oestrus cycle on endothelial NO-mediated relaxation induced by ACh in rat mesenteric arteries. (a) Typical traces illustrating the relaxant responses to ACh during 10 μM Phe-induced contraction in control (sham-operated) rat mesenteric arteries in the presence of 10 μM indomethacin, 500 nM apamin and 100 nM ChTX. Right panel shows that the remaining relaxation to ACh was abolished by further treatment with 100 μM L-NOARG. Indomethacin, apamin, ChTX and L-NOARG were added to the bath 15–20 min before application of Phe. (b) Concentration-response curves for ACh-induced relaxation in mesenteric arteries from rats that were sham-operated, ovariectomized, and ovariectomized and 17β-oestradiol-treated. (c) Concentration-response curves for ACh-induced relaxation in mesenteric arteries from oestrus and diestrus rats. All curves were constructed in the presence of indomethacin, apamin and ChTX. The points are means of 6–11 experiments; the vertical lines show s.e.mean. Responses are expressed as per cent relaxation of Phe-induced contraction. Statistically significant differences were not found between the concentration-response curves of the experimental groups.

Figure 4

Influences of ovariectomy and oestrus cycle on EDHF-mediated relaxation induced by ACh in rat mesenteric arteries. (a) Typical traces illustrating the relaxant responses to ACh during 300 nM Phe-induced contraction in control (sham-operated) rat mesenteric arteries in the presence of 10 μM indomethacin and 100 μM L-NOARG. Right panel shows that the remaining relaxation to ACh was abolished by further treatment with 500 nM apamin and 100 nM ChTX. Indomethacin, L-NOARG, apamin and ChTX were added to the bath 15–20 min before application of Phe. (b) Concentration-response curves for ACh-induced relaxation in mesenteric arteries from rats that were sham-operated, ovariectomized, and ovariectomized and 17β-oesradiol-treated. (c) Concentration-response curves for ACh-induced relaxation in mesenteric arteries from oestrus and diestrus rats. All curves were constructed in the presence of indomethacin and L-NOARG. The points are means of 6–12 experiments; vertical lines show s.e.mean. Responses are expressed as per cent relaxation of Phe-induced contraction.

Figure 5

Effects of indomethacin, L-NOARG and apamin plus ChTX on membrane hyperpolarization induced by ACh in control (sham-operated) rat mesenteric arteries. Actual recordings of the hyperpolarizing responses to 1 μM ACh before (left panels) and after (right panels) treatment with 10 μM indomethacin (a), 100 μM L-NOARG (b) or 500 nM apamin plus 100 nM ChTX (c) are shown.

Figure 6

Influences of ovariectomy and oestrus cycle on endothelium-dependent hyperpolarization induced by ACh in rat mesenteric arteries. Actual recordings of the hyperpolarizing responses to 1 μM ACh in mesenteric arteries from sham-operated (a), ovariectomized (b), ovariectomized and 17β-oestradiol-treated (c), and diestrus (d) rats are shown. (e) Concentration-response curves for ACh-induced hyperpolarization in mesenteric arteries from rats that were sham-operated, ovariectomized, and ovariectomized and 17β-oestradiol-treated. (f) Concentration-response curves for ACh-induced hyperpolarization in mesenteric arteries from oestrus and diestrus rats. The points are means of 11–14 experiments; vertical lines show s.e.mean.

Figure 7

Influences of ovariectomy and oestrus cycle on endothelium-independent hyperpolarization induced by pinacidil in rat mesenteric arteries. Actual recordings of the hyperpolarizing responses to 10 μM pinacidil in mesenteric arteries from sham-operated (a), ovariectomized (b), ovariectomized and 17β-oestradiol-treated (c), and diestrus (d) rats are shown.