The calcium ionophore A23187 induces endothelium-dependent contractions in femoral arteries from rats with streptozotocin-induced diabetes

Abstract

Background and purpose: To study the importance of endothelium-derived contracting factors (EDCFs) in arteries of rats with type I diabetes.

Experimental approach: Rat femoral arteries were collected four or twelve weeks after induction of diabetes with streptozotocin. Rings, with or without endothelium, were suspended in organ chambers for isometric tension measurement. COX protein levels were determined by Western blotting.

Key results: Four weeks after the injection of streptozotocin, the endothelium-dependent relaxations (during contractions to phenylephrine) to A23817 were attenuated, but the endothelium-dependent contractions (quiescent preparations) to the ionophore were augmented. Indomethacin and S18886 prevented the endothelium-dependent contractions, while dazoxiben reduced them in rings from streptozotocin-treated rats, suggesting that thromboxane A2, activating TP- receptors, is involved. Twelve weeks after the injection of streptozotocin, the changes in endothelium-dependent relaxations and contractions to A23187 were even more noticeable. The protein expression of COX-1 was increased in femoral arteries of the diabetic rats. Valeryl salicylate and SC560 inhibited the contractions, suggesting that the EDCFs are produced by COX-1. At that time, a combination of S18886 with EP1-blockers was required to abolish the contractions, suggesting that the EDCFs involved act at both TP- and EP-receptors. Rings without endothelium from streptozotocin-treated rats exhibited a reduced maximal contraction to potassium chloride and U46619, combined with hyper-responsiveness to the latter, suggesting that more prolonged diabetes also alters the responsiveness of vascular smooth muscle.

Conclusion and implications: The production of EDCFs is progressively increased in the course of type I diabetes. Eventually, the disease also damages vascular smooth muscle.

Figure 1

Relaxations to A23187 in femoral arteries from control and streptozotocin-treated rats after 4 (a and b) and 12 (c and d) weeks. Data shown are means±s.e.m. (vertical lines); n=7. ^*Statistically significant difference from control rings (P<0.05). ^#Statistically significant difference from rings of control rats under identical conditions (P<0.05). % indicates a statistically significant difference from rings of 4 weeks streptozotocin-treated rats under identical conditions (P<0.05).

Figure 2

Contractions to A23187 in femoral arteries from control and streptozotocin-treated rats in the presence of L-NAME (0.3 mM) after 4 (a and b) and 12 (c and d) weeks. Data shown are means±s.e.m. (vertical lines); n=7. ^*Statistically significant difference from control rings (P<0.05). ^#Statistically significant difference from rings of control rats under identical conditions (P<0.05).

Figure 3

Effects of the inhibitor of thromboxane synthase dazoxiben (10 μM) on the response to A23187, in the presence of L-NAME (0.3 mM), in femoral arteries with endothelium of rats 4 weeks (a) and 12 weeks (b) after injection of streptozotocin. Data shown are means±s.e.m. (vertical lines); n=7. ^*Statistically significant difference from rings under control conditions (P<0.05).

Figure 4

Effects of inhibitors of cyclooxygenase on the response to A23187 in quiescent femoral arteries of rats 12 weeks after the injection of streptozotocin. The experiments were performed in the presence of L-NAME (0.3 mM). Data shown as means±s.e.m. (vertical lines); n=7–8. ^*Statistically significant difference from rings (with endothelium) under control conditions (COX-1 inhibitors: valeryl salicylate (a) and SC560 (b); COX-2 inhibitor: NS-398 (a)).

Figure 5

Effects of antagonists of TP and EP₁ receptors on the response to A23187 in quiescent femoral arteries 12 weeks after the injection of streptozotocin. The experiments were performed in the presence of L-NAME (0.3 mM). Data shown are means±s.e.m. (vertical lines); n=6–9. ^*Statistically significant difference from rings under control conditions (P<0.05).

Figure 6

Response to U46619 in rings of femoral arteries without endothelium from control rats and those 4 (a) and 12 (b) weeks after an injection of streptozotocin. Data shown are means±s.e.m. (vertical lines); n=7. ^*Statistically significant difference from rings of control rats (P<0.05).

Figure 7

The presence of COX-1 (a and b) and COX-2 (c and d) in intact femoral artery from control and streptozotocin-treated rats, 4 weeks and 12 weeks after the injection of streptozotocin. Data presented are a percentage of control and shown as means±s.e.m.; n=6. ^*Statistically significant difference from control rats (P<0.05).