Role of impaired endothelial cell Ca(2+) signaling in uteroplacental vascular dysfunction during diabetic rat pregnancy

Abstract

Diabetes mellitus in pregnancy is associated with impaired endothelium-mediated dilatation of maternal arteries, although the underlying cellular mechanisms remain unknown. In this study, we hypothesized that diabetes during rat gestation attenuates agonist-induced uterine vasodilation through reduced endothelial cell (EC) Ca(2+) elevations and impaired smooth muscle cell (SMC) hyperpolarization and SMC intracellular Ca(2+) concentration ([Ca(2+)]i) responses. Diabetes was induced by an injection of streptozotocin to second-day pregnant rats and confirmed by the development of maternal hyperglycemia. Control rats were injected with a citrate buffer. Fura-2-based measurements of SMC [Ca(2+)]i or microelectrode recordings of SMC membrane potential were performed concurrently with dilator responses to ACh in uteroplacental arteries from control and diabetic pregnant rats. Basal levels of EC [Ca(2+)]i and ACh-induced EC [Ca(2+)]i elevations in pressurized vessels and small EC sheets were studied as well. Diabetes reduced ACh-induced vasodilation due to a markedly impaired EDHF-mediated response. Diminished vasodilation to ACh was associated with attenuated SMC hyperpolarization and [Ca(2+)]i responses. Basal levels of EC [Ca(2+)]i and ACh-induced EC [Ca(2+)]i elevations were significantly reduced by diabetes. In conclusion, these data demonstrate that reduced endothelium-mediated hyperpolarization contributes to attenuated uteroplacental vasodilation and SMC [Ca(2+)]i responses to ACh in diabetic pregnancy. Impaired endothelial Ca(2+) signaling is in part responsible for endothelial dysfunction in the uterine resistance vasculature of diabetic rats. Pharmacological improvement of EC Ca(2+) handling may provide an important strategy for the restoration of endothelial function and enhancement of maternal blood flow in human pregnancies complicated by diabetes.

Fig. 1.

Smooth muscle cell (SMC) intracellular Ca²⁺ concentration ([Ca²⁺]_i) and dilator responses of uteroplacental radial arteries to ACh are impaired in diabetic pregnancy. A and B: representative changes in SMC [Ca²⁺]_i and diameter of arteries from control (A) and diabetic (B) rats induced by cumulative application of ACh. A: application of phenylephrine (PE) resulted in an elevation of SMC [Ca²⁺]_i from 66 to 332 nM and in a reduction of arterial diameter from 164 to 84 μm (control). Administration of ACh reduced the SMC [Ca²⁺]_i response and dilated the artery in a concentration-dependent manner. B: application of PE increased SMC [Ca²⁺]_i from 101 to 401 nM and reduced the diameter of the diabetic artery from 174 to 75 μm. Dotted lines show the diameters of maximally dilated control (168 μm) and diabetic (175 μm) arteries in response to 10 μM diltiazem and 100 μM papaverine. Solid horizontal lines depict the time of exposure of arteries to PE and ACh. “T” and “S” indicate transient and sustained decreases in SMC [Ca²⁺]_i responses and vasodilation, respectively. T is the maximal change in the ACh response calculated during 15–20 s at the end of first min of ACh application. S was calculated during the last 15–20 s of the 3-min application of each concentration of ACh. ACh-induced vasodilation was expressed as the percentage of maximal dilatation induced by papaverine and diltiazem (D_max).

Fig. 2.

Decreased vasodilator responsiveness and sensitivity to ACh in uteroplacental arteries of diabetic pregnant rats. A and B: summary graphs showing the degree of T and S vasodilation as a function of ACh concentrations in arteries from control and diabetic rats. ACh-induced vasodilation is expressed as D_max. *Significantly different compared with the respective control group at P < 0.05 (by two-way repeated-measures ANOVA). C and D: bar graphs showing the significant increase in the concentration of ACh required for half-maximal T and S dilatation (EC₅₀) of uterine arteries of diabetic rats [−6.69 ± 0.05 and −6.67 ± 0.07 log(ACh); in M] compared with control rats [−7.03 ± 0.12 and −7.05 ± 0.12 log(ACh), in M]. *Significantly different compared with the respective control group at P < 0.05 (by unpaired Student's t-test). Numbers in parentheses indicate the numbers of arteries tested.

Fig. 3.

Diabetes in rat pregnancy markedly impairs EDHF-mediated uteroplacental vasodilation to ACh. A and B: summary graphs showing the degree of T and S EDHF-mediated vasodilation as a function of ACh concentrations in arteries from control and diabetic rats pretreated with N-nitro-l-arginine (l-NNA; 200 μM) and indomethacin (10 μM). ACh-induced vasodilation is expressed as D_max. *Significantly different compared with the respective control group at P < 0.05 (by two-way repeated-measures ANOVA). C and D: bar graphs showing the marked increase in ACh EC₅₀ concentrations for T and S vasodilation in uterine arteries of diabetic [−6.29 ± 0.15 and −6.42 ± 0.21 log(ACh), in M] versus control [−6.89 ± 0.09 and −6.99 ± 0.13 log(ACh), in M] rats. *Significantly different compared with the respective control group at P < 0.05 (by unpaired Student's t-test). Numbers in parentheses indicate the numbers of arteries tested.

Fig. 4.

Diabetic pregnancy is associated with reduced SMC [Ca²⁺]_i responses to ACh. A and B: graphs showing diabetes-induced effects on SMC [Ca²⁺]_i responses to ACh in intact (A) and l-NNA- and indomethacin-treated (B) uteroplacental arteries. *Significantly different compared with the respective control group at P < 0.05 (by two-way repeated-measures ANOVA).

Fig. 5.

Impairment of SMC hyperpolarization and associated dilation of uteroplacental arteries to ACh in rat diabetic pregnancy. A: original tracings showing membrane potential with oscillatory activity and diameter changes recorded from a pressurized artery of a control rat before and on minutes 8–9 of application of ACh. B: original tracings of membrane potential and lumen diameter changes before and on minutes 6–7 of ACh administration in an artery from a diabetic pregnant rat. Arteries were preconstricted with PE before ACh was tested. Membrane potential and arterial diameter axes are scaled to show actual values for tracings in A and B. C: bar graphs showing resting membrane potential values, PE-induced depolarization, and hyperpolarizing effects of 3 × 10⁻⁷M and 10⁻⁶M ACh in arteries of control and diabetic rats. D: bar graphs showing vasodilator responses of arteries from control and diabetic rats associated with the ACh-induced hyperpolarization shown in C. Vasodilation is expressed as D_max. *Significantly different compared with the respective control group at P < 0.05 (by two-way repeated-measures ANOVA).

Fig. 6.

Attenuation of ACh-induced endothelial cell (EC) [Ca²⁺]_i elevations in pressurized uteroplacental arteries of diabetic rats. A and B: representative changes in EC [Ca²⁺]_i in response to increasing concentrations of ACh in arteries from a control rat (A) and a diabetic rat (B). Mean values for T and S responses were calculated by averaging all data points over 5–10 s of the peak response (T) and over last 15–20 s of the sustained response (S) to each concentration of ACh. C and D: bar graphs showing diabetes-induced impairment of T and S EC [Ca²⁺]_i responses to ACh. Incremental changes in EC [Ca²⁺]_i were calculated after the subtraction of basal [Ca²⁺]_i levels measured before the administration of ACh. *Significantly different compared with the respective control group at P < 0.05 (by two-way repeated-measures ANOVA).

Fig. 7.

Experimental diabetes in pregnancy impairs ACh-induced EC [Ca²⁺]_i responses in uteroplacental sheets of ECs. A and B: original recordings of EC [Ca²⁺]_i elevations in response to increasing concentrations of ACh in endothelial sheets dissociated from arteries of a control rat (A) and a diabetic rat (B). Mean values for T and S responses were calculated by averaging all data points over 5–10 s of the peak response (T) and over last 15–20 s of the sustained response (S) to each concentration of ACh. C and D: bar graphs showing diabetes-induced impairment of T and S EC [Ca²⁺]_i responses to ACh. Incremental changes in EC [Ca²⁺]_i were calculated after the subtraction of basal [Ca²⁺]_i levels measured before the administration of ACh. *Significantly different compared with the respective control group at P < 0.05 (by two-way repeated-measures ANOVA).