[Mechanism of 17β-estrogen on intracellular free calcium regulation in smooth muscle cells at the endometrial-myometrial interface in uteri with adenomyosis].

OBJECTIVE

To investigate the regulation mechanism of estrogen on the free calcium of smooth muscle cells at the endometrial-myometrial interface (EMI) in uteri with adenomyosis.

METHODS

From September 2011 to November 2012, 59 uterine myometrial specimens were obtained from 59 cases underwent hysterectomy, including 28 adenomyosis patients as adenomyosis (ADS) group and 31 patients with cervical intraepithelial neoplasia III as control group. EMI smooth muscle cells were cultured and loaded with calcium ion fluorescent probe fluo-4/AM. After treated with trisphosphate (IP3) receptor antagonist, blocker of sarcoplasmic reticulum calcium-adenosine triphosphate (ATP), depleted agent of the ryanodine receptor-operated Ca(2+), inhibitor of L-type calcium channel, inhibitor of Na(+)-Ca(2+) exchanger, the labeled cells were stimulated with estrogen. The changes of intracellular Ca(2+) fluorescence intensity were detected by laser scanning microscopy. The changes of intracellular Ca(2+) concentration was indicated by △FCa(2+).

RESULTS

(1) Under normal calcium conditions, after the stimulation of estrogen, intracellular Ca(2+) fluorescence intensity in ADS group and control group both increased than those without estrogen. The △FCa(2+) in ADS group was 384 ± 26, and in the control group △FCa(2+) was 235 ± 20. The △FCa(2+) in ADS group was higher than that in the control (P < 0.01). Without calcium conditions, the △FCa(2+) in ADS group was 207 ± 17, and in the control group △FCa(2+) was 221 ± 19. The △FCa(2+) in ADS group was almost the same with the increase in the control (P = 0.731). The △FCa(2+) in ADS group was significantly decreased compared with the calcium condition (P < 0.01). However, there was no significant difference in the control between with and without calcium conditions (P = 0.060). (2) After treated with IP3 receptor antagonist, blocker of sarcoplasmic reticulum calcium-ATP, depleted agent of the ryanodine receptor-operated Ca(2+), the △F[Ca(2+)]i in both groups were significantly reduced (P < 0.05), the increase in ADS group was significantly higher than that in the control (P < 0.05). (3)After treated with inhibitor of L-type calcium channel, the △FCa(2+) in ADS group was 211 ± 19, while in the control group △FCa(2+) was 203 ± 16, and there was no significantly increased intracellular Ca(2+) in both groups (P > 0.05). But, the △FCa(2+) in ADS group was significantly reduced after treatment compared to before treatment, (211 ± 19 vs 384 ± 28; P = 0.001). The increase in control group was almost the same with before (203 ± 16 vs 234 ± 22, P = 0.141). (4) After treated with inhibitor of Na(+)-Ca(2+) exchanger, the △FCa(2+) in ADS group was 357 ± 24 and in the control △FCa(2+)was 209 ± 19. The increase in ADS group was significant higher than that in the control (P = 0.000). Compared with △FCa(2+) on the condition without treating with inhibitor of Na(+)-Ca(2+) exchanger, △FCa(2+) was 363 ± 21 in ADS group and △FCa(2+) was 237 ± 20 in control group after treatment. When compared with before treatment, there was no significant difference in both groups (P > 0.05).

CONCLUSIONS

The increase of intracellular Ca(2+) induced by estrogen at EMI smooth muscle cells in adenomyosis patients was mostly from the release of arcoplasmic reticulum, and also from the Ca(2+) influx controlled by L-type calcium channel. The increase of Ca(2+) inducing abnormal contraction of EMI muscle may have relationship with the development of adenomyosis.