Role of increased endothelin-1 on apoptosis of cerebral cortex neurons of rats

Anding XU; Wanyang YANG; Zihua ZENG; Jingfang DI; Haifeng MIAO; Yijuan WU; Wenyan ZHUO

ABSTRACT

BACKGROUND:

Endothelin(ET) -1 is a peptide with potent actions on blood vessels and nerve system. Its expression increases in the central nervous system(CNS) in a variety of pathological conditions, inducing harmful effects on the nervous tissue. However it is not clearly elucidated whether the over-expressed ET-1 can directly induce neuronal apoptosis.

OBJECTIVE:

To investigate whether ET-1 can directly induce apoptosis in primarily cultured brain neurons of rat, and which ET receptor subtype(s) is involved in this action.

DESIGN:

Completely randomized and controlled experimental study based on cells.

SETTING:

Neurological department in a university hospital, pathological department of a university and laboratory center of tissue transplantation and immunology, life science and technology college.MATERIALS This study was completed in the Pathology Department, the Institute of Tissue Transplantation and Immunology, the Life Science and Technology College of Jinan University. The subjects were primarily-cultured neurons obtained from cerebral cortex of newborn rats that were provided by the Experimental Animal Center of the Medical College, Sun Yat-sen University.

INTERVENTIONS:

After culturing for five days, the neurons were treated with ET-1 (0. 2 nmol/L and 20 nmol/L) for 24 hours. Apoptotic neurons were semi-quantitatively measured with Annexin V and Hoechst 33258 staining respectively. ET-1(20 nmol/L), with BQ123(a selective antagonist for ET receptor A, 1 mmol/L) or with BQ788(a selective antagonist for ET receptor B, 1 mmol/L), was added respectively into the cultures simultaneously. And the apoptotic neurons were quantitatively measured with flow cytometry 24 hours later. Equal amount of PBS, instead of ET-1, waw added into the control subjects.MAIN OUTCOME

MEASURES:

The effect of ET-1 on apoptosis rate of cultured rat cortical neurons, and the ET receptor subtypes involved in this action.

RESULTS:

Twenty-four hours after treated with 0.2 nmol/L ET-1, the Annexin-V, and Hoechest 33258 positive stained cell rates[ (23.00 ± 9.96)%,(9.82 ±0.95)% ] were of no difference as compared with those of the controls[ (13.50 ± 3.35)%, (8.21 ± 2. 17)% ]. By contrast, after incubation with the higher dose of ET-1 (20 nmol/L), significant higher rate of apoptosis was measured in Annexin V staining[(50.50 ± 10.78)%, P=0.01, n=4] and Hoechest 33258 staining[(13.78±1.52)%, P= 0. 000, n = 8] . Analyzed with flow cytometry, the apoptosis rate was (0.20±0. 15)% in the control group, (26. 11 ±3.28)% in 20 nmol/LET-1 group, and(13.58 ±4. 92)% in BQ123 +ET-1 and(9.99 ±3.30)% in BQ788 +ET-1 respectively, indicating that BQ123 and BQ788 partially-blocked the apoptosis effect of ET-1 on. cultured neurons(BQ123 + ET-1 vs ET-1, P = 0. 005; BQ788 + ET-1 vs ET-1, P = 0. 001, n = 4, respectively).

CONCLUSION:

The higher dose of ET-1 (20 nmol/L) can directly induce apoptosis of primarily-cultured cerebral neurons of rats. The effect of ET-1 inducing neuronal apoptosis may be mediated via both ET receptors A and B.