Expression of steroidogenic enzyme messenger ribonucleic acid and cortisol production in adrenocortical cells isolated from halothane-sensitive and halothane-resistant pigs
✍ Scribed by Hui-Sheng Huang; Ming-Che Wu; Pi-hsueh Shirley Li
- Book ID
- 101340615
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 307 KB
- Volume
- 79
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
Stress susceptibility in pigs is inherited by a single recessive gene (Hal n ), and homozygous individuals can be identified by exposure to halothane anesthesia. Previous studies have shown that in stress-susceptible pigs, exposure to a high ambient temperature resulted in a twofold increase in corticotropin (ACTH) and lower plasma cortisol. To determine whether there is a fundamental difference in adrenocortical function between halothane-sensitive (HAL-S) and halothaneresistant (HAL-R) pigs, independent of other factors influencing the hypothalamic-pituitary-adrenal (HPA) axis, we compared cortisol responses to ACTH and 8-bromo-cyclic AMP (8-Br-cAMP) in HAL-S and HAL-R pig adrenocortical cells in vitro. We also determined directly the accumulation of four different mRNAs encoding cholesterol side-chain cleavage cytochrome P450 (P450 scc ), 17␣-hydroxylase cytochrome P450 (P450 17␣ ), 21-hydroxylase cytochrome P450 (P450 c21 ) and 11-hydroxylase cytochrome P450 (P450 11 ) in HAL-S pig adrenal cells and compared them to HAL-R pigs. A time-and dose-dependent increase in medium content of cortisol and cAMP was observed after ACTH treatment. 8-Br-cAMP also caused a time-and dose-dependent increase in cortisol production in the medium. Addition of ACTH or 8-Br-cAMP to HAL-S and HAL-R male Lanyu small-ear miniature pig adrenocortical cells increased cortisol production in a dose-and time-related manner. However, cells isolated from HAL-S pigs had a lower cortisol production in response to ACTH or 8-Br-cAMP compared to those from HAL-R pigs. Treatment of cultured cells with 8-Br-cAMP (0.5 mM) for 18 h resulted in a significant increase in P450 scc , P450 17␣ , P450 c21 , and P450 11 mRNA levels. In the absence of 8-Br-cAMP, the four genes were expressed constitutively in both HAL-S and HAL-R pig adrenal cells. Densitometric scanning of the autoradiograph indicated that the relative amounts of P450 scc and P450 17 ␣ mRNAs in HAL-S pig adrenal cells were between 48% and 53% of those detected in HAL-R pig adrenal cells (P Ͻ 0.05). No difference in the amounts of P450 c21 and P450 11 was seen in HAL-S and HAL-R pig adrenal cells. Addition of 8-Br-cAMP (0.5 mM) resulted in a uniform increase in the levels of all four P450 mRNAs in both HAL-S and HAL-R pig adrenal cells. However, the amounts of P450 scc mRNA in HAL-S pig adrenal cells were 67% (P Ͻ 0.05) of those measured in HAL-R pig adrenal cells, whereas the amounts of P450 17␣ , P450 c21 , and P450 11 mRNAs were similar in these cells. Our data suggest an HPA axis defect in HAL-S pigs at the adrenal level. This defect appears to be at the level of P450scc gene expression, which could be partially related to reduced cortisol production by ACTH stimulation.