| Summary: | 博士 === 高雄醫學大學 === 醫學研究所 === 90 === Atrial natriuretic peptide (ANP) is a peptide not only with potent diuretic, natriuretic and vasorelaxant effects but also with potent aldosterone and other steroid hormone inhibitory activities. In the past few years, we demonstrated that immunoreactive ANP substance was present in the secretory granule of human adrenal pheochromocytoma cells. We also found that ANP was synthesized in the human adrenal medulla, and such medullary ANP synthesis increased in the patients with hypermineralocorticoidism. However, no evidence exists that adrenal medullary ANP affects the zona glomerulosa synthesis of aldosterone. The three subtypes of ANP receptors have been found in both adrenal cortical and medullary cells. Therefore, we hypothesize that ANP can be synthesized in the adrenal cortex in addition to the adrenal medulla, and adrenal ANP expression can be regulated in response to volume overloading. In additon, the expression of adrenal neuronal nitric oxide synthase (nNOS) and changes in mRNA of key proteins or enzymes of adrenal hormone synthesis under volume change are still unclear. Based on the above mentioned background, we performed several continuous studies with the aims to investigate the following questions.
(1) To re-evaluate the sites of ANP synthesis and to explore whether the ANP secretory granules are exist in the zona glomerulosa.
(2) To further evaluate the role of adrenal ANP in body fluid homeostasis, we investigated the changes in adrenal ANP in rats receiving DOCA-salt treatment.
(3) To investigate whether adrenal neuronal nitric oxide synthase (nNOS) expression and mRNA of key proteins or enzymes of adrenal hormone synthesis are responsive to alterations in body volume, including water deprivation-induced volume deficiency and DOCA-salt treatment-induced volume expansion.
The results in this study reveal:
1. In adrenal glands both in normal rats and human, we found that not only adrenal medulla but also adrenal zona glomerulosa are the sites of ANP synthesis. ANP secretory granules are found in the zona granulosa cells. Aldosterone-producing adenoma, originally from zona glomerulosa, is also a site of ANP synthesis.
2. In situ hybridization and immunohistochemical studies showed that adrenal ANP mRNA and ANP-like immunoreactivities were mainly localized in the zona glomerulosa and medulla of vehicle-treated rats. DOCA-salt treatment activated ANP mRNA and peptide expression in all adrenal zones, especially in the zona fasciculate/reticularis from 12 hours to the entire 8-days study period. Using a semiquantitative RT-PCR technique, the relative quantities of ANP mRNA in the adrenals of the DOCA-salt-treated group were significantly increased from 1 to 8 days, whereas the adrenal weights of DOCA-salt-treated rats were significantly decreased from day 2 to day 8.
3. Using a semi-quantitative multiple RT-PCR technique, the relative quantities of nNOS mRNA as well as the tyrosine hydroxylase (TH) mRNA and phenylethanolamine N-methyltransferase (PNMT) mRNA in the adrenals of the water-deprived group significantly increased from 12 h to 4 days, whereas the steroidogenic acute regulatory protein (StAR) mRNA showed no significant change during the same study period. In situ hybridization and immunohistochemical studies showed that water deprivation activated adrenal nNOS mRNA and protein expression in the medulla. Four days after water deprivation, nNOS protein expression, determined by Western blot, increased significantly in the adrenal gland.
4. The relative quantities of nNOS mRNA as well as the PNMT mRNA and StAR mRNA in the adrenals of the DOCA-salt-treated group significantly decreased, whereas the steroid acute regulatory protein TH mRNA showed no significant change during the same study period. In situ hybridization and immunohistochemical studies showed that DOCA-salt treatment inactivated adrenal nNOS mRNA and protein expression in the medulla. Eight days after DOCA-salt treatment, nNOS protein expression determined by Western blot decreased significantly in the adrenal gland.
In conclusion, our results are the first to indicate that ANP is synthesized not only in the adrenal medulla but also in the adrenal cortex, and their syntheses are markedly increased in DOCA-salt-treated rats. These results imply that adrenal ANP may participate in the intraadrenal regulation of adrenal function on water-electrolyte homeostasis in an autocrine or paracrine manner. The augmentation in nNOS gene expression in the adrenal glands of water deprived rats and the decrement after DOCA-salt treatment, particularly in the adrenal medulla, may participate in the adrenal regulatory pathways. It is reasonable to propose that NO derived from the adrenal nNOS may locally preserve adrenal blood flow through counteracting the circulating vasoconstrictive substance effect to help the autoregulation of the adrenal gland.
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