• Angiotensin II Regulation of Aldosterone Synthase

      Nogueira, Edson da F.; Department of Physiology (2009-07)
      Angiotensin II (Ang II) is the major physiological regulator of aldosterone production acting acutely to stimulate aldosterone biosynthesis and chronically to increase the capacity of the adrenals to produce aldosterone. Aldosterone is principally synthesized in the zona glomerulosa of the adrenal by a series of enzymatic reactions leading to the conversion of cholesterol to aldosterone. The major goal of our study was to define the Ang II-induced mechanisms regulating the expression of aldosterone synthase (CYP11B2) in adrenocortical cells. We approached the analysis of the protein synthesis-dependent regulation of this enzyme by defining, through microarray and real time PCR analysis, the transcription factors that are rapidly induced by Ang II incubation of adrenocortical cell models from three species (human, bovine, and rat). The gene list generated by this comparison included: ATF3, BTG2, NR4A1, NR4A2, NR4A3, EGR1, FOS, FOSB, and JUNB. Importantly, pretreatment of H295R cells with cycloheximide had no effect on Ang II induction of these genes, suggesting that they are direct targets of Ang II signaling. Co-transfection studies, used to investigate the role of these transcription factors in the regulation of CYP11B2, determined that out of the nine transcription factors listed above, only the NGFI-B family members (NGFI-B, NURR1, and NOR1) increased expression of CYP11B2. The importance of NGFI-B in the regulation of CYP11B2 was confirmed by the decrease in CYP11B2 expression in the presence of a dominant-negative (DN)- NGFI-B. A pharmacological approach used to characterize the Ang II pathways regulating transcription of NGFI-B family genes suggested that Ang II binding to the AT1R increases activity of protein kinase C (PKC), Ca -dependent calmodulin kinases (CaMK), and SRC kinase (SRC), which act to regulate the expression of the family of NGFI-B genes as well as CYP11B2. In the current study we also analyzed protein synthesis-independent mechanisms regulating CYP11B2 expression. We studied the role of the ATF/CREB family of transcription factors (ATF1, ATF2, CREB, and CREM), which may bind the cAMP response element (CRE) in the promoter region of the CYP11B2 gene. Importantly, analysis of these transcription factors in the human H295R adrenocortical cell line revealed very low expression of CREB in comparison to the other CRE-binding proteins herein studied. We investigated Ang II-induced phosphorylation of these transcription factors, their binding to the promoter region of CYP11B2, and their effect on CYP11B2 expression. Ang II time-dependently induced phosphorylation of ATF1, ATF2, and CREM in H295R cells. The association of these transcription factors with the CYP11B2 promoter region was induced by Ang II and K+. Transfection of siRNA for ATF1, ATF2, and CREM significantly reduced CYP11B2 expression in Ang II-stimulated conditions. Expression of NURR-1 alone or with constitutively active ATF1, ATF2, CREB, and CREM increased the promoter activity of CYP11B2 in H295R cells. In summary, Ang II rapidly induces expression of newly synthesized transcription factors as well as the phosphorylation of transcription factors already present in the adrenocortical cell. These events are followed by increased CYP11B2 expression and, therefore, represent important mechanisms to increase the adrenal capacity to produce aldosterone.
    • Increased S-nitrosylation Impairs Contraction and Relaxation in Mouse Aorta

      Choi, Hyehun; Department of Physiology (2011-06)
      S-Nitrosylation is a ubiquitous protein modification in redox-based signaling. This modification uses nitric oxide (NO) to forms S-nitrosothiol (SNO) on cysteine residues. Thioredoxin (Trx) and Trx reductase (TrxR) play a role in limiting Snitrosylation. We hypothesized overall that S-nitrosylation of intracellular signaling molecules impairs contraction and relaxation of vascular smooth muscle cells. Aortic rings from C57BL/6 mice were used to measure vascular contraction and relaxation. The rings were treated with TrxR inhibitors, auranofin or 1-chloro-2,4-dinitrobenzene (DNCB), and/or NO donors, propylamine propylamine NONOate (PANOate) or S-nitrosocysteine (CysNO), to increase Snitrosylation. Contractile responses of aortic rings to phorbol-12,13-dibutyrate (PDBu), a PKC activator, were attenuated by auranofin, DNCB, PANOate, and CysNO. PKCa S-nitrosylation was increased by a TrxR inhibitor and CysNO; concomitantly, PKCa activity and downstream signaling were inhibited as compared to control protein. Vascular relaxation in aortic rings from normotensive (Sham) and angiotensin II (Angll)-induced hypertensive mice was measured after contraction with phenylephrine in the presence or absence of DNCB. DNCB reduced relaxation to acetylcholine (ACh) compared to vehicle, but the antioxidants, apocynin and tempol, normalized DNCB-induced impaired relaxation to ACh in sham aorta. Soluble guanylyl cyclase (sGC) S-nitrosylation was increased by DNCB, and sGC activity (cyclic GMP assay) was reduced in sham aorta. In aortic rings from Angll-treated mice, DNCB did not change relaxation to ACh compared to vehicle. DNCB decreased relaxation to sodium nitroprusside (SNP) in aortic rings from both sham and Angll mice. Total protein S-nitrosylation was enhanced in Angll aorta compared to sham, and TrxR activity was inhibited in Angll aorta compared to sham. These data suggest that PKC is inactivated by S-nitrosylation and this modification inhibits contractile responses to PDBu. TrxR inhibition reduces vascular relaxation via increasing oxidative stress and sGC S-nitrosylation. In Angll-induced hypertensive mice, augmented S-nitrosylation is associated with impaired vasodilation. Thus, TrxR and Snitrosylation may provide a critical mechanism in hypertension associated with abnormal vascular reactivity.
    • Protein Kinase D Restrains Angiotensin II-Induced Aldosterone Secretion in Primary Adrenal Glomerulosa Cells

      Shapiro, Brian A.; Department of Physiology (2007-07)
      Misregulation of the renin-angiotensin II (Angll)-aldosterone (Aldo) system is a key feature of cardiovascular disease. A focus of study in this system is the Angll-elicited secretion of Aldo from the adrenocortical zona glomerulosa. An excellent model in which to study this phenomenon is primary cultures of bovine adrenal glomerulosa (AG) cells. These cells secrete detectable quantities of Aldo in response to secretagogues, such as Angll, elevated potassium (K+), adrenocorticotrophic hormone (ACTH) and phorbol 12-myristate 13-acetate (PMA), within 30 minutes. The serine (Ser)/threonine kinase protein kinase D (PKD) is reported to be activated by Angll in several systems, including the adrenocortical carcinoma cell line NCI H295R, and is thought to have a positive role in chronic (24 hours) Angll-evoked Aldo secretion. Because the role of PKD in acute Angll-elicited Aldo secretion has never been examined in a primary culture system, we undertook to study the role of PKD in acute (minutes to one hour) Aldo secretion. Thus, Angll (10 nM) and PMA (100 nM), but not elevated K+ (15 mM) and ACTH (10 nM), induced phosphorylation of PKD on Ser910, a marker of PKD activation, in primary bovine AG cells. This finding was confirmed by an in vitro kinase activity assay. Angll and PMA were also able to induce PKD activation in H295R cells. Furthermore, this activation was concentration dependent, and was rapidly induced (by 5 min). PKD activation was dependent on Angll type 1 (AT-1), but not AT-2 receptor, signaling, and was independent of tyrosine kinase signaling. Finally, we introduced, via adenovirus transduction, wild-type PKDwt and dominant negative PKDS738/742A constructs into primary AG cells and monitored Angll-evoked Aldo secretion. PKDwt -transduced AG cells exhibited decreased Angll-stimulated Aldo secretion, while in the PKDS738A742A - infected AG cells Angll-stimulated Aldo was enhanced. Thus, we hypothesize that PKD has an anti-secretory role in Angll-induced acute Aldo secretion.