Development of a high-throughput assay for aldosterone synthase inhibitors using high-performance liquid chromatography-tandem mass spectrometry.

Aldosterone plays a key role in the pathogenesis of hypertension, congestive heart failure, and chronic kidney disease. Aldosterone biosynthesis involves three membrane-bound enzymes: aldosterone synthase, adrenodoxin, and adrenodoxin reductase. Here, we report the development of a mass spectrometry-based high-throughput whole cell-based assay for aldosterone synthesis. A human adrenal carcinoma cell line (H295R) overexpressing human aldosterone synthase cDNA was established. The production of aldosterone in these cells was initiated with the addition of 11-deoxycorticosterone, the immediate substrate of aldosterone synthase. An automatic liquid handler was used to gently distribute cells uniformly to well plates. The adaption of a second automated liquid handling system to extract aldosterone from the cell culture medium into organic solvent enabled the development of 96- and 384-well plate formats for this cellular assay. A high-performance liquid chromatography-tandem mass spectrometry method was established for the detection of aldosterone. Production of aldosterone was linear with time and saturable with increasing substrate concentration. The assay was highly reproducible with an overall average Z' value=0.49. This high-throughput assay would enable high-throughput screening for inhibitors of aldosterone biosynthesis.

Identification and characterization of hamster stearoyl-CoA desaturase isoforms.

Stearoyl-CoA desaturase (SCD) catalyzes the formation of monounsaturated fatty acids from saturated fatty acids. It plays a key role in lipid metabolism and energy expenditure in mammals. In mice, four SCD isoforms (SCD1-4) have been identified. Here we report the identification of cDNA sequences corresponding to SCD1, SCD2 and SCD3 of golden hamster. The deduced amino acid sequences of these hamster SCD (hmSCD) isoforms display a high degree of homologies to their mouse counterparts (mouse SCD). Polyclonal antibodies specific to rodent SCDs detected proteins of predicted size in the human embryonic kidney 293 cells transfected with hmSCD cDNAs. Microsome fractions prepared from these cells also displayed increased SCD activity versus cells transfected with vector alone. Real-time reverse transcription-polymerase chain reaction analysis revealed the highest expression of hmSCD1 in liver and adipose tissue, while the highest hmSCD2 expression was detected in the brain. Very low levels of hmSCD3 mRNA can be detected in the tissues tested. This report is the first description of three SCD isoforms in the hamster and will provide useful tools in the further study of fatty acids metabolism in this species.

Identification and expression of novel isoforms of human stromal cell-derived factor 1.

Stromal Cell-derived factor 1 (SDF-1) is a CXC chemokine that binds to the CXCR4 receptor. Recent publication indicates that the SDF-1/CXCR4 signaling pathway plays a pivotal role during development and in many patho-physiological conditions including hematopoiesis, blood vessel formation, cancer metastasis, angiogenesis and HIV infection. Two human SDF-1 isoforms, SDF-1alpha and SDF-1beta, have been reported to date. Here we report the identification of four additional human SDF-1 isoforms derived from alternative splicing events, SDF-1gamma, SDF-1delta, SDF-1epsilon and SDF-1phi. These SDF-1 splice variants all share the same first three exons but contain different fourth exons. The human SDF-1 gene spans over 88 kilobase-pairs on chromosome 10. Using the semi-quantitative RT-PCR method, we determined the tissue distribution of these SDF-1 isoforms. SDF-1alpha and SDF-1beta share similar expression patterns and the highest expression were detected in liver, pancreas and spleen. SDF-1gamma seems to be the human orthologue of recently isolated rat SDF-1gamma, and its expression was only detected in the heart. SDF-1delta expression can be detected in several adult tissues but the highest expression was detected in fetal liver. When transfected into HEK293 cells, all the SDF-1 isoforms can be detected as secreted proteins in the cell culture media. The conditioned media from transfected cells can stimulate cell migration in a CXCR4-dependent manner. These data suggest that the novel SDF-1 splice variants encode functional proteins.

Protein kinase C betaII activation induces angiotensin converting enzyme expression in neonatal rat cardiomyocytes.

OBJECTIVE: Members of the protein kinase C (PKC) family are important mediators of cell signaling underlying multiple aspects of myocardial function. Activation of the betaII isoform of PKC is thought to be involved in the development of congestive heart failure. To investigate the biological effect of PKC-betaII, we measured gene expression of angiotensin converting enzyme (ACE) and angiotensin II (AngII) receptors AT(1A) and AT(1B) in cardiomyocytes overexpressing PKC-betaII. METHODS: An adenovirus construct expressing PKC-betaII was introduced into cultured neonatal rat ventricular myocytes (NRVMs). Western blot and in situ kinase assay was used to measure PKC-betaII level and activity in NRVMs. Real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to measure the mRNA levels of several genes following PMA stimulation of either un-infected or ad-PKC-betaII infected cells. RESULTS: Our data show that activation of PKC-betaII in cardiomyocytes leads to elevated expression of angiotensin-converting enzyme (ACE) gene. Treatment of adeno-PKC-betaII infected cardiomyocytes with phorbol 12-myristate 13-acetate (PMA) resulted in an 8-fold increase of ACE mRNA expression, whereas ACE mRNA levels only increased around 2-fold in uninfected or adeno-GFP (green fluorescent protein) infected cardiomyocytes with similar PMA treatment. The induction of ACE mRNA was blocked by the PKC-beta-specific antagonist LY379196. No significant change of angiotensin II receptors AT1a and AT1b could be detected in the cardiomyocytes expressing PKC-betaII. CONCLUSION: These data indicate that ACE is a transcription target of PKC-betaII activation in cardiomyocytes, and also suggest a mechanism for the involvement of PKC in cardiac hypertrophy and fibrosis through increased activity of angiotensin converting enzyme in the myocardium.