G. Satyanarayana Reddy
Brown University
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Featured researches published by G. Satyanarayana Reddy.
The Journal of Steroid Biochemistry and Molecular Biology | 1997
Heide S. Cross; Meinrad Peterlik; G. Satyanarayana Reddy; Inge Schuster
1Alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and its synthetic analogues exhibit structure-related variations in their growth inhibitory actions in human colon adenocarcinoma-derived Caco-2 cells. Because this might be caused by differences in resistance against metabolic degradation, we used high performance liquid chromatography (HPLC) analysis to investigate pathways of vitamin D metabolism in two different Caco-2 cell clones. Importantly, when Caco-2 cells were incubated with tritium-labelled 25-hydroxyvitamin D3 (25(OH)D3) for up to 2 h they produced almost exclusively a metabolite, which was identified as 1alpha,25(OH)2D3 by co-chromatography with the synthetic standard in two different HPLC systems, and by a radioligand assay showing an identical binding affinity to the intestinal nuclear vitamin D receptor. Expression of the 25(OH)D3-1alpha-hydroxylase appears to be constitutive because almost identical enzyme activities are observed in any growth phase. 1Alpha,25(OH)2D3 can also activate side chain metabolism in Caco-2 cells: thereby, 1alpha,25(OH)2D3 or 25(OH)D3 are metabolized through the C-24 oxidative pathway into 1alpha,24(R),25(OH)3D3 and 24(R),25(OH)2D3, respectively, which undergo sequential metabolism into 1alpha,25(OH)2-24oxo-D3 and 24-oxo-25(OH)D3. Through C-23 oxidation these intermediary metabolites are further converted into 1alpha,23,25(OH)3-24-oxo-D3 and 23,25(OH)2-24-oxo-D3. Also direct C-23 oxidation of the substrates 1alpha,25(OH)2D3 and 25(OH)D3 generates 1alpha,23(S),25(OH)3D3 and 23(S),25(OH)2D3, respectively. In summary, our results demonstrated the presence of distinct pathways of vitamin D metabolism in Caco-2 cells: apart from metabolizing 1alpha,25(OH)2D3 along the C-24 and C-23 oxidative pathways, Caco-2 cells are able to synthesize 1alpha,25(OH)2D3 from 25(OH)D3 through constitutive expression of 25(OH)D3-1alpha-hydroxylase activity. The relevance of this finding for the intrinsic growth control of neoplastic colonocytes is discussed.
Journal of Cellular Biochemistry | 1997
Moray J. Campbell; G. Satyanarayana Reddy; H. Phillip Koeffler
The seco‐steroid hormone, 1α,25 dihydroxyvitamin D3 (1α,25(OH)2D3) binds to a specific nuclear receptor that acts as a ligand‐inducible transcription factor. The resulting genomic effects include partial arrest in G0/G1 of the cell cycle and induction of differentiation; these effects have been observed in various types of cancer. Recently, we produced enzymatically the natural 24‐oxo metabolites of 1α,25(OH)2D3 and two of its potent synthetic analogs (1α,25‐(OH)2‐16‐ene‐D3 and 1α,25‐(OH)2‐20‐epi‐D3) using a rat kidney perfusion system. We have found that the 24‐oxo metabolites of both 1α,25(OH)2D3 and its analogs have either the same or greater antiproliferative activity against various cancer cells as their parental compounds. Notably, two cell lines (DU‐145 (prostate cancer) and MDA‐MB‐436 [breast cancer]) that were extremely resistant to the antiproliferative effects of vitamin D3 analogs displayed greater sensitivity towards the 24‐oxo metabolite of the vitamin D3 analog. Similarly, the 24‐oxo metabolites had the capacity to induce differentiation and apoptosis and to diminish the proportion of cells in S phase. Most interestingly, while the analog 1α,25(OH)2‐20‐epi‐D3 induced expression of BRCA1 in MCF‐7 breast cancer cells; its 24‐oxo metabolite dramatically suppressed BRAC1 expression. Thus, we have shown for the first time that the various biological activities produced by the hormone 1α,25(OH)2D3 and some of its analogs may represent a combination of actions by the hormone 1α,25(OH)2D3 and its natural 24‐oxo metabolites. J. Cell. Biochem. 66:413–425, 1997.
Molecular Genetics and Metabolism | 2002
Virender K. Rehan; John S. Torday; Sara Peleg; Lynn Gennaro; Paul Vouros; James F. Padbury; D.Sunita Rao; G. Satyanarayana Reddy
Pulmonary alveolar type II cells have been shown to be a possible target for the secosteroid hormone, 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], during perinatal transition. At present, there is great interest to isolate and identify the metabolites of 1alpha,25(OH)2D3 produced in its target tissues and to determine the contribution of each individual metabolite of 1alpha,25(OH)2D3 to the final expression of the pleiotropic actions attributed to 1alpha,25(OH)2D3. Of all the known metabolites of 1alpha,25(OH)2D3, 1alpha,25(OH)2-3-epi-D3 has gained most attention as it is produced only in specific tissues and possesses significant activity in tissues in which it is produced. Furthermore, in vivo studies indicate that this metabolite when compared to 1alpha,25(OH)2D3 is less calcemic. Therefore, we performed the present study to identify production of 1alpha,25(OH)2-3-epi-D3 in alveolar type II cells, and to evaluate its effect on surfactant synthesis. We incubated NCI-H441 cells, an alveolar type II cell line, with 1alpha,25(OH)2D3 and demonstrated that these cells metabolize 1alpha,25(OH)2D3 to various previously well-characterized polar metabolites, and to a less polar metabolite which was unequivocally identified as 1alpha,25(OH)2-3-epi-D3 by GC/MS and HPLC analysis. Further, biological activity studies in H441 cells indicated that 1alpha,25(OH)2-3-epi-D3 possesses significant activity in terms of its ability: (i) to increase surfactant phospholipid synthesis, (ii) to induce surfactant SP-B mRNA gene expression, and (iii) to increase surfactant SP-B protein synthesis. However, the activity of 1alpha,25(OH)2-3-epi-D3 when compared to 1alpha,25(OH)2D3 in generating VDR-mediated transcriptional activity in ROS 17/2.8 cells transfected with human osteocalcin VDRE/growth hormone gene construct, was significantly reduced. The high metabolic stability of 1alpha,25(OH)2-3-epi-D3, as previously proposed by us, may be a possible explanation for the high in vitro activity in spite of the reduced VDR-mediated transcriptional activity. In summary, we report for the first time the pathways of 1alpha,25(OH)2D3 metabolism in pulmonary alveolar type II cells and indicate that 1alpha,25(OH)2-3-epi-D3, a natural intermediary metabolite of 1alpha,25(OH)2D3 possesses significant activity in stimulating surfactant synthesis in alveolar type II cells.
Steroids | 2001
Milan R. Uskokovic; Anthony W. Norman; Percy S. Manchand; George P. Studzinski; Moray J. Campbell; H. Phillip Koeffler; Atsuko Takeuchi; Mei-Ling Siu-Caldera; D.Sunita Rao; G. Satyanarayana Reddy
The secosteroid hormone 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway is initiated by hydroxylation at C-24 of the side chain and leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways are initiated by hydroxylations at C-23 and C-26 of the side chain and lead to the formation of the end product, calcitriol lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 of the A-ring to form 1alpha,25(OH)(2)-3-epi-D(3). A rational design for the synthesis of potent analogs of 1alpha,25(OH)(2)D(3) is developed based on the knowledge of the various metabolic pathways of 1alpha,25(OH)(2)D(3). Structural modifications around the C-20 position, such as C-20 epimerization or introduction of the 16-double bond affect the configuration of the side chain. This results in the arrest of the C-24 hydroxylation initiated cascade of side chain modifications at the C-24 oxo stage, thus producing the stable C-24 oxo metabolites which are as active as their parent analogs. To prevent C-23 and C-24 hydroxylations, cis or trans double bonds, or a triple bond are incorporated in between C-23 and C-24. To prevent C-26 hydroxylation, the hydrogens on these carbons are replaced with fluorines. Furthermore, testing the metabolic fate of the various analogs with modifications of the A-ring, it was found that the rate of C-3 epimerization of 5,6-trans or 19-nor analogs is decreased to a significant extent. Assembly of all these protective structural modifications in single molecules has then produced the most active vitamin D(3) analogs 1alpha,25(OH)(2)-16,23-E-diene-26,27-hexafluoro-19-nor-D(3) (Ro 25-9022), 1alpha,25(OH)(2)-16,23-Z-diene-26,27-hexafluoro-19-nor-D(3) (Ro 26-2198), and 1alpha,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-19-nor-D(3) (Ro 25-6760), as indicated by their antiproliferative activities.
Journal of Cellular Biochemistry | 2008
Rachamallu Aparna; Jagu Subhashini; Karnati R. Roy; G. Satyanarayana Reddy; Matthew Robinson; Milan R. Uskokovic; Gorla V. Reddy; Pallu Reddanna
Inducible cyclooxygenase‐2 (COX‐2) has been implicated to play a role in inflammation and carcinogenesis and selective COX‐2 inhibitors have been considered as anti‐inflammatory and cancer chemopreventive agents. 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3), the active hormonal form of vitamin D3 also has been considered to be a cancer chemopreventive agent in addition to its important role in maintaining calcium homeostasis. Based on these observations, we studied the direct effect of 1α,25(OH)2D3 and one of its less calcemic synthetic analogs, 1α,25(OH)2‐16‐ene‐23‐yne‐D3 on the activity of both COX‐1 and COX‐2 in an in vitro enzyme assay. Preliminary data indicated that both 1α,25(OH)2D3 and 1α,25(OH)2‐16‐ene‐23‐yne‐D3 inhibited selectively the activity of COX‐2 with no effect on the activity of COX‐1. Out of the two compounds, 1α,25(OH)2‐16‐ene‐23‐yne‐D3 was found to be more effective with an IC50 of 5.8 nM. Therefore, the rest of the experiments were performed using 1α,25(OH)2‐16‐ene‐23‐yne‐D3 only. 1α,25(OH)2‐16‐ene‐23‐yne‐D3 inhibited the proliferation of lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) with a reduction in the expression of COX‐2 along with other inflammatory mediators like inducible nitric oxide synthase (iNOS) and interleukin‐2 (IL‐2). Furthermore, 1α,25(OH)2‐16‐ene‐23‐yne‐D3 also inhibited carrageenan induced inflammation in an air pouch of a rat and effectively reduced the expression of COX‐2, iNOS, and IL‐2 in the tissues of the same air pouch. In both cases, 1α,25(OH)2‐16‐ene‐23‐yne‐D3 did not show any effect on the expression of COX‐1. In summary, our results indicate that 1α,25(OH)2‐16‐ene‐23‐yne‐D3, a less calcemic vitamin D analog, exhibits potent anti‐inflammatory effects and is a selective COX‐2 inhibitor. J. Cell. Biochem. 104: 1832–1842, 2008.
Steroids | 2001
G. Satyanarayana Reddy; K.Raman Muralidharan; William H. Okamura; Kou-Yi Tserng; John A. McLane
We previously reported that 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is metabolized into 1α,25-dihydroxy-3-epi-vitamin D3 [1α,25(OH)2-3-epi-D3] in primary cultures of neonatal human keratinocytes. We now report that 1α,25(OH)2-3-epi-D3 itself is further metabolized in human keratinocytes into several polar metabolites. One of the polar metabolite was unequivocally identified as 1α,23,25-trihydroxy-3-epi-vitamin D3 by mass spectrometry and its sensitivity to sodium periodate. Three of the polar metabolites were identified as 1α,24,25-trihydroxy-3-epi-vitamin D3, 1α,25-dihydroxy-24-oxo-3-epi-vitamin D3 and 1α,23,25-trihydroxy-24-oxo-3-epi-vitamin D3 by comigration with authentic standards on both straight and reverse phase HPLC systems. In addition to the polar metabolites, 1α,25(OH)2-3-epi-D3 was also metabolized into two less polar metabolites. A possible structure of either 1αOH-3-epi-D3-20,25-cyclic ether or 1αOH-3-epi-D3-24,25-epoxide was assigned to one of the less polar metabolites through mass spectrometry. Thus, we indicate for the first time that 1α,25(OH)2-3-epi-D3 is metabolized in neonatal human keratinocytes not only via the same C-24 and C-23 oxidation pathways like its parent, 1α,25(OH)2D3; but also is metabolized into a less polar metabolite via a pathway that is unique to 1α,25(OH)2-3-epi-D3.
Journal of Chromatography A | 1993
Bernice Yeung; Paul Vouros; G. Satyanarayana Reddy
A mass spectrometric method for the detection of vitamin D3 metabolites is described. This method involves the derivatization of the metabolites by cycloaddition with 4-phenyl-1,2,4-triazoline-3,5-dione, followed by their characterization by continuous-flow fast atom bombardment (CF-FAB) tandem mass spectrometry (MS-MS) and high-performance liquid chromatography (HPLC). Using HPLC, this derivatization has been shown to increase the UV detectability of 25-hydroxyvitamin D3 by about 5-fold. The FAB spectra of the adducts are dominated by peaks corresponding to a protonated molecule and a fragment ion derived in part from the loss of the side chain. Multiple reaction monitoring (MRM) of this transition by MS-MS may be utilized for trace level analysis of vitamin D metabolites. Sample introduction by flow injection yields detection limits in the low nanogram to high picogram range, whereas the use of on-line capillary LC has been found to decrease the detection limits to the low picogram level.
Journal of Cellular Biochemistry | 2005
Alex J. Brown; Andrew S. Weiskopf; Paul Vouros; Gino J. Sasso; Milan R. Uskokovic; Guochun Wang; G. Satyanarayana Reddy
Since our original demonstration of the metabolism of 1α,25(OH)2D3 into 1α,25(OH)2‐3‐epi‐D3 in human keratinocytes, there have been several reports indicating that epimerization of the 3 hydroxyl group of vitamin D compounds is a common metabolic process. Recent studies reported the metabolism of 25OHD3 and 24(R),25(OH)2D3 into their respective C‐3 epimers, indicating that the presence of 1α hydroxyl group is not necessary for the 3‐epimerization of vitamin D compounds. To determine whether the presence of a 25 hydroxyl group is required for 3‐epimerization of vitamin D compounds, we investigated the metabolism of 1αOHD3, a non‐25 hydroxylated vitamin D compound, in rat osteosarcoma cells (ROS 17/2.8). We noted metabolism of 1αOHD3 into a less polar metabolite which was unequivocally identified as 1αOH‐3‐epi‐D3 using the techniques of HPLC, GC/MS, and 1H‐NMR analysis. We also identified 1αOH‐3‐epi‐D3 as a circulating metabolite in rats treated with pharmacological concentrations of 1αOHD3. Thus, these results indicated that the presence of a 25 hydroxyl group is not required for 3‐epimerization of vitamin D compounds. Furthermore, the results from the same studies also provided evidence to indicate that 1αOH‐3‐epi‐D3, like 1αOHD3, is hydroxylated at C‐25. We then evaluated the biological activities of 1αOH‐3‐epi‐D3. Treatment of normal rats every other day for 7 days with 2.5 nmol/kg of 1αOH‐3‐epi‐D3 did not raise serum calcium, while the same dose of 1αOHD3 increased serum calcium by 3.39 ± 0.52 mg/dl. Interestingly, in the same rats which received 1αOH‐3‐epi‐D3 we also noted a reduction in circulating PTH levels by 65 ± 7%. This ability of 1αOH‐3‐epi‐D3 to suppress PTH levels in normal rats without altering serum calcium was further tested in rats with reduced renal function. The results indicated that the ED50 of 1αOH‐3‐epi‐D3 for suppression of PTH was only slightly higher than that of 1α,25(OH)2D3, but that the threshold dose of the development of hypercalcemia (total serum Ca > 10.5 mg/dl) was nearly 80 times higher. These findings indicate that 1αOH‐3‐epi‐D3 is a highly selective vitamin D analog with tremendous potential for treatment of secondary hyperparathyroidism in chronic renal failure patients.
Biochemical Pharmacology | 1995
Bernice Yeung; Paul Vouros; Mei-Ling Siu-Caldera; G. Satyanarayana Reddy
1,25-Dihydroxy-16-ene vitamin D3 is a synthetic analog of 1,25-dihydroxyvitamin D3, the most physiologically active metabolite of vitamin D3. The renal metabolism of 1,25-dihydroxy-16-ene vitamin D3 had been studied previously using a perfused rat kidney system [Reddy et al., Bioorg Med Chem Lett 3: 1879-1884, 1993], and its C-24 oxidative metabolic pathway had been found to be different from that of 1,25-dihydroxyvitamin D3 by HPLC. To further delineate the differences between the C-24 oxidative metabolic pathways of 1,25-dihydroxyvitamin D3 and 1,25-dihydroxy-16-ene vitamin D3 in this present study we investigated the C-24 oxidation pathway of 1,25-dihydroxy-16-ene vitamin D3 using a novel detection approach based on on-line capillary liquid chromatography coupled to electrospray tandem mass spectrometry. Two types of tandem mass spectrometric detection were employed to characterize the metabolites in the kidney perfusate: (a) the preliminary screening of metabolites by parent scan, which led to the tentative discovery of the production of 1,23,25-trihydroxy-24-oxo-16-ene vitamin D3, a new metabolite of 1,25-dihydroxy-16-ene vitamin D3, and (b) the pharmacokinetic studies of the substrate, 1,25-dihydroxy-16-ene vitamin D3 and its metabolites by multiple reaction monitoring. In the latter, the mass spectrometric sensitivity for quantification was found to be about 20-fold better than UV detection. The current work concluded that the C-24 oxidative metabolic pathway of 1,25-dihydroxy-16-ene vitamin D3 closely mimicked that of its natural counterpart. Furthermore, the use of mass spectrometry permitted the clearance rate of the starting substrate to be studied at a more physiological level (ng/mL or submicromolar level), which had not been possible previously by HPLC-UV detection.
American Journal of Physiology-endocrinology and Metabolism | 1999
Wen Yang; Peter A. Friedman; Rajiv Kumar; John L. Omdahl; Brian K. May; Mei Ling Siu-Caldera; G. Satyanarayana Reddy; Sylvia Christakos
Previous studies using microdissected nephron segments reported that the exclusive site of renal 25-hydroxyvitamin D3-24-hydroxylase (24OHase) activity is the renal proximal convoluted tubule (PCT). We now report the presence of 24OHase mRNA, protein, and activity in cells that are devoid of markers of proximal tubules but express characteristics highly specific for the distal tubule. 24OHase mRNA was undetectable in vehicle-treated mouse distal convoluted tubule (DCT) cells but was markedly induced when DCT cells were treated with 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]. 24OHase protein and activity were also identified in DCT cells by Western blot analysis and HPLC, respectively. 8-Bromo-cAMP (1 mM) or parathyroid hormone [PTH-(1-34); 10 nM] was found to potentiate the effect of 1, 25(OH)2D3 on 24OHase mRNA. The stimulatory effect of cAMP or PTH on 24OHase expression in DCT cells suggests differential regulation of 24OHase expression in the PCT and DCT. In the presence of cAMP and 1, 25(OH)2D3, a four- to sixfold induction in vitamin D receptor (VDR) mRNA was observed. VDR protein, as determined by Western blot analysis, was also enhanced in the presence of cAMP. Transient transfection analysis in DCT cells with rat 24OHase promoter deletion constructs demonstrated that cAMP enhanced 1, 25(OH)2D3-induced 24OHase transcription but this enhancement was not mediated by cAMP response elements (CREs) in the 24OHase promoter. We conclude that 1) although the PCT is the major site of localization of 24OHase, 24OHase mRNA and activity can also be localized in the distal nephron; 2) both PTH and cAMP modulate the induction of 24OHase expression by 1,25(OH)2D3 in DCT cells in a manner different from that reported in the PCT; and 3) in DCT cells, upregulation of VDR levels by cAMP, and not an effect on CREs in the 24OHase promoter, is one mechanism involved in the cAMP-mediated modulation of 24OHase transcription.Previous studies using microdissected nephron segments reported that the exclusive site of renal 25-hydroxyvitamin D3-24-hydroxylase (24OHase) activity is the renal proximal convoluted tubule (PCT). We now report the presence of 24OHase mRNA, protein, and activity in cells that are devoid of markers of proximal tubules but express characteristics highly specific for the distal tubule. 24OHase mRNA was undetectable in vehicle-treated mouse distal convoluted tubule (DCT) cells but was markedly induced when DCT cells were treated with 1,25 dihydroxyvitamin D3[1,25(OH)2D3]. 24OHase protein and activity were also identified in DCT cells by Western blot analysis and HPLC, respectively. 8-Bromo-cAMP (1 mM) or parathyroid hormone [PTH-(1-34); 10 nM] was found to potentiate the effect of 1,25(OH)2D3on 24OHase mRNA. The stimulatory effect of cAMP or PTH on 24OHase expression in DCT cells suggests differential regulation of 24OHase expression in the PCT and DCT. In the presence of cAMP and 1,25(OH)2D3, a four- to sixfold induction in vitamin D receptor (VDR) mRNA was observed. VDR protein, as determined by Western blot analysis, was also enhanced in the presence of cAMP. Transient transfection analysis in DCT cells with rat 24OHase promoter deletion constructs demonstrated that cAMP enhanced 1,25(OH)2D3-induced 24OHase transcription but this enhancement was not mediated by cAMP response elements (CREs) in the 24OHase promoter. We conclude that 1) although the PCT is the major site of localization of 24OHase, 24OHase mRNA and activity can also be localized in the distal nephron; 2) both PTH and cAMP modulate the induction of 24OHase expression by 1,25(OH)2D3in DCT cells in a manner different from that reported in the PCT; and 3) in DCT cells, upregulation of VDR levels by cAMP, and not an effect on CREs in the 24OHase promoter, is one mechanism involved in the cAMP-mediated modulation of 24OHase transcription.