Fig. 1.

Vascular tissue and vascular smooth muscle cells (VSMCs) express 2 isoforms of glucose-6-phosphate dehydrogenase (G6PD). A: Western blot analyses were performed using homogenates obtained from the human internal mammary artery (HIMA), bovine coronary artery (BCA), cultured human coronary artery smooth muscle (HCASMC), and cultured bovine coronary artery smooth muscle (BCASMC) cells, respectively. Proteins were separated on 9% SDS-PAGE and immunoblotted using rabbit anti-G6PD antibody. Two distinct bands (a fast and slow migrating) of G6PD were observed as shown in the representative Western blot of 4 different experiments. B: 2-step RT and PCR were performed with total RNA isolated from HCASMC and BCASMC. OligodT (OdT) and random hexamers (RHx) were used for the RT, and specific primers for G6PD₅₄₅ (1,638 bp; NM_000402.4) were used for the PCR amplification. The PCR products were run on 1% agarose gel along with a DNA marker (M). C: G6PD₅₄₅ mRNA/cDNA isolated from HCASMCs and BCASMCs, respectively, exhibited 100% homology with the human (accession no. NM_000402.4; C,i) and bovine (accession No. NM_001244135.1; C,ii) G6PD₅₄₅ mRNA, reported in GenBank. Alignment of the first 93 nucleotides (5′-end) of the human and bovine G6PD₅₄₅ and G6pd₅₄₅ genes showed the presence of 2 ATG/start codons, located 90 nucleotides apart. C,iii: alignment of the first 30, NH₂-terminal amino acids between the human (NP_000393.4) and bovine (NP_001231064.1) orthologs of G6PD₅₄₅ protein shows 63.3% homology across the 2 species. Hu, human; gb, Genbank; Bv, bovine. D and E: bovine coronary arteries smooth muscle cells (BCASMCs) were transfected with Smartpool-G6PD-siRNAs (D) or G6PD₅₄₅-siRNA (E) separately and scramble/nontargeting (NT) siRNA sequence for 72 h. Western blot analyses were performed using rabbit anti-G6PD antibody. β-Actin (ACTB) was used as a loading control. UT, untransfected. A representative of 4 different Western blot experiments is shown. F: BCASMCs were cultured in serum-free or 10% serum containing cell culture media. Western blot analyses were performed using rabbit anti-G6PD antibody. The slow migrating G6PD isoform was not expressed in SMCs cultured in serum-free media; n = 3 in each group. G: basal G6PD activity was measured in SMCs cultured in serum-free and 10% serum containing media. The activity increased (*P < 0.05) by culturing the BCASMCs in 10% serum as compared to serum-free condition; n = 5 in each group; note overlapping circles represent number of experiments.