Evidence for Functional Heterogeneity of Circulating B-Type Natriuretic Peptide
Faquan Liang, MD, PhD*,
Jessica O’Rear*,
Ute Schellenberger, PhD*,
Lungkuo Tai, PhD*,
Michael Lasecki*,
George F. Schreiner, MD, PhD*,
Fred S. Apple, PhD ,
Alan S. Maisel, MD ,
N. Stephen Pollitt, PhD* and
Andrew A. Protter, PhD*,*
* Scios, Inc., Fremont, California
Hennepin County Medical Center and University of Minnesota School of Medicine, Minneapolis, Minnesota
Veteran’s Affairs Medical Center, San Diego, California.
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Figure 2 Western Blot Analysis of Immunoreactive BNP Species in Plasma From HF Patients
(A) Plasma samples from 5 HF patients were incubated with BNP antibody-conjugated beads for 16 h at 4°C. A control sample with 1 ng recombinant proBNP and 3 ng BNP (lane 1) and the immunoprecipitates (lanes 2 to 6) from HF individuals were subjected to Western blot analysis. The BNP concentration in the patients’ plasma as determined by the Biosite Triage point-of-care assay is indicated. (B) Recombinant proBNP (lanes 1 to 3) and the immunoprecipitates (lanes 4 to 6) from an HF patient were incubated with or without deglycosylation enzymes (E) for 8 h, then subjected to Western blot analysis. BNP = B-type naturiuretic peptide; HF = heart failure.
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Figure 3 Comparison of proBNP With BNP on Stimulation of Intracellular cGMP Accumulation in Vascular Endothelial and Smooth Muscle Cells
Human aortic endothelial cells (HAEC), human coronary artery endothelial cells (HCAEC), human umbilical endothelial cells (HUVEC), and human aortic smooth muscle cells (HASMC) were preincubated and treated with increasing concentrations of BNP or proBNP for 10 min. Cells were lysed, and the levels of cGMP in the lysates were measured. Values are mean ± standard deviation. BNP = B-type naturiuretic peptide; cGMP = cyclic guanosine monophosphate.
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Figure 4 Biological Activity of Glycosylated Mammalian Cell-Derived proBNP With Unglycosylated Bacteria-Derived proBNP on Cellular Synthesis of cGMP in HAECs and CHO-GCA Cells
Cells were incubated with 0.1 mmol/l 3-isobutyl-1-methylxanthine for 1 h, treated with increasing concentrations of proBNP expressed either from CHO cells or from Escherichia coli for 10 min, and lysed for measurement of intracellular cGMP levels. BNP = B-type naturiuretic peptide; cGMP = cyclic guanosine monophosphate; CHO = Chinese hamster ovary; GCA = guanylyl cyclase receptor A; HAEC = human aortic endothelial cells.
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Figure 5 Protease Inhibition to Block Processing Does Not Affect proBNP Bioactivity
Cells were preincubated with 0.1 mmol/l 3 = isobutyl = 1 = methylzanthine for 1 h, then treated with the indicated concentrations of proBNP in the presence or absence of a protease inhibitor cocktail for 10 min. Cells were lysed and intracellular cGMP levels determined. BNP = B-type naturiuretic peptide; cGMP = cyclic guanosine monophosphate.
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Figure 6 Triage BNP and Advia Centaur BNP Tests Detect Both BNP and proBNP
BNP and recombinant (mammalian cell-derived) proBNP were added to normal human plasma. The levels of plasma BNP or proBNP were measured by Triage BNP test (A) or Advia Centaur test (B). BNP = B-type naturiuretic peptide.
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Figure 7 Elecsys 2010 proBNP Test Detects proBNP
BNP and recombinant (Mammalian cell-derived) proBNP added to normal human plasma. The levels of plasma BNP or proBNP were measured by the NT-proBNP test. BNP = B-type naturiuretic peptide.
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