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Increased expression of constitutive nitric oxide synthase III, but not inducible nitric oxide synthase II, in human heart failure ¹

Birgitt Stein, PhD^*, Thomas Eschenhagen, MD^*, Jochen Rüdiger, MS^*, Hasso Scholz, MD^*, Ulrich Förstermann, MD, PhD and Ingolf Gath, PhD

^* Institüt für Experimentelle und Klinische Pharmakologie ünd Toxikologie, Abteilung Pharmakologie, Universitäts-Krankenhaus Eppendorf, Universität Hamburg, Hamburg, Germany
Pharmakologisches Institut, Universität Johannes-Gutenberg, Mainz, Germany

View larger version (57K): [in a new window]   Figure 1 RNase protection analysis with antisense-RNA probes specific for human NOS II and NOS III. In lane 1 the unhybridized NOS II and NOS III antisense-probes are shown. Lanes 2 to 14 show hybridizations of the respective RNA samples with both antisense-probes: 2, total RNA from yeast (negative control); 3 to 10, increasing amounts of NOS II and NOS III sense-RNA (3 to 4: 0.5 pg, 5 to 6: 2.5 pg, 7 to 8: 5 pg, 9: 10 pg, 10: 20 pg); 11 to 12, total RNA from NF human hearts; 13 to 14, total RNA from failing human hearts.

View larger version (88K): [in a new window]   Figure 2 RNase protection analysis for the quantification of NOS II- and NOS III-mRNA levels in total RNA from left and right ventricular tissue of NF and failing human hearts from patients with dilated cardiomyopathy (dCMP), ischemic cardiomyopathy (iCMP) or postmyocarditis cardiomyopathy (mCMP). Shown is a representative autoradiogram. Lanes indicate: 1, length marker; 2 to 5, unhybridized antisense-RNA probes; 2, NOS II (619 nt); 3, NOS III (505 nt); 4, Gs (403 nt); 5, all three probes combined; 6 to 23, hybridization of the respective RNA samples with all three antisense-RNA probes; 6, NOS II (587 nt) and NOS III sense-RNA (462 nt); 7, 10 µg total RNA from yeast (negative control), 8 to 22, 20 µg total RNA from NF (8,15,16) and failing human hearts of patients with dCMP (10,12,18,20), iCMP (9,11,14,17) or mCMP (13,19,21,22); 23, 3 µg total RNA from stimulated human hepatocytes (NOS II positive control, 570 nt). Hybridization of total RNA from ventricular tissue with NOS II antisense-RNA, but not with NOS II antisense-RNA, yielded a protected fragment of the expected size (433 nt for NOS III). Sizes of antisense-RNA probes and protected fragments are given in parentheses in the legend.

View larger version (24K): [in a new window]   Figure 3 NOS III-mRNA levels in nonfailing (NF) and failing (F) human hearts from patients with dilated cardiomyopathy (dCMP), ischemic cardiomyopathy (iCMP) and postmyocarditis cardiomyopathy (mCMP) depicted as histogram (A) presenting means ± SEM in pg/µg total RNA, or as scatterdiagram (B) showing the individual pixel values. F depicts summarized data from all failing hearts. *p < 0.05 versus NF.

View larger version (42K): [in a new window]   Figure 4 Western blots of CHAPS-solubilized protein from nonfailing (NF) and failing human heart tissues from patients with dilated cardiomyopathy (dCMP), ischemic cardiomyopathy (iCMP) and postmyocarditis cardiomyopathy (mCMP). Electrophoreses were performed in 7.5% resolving gels and blots were immunostained with anti-NOS II antibody (upper panel) or anti-NOS III antibody (lower panel). For normalization of the NOS staining signal, blots were simultaneously immunostained with an antibody to beta-tubulin. Positive control proteins were included (CHAPS-solubilized homogenates, from lipopolysaccharide-induced RAW 264.7 macrophages for NOS II and from EA.hy 926 endothelial cells for NOS III). Protein samples were applied as follows: heart muscle samples (75 µg protein/lane), RAW 264.7 macrophages and EA.hy 926 endothelial cells (20 and 40 µg protein/lane, respectively). Results are representative of four independent experiments with identical results.

View larger version (65K): [in a new window]   Figure 5 Immunohistochemical localization of endothelial-type NOS III in human heart. (A) Staining of blood vessels within the myocardium. The anti-NOS III antibody selectively stained vascular endothelium (dCMP = idiopathic dilated cardiomyopathy; iCMP = ischemic cardiomyopathy; mCMP = postmyocarditis cardiomyopathy and NF = nonfailing heart). (B) Staining of cardiomyocytes (NF, dCMP, iCMP and mCMP) on the same slices as the blood vessels in A. Incubations with a rabbit nonimmune serum gave no staining in the myocardium (Neg, negative control, slice object: mCMP). The average diameter of cardiomyocytes from failing hearts was increased compared to NF hearts. Magnification in A and B 400x. Photomicrographs are representative of at least four experiments with identical results.