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Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes

Stephen M. Dodge, MD^*,a,b,c, Michael A. Beardslee, MD^a,b,c, Bruce J. Darrow, MD, PhD^,a,b,c, Karen G. Green, BA^a,b,c, Eric C. Beyer, MD, PhD^,a,b,c and Jeffrey E. Saffitz, MD, PhD, FACC^a,b,c

^a Department of Pathology, Washington University School of Medicine, Saint Louis, Missouri, USA
^b Department of Medicine, Washington University School of Medicine, Saint Louis, Missouri, USA
^c Department of Pediatrics, Washington University School of Medicine, Saint Louis, Missouri, USA

View larger version (21K): [in a new window]   Figure 1 A, Representative immunoblot comparing the amount of Cx43 in control cultures and cultures treated for 6 or 24 h with angiotensin II (A-II) or for 24 h with dibutyryl cAMP (db-cAMP). Hatch marks to the right of the numbers in A indicate the positions of molecular weight markers from 66 to 14 kDa. B, Densitometric quantification of immunoblot band intensities in cultures treated with angiotensin (A-II) for 6 or 24 h or dibutyryl cAMP (db-cAMP) for 6 h. The angiotensin effect was blocked by losartan (Los). The values (means ± SD) from treated cultures have been normalized to values in control cultures in each of four separate experiments. Asterisks indicate p < 0.05 compared with controls.

View larger version (96K): [in a new window]   Figure 2 Representative confocal microscopic images of Cx43 immunoreactive signal in control myocytes (left panel) and myocytes exposed to angiotensin II for 24 h (right panel). The gap junctional signal appears as bright, punctate spots concentrated at sites of cellular apposition. A greater number and larger size of spots is apparent in the treated cultures.

View larger version (12K): [in a new window]   Figure 3 Quantitative confocal analysis of Cx43 immunofluorescence signal in cultures treated for 24 h with angiotensin II (A-II) in the presence or absence of losartan (Los). Values (means ± SD) have been normalized to values in control cultures in each of four separate experiments. Asterisks indicate p < 0.02 compared with controls.

View larger version (153K): [in a new window]   Figure 4 Representative electron micrographs of sites of intercellular connections in control myocytes (A) and myocytes exposed to angiotensin II for 24 h (B). Individual gap junction profiles are identified by arrows. Bar = 1.0 µm.

View larger version (22K): [in a new window]   Figure 5 A, Representative immunoprecipitation assay showing the amount of radioactivity incorporated into Cx43 during a 2-h interval of metabolic labeling in control cultures or in cultures treated with angiotensin II (A-II) for 6 or 24 h, angiotensin plus losartan (A-II + Los) for 24 h, or dibutyryl cAMP (db-cAMP) for 6 or 24 h. B, Densitometric quantification of signal intensity in immunoprecipitation studies of control cultures or cultures treated with angiotensin II (A-II) (n = 4 for each), angiotensin II plus losartan (A-II + Los) (n = 4) or dibutyryl cAMP (db-cAMP) (n = 2). Values (means ± SD) have been normalized to control values in each experiment. Asterisks indicate p < 0.03.