Mary O. Gray

Angiotensin II stimulates cardiac myocyte hypertrophy via paracrine release of TGF-β1 and endothelin-1 from fibroblasts

OBJECTIVE We sought to determine whether angiotensin II (Ang II) promotes hypertrophy of cardiac directly or via paracrine mechanisms mediated by cardiac fibroblasts. METHODS We studied neonatal rat cardiac myocytes and fibroblasts in culture as a model system. Paracrine effects of Ang II were identified using conditioned medium and co-culture experiments. RESULTS Ang II type 1 (AT1) receptors responsible for myocyte growth localized to fibroblasts in radioligand binding, emulsion autoradiography, Western analysis, and immunofluorescence staining experiments. The bulk of AT1 receptor binding in myocyte cultures (1343 +/- 472 sites/cell) was to Ang II receptors on contaminating fibroblasts (9747 +/- 2126 sites/cell). Ang II induced significant paracrine trophic effects on myocytes in conditioned medium (40% increase in protein synthesis over control) and co-culture (4-fold increase over control) experiments. TGF-beta 1 and endothelin-1 were paracrine mediators of hypertrophy in neutralization experiments. CONCLUSIONS Ang II stimulates cardiac myocyte hypertrophy via paracrine release of TGF-beta 1 and endothelin-1 from cardiac fibroblasts in a neonatal rat cell culture model.

An Improved Permeabilization Protocol for the Introduction of Peptides Into Cardiac Myocytes: Application to Protein Kinase C Research

We have developed an improved, less disruptive procedure for the transient permeabilization of neonatal cardiac myocytes using saponin. The method allows delivery of peptides to a high percentage of cells in culture without effects on long-term cell viability. Permeation was confirmed microscopically by cellular uptake of a fluorescently labeled peptide and biochemically by uptake of 125I-labeled calmodulin and a 20-kD protein kinase C epsilon fragment into the cells. The intracellular molar concentration of the introduced peptide was approximately 10% of that applied outside. We found no significant effects of permeabilization on spontaneous, phorbol ester-modulated, or norepinephrine-modulated contraction rates. Similarly, the expression of c-fos mRNA (measured 30 minutes after permeabilization) and the incorporation of [-14C]phenylalanine following agonist stimulation (measured 3 days after permeabilization) were not altered by saponin permeabilization. Finally, permeabilization of cells in the presence of a protein kinase C pseudosubstrate peptide, but not a control peptide, inhibited phorbol ester-induced [14C]phenylalanine incorporation into proteins by 80%. Our results demonstrate a methodology for the introduction of peptides into neonatal cardiac myocytes that allows study of their actions without substantial compromises in cell integrity.

Effect of anoxia/reperfusion on the reversible active/de-active transition of NADH-ubiquinone oxidoreductase (complex I) in rat heart.

The multi-subunit mammalian NADH-ubiquinone oxidoreductase (complex I) is part of the mitochondrial electron transport chain and physiologically serves to reduce ubiquinone with NADH as the electron donor. The three-dimensional structure of this enzyme complex remains to be elucidated and also little is known about the physiological regulation of complex I. The enzyme complex in vitro is known to exist as a mixture of active (A) and de-active (D) forms [Biochim. Biophys. Acta 1364 (1998) 169]. Studies are reported here examining the effect of anoxia and reperfusion on the A/D-equilibrium of complex I in rat hearts ex vivo. Complex I from the freshly isolated rat heart or after prolonged (1 h) normoxic perfusion exists in almost fully active form (87+/-2%). Either 30 min of nitrogen perfusion or global ischemia decreases the portion of active form of complex I to 40+/-2%. Upon re-oxygenation of cardiac tissue, complex I is converted back predominantly to the active form (80-85%). Abrupt alternation of anoxic and normoxic perfusion allows cycling between the two states of the enzyme. The possible role in the physiological regulation of complex I activity is discussed.

Cross Talk Between Angiotensin AT1 and α1-Adrenergic Receptors : Angiotensin II Downregulates α1a-Adrenergic Receptor Subtype mRNA and Density in Neonatal Rat Cardiac Myocytes

Abstract Signaling mediated by the angiotensin (Ang) II and α 1 -adrenergic receptor (α 1 -AR) pathways is important for cardiovascular homeostasis. However, it is unknown whether Ang II has any direct effect on α 1 -AR expression and signaling in cardiac myocytes. In the present study, we determined α 1 -AR subtype mRNA levels by RNase protection; receptor density by competition binding with 5-methylurapidil; and α 1 -AR mediated c- fos expression by Northern blot analysis. We found that Ang II had no effect on α 1b - and α 1d -AR mRNA levels but decreased the α 1a -AR mRNA level in a time- and dose-dependent manner. The maximal effect occurred at 6 hours with 100 nmol/L Ang II (40.0±8.2% reduction, n=4, P 1a -AR mRNA level induced by Ang II is mediated by the Ang II AT 1 receptor subtype and is associated with decreased stability of α 1a -AR mRNA. Corresponding to the changes in the α 1a -AR mRNA level, Ang II (100 nmol/L, 24 hours) reduced the density of high-affinity sites for 5-methylurapidil (α 1A -AR) by 29% (56.5±6.4 versus 79.0±11.6 fmol/mg protein, n=4, P 1 -AR stimulated c- fos induction, which could be blocked by 5-methylurapidil but not by chloroethylclonidine, was attenuated by Ang II preincubation (100 nmol/L, 24 hours). We conclude that there is previously undescribed cross talk between AT 1 receptors and α 1 -ARs. Ang II selectively downregulates α 1a -AR subtype mRNA and its corresponding receptor as well as α 1a -AR mediated expression of the immediate-early gene c- fos in cardiac myocytes.