Identification of G protein-coupled signaling pathways in cardiac fibroblasts: cross talk between G(q) and G(s).

Cardiac fibroblasts (CFs) are an important cellular component of myocardial responses to injury and to hypertrophic stimuli. We studied G protein-coupled receptors to understand how CFs integrate signals that activate G(q), G(s), and G(i). We predicted that the second messenger pathways present in CFs were distinct from those in cardiac myocytes and that unique signaling interactions existed in the CFs. ANG II, bradykinin, ATP, and UTP stimulated inositol phosphate (IP) production 2.2- to 7-fold. Each of these agonists elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) via release from the intracellular Ca(2+) storage compartment. Endothelin-1 (ET-1), carbachol, and norepinephrine failed to increase either IP production or [Ca(2+)](i). Although agonists that activated IP and Ca(2+) transients had no effect on cAMP production when administered alone, these agents potentiated the beta(2)-adrenergic response two- to fourfold. Hormones known to inhibit adenylyl cyclase activity in cardiac myocytes, such as ET-1 and carbachol, failed to lower the beta-adrenergic response in fibroblasts. Order of potency and inhibitor data indicate that the functional receptor subtypes in these cells are beta(2), P2Y(2), and AT(1) for isoproterenol, ATP, and ANG II, respectively. We conclude that CFs express functional G protein-linked receptors that couple to G(q) and G(s), with little or no coupling to G(i). The expression of receptors and their coupling to G(q)- but not to G(i)-linked responses distinguishes the signaling in CFs from that in myocytes. Furthermore, agonists that activate G(q) in CFs potentiate stimulation of G(s), an example of signaling cross talk not observed in adult myocytes. These data suggest that G protein-mediated signaling in CFs is unique and may contribute to the specificity of hormone and drug action on individual cell types within the heart.

Endothelin ETA receptor regulates signaling and ANF gene expression via multiple G protein-linked pathways.

We have characterized the interaction of endothelin (ET) with cultured neonatal rat ventricular myocytes. Binding studies indicate a single population of ETA receptors [53,000 sites/cell, apparent dissociation constant (Kd) for ET-1 approximately 0.07 nM]. Analysis of mRNA levels for ET receptors using 35 cycles of reverse transcriptase-polymerase chain reaction demonstrates the presence of only ETA-receptor message. Studies with ET-1 and a variety of congeners and antagonists indicate that ETA receptors couple to both the stimulation of phosphoinositide turnover and the inhibition of adenylyl cyclase. In myocytes transfected with an atrial natriuretic factor (ANF) promoter linked to a luciferase reporter gene, ET-1 stimulates luciferase expression through an ETA receptor. These data indicate that the ETA receptor is the exclusive receptor on neonatal ventricular myocytes and that this receptor couples to both phosphoinositide hydrolysis and adenylyl cyclase. ET-1 also induces a threefold increase in mitogen-activated protein kinase (MAPK) activity, an effect that is not sensitive to pertussis toxin (PTx). By contrast, ET-stimulated ANF-luciferase expression is partially inhibited by treatment of cells with PTx, suggesting that both PTx-sensitive (Gi) and PTx-insensitive (Gq) pathways mediate the effects of ET-1 on ANF gene expression in neonatal myocytes and that hormonal regulation of ANF expression may utilize pathways in addition to the activation of MAPK.

Expression of renin and angiotensin-converting enzyme in human hearts.

To understand the significance of the tissue renin-angiotensin system in the heart, we examined the expression of renin and angiotensin-converting enzyme (ACE) in autopsied human hearts. Samples were taken from organs obtained at autopsy from 15 patients without heart disease and 3 patients with heart disease (old myocardial infarctions, acute myocardial infarctions, and hypertrophic cardiomyopathy). We examined the expression of renin and ACE mRNA by using the reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR showed the expression of renin in the right atria in all patients. However, expression of renin mRNA in the left ventricles was not found in any of the 15 hearts without heart disease. In contrast, renin mRNA was detected in the left ventricles in hearts with heart disease. ACE mRNA was detected in both the atria and the ventricles in normal hearts, and its expression did not alter in diseased hearts. These findings suggest that renin mRNA is expressed mainly in the right atria in normal hearts, but that its expression in the left ventricle can be activated in some pathological conditions.