ABSTRACT
Endocardial cushion tissue is formed by an epithelial-mesenchymal transformation of endocardial cells, a process which results from an inductive interaction between the myocardium and endocardium within the atrioventricular (AV) and outflow tract (OT) regions of the heart. We report here that a protein previously found to be required for myocardially induced transformation of endocardial cells in vitro, ES/130, is highly expressed within the AV and OT regions not only by myocardial cells, but also by the endocardium and its mesenchymal progeny. Given these findings and others, we have tested the hypothesis that endocardial cushion tissue secretes factors which autoregulate its transformation to mesenchyme. Endocardial cushion tissue was cultured and its conditioned growth medium was harvested and applied to nontransformed endocardial cells maintained in the absence of the inductive myocardium. This treatment resulted in endocardial cell invasion into three-dimensional collagen gels plus increased expression of proteins associated with endocardial cell transformation in vivo. Whereas endocardial cushion tissue was found to express ES/130 protein in vivo and in vitro, minimal detection of ES/130 in its conditioned growth medium was observed in immunoblots. Attempts to inhibit the mesenchyme-promoting activity of the conditioned medium with ES/130 antisense were unsuccessful. However, strong intracellular ES/130 expression was detected in endocardial cells, and this expression correlated with the ability of endocardial cells to transform. For example, the minority of endocardial cultures that failed to transform in response to conditioned medium treatment also failed to undergo increased expression of ES/130. These observations are interpreted to suggest that (i) endocardial cushion tissue secretes factors that promote its transformation to mesenchyme, and (ii) while endocardial cushion tissue appears to signal through secretion of factors other than or in addition to ES/130, intracellular ES/130 expression nevertheless may be a target endocardial cell response required for endocardial cell transformation.
Subject(s)
Avian Proteins , Chick Embryo/embryology , Embryonic Induction , Endocardium/embryology , Extracellular Matrix Proteins/metabolism , Signal Transduction/physiology , Animals , Cells, Cultured , Culture Media, Conditioned/metabolism , Culture Media, Conditioned/pharmacology , Endocardium/drug effects , Endocardium/metabolism , Extracellular Matrix Proteins/genetics , Heart Septum/embryology , Mesoderm/cytology , Mesoderm/metabolism , Oligonucleotides, Antisense/pharmacology , RNA, Messenger/metabolismABSTRACT
ES/130 is a novel 130-kDa protein that has been linked previously to the transformation of endocardial endothelium into cushion mesenchyme. In the present study we report the localization of protein and mRNA for ES/130 in stages 7-plus through 20 chick embryos and present functional data related to a potential mechanism for ES/130. The temporal and spatial regulation of ES/130 expression suggests that this epithelial-to-mesenchymal transformation is a result of homogenetic induction. Functional studies indicate that myocardially derived ES/130 elicits expression of this protein by target AV endothelial cells, which is linked to a signal transduction cascade. The localization of ES/130 to other sites of inductive interactions (e.g., limb bud ectoderm, gut, and notochord) implies that this protein may have a more widespread importance to embryogenesis beyond its involvement in cardiac cushion tissue formation.
Subject(s)
Avian Proteins , Extracellular Matrix Proteins/metabolism , Heart/embryology , Mesoderm/physiology , Animals , Base Sequence , Cells, Cultured , Chick Embryo , Down-Regulation , Embryonic Induction , Endothelium/cytology , Endothelium/embryology , Extracellular Matrix Proteins/genetics , Gene Expression Regulation, Developmental , Immunohistochemistry , In Situ Hybridization , Molecular Sequence Data , Morphogenesis , RNA, Messenger/metabolism , Signal Transduction , Time FactorsABSTRACT
Electrophysiological approaches using intracellular microelectrode techniques have failed to critically test the hypothesis that cyclic AMP (cAMP) mediates the slow inhibitory postsynpatic potential (IPSP). The slow IPSP is not readily elicited, and the resting membrane potential is relatively insensitive to application of catecholamines and adenine nucleotides. However, comprehensive studies of voltage-dependent events in postganglionic neurons reveal three Ca2+-dependent potentials that are quite sensitive to catecholamines and adenine nucleotides. The hyperpolarizing afterpotential, the action potential shoulder, and the Ca2+ spike are all inhibited by alpha-adrenergic agonists, adenosine, and cAMP. We have proposed that simulation of alpha-adrenergic and adenosine receptors on the post-synaptic membrane results in antagonism of an inward Ca2+ current. Further experimentation is necessary to determine if cAMO acts as a second messenger or only by activating an adenosine receptor. Preliminary studies suggest that catecholamines and adenine nucleotides have similar and potent actions on the terminals of preganglionic axons. Here, inhibition of Ca2+ influx results in reduced acetylcholine release but facilitates high-frequency cholinergic transmission. More quantitative biophysical and pharmacological studies are required to better characterize the synaptic mechanisms in sympathetic ganglia.
