The bulk of muscle cells of the mammalian heart atria contain elements found in endocrine cells. ... more The bulk of muscle cells of the mammalian heart atria contain elements found in endocrine cells. These elements include a highly developed Golgi complex, a relatively high proportion of rough endoplasmic reticulum and membrane-bound granules referred to as specific atrial granules. Morphologically as well as histochemically these granules resemble storage granules known to store polypeptide hormones (1,2).
To define the role of Irx4, a member of the Iroquoisfamily of homeobox transcription factors in m... more To define the role of Irx4, a member of the Iroquoisfamily of homeobox transcription factors in mammalian heart development and function, we disrupted the murine Irx4 gene. Cardiac morphology in Irx4-deficient mice (designatedIrx4 Δex2/Δex2) was normal during embryogenesis and in early postnatal life. AdultIrx4 Δex2/Δex2 mice developed a cardiomyopathy characterized by cardiac hypertrophy and impaired contractile function. Prior to the development of cardiomyopathy,Irx4 Δex2/Δex2 hearts had abnormal ventricular gene expression: Irx4-deficient embryos exhibited reduced ventricular expression of the basic helix-loop-helix transcription factor eHand (Hand1), increasedIrx2 expression, and ventricular induction of an atrial chamber-specific transgene. In neonatal hearts, ventricular expression of atrial natriuretic factor and α-skeletal actin was markedly increased. Several weeks subsequent to these changes in embryonic and neonatal gene expression, increased expression of hypertrophic m...
The development and long-term performance of a radioimmunoassay method for cardionatrin (C I = AN... more The development and long-term performance of a radioimmunoassay method for cardionatrin (C I = ANF 99-126) is described. The method was evaluated for accuracy, specificity and precision using different protocols and in various species. The antiserum raised against C I cross-reacts 100% with both human and rat C I and 122% with cardionatrin IV (C IV = ANF 1-126). Dextran-coated charcoal (DCC) and double antibody (DA) disequilibrium protocols were used for the separation of free from antibody-bound radioactivity. The sensitivity, coefficient of variation within assay and between assay for DCC was 4.6 pg/tube, 3.1% and 13.8% respectively, and 1.5 pg/tube, 3.8% and 9.1% for the DA. The mean normal plasma C I levels in human, rat and dog after plasma acidification and extraction using the DA method was 49.7 +/- 4.0; 253.6 +/- 19.6 and 59.9 +/- 3.4 pg/mL respectively.
Canadian Journal of Physiology and Pharmacology, 2001
Under physiological conditions, the endocrine heart contributes to the maintenance of cardiovascu... more Under physiological conditions, the endocrine heart contributes to the maintenance of cardiovascular homeostasis through the polypeptide hormones ANF and BNP, which are members of the natriuretic peptide (NP) family. Given that NPs are of interest from the basic and clinical points of view, the genetic expression and secretion of ANF and BNP as well as the nature of the interaction of these hormones with their receptors has been the subject of extensive studies since the discovery of ANF in 1980. Following hemodynamic overload, increased secretion of NPs by the heart can be seen. This change may occur without an increase in gene expression as observed for atrial NPs following acute volume expansion, or it can occur with an increase in both ANF and BNP gene expression in atria only as seen in mineralocorticoid escape during which it is obvious that a critical decrease in hormone stores must be reached before transcriptional activation occurs. Chronic hemodynamic pressure or volume ov...
Several laboratories have reported the isolation and amino acid sequence of peptides from mammali... more Several laboratories have reported the isolation and amino acid sequence of peptides from mammalian atria which are potent diuretic, natriuretic and vasodilatory agents (Flynn et al., 1983; Seidah et al., 1984; Atlas et a/., 1984; Currie et al., 1984; Kangawa & Matsuo, 1984; Misono et al., 1984). All of the peptides sequenced so far are either extended or truncated versions of a 28 residue disulphide-looped structure (cardionatrin I), whose sequence was reported initially from this laboratory (Flynn et al., 1983). It has now been shown that these biologically active peptides derive from the C-terminal region of a common precursor (procardionatrin or proatrialnatriuretic factor) whose structure was determined by cDNA sequencing (Yamanaka eC al., 1984; Maki et al., 1984; Oikawa et al., 1984; Seidmann et al., 1984; Flynn et al., 1985). It remains to be established which of the various peptides isolated to date are the natural cleavage products of the pro-protein. Using extraction conditions known to inhibit proteolysis (Bennett et al., 1981) we have not found the multitude of different peptides reported by other laboratories (Currie et al., 1984; Seidah et al., 1984). Frozen rat atria were ground with solid COz chips and homogenized in 1Ovol. of 1 .O M-acetic acid/l .O M-HCI/I% (w/v) NaCl. After centrifugation of the extracts the supernatants were passed through pre-wetted Sep-Pak (Waters) cartridges. Each cartridge was washed with 0.1% (v/v) trifluoroacetic acid (TFA) and eluted with 3ml of 80% (v/v) acetonitrile in 0.1% TFA. The combined cartridge eluates were freezedried, dissolved in 5 ml of 1.0111-acetic acid/l% (w/v) NaCl and fractionated on a Biol-Gel P10 column using the same solution. The fraction pools indicated by the bars in Fig. 1 contained diuretic and natriuretic activity when assayed by the procedure of de Bold (1982). These fraction pools were then further processed by reverse-phase h.p.l.c., as recently described (Flynn et al., 1985). This procedure showed that each of the areas of activity denoted in Fig. 1 contained a single peptide. Each of these peptides was then subjected to protein sequencing with an Applied Biosystems Gas Phase Model A70A Sequencer. The sequence of the peptide purified by reverse-phase h.p.1.c. from the area corresponding to CI in Fig. 1 was that of cardionatrin I (residues 123-150 of the pro-protein) sequenced previously by us (Flynn et al., 1983). The sequence of CII could not be determined because insufficient amounts were present. Amino acid sequence determination of CIII located the N-terminus of this peptide at position 73 of the pro-protein. The N-terminal sequence of CIV begins at Asp-25, which is the cleavage point for the removal of the signal sequence. Amino acid compositional analysis of CIII and CIV indicated that these peptides, like cardionatrin I , extend up to and include Tyr-150. Cardionatrin IV, therefore, is procardionatrin (proatrialnatriuretic factor) minus the two terminal arginine residues. Thus, four peptides, CIV and three others all derived from CIV, are the only peptides present in acid extracts of atrial muscle. When the fractions obtained in Fig. 1 were assayed by a
Background In hypertension with cardiac hypertrophy, the specific contributions to increased prod... more Background In hypertension with cardiac hypertrophy, the specific contributions to increased production of the cardiac natriuretic peptides (NP) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) by load and the hypertrophic process are not known. In the present work we determine ANF and BNP synthesis and secretion in the aortic-banded rat treated with dosage schedules of the ACE inhibitor ramipril that result in the prevention or regression of both hypertension and hypertrophy (high dosage) or in the prevention or regression of hypertrophy alone with persistent hypertension (low dosage). Myosin heavy chain (MHC) isoform switch was studied as an indicator of ventricular cardiocyte hypertrophy as well as the levels of collagen III mRNA as a measure of changes in extracellular matrix. Methods and Results Ramipril was administered for 6 weeks just after suprarenal aortic banding, or rats were banded for 6 weeks, after which ramipril was administered during the followin...
Techniques developed over the years in our laboratory for the study of tissue expression, storage... more Techniques developed over the years in our laboratory for the study of tissue expression, storage, and secretion of the cardiac hormones ANF, BNP, and CNP are described below. They have proven highly reliable in our hands when the steps outlined are followed as described. Given the generic nature of the procedures, these should be applicable to other polypeptides.
The cardiac natriuretic peptides atrial natriuretic factor (ANF) and brain natriuretic peptide (B... more The cardiac natriuretic peptides atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are discoordinately regulated in myocardial inflammation associated with acute allograft rejection in humans and during in vitro exposure of cardiocyte cultures to some proinflammatory cytokines. We used experimental autoimmune myocarditis (EAM) to determine whether the discoordinate regulation of ANF and BNP was specific to the situations above or was generally associated with other types of myocardial inflammation. The dependency of this process to angiotensin signaling was also determined, given that previous work demonstrated beneficial effects of the angiotensin receptor blocker olmesartan in myocarditis. Histopathological changes, plasma and cardiac ANF, BNP, and selected cytokines gene expression as well as plasma cytokine levels using a cytokine array were determined in EAM, angiotensin receptor blocker-treated, and control rats. It was found that EAM specifically increases B...
Atrial cardiocytes in the heart of mammals produce in a regulated manner the polypeptide hormones... more Atrial cardiocytes in the heart of mammals produce in a regulated manner the polypeptide hormones atrial natriuretic factor (ANF, ANP) and brain natriuretic peptide (BNP). The biological actions of ANF and BNP are similar; they include the modulation of systems that tend to increase extracellular fluid volume and blood pressure, such as the renin-angiotensin system and the sympathetic nervous system. Additionally, both hormones have potent growth-regulating properties. ANF and BNP signal by activating membrane-bound guanylyl cyclase receptors, leading to an increase in intracellular cGMP and thus affecting the activity of cGMP-regulated enzymes and ion channels. Under chronic hemodynamic overload, cardiac ANF and BNP synthesis and secretion are increased. This increase is viewed as a cardioprotective mechanism, given the beneficial effects of ANF and BNP on cardiac preload, afterload and cardiovascular growth. As discussed in this review, some basic facts regarding the synthesis and secretion of ANF and BNP and their peripheral effects remain to be clarified. Nevertheless, at the clinical level, the elevation of circulating ANF and BNP in heart failure or following acute coronary syndromes has been shown to have diagnostic and prognostic implications. Moreover, these peptides themselves hold promise as therapeutic agents in the treatment of heart failure. Additional pharmaceutical applications might be gleaned from current preclinical and clinical studies showing beneficial effects of ANF or BNP in the treatment of hypertension, bronchospasm and in tissue remodeling following acute myocardial infarction.
The increase in natriuretic peptides (NP), atrial natriuretic factor (ANF), and brain natriuretic... more The increase in natriuretic peptides (NP), atrial natriuretic factor (ANF), and brain natriuretic peptide (BNP) production and release by cardiocytes that occurs in hypertension has been considered to be a compensatory mechanism against ventricular overload. Studies on NP production ...
The cardiac natriuretic peptides (NPs) atrial natriuretic factor (ANF) and brain natriuretic pept... more The cardiac natriuretic peptides (NPs) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are polypeptide hormones synthesized, stored, and secreted by cardiac muscle cells (cardiocytes). The NPs modulate extracellular fluid volume and blood pressure and have potent growth-regulating properties, which make them of great interest for cardiac remodeling in acute myocardial infarction and congestive heart failure. We have observed that the production of NP can be coordinately or discoordinately regulated. In the former type, muscle stretch-elicited secretion triggers signals mediated by Gi/o protein, whereas agonists such as endothelin 1 independently signal through Gq. Discoordinated regulation is observed following stimulations by some cytokines, which selectively up-regulate BNP. This regulation takes place at the translational and transcriptional levels and is dependent on a p38 signaling pathway. Further details of processes regulating NP secretion need to be defined to develop a comprehensive view of the endocrine function of the heart. Nevertheless, translational research in the area of NPs has demonstrated the usefulness of these hormones as a marker of disease and as potential therapeutic agents. The latter application of NP is particularly attractive given that ANF and BNP possess pharmacologic actions that require polypharmacy in the treatment of acute myocardial infarction and congestive heart failure.
Adult rat ventricular cardiocytes, when cocultured with epicardial mesothelial cells (EMC), demon... more Adult rat ventricular cardiocytes, when cocultured with epicardial mesothelial cells (EMC), demonstrate remarkable plasticity of phenotype accompanied by a significant increase in cardiocyte contractile protein content, suggesting that a factor with growth-promoting properties may take part in EMC-adult rat ventricular cardiocyte interactions. Endothelin (ET) has been shown to induce cell hypertrophy, including enhancement of expression of muscle-specific genes. We investigated the ability of EMC to synthesize and release ET. By light microscopy, specific immunostaining, with either ET-1 or Big ET-1 antibodies, was visualized in EMC as a fine punctate distributed throughout the cytoplasm. Reverse phase-high performance liquid chromatography (HPLC) of epicardial mesothelial cells conditioned medium showed several peaks of immunoreactive ET. The major peak eluted with the same retention time as that of ET-1. By Northern blot analysis, a specific 2.3-kilobase (kb) mRNA species was detected by hybridization to a cDNA insert encoding for rat prepro-ET-1. ET accumulated in the culture medium in a time-dependent manner, whereas cell content remained comparatively low. Angiotensin II (AII) dose-dependently stimulated release of immunoreactive ET into the culture medium.
The bulk of muscle cells of the mammalian heart atria contain elements found in endocrine cells. ... more The bulk of muscle cells of the mammalian heart atria contain elements found in endocrine cells. These elements include a highly developed Golgi complex, a relatively high proportion of rough endoplasmic reticulum and membrane-bound granules referred to as specific atrial granules. Morphologically as well as histochemically these granules resemble storage granules known to store polypeptide hormones (1,2).
To define the role of Irx4, a member of the Iroquoisfamily of homeobox transcription factors in m... more To define the role of Irx4, a member of the Iroquoisfamily of homeobox transcription factors in mammalian heart development and function, we disrupted the murine Irx4 gene. Cardiac morphology in Irx4-deficient mice (designatedIrx4 Δex2/Δex2) was normal during embryogenesis and in early postnatal life. AdultIrx4 Δex2/Δex2 mice developed a cardiomyopathy characterized by cardiac hypertrophy and impaired contractile function. Prior to the development of cardiomyopathy,Irx4 Δex2/Δex2 hearts had abnormal ventricular gene expression: Irx4-deficient embryos exhibited reduced ventricular expression of the basic helix-loop-helix transcription factor eHand (Hand1), increasedIrx2 expression, and ventricular induction of an atrial chamber-specific transgene. In neonatal hearts, ventricular expression of atrial natriuretic factor and α-skeletal actin was markedly increased. Several weeks subsequent to these changes in embryonic and neonatal gene expression, increased expression of hypertrophic m...
The development and long-term performance of a radioimmunoassay method for cardionatrin (C I = AN... more The development and long-term performance of a radioimmunoassay method for cardionatrin (C I = ANF 99-126) is described. The method was evaluated for accuracy, specificity and precision using different protocols and in various species. The antiserum raised against C I cross-reacts 100% with both human and rat C I and 122% with cardionatrin IV (C IV = ANF 1-126). Dextran-coated charcoal (DCC) and double antibody (DA) disequilibrium protocols were used for the separation of free from antibody-bound radioactivity. The sensitivity, coefficient of variation within assay and between assay for DCC was 4.6 pg/tube, 3.1% and 13.8% respectively, and 1.5 pg/tube, 3.8% and 9.1% for the DA. The mean normal plasma C I levels in human, rat and dog after plasma acidification and extraction using the DA method was 49.7 +/- 4.0; 253.6 +/- 19.6 and 59.9 +/- 3.4 pg/mL respectively.
Canadian Journal of Physiology and Pharmacology, 2001
Under physiological conditions, the endocrine heart contributes to the maintenance of cardiovascu... more Under physiological conditions, the endocrine heart contributes to the maintenance of cardiovascular homeostasis through the polypeptide hormones ANF and BNP, which are members of the natriuretic peptide (NP) family. Given that NPs are of interest from the basic and clinical points of view, the genetic expression and secretion of ANF and BNP as well as the nature of the interaction of these hormones with their receptors has been the subject of extensive studies since the discovery of ANF in 1980. Following hemodynamic overload, increased secretion of NPs by the heart can be seen. This change may occur without an increase in gene expression as observed for atrial NPs following acute volume expansion, or it can occur with an increase in both ANF and BNP gene expression in atria only as seen in mineralocorticoid escape during which it is obvious that a critical decrease in hormone stores must be reached before transcriptional activation occurs. Chronic hemodynamic pressure or volume ov...
Several laboratories have reported the isolation and amino acid sequence of peptides from mammali... more Several laboratories have reported the isolation and amino acid sequence of peptides from mammalian atria which are potent diuretic, natriuretic and vasodilatory agents (Flynn et al., 1983; Seidah et al., 1984; Atlas et a/., 1984; Currie et al., 1984; Kangawa & Matsuo, 1984; Misono et al., 1984). All of the peptides sequenced so far are either extended or truncated versions of a 28 residue disulphide-looped structure (cardionatrin I), whose sequence was reported initially from this laboratory (Flynn et al., 1983). It has now been shown that these biologically active peptides derive from the C-terminal region of a common precursor (procardionatrin or proatrialnatriuretic factor) whose structure was determined by cDNA sequencing (Yamanaka eC al., 1984; Maki et al., 1984; Oikawa et al., 1984; Seidmann et al., 1984; Flynn et al., 1985). It remains to be established which of the various peptides isolated to date are the natural cleavage products of the pro-protein. Using extraction conditions known to inhibit proteolysis (Bennett et al., 1981) we have not found the multitude of different peptides reported by other laboratories (Currie et al., 1984; Seidah et al., 1984). Frozen rat atria were ground with solid COz chips and homogenized in 1Ovol. of 1 .O M-acetic acid/l .O M-HCI/I% (w/v) NaCl. After centrifugation of the extracts the supernatants were passed through pre-wetted Sep-Pak (Waters) cartridges. Each cartridge was washed with 0.1% (v/v) trifluoroacetic acid (TFA) and eluted with 3ml of 80% (v/v) acetonitrile in 0.1% TFA. The combined cartridge eluates were freezedried, dissolved in 5 ml of 1.0111-acetic acid/l% (w/v) NaCl and fractionated on a Biol-Gel P10 column using the same solution. The fraction pools indicated by the bars in Fig. 1 contained diuretic and natriuretic activity when assayed by the procedure of de Bold (1982). These fraction pools were then further processed by reverse-phase h.p.l.c., as recently described (Flynn et al., 1985). This procedure showed that each of the areas of activity denoted in Fig. 1 contained a single peptide. Each of these peptides was then subjected to protein sequencing with an Applied Biosystems Gas Phase Model A70A Sequencer. The sequence of the peptide purified by reverse-phase h.p.1.c. from the area corresponding to CI in Fig. 1 was that of cardionatrin I (residues 123-150 of the pro-protein) sequenced previously by us (Flynn et al., 1983). The sequence of CII could not be determined because insufficient amounts were present. Amino acid sequence determination of CIII located the N-terminus of this peptide at position 73 of the pro-protein. The N-terminal sequence of CIV begins at Asp-25, which is the cleavage point for the removal of the signal sequence. Amino acid compositional analysis of CIII and CIV indicated that these peptides, like cardionatrin I , extend up to and include Tyr-150. Cardionatrin IV, therefore, is procardionatrin (proatrialnatriuretic factor) minus the two terminal arginine residues. Thus, four peptides, CIV and three others all derived from CIV, are the only peptides present in acid extracts of atrial muscle. When the fractions obtained in Fig. 1 were assayed by a
Background In hypertension with cardiac hypertrophy, the specific contributions to increased prod... more Background In hypertension with cardiac hypertrophy, the specific contributions to increased production of the cardiac natriuretic peptides (NP) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) by load and the hypertrophic process are not known. In the present work we determine ANF and BNP synthesis and secretion in the aortic-banded rat treated with dosage schedules of the ACE inhibitor ramipril that result in the prevention or regression of both hypertension and hypertrophy (high dosage) or in the prevention or regression of hypertrophy alone with persistent hypertension (low dosage). Myosin heavy chain (MHC) isoform switch was studied as an indicator of ventricular cardiocyte hypertrophy as well as the levels of collagen III mRNA as a measure of changes in extracellular matrix. Methods and Results Ramipril was administered for 6 weeks just after suprarenal aortic banding, or rats were banded for 6 weeks, after which ramipril was administered during the followin...
Techniques developed over the years in our laboratory for the study of tissue expression, storage... more Techniques developed over the years in our laboratory for the study of tissue expression, storage, and secretion of the cardiac hormones ANF, BNP, and CNP are described below. They have proven highly reliable in our hands when the steps outlined are followed as described. Given the generic nature of the procedures, these should be applicable to other polypeptides.
The cardiac natriuretic peptides atrial natriuretic factor (ANF) and brain natriuretic peptide (B... more The cardiac natriuretic peptides atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are discoordinately regulated in myocardial inflammation associated with acute allograft rejection in humans and during in vitro exposure of cardiocyte cultures to some proinflammatory cytokines. We used experimental autoimmune myocarditis (EAM) to determine whether the discoordinate regulation of ANF and BNP was specific to the situations above or was generally associated with other types of myocardial inflammation. The dependency of this process to angiotensin signaling was also determined, given that previous work demonstrated beneficial effects of the angiotensin receptor blocker olmesartan in myocarditis. Histopathological changes, plasma and cardiac ANF, BNP, and selected cytokines gene expression as well as plasma cytokine levels using a cytokine array were determined in EAM, angiotensin receptor blocker-treated, and control rats. It was found that EAM specifically increases B...
Atrial cardiocytes in the heart of mammals produce in a regulated manner the polypeptide hormones... more Atrial cardiocytes in the heart of mammals produce in a regulated manner the polypeptide hormones atrial natriuretic factor (ANF, ANP) and brain natriuretic peptide (BNP). The biological actions of ANF and BNP are similar; they include the modulation of systems that tend to increase extracellular fluid volume and blood pressure, such as the renin-angiotensin system and the sympathetic nervous system. Additionally, both hormones have potent growth-regulating properties. ANF and BNP signal by activating membrane-bound guanylyl cyclase receptors, leading to an increase in intracellular cGMP and thus affecting the activity of cGMP-regulated enzymes and ion channels. Under chronic hemodynamic overload, cardiac ANF and BNP synthesis and secretion are increased. This increase is viewed as a cardioprotective mechanism, given the beneficial effects of ANF and BNP on cardiac preload, afterload and cardiovascular growth. As discussed in this review, some basic facts regarding the synthesis and secretion of ANF and BNP and their peripheral effects remain to be clarified. Nevertheless, at the clinical level, the elevation of circulating ANF and BNP in heart failure or following acute coronary syndromes has been shown to have diagnostic and prognostic implications. Moreover, these peptides themselves hold promise as therapeutic agents in the treatment of heart failure. Additional pharmaceutical applications might be gleaned from current preclinical and clinical studies showing beneficial effects of ANF or BNP in the treatment of hypertension, bronchospasm and in tissue remodeling following acute myocardial infarction.
The increase in natriuretic peptides (NP), atrial natriuretic factor (ANF), and brain natriuretic... more The increase in natriuretic peptides (NP), atrial natriuretic factor (ANF), and brain natriuretic peptide (BNP) production and release by cardiocytes that occurs in hypertension has been considered to be a compensatory mechanism against ventricular overload. Studies on NP production ...
The cardiac natriuretic peptides (NPs) atrial natriuretic factor (ANF) and brain natriuretic pept... more The cardiac natriuretic peptides (NPs) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are polypeptide hormones synthesized, stored, and secreted by cardiac muscle cells (cardiocytes). The NPs modulate extracellular fluid volume and blood pressure and have potent growth-regulating properties, which make them of great interest for cardiac remodeling in acute myocardial infarction and congestive heart failure. We have observed that the production of NP can be coordinately or discoordinately regulated. In the former type, muscle stretch-elicited secretion triggers signals mediated by Gi/o protein, whereas agonists such as endothelin 1 independently signal through Gq. Discoordinated regulation is observed following stimulations by some cytokines, which selectively up-regulate BNP. This regulation takes place at the translational and transcriptional levels and is dependent on a p38 signaling pathway. Further details of processes regulating NP secretion need to be defined to develop a comprehensive view of the endocrine function of the heart. Nevertheless, translational research in the area of NPs has demonstrated the usefulness of these hormones as a marker of disease and as potential therapeutic agents. The latter application of NP is particularly attractive given that ANF and BNP possess pharmacologic actions that require polypharmacy in the treatment of acute myocardial infarction and congestive heart failure.
Adult rat ventricular cardiocytes, when cocultured with epicardial mesothelial cells (EMC), demon... more Adult rat ventricular cardiocytes, when cocultured with epicardial mesothelial cells (EMC), demonstrate remarkable plasticity of phenotype accompanied by a significant increase in cardiocyte contractile protein content, suggesting that a factor with growth-promoting properties may take part in EMC-adult rat ventricular cardiocyte interactions. Endothelin (ET) has been shown to induce cell hypertrophy, including enhancement of expression of muscle-specific genes. We investigated the ability of EMC to synthesize and release ET. By light microscopy, specific immunostaining, with either ET-1 or Big ET-1 antibodies, was visualized in EMC as a fine punctate distributed throughout the cytoplasm. Reverse phase-high performance liquid chromatography (HPLC) of epicardial mesothelial cells conditioned medium showed several peaks of immunoreactive ET. The major peak eluted with the same retention time as that of ET-1. By Northern blot analysis, a specific 2.3-kilobase (kb) mRNA species was detected by hybridization to a cDNA insert encoding for rat prepro-ET-1. ET accumulated in the culture medium in a time-dependent manner, whereas cell content remained comparatively low. Angiotensin II (AII) dose-dependently stimulated release of immunoreactive ET into the culture medium.
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