Differential regulation of cardiac angiotensin converting enzyme binding sites and AT1 receptor density in the failing human heart

BACKGROUND: The regulation and interaction of ACE and the angiotensin II (Ang II) type I (AT1) receptor in the failing human heart are not understood. METHODS AND RESULTS: Radioligand binding with 3H-ramiprilat was used to measure ACE protein in membrane preparations of hearts obtained from 36 subjects with idiopathic dilated cardiomyopathy (IDC), 8 subjects with primary pulmonary hypertension (PPH), and 32 organ donors with normal cardiac function (NF hearts). 125I-Ang II formation was measured in a subset of hearts. Saralasin (125I-(Sar1,Ile8)-Ang II) was used to measure total Ang II receptor density. AT1 and AT2 receptor binding were determined with the AT1 receptor antagonist losartan. Maximal ACE binding (Bmax) was 578+/-47 fmol/mg in IDC left ventricle (LV), 713+/-97 fmol/mg in PPH LV, and 325+/-27 fmol/mg in NF LV (P<0.001, IDC or PPH versus NF). In IDC, PPH, and NF right ventricles (RV), ACE Bmax was 737+/-78, 638+/-137, and 422+/-49 fmol/mg, respectively (P=0.02, IDC versus NF; P=0.08, PPH versus NF). 125I-Ang II formation correlated with ACE binding sites (r=0.60, P=0.00005). There was selective downregulation of the AT1 receptor subtype in failing PPH ventricles: 6.41+/-1.23 fmol/mg in PPH LV, 2.37+/-0.50 fmol/mg in PPH RV, 5.38+/-0.53 fmol/mg in NF LV, and 7.30+/-1.10 fmol/mg in NF RV (P=0.01, PPH RV versus PPH LV; P=0.0006, PPH RV versus NF RV). CONCLUSIONS: ACE binding sites are increased in both failing IDC and nonfailing PPH ventricles. In PPH hearts, the AT1 receptor is downregulated only in the failing RV.     

Regulation of the mRNA-binding protein AUF1 by activation of the beta-adrenergic receptor signal transduction pathway

In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors ( beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreased (approximately 40%) beta2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to beta1-AR 3'-untranslated region mRNA.     

Effects of thyroid hormone on cardiac beta-adrenergic responsiveness in conscious baboons

BACKGROUND: Many of the cardiovascular manifestations of thyroid hormone excess resemble those produced by sympathoadrenal stimulation. The objective of this study was to determine the effects of thyroid hormone excess on myocardial beta-adrenergic expression and responsiveness to infused agonists in the primate heart. METHODS AND RESULTS: The responses of left ventricular isovolumic contraction (dP/dt(max)) and relaxation (tau) during graded dobutamine infusion were studied both before and after 4 weeks of thyroid hormone administration in 8 chronically instrumented baboons. At matched (atrially paced) heart rates, thyroid hormone significantly increased resting dP/dt(max) (3073+/-1034 versus 2318+/-829 mm Hg/s, P<.05) and decreased tau (24.0+/-5.5 versus 28.2+/-5.4 ms, P<.05). The change from baseline for dP/dt(max) and tau in response to beta1-adrenergic stimulation was significant at each dobutamine dose (2.5 to 10 microg x kg(-1) x min(-1)), but when expressed as a percent change, it was similar before versus after thyroid hormone. Similar changes were found when beta2-adrenergic stimulation was produced by terbutaline infusion in three additional baboons. beta-Adrenergic receptor (betaAR) expression was higher in five thyroxine-treated than in five control baboons (37.4+/-1.2 versus 15.7+/-3.2 fmol/mg, P<.001), and this was due to a greater increase in the beta2AR (5.9+/-1.5 to 20.6+/-1.2 fmol/mg, P<.001) than the beta1AR (9.7+/-1.7 to 16.8+/-0.1 fmol/mg, P<.01) subtype. CONCLUSIONS: In the primate heart, thyroid hormone produces positive inotropic and lusitropic effects in the resting state and upregulates both beta1AR and beta2AR, with the beta2AR increase predominating. At equivalent rates, however, thyroid hormone excess does not appear to enhance the sensitivity of left ventricular contractility and relaxation to either beta1- or beta2-adrenergic stimulation.     

Global increases in seizure susceptibility in mice lacking 5-HT2C receptors: a behavioral analysis

OBJECTIVE: Vasodilation by beta-adrenergic receptors of smooth muscle cells appears to be impaired early after the onset of hypercholesteremia. The aim of this study was to analyze the modulation of beta-adrenergic receptor density and adenylyl cyclase activity in the presence of moderately elevated concentrations of LDL. The effects of beta 1- and beta 2-adrenergic receptor antagonists on LDL-induced receptor changes were studied. METHODS AND RESULTS: Media explants of porcine coronary arteries were incubated with moderately elevated LDL concentrations (0.7-3.9 mmol/l). The density of beta-adrenergic receptors was determined in plasma membranes using the radioligand [125I]iodocyanopinodolol. LDL (3.9 mmol/l) resulted in a decrease of beta-adrenergic receptor density (control 137 +/- 5 vs. 89 +/- 7 fmol/mg protein, P < 0.01). After removal of LDL and cultivation for an additional 3 days beta-adrenergic receptors increased to 129 +/- 5 fmol/mg. In the presence of the beta 1- or beta 2-adrenergic receptor antagonists the LDL-mediated decrease was inhibited. Addition of metoprolol after 3 days of LDL incubation caused a restoration of receptor density. The basal, isoproterenol- and forskolin-stimulated adenylyl cyclase activities were increased after LDL incubation by 180, 110 or 80%, respectively. CONCLUSION: Moderately elevated LDL levels decreased beta-adrenergic receptor density while adenylyl cyclase activity was simultaneously increased. beta 1- or beta 2-adrenergic receptor antagonists prevented this receptor decrease and might preserve the beta-adrenergic receptor density in the presence of moderately elevated LDL levels.     

Changes in gene expression in the intact human heart. Downregulation of alpha-myosin heavy chain in hypertrophied, failing ventricular myocardium

Using quantitative RT-PCR in RNA from right ventricular (RV) endomyocardial biopsies from intact nonfailing hearts, and subjects with moderate RV failure from primary pulmonary hypertension (PPH) or idiopathic dilated cardiomyopathy (IDC), we measured expression of genes involved in regulation of contractility or hypertrophy. Gene expression was also assessed in LV (left ventricular) and RV free wall and RV endomyocardium of hearts from end-stage IDC subjects undergoing heart transplantation or from nonfailing donors. In intact failing hearts, downregulation of beta1-receptor mRNA and protein, upregulation of atrial natriuretic peptide mRNA expression, and increased myocyte diameter indicated similar degrees of failure and hypertrophy in the IDC and PPH phenotypes. The only molecular phenotypic difference between PPH and IDC RVs was upregulation of beta2-receptor gene expression in PPH but not IDC. The major new findings were that (a) both nonfailing intact and explanted human ventricular myocardium expressed substantial amounts of alpha-myosin heavy chain mRNA (alpha-MHC, 23-34% of total), and (b) in heart failure alpha-MHC was downregulated (by 67-84%) and beta-MHC gene expression was upregulated. We conclude that at the mRNA level nonfailing human heart expresses substantial alpha-MHC. In myocardial failure this alteration in gene expression of MHC isoforms, if translated into protein expression, would decrease myosin ATPase enzyme velocity and slow speed of contraction.     

Tocolytic therapy with fenoterol induces selective down-regulation of beta-adrenergic receptors in human myometrium

Tocolytic therapy with beta-adrenergic receptor agonists is a standard regimen to prevent preterm birth. Agonists exposure of beta-adrenergic receptors causes receptor desensitization in other organs, and this may limit the therapeutic value of beta-adrenergic receptor agonists. To study the effects of prolonged beta-adrenergic agonist treatment in human myometrium, we obtained biopsies during Caesarean section of 14 pregnant patients who had received fenoterol for at least 5 days and 14 untreated pregnant controls. The densities of total beta-adrenergic receptors, which are mainly of the beta 2-subtype as assessed by [125I]iodo-cyanopindolol binding in crude membrane fractions, were more than 50% smaller in women receiving fenoterol, whereas alpha 2-adrenergic receptor densities were similar. Gs and Gi G-protein alpha-subunit densities were unaltered as assessed by Western blotting and pertussis toxin-catalyzed [32P]ADP-ribosylation. beta-Adrenergic receptor kinase (beta ARK) activity, as determined using bovine rhodopsin as the substrate, was the same in the two groups. Adenylyl cyclase activities in the presence of guanine nucleotides, NaF, forskolin, or Mn+2 were also not altered by fenoterol treatment. The messenger RNA (mRNA) concentrations of beta 2-adrenergic receptors, beta ARK-I and glyceraldehyde-3-phosphate dehydrogenase (as a reference), as determined by quantitative PCR, were unaffected by fenoterol treatment. We conclude that tocolysis with fenoterol results in a selective down-regulation of myometrial beta-adrenergic receptors, which is not associated with a reduction in the respective mRNA concentrations or alterations of alpha 2-adrenergic receptors, Gs and Gi alpha-subunits, or beta ARK activity or mRNA.     

Influence of RF capacitive heating on the alpha 1-adrenergic receptors of rat prostates

The aim of this study was to find out the influence of radiofrequency (RF) capacitive heating on the alpha 1-adrenergic receptor of rat prostates. The prostates of 30-week-old Wistar rats were submitted to a 1-hour single session of RF capacitive heating at 45 degrees C. The ventral prostates that were submitted to heating were compared to those of other rats that were not submitted to heating. In order to determine the density of alpha 1-adrenergic receptors in rat ventral prostates, binding assays for alpha 1-adrenergic receptor were performed with [3H]prazosin in membrane preparations. The receptor density in the control group was 27.07 +/- 3.75 fmol/mg protein. The alpha 1-adrenergic receptor density (Bmax) in the thermotherapy group was 17.91 +/- 5.15 fmol/mg protein. A remarkable decrease in the density of alpha 1-adrenergic receptors was observed in the rat prostates of the thermotherapy group. In conclusion, the present results demonstrate that heating the rat prostate by RF capacitive heating damages the alpha 1-adrenergic receptors.     

Troponin T mRNA and protein isoforms in the human left ventricle: pattern of expression in failing and control hearts

We and others have previously cloned several cDNAs of human cardiac troponin T (cTnT), demonstrating the multiplicity of cTnT isoforms in the human heart. Four of them named cTnT1, 2, 3 and 4 result from a combinatorial alternative inclusion of 30- and 15-nucleotides in the 5' coding region of the cDNAs. In failing human ventricles, increased expression of cTnT4 has been reported at the protein level. More recent RT-PCR experiments showed increased expression of fetal-type splicing products in the 5' region, one of them corresponding to cTnT1. To clarify this issue, we examined the accumulation of the 4 cTnT mRNA and protein species in left ventricular specimens at the time of heart transplantation, and in control left ventricular samples using RNase protection and Western blotting. In all samples, cTnT3 was the major mRNA isoform, cTnT4 a minor isoform while cTnT1 and cTnT2 mRNAs were present but barely detectable. At the protein level, cTnT3, 4 and 1 were detected with the same relative abundance as that seen at the mRNA level. In addition, we detected a fourth TnT species of very low abundance corresponding either to a skeletal or to a "short" cardiac TNT isoform. Compared to controls, increased levels of cTnT4 mRNA and protein were detected in only half the failing ventricles independently of the cause of failure, suggesting that this increase may not be a general characteristic of left ventricular failure but instead could be related to stress. Unexpectedly, we found a decrease in cTnT1 protein expression in all failing ventricular samples studied, compared to controls.     

Deficient muscle carnitine transport in primary carnitine deficiency

The effects of conditions that either increase or decrease heart rate on the pharmacological properties of adenosine receptors in cultured rat myocytes were examined. Levels of A1 adenosine receptors, following prolonged treatment with electrical stimulation (ES) or the antiarrhythmic drug amiodarone, were determined using radioligand binding with the specific A1 receptor antagonist [3H]1,3-dipropyl-8-cyclopentylxanthine (CPX). The effects of lowering temperature were also explored. Exposure to amiodarone for 4 days reduced the density of A1 receptors by 19% (from 24.7 +/- 0.4 to 20.09 +/- 0.3 fmol/dish) and inhibited the rate of contraction by 60% (from 188 +/- 16 to 76 +/- 30 beats/min), without changing the receptor affinity, protein content, creatine kinase (CK) activity or cell number. Electrical stimulation at 25 degrees C elevated the density of A1 adenosine receptors by 185% (from 4.1 +/- 0.4 to 11.69 +/- 2.1 fmol/dish). Four days of reduced temperature (from 37 degrees C to either 30 or 25 degrees C) lowered the density of A1 adenosine receptors by 69 or 86%, respectively (from 24.1 +/- 1.2 to 7.4 +/- 0.4 or 3.4 +/- 0.3 fmol/dish), with no significant change in the receptor affinity, activity of CK, or lactate dehydrogenase (LDH), protein content or cell number. The observed up- and down-regulation of A1 adenosine receptors in primary myocyte cultures in response to conditions that exogenously alter the rate of contraction, is indicative of the role of adenosine receptors in adaptation of heart cells to stress.     

Nuclear matrix proteins and their potential applications to diagnostic pathology

The sympathetic nervous system controls lipolysis in fat by activation of four adrenergic receptors: beta1, beta2, beta3, and alpha2. During pregnancy, maternal metabolism presents anabolic and catabolic phases, characterized by modifications of fat responsiveness to catecholamines. The contributions of the four adrenergic receptors to adipocyte responsiveness during pregnancy have never been studied. Our aim was to evaluate the influence of pregnancy on adrenergic receptor-mediated lipolysis in rabbit white adipocytes. Functional studies were performed using subtype-selective and non-selective adrenergic receptor agonists. Overall adrenergic responsiveness was measured with the physiological agonist epinephrine. Non-adrenergic agents were used to evaluate different steps of the lipolytic cascade. The alpha2- and beta1/beta2-adrenergic receptor numbers were determined with selective radioligands. Non-adrenergic agents revealed that pregnancy induced an intracytoplasmic modification of the lipolytic cascade in inguinal but not in retroperitoneal adipocytes. Pregnancy induced an increase in beta1- and specially beta3-mediated lipolysis. The amounts of adipocyte beta1/beta2- and alpha2-adrenergic receptors were increased in pregnant rabbits. Epinephrine effects revealed an increased contribution of alpha2-adrenergic receptor-mediated antilipolysis in adipocytes from pregnant rabbits. These results indicate that pregnancy regulates adipocyte responsiveness to catecholamines mainly via the alpha2- and beta3-adrenergic pathways. Pregnancy induces an intracytoplasmic modification of the lipolytic cascade, probably via hormone-sensitive lipase, with differences according to fat location.-Bousquet-Mélou, A., C. Mu?oz, J. Galitzky, M. Berlan, and M. Lafontan. Pregnancy modifies the alpha2-beta-adrenergic receptor functional balance in rabbit fat cells.     

Angiotensin II type 2 receptor is upregulated in human heart with interstitial fibrosis, and cardiac fibroblasts are the major cell type for its expression

The expression pattern of angiotensin (Ang) II type 2 receptor (AT2-R) in the remodeling process of human left ventricles (LVs) remains poorly defined. We analyzed its expression at protein, mRNA, and cellular levels using autopsy, biopsy, or operation LV samples from patients with failing hearts caused by acute (AMI) or old (OMI) myocardial infarction and idiopathic dilated cardiomyopathy (DCM) and also examined functional biochemical responses of failing hearts to Ang II. In autopsy samples from the nonfailing heart group, the ratio of AT1-R and AT2-R was 59% and 41%, respectively. The expression of AT2-R was markedly increased in DCM hearts at protein (3.5-fold) and mRNA (3.1-fold) levels compared with AMI or OMI. AT1-R protein and mRNA levels in AMI hearts showed 1.5- and 2.1-fold increases, respectively, whereas in OMI and DCM hearts, AT1-R expression was significantly downregulated. AT1-R-mediated response in inositol phosphate production was significantly attenuated in LV homogenate from failing hearts compared with nonfailing hearts. AT2-R sites were highly localized in the interstitial region in either nonfailing or failing heart, whereas AT1-R was evenly distributed over myocardium at lower densities. Mitogen-activated protein kinase (MAPK) activation by Ang II was significantly decreased in fibroblast compartment from the failing hearts, and pretreatment with AT2-R antagonist caused an additional significant increase in Ang II-induced MAPK activity (36%). Cardiac hypertrophy suggested by atrial and brain natriuretic peptide levels was comparably increased in OMI and DCM, whereas accumulation of matrix proteins such as collagen type 1 and fibronectin was much more prominent in DCM than in OMI. These findings demonstrate that (1) AT2-R expression is upregulated in failing hearts, and fibroblasts present in the interstitial regions are the major cell type responsible for its expression, (2) AT2-R present in the fibroblasts exerts an inhibitory effect on Ang II-induced mitogen signals, and (3) AT1-R in atrial and LV tissues was downregulated during chronic heart failure, and AT1-R-mediated functional biochemical responsiveness was decreased in the failing hearts. Thus, the expression level of AT2-R is likely determined by the extent of interstitial fibrosis associated with heart failure, and the expression and function of AT1-R and AT2-R are differentially regulated in failing human hearts.     

The effect of combretastatin A-4 disodium phosphate in a C3H mouse mammary carcinoma and a variety of murine spontaneous tumors

OBJECTIVE: Human myometrium contains both beta1-adrenergic and beta2-adrenergic receptors. This study was designed to assess the importance of each beta-adrenergic receptor subtype in relaxation of human myometrial muscle strips. STUDY DESIGN: Radioligand binding studies were used to establish the presence of each beta-adrenergic receptor subtype, whereas highly selective beta1-antagonists and beta2-antagonists were used to assess the contribution of beta-adrenergic receptor subtypes to myometrial relaxation after exposure to (-)-isoproterenol. RESULTS: Membranes prepared from myometrium contained 82% +/- 4% beta2-adrenergic receptors. After contraction produced by exposure to potassium chloride (35 mmol/L), isoproterenol produced relaxation with half maximal effect at 0.02 micromol/L and a maximal relaxation of 52% +/- 3%. Beta1-antagonist CGP-20712A had no significant effect, whereas beta2-antagonist ICI-118551 produced a characteristic rightward shift of the isoproterenol concentration-relaxation relationship. CONCLUSIONS: Although both beta1-adrenergic receptors and beta2-adrenergic receptors are present in human myometrial tissue at term, relaxation by nonselective beta-agonist isoproterenol is mediated exclusively by beta2-adrenergic receptors.     

Transition probability cell cycle model. Part I--Balanced growth

In this study, we have identified and characterized functional alpha2-adrenergic receptor (alpha2-AR) subtypes in human corpus cavernosum and in cultured human corpus cavernosum smooth muscle cells. Analysis of total RNA, isolated from whole corpus cavernosum tissue and smooth muscle cells, by RNase protection assays, demonstrated expression of mRNA for alpha2A, alpha2B, and alpha2C adrenergic receptor subtypes in whole tissue and alpha2A and alpha2C subtypes in cultured smooth muscle cells. Binding studies with [3H]RX821002 (a highly selective and specific ligand for alpha2-adrenergic receptor) in isolated membrane fractions of human corpus cavernosum smooth muscle cells, demonstrated specific alpha2-AR binding sites with high affinity (Kd = 0.63 nM) and limited capacity (25-30 fmol/mg protein). Binding of [3H]RX821002 was displaced with the nonselective alpha-AR antagonist, phentolamine, and with the alpha-AR agonist, norepinephrine, in a dose-dependent manner, but not by the selective alpha1-AR agonist, phenylephrine. Binding of [3H]rauwolscine was also displaced by phentolamine. UK 14,304, a selective alpha2-AR agonist, inhibited forskolin-induced cyclic adenosine monophosphate (cAMP) synthesis in cultured human corpus cavernosum smooth muscle cells and induced dose-dependent contractions of tissue strips in organ bath chambers. UK 14,304-induced contractions were inhibited with alpha2-AR selective antagonists, rauwolscine and delquamine (RS 15385-197). These observations suggest that in human corpus cavernosum, norepinephrine (NE) and epinephrine may activate postsynaptic alpha2-AR subtypes, in addition to activating alpha1-AR subtypes, on smooth muscle cells, contributing to local control of human corpus cavernosum smooth muscle tone, in vivo.     

Increased availability and open probability of single L-type calcium channels from failing compared with nonfailing human ventricle

BACKGROUND: The role of the L-type calcium channel in human heart failure is unclear, on the basis of previous whole-cell recordings. METHODS AND RESULTS: We investigated the properties of L-type calcium channels in left ventricular myocytes isolated from nonfailing donor hearts (n= 16 cells) or failing hearts of transplant recipients with dilated (n=9) or ischemic (n=7) cardiomyopathy. The single-channel recording technique was used (70 mmol/L Ba2+). Peak average currents were significantly enhanced in heart failure (38.2+/-9.3 fA) versus nonfailing control hearts (13.2+/-4.5 fA, P=0.02) because of an elevation of channel availability (55.9+/-6.7% versus 26.4+/-5.3%, P=0.001) and open probability within active sweeps (7.36+/-1.51% versus 3.18+/-1.33%, P=0.04). These differences closely resembled the effects of a cAMP-dependent stimulation with 8-Br-cAMP (n= 11). Kinetic analysis of the slow gating shows that channels from failing hearts remain available for a longer time, suggesting a defect in the dephosphorylation. Indeed, the phosphatase inhibitor okadaic acid was unable to stimulate channel activity in myocytes from failing hearts (n=5). Expression of calcium channel subunits was measured by Northern blot analysis. Expression of alpha1c- and beta-subunits was unaltered. Whole-cell current measurements did not reveal an increase of current density in heart failure. CONCLUSIONS: Individual L-type calcium channels are fundamentally affected in severe human heart failure. This is probably important for the impairment of cardiac excitation-contraction coupling.