Rhoda E. Kuc

Characterization and localization of endothelin receptor subtypes in the human atrioventricular conducting system and myocardium.

The characterization and localization of endothelin A (ETA) and endothelin B (ETB) receptors have been determined in tissue sections of the human atrioventricular conducting system, surrounding regions of atrial and ventricular myocardium, and the left ventricular free wall by use of radioligand binding, polymerase chain reaction, and in situ hybridization. Selective ETA (BQ123) and ETB (BQ3020) compounds in conjunction with [125I]endothelin-1 revealed the presence of ETA and ETB receptors in the left ventricular free wall (BQ123: 57 +/- 5% ETA, 43 +/- 2% ETB, n = 3; BQ3020: 67 +/- 3% ETA, 33 +/- 3% ETB, n = 3). Autoradiography using [125I]endothelin-1 in the absence or presence of BQ3020, BQ123, or endothelin-1 showed ETA and ETB receptors localized to atrial and ventricular myocardium, the atrioventricular conducting system, and endocardial cells. There was a higher proportion of ETB receptors in the atrioventricular node and the penetrating and branching bundles of His than in the surrounding intervent...

Orphan‐receptor ligand human urotensin II: receptor localization in human tissues and comparison of vasoconstrictor responses with endothelin‐1

We have determined the distribution of receptors for human urotensin‐II (U‐II) in human and rat CNS and peripheral tissues. In rat, [125I]‐U‐II binding density was highest in the abducens nucleus of brainstem (139.6±14 amol mm−2). Moderate levels were detected in dorsal horn of spinal cord and lower levels in aorta (22.5±6 amol mm−2). In human tissues density was highest in skeletal muscle and cerebral cortex (∼30 amol mm−2), with lower levels (<15 amol mm−2) in kidney cortex and left ventricle. Little binding was identified in atria, conducting system of the heart and lung parenchyma. Receptor density was less in human coronary artery smooth muscle (14.6±3 amol mm−2, n=10) than rat aorta with no significant difference between normal and atherosclerotic vessels. In human skeletal muscle [125I]‐U‐II bound to a single receptor population with KD=0.24±0.17 nM and Bmax=1.97±1.1 fmol mg−1 protein (n=4). U‐II contracted human coronary, mammary and radial arteries, saphenous and umbilical veins with sub‐nanomolar EC50 values. U‐II was 50 times more potent in arteries and <10 times more potent in veins than endothelin‐1 (ET‐1). The maximum response to U‐II (∼20% of control KCl) was significantly less than to ET‐1 (∼80% KCl). In contrast, in rat aorta, U‐II and ET‐1 were equipotent with similar maximum responses. This is the first report of high affinity receptors for [125I]‐U‐II in human CNS and peripheral tissues. This peptide produces potent, low efficacy, vasoconstriction in human arteries and veins. These data suggest a potential role for U‐II in human physiology.

Endothelin ETA and ETB mRNA and receptors expressed by smooth muscle in the human vasculature: majority of the ETA sub-type.

1 We measured the ratio of ETA and ETB sub‐types in the media (containing mainly smooth muscle) of human cardiac arteries (aorta, pulmonary and coronary), internal mammary arteries and saphenous veins.

Human endothelin receptors characterized using reverse transcriptase-polymerase chain reaction, in situ hybridization, and subtype-selective ligands BQ123 and BQ3020 : evidence for expression of ETB receptors in human vascular smooth muscle

Our aim was to characterize and determine the function of endothelin (ET) receptor subtypes in human vascular tissue. Reverse transcriptase-polymerase chain reaction with nested oligonucleotide primers detected the presence of mRNA encoding both ETA and ETB receptors in the media from aorta and pulmonary and coronary arteries. In situ hybridization confirmed the presence of mRNA for both subtypes in the media of coronary arteries. Saturation binding assays using 125I-ET-1 found a single population of high-affinity ET receptors (n = three patients, +/- SEM) in aorta (Kd = 0.507 +/- 0.020 nM; Bmax = 9 +/- 4 fmol/mg protein) and pulmonary (Kd = 0.845 +/- 0.245 nM; Bmax = 15 +/- 10 fmol/mg protein) and coronary arteries (Kd = 0.141 +/- 0.020 nM; Bmax = 71 +/- 21 fmol/mg protein). Using media from coronary arteries, the ETA-selective ligand BQ123 (cyclo[D-Asp-L-Pro-D-Val-L-Leu-D-Trp]) and the ETB-selective ligand BQ3020 (Ala11,15-Ac-ET-1[6-21]) both produced biphasic competition binding curves against 125I-ET-1, confirming the presence of high- and low-affinity sites corresponding to the two subtypes: BQ123 (KdETA = 0.85 +/- 0.03 nM; KdETB = 7.58 +/- 2.27 microM; ETA/ETB, 87%:13%) and BQ3020 (KdETA = 0.22 +/- 0.04 microM; KdETB = 0.77 +/- 0.34 nM; ETA/ETB, 62%:38%). BQ123 (0.1 microM) caused a significant parallel rightward shift of ET-1-induced vasoconstriction of coronary arteries in vitro, but BQ3020 and Ala1,3,11,15-ET-1 failed to show any agonist activity when tested at concentrations of < or = 3 microM in three vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular distribution of immunoreactive urotensin-II in human tissues with evidence of increased expression in atherosclerosis and a greater constrictor response of small compared to large coronary arteries

We detected urotensin-II-like immunoreactivity in the endothelium of normal human blood vessels from heart, kidney, placenta, adrenal, thyroid and umbilical cord. Immunoreactivity was also detected in endocardial endothelial and kidney epithelial cells. In atherosclerotic coronary artery, immunoreactivity localized to regions of macrophage infiltration. Urotensin-II constricted human atherosclerotic epicardial coronary arteries with pD2=10.58 +/- 0.46 (mean +/- S.E.M.) and Emax=11.4 +/- 4.2% KCl and small coronary arteries with pD2=9.25 +/- 0.38 and Emax=77 +/- 16% KCl. Small coronary arteries clearly exhibited a greater maximum response to urotensin-II than epicardial vessels. This enhanced responsiveness may be of importance in heart failure, where circulating concentrations of U-II are increased, or in atherosclerosis where focally up-regulated urotensin-II production may act down stream to produce significant vasospasm, compromising blood flow to the myocardium. We conclude that urotensin-II is a locally released vasoactive mediator that may be an important regulator of blood flow particularly to the myocardium and may have a specific role in human atherosclerosis.

Kisspeptins: a multifunctional peptide system with a role in reproduction, cancer and the cardiovascular system

Orphan G‐protein‐coupled receptors that have recently been paired with their cognate ligand are an often untapped resource for novel drug development. The KISS1 receptor (previously designated GPR54) has been paired with biologically active cleavage peptides of the KiSS‐1 gene product, the kisspeptins (KP). The focus of this review is the emerging pharmacology and physiology of the KP. Genetic linkage analysis in humans revealed that mutations in KISS1 (GPR54, AXOR12 or hOT7T175) result in idiopathic hypogonadotrophic hypogonadism and knockout mouse studies confirmed this finding. Identification of KISS1 (GPR54) as a molecular switch for puberty subsequently led to the discovery that KP activate the GnRH cascade. Prior to the role of KISS1 (GPR54) in puberty being described, KP had been shown to be inhibitors of tumour metastasis across a range of cancers. Subsequently the mechanism of this inhibition has been suggested to be via altered cell motility and adhesiveness. PCR detected highest expression of KP and KISS1 (GPR54) in placenta, and changes in KP levels throughout pregnancy and expression in trophoblasts suggests a role in placentation. Placentation and metastasis are invasive processes that require angiogenesis. Investigation of KISS1 (GPR54) and KP in vasculature revealed discrete localisation of KISS1 (GPR54) to blood vessels prone to atherosclerosis and a potent vasoconstrictor action. A role for KP has also been shown in whole body homeostasis. KP are multifunctional peptides and further investigation is required to fully elucidate the complex pathways regulated by these peptides and how these pathways integrate in the whole body system.

Vasoconstrictor endothelin receptors characterized in human renal artery and vein in vitro.

1 We have identified the endothelin receptors present in the media of human main stem renal artery and vein and characterized the subtypes mediating vasoconstriction in these blood vessels in vitro. 2 Messenger RNA encoding both ETA and ETB receptors was identified in the smooth muscle layer of human renal artery and vein by reverse transcriptase‐polymerase chain reaction assay. In cryostat‐cut cross‐sections of both vessels autoradiographical visualisation suggested a majority of ETA receptors. Intense binding was obtained to the non‐selective ligand [125I]‐ET‐1 and the ETA‐selective [125I]‐PD151242 but only weak labelling of sites by the ETB‐selective [125I]‐BQ3020. 3 ET‐1 potently constricted renal artery and vein preparations with EC50 values of 4.06 nm and 1.00 nm, respectively. Sarafotoxin 6b was approximately ten times less potent than ET‐1 with EC50 values of 36.3 nm and 13.8 nm respectively. In the renal artery, ET‐3 and sarafotoxin 6c showed little or no activity up to 300 nm. Responses to these peptides were more variable in the renal vein. Preparations from three individuals did not respond to ET‐3 but in three further cases, although ET‐3 was much less potent than ET‐1, full dose‐response curves were obtained. S6c elicited dose‐related contractions in vein preparations from 5/6 individuals and although more potent than ET‐1, the maximum response was 30–60% of that obtained to ET‐1. 4 ET‐1‐induced vasoconstriction of renal artery and vein was antagonized by the ETA‐selective, BQ123 (3–10μm). The dose‐response curves to ET‐1 were displaced in a parallel rightward fashion with no attenuation of the maximum responses. pA2 values were estimated to be 6.8 ± 0.1 and 6.8 ± 0.4 for artery and vein respectively. 5 These data suggest that mRNA encoding both ETA and ETB receptors is present in the media of human main stem renal artery and vein. However, autoradiographical studies indicate that the majority of ET receptors expressed are of the ETA subtype. The relative potencies of ET‐1 and ET‐3 as vasoconstrictors of renal blood vessels in vitro is consistent with this being an ETA‐mediated response, and therefore whilst responses to S6c indicate that constrictor ETB receptors may be present in renal veins from some individuals these are likely to be of less importance in these blood vessels.

[125I]-PD 151242: a selective radioligand for human ETA receptors

Our aim was to synthesize a new endothelin ETA selective radioligand, [125I]‐PD 151242 and characterize the compound in human vascular tissue. Binding of [125I]‐PD151242 to sections of human aorta was time‐dependent and reached equilibrium after 120 min at 23°C with an association rate constant of 1.26 ± 0.17 × 108 M−1 min−1 (n = 3 individuals ± s.e.mean). The binding was reversible at 23°C with an observed dissociation rate constant of 0.0025 ± 0.0006 min−1 (n = 3). Saturation binding assays using [125I]‐PD151242 revealed a single population of high affinity ET receptors (n = 3) in aorta (KD = 0.76 ± 0.17 nM; Bmax = 5.98 ± 1.56 fmol mg−1 protein), pulmonary (KD = 1.75 ± 0.20 nM; Bmax = 12.78 ± 1.39 fmol mg−1 protein) and coronary arteries (KD = 0.51 ± 0.07 nM; Bmax = 44.9 ± 1.67 fmol mg−1 protein). ETA selective ligands competed for [125I]‐PD151242 binding in aorta with nanomolar affinity (BQ123, KD = 0.41 ± 0.26 nM; FR139317, KD = 0.55 ± 0.11 nM) whereas the ETB selective compound, BQ3020, competed with micromolar affinity (KD = 1.36 ± 0.25 μm). In isolated coronary arteries, PD151242 was a functional antagonist and caused a significant, parallel rightward shift of the ET‐1 dose‐response curve with a pA2 value of 5.92 (n = 5) and a slope of unity. The high affinity and selectivity of [125I]‐PD151242 for ETA receptors will facilitate the characterization of this sub‐type in human tissues.

Modulation of the apelin/APJ system in heart failure and atherosclerosis in man

Background and purpose:  The aim of this study was to determine whether the apelin/APJ system is altered in human cardiovascular disease by investigating whether the expression of apelin or its receptor is altered at the protein level.

Novel Snake Venom Ligand Dendroaspis Natriuretic Peptide Is Selective for Natriuretic Peptide Receptor-A in Human Heart: Downregulation of Natriuretic Peptide Receptor-A in Heart Failure

The natriuretic peptides are considered to be cardioprotective; however, their receptors have not been identified in human myocardium using radiolabeled analogs. Dendroaspis natriuretic peptide (DNP) has been recently identified as a new member of this peptide family and is thought to be less susceptible to enzymatic degradation. Therefore, we have developed the novel radiolabeled analog [125I]-DNP and used this to localize high-affinity (KD=0.2 nmol/L), saturable, specific binding sites in adult human heart (n=6) and coronary artery (n=8). In competition binding experiments, atrial natriuretic peptide and brain type natriuretic peptide had greater affinity for [125I]-DNP binding sites than C-type natriuretic peptide and the natriuretic peptide receptor (NPR)-C ligand, cANF. This rank order of potency suggested binding of [125I]-DNP was specific to NPR-A. Messenger RNA encoding NPR-A was identified in left ventricle and coronary artery smooth muscle, and expression was confirmed by immunocytochemical studies at the protein level. In addition, fluorescence dual labeling immunocytochemistry localized NPR-A protein to cardiomyocytes, endocardial endothelial cells, and smooth muscle of intramyocardial vessels. Importantly, we demonstrated a significant downregulation in the density of NPR-A in heart and coronary artery of patients with ischemic heart disease that may explain, in part, the attenuated natriuretic peptide response reported in this patient group.