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Experimental Biology and Medicine 231:741-745 (2006)
© 2006 Society for Experimental Biology and Medicine

BASIC BIOLOGY

Quantification of Endothelin Receptor Subtypes in Peripheral Tissues Reveals Downregulation of ETA Receptors in ETB-Deficient Mice

Rhoda E. Kuc1, Janet J. Maguire and Anthony P. Davenport

Clinical Pharmacology Unit, University of Cambridge, Level 6, Addenbrooke’s Centre for Clinical Investigation, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom

1To whom requests for reprints should be addressed at Clinical Pharmacology Unit, University of Cambridge, Level 6, Addenbrooke’s Centre for Clinical Investigation, Box 110, Addenbrooke’s Hospital, Cambridge CB2 2QQ, UK. E-mail: rek22{at}medschl.cam.ac.uk

Abstract

We have previously shown that in homozygous endothelin (ET)B– /– deficient mice, ETA receptor density is significantly downregulated in the brain by 45%. In these mice, plasma ET-1 levels are elevated. Our aim was to use quantitative autoradiography to establish the distribution of ET receptor subtypes in peripheral tissues from wild-type mice and to measure the density of the ETA subtype in ETB– /– knockout animals. Our second aim was to test whether deletion of ETB receptors, which is associated with elevated plasma levels of ET-1, would also reduce ETA expression in the periphery. In longitudinal sections from wild-type mice, the highest densities of ETA receptors localized to major organs including the ventricle of the heart, lung, and liver parenchyma. High densities of ETA receptors were detected in the smooth muscle layer of the vasculature such as intrarenal vessels as well as the smooth muscle layer and epithelial cells of the gastrointestinal tract. In these tissues, the ETA subtype was more abundant, representing between 60% and 100% of the ET receptors. ETB receptors predominated in the medulla of kidney, with high densities also localizing to glomeruli within the cortex and to the sinusoids from the liver. Lower densities of ETB receptors were also present in the lung, heart, liver, and the smooth muscle layer of the gastrointestinal tract. In ETB– /– knockout mice, ETB receptors were not detected as expected by either ligand binding or immunocytochemistry. The pattern of ETA receptor distribution in the ETB– /– knockout mice was similar to the controls, but the density of ETA receptors was significantly reduced in the lung by 39%. Diminished responses to the endogenous agonist after repeated stimulation are an important feature of G-protein signaling, preventing potential damage to the overstimulated cell, and it is likely that downregulation occurs in response to higher circulating levels of ET-1.

Key Words: image analysis • knockout mouse • quantitative autoradiography • radioligand binding

The structure of endothelin-1 (ET-1) is unique among the mammalian bioactive peptides in containing two disulfide bridges, especially the bridge between Cys1 and Cys15 residues, which may render the N-terminus less susceptible to aminopeptidases. This can confer increased resistance to degradation of the peptide by enzymatic pathways. ET-1 remains one of the most potent constrictors of human vessels with an unusually long-lasting action (1). Effective removal from the circulation of ET-1 by non-enzymatic pathways such as receptor internalization may be crucial, particularly under pathophysiological conditions, when circulating plasma levels may be elevated.

An important emerging role of the ETB subtype is to function as a clearing receptor, removing ET-1 from the circulation (2, 3). We have recently shown using positron emission tomography to dynamically image ET receptors in vivo, that ETB receptors in lungs, kidney, and, to a lesser extent, liver, efficiently clear 18F-labeled ET-1 from the circulation to prevent the peptide binding to the heart (4). We speculated that ETB receptors clear ET-1 from the circulation, thus protecting the heart from potentially deleterious actions of ET-1, mediated via the ETA subtype. We have also shown that in human blood vessels removed from patients with pathophysiological conditions known to elevate plasma levels of ET-1, smooth muscle ETA receptors are downregulated. We speculated that this may be the result of an adaptive response to high levels of agonist (5), because, unlike many transmitters, the ET system is characterized by a lack of ETA receptor reserve (6). In agreement, in a rat model of acute hypertension in which adenovirus transfer of the pre-proET-1 gene significantly increased plasma ET-1 levels after 4 days, there was a significant compensatory downregulation of ETA receptor density by 50%, correlating with reduced constrictor activity of ET-1 in peripheral vessels (7).

Genetic disruption of the ETB receptor in mice also significantly increases blood pressure, and circulating ET-1 levels are doubled in heterozygotes compared with wild-type controls (8). Homozygote ETB– /– knockout mice are viable at birth and can survive for up to 8 weeks, although they display aganglionic megacolon as a result of absence of ganglion neurons together with a pigmentary disorder in their coats (8–11). We have previously shown there is a marked 45% downregulation of ETA receptors in response to ETB gene deletion in brains from these ETB– /– knockout mice (12). Our aim was to use quantitative autoradiography to establish the distribution of ET subtypes in peripheral tissues from wild-type mice and to measure the density of the ETA subtype in ETB– /– knockout mice, to test whether deletion of ETB receptors, leading to reduced plasma clearance, would also reduce ETA expression in the periphery.

Materials and Methods

Quantitative Autoradiography ETB Knockout Mouse Model.
After euthanasia (carried out in compliance with the NIH Guide for the Care and Use of Laboratory Animals and local and federal animal welfare laws) (10), representative longitudinal cryostat sections (30 µm) were cut from the midline at a level to include the heart, lungs, liver, and kidney from the torsos of four male WT+/+ (control) mice, aged 29 ± 3 days and four male ETB– /– receptor knockout mice, age 25 ± 2 days. The autoradiographic distribution of all ET receptors was determined as previously described (13) by incubating sections for 2 hrs at 23° C with [125I]-ET-1 (0.1 nM). ETA receptors were visualized by incubating adjacent sections with [125I]-ET-1 (0.1 nM) in the presence of either 0.1 µM BQ3020 (a concentration calculated to block binding of the radiolabel to the ETB subtype) or 0.1 µM BQ123 to detect ETB. Nonspecific binding (NSB) was defined by incubating an additional adjacent section with the radioligand in the presence of unlabeled ET-1 (1 µM). After incubation and washing in ice-cold Tris-HCl buffer to break the equilibrium, the dried sections were apposed to radiation sensitive film, together with calibrated 125I standards from 5 to 7 days, before developing the films. The resulting autoradiograms were analyzed using computer-assisted densitometry (Quantimet 970, Leica, Milton Keynes, UK) as previously described (13). Briefly, each autoradiographic image was digitized. A cursor was used to delineate regions of interest and the integrated optical density measured. When all measurements had been made for a particular section, the threshold for detecting the autoradiogram was increased to produce a template that was used to align the autoradiographic image of an adjacent section used to define the NSB. The second image was digitally subtracted from the first to measure the amount of specific binding. The resulting optical densities were converted to the amount of specifically bound radioligand in amol/mm2 by interpolation from the standards curve.

Histology and Immunocytochemistry.
After exposure to film, the radiolabeled sections were stained with hematoxylin and eosin to facilitate histologic identification of structures in the autoradiograms measured by image analysis. To confirm complete deletion of the ETB– /– receptor in the knockout mice, immunocytochemistry was carried out by incubating adjacent cryostat sections (30 µm) as previously described (14) using site-directed rabbit antisera raised to the receptor protein sequence, ETB(302–313). Primary antisera were visualized by the peroxidase-antiperoxidase technique.

Results

Distribution of ETA and ETB Receptors in WT+/+ Mice.
The distribution of ETA and ETB subtypes in representative longitudinal sections cut at the level of the heart from torso of a WT+/+ mouse is shown in Figure 1Go and with measured receptor densities in Table 1Go. The highest densities of ETA receptors localized to major organs, including the lung (parenchyma), heart (ventricle), and liver (parenchyma). Where resolution was sufficient in the autoradiograms (Fig. 2A and 2CGo, Table 1Go), measurements were also made in more discrete regions that comprise mainly one cell type, revealing high densities of ETA receptors in the smooth muscle layer of the vasculature, such as intrarenal vessels and the smooth muscle layer and epithelial cells of the gastrointestinal tract. In these tissues, the ETA subtype was more abundant, representing between 60% and 100% of the ET receptors.


Figure 1
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  		Figure 1. Representative autoradiograms showing localization of ET receptors in consecutive longitudinal cryostat sections cut through the midline of the torso of male WT+/+ control (upper panel) or homozygous ETB– /– knockout mice (lower panel). To visualize all ET receptors (A, E; total), sections were incubated with 0.1 nM [125I]-ET-1 alone. Adjacent sections were incubated with the label plus 1 µM ET-1 to define the NSB (B, F). In C and G, a concentration of ETB ligand, BQ3020 (200 nM) was calculated to selectively block [125I]-ET-1 binding to ETB receptors to reveal the ETA distribution. In D and H, the concentration of an ETA selective ligand, BQ123 (200 nM), was calculated to block [125I]-ET-1 binding to this subtype to visualize the ETB distribution. In the control mouse (D), high densities of ETB binding were detected in kidney and lungs, with lower levels in the liver. In the ETB knockout, binding could not be detected, as expected, above the NSB, confirming the targeted disruption of the ETB gene in all tissues examined. ant, anterior; post, posterior; g, gut; h, heart; k, kidney; l, lung; li, liver. Scale bar = 10 mm.

 

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  		Table 1. ETA and ETB Receptor Density in WT+/+ Control Mouse Tissuesa
 

Figure 2
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  		Figure 2. Representative autoradiographic images at higher magnification of ET receptor subtype distribution in the WT+/+ control mouse. (A) High densities of ETA receptors are present in heart, liver, and lungs as well as the smooth muscle and epithelial cell layer of the gastrointestinal tract. ETA receptors localize to the vasculature such as intrarenal vessels indicated by arrows (C) in both renal cortex and medulla. The ETB subtype localized to endothelial cell-like sinusoids in liver indicated by arrows (B) with lower densities in lung parenchyma, ventricle of the heart, and to the smooth muscle layer of the gastrointestinal tract but not to the epithelial layer. ETB receptors predominated in the medulla of kidney (D), with high densities also localizing to glomeruli (arrows) within the cortex. g, gut; h, heart; l, lung; li, liver, c, cortex, m, medulla. Scale bar = 5 mm.

 
ETB receptors predominated in the medulla of kidney (Fig. 2DGo, Table 1Go), with high densities also localizing to glomeruli within the cortex and to the endothelial cell-like sinusoids from the liver (Fig. 2BGo). Lower densities of ETB receptors were also present in the lung (parenchyma), heart (ventricle), liver (parenchyma), and the smooth muscle layer of the gastrointestinal tract but not to the epithelial layer (Table 1Go). In the smooth muscle layer of vessels from the major organs visualized in the autoradiograms, ETB binding was difficult to detect. Binding to endothelial cells in the vasculature is below the level for detection by film autoradiography and could not be measured.

Quantification of ETA Receptors in Peripheral Tissues from ETB– /– Knockout Mice.
Specific binding of the ETB ligand could not be measured above the level of the NSB in all of the knockout animals (Fig. 1Go). No staining was detected using site-directed antisera to the ETB receptor, confirming the successful disruption of the protein as expected. ETA receptor distribution (Fig. 1Go) in the ETB– /– knockout mice was similar to the controls, but the density of ETA receptors was significantly reduced in lung by 39%, although no differences were found in the heart or liver (Fig. 3Go).


Figure 3
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  		Figure 3. Comparison of ETA receptor density in peripheral tissues from ETB– /– knockout mice compared with WT+/+ control mice. For each organ, bars represent the mean of 4 mice ± SEM (*P < 0.003, Mann-Whitney U test).

 
Discussion

Although mice are now widely used to further understand the role of this peptide through target disruption or cell-specific overexpression of key components in the ET system (11), the distribution of ET receptor subtypes has not been extensively studied in this species using whole-body autoradiography. In peripheral tissues, the highest density of ET receptors was present in the lungs as in humans (15) and contained a high proportion of the ETB subtype, mainly present on endothelial cells (16), consistent with a function in clearing ET-1 from the circulation (4). The sinusoids forming the capillary bed of the liver also express ETB receptors, which are lined with endothelial cells, consistent with a role in removing ET-1 from the circulation, whereas the parenchyma is mainly ETA. The kidney also efficiently clears ET-1 from the circulation, and a high density of ETB binding localized to the glomeruli of the renal cortex and to the medulla. The distribution of the two subtypes in mouse kidney is similar to human renal tissues (17, 18), with comparable ratios of ETA:ETB receptors for example in the medulla. The major difference is the high density of ETB receptors in glomeruli from mice, that are also present in rats, but absent in humans (17).

The ratio of ETA:ETB receptors in the ventricle of the mouse heart from the strain used in this study (129/Sv-Ednrb) (10) is comparable to other mice with a different genetic background, C57/CL6J (19), and to humans (20). ETA receptors are also the principal isoform in the smooth muscle layer of mouse vasculature such as intrarenal arteries, whereas this is reversed in the airway smooth muscle in which ETB receptors predominate (10) and form a significant proportion of ET receptors in smooth muscle from the gut. In the tissues examined, the pattern and ratio of ET receptor subtype expression is similar to that seen in man suggesting that disruption of ET receptor subtypes targeted to specific cells using techniques such as the Cre/lox system (16, 21) may have relevance to the development of animal models of human disease.

Interestingly, in tissue where the ETA receptors predominated (heart and liver), no change was detected in ETA receptor density in ETB –/– receptor knockout mice. However, in lung parenchyma where the ratio of ETA to ETB receptors is almost equal, suggesting they could be expressed together in the same cell and might be linked by dimerization, there was a significant reduction in ETA receptor density. A second possibility to explain down-regulation of ETA receptors in ETB-rich tissues is that evidence is emerging that ETB receptor signaling is crucial for embryonic development. Deletion of the ETB receptor may therefore alter the survival of ETA-expressing cells, although the evidence in peripheral tissues is that this deletion mainly affects enteric neurons and melanocytes. However, diminished responses to the endogenous agonist after repeated stimulation are an important feature of G-protein signaling, preventing potential damage to the overstimulated cell. A more likely explanation is that the downregulation that has been observed in the lung tissue occurs in response to higher circulating levels of ET-1.

Footnotes

This study was supported by the British Heart Foundation.

Received for publication September 29, 2005. Accepted for publication November 7, 2005.

References

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