0013-7227/92/1304-1885$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine

Vol. 130, No. 4 Printed in U.S.A.

Society

Distinct Tissue Distribution Two Messenger Ribonucleic Subtypes of Rat Endothelin SEIJI HORI, SHIGETADA

YASATO KOMATSU, NAKANISHI

RYUICHI

and Cellular Localization Acids Encoding Different Receptors*

SHIGEMOTO,

NOBORU

MIZUNO,

of

AND

Institute for Immunology (S.H., Y.K., S.N.) and the Department of Morphological Brain Science (R.S., N.M.), Kyoto University Faculty of Medicine, Kyoto 606, Japan

ABSTRACT.

Endothelins (ETs) are very potent vasoconstrictive peptides and have diverse functions in both vascular and nonvascular tissues. This investigation concerns the tissue distribution and cellular localization of rat mRNAs encoding two different subtypes of ET receptors (ET* and ETs). We isolated 46 cDNA clones from a rat lung cDNA library by hybridization with the bovine ETA cDNA. The characterization of these cDNA clones indicated that they represent either the ET* or ETs cDNA. In situ and blot hybridization analyses revealed that the rat ETA mRNA is predominantly expressed in vascular smooth muscle cells of a variety of tissues, bronchial smooth muscle cells, myocardium, and the pituitary gland. There is no signifi-

cant expression of ETs mRNA in vascular smooth muscle cells, and ETA, thus, plays a primary role in ET-induced vascular contraction. ETs mRNA is more widely distributed in various cell types of many tissues. Its prominent expression is seen in glial cells throughout the brain regions, epithelial cells of the choroid plexus, ependymal cells lining the ventricle, myocardium, endothelial cells of glomeruli, and epithelial cells of the thin segments of Henle’s loops. Our investigation demonstrates that the mRNAs for the two subtypes of rat ET receptors show specialized expression patterns of cell types in both brain and peripheral tissues. (Endocrinology 130: l&85-1895,1992)

E

NDOTHELINS (ETs) are members of the family of peptides consisting of 21 amino acid residues with 2 interconnecting disulfide linkages (1, 2). ET-l was first identified as a potent vasoconstrictor produced by vascular endothelial cells (l), and the existence of ET-2 and ET-3 was subsequently predicted after the isolation of genes related to ET-l (3,4). The mammalian ETs closely resemble the sarafotoxin class of snake venom peptides, which produce coronary vasoconstriction (2,5). The members of the ET family show a variety of biological activities in both vascular and nonvascular tissues, including hemodynamic, cardiac, pulmonary, and renal effects (1, 3, 4, 6-9); the modulation of neural functions (10); and cell mitogenesis (11). The involvement of ETs in the pathology of some human diseases has been suggested by studies of animal models and humans. Especially, evidence has been presented indicating that ETs might be involved in essential hyperten-

sion (12), acute myocardial infarction (13), renal failure (14), and subarachnoid hemorrhage (15). The three ETs possess a common spectrum of biological activities, but their biological potencies differ, depending on the pharmacological preparations tested (4, 8). These studies as well as ligand-binding experiments suggested that there .are multiple ET receptors (ETRs) (16). Recently, the isolation and characterization of cDNA clones encoding ETRs have been reported from several laboratories. Our group cloned a bovine ETR with the rank order of binding affinities of ET-l > ET2 > ET-3 (17). The receptor cloned by Lin et al. (18) from A10 rat vascular smooth muscle cells showed a similarity in both its amino acid sequence and its peptidebinding selectivity with the above receptor, and thus, it was found to represent the rat counterpart of the bovine receptor. Sakurai et al. (19), on the other hand, reported the cloning of a different subtype of the rat receptor, and this receptor showed a lower sequence similarity with the above receptors and a different pattern of binding selectivity, with comparably high affinities for the three ETs. Thus, there are at least two subtypes of ETRs, termed ETA and ETn (20). Both ETA and ETx show structural characteristics, possessing seven putative transmembrane segments similar to other members of G-protein-

Received November 18,199l. Address requests for reprints to: Shigetada Nakanishi, Institute for Immunology, Kyoto University Faculty of Medicine, Yoshida, Sakyoku, Kyoto 606, Japan. *This work was supported in part by research grants from the Ministry of Education, Science, and Culture of Japan; the Ministry of Health and Welfare of Japan; the Yamanouchi Foundation for Research on Metabolic Disorders; and the Uehara Memorial Foundation. 1885

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1886

ET RECEPTOR

mRNA

coupled receptors, and function by mediating intracellular signal transduction through G-proteins (17-19). An understanding of cell-specific expression patterns of the multiple ETRs is important for assessing the disparate functional roles of the receptors. The tissue distribution of ET-binding sites has been investigated by radioligand binding and autoradiography (21). However, because ETA and ETs show a comparably high affinity to ET-l, it is difficult to distinguish the localization of different receptor subtypes by autoradiographic ligandbinding techniques. In this investigation we first isolated two types of cDNA clones for rat ETRs from a rat lung cDNA library by cross-hybridization with the bovine ETA cDNA. Although the deduced amino acid sequences for the two receptors showed minor differences from those reported by Lin et al. (18) and Sakurai et al. (19), the two receptors were characterized as ETA and ETB on the basis of the nucleotide sequence determination and the ligand-binding properties of these receptors. We investigated expression sites of the two mRNAs by RNA blot and in situ hybridization analyses. We here report that the ETA and ETs mRNAs show distinct expression patterns in cell types of both the central nervous system and peripheral tissues. Materials

and Methods

Materials

Materials were obtained from the following sources:Superscript reverse transcriptase from Bethesda ResearchLaboratories (Gaithersburg, MD); XZAPII from Stratagene (La Jolla, CA); ET-l, ET-2, ET-3, and sarafotoxin S6b (SRTX S6b)from Peptide Institute (Osaka,Japan); lz51-labeled BoltonHunter ET-1 ( [iz51]ET-1; 74Tera Bequerel/mmol), nylon membrane, [35S]CTP(30 Tera Bequerel/mmol), and /3Max film from Amersham (Tokyo, Japan); NTB2 from Eastman Kodak (Rochester,NY); and OCT compound from Miles (Elkhart, IN). Other reagentswere describedpreviously (22). cDNA cloning of rat ETRs

A rat lung cDNA library was constructed according to the proceduresdescribedpreviously (22). Rat lung poly(A)+ RNA was subjectedto centrifugation of sucrosedensity gradient (5 25%) (22). A fraction (2-4 kilonucleotides) giving potent ETR expressionin Xenopus oocytes was identified by electrophysiological measurements(22) and was used for the cDNA synthesis. A mixture of double stranded cDNAs synthesized was inserted into the EcoRI site of XZAPII vector DNA. Approximately 1 x lo5 phageclonesof the cDNA library were screened by hybridization with the 969-basepair (bp) NcoI-EcoRI fragment derived from the bovine ETA cDNA clone (pBETR1) (17).Forty-six hybridization-positive cloneswere identified and isolated by repeated plaque purification. Each of the phage clonescontaining the cDNA insert wasconverted to the cDNA plasmidby rescueexcision. The resultant plasmid cloneswere classified into 2 groups, consisting of 10 and 36 clones, by

DISTRIBUTION

Endo. Voll30.

1992 No 4

restriction enzyme and blot hybridization analyses.Two clones (pRlA9 and pRlAlG), each representing1 of the 2 groups,were transcribed in vitro and tested for the expressionof ETR in Xenopus oocytes. Both cloneswere found to induce an electrophysiological responseto application of ET-l. The cDNA inserts of both cloneswere subjectedto sequencedetermination of both strands by the chain termination method (23). ETR cDNA expression

in COS cells and peptide

binding

assay

The XhoI-Not1 fragments of pRlA9 and pRlA16 were subclonedinto the eukaryotic expressionvector CDM8 (24). Transient expressionof an individual ETR in COS cells by transfection of the resultant receptor cDNACDM8 plasmid and isolation of crude membranesof the DNA-transfected cells were describedpreviously (22). Ligand binding wasperformed as previously described(22); cell membranes(0.8 pg/ml) were incubated with various concentrations (saturation experiments) or 50 pM (competition experiments) of [‘251]ET-1 in 0.25 ml binding solution (17). Each experiment wascarried out in triplicate. Nonspecific binding was identified as binding activity in the presenceof 1 pM ET-l and wassubtracted from total binding activity for determination of specificbinding. The specific binding activity amounted to 90-92% of the total binding activity. The theoretical curves for displacementof [‘251]ET-1 binding by ET isopeptides and SRTX S6b were drawn by nonlinear least squaresanalysis, as previously described(22). RNA blot hybridization

analysis

Poly(A)+ RNAs were isolated from various tissuesof adult femaleSprague-Dawleyrats, as describedpreviously (22). RNA blot hybridization analysis [5 pg poly(A)+ RNA each] was carried out as describedpreviously (22). The 1055-bp HphI fragment and the 1495-bpEcoTlU fragment were excisedfrom pRlA9 and pRlA16, respectively, and used as probes for hybridization under high stringency conditions; hybridization was carried out at 42 C in the solution previously described(22), and filter washingwasperformed at 50 C in a solution containing 0.1 x saline sodium citrate (SSC; 1 x SSC contains 150 mM NaCl and 15 mM sodiumcitrate) and 0.1% sodiumdodecyl sulfate. The size markers usedwere rat ribosomalRNAs. In situ hybridization

analysis

The 1735-bpSacI-XbaI fragment of pRlA9 and the 1495-bp EcoT141 fragment of pRlA16 were subcloned into multiple cloning sites of pBluescript. Antisense RNAs were synthesized in uitro by T7 RNA polymerasein the presenceof [35S]CTP. The resultant RNAs were hydrolyzed in 0.1 M sodiumbicarbonate buffer (pH 10.6) to make approximately 150-nucleotide fragments. Various tissuesof male or female Sprague-Dawley rats (6-10 weeks old) were frozen and embeddedin OCT compound.Nervous tissuesand peripheral tissueswerecut into sectionsof lo- and 7-pm thickness on a cryostat, respectively. Sectionswere fixed with 4% formaldehyde, treated with 0.25% acetic anhydride, dehydrated, and incubatedwith an 35S-labeled antisenseRNA probe in the hybridization solution at 55 C for 4-6 h (25). Hybridized sections were washedwith 2 x SSC containing 10 mM &mercaptoethanol at room temperature,

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ET RECEPTOR

mRNA

treated with RNase-A (20 pg/ml) for 30 min, and then washed with 0.1 x SSC at 60 C. The sections were dehydrated and exposed to /3Max film for 5-7 days or dipped into NTB2 diluted 1:l with distilled water, developed after 4-8 weeks of exposure, and counterstained with cresylviolet (nervous tissues) or hematoxylin-eosin (peripheral tissues).

Results Molecular

cloning of rat ETR cDNAs

We initiated this investigation to examine the diversity

of the ETR family and, therefore, screened a rat lung cDNA library by cross-hybridization with the bovine ETA cDNA probe. We isolated 46 hybridization-positive clones from approximately 1 x lo5 cDNA clones and classified these clones into 2 groups on the basis of restriction mapping and DNA blot hybridization analyses. Groups 1 and 2 consist of 10 and 36 clones, respectively, and no other clone indicating a different restriction map or a distinct hybridization pattern was identified among these 46 clones. pRlA9 and pRlA16 were selected as representative clones of groups 1 and 2, respectively, and their nucleotide sequences were determined. pRlA16 showed the nucleotide sequence virtually identical to that of the ETa cDNA reported by Sakurai et al. (19), while pRlA9 turned out to correspond to the ETA cDNA reported recently by Lin et al. (18). However, there were some nucleotide differences resulting in amino acid substitutions between our cDNA clones and those reported by the other groups. The amino acid sequence deduced from pRlA9 showed substitutions of Asn (AAC) at residue 46 for Asp (GAC), and Gln (CAA) at residue 53 for Arg (CGA), while the sequence predicted from pRlA16 displayed Ser-Ser-Ala-Pro at residues 66-69 in place of Phe-Arg-Thr at residues 66-68 reported by Sakurai et al. (19) (see Fig. 1). The latter change was due to a shift of the reading frame resulting from the addition of three nucleotides as follows: SerSerAlaPro TETCCGCMCT

PheAr gThr us. TTCCGCACT.

The ETz sequence deduced from our cDNA clone is, thus, one amino acid longer than that reported by Sakurai et al. (19) and is more closely related to the human ETn sequence at the amino acid-substituted portion (2628). The other nucleotide differences in the ETn cDNA clones were an A/C substitution 28 nucleotide residues up-stream from the translation initiation codon and a synonymous valine codon (GTA/GT@ at amino acid residue 113. On the basis of the amino acid sequences of rat ETA and ET* deduced from our cDNA clones, Figure 1 shows the amino acid sequence alignment of the two types of ETRs of different animal species (17, 26, 29). The ETRs

DISTRIBUTION

1887

show structural characteristics common to the members of G-protein-coupled receptors as follows (30): the presence of seven hydrophobic, presumably membrane-spanning domains; potential N-glycosylation sites at the amino-terminal extracellular domains following a putative signal peptide; and several possible phosphorylation sites at the cytoplasmic domains. The amino acid sequences of the ETRs are more homologous among the same receptor subtypes of different animal species than between the different receptor subtypes of the same animal species. The overall homology of the ETRs is as follows: rat ETA us. bovine ETA, 91.1%; rat ETA us. human ETA, 92.7%; rat ETz us. human ETa, 88.7%; and rat ETA us. rat ETn, 53.3%. Characterization of cloned rat ETRs

In both cDNA clones, injection of the mRNA synthesized in vitro from the cloned cDNA into Xenopus oocytes elicited potent electrophysiological responses to ET-l (data not shown), indicating that both types of the cDNA clones encode functional receptor proteins. To determine the binding characteristics of the two cloned receptors more accurately, we investigated the ligandbinding property of each receptor by DNA transfection and transient expression of the receptor cDNA in mammalian COS cells. Binding of [‘251]ET-1 to membranes derived from receptor-expressing cells was saturable in both cases, with a dissociation constant (KJ of 0.11 nM for ETA and 0.04 nM for ETa (Fig. 2, A and B). No such binding of [lz51]ET-1 was detected on membranes prepared from untransfected cells or cells transfected with the vector DNA alone (data not shown). The Ki values of the two receptors for three ET isopeptides and SRTX S6b were calculated by ligand binding competition experiments (Fig. 2, C and D). The Ki values of ETA for ET-l, ET-2, ET-3, and SRTX S6b were 0.27, 1.2, 638, and 23.2 nM, respectively, while those of ETa for the corresponding peptides were 71, 113, 113, and 90 PM, respectively. Thus, rat ETA, like its bovine counterpart, shows a high affinity for both ET-1 and ET-2, but not for ET-3, whereas ETz has a similar affinity for all three ETs and SRTX S6b. RNA blot hybridization analysis of ET, and ETB mRNAs

Relative levels of the two ETR mRNAs were analyzed in the brain and various peripheral tissues by RNA blot hybridization (Fig. 3). The analysis of ETA mRNA gave rise to two hybridization bands with estimated mRNA sizes of about 5.2 and 4.2 kilonucleotides. The two different sizes of the ETA mRNA are probably derived from the same gene, because both bands were seen under high stringency filter-washing conditions. The analysis of ETn mRNA yielded a single band with an estimated

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ET RECEPTOR

1888

rat bovine human rat human

ET% EP EP Elb En

mRNA DISTRIBUTION

Endo* Vol130*No4

~~~iii;~~LAsf~~~~-~N :METFWLRLSFWVALVGGVISbN ~~-~.T.~-~.Ckkl?.S~-~~~~~~~~~-~N ._____._-________.________ ~MQSSASRCGRALVALit-&~~ti&KRGFPPAQATPSLLGT iMQPPPSLCGRALVALVLACGLSRIWG$ERGFPPDRATP-LLQT L.............~-----------------------------

STNLSNHVDDFTTFRGTELSFLVTTHQPTNLVLPSNGSMH

( 66)

HLKQRREVAKTVFCL

FIG. 1. Comparison of the amino acid sequences of five ETRs. The amino acid sequences of rat ET* and ETs am taken from those deduced from the nucleotide sequences of our cDNA clones (pRlA9 and pRlA16). The amino acid substitutions between our sequences and those reported by Sakurai et al. (19) and Lin et al. (18) are underlined. The sequences of the five ETRs [rat ET.., and ETa, bovine ETA (17), human ET, (29), and human ETs (26-28)] are aligned with one-letter notation by inserting gaps (-) to achieve maximum homology. The amino acids identical in all five sequences are enclosed by a solid line. Positions of the putative transmembrane segments of I-VII are indicated above the amino acid sequences; the termini of each segment are tentatively assigned on the basis of comparison of hydrophobicity profiles of the five ETRs. The putative signal peptide at the amino-terminus of each receptor is enclosed by a dotted line. A, Potential N-glycosylation sites; 0, possible phosphorylation sites.

mRNA size of about 4.7 kilonucleotides. Both ETA and ETa mRNAs are distributed in a variety of rat tissues. Expression levels of ETA mRNA are very high in the lung and the cardiac atrium and ventricle. The mRNA levels are also high in the liver and uterus, but are relatively low in the brain and gut. Levels of ETz mRNA, on the other hand, are the highest in the cerebellum and also high in the cerebrum and brain stem, lung, and colon. In situ hybridization analysis of ETA and ETB mRNAs

The regional distribution and cellular localization of the ETA and ETx mRNAs were investigated in tissue sections of rat brain and many other peripheral tissues by in situ hybridization analysis. 35S-Labeled riboprobes derived from the ETA and ETa cDNAs were used for analysis of the two ETR mRNAs. The specific hybridization of each probe under our experimental conditions was confirmed by no significant hybridization in parallel

experiments using the same probe in the presence of an excess of unlabeled probe. Furthermore, the two cRNA probes for ETA and ETe mRNAs showed completely different patterns of hybridization, indicating that the cRNA probe used did not mutually cross-hybridize to the mRNA for the other subtype of ETR. The results of in situ hybridization are presented in Figs. 4-8, and the relative expression levels and the cellular localization of the two ETR mRNAs in the brain and peripheral tissues are summarized in Table 1. In the following section, we describe the distribution and cellular localization of the two ETR mRNAs in the brain and peripheral tissues. Brain. A macroscopic

visualization of in situ hybridization for ETz mRNA revealed a wide distribution of a hybridization signal of ETa mRNA in many regions of the brain (Fig. 4, B and D). Autoradiographic grains were particularly concentrated in the cerebellum. High levels of hybridization were also seen in the diencephalon,

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ET

RECEPTOR

mRNA

DISTRIBUTION

1889

was similarly observed in smooth muscle layers of the aorta (Fig. 6, E and F), whereas no such labeling of ETz mRNA was detected in the aorta (data not shown).

12

11

10 9 6 -log [peptide]

12

11 10 9 6 -log [peptide]

7 6 (M)

5

D

B

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55 1 La s-m

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kJ E&d &mnorrtlg Pr%)

:0

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f’ 0

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f”11

0.6 ET-I (IIM)

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2100 ?60 e C

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FIG. 2. Saturation isotherms and displacements of specific [1261]ET-1 binding to membranes of cells transfected with rat ETA, and ETs cDNAs. Saturation isotherms of [‘?]ET-1 to membranes prepared from ETA- and ETs-expressing cells are indicated in A and B, respectively. Inset, Scatchard plot of [1261]ET-1 binding. Displacement of specific binding of [iz61]ET-1 to membranes prepared from ET*- and ETs-expressing cells are indicated in C and D, respectively. The unlabeled peptides added to the binding assay are as follows: 0, ET-l; 0, ET-2; n , ET-3; and Cl, SRTX S6b.

mesencephalon, and lower brain stem. In contrast, no significant hybridization signal over a nonspecific signal was detected for ETA mRNA in the brain regions by macroscopic analysis, with the exception of a diffusely distributed signal observed in the anterior pituitary (Fig. 4, A and C). Brightfield photomicrographs for the analysis of ETA mRNA at high magnification revealed that autoradiographic silver grains were highly and restrictedly located at smooth muscle cells of blood vessels in the brain (Fig. 5A). In contrast, no significant labeling of ETz mRNA was seen in blood vessel cells (Fig. 5B), and the majority of silver grains for ETz mRNA were present over glial cells in many brain regions (Figs. 4 and 5, B and C). Prominent labeling of ETn mRNA was observed in Purkinje cell layers of the cerebellum (Fig. 4B). At a higher magnification, this labeling was mainly located in glial cells, especially Bergmann glial cells, but not in Purkinje cells (Fig. 5D). A high density of a hybridization signal of ETe mRNA was also seen in epithelial cells of the choroid plexus (Fig. 5E) and ependymal cells lining ventricles (Fig. 5F). Heart and aorta. Homogeneous

hybridization signals for both ETA and ETB mRNAs were observed throughout the myocardium (Fig. 6, A and B). In addition, filamentous or twisted structures of hybridization signal were observed in a macroscopic autoradiograph of ETA mRNA (Fig. 6A), and these structures corresponded to highly labeled smooth muscle layers of coronary vessels (Fig. 6, C and D). A high density of silver grains for ETA mRNA

Lung. Hybridization signals for both ETA and ETz mRNAs were observed in a patchy distribution throughout lung parenchyma (Fig. 7, A and B). Because of the histological limitations due to hybridization treatment, definitive identification of cell types expressing either ETA or ETz mRNA was difficult by high resolution with brightfield photomicrographs. Besides the above-mentioned expression sites of ETA mRNA, high levels of expression of this mRNA were observed in smooth muscle layers of bronchi and blood vessels (Fig. 7C). Adrenal gland.

A strong hybridization signal for ETA mRNA was observed at the boundary of the adrenal cortex and medulla, whereas a hybridization signal for ETn mRNA was diffusely distributed in both adrenal cortex and medulla (Fig. 7, D and E). Silver grains for ETA mRNA were located over blood vessel cells at the corticomedullary junction where the cortical capillaries empty into large medullary sinuses (Fig. 7F). Kidney. In situ hybridization

of kidney sections revealed distinct macroautoradiographic patterns of the two ETR mRNAs (Fig. 8, A and B). A large number of punctate hybridization signals observed for ETA mRNA (Fig. 8A) corresponded to glomeruli where silver grains were confined to afferent and efferent glomerular arterioles (Fig. BC). Longitudinal bands were also observed for ETA mRNA (Fig. 8A), indicating high levels of expression of the ETA mRNA in renal arteries (Fig. 8E). The ETA mRNA is, thus, greatly and restrictedly expressed in a variety of renal vessels possessing smooth muscle cells. In the analysis of ETz mRNA, more marked punctate signals were observed in the renal cortex, and silver grains corresponding to this signal were present in the glomerulus rather than over the glomerular arterioles (Fig. 8D). In the glomerulus, silver grains were located over the endothelial cells, but their presence in podocytes and mesangial cells cannot be excluded (Fig. 8D). In the inner stripe of outer medulla, high levels of hybridization signals of ETn mRNA were seen as longitudinal bands, which correspond to vasa recta bundles (Fig. 8B). Relatively homogeneous hybridization signals of ETz mRNA covered the inner medulla, and at this region, silver grains were located over epithelial cells of thin segments of Henle’s loops and also probably interstitial cells and capillary endothelial cells, but not over epithelial cells of collecting ducts (Fig. 8F). Others. Sections

of the liver, like other tissue sections, showed high densities of silver grains for ETA mRNA in smooth muscle layers of blood vessels (data not shown). As described in the RNA hybridization analysis (Fig. 3),

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ET RECEPTOR

1890

A

mRNA DISTRIBUTION

\

\ v>:s,..~ $$.., i i

B

1992 No 4

1 2 3 4 5 6 7 8 9101112 \\.

FIG. 3. RNA blot hybridization analyses of poly(A)+ RNAs from various tissues. The analyses of ETa and ETs mRNAs are indicated in A and B. In both A and B, the poly(A)+ RNAs used were isolated from the following tissues: 1, cerebrum and brain stem; 2, cerebellum; 3, lung; 4, cardiac atrium; 5, cardiac ventricle; 6, stomach; 7, liver; 8, adrenal gland; 9, kidney; 10, small intestine; 11, colon; and 12, uterus.

Endo. Voll30.

-Origin

0

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nals for both mRNAs were unusually high in sections of these tissues for unknown reasons, definitive identification of expression sites of the two ETR mRNAs in these tissues was difficult. Discussion

FIG. 4. The regional distribution of ETA and ETs mRNAs in rat brain. A and B are photomicrographs of film autoradiograms from sagittal sections hybridized with antisense %l-labeled riboprobes for ET, and ETa mRNAs, respectively. C and D are autoradiographs of the adjacent sections of A and B, respectively, as control experiments using the same probe in the presence of an excess of unlabeled probe. pit, Anterior pituitary gland; Cx, cerebral cortex; Cb, cerebellum. Bar, 4 mm.

ETA mRNA was highly expressed in the uterus, while ETa mRNA was enriched in the small intestine and colon. However, because nonspecific hybridization sig-

In this paper we describe the characterization of two different subtypes of rat ETRs and the tissue distribution and cellular localization of their mRNAs on the basis of the molecular cloning of the functional cDNAs encoding these receptor subtypes. In initial studies we examined the existence of multiple subtypes of rat ETRs by characterizing cDNA clones that hybridized to the previously isolated bovine ETA cDNA clone. The 46 cDNA clones isolated were classified into 2 groups, and there were no other cDNA clones indicating the existence of an additional subtype of ETR in the isolated cDNA clones. The deduced amino acid sequences of the 2 groups of cDNA clones show 7 transmembrane-spanning domains, and these 2 receptors, therefore, belong to the members of Gprotein-coupled receptors. Although there are some

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ET RECEPTOR TABLE

1. Distribution

and cellular localization

mRNA DISTRIBUTION

1891

of rat ETA and ETa mRNAs

Northern blot

In situ hybridization

Cerebrum and brain stem

ETA +

+++

Smooth muscle cells of blood vessels

Glial cells in brain stem, epithelial cells of choroid plexus, ependyma1 cells lining ventricles

Cerebellum

+

+++

Smooth muscle cells of blood vessels

Bergmann glia and other glial cells

Lung

+++

+++

Smooth muscle cells of blood vessels and bronchi

NI

Heart Atrium

+++

++

Myocardial cells, smooth cells of coronary arteries

muscle

Myocardial cells

+++

++

Myocardial cells, smooth cells of coronary arteries

muscle

Myocardial cells

Stomach

+

+

NI

NI

Liver

++

+

Smooth muscle cells of blood vessels

NI

Adrenal gland

+

++

Corticomedullary

NI

Kidney

+

++

Smooth muscle cells of blood vessels including glomerular arterioles

Endothelial cells of glomeruli, vasa recta bundles, thin segments of Henle’s loops

Small intestine

+

++

NI

NI

Colon

+

+++

NI

NI

Uterus

++

+

NI

NI

Ventricle

ETB

ETA

ETB

junction

Expression levels and cellular localization of ETA and ETs mRNAs are summarized on the basis of the results presented in Figs. 3-8. +++, High levels of expression; ++, moderate levels of expression; +, low levels of expression; NI, definitive identification of cell types expressing ETR mRNAs was unsuccessful due to either the difficulty of morphological resolution or high levels of nonspecific hybridization.

amino acid variations in both of our cDNA clones from those reported by Sakurai et al. (19) and Lin et al. (18), the characterization of our cloned receptors in DNAtransfected cells demonstrated that they represent 2 subtypes of ETRs, termed ETA and ETB. Interestingly, a similar screening of a human placenta cDNA library y cross-hybridization with the bovine ETA cDNA allowed the isolation of human ETA and ETB cDNA clones, but not others (26, 29). The subtypes of a certain family of G-protein-coupled receptors, however, usually exist in more than the ligand species for the corresponding receptor family (30,31). It, thus, still remains possible that an additional subtype of ETR, other than ETA and ETz, may exist in the mammalian ET system. Previous autoradiographic studies of ET-l-binding sites indicated that the ET-binding sites are widely distributed not only in arterial smooth muscle cells, but also in various other organs (21). This distribution of ETbinding sites is in good agreement with the sum of the expression si-5s of the ETA and ETB mRNAs reported

in this paper. However, our in situ hybridization and RNA blot hybridization analyses unequivocally demonstrated that the two ETR mRNAs show distinct expression patterns of cell types in the brain and peripheral tissues. The ETA mRNA is predominantly expressed in vascular smooth muscle layers of a variety of tissues, including brain blood vessels, coronary arteries, aorta, and afferent and efferent arterioles of glomeruli. Numerous lines of evidence have indicated that ET-l has the most potent contracting activity for various vascular smooth muscles and, thus, plays a key role in controlling blood supply in the brain, heart, kidney, and other tissues (2). The ETA mRNA, which is prominently and ubiquitously distributed in vascular smooth muscle layers, is, thus, responsible for evoking a potent vasoconstriction of ET-l to control the blood supply in many tissues. The ETA mRNA is also highly expressed in smooth muscle cells of bronchi, and this receptor is similarly involved in the contraction of the pulmonary tract by ET-l. A high density of ETA mRNA was also observed in the

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1892

ET

RECEPTOR

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DISTRIBUTION

Endo - 1992 Voll30. No 4

FIG. 5. The cellular localization of ETA and ETs mRNAs in the brain. Brightfield photomicrographs of emulsion-dipped sections for the analyses of ET* and ETs mRNAs are illustrated in A and B-F, respectively. High densities of autoradiographic silver grains for ETA mRNA, as shown in A, were observed in smooth muscle layers of blood vessels throughout the brain regions. Autoradiographic grains for ETs mRNA are seen in blood vessel-associated glial cells (B), glial cells of other regions (C), Bergmann glial cells of cerebellum (D), epithelial cells of the choroid plexus (E), and ependymal cells lining the ventricle (F). Black arrows, Glial cells; white arrowheads, neuronal cells; m, molecular layer; p, Purkinje cell layer; g, granular layer; ChP, choroid plexus; 3V, third ventricle. Bars, 25 pm.

anterior pituitary. It has been reported that ET-l is very active in the stimulation of gonadotropin release in pituitary cells (32). Thus, ETA plays a major role in smooth muscle contraction and may also have some particular functions, such as that of gonadotropin release. ETB mRNA is more widely distributed in cell types of a variety of tissues. A number of the ET functions that cannot simply be explained by its action on vascular mooth muscles have been reported on those tissues expressing high levels of ETa mRNA. ETB mRNA is expressed in glial cells of many brain regions as well as in epithelial cells of the choroid plexus and ependymal cells of the ventricles. Because ETs have a mitogenic activity on primary cultured glial cells (ll), ETa may be involved in potentiating glial proliferation during development or reactive gliosis. Glial cells also produce diffusible mediators, such as nitric oxide, in response to ET (33). ETB could, thus, participate in the modulation of a neuronal function of the central nervous system. ETn mRNA is distinctly localized in glomeruli and inner

medulla of the kidney. It has been reported that iv ET-l perfusion causes profound diuresis and natriuresis despite causing no significant change in the glomerular filtration rate (9). Thus, ETn could play an important role in the control of sodium and water transport in the kidney. Similarly, ETs may have a regulatory function on water and sodium reabsorption in the choroid plexus and ventricles. In the heart, both ETA and ETa mRNAs were diffusely distributed throughout the myocardium of the ventricle and atrium. ET-l induces chronotropic and inotropic effects on the myocardium (7, 34), which are thought to result from the interaction of ET-l with the myocardial ETR. However, which subtype of the ETRs (or both) is involved in inducing the chronotropic and inotropic effects remains to be determined. Our recent transfection experiments using Chinese hamster ovary cells indicated that both ETA and ETB are coupled to the stimulation of inositol phosphate formation and, thus, result in increasing intracellular Ca2+ mobilization (Aramori, I., and S. Nakanishi, unpub-

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ET RECEPTOR

mRNA

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1893

FIG. 6. The regional and cellular localization of ETR mRNAs in the heart and aorta. B shows the analysis of ETs mRNA, while other series indicate those of ETA mRNA. Photomicrographs of film autoradiograms from sections of the heart are displayed in A and B. In A, intense hybridization signals over the coronary arteries are marked by arrows. Photomicrographs of a darktield view (C) and a brightfield view (D) covering the coronary artery region indicate high densities of silver grains for ETA mRNA in smooth muscle layers of the coronary artery. By brightfield photomicrographs, high densities of silver grains for ETA mRNA are observed in smooth muscle layers of aorta (E), whereas no significant grains are seen in an adjacent section hybridized in the presence of excess of unlabeled probe (F). Bars, 2 mm for A, 250 pm for C, and 25 wrn for E.

FIG. 7. The regional and cellular localization of ETA and ETs mRNAs in the lung and adrenal gland. The analyses of ET, mRNA are shown in A, C, D, and F, while those of ETs mRNA are displayed in B and E. Photomicrographs of film autoradiograms from sections of the lung and adrenal gland are illustrated in A-B and D-E, respectively. High densities of autoradiographic grains for ETA mRNA in smooth muscle layers of both bronchus (br) and blood vessel (ve) of the lung (C) and at the corticomedullary junction of the adrenal gland (as indicated by arrows in F) are shown by brightfield photomicrographs of emulsion-dipped sections of these tissues. ctx, Adrenal cortex; med, adrenal medulla. Bars, 1 mm for A and D, 70 pm for C, and 25 pm for F.

lished observation). This transfection system also indicated that the two receptors have different effects on the CAMP signal transduction cascades. ETA showed a rapid and marked stimulation of CAMP formation in response to ET interaction, whereas ETz displayed considerable

inhibition of forskolin-stimulated CAMP accumulation. The responses of both receptors were in complete agreement with the ET binding selectivity of each subtype of the receptors. Thus, our investigations demonstrate that the ETRs are diversified not only by specializing expres-

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ET RECEPTOR

mRNA

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Endo. Voll30.

1992 No 4

FIG. 8. The regional and cellular localization of ETA and ETs mRNAs in the kidney. The analyses of ETA mRNA are shown in A, C, and E, while those of ETz mRNA are displayed in B, D, and F. A and B, Photomicrographs of film autoradiograms; C-F, brightfield photomicrographs of emulsion-dipped sections. Cx, Cortex; OM, outer medulla; IM, inner medulla; ar, glomerular arteriole; gl, glomerulus; He, thin segments of Henle’s loop; CD, collecting duct. Autoradiographic grains for ETA mRNA are confined to afferent and efferent glomerular arterioles (C) and renal artery (E), while those for ETa mRNA are intensely observed in the glomerulus (D) and over thin segments of Henle’s loop (F). Bars, 1 mm for A, and 25 pm for C and E.

sion patterns of the individual receptors, but also by mediating distinct signal transduction. Acknowledgments We are grateful to Tetsuo Sugimoto for helpful and Akira Uesugi for photographic assistance.

discussion,

References 1. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T 1988 A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411-415 2. Yanagisawa M, Masaki T 1989 Molecular biology and biochemistry of the endothelins. Trends Pharmacol Sci 10:374-378 3. Yanagisawa M, Inoue A, Ishikawa T, Kasuya Y, Kimura S, Kumagaye S, Nakajima K, Watanabe TX, Sakakibara S, Goto K, Masaki T 1988 Primary structure, synthesis, and biological activity of rat endothelin, an endothelium-derived vasoconstrictor peptide. Proc Nat1 Acad Sci USA 85:6964-6967

4. Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T 1989 The human endothelin family: three structurally and pharmacologically distinct isopeptide predicted by three separate genes. Proc Nat1 Acad Sci USA 86:2863-2867 5. Kloog Y, Ambar I, Sokolovsky M, Kochva E, Wollberg Z, Bdolah A 1988 Sarafotoxin, a novel vasoconstrictor peptide: phosphoinositide hydrolysis in rat heart and brain. Science 242:268-270 6. de Nucci G, Thomas R, D’Orleans-Juste P, Antunes E, Walder C, Warner TD, Vane JR 1988 Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Nat1 Acad Sci USA 85:9797-9800 7. Ishikawa T, Yanagisawa M, Kimura S, Goto K, Masaki T 1988 Positive inotropic action of novel vasoconstrictor peptide endothelin on guinea pig atria. Am J Physiol255:H970-H973 8. Spokes RA, Ghatei MA, Bloom SR 1989 Studies with endothelin3 and endothelin-1 on rat blood pressure and isolated tissues: evidence for multiple endothelin receptor subtypes. J Cardiovasc Pharmacol13:S191-S192 9. Harris PJ, Zhuo J, Mendelsohn FAO, Skinner SL 1991 Haemodynamic and renal tubular effects of low doses of endothelin in anaesthetized rats. J Physiol433:25-39 10. Samson WK, Skala K, Huang F-LS, Gluntz S, Alexander B,

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ET

11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22. 23.

RECEPTOR

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Gomez-Sanchez CE 1991 Central nervous system action of endothelin-3 to inhibit water drinking in the rat. Brain Res 539: 347-351 MacCumber MW, Ross CA, Snyder SH 1990 Endothelin in brain: receptors, mitogenesis, and biosynthesis in glial cells. Proc Nat1 Acad Sci USA 87:2359-2363 Saito Y, Nakao K, Mukoyama M, Imura H 1990 Increased plasma endothelin level in natients with essential hypertension. N Engl J __ Med 322:205 Salminen K, Tikkanen I, Saijonmaa 0, Nieminen M, Fyhrquist F, Frick MH 1989 Modulation of coronarv tone in acute mvocardial infarction by endothelin. Lancet 2~747 Tomita K, Ujiie K, Nakanishi T, Tomura S, Matsuda 0, Ando K, Shichiri M, Hirata Y, Marumo F 1989 Plasma endothelin levels in patients with acute renal failure. N Engl J Med 321:1127 Masaoka H, Suzuki R, Hirata Y, Emori T, Marumo F, Hirakawa K 1989 Raised plasma endothelin in aneurysmal subarachnoid haemorrhage. Lancet 2:1402 Martin ER, Brenner BM, Ballermann BJ 1990 Heterogeneity of cell surface endothelin receptors. J Biol Chem 265:14044-14049 Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S 1990 Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348730-732 Lin HY, Kaji EH, Winkel GK, Ives HE, Lodish HF 1991 Cloning and functional expression of a vascular smooth muscle endothelin 1 receptor Proc Nat1 Acad Sci USA 88:3185-3189 Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, Masaki T 1990 Cloning of a cDNA encoding a nonisopeptide-selective subtype of the endothelin receptor. Nature 348732-735 Vane J 1990 Endothelins come home to roost. Nature 348673 Koseki C. Imai M. Hirata Y. Yanaaisawa M. Masaki T 1989 Autoradiographic distribution in rat tissues of’ binding sites for endothelin: a neuropeptide? Am J Physiol256R858R866 Shigemoto R, Yokota Y, Tsuchida K, Nakanishi S 1990 Cloning and expression of a rat neuromedin K receptor cDNA. J Biol Chem 265:623-628 Sanger F, Nickelen S, Coulson AR 1977 DNA sequencing with chain-terminating inhibitors. Proc Nat1 Acad Sci USA 74:

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5463-5467 24. Seed B 1987 An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. Nature 329840842 25. Masu M, Tanabe Y, Tsuchida K, Shigemoto R, Nakanishi S 1991 Sequence and expression of a metabotropic glutamate receptor. Nature 349760-765 26. Ogawa Y, Nakao K, Arai H, Nakagawa 0, Hosoda K, Suga S, Nakanishi S, Imura H 1991 Molecular cloning of a non-isopeptideselective human endothelin receptor. Biochem Biophys Res Commun 178248-255 27. Nakamuta M, Takayanagi R, Sakai Y, Sakamoto S, Hagiwara H, Mizuno T, Saito Y, Hirose S. Yamamoto M. Nawata H 1991 Cloning and sequence analysis .of a cDNA encoding human nonselective type of endothelin receptor. Biochem Biophys Res Commun 177:34-39 28. Sakamoto A, Yanagisawa M, Sakurai T, Takuwa Y, Yanagisawa H, Masaki T 1991 Cloning and functional expression of human cDNA for the ETs endothelin receptor. Biochem Biophys Res Commun 178656-663 29. Hosoda K, Nakao K, Arai H, Suga S, Ogawa Y, Mukoyama M, Shirakami G, Saito Y, Nakanishi S, Imura H 1991 Cloning and expression of human endothelin-1 receptor cDNA. FEBS Lett 28223-26 30. Nakanishi S, Ohkubo H, Kakixuka A, Yokota Y, Shigemoto R, Sasai Y, Takumi T 1990 Molecular characterization of mammalian tachykinin receptors and a possible epithelial potassium channel. Recent Prog Horm Res 4659-84 31. Dohlman HG, Thorner J, Caron MG, Lefkowitz RJ 1991 Model systems for the study of seven-transmembrane-segment receptors. Annu Rev Biochem 60:653-688 32. Stojilkovic SS, Merelli F, Iida T, Krsmanovii: LZ, Catt KJ 1990 Endothelin stimulation of cytosolic calcium and gonadotropin secretion in anterior nituitarv cells. Science 2481663-1666 33. Reiser G 1990 Endothelin and a Ca’+ ionophore raise cyclic GMP levels in a neuronal cell line via formation of nitric oxide. Br J Pharmacol 101:722-726 34. Ishikawa T, Yanagisawa M, Kimura S, Goto K, Masaki T 1988 Positive chronotropic effects of endothelin, a novel endotheliumderived vasoconstrictor peptide. Pfluegers Arch 413:108-110

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