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Vol. 51, Issue 3, 403-438, September 1999

Autocrine-Paracrine Endothelin System in the Physiology and Pathology of Steroid-Secreting Tissues

Gastone G. Nussdorfer1, Gian Paolo Rossi, Ludwik K. Malendowicz and Giuseppina Mazzocchi

Department of Human Anatomy and Physiology, Section of Anatomy (G.G.N., G.M.); and Department of Experimental and Clinical Medicine, University of Padua, Padua, Italy (G.P.R.); and Department of Histology and Embryology, School of Medicine, Poznan, Poland (L.K.M.)

I. Introduction
II. Biology of Endothelins and Steroid-Secreting Cells
    A. Endothelins and their Receptors
        1. Endothelins.
        2. Endothelin Receptors.
    B. Steroid-Secreting Cells
        1. Adrenocortical Cells.
        2. Leydig Cells.
        3. Granulosa and Thecal Cells and Lutein Cells.
III. Endothelin Biosynthesis in Steroid-Secreting Tissues
    A. Gene Expression Studies
        1. Adrenal Gland.
        2. Testis.
        3. Ovary.
    B. Immunocytochemical Studies
        1. Adrenal Gland.
        2. Testis.
        3. Ovary.
    C. Summary
IV. Endothelin Receptor Subtypes in Steroid-Secreting Tissues
    A. Gene Expression Studies
        1. Adrenal Gland.
        2. Testis.
        3. Ovary.
    B. Saturation and Inhibition Binding Studies
        1. Adrenal Gland.
        2. Testis.
        3. Ovary.
    C. Immunocytochemical Studies
        1. Adrenal Gland.
        2. Testis.
        3. Ovary.
    D. Autoradiographic Studies
        1. Adrenal Gland.
        3. Ovary.
    E. Summary
V. Effects of Endothelins on the Secretory Activity of Steroid-Secreting Cells
    A. Hormone Secretion
        1. Adrenal Cortex.
        2. Testis.
        3. Ovary.
    B. Mechanisms of Action and Receptor Subtypes Involved
        1. Adrenal Cortex.
        2. Testis.
        ii. Stimulation of Cyclooxygenase-Dependent Cascade.
        3. Ovary.
        4. Other Possible Mechanisms.
    C. Summary
VI. Effects of Endothelins on the Growth of Steroid-Secreting Cells
    A. Cell Hypertrophy and Proliferation
        1. Adrenal Cortex.
        2. Testis.
        3. Ovary.
    B. Mechanisms of Action and Receptor Subtypes Involved
        1. Adrenal Cortex.
        2. Ovary.
    C. Summary
VII. Regulation of Endothelin Synthesis and Release in Steroid-Secreting Tissues
    A. Adrenal Gland
        1. Increase in Blood Flow.
        2. Effect of Nitric Oxide.
    B. Testis
    C. Ovary
    D. Summary
VIII. Involvement of Endothelins in the Pathophysiology of Steroid-Secreting Tissues
    A. Adrenal Gland
        1. Aldosterone-Producing Adenomas.
        2. Glucocorticoid- and Sex Steroid-Secreting Tumors.
        3. Adrenocortical Carcinomas.
        4. Idiopathic Hyperaldosteronism.
    B. Testis
    C. Ovary
IX. Conclusions and Perspectives
Acknowledgments
References


    I. Introduction
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Endothelins (ETs)2 are a family of endogenous peptides, mainly secreted by endothelial cells, which exert a potent vasoconstrictor and pressor activity, acting through two classes of receptors named ETA and ETB. ETs were originally isolated from porcine aortic endothelium (Yanagisawa et al., 1988; Inoue et al., 1989), but subsequent studies revealed that ETs are synthesized and their receptors are present in a variety of tissues, where they play important physiological and pathophysiological roles. At present, ETs are thought to regulate the cardiovascular apparatus and blood pressure, kidney excretion, pulmonary function, endocrine gland secretion, and the development of tissues derived from embryonic neural crest lineage and of the central and peripheral nervous systems. The ability of all of these tissues to synthesize ETs and the fact that blood concentrations of these peptides, not exceeding the picomolar range under both physiological and pathophysiological conditions (Kohno et al., 1990a; Weitzberg et al., 1991; Letizia et al., 1996; Maeda et al., 1997; Herold et al., 1998; Kobayashi et al., 1998; Makino and Kamata, 1998), are well below those able to evoke appreciable biological effects, led to the contention that ETs almost exclusively act through autocrine/paracrine mechanisms.

ETs are known to be involved in the functional regulation of neuroendocrine axes, among which are the hypothalamo-pituitary-adrenal and the hypothalamo-pituitary-gonadal axes, acting on either their central or peripheral branch. Evidence has been accumulated that indicates that the ET genes and their receptors are expressed in both adrenal glands and endocrine components of the gonads, the function of which they variously modulate. Because adrenocortical cells, Leydig interstitial cells of the testis, and ovarian granulosa/lutein cells possess common morphological and functional characteristics, they are frequently called steroid-secreting cells. Steroid-secreting cells produce cholesterol-derived hormones through complex multistep pathways involving enzymes located in both mitochondria and smooth endoplasmic reticula (SER) and release newly formed steroid hormones constitutively as soon as they are produced (i.e., without the possibility of any cytoplasmatic storage). Despite these similarities, steroid-secreting cells display marked differences in their response to ETs.

Since their discovery in the late 1980s, ETs and their receptors have been the topic of several excellent review articles, emphasizing their possible role in health and disease (LeMonnier de Gouville et al., 1989; Simonson and Dunn, 1990, 1991; Sakurai et al., 1992; Battistini et al., 1993; Haynes and Webb, 1993; Haynes et al., 1993; Lotersztajn, 1993; Bax and Saxena, 1994; Rubanyi and Polokoff, 1994; Goto et al., 1996; Levin, 1996a; Ohlstein et al., 1996; Schiffrin, 1996; Dashwood et al., 1997; Webb, 1997; Webb and Meek, 1997; Stjernquist, 1998; Webb et al., 1998). Because of the very potent vasoconstrictor effects of ETs, a large part of these surveys dealt with the cardiovascular system. The possible involvement of ETs in the functional regulation of the endocrine system also has been surveyed, although far less extensively (MacRae and Bloom, 1992; Kennedy et al., 1993; Masaki, 1993; Takuwa, 1993; Naruse et al., 1994a; Inagami et al., 1995; Stojilkovic and Catt, 1996; Ehrhart-Bornstein et al., 1998). However, only a few surveys specifically dealt with the autocrine/paracrine role played by ETs in the functional regulation of steroid-secreting tissues. Of the latter, two concerned the adrenal cortex (Nussdorfer et al., 1997a; Remy-Jouet et al., 1998), and two others concerned the endocrine gonads, although marginally (Saez, 1994; Stojilkovic, 1996).

After a brief summary of the current knowledge of the molecular and cell biology of ETs and steroid-secreting tissues, in the following sections of this survey we will describe and discuss findings indicating that locally synthesized ETs are involved in the autocrine/paracrine control of the secretion and growth of steroid-secreting tissues. Then we shall review the possible mechanisms underlying these actions of ETs, as well as those regulating the local release of ETs. Finally, evidence for the possible involvement of ETs in the pathophysiology of steroid-secreting tissues will be surveyed.


    II. Biology of Endothelins and Steroid-Secreting Cells
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A. Endothelins and their Receptors

1. Endothelins. ETs are peptides formed by 21 amino acids, with four cysteine residues forming two intramolecular disulfide bonds. The ET family includes three distinct isoforms, ET-1, ET-2, and ET-3. Their amino acid sequences are very similar, but their tissue distribution differs considerably. Interestingly, soon after the first publication on ETs, the structure of peptides isolated from the venom of the Israeli burrowing asp Actrapsis engaddensis, termed sarafotoxins (STXs), was shown to have remarkable similarities to ETs (Kloog and Sokolovsky, 1989). The four STXs known so far (STXa, STXb, STXc, and STXd) have 21-amino acid residues and possess considerable sequence homology to ETs in their carboxyl terminus, which is crucial for binding to their specific receptors (Fig. 1). Although all ETs of the family are synthesized as prepro(pp) proteins of about 200 amino acids, which share a high degree of sequence homology, and a two-step processing pathway, they are encoded by three distinct genes.



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  		Fig. 1.   Amino acid sequence of human ETs and STXc. Amino acid substitutions with respect to ET-1 are indicated.

a. ENTOTHELIN GENES AND THEIR REGULATION. Three genes encoding ET-like sequences were identified in the mammalian genome and shown to encode the precursors of ETs, ppET-1, ppET-2, and ppET-3 (Inoue et al., 1989). Subsequent studies mapped the three genes in the human genome on chromosomes 6, 1, and 20, respectively (Bloch et al., 1989a,b,1991; Hoehe et al., 1993). Detailed molecular investigation of the ppET gene promoter regions demonstrated sequences necessary for high levels of transcription (Lee et al., 1990, 1991), as well as for the specificity of tissue expression (Benatti et al., 1994). It was also established that several extracellular factors could influence ET production both positively and negatively. Factors activating the phospholipase (PL) C/protein kinase (PK) C pathway may enhance ET-1 production, through binding of the proto-oncogenes jun and fos to the activator protein-1 (AP-1) transcription regulatory element of the ppET-1 promoter. These factors include insulin, thrombin, oxidized low density lipoprotein, angiotensin-II (ANG-II), vasopressin, and ET-1 itself (Kohno et al., 1990b, 1992; Emori et al., 1991, 1992; Lee et al., 1991; Boulanger et al., 1992; Benatti et al., 1994). Factors, possibly acting through the cGMP-mediated pathway and thought to inhibit ET-1 production, include atrial natriuretic peptide (ANP), parathyroid hormone-related protein, and lipopolysaccharide (Fujisaki et al., 1995; Jiang et al., 1996; Kaddoura et al., 1996; Wada et al., 1996). Shear stress ambivalently affects ppET-1 gene transcription, with low stress (<2 dyne/cm2) and high stress (>6 dyne/cm2) enhancing and blunting it, respectively (Malek and Izumo, 1992; Kuchan and Frangos, 1993; Malek et al., 1993, 1997; 1994; Davies, 1995). Because inhibitors of either nitric oxide (NO) synthesis or guanylate cyclase abolished this latter effect, it was suggested that high shear stress releases NO, which in turn would inhibit ET-1 production via cGMP generation (Rubanyi and Polokoff, 1994). Because Ca2+ chelators also blunt ET-1 release from endothelial cells (Emori et al., 1992), it is likely that cGMP-induced inhibition of ET-1 is due to its ability to impair intracellular Ca2+ availability.

b. BIOSYNTHESIS. All biologically active isopeptides of the family are generated via a two-step processing (Fig. 2), which first involves sequential cleavage by signal peptide peptidases and furin-like convertases to form the physiologically inactive ET precursors, the big-ETs (for references, see Rubanyi and Polokoff, 1994). The big-ETs are polypeptides of 37 to 41 amino acids, which show species-specific differences in their C-terminal 22-41 sequence. They are cleaved exactly at the Trp21-Val/Ile22 peptide bond to form physiologically active ETs by a specific endopeptidase referred to as endothelin-converting enzyme (ECE). After its first cloning from bovine tissues, the cDNA species coding for two ECE forms, termed ECE-1 and ECE-2, have been isolated (for review, see Schweizer et al., 1997). Both ECEs belong to a new family of zinc metalloendopeptidases, which are inhibited by phosphoramidon, and display remarkable structural similarities with a short N-terminal domain, a single transmembrane segment, and a large C-terminal part, including the active site of the enzyme. Very recently, the purification of a novel ECE specific for big-ET-3 from bovine iris was reported (Hasegawa et al., 1998). The genomic organization of the human ECE-1 gene, which is localized on chromosome 1p36 (Valdenaire et al., 1995) and more precisely on 1p36.2.3 (Albertin et al., 1996), has recently been unraveled. Shear-stress response elements have been identified on the regulatory region of the human ECE-1 gene, which provides a rational basis for the coupling of shear stress with ET-1 synthesis (Valdenaire et al., 1995). Moreover, an ETB-mediated suppressive effect of ET-1 on ECE-1 mRNA expression has been demonstrated in cultured rat pulmonary endothelial cells, thereby suggesting the existence of a negative feedback mechanism regulating ET-1 synthesis (Naomi et al., 1998). Although the ET biosynthetic pathway involving ECEs is the predominant one and the most widely investigated, there is the possibility that big ETs are also specifically cleaved by chymase at the Tyr31-Gly32 bond (Nakano et al., 1997; Fig. 2). This cleavage would lead to novel ET(1-31) isopeptides, which were shown in vitro to evoke a less potent but slower and longer-lasting contraction of porcine coronary artery and rat aorta than ETs(1-21). Interestingly, at variance with ET-1, the vasoconstrictor effect of ETs(1-31) was inhibited by either the ETA receptor antagonist BQ-485 or the ETB receptor antagonist BQ-788, which suggests that these novel peptides might act in a different manner from classic ETs, possibly through different receptors (Kishi et al., 1998).



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  		Fig. 2.   Biosynthesis and processing of ET-1 from ppET-1. SP, signal peptide.

c. CLEARANCE. The plasma half-life of ET-1 in mammals is less than 1.5 min because it is efficiently cleared by splanchnic, renal, and pulmonary vascular beds (Hemsen et al., 1995; Kobayashi et al., 1998). On binding to its receptors on cell membrane, ET-1 undergoes internalization followed by rapid degradation, through still incompletely understood enzymatic pathways (Perez-Vizcaino et al., 1995). Because the plasma levels of ET-1 were increased by both mixed ETA/ETB- and ETB-specific, but not ETA-specific receptor antagonists, it has been proposed that the ETB receptor may play a role as a clearance receptor (Fukuroda et al., 1994; Teerlink et al., 1994; Gandhi et al., 1998; Kobayashi et al., 1998). This role of ETB receptors needs to be taken into account in the interpretation of results of experiments using ETB-specific antagonists, where a potentiation of ETA-mediated effects is likely to supervene.

2. Endothelin Receptors. Within 2 years of the initial report of ET discovery, the genes encoding two different specific receptors termed ETA and ETB, were identified and cloned (Arai et al., 1990; Sakurai et al., 1990; Lin et al., 1991). ET-1 is deemed to exert its effects via the activation of these main receptor subtypes, although molecular variants, most likely deriving from alternative splicing, have been reported (Elshourbagy et al., 1996; Miyamoto et al., 1996).

a. BIOCHEMISTRY AND MOLECULAR BIOLOGY. The ETA and ETB receptors at first were differentiated pharmacologically based on their affinity for the isopeptides of the ET family. The ETA receptor is characterized by its very high (subnanomolar) affinity for ET-1 and ET-2 and a 70- to 100-fold lower affinity for ET-3. The ETB receptor is non-isopeptide-selective, as it binds with high and similar affinity to all isopeptides of the ET family. As mentioned above, ETA and ETB receptors are coded by two different genes, which are structured into eight and seven exons and have been mapped on chromosomes 4 and 13, respectively. Because these genes show considerable similarities in their structural organization (Elshourbagy et al., 1996), they are thought to derive from a common ancestral precursor gene. Both receptors belong to the superfamily of the G protein-coupled receptors and entail seven transmembrane domains, an extracellular N terminus, and an intracytoplasmic C terminus. The two receptors differ not only in the N terminus, but also in the C terminus, which is crucial for their coupling to G proteins (see below), and, therefore, for their divergent cellular effects. In contrast, they share a high degree of homology in the seven highly conserved transmembrane domains. Based on their different affinity for selective ligands, the existence of different subtypes of ETA and ETB receptors (provisionally named ETA1, ETA2, ETA3, etc. and ETB1, ETB2, etc.) has been postulated (Table 1). However, only two genes encoding for the aforementioned two major ETA and ETB receptor subtypes exist in the mammalian genome. Thus, although a classification of ETA and ETB in pharmacologically distinguishable subtypes has been proposed, it has not gained universal acceptance because their identification at a molecular level in mammals has been elusive thus far. However, the heterogeneity of ET receptors should be taken into account in the interpretation of different results of studies that will be reviewed in this paper. Screening of amphibian libraries has revealed the existence of two additional ET receptor subtypes. ETC and ETAX receptor genes were identified and cloned from octopus melanophores and heart, respectively (Karne et al., 1993; Kumar et al., 1993).


                              
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  		TABLE 1
Main antagonists and agonists of endothelin receptors

b. G PROTEIN COUPLING AND SIGNAL TRANSDUCTION. Binding of ET isopeptides to ETA and ETB heptahelical G protein-coupled receptors results in activation of at least three classes of Galpha proteins (Galpha s, Galpha i, and Galpha q; Levin, 1996a; Miyauchi and Masaki, 1999). Interestingly, different domains of the ET-receptor structure are required for the coupling of different Galpha proteins. Galpha protein coupling is associated to the activation of the second messenger system. Galpha s coupling results in the activation of adenylate cyclase; Galpha i activation leads to the inhibition of adenylate cyclase and probably the activation of voltage-gated Ca2+ channels. Galpha q coupling most likely is linked to activation of PLC and a nonselective cation channel. Other possible signaling mechanisms of ETA and ETB receptors, including PLA2 and PLD activation (for review, see Douglas and Ohlstein, 1997), will be discussed in the following sections of this review.

c. RECEPTOR ANTAGONISTS. Two approaches to inhibit the ET system have been pursued, the blockade of ET receptors with selective or mixed ETA/ETB antagonists (for review, see Ferro and Webb, 1996; Gray and Webb, 1996), and, more recently, the inhibition of ECE-1 (Turner et al., 1998). A list of the ET receptor antagonists that have been developed and/or are currently pursued as potential therapeutic agents, as well as of the main ET receptor agonists, is shown in Table 1.

B. Steroid-Secreting Cells

Steroid-secreting cells possess common morphological features. This reflects their ability to synthesize steroid hormones starting from cholesterol, which may be taken up from circulating lipoproteins and also locally synthesized through a series of enzymes located on mitochondrial cristae and SER membranes. These cells display variously shaped mitochondria that always contain tubular or tubulovesicular cristae, a well developed SER, only a few profiles of rough endoplasmic reticulum, and a variable number of lipid droplets. The number of lipid droplets depends on the functional status of the cells because they contain cholesterol available for steroid-hormone production (for review, see Nussdorfer, 1986; Vinson et al., 1992; Sawyer, 1995; Russel, 1996).

The pathways of steroid hormone biosynthesis in the adrenal cortex and gonadal cells are depicted in Fig. 3. The first step of this process, which is selectively activated by the main agonists, is the hydrolysis of cholesterol esters stored in lipid droplets into free cholesterol, which is transported into mitochondria, where it is converted to pregnenolone by hydroxylating enzymes cleaving its side chain (P450scc). This is the rate-limiting step of steroid synthesis and is rather complex and highly regulated. To summarize, it involves the sterol carrier protein 2- and steroidogenic acute regulatory protein-mediated transport of cholesterol to the outer mitochondrial membrane and its translocation to the P450scc located on the inner membrane, a process in which peripheral benzodiazepine receptors located on the outer mitochondrial membrane seem to play a major role (Pfeifer et al., 1993; Stocco, 1996; Stocco and Clark, 1996,1997; Papadopoulos et al., 1997; Kallen et al., 1998). Newly formed pregnenolone leaves mitochondria and reaches SER, where 3beta -hydroxysteroid dehydrogenase (3beta -HSD) transforms it into progesterone. Pregnenolone and progesterone may be converted in the SER to 17-hydroxypregnenolone and 17-hydroxyprogesterone by P450c17 (17alpha -hydroxylase). At this point, the pathway of steroid synthesis differs in various steroid-secreting cells, i.e., adrenocortical cells, testis Leydig cells, granulosa and thecal cells of ovarian follicles, and lutein cells (either of granulosa or thecal origin; for review, see Erickson et al., 1985; Nussdorfer, 1986; Amsterdam and Romensch, 1987; Miller, 1988; Niswender and Nett, 1988; Fraser, 1992; Hanukoglu, 1992; Gibori, 1993; Saez, 1994; Sawyer, 1995; Payne and O'Shaughnessy, 1996; Boon et al., 1997).



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  		Fig. 3.   Pathway of hormone synthesis and degradation in steroid-secreting cells. Steps occurring in mitochondria and SER are indicated by different arrows.

1. Adrenocortical Cells. P450c21 (21-hydroxylase), located in the SER, converts progesterone and 17-hydroxyprogesterone to 11-deoxycorticosterone and 11-deoxycortisol, respectively. These two steroids penetrate the mitochondria, where P450c11 (11beta -hydroxylase) transforms them into corticosterone and cortisol, which are the glucocorticoids produced by the zona fasciculata/reticularis (ZF/R) of rodents and other mammals, respectively. P450c11 also possesses 18-hydroxylase activity, thereby producing little amounts of 18-hydroxy-11-deoxycorticosterone and 18-hydroxy-11-deoxycortisol. In these zones, and especially in the zona reticularis (ZR), a P450c17-coupled C17,20-lyase located in SER may convert 17-hydroxypregnenolone and 17-hydroxypregnenolone into adrenal androgens, i.e., dehydroepiandrosterone (DHEA) and androstenedione. In the zona glomerulosa (ZG), corticosterone is converted to 18-hydroxycorticosterone and then aldosterone by intramitochondrial P450c18 (a mixed hydroxylase-dehydrogenase, also named aldosterone synthase). Of interest, mammalian adrenocortical cells also express the 11beta -hydroxysteroid dehydrogenase (11beta -HSD) gene and possess 11beta -HSD activity (Yang and Matthews, 1995; Musajo et al., 1996; Roland and Funder, 1996; Shimojo et al., 1996; Smith et al., 1997; Mazzocchi et al., 1998c). This enzyme, located in the SER (Náray-Fejes-Toth et al., 1998), catalyzes the conversion of the glucocorticoids cortisol and corticosterone into their corresponding inactive forms, cortisone and 11-dehydrocorticosterone, respectively (for review, see Funder, 1995, 1996). Evidence has been provided that indicates that 11beta -HSD activity is negatively modulated by the rise in the intra-adrenal concentration of non-11beta -hydroxylated steroid hormones (Musajo et al., 1996; Morita et al., 1997; Mazzocchi et al., 1998c). The main agonists involved in the physiological regulation of the secretory activity of adrenocortical cells are adrenocorticotropic hormone (ACTH), ANG-II, and K+ (for review, see Quinn and Williams, 1992; Vinson et al., 1992; Ganguly and Davis, 1994). ACTH stimulates both ZG and ZF/R cells, acting through specific receptors mainly coupled with the adenylate cyclase/PKA-dependent signaling pathway. ANG-II stimulates zona glomerulosa cells, acting via AT1 receptors mainly coupled with PLC-dependent cascade. PLC catalyzes the breakdown of phosphatidylinositol to inositol-triphosphate (IP3) and diacylglycerol (DAG). DAG activates PKC, and IP3 enhances Ca2+ release from intracellular stores, thereby raising intracellular (cytosolic) calcium concentration ([Ca2+]i), which in turn activates PKC. There is evidence that ANG-II may also activate tyrosine kinase (TK)-dependent cascade. In humans and calves, ANG-II is also able to stimulate ZF/R cells secretion (Bird et al., 1992; Ouali et al., 1992; Clyne et al., 1993; Lebrethon et al., 1994). There is general agreement that ACTH and ANG-II are able to activate both the cyclooxygenase (CO) and lipoxygenase (LO) pathways of arachidonic acid, leading to the production of prostaglandins (PGs) and 12-hydroxyeicosatetraneoic acid, respectively. Arachidonic acid may be released from plasma membrane phospholipids by the action of PLA2 or it may derive from DAG. K+ electively depolarizes plasma membrane of ZG cells, thereby opening voltage-gated Ca2+ channels and raising [Ca2+]i. The main pathways involved in the signaling mechanism of steroid-secreting-cell agonists and their possible interrelationships are depicted in Fig. 4, and the most widely employed selective stimulators and inhibitors of the various steps of the signaling cascades are shown in Table 2.



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  		Fig. 4.   Main pathways in the signaling mechanisms of agonists in steroid-secreting cells. AC, adenylate cyclase; CM, calmodulin; G, G protein; PCh, phosphatidyl choline; PIP2, phosphatidylinositol biphosphate; R, receptor. Other abbreviations are indicated in the text.


                              
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  		TABLE 2
Main inhibitors and activators of the signaling cascades

2. Leydig Cells. 17-Hydroxypregnenolone and 17-hydroxyprogesterone are converted into DHEA and androstenedione by P450c17/17-20-lyase, as occurs in adrenal ZR. DHEA, in turn, is transformed to androstenedione by 3beta -HSD. A 17beta -HSD, located in the SER, then transforms androstenedione to testosterone, the main androgen produced by the testis. Testosterone may be partially inactivated by 5alpha -reductase, which transforms it into 5alpha -dihydrotestosterone, or in small amounts aromatized to estradiol by P450arom. All of these last steps occur in the SER. Luteinizing hormone (LH) is absolutely required for the maintenance of Leydig cell-specific functions and is the main secretagogue of testosterone, at least under physiological conditions (for review, see Saez, 1994). LH acts through specific LH/human chorionic gonadotropin (hCG) G protein-coupled receptors, the main signaling mechanism of which is the activation of adenylate cyclase/PKA cascade. Evidence also is available that PLC- and PLA2-dependent cascades may play a role in transducing LH secretagogue signals (for review, see Cooke, 1996; Fig. 4).

3. Granulosa and Thecal Cells and Lutein Cells. The pathway of steroid synthesis, leading to the production of progesterone and estrogens, is the same as described above and is strictly controlled by follicle-stimulating hormone (FSH) and LH. Again, progesterone may be inactivated by 20alpha -HSD and 5alpha -reductase. Granulosa cells are exclusively provided with FSH receptors, whereas thecal cells (located in the theca interna) possess LH-hCG receptors, both receptors being mainly coupled with the adenylate cyclase/PKA signaling cascade (Fig. 4). Thecal cells under the influence of LH are stimulated to differentiate and produce androstenedione and little amounts of testosterone. These hormones diffuse through the basement lamina, reaching granulosa cells, which, under the influence of FSH, aromatize them to estradiol and estrone. Estrogens diffuse into the capillaries of the theca interna or are poured in the intracellular spaces of the granulosa. The rise in the level of circulating estrogens results in the preovulatory surge of LH, and the increasing intrafollicular concentration of estrogens stimulates proliferation of granulosa cells, which in the preovulatory period acquire LH receptors. Under the influence of LH, the production of estrogens of granulosa cells declines, and their main secretory product becomes progesterone. After ovulation, steroid- secreting cells of the ovarian follicle differentiate into granulosa lutein cells and thecal lutein cells, which both secrete large amounts of progesterone, the elevated levels of LH greatly enhancing in them the expression of P450scc and 3beta -HSD genes (for review, see Erickson et al., 1985; Hanukoglu, 1992; Hsueh and Billig, 1995).


    III. Endothelin Biosynthesis in Steroid-Secreting Tissues
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Much evidence indicates that ETs are synthesized in the mammalian adrenal gland, testis, and ovary. At present, no data are available on this matter as far as lower vertebrates are concerned.

A. Gene Expression Studies

1. Adrenal Gland. Following the preliminary findings of low-abundance expression of ET-1 mRNA in the porcine adrenal gland (Nunez et al., 1990), numerous studies were carried out in tissues harvested from humans and rats that used reverse transcription-polymerase chain reaction (RT-PCR) and Southern hybridization with a 21-mer 35S-labeled oligonucleotide probe complementary to a sequence within the cDNA between the primers.

a. HUMAN ADRENALS. The presence of ppET-1 mRNA was first demonstrated in homogenates of adrenals from patients with aldosterone-producing adenomas (APA) causing Conn's syndrome who were treated by adrenalectomy (Imai et al., 1992). Using Northern blotting of poly(A)+ RNA and hybridization with a specific cDNA probe for the ET-1 gene, ppET-1 mRNA was detected in the normal cortical tissue surrounding the tumor. These results were confirmed by Rossi et al. (1994, 1995b), who studied the ppET-1 gene expression by RT-PCR on homogenates of a large number of normal adrenals obtained from patients undergoing unilateral adrenalectomy for kidney cancer. However, the use of tissue homogenates prevented definitive conclusions concerning the parenchymal or extraparenchymal location of the ppET-1 gene, and the same was true for the ECE-1 gene, whose expression was demonstrated in homogenates of both adrenocortical and adrenomedullary tissues (Rossi et al., 1995a). To address this issue, Rossi et al. (1997b) used dispersed and purified adrenocortical cells. RT-PCR evidenced the expression of ppET-1 and ECE-1 genes in these preparations, thereby making it likely that human adrenocortical cells are able to synthesize ET-1. These findings were confirmed by Mathieu et al. (1988) by using primary cultures of human adrenal cells enriched in ZG cells. Although ETs are known to play an important role in the development of neural crest-derived cells (Kurihara et al., 1994; Brand et al., 1998) and ET mRNAs have been detected in pheochromocytomas (Amico et al., 1993; Kon, 1993), ET genes do not appear to be expressed in normal adrenal medulla.

b. RAT ADRENALS. The expression of the ppET-1 gene was demonstrated by RT-PCR in both homogenates and dispersed ZG or ZF/R cells (Belloni et al., 1996c). The presence of ET gene products in adrenal medulla has not been reported.

2. Testis. ET-1 protein and ppET-1 mRNA have been detected in homogenates of the rat testis (Matsumoto et al., 1989; Sakurai et al., 1991). This finding was confirmed in the human testis by in situ hybridization (Ergun et al., 1998).

3. Ovary. a. PRIMATE OVARY. ppET-1 mRNA has been demonstrated by Northern blot analysis in the ovarian homogenates. Subsequent in situ hybridization showed its presence in the granulosa cells, but not in the thecal compartment of follicles at different stages of maturation (Magini et al., 1996). The hybridization signal was especially prominent in the granulosa cells of the tertiary follicles, and also detectable in the endothelial cells of the blood vessels. Western blot analysis revealed the presence of ECE-1 in the menstrual corpora lutea, the enzyme being more abundant in early and midluteal phases than in the late luteal phase (Yoshioka et al., 1998). Karam et al. (1999) confirmed the presence of ECE-1 gene expression in the theca interna cells of secondary, tertiary, and atretic follicles, as well as in the corpora lutea of human and monkey ovary; however, ET-1 mRNA was observed only in the endothelial cells of blood vessels. The presence of ET-like immunoreactivity (LI) in the follicular fluid has been studied by several groups. Earlier radioimmunoassay measurements gave rather inhomogenous results, with ET-1 concentration ranging from 12 to 200 fmol/ml in mature follicles, and from 0.2 to 0.4 pmol/ml in immature ones (Kamada et al., 1993b; Abae et al., 1994; Kubota et al., 1994). However, an inverse correlation between ET-1 concentration and the maturity of follicles has not been confirmed by other investigators (Schiff et al., 1993; Magini et al., 1996; Sudik et al., 1996). Haq et al. (1996) reported the presence of ET-1-LI, ET-2-LI and big-ET-1-LI in the follicular fluid obtained from mature follicles at the time of oocyte aspiration in women undergoing ovulation induction by human menopausal gonadotropin. The mean concentrations of the three peptides were 6.8, 12.6, and 8.2 fmol/ml, respectively. Sudik et al. (1996) studied the level of ET-1 and ET-2 in follicular fluids of 57 women undergoing an in vitro fertilization-embryo transplantation (IVFET) program. ET-1-LI was detected in all samples (7.6 ± 4.8 fmol/ml), and ET-2-LI in about 68% of samples (5.5 ± 6.3 fmol/ml). A significant negative correlation existed between ET-2 concentration and follicle size. No obvious correlation was observed between ET concentration and estradiol, progesterone, and testosterone content in the follicular fluid (Kamada et al., 1993b; Schiff et al., 1993; Sudik et al., 1996). These findings strongly suggest the involvement of ETs in the follicular development. In keeping with this contention are the observations of Stones et al. (1996), who studied ET release from isolated perfused premenopausal ovaries. Of the 12 studied, five released ETs, and four of these contained either a developing follicle or a corpus luteum. Seven of the eight inactive ovaries did not release ETs.

b. CALF OVARY. Northern blot hybridization revealed the expression of ET-1 gene in ovary homogenates (Fujii et al., 1991). ET-1 mRNA and protein levels in the bovine corpora lutea depended on the stage of estrous cycle (Girsh et al., 1996a,b). In young corpora lutea (day 5) they were 2.9 ± 0.3 U and 6.8 ± 1.2 fmol/mg, respectively. In old corpora lutea (day 18) 7.4 ± 0.5 U and 19.5 ± 1.9 fmol/mg. Accordingly, plasma ET-1 concentration increased around luteolysis and estrus (Ohtani et al., 1998).

c. PIG OVARY. The expression of ppET-1 mRNA and the presence of ET-1-LI have been demonstrated in cultured granulosa cells from small or medium follicles (Kamada et al., 1993a; Kubota et al., 1994). The concentration of ET-1-LI in the follicular fluid was 3.6 to 5.7 fmol/ml (Iwai et al., 1991).

d. RAT OVARY. The ET-1 content in the ovary of adult animals was very elevated (1.2 ± 0.2 pmol/g; Usuki, 1991; Usuki et al., 1991c). High concentrations of ET-1-LI have been also detected in the corpora lutea of superovulated rats and follicles (Usuki et al., 1991b, c, 1992). The fact that these concentrations where higher than those present in the blood indicates the local synthesis of ETs.

B. Immunocytochemical Studies

The presence of immunoreactive ETs in steroid-secreting tissues has been investigated by the use of specific polyclonal and monoclonal antibodies against ETs risen in different species (Kondoh et al., 1990; Traish et al., 1992).

1. Adrenal Gland. All of the studies were carried out in normal or pathologic human glands. Li et al. (1994) reported the presence of ET-1-LI especially in the ZF, where about 50% of parenchymal cells were positive. Immunostained cells were few in the ZG and ZR, and absent in the vascular elements of the gland. Immunostaining appeared in the form of grains, vacuoles, or membranes. Subsequent electron microscopic studies (Li et al., 1995a) showed that vacuoles and grains corresponded to ET-1 located around lipid droplets and in endoplasmic reticulum-mitochondria, respectively, whereas ET-1 found in membranes was probably bound to its receptors. More recently, Li et al. (1999) confirmed the distribution of ET-1-positive cells in the human adrenal, but reported that the extracted ET-1-like protein had a molecular mass of 9 kDa, i.e., lower than that of ppET-1 (21 kDa) and higher than that of ET-1 (2.5 kDa). The functional relevance of this finding remains to be elucidated, although it could suggest alternative post-translational processing of big-ET-1 in the human adrenals (see Section IIA). According to Hiraki et al. (1997), ET-1/big-ET-1-LI was present throughout all of the cortex, although more abundant in the ZF, with ET-3/big-ET-3-LI being very limited in the cortical cells. All of these investigators agree that ET-LI was absent in adrenal medulla, although ET-1-LI and ET-3-LI were detected in pheochromocytomas (Sone et al., 1991; Watanabe et al., 1997). Contrasting findings were obtained by Davenport et al. (1996,1998), who reported ET-1/big-ET-1-LI and ECE-1-LI to be exclusively confined to endothelial cells of the smaller resistance vessels of the pericapsular arterial plexus and the central vein, and ET-3/big-ET-3-LI in the adrenal medulla.

2. Testis. According to Fantoni et al. (1993), in 20-day-old rats, Leydig cells did not contain immunocytochemically detectable ET-1-LI, whereas Sertoli cells were positive. ET-1-LI was detectable in Leydig cells, as well as in Sertoli and endothelial cells, of adult rat testis (Fantoni et al., 1993; Collin et al., 1996).

3. Ovary. The presence of immunostaining for ET-1 in the human ovarian cortex has been demonstrated by Magini et al. (1996). ET-1-LI was located in the wall of follicles at different stages of maturation, especially in the cytoplasm of granulosa cells and in the endothelial cells of the thecal capillaries. Follicles at the early stage of development were deprived of ET-1-LI. In contrast, Karam et al. (1999) reported the presence of ET-1-LI exclusively in the blood vessels of the human and monkey ovaries. ECE-1 was immunocytochemically detected in the human ovary (Yoshioka et al., 1998): in the preovulatory follicles, immunostaining was weak in granulosa cells and moderate in theca interna cells; in both menstrual and pregnant corpora lutea, abundant immunostaining was contained in both types of lutein cells, thereby suggesting that ECE-1 expression increases during luteinization.

C. Summary

RT-PCR demonstrated the expression of the ET-1 and ECE-1 genes in the mammalian adrenal glands and ovary (developing corpora lutea), and of the ET-1 gene in the Leydig cells of the testis. Radioimmunoassay and immunocytochemistry detected ET-LI in all three steroidogenic tissues.
    IV. Endothelin Receptor Subtypes in Steroid-Secreting Tissues
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The gene expression, localization, and binding properties of ETA- and ETB-receptor subtypes have been investigated only in steroidogenic tissues of mammals by both biochemical and morphological techniques.

A. Gene Expression Studies

1. Adrenal Gland. a. HUMAN ADRENALS. The expression of the ET receptor mRNAs was first detected by Northen blotting analysis in homogenates of normal cortical tissue surrounding APAs. However, the results of the 125I-ET-1 competitive displacement binding were consistent with the presence of only a single class of receptors (Imai et al., 1992). The presence of both ETA- and ETB-subtype mRNAs was conclusively demonstrated by Rossi et al. (1994, 1995b). They were able to detect by RT-PCR the expression of both receptor-subtype genes in homogenates of a large number of histologically normal adrenal cortices of patients adrenalectomized for kidney cancer. These findings were subsequently confirmed by Davenport et al. (1996). Further studies showed the presence of both ETA- and ETB-receptor mRNAs in isolated and purified normal human adrenocortical cells (Rossi et al., 1997b).

b. RAT ADRENALS. ET-receptor mRNAs were detected by in situ hybridization by Hori et al. (1992). The hybridization signal for ETB mRNA was diffusely distributed in both cortex and medulla, whereas that for ETA mRNA was almost exclusively restricted to the cortico-medullary border, in the blood vessels of this region. To localize the ETA and ETB genes in the different adrenocortical zones, Belloni et al. (1996c) investigated by RT-PCR both homogenates and dispersed ZG and ZF/R cells. Although they found the specific mRNA for both receptor subtypes in tissue homogenates, they could detect only the ETB-receptor mRNA in both dispersed adrenocortical-cell types. This last finding strongly suggested the extraparenchymal expression of the ETA gene in the adrenal cortex of this species.

2. Testis. Maggi et al. (1995) detected by Northen blot analysis the specific mRNAs for ETA and ETB receptors in homogenates of human testis, with the level of ETA expression being about 7-fold higher than that of ETB, a finding consistent with the reported lack of ETB-receptor mRNA in the rat testis (Sakurai et al., 1990).

3. Ovary. In cultured luteinized human granulosa cells obtained from patients undergoing an IVFET program, Northern blot analysis revealed the expression of both ETA- and ETB-receptor genes. The ETA-receptor mRNA was much more abundant than that of ETB receptors (Kamada et al., 1995). However, in situ hybridization studies showed the presence of both ETA- and ETB-receptor mRNAs only in the vascular component of the human and monkey ovaries (Karam et al., 1999). The mRNAs of both ET-receptor subtypes were detected in the rat ovary, but in this case, ETB expression prevailed over that of ETA (Iwai et al., 1993). In situ hybridization showed that ETB mRNA was abundant in the granulosa cells of developing follicles and absent in atretic and preantral ones. No hybridization signals were observed in the thecal cells.

B. Saturation and Inhibition Binding Studies

1. Adrenal Gland. a. HUMAN ADRENALS. Earlier studies showed that in human adrenocortical membranes, 125I-ET-1 saturation isotherms attained an equilibrium after 30 min at 30°C; competitive displacement by ET-2 and ET-3 was consistent with the presence of a single class of high-affinity binding sites (Nunez et al., 1990). However, different findings were obtained by Rossi et al. (1994), who were able to demonstrate the presence of both ETA- and ETB-receptor subtypes, and to characterize their density and binding properties.

b. CALF ADRENALS. In bovine ZG cell cultures, 125I-ET-1 binding was time-dependent, was saturable, and reached an apparent equilibrium after 60 min at room temperature (Cozza et al., 1989). Scatchard analysis revealed the presence of a single class of high-affinity binding sites with an apparent Kd for ET-1 of 100 pM and a Bmax of 52,500 receptors/cell. ET-3 was only about 40% as potent as ET-1. Further investigation (Gomez-Sanchez et al., 1990) showed that although ET-1 and STXb were equipotent in their aldosterone secretagogue action on ZG cell cultures, STXb displaced 125I-ET-1 binding with only 3% of the ET-1 potency. This discrepancy suggested the possibility of the presence of a second adrenal receptor for ET-1, a hypothesis that was further supported by the observation that STXb was more effective than ET-1 in down-regulating the higher-affinity, lower-capacity binding site.

c. RAT ADRENALS. A high density of 125I-ET-1 binding sites, which was surpassed only by that found in the lung, was reported in the adrenal gland by both in vitro and in vivo studies, with the adrenal medulla being much more heavily labeled than the cortex (Koseki et al., 1989a,b). More recent investigations (Kapas et al., 1996) demonstrated 125I-ET-1 binding in the cortex, which was saturable at from 75 to 120 and from 175 to 200 pM in the ZG and ZF/R, respectively. Scatchard analysis revealed the presence of two distinct populations of binding sites in the ZG: high-affinity (Kd, 1.87 nM; Bmax, 535 fmol/106 cells) and low-affinity (Kd, 10.25 nM; Bmax, 1047 fmol/106 cells) binding sites; conversely, ZF/R contained only a single class of low-affinity binding sites (Kd, 4.95 nM; Bmax, 521 fmol/106 cells). BQ-123 inhibited 125I-ET-1 binding in the ZG with a potency comparable to that of ET-1, whereas RES-701-1 was less effective; the reverse occurred in the ZF/R. Kapas et al. (1996) concluded that the ETA receptors predominate in rat ZG and ETB receptors in the inner cortical zones.

2. Testis. The presence of a single class of ET binding sites has been described in rat testis homogenates (Sakaguchi et al., 1992) and dispersed Leydig cells (Fantoni et al., 1993). Scatchard analysis demonstrated a Bmax of 250 and 13 pmol/mg of protein, respectively. Competition curves showed the following rank of affinity: ET-1 = ET-2 (Kd, 0.6 nM) >> ET-3 (Kd, 6 nM) >> big-ET-1, thereby suggesting that the binding sites were ETA receptors. A single class of ET-1 binding sites (Kd, 1 nM; Bmax, 59 fmol/106 cells) was also reported in the murine tumor cell line MA-10 (Ergul et al., 1993).

3. Ovary. a. HUMAN OVARY. Two distinct populations of ET-binding sites were found in the human ovary, with the following binding parameters: Bmax, 5309 versus 50 fmol/mg of protein; Kd for ET-1, 0.88 versus 0.02 nM; Kd for ET-3, 344 versus 0.01 nM; Kd for BQ-123, 5 versus >10,000 nM; and Kd for IRL-1620, > 10,000 versus 14 nM. These data clearly indicated that the two classes of binding sites were ETA and ETB receptors, respectively, ETA being about 100-fold more abundant than ETB (Mancina et al., 1997).

b. CALF OVARY. A single class of high-affinity ET binding sites has been demonstrated in cultured lutein cells by Girsh et al. (1996a). Scatchard analysis indicated a Bmax of from 38 to 50 fmol/105 cells, and a Kd for ET-1 of from 0.2 to 0.5 nM. The Kd for ET-3 was 3 orders of magnitude less. This finding, along with the selective displacement of 125I-ET-1 binding by BQ-123, suggested that the binding sites were ETA receptors. However, the inhibition curve of ET-1 binding by ET-3 was biphasic in the whole corpus luteum-derived cells, probably due to the presence of ETB receptors in the endothelial cells.

c. PIG OVARY. The presence of a single class of high-affinity binding sites for both ET-1 and ET-3 was demonstrated in cultured granulosa cells (Kamada et al., 1992) and membranes of granulosa cells harvested from small- or medium-sized follicles (Kubota et al., 1994): Bmax and Kd were 1.84 pmol/mg of protein and 0.6 nM, respectively. Similar results were obtained more recently by Flores et al. (1995). All of the investigators agree that these binding sites are predominantly ETA receptors.

d. RAT OVARY. In dispersed or cultured granulosa cells obtained from immature rat ovaries, a single class of high-affinity ET binding sites was found, which displayed similar affinity for ET-1, ET-2, and ET-3 (Tedeschi et al., 1994; Otani et al., 1996). Scatchard analysis demonstrated a Bmax of 2.5 fmol × 105 cell and a Kd of 0.23 nM. Otani et al. (1996) also showed that 125I-ET-1-specific binding was concentration-dependently increased by FSH, decreased by LH, and unaffected by 10-7 M estradiol, androstenedione, or progesterone. A promoting effect of FSH on ET-1 binding of granulosa cells was also observed by Usuki et al. (1998). Subsequent equilibrium-binding studies with 125I-Tyr13-ET-1 and the ETB receptor agonist 125I-IRL-1620 provided evidence that rat granulosa cells possessed both ETA and ETB receptors, and that FSH predominantly increased the number and affinity of the ETA subtype.

C. Immunocytochemical Studies

The lack of widely available antibodies specific for each ET-receptor subtype until now has prevented extensive investigations.

1. Adrenal Gland. Hagiwara et al. (1993) studied the localization of the ETB receptor in the bovine adrenals and found prominent staining exclusively in the endothelial cells of inner zones. More recently, using antibodies against ETA (amino acid residues 59-69) and ETB (residues 420-433) raised in rabbits, Hiraki et al. (1997) observed the presence of ETB receptors especially in the ZG of the bovine adrenal cortex; whole ETA immunostaining was only occasionally seen in the cortex. The adrenal medulla did not show any staining with either antiserum. The discrepancy of these findings with the distribution of ETA- and ETB-receptor mRNAs may be explained by assuming that ET receptor density is not high enough to be visualized with this technique.

2. Testis. Using a specific antiserum raised against ETB receptors, Hagiwara et al. (1993) showed that in the bovine testis, the percentage of immunoprecipitable ET-1 binding sites was only 5%, thereby making it likely that 95% of binding sites were ETA receptors. However, the presence of both ETA and ETB receptors has been reported recently in human Leydig cells (Ergun et al., 1998).

3. Ovary. The exact location of ET-receptor subtypes in the ovarian follicles and corpus luteum has not been demonstrated by immunocytochemistry.

D. Autoradiographic Studies

1. Adrenal Gland. a. HUMAN ADRENALS. Using in vitro labeling and quantitative densitometry, Davenport et al. (1989) showed that 125I-ET-1 binding was about 2-fold higher in the outer cortex than in the ZF/R. The kind and distribution of ETA- and ETB-receptor subtypes has been investigated by the use of selective ligands (Belloni et al., 1994; Rossi et al., 1994). Total 125I-ET-1 (10-9 M) binding was intense in the ZG, whereas in the inner cortex it appeared weak and mainly confined between parenchymal-cell cords; the muscular wall of the extracapsular arterioles was heavily labeled. BQ-123 (10-7 M) eliminated labeling in the vessels and markedly decreased it in the ZG without affecting binding in the ZF; in contrast, BQ-788 and STXc (10-7 M) lowered labeling in the ZG and completely inhibited it in the ZF without changing binding in the capsular vessels. Collectively, these findings allowed these investigators to conclude that ZG was provided with both ETA and ETB receptors, and ZF with only ETB receptors. More recently, Davenport et al. (1996) labeled ETA and ETB receptors using 125I-PD-151242 and 125I-BQ-3020, respectively, and performed saturation-binding assay with computer-assisted densitometric analysis of autoradiograms. ETA receptors were present only in the ZG (Kd, 140 pM; Bmax, 70 fmol/mg of protein). In contrast, ETB receptors were found throughout the entire gland: ZG (Kd, 101 pM; Bmax, 63 fmol/mg of protein), ZF (Kd, 145 pM; Bmax, 68 fmol/mg of protein), ZR (Kd, 118 pM; Bmax, 72 fmol/mg of protein), and the adrenal medulla (Kd, 145 pM; Bmax, 76 fmol/mg of protein).

b. RAT ADRENALS. Earlier studies showed that, like in humans, 125I-ET-1 labeling in rats was higher in the outer than the inner adrenal layers (Davenport et al., 1989; Kohzuki et al., 1989, 1991). Kohzuki et al. (1991) also calculated by quantitative densitometry a Ka for ET-1 binding of 7.1 and 9.5 × 10-9 M in the ZG and the adrenal medulla, respectively. Although Koseki et al. (1989a,b) reported heavier labeling of the medulla than of the cortex after in vivo i.v. injection of 125I-ET-1, Neuser et al. (1989, 1991), using whole-body autoradiography, showed about equal 125I-ET-1 enrichments in the two zones. A diffuse labeling of the entire gland was also observed after the injection of 125I-big-ET-1 (Neuser et al., 1991). The use of selective ligands of ETA and ETB receptors and computer-assisted densitometry allowed Belloni et al. (1995, 1996c) to show that rat adrenal possessed the same receptor-subtype distribution as human glands. Subsequent studies (Belloni et al., 1997) not only confirmed the previous results, but also demonstrated that 125I-ET-3 and 125I-BQ-3020 binding was present in both the cortex and the medulla and was completely displaced by BQ-788, but not by BQ-123. Taken together, the above surveyed findings clearly indicate the presence of both ETA and ETB receptors in the ZG and the adrenal medulla, and of only ETB receptors in the ZF/R.

2. Testis. 125-I-ET-1 binding sites were autoradiographically demonstrated in Leydig and peritubular myoid cells of the rat testis (Sakaguchi et al., 1992; Fantoni et al., 1993; Maggi et al., 1995; Belloni et al., 1996a). Binding of Sertoli cells and seminiferous tubules was very scarce. Belloni et al. (1996a) provided evidence that 125I-ET-1 binding was displaced by BQ-123 and unaffected by BQ-788 and STXc, thereby confirming that Leydig cells were almost exclusively provided with ETA receptors.

3. Ovary. In situ 125I-ET-1- and 125I-ET-3-binding inhibition studies showed that the majority of ETA and ETB receptors were present in the blood vessels of the human ovary. ETA receptors were located in the theca interna cells of the ovulatory follicles, in close proximity to the granulosa layer, the cells of which were weakly labeled. ETB receptors were absent in human follicles (Mancina et al., 1997). 125I-ET-1 binding sites were autoradiographically demonstrated in the granulosa cell layer of preovulatory follicles and in the vascular component of corpora lutea of the pig ovary; binding was absent in the granulosa cells of preantral follicles and in lutein cells (Flores et al., 1995). Displacement binding studies with selective ligand indicated that ET receptors were of the ETA subtype. Cultured granulosa cells collected from large antral follicles retained their ET-1 binding capacity at a higher rate than that of cells obtained from immature follicles (Flores et al., 1995). In the pregnant mare serum- and hCG-treated rat ovaries, 125I-ET-1 binding sites were exclusively detected in the granulosa cells, especially of the antral follicles. They were absent in the thecal and lutein cells, as well as in the atretic follicles (Otani et al., 1996). Mancina et al. (1997) confirmed this observation and showed that ET binding sites were ETB receptors by using selective ligands.

E. Summary

RT-PCR demonstrated the expression of both ETA- and ETB-receptor genes in the mammalian adrenal glands. The expression of ETA gene prevails over that of ETB gene in the testis and ovary. Saturation- and inhibition-binding studies showed that adrenal glands are provided with two classes of ET receptors, whereas testis and ovary almost exclusively possess ETA receptors. Immunocytochemical and radioautographic studies confirmed these findings, and additionally demonstrated that adrenal ZG and medulla contain both ETA and ETB receptors, and inner adrenocortical zones contain only ETB receptors.
    V. Effects of Endothelins on the Secretory Activity of Steroid-Secreting Cells
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A. Hormone Secretion

1. Adrenal Cortex. A potent secretagogue action of ETs has been firmly demonstrated on both mineralocorticoid and glucocorticoid hormones.

a. HUMAN ADRENOCORTICAL CELLS. By using a mixture of dispersed human ZG and ZF/R cells, Hinson et al. (1991c) observed a marked stimulatory action of both ET-1 and ET-3 on basal aldosterone and cortisol secretions. Minimal effective concentrations were 10-14/10-13 M. The maximal effective concentration was 10-7 M, which elicited 5- and 11- to 30-fold rises in aldosterone and cortisol, respectively. These findings with ET-1 were confirmed recently by studies showing that 10-9 M ET-1 elicited 5- and 3-fold increases in aldosterone and cortisol output (Rossi et al., 1997b). Moreover, these authors showed a marked pregnenolone secretagogue effect of ET-1, thereby indicating that the peptide enhanced both early and late steps of steroid synthesis (Fig. 3). Less-marked stimulatory effects of ET-1 were observed by using adrenal slices obtained from kidney donors or fragments of normal adrenal tissue adjacent to APAs (Zeng et al., 1992). In those tissues, aldosterone secretion was increased, with minimal and maximal effective concentrations of 10-11 M (30% rise) and 10-8 M (60% rise), respectively. In vivo studies carried out in human volunteers did not show any effect of ET-1 infusion on both plasma renin activity (PRA) and basal aldosterone or cortisol plasma concentrations (Vierhapper et al., 1990, 1995). However, the i.v. infusion of ET-1, at a rate of 2 pmol/kg · min for 15 min and 1 pmol/kg · min for the subsequent 105 min, evoked a 75% potentiation of plasma aldosterone response to the bolus injection of 0.25 mg ACTH.

b. BOVINE ADRENOCORTICAL CELLS. Using cultured bovine ZG and ZF cells, Rosolowsky and Campbell (1990) were unable to observe any effect of ET-1 on either basal aldosterone and cortisol secretion or ANG-II-stimulated aldosterone release over a range of concentrations from 10-11 to 10-6 M. However, they showed a clear-cut potentiating action of ET-1 on both ACTH- and dibutyryl (db) cAMP (cAMP)-stimulated aldosterone release. The maximal effective concentration was 10-8 M (about 50% increase). Of interest, a 2-day exposure to ACTH enabled cultured ZG cells to respond to 5 × 10-8 M ET-1 by raising their basal release of aldosterone. This finding makes likely the possibility that ACTH sensitized ZG cells to ET-1 via the activation of adenylate cyclase. Quite different results were obtained by Cozza and Gomez-Sanchez (Cozza et al., 1989, 1992; Cozza and Gomez-Sanchez, 1990, 1993). They found that ET-1 concentration-dependently increased basal aldosterone secretion of cultured ZG cells, with minimal and maximal effective concentrations being 10-10 M (2-fold rise) and 10-8 M (4-fold rise), respectively. Big-ET-1 and ET-3 were less effective than ET-1, whereas ET-2 and STXb were equipotent with ET-1. ET-1 also potentiated maximal ANG-II-stimulated aldosterone secretion (10-10 M and 10-8 M ET-1; 70 and 100%, respectively), acting on both early and late steps of steroid synthesis. ET-1 also potentiated the aldosterone response to ACTH, although to a lesser extent than that to ANG-II, but it did not affect the response to K+.

c. DOG AND RABBIT ADRENOCORTICAL CELLS. ET-1 infusion (i.v. 30 ng/kg · min) induced a sizeable increase in both PRA and plasma aldosterone concentration (PAC) in the dog (Goetz et al., 1988; Miller et al., 1989, 1993). ET-1 enhanced basal aldosterone but not corticosterone secretion of dispersed rabbit adrenocortical cells. The response was moderate (maximal effective concentration elicited a 26% rise), although ZG cells displayed a very high sensitivity to ET-1 (EC50, 5 × 10-14 M).

d. RAT ADRENOCORTICAL CELLS. Consistent evidence indicates that ETs enhance basal aldosterone and corticosterone production by dispersed ZG and ZF/R cells, respectively. However, marked differences were reported with regards to the intensity of the response and the sensitivity to the peptides. The minimal effective concentration of ET-1 ranged from 10-14 to 10-10 M. The maximal effective concentration (10-8/10-7 M) evoked rises varying from 50% (Woodcock et al., 1990b) to 2- or 3-fold (Mazzocchi et al., 1990a; Belloni et al., 1995, 1996c, 1997) or even to 15- or 30-fold (Hinson et al., 1991a,b