Progesterone receptor in the chick oviduct: an immunohistochemical study with antibodies to distinct receptor components.

We performed immunohistochemical studies of chicken oviduct after different fixation procedures, by using antibodies against the progesterone receptor: polyclonal antibodies IgG-G3 against the "8S" form (an oligomere containing progesterone-binding and nonprogesterone-binding units), polyclonal antibodies IgG-RB against the progesterone-binding B subunit, and monoclonal BF4 against the non-progesterone-binding 90,000-mol-wt protein component. Chickens were immature animals with or without estrogen priming, and with or without progesterone treatment. The antibodies were revealed by means of an immunoperoxidase technique that used the avidin-biotin-peroxidase complex, and controls were performed by presaturation of antibodies with the purified 8S-progesterone receptor, the B subunit, and 90,000-mol-wt protein. The progesterone receptor was detected not only in well-characterized target tissues, i.e., in glands and luminal epithelium, but also in stromal cells (some displayed the strongest reaction), in mesothelium, and in fibers of smooth muscles. Only in cell nuclei, whether or not the animals received an injection of progesterone was an antigen revealed corresponding to the B subunit (and/or to the A subunit, because there is immunoreactivity of IgG-RB with both hormone-binding subunits A and B). The 90,000-mol-wt protein was revealed in both cytoplasm and nuclei. These immunohistological data suggest that the concept of steroid action that necessarily involves the original formation of the hormone-receptor complexes in the cytoplasm before translocation to the nucleus, may have to be revised.

Ectopic expression of serotonin7 receptors in an adrenocortical carcinoma co-secreting renin and cortisol.

Abnormal expression of membrane receptors has been previously described in benign adrenocortical neoplasms causing Cushing's syndrome. In particular, we have observed that, in some adreno corticotropic hormone (ACTH)-independent macronodular adrenal hyperplasia tissues, cortisol secretion is controlled by ectopic serotonin(7) (5-HT(7)) receptors. The objective of the present study was to investigate in vitro the effect of serotonin (5-hydroxy tryptamine; 5-HT) on cortisol and renin production by a left adrenocortical carcinoma removed from a 48-year-old female patient with severe Cushing's syndrome and elevated plasma renin levels. Tumor explants were obtained at surgery and processed for immunohistochemistry, in situ hybridization and cell culture studies. 5-HT-like immunoreactivity was observed in mast cells and steroidogenic cells disseminated in the tissue. 5-HT stimulated cortisol release by cultured cells. The stimulatory effect of 5-HT on cortisol secretion was suppressed by the 5-HT(7) receptor antagonist SB269970. In addition, immunohistochemistry showed the occurrence of 5-HT(7) receptor-like immunoreactivity in carcinoma cells. mRNAs encoding renin as well as renin-like immunoreactivity were detected in endothelial and tumor cells. Cell incubation studies revealed that the adrenocortical tissue also released renin. Renin production was inhibited by 5-HT but was not influenced by ACTH and angiotensin II (Ang II). In conclusion, the present report provides the first demonstration of ectopic serotonin receptors, i.e. 5-HT(7) receptors, in an adrenocortical carcinoma. Our results also indicate that 5-HT can influence the secretory activity of malignant adrenocortical tumors in an autocrine/paracrine manner. The effects of 5-HT on adrenocortical tumor cells included a paradoxical inhibitory action on renin production and a stimulatory action on cortisol secretion involving 5-HT(7) receptors.

Ontogeny of endothelins-1 and -3, their receptors, and endothelin converting enzyme-1 in the early human embryo.

The targeted gene inactivation of endothelins-1 and -3 (ET-1 and ET-3) and of one of their receptors, ETB, in the mouse causes severe defects in the embryonic development. These defects, cardiovascular and craniofacial malformations for ET-1, and colonic agangliogenesis associated with skin pigmentation anomalies for ET-3 and the ETB receptor, reproduce pathological phenotypes due to natural mutations of the same genes in the mouse and the human. The mutant phenotypes have been causatively linked to deficient migration/proliferation/differentiation of neural crest cells, i.e., neurocristopathies. To bring new insight about the exact roles of ETs in development and the involvement of neural crest cells in these processes, we have explored, by in situ hybridization, the ontogeny in the early human embryo of the ET system (ET-1 and ET-3, ETA and ETB receptors, ET converting enzyme-1). ET receptor mRNA expression in neural crest cells starts at 3 wk of gestation and continues during the entire period studied (up to 6 wk of gestation). During this period, ETA expression progressively spreads to undifferentiated mesodermal components of various structures and organs (head and axial skeleton, lateral and ventral subdermal mesoderm), whereas ETB expression remains more restricted to fewer differentiated cells (neural tube, sensory and sympathetic ganglia, endothelium). Some of these tissues and structures that express either one of the receptors do not appear to be of neural crest origin. In the digestive tract and the cardiovascular area, the present observations on the sources of ETs and their target cells in the young embryo provide the basis for a dynamic interpretation of the results of gene targeting of the mouse and the human phenotypes, and point to other possible roles of ETs in other ontogenetic processes. The results support the concept of local, rather than hormonal, interactions between the sources and targets of ETs during development.

Glucocorticoid-suppressible hyperaldosteronism and adrenal tumors occurring in a single French pedigree.

Glucocorticoid-suppressible hyperaldosteronism is a dominantly inherited form of hypertension believed to be caused by the presence of a hybrid CYP11B1/CYP11B2 gene which has arisen from an unequal crossing over between the two CYP11B genes in a previous meiosis. We have studied a French pedigree with seven affected individuals in which two affected individuals also have adrenal tumors and two others have micronodular adrenal hyperplasia. One of the adrenal tumors and the surrounding adrenal tissue has been removed, giving a rare opportunity to study the regulation and action of the hybrid gene causing the disease. The hybrid CYP11B gene was demonstrated to be expressed at higher levels than either CYP11B1 or CYP11B2 in the cortex of the adrenal by RT-PCR and Northern blot analysis. In situ hybridization showed that both CYP11B1 and the hybrid gene were expressed in all three zones of the cortex. In cell culture experiments hybrid gene expression was stimulated by ACTH leading to increased production of aldosterone and the hybrid steroids characteristic of glucocorticoid-suppressible hyperaldosteronism. The genetic basis of the adrenal pathologies in this family is not known but may be related to the duplication causing the hyperaldosteronism.

Expression of the chicken angiotensin II receptor: atypical pattern compared to its mammalian homologues.

We have recently cloned and characterized pharmacologically a chicken angiotensin II receptor (cAT). To evaluate its putative role in developmental processes, we investigated its spatio-temporal distribution in the chicken embryo up to E14. The cAT mRNA is expressed in a developmental manner in the mesonephros and allantois, as well as in the heart, branchial arches or limbs. These results, the first to report the embryonic distribution of an angiotensin receptor in a non-mammalian species, show that its expression pattern does not correspond to either one of the two angiotensin receptor types in mammalian species.

Krit1/cerebral cavernous malformation 1 mRNA is preferentially expressed in neurons and epithelial cells in embryo and adult.

Cavernous malformations are capillaro-venous lesions mostly located within the central nervous system (CCM/OMIM#116860) and occasionally within the skin and/or retina. They occur as a sporadic or hereditary condition. Three CCM loci have been mapped, and the sole gene identified so far, CCM1, has been shown to encode KRIT1, a protein of unknown function. In an attempt to get some insight on the relationship between KRIT1 mutations and CCM lesions, we investigated Krit1 mRNA expression during mouse development from E7.5 to E20.5 and in adult tissues, of both mouse and human origin. A ubiquitous Krit1 mRNA expression was detected from E7.5 up to E9.5. Then, it became progressively restricted from E10.5 to E12.5, to become detectable later essentially in the nervous system and various epithelia. Strong labelling was observed in neurons in the brain, cerebellum, spinal cord, retina and dorsal root ganglia. In epithelia, Krit1 mRNA expression was detected in differentiating epidermal, digestive, respiratory, uterine and urinary epithelia. A similar pattern of expression persisted in mouse and man adult nervous system and epithelia. Unexpectedly, in vascular tissues, expression of Krit1 was detected only in large blood vessels of the embryo.

Adipose angiotensinogen is involved in adipose tissue growth and blood pressure regulation.

White adipose tissue and liver are important angiotensinogen (AGT) production sites. Until now, plasma AGT was considered to be a reflection of hepatic production. Because plasma AGT concentration has been reported to correlate with blood pressure, and to be associated with body mass index, we investigated whether adipose AGT is released locally and into the blood stream. For this purpose, we have generated transgenic mice either in which adipose AGT is overexpressed or in which AGT expression is restricted to adipose tissue. This was achieved by the use of the aP2 adipocyte-specific promoter driving the expression of rat agt cDNA in both wild-type and hypotensive AGT-deficient mice. Our results show that in both genotypes, targeted expression of AGT in adipose tissue increases fat mass. Mice whose AGT expression is restricted to adipose tissue have AGT circulating in the blood stream, are normotensive, and exhibit restored renal function compared with AGT-deficient mice. Moreover, mice that overexpress adipose AGT have increased levels of circulating AGT, compared with wild-type mice, and are hypertensive. These animal models demonstrate that AGT produced by adipose tissue plays a role in both local adipose tissue development and in the endocrine system, which supports a role of adipose AGT in hypertensive obese patients.

Effects of mineralocorticoid receptor gene disruption on the components of the renin-angiotensin system in 8-day-old mice.

Targeted disruption of mineralocorticoid receptor (MR) gene results in pseudohypoaldosteronism type I with failure to thrive, severe dehydration, hyperkalemia, hyponatremia, and high plasma levels of renin, angiotensin II, and aldosterone. In this study, mRNA expression of the different components of the renin-angiotensin system (RAS) were evaluated in liver, lung, heart, kidney and adrenal gland to assess their response to a state of extreme sodium depletion. Angiotensinogen, renin, angiotensin-I converting enzyme, and angiotensin II receptor (AT1 and AT2) mRNA expressions were determined by Northern blot and RT-PCR analysis. Furthermore, in situ hybridization and immunohistochemistry allowed us to identify the cell types involved in the variation of the RAS component expression. In the heterozygous mice (MR+/-), compared with wild-type mice (MR+/+), there was no significant variation of any mRNA of the RAS components. In MR knockout mice (MR-/-), compared with wild-type mice, there were significant increases in the expression level of several RAS components. In the liver, angiotensinogen and AT1 receptor mRNA expressions were moderately stimulated. In the kidney, renin mRNA was increased up to 10-fold and in situ hybridization showed a marked recruitment of renin-producing cells; however, the levels of angiotensin-I converting enzyme mRNA and AT1 mRNA were not changed. Interestingly, in adrenal gland, renin expression was also strongly up-regulated in a thickened zona glomerulosa, whereas AT1 mRNA expression remained unchanged. Altogether, these results demonstrate that in the MR knockout mice model, RAS component expressions are differentially altered, renin being the most stimulated component. Angiotensinogen and AT1 in the liver are also increased, but the other elements of the RAS are not affected.

Regulation by estradiol of the progesterone receptor in the hypothalamus and pituitary: an immunohistochemical study in the chicken.

An antibody (IgG-RB) against the chick oviduct progesterone receptor (PR) was used to study the regulation of PR by estrogen in chicken pituitary and hypothalamus. PR was revealed exclusively in cell nuclei, whatever the hormonal state, including the absence of progesterone. In immature untreated chickens, PR was not detected in the pars distalis, while the pars tuberalis, preoptic area, and infundibular region of hypothalamus showed IgG-RB-immunoreactive cells or neurons. Estrogenic stimulation induced the appearance of PR in cells of the pars distalis and increased the immunoreactivity in the hypothalamus of young chickens of both sexes. After hormonal withdrawal, PR immunostaining returned to the level in untreated immature animals. In young hens, before they laid the first egg, PR appears progressively in pars distalis cells during the pubertal period. Antibodies to pituitary hormones (LH, FSH, GH, PRL, and ACTH) were used to characterize the secretory properties of PR-containing cells. LH- and FSH-immunoreactive cells were localized throughout the pars distalis in untreated animals. After estradiol treatment of young sexually immature chickens, immunostaining of LH and FSH was strongly reduced, up to extinction, in many gonadotropic cells, and only few PR-containing cells demonstrated some immunoreactivity in the cytoplasm. In contrast, in juvenile hens, a majority of PR-containing cells were identified as LH immunoreactive, that is gonadotrophs. There are probably two reasons for the paucity of doubly reactive cells in estradiol-treated chickens. One is technical, the optimal fixation time for both LH (greater than 20 h) and PR (less than 7 h) makes it practically impossible to reveal the hormone and the receptor at the same time. The other is related to the physiology of the system, which involves the simultaneous decrease in LH immunoreactivity and the PR induction in gonadotrophs by estradiol. The presence of PR in gonadotrophs suggests a direct feedback mechanism of sex steroids on pituitary cells in addition to the indirect effect through GnRH modulation. The hormonal content of PRL-, GH-, and ACTH-producing cells was not modified by estradiol treatment, as judged by the appropriate immunoreactivity, and their nuclei did not display PR. However, PRL cells and PR-containing cells were frequently present near each other within the same cell cords.

Co-expression of type 1 angiotensin II receptor (AT1R) and renin mRNAs in juxtaglomerular cells of the rat kidney.

Physiological and ligand binding studies have shown that Angiotensin II (AII) exerts various functions along different segments of the nephron, via the type-1 receptor (AT1R), resulting in the control of glomerular filtration rate (GFR) and water and salt homeostasis. We have used the recently cloned AT1R cDNA to localize, by in situ hybridization, the cells expressing AT1R mRNA in the rat kidney. On serial sections, juxtaglomerular (JG) renin secreting cells, identified by hybridization with a renin cRNA probe, also co-express AT1R mRNA. The co-expression of AT1R and renin mRNAs in the same cells documents visually the direct feedback control of AII on renin secretion. AT1R mRNA was also present in known target cells for AII: proximal convoluted tubule, mesangium and vasa recta.

Progesterone receptor in the chick bursa of fabricius: characterization and immunohistochemical localization.

A high affinity progesterone-binding site was studied in the chick bursa of Fabricius. The dissociation constant for progesterone was 1.4 nM, and the concentration of progesterone-binding sites increased with estradiol treatment. In estradiol-treated bursas, the receptor concentration was about 240 fmol/mg protein. The binding site was specific for progestins, with the following order of affinities: ORG 2058 greater than progesterone greater than promegestone. Androgens, dexamethasone, and estradiol were weak competitors for progesterone binding in the bursa cytosols from estradiol-treated chicks. Immunoglobulin G fraction of antiserum (immunoglobulin G-RB) raised in rabbit against the B-subunit of chick oviduct progesterone receptor (PR) was used for an immunohistochemical study. The PR was found only in the interfollicular cells, which were most probably nonlymphoid cells. Staining was localized exclusively in the elongated nuclei of these cells. No staining was seen in the bursal epithelium or inside the lymphoid follicles. The results indicate that the interfollicular cells of the bursa contain specific PRs which are under estrogen regulation as in the oviduct. Thus, these cells might be under direct progesterone regulation.

Nuclear localization of progesterone receptor before and after exposure to progestin at low and high temperatures: autoradiographic and immunohistochemical studies of chick oviduct.

The intracellular location of progesterone receptor and tritiated progestin was assessed in chick oviduct before and after in vitro exposure to 5 nM [3H]Org 2058 ([6,7-3H]16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione) for 5 or 45 min at 4 or 37 C. The experiments were designed to allow the intracellular localization of occupied and unoccupied receptor in relatively intact tissue. Autoradiography and immunohistochemistry were used to localize [3H]Org 2058 and the progesterone receptor, respectively. Autoradiograms showed radiolabeled progestin concentrated in oviduct cell nuclei not only after incubation at 37 C, but after incubation at 4 C as well. Cytoplasmic concentration was never observed. Immunostaining revealed progesterone receptor always located in cell nuclei, regardless of temperature or time of exposure to labeled ligand or whether the tissue was exposed to progestin. The results indicate that the chick oviduct nuclear progesterone receptor does not undergo a temperature-dependent translocation from cytoplasm to nucleus upon binding ligand.

Angiotensin II type 2 receptor mRNA expression in the developing cardiopulmonary system of the rat.

Recent studies have shown that angiotensin II has a trophic action on the heart. The presence of two types of angiotensin II receptors, type 1 (AT1) and type 2 (AT2), has been reported in the rat heart. This in situ hybridization study describes the tissue and cell location of AT2 receptor mRNA in the developing rat cardiopulmonary system, from 15 days of gestation to adulthood. Expression of AT1A receptor mRNA was studied in parallel for direct comparison. The aortic arch and pulmonary artery expressed high levels of AT2 receptor mRNA from 15 days of gestation up until 15 days postpartum, whereas expression of this mRNA was observed only just before and after birth in the coronary arteries. AT2 receptor mRNA was not detected in any cardiac muscle of the fetus, neonate, or adult. The annulus of all four heart valves expressed AT2 mRNA from 21 days of gestation until 10 days postpartum, but no labeling was seen in the valve leaflets. The subendocardial atrial tissue showed a high level of AT2 receptor mRNA expression during the early postnatal period, but no expression was observed in the atrial myocytes from fetal stages to adulthood. The bronchi and trachea, but not the lung parenchyma, showed a high level of AT2 receptor mRNA expression starting from 17 days of gestation until 10 days postpartum. AT2 receptor mRNA expression in the cardiopulmonary system is therefore transient, developmentally regulated, and mostly located in vascular structures. By these three characteristics, its expression contrasts with that of AT1A, which is continuously expressed in the cardiac muscle to adulthood. This spatiotemporal pattern of expression of angiotensin II receptor mRNAs during development suggests a possible role for angiotensin II in organogenesis.

Tissue-specific expression of type 1 angiotensin II receptor subtypes. An in situ hybridization study.

The angiotensin II type 1 (AT1) receptor in murine species exists as two isoforms (AT1A and AT1B) encoded by two different genes. Both subtypes have a 9/10 homology in the coding sequence of their mRNA. We examined organs of adult rats (liver, pituitary gland, adrenal gland, kidney, heart, and lung) to study the differential expression of these two genes in target tissues for angiotensin II. AT1A and AT1B mRNAs were detected by in situ hybridization using specific riboprobes for the 3' noncoding region of the mRNAs that have the lowest homology (approximately 6/10). Only AT1A was expressed in the liver, heart, and lung, and only AT1B was expressed in the anterior pituitary, where most cells were positive. In the adrenal gland, AT1A mRNA was detected in the zona glomerulosa and medulla and AT1B in the glomerulosa. In the kidney, AT1A mRNA was the predominant isoform (mesangial and juxtaglomerular cells, proximal tubules, vasa recta, and interstitial cells), but AT1B was also detected in mesangial and juxtaglomerular cells and in the renal pelvis. The results of this in situ detection suggest a tissue-selective regulation of AT1A and AT1B mRNAs. This tissue specificity may constitute a prerequisite condition if the two angiotensin II receptor subtypes, which are pharmacologically similar, are to selectively modulate the various effects of angiotensin II in the different target tissues.

Distribution of type 1 angiotensin II receptor subtype messenger RNAs in the rat fetus.

The localization of the two type 1 angiotensin II receptor subtype (AT1A and AT1B) messenger RNAs in the 19-day-old rat fetus was studied by in situ hybridization. AT1 receptor mRNAs were detected in target organs of the renin-angiotensin system such as the kidney, adrenal gland, liver, heart, large arteries, and pituitary gland. In addition, angiotensin II receptors were present in specialized mesenchymal cells surrounding the cartilage, in the pericardium, in the lung, and in the undifferentiated mesenchymal tissue. The AT1A subtype was predominant in all tissues and organs except the adrenal cortex and glomeruli in the kidney, which expressed both AT1A and AT1B mRNAs. The widespread distribution of AT1 receptors in tissues and organs involved in hydromineral equilibrium and blood pressure regulation shows that during fetal development angiotensin II may already act as a regulator of the cardiovascular system. An effect on cellular differentiation and/or proliferation via AT1 receptors is also suggested by their location in several mesenchymes.

Gene expression and tissue localization of the two isoforms of angiotensin I converting enzyme.

Angiotensin converting enzyme exists in two different isoforms, somatic and germinal, whose respective distributions and intracellular localizations have not been precisely determined. The differing biochemical and molecular characteristics of the two isozymes allowed the preparation of antibodies specific for each of the two angiotensin converting enzyme isoforms and of two nucleic acid probes, one of which was specific for the germinal isoform. Immunohistochemistry and in situ hybridization were used to determine the cell distribution of, respectively, the two isoforms and their corresponding messenger RNAs in the classically studied tissues of male adult humans and marmosets. Results provided by the two different methods were always concordant and were identical in the two species. The somatic angiotensin converting enzyme form was expressed uniquely in somatic tissues (vascular endothelial cells and at the brush border of renal proximal convoluted tubule, jejunal villus, and epididymal duct epithelia), and the germinal form was expressed uniquely in germinal cells with a precise stage-specific pattern, starting in round spermatids and finishing in spermatozoa. In situ hybridization documented the presence of somatic angiotensin converting enzyme messenger RNA in renal tubule epithelium, jejunal enterocytes, and epididymal epithelium and demonstrated that there was no direct correlation between the levels of angiotensin converting enzyme messenger RNA and the enzyme it encodes for, i.e., angiotensin converting enzyme, in a given epithelium. The significance of the ultraselective expression of germinal angiotensin converting enzyme and of its specific messenger RNA at a very precise stage of spermatogenesis remains uncertain.

Coexpression of renin, angiotensinogen, and their messenger ribonucleic acids in adrenal tissues.

The presence of the two components of the renin-angiotensin system (RAS) has been systematically investigated in human normal and pathological adrenal tissues with two aims: 1) the detection of renin and especially angiotensinogen, which has not been reported before; and 2) to study possible differences in the coexpression of renin and angiotensinogen in tissue of cortical and medullary origin. The relative levels of renin and angiotensinogen mRNAs were determined by Northern blot analysis in normal (n = 5) and pathological adrenal tissues of cortical (n = 23) and medullary (n = 10) origin. Renin, prorenin, and angiotensinogen levels were also measured. Renin concentrations in normal and pathological adrenals were around 30-fold higher than those in the plasma of normal subjects, except for a Cushing's adenoma, which contains an extremely high renin content. Renin accounted for 56% of the total renin in normal adrenals and up to 87% in neoplastic tissues. This high proportion of renin indicates a likely conversion of prorenin to renin within these tissues. Renin mRNA was detected in each group of adrenal tissues. There was a significant correlation between the concentration of renin and its mRNA (r = 0.75; P less than 0.05). Angiotensinogen and its mRNA were detected in all normal and pathological adrenals. Compared to normal adrenal tissues, the relative amount of angiotensinogen mRNA was significantly higher in pheochromocytomas. However, the increased mRNA level in these tissues was not accompanied by a parallel increase in tissue angiotensinogen levels. Since the translational efficiency of angiotensinogen was verified by in vitro cell-free translation, the low level of angiotensinogen compared to the relatively high amount of its mRNA suggests a lack of storage of this protein in adrenal cells, as in liver cells. This study demonstrates that renin and angiotensinogen are coexpressed in normal and pathological tissues. Tissues of different cellular origin (zona glomerulosa, fasciculata, and medullary tissue), were able to express, store, and process renin and synthesize angiotensinogen. There was no obvious relationship between the expression of these proteins and the pathophysiology of the adrenal gland.

Variable expression of the V1 vasopressin receptor modulates the phenotypic response of steroid-secreting adrenocortical tumors.

We studied the putative role of the vasopressin receptors in the phenotypic response of steroid-secreting adrenocortical tumors. A retrospective analysis of a series of 26 adrenocortical tumors responsible for Cushing's syndrome (19 adenomas and 7 carcinomas) showed that vasopressin (10 IU, i.m., lysine vasopressin) induced an ACTH-independent cortisol response (arbitrarily defined as a cortisol rise above baseline of 30 ng/mL or more) in 7 cases (27%). In comparison, 68 of 90 patients with Cushing's disease (76%) had a positive cortisol response. We then prospectively examined the expression of vasopressin receptor genes in adrenocortical tumors of recently operated patients (20 adenomas and 19 adrenocortical carcinomas). We used highly sensitive and specific quantitative RT-PCR techniques for each of the newly characterized human vasopressin receptors: V1, V2, and V3. The V1 messenger ribonucleic acid (mRNA) was detected in normal adrenal cortex and in all tumors. Its level varied widely between 2.0 x 10(2) and 4.4 x 10(5) copies/0.1 microgram total RNA, and adenomas had significantly higher levels than carcinomas, although there was a large overlap. Among the 6 recently operated patients who had been subjected to the vasopressin test in vivo, the tumor V1 mRNA levels were higher in the 4 responders (9.5 x 10(3) to 5.0 x 10(4)) than in the 2 nonresponders (2.0 x 10(2) and 1.8 x 10(3)). One adenoma that had a brisk cortisol response in vivo, also had in vitro cortisol responses that were inhibited by a specific V1 antagonist. In situ hybridization showed the presence of V1 mRNA in the normal human adrenal cortex where the signal predominated in the compact cells of the zona reticularis. A positive signal was also present in the tumors with high RT-PCR V1 mRNA levels; its distribution pattern was heterogeneous and showed preferential association with compact cells. RT-PCR studies for the other vasopressin receptors showed a much lower signal for V2 and no evidence for V3 mRNA. We could not establish whether the V2 mRNA signal observed in normal and tumoral specimens was present within adrenocortical cells or merely within tissue vessels. We conclude that the vasopressin V1 receptor gene is expressed in normal and tumoral adrenocortical cells. High, and not ectopic, expression occurs in a minority of tumors that become directly responsive to vasopressin stimulation tests.

Cloning and expression pattern of EPAS1 in the chicken embryo. Colocalization with tyrosine hydroxylase.

EPAS1 is a hypoxia-inducible transcription factor, highly expressed in vasculature and recently shown to be necessary for catecholamine production during embryogenesis. We report here the cloning and detailed expression pattern of this factor in the chicken embryo. We show that chicken EPAS1 presents an overall identity of 76% with the human sequence and that it is strongly expressed in the blood vessel wall, mostly in endothelial cells, but also in vascular smooth muscle cells. Moreover, we report non-vascular expression sites: liver, kidney, and, quite interestingly, cells of the sympathetic nervous system where EPAS1 is coexpressed with one of its putative target genes, the tyrosine hydroxylase. EPAS1 could therefore represent the link between the vascular system and the sympathetic nervous system, both sensitive to hypoxia.

Molecular cloning, expression and tissue distribution of a chicken angiotensin II receptor.

A cDNA encoding a chicken angiotensin II receptor from adrenal gland was isolated to serve as a molecular tool to study the role of AngII in avian embryonic development. This cDNA, sharing a high homology with another avian receptor (turkey), encodes a protein of 359 amino acids with 75% sequence identity with the mammalian type 1 receptor. Transient expression has revealed pharmacological properties distinct from mammalian receptors and a functional coupling leading to the increase in inositol phosphate production. The AngII receptor mRNA is expressed in classical target organs for AngII (adrenal gland, heart, kidney) and, interestingly, in endothelial cells where it may mediate the peculiar vasorelaxation effect of AngII in the chicken.