Mineralocorticoid effector mechanism in human mononuclear leukocytes

Mineralocorticoid receptors and mineralocorticoid effector mechanism were determined in mononuclear leukocytes (MNL) from normal subjects. The hierarchy of affinities of competitors for the receptor was similar to that described in other non-classical target tissues for aldosterone. In spite of the relative high affinity of cortisol for the receptor, these binding sites are occupied in vivo by aldosterone and play a mineralocorticoid effect in terms of electrolyte content of the cells. The effect of aldosterone is to prevent the loss of electrolytes due to incubation in medium alone and this action is reversed by addition of actinomycin D. In addition, the incubation of the MNL with aldosterone plus human alpha ANP leads to complete block of the action of aldosterone alone. This effect is not mediated by binding of alpha ANP to mineralocorticoid receptors but is probably related to a some postereceptorial effect of aldosterone at the level of plasma membrane. We conclude that the model of MNL is a good tool for studying mineralocorticoid receptors regulation and consequent effector mechanism in humans.     

Immunofluorescence of mineralocorticoid receptors in peripheral lymphocytes: Presence of receptor-like activity in patients with the autosomal dominant form of pseudohypoaldosteronism, and its absence in the recessive form

Pseudohypoaldosteronism is a syndrome characterized by salt wasting and a failure to thrive due to the resistance towards the action of aldosterone. Aldosterone levels and plasma renin activity are extremely elevated and aldosterone binding sites in peripheral mononuclear leukocytes have regularly shown to be reduced or absent. Sporadic as well as familial cases have been identified and an autosomal dominant as well as an autosomal recessive mode of inheritance has been described. A defect in the aldosterone receptor has been postulated, however, molecular genetic analysis in selected patients has not revealed a mutation in the sequence of the coding region of the cDNA of the mineralocorticoid receptor gene. In the present study we have used a fluorescence-labeled antibody to detect possible receptor expression in monocytes from patients with various clinical forms of pseudohypoaldosteronism. Patients with the sporadic as well as with the autosomal dominant form were clearly immunopositive despite being negative in terms of aldosterone receptor binding. In contrast in two patients with the autosomal recessive form there was no detectable receptor protein, consistent with the results obtained in the aldosterone binding studies. These results suggest that the pathogenesis of pseudohypoaldosteronism is heterogeneous not only regarding the mode of inheritance but also in terms of receptor binding. Thus, in a subgroup of patients the inability of the receptor to bind ligand may be due to a defect involving other, probably cellular factors rather than a deficiency or a defect in the mineralocorticoid receptor system itself.     

Mineralocorticoid receptor gene expression in the gastrointestinal tract: distribution and ontogeny

The gastrointestinal tract is a well characterized target tissue for aldosterone, where it regulates electrolyte transport, particularly in the descending colon. Previous studies have demonstrated the presence of aldosterone receptors in gastrointestinal tissues. We have used specific cRNA probes for the rat mineralocorticoid receptor to explore both the distribution and ontogeny of mineralocorticoid receptor gene expression in the gastrointestinal tract. Mineralocorticoid receptor gene expression is found throughout the small and large intestine, but is absent from the stomach. The highest levels are observed in the distal colon, and significant expression is found in the duodenum; in both tissues levels of expression are higher than those in kidney. In both the developing duodenum and colon, mineralocorticoid receptor gene expression precedes the development of the full physiological response to aldosterone. These findings emphasise the colon as an important target tissue for aldosterone, and raise the question of potential roles for aldosterone in the duodenum.     

A genomewide scan identifies novel early-onset primary open-angle glaucoma loci on 9q22 and 20p12

Glaucoma is a leading cause of blindness worldwide. The disease is characterized by a degeneration of the optic nerve, which is usually associated with elevated intraocular pressure. The common form of adult-onset primary open-angle glaucoma is inherited as a complex trait, whereas the rarer early-onset juvenile open-angle glaucoma (JOAG) exhibits autosomal dominant inheritance. Of all cases of JOAG, approximately 10%-20% are caused by mutations in the myocilin gene. We have identified 25 pedigrees that are affected with typical JOAG and that demonstrate autosomal dominant inheritance. We sequenced the myocilin gene in probands from each family and found mutations in 8% of this population. To identify novel genes responsible for JOAG, we used families that did not have myocilin mutations for a genomewide screen. Markers located on chromosomes 9q22 and 20p12 showed evidence for linkage, identifying two novel loci for early-onset open-angle glaucoma.     

Evidence that paternal expression of the epsilon-sarcoglycan gene accounts for reduced penetrance in myoclonus-dystonia

Myoclonus-dystonia (M-D) is a movement disorder characterized by rapid muscle contractions and sustained twisting and repetitive movements and has recently been associated with mutations in the epsilon-sarcoglycan gene (SGCE). The mode of inheritance is autosomal dominant with reduced penetrance upon maternal transmission, suggesting a putative maternal imprinting mechanism. We present an apparently sporadic M-D case and two patients from an M-D family with seemingly autosomal recessive inheritance. In both families, we detected an SGCE mutation that was inherited from the patients' clinically unaffected fathers in an autosomal dominant fashion. Whereas, in the first family, RNA expression studies revealed expression of only the mutated allele in affected individuals and expression of the normal allele exclusively in unaffected mutation carriers, the affected individual of the second family expressed both alleles. In addition, we identified differentially methylated regions in the promoter region of the SGCE gene as a characteristic feature of imprinted genes. Using a rare polymorphism in the promoter region in a family unaffected with M-D as a marker, we demonstrated methylation of the maternal allele, in keeping with maternal imprinting of the SGCE gene. Loss of imprinting in the patient with M-D who had biallelic expression of the SGCE gene was associated with partial loss of methylation at several CpG dinucleotides.     

A homozygous missense mutation in SCNN1A is responsible for a transient neonatal form of pseudohypoaldosteronism type 1

Pseudohypoaldosteronism type 1 (PHA1) is a monogenic disorder of mineralocorticoid resistance characterized by salt wasting, hyperkalemia, high aldosterone levels, and failure to thrive. An autosomal recessive form (AR-PHA1) is caused by mutations in the epithelial sodium channel ENaC with usually severe and persisting multiorgan symptoms. The autosomal dominant form of PHA1 (AD-PHA1) is due to mutations in the mineralocorticoid receptor causing milder and transient symptoms restricted to the kidney. We identified a homozygous missense mutation in the SCNN1A gene (c.727T>C/p.Ser(243)Pro), encoding α-subunit of ENaC (α-ENaC) in a prematurely born boy with a severe salt-losing syndrome. The patient improved rapidly under treatment, and dietary salt supplementation could be stopped after 6 mo. Interestingly, the patient's sibling born at term and harboring the same homozygous Ser(243)Pro mutation showed no symptom of salt-losing nephropathy. In vitro expression of the αSer(243)Pro ENaC mutant revealed a slight but significant decrease in ENaC activity that is exacerbated in the presence of high Na(+) load. Our study provides the first evidence that ENaC activity is critical for the maintenance of salt balance in the immature kidney of preterm babies. Together with previous studies, it shows that, when the kidney is fully mature, the severity of the symptoms of AR-PHA1 is related to the degree of the ENaC loss of function. Finally, this study identifies a novel functional domain in the extracellular loop of ENaC.     

Homozygosity mapping of a Weill-Marchesani syndrome locus to chromosome 19p13.3-p13.2

Weill-Marchesani syndrome (WMS) is a rare disease characterized by short stature, brachydactyly, joint stiffness, and characteristic eye abnormalities, including microspherophakia, ectopia lentis, and glaucoma. Both autosomal recessive and autosomal dominant modes of inheritance have been described in association with WMS. We have performed a genome-wide search in two large consanguineous families of Lebanese and Saudian origin consistent with an autosomal recessive mode of inheritance. Here, we report the linkage of the disease gene to chromosome 19p13.3-p13.2 (Zmax=5.99 at theta=0 at locus D19S906). A recombination event between loci D19S905 and D19S901 defines the distal boundary, and a second recombination event between loci D19S221 and D19S840 defines the proximal boundary of the genetic interval encompassing the WMS gene (12.4 cM). We hope that our ongoing studies will lead to the identification of the disease-causing gene.     

Paradoxical involvement of glucocorticoid receptors in the aldosterone-induced impairment of ACTH secretion from perifused pituitary glands

The present investigation was aimed at examining whether interaction of aldosterone with specific mineralocorticoid receptors at the level of the pituitary gland may account for the inhibitory effect of that steroid on ACTH secretion. By using pituitaries from neonatal rats, which we show to completely lack specific mineralocorticoid receptors but to contain a functional glucocorticoid receptor system, we demonstrated the persistence of aldosterone-induced inhibition of ACTH release from perifused glands. Conversely, when the glucocorticoid receptors sites were blocked in pituitaries from mature rats by means of a potent antiglucocorticoid (RU 38486), thus leaving unaltered mineralocorticoid binder, aldosterone no longer dampened hormonal output. We conclude that the latter steroid affected corticotropic activity by interacting not with its proper and specific receptor, but rather with the glucocorticoid binding sites.     

Effect of adrenalectomy and aldosterone on the modulation of mineralocorticoid receptors in rat kidney

Adrenalectomized rat kidney is commonly used for the study of mineralocorticoid mechanism of action in mammals. In this model, aldosterone is known to bind to two classes of binding sites: type I (mineralocorticoid) and type II (glucocorticoid). The study of the aldosterone binding in normal rat kidney requires the elimination of endogenous hormones bound to each type of receptor. Thus, a suitable technique was developed using in situ perfusion of the kidneys. The efficacy of this method was of about 85 to 90% at the level of both cytoplasm and nucleus. Aldosterone binding capacity was checked in normal rat kidney after in situ perfusion and was found to be 300 to 500% lower than in adrenalectomized rat kidney, both in cytoplasm and nuclei. Computer analysis of aldosterone binding parameters in the cytoplasm (30,000 X g supernatant) of rat kidney suggested that adrenalectomy might induce an important rise in the number of mineralocorticoid receptors (congruent to 260%). An increase in the number of glucocorticoid receptors was also observed but appeared to be lower. Aldosterone, when perfused during 24 h in adrenalectomized rats, lowered the number of type I sites to the same level as observed in normal rat kidney. This effect was fully reversible after interruption of aldosterone perfusion. These results suggested an aldosterone-induced down regulation of mineralocorticoid receptors.     

Mineralocorticoid receptors and hypertension

Mineralocorticoid receptors (MR) have equal affinity for the mineralocorticoid aldosterone, and the physiological glucocorticoids cortisol and corticosterone. In epithelial tissues in vivo, MR are protected against glucocorticoid occupancy by the enzyme 11β-hydroxysteroid dehydrogenase, allowing access by the lower circulating levels of the physiological mineralocorticoid aldosterone. In non-epithelial tissues, including the heart and most areas of the central nervous system, MR are not so protected, and their physiological ligand is cortisol/corticosterone. Intracerebroventricular infusion studies have shown that aldosterone occupancy of such unprotected circumventricular MR is necessary for mineralocorticoid hypertension, and the hypertensinogenic effects of peripherally infused aldosterone can be blocked by intracerebroventricular infusion of the MR antagonist RU28318. Prolonged (8 weeks) administration of mineralocorticoids to salt-loaded rats has been shown to be followed by hypertension, cardiac hypertrophy and cardiac fibrosis. Whether the hypertrophy and fibrosis reflect primary effects of aldosterone via cardiac MR, or effects secondary to occupancy of protected, epithelial MR, remains to be determined, as does the mechanism of action of salt loading in this model of mineralocorticoid hypertension.     

Isolation and identification of an endogenous metabolite of 18-oxocortisol from human urine

The naturally occurring mineralocorticoid agonist, 18-oxocortisol, is secreted in increased amounts in two hypertensive syndromes. One is primary aldosteronism and the other a genetic disorder first described by Sutherland and co-workers in which aldosterone secretion is ACTH-dependent and the mode of inheritance is autosomal dominant. 18-Hydroxy and -oxocortisol are the components of the cortisol oxidation pathway which arise when cortisol becomes an alternate substrate for corticosterone methyl oxidase. This enzyme system normally resides in the glomerulosa zone of the mammalian adrenal cortex. In an effort to account for a larger fraction of 18-oxocortisol and provide a reliable index of its secretion and of the expression of the cortisol C-18 oxidation pathway, metabolites were sought in the urine of a patient with the ACTH-dependent autosomal dominant form of aldosteronism. Using a variant of the technique of reverse isotope dilution, a pool of [3H]-labeled urinary metabolites form a normal subject was mixed with the patient's urine and subjected to customary methods of hydrolysis for urinary steroids. The radiolabeled glucuronide fraction was the most abundant and was subjected to repeated HPLC fractionation to yield the predominant component. The evidence from gas chromatography-mass spectrometry indicated that this metabolite was a tetrahydro derivative. The structure of the isolated tetrahydro 18-oxocortisol was confirmed by a biosynthesis of a reference standard from 18-oxocortisol and a 5 beta-pregnane reductase preparation.     

Idiopathic hemochromatosis: demonstration of homozygous-heterozygous mating by HLA typing of families

Summary In five families with idiopathic (hereditary) hemochromatosis, clinical and biochemical expression of the disease occurred in offspring of probands, suggesting an autosomal dominant mode of inheritance. However, HLA typing of subjects indicated that a homozygous-heterozygous mating almost certainly had occurred in four of the five families, resulting in homozygous offspring. Thus, in these families inheritance of the hemochromatosis trait was best explained in terms of an autosomal recessive or intermediate mode of inheritance. This study demonstrates the value of HLA typing in identifying homozygous-heterozygous matings in hemochromatosis families.     

Arterial smooth muscle effects of aldosterone and vasopressin: action on ionic fluxes

We have shown previously that aldosterone injected s.c. to adrenalectomized rats has a mineralocorticoid specific action on the transmembrane movements of sodium and potassium from the rat tail artery. These effects appeared to be partly due to an unknown humoral factor. Indeed, the late in vivo effects of aldosterone on 22Na and 86Rb effluxes are suppressed or reduced after in vitro exposure to the hormone. In rats perfused with a specific antagonist of the pressor effect of vasopressin, the in vitro administration of aldosterone induced a kinetic action similar to that observed after in vitro exposure to the mineralocorticoid. Vasopressin exerts a direct action on 22Na and 86Rb effluxes. These effects were correlated in the time with the late in vivo effects of aldosterone. Moreover, vasopressin appears to potentiate the in vitro effects of aldosterone on 22Na and 86Rb effluxes. It is not yet possible to ascertain if this effect is additive or permissive.     

A three generations family with blepharo-naso-facial malformations suggestive of Pashayan syndrome

Blepharo-naso-facial syndrome, described by Pashayan et al. (10), is characterized by telecanthus, lateral displacement and stenosis of lacrimal puncta, bulky nose, mask-like facies, trapezo?dal upper lip, torsion dystonia and mental retardation. We report on a family with this rare malformation syndrome, confirming the existence of this syndrome and its dominant inheritance. The proband had a fleshy nose, a prominant nose bridge, an hypoplastic midface, telecanthus with temporal displacement of puncta, lacrimal excretory obstruction. CNS torsion dystonia, increased deep tendon reflexes, Babinski reflexes, poor coordination and joint laxity. The proband's mother, brother and maternal grandfather also showed manifestations of the syndrome. The proband and his brother had delayed developmental milestones. Hearing impairment was present in the proband, his mother and his grandfather but was absent in the proband's brother. The blepharonasofacial syndrome was described by Pashayan et al. (10) in four members of one family, two male and one female sib and their mother. Two other sibs were unaffected. Many of the features of the blepharo-facio-nasal syndrome also occur in other well known syndromes i.e. Waardenburg syndrome. The pedigrees of the family of Pashayan et al. (10) and of our family are compatible with Mendelian dominant inheritance, either autosomal or X-linked. X-linked dominant inheritance cannot be ruled out except by male-to-male transmission, which does not occur in these families. Pashayan et al. (10) suggested that an autosomal gene with variable expressivity appears more likely. More families are needed for defining the transmission of the condition and for mapping the gene involved in the blepharo-naso-facial syndrome.     

Aldosterone impairs insulin responsiveness in U-937 human promonocytic cells via the downregulation of its own receptor

In an earlier study, we have reported an inhibition of insulin receptor (IR) mRNA levels and insulin binding by aldosterone in U-937 human promonocytic cells. In the present extension of our studies, we demonstrate that this inhibition by aldosterone had no effects on basal glucose transport or on basal thymidine incorporation into DNA, while the cell responsiveness reflected by the maximal response to insulin was decreased by 23% for glucose transport and by 31% for DNA synthesis after the aldosterone treatment. We also prove that this inhibition of the insulin response by aldosterone is mediated by a downregulation of the levels of mineralocorticoid receptors (MRs) (50% decrease) and their mRNA (50% decrease). In addition, the mineralocorticoid antagonist spironolactone reversed the decrease in MR mRNA levels elicited by aldosterone, which suggests the involvement of this receptor in the process.     

关于笑窝遗传方式的探讨

为了探讨笑窝的遗传方式,对２６个笑窝家系进行了调查和分析,初步认主笑窝为常染色体不规则显性遗传。     

Cross-reactivity of a monoclonal antiglucocorticoid receptor antibody BuGR1 with glucocorticoid and mineralocorticoid receptors of various species

The reactivity of a monoclonal antibody BuGR1, raised against glucocorticoid receptors of rat liver, with glucocorticoid and mineralocorticoid receptors of mammalian (rabbit) and amphibian (A6 cells) origin was examined. The glucocorticoid receptors of rabbit kidney and liver and of A6 cells were labeled with tritiated dexamethasone. The mineralocorticoid receptors were labeled with tritiated aldosterone in the presence or absence of RU26988, depending on whether aldosterone was bound to glucocorticoid receptors (A6 cells) or not (rabbit kidney), in addition to its binding to mineralocorticoid receptors. BuGR1 did not recognize mineralocorticoid receptors of A6 cells and rabbit kidney. BuGR1 cross-reacted with glucocorticoid receptors of rabbit liver and kidney but not of A6 cells, suggesting that the domain of glucocorticoid receptors recognized by BuRG1 could be present only in the mammalian species. The findings indicate that BuGR1 shows species differences as well as receptor class specificity.     

Mode of inheritance in familial cases of primary gonadotropic deficiency

The mode of inheritance of primary gonadotropic deficiency was studied in 38 children and adolescents. 92% of this population was male with high frequencies of undescended testes (80%) and micropenis (31%). Anosmia was present in 61% of the patients aged more than 5 years and was a frequent genetic marker in the families. Inheritance was matrilineal in 18, X-linked dominant or autosomal dominant in 6. In 13 cases, the transmission was patrilineal and evoked autosomal dominant inheritance. An autosomal recessive transmission was likely in 7 patients. The data agree with the suggestion of multiple modes of inheritance of congenital gonadotropic deficiency, and clearly show the wide range of expressivity of the disorder.