Salt-Sensitive Hypertension in GR+/- Rats Is Accompanied with Dysregulation in Adrenal Soluble Epoxide Hydrolase and Polyunsaturated Fatty Acid Pathways.

Mutations within the glucocorticoid receptor (GR) gene locus lead to glucocorticoid resistance which is characterized by several clinical symptoms such as adrenal gland hyperplasia and salt-sensitive hypertension, although the underlying mechanisms are still unknown. We studied GR haploinsufficient (GR+/-) Sprague Dawley rats which, on a standard diet, showed significantly increased plasma aldosterone and corticosterone levels and an adrenocortex hyperplasia accompanied by a normal systolic blood pressure. Following a high salt diet, these rats developed salt-sensitive hypertension and maintained elevated enzyme-soluble epoxide hydrolase (sEH) in adrenal glands, while sEH was significantly decreased in wild-type rats. Furthermore, GR+/- rats showed dysregulation of the equilibrated linoleic and arachidonic acid pathways, with a significant increase of less active metabolites such as 8,9-DiHETrE. In Sprague Dawley rats, GR haploinsufficiency induced steroid disturbances, which provoked hypertension only in combination with high salt intake, which was accompanied by disturbances in sEH and fatty acid metabolism. Our results suggest that sEH inhibition could be a potential target to treat hypertension in patients with GR haploinsufficiency.

Dietary sodium restriction sex specifically impairs endothelial function via mineralocorticoid receptor-dependent reduction in NO bioavailability in Balb/C mice.

Recent findings from our group demonstrated that females exhibit higher endothelial mineralocorticoid receptor (MR) expression than males, which predisposes them to aldosterone-mediated endothelial dysfunction in the context of metabolic disorders. However, whether the endothelium of female mice presents a higher propensity to MR-mediated dysfunction than that of males in the absence of comorbidities remains unknown. We therefore sought to investigate whether increasing aldosterone production endogenously with sodium restriction impairs endothelial function in otherwise healthy female mice. We fed male and female Balb/C mice a normal (0.4% NaCl; NSD) or sodium-restricted diet (0.05% NaCl; SRD) for 4 wk. Females exhibited higher baseline endothelial function (relaxation to acetylcholine) and lower vascular contractility (constriction to phenylephrine, serotonin, and KCl). However, SRD impaired endothelial-dependent relaxation and increased vascular contractility in female mice, effectively ablating the baseline sex difference. Female sex also increased baseline adrenal CYP11B2 expression; however, SRD significantly enhanced CYP11B2 expression in male and female mice and ablated the sex difference. Nitric oxide synthase (NOS) inhibition with Nω-nitro-l-arginine methyl ester hydrochloride eliminated both sex as well as diet-induced differences in endothelial dysfunction. In accordance, females demonstrated higher vascular endothelial NOS expression at baseline, which SRD significantly decreased. In addition, SRD diminished vascular NOX4 expression in female mice only. MR blockade with spironolactone-protected female mice from decreases in endothelial-dependent relaxation but not increases in vascular contractility. Utilizing sodium restriction as a method to increase plasma aldosterone levels in healthy female mice, we demonstrated that female mice are more susceptible to vascular damage via MR activation in the vascular endothelium only.NEW & NOTEWORTHY Female sex confers improved endothelial relaxation and vascular constriction responses in female Balb/C mice compared with males under baseline conditions. Sodium restriction impairs endothelial function, which is nitric oxide dependent, and increases vascular contractility in association with reduced vascular endothelial nitric oxide synthase and NOX4 expression in female mice ablating the baseline sex difference. Mineralocorticoid receptor antagonism ablates sodium restriction-induced endothelial dysfunction, but not increased vascular contractility, in female mice.

The Angiotensin-(1-12)/Chymase axis as an alternate component of the tissue renin angiotensin system.

The identification of an alternate extended form of angiotensin I composed of the first twelve amino acids at the N-terminal of angiotensinogen has generated new knowledge of the importance of noncanonical mechanisms for renin independent generation of angiotensins. The human sequence of the dodecapeptide angiotensin-(1-12) [N-Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10-Val1-Ile12-COOH] is an endogenous substrate that in the rat has been documented to be present in multiple organs including the heart, brain, kidney, gut, adrenal gland, and the bone marrow. Newer studies have confirmed the existence of Ang-(1-12) as an Ang II-forming substrate in the blood and heart of normal and diseased patients. Studies to-date document that angiotensin II generation from angiotensin-(1-12) does not require renin participation while chymase rather than angiotensin converting enzyme shows high catalytic activity in converting this tissue substrate into angiotensin II directly.

Phosphodiesterase-2 inhibitor reverses post-traumatic stress induced fear memory deficits and behavioral changes via cAMP/cGMP pathway.

Phosphodiesterase 2 is one of the phosphodiesterase (PDEs) family members that regulate cyclic nucleotide (namely cAMP and cGMP) concentrations. The present study determined whether PDE2 inhibition could rescue post-traumatic stress disorder (PTSD)-like symptoms. Mice were subjected to single prolonged stress (SPS) and treated with selective PDE2 inhibitor Bay 60-7550 (0.3, 1, or 3 mg/kg, i.p.). The behavioral tests such as forced swimming, sucrose preference test, open field, elevated plus maze, and contextual fear paradigm were conducted to determine the effects of Bay 60-7550 on SPS-induced depression- and anxiety-like behavior and fear memory deficits. The results suggested that Bay 60-7550 reversed SPS-induced depression- and anxiety-like behavior and fear memory deficits. Moreover, Bay 60-7550 prevented SPS-induced changes in the adrenal gland index, synaptic proteins synaptophysin and PSD95 expression, PKA, PKG, pCREB, and BDNF levels in the hippocampus and amygdala. These effects were completely prevented by PKG inhibitor KT5823. While PKA inhibitor H89 also prevented Bay 60-7550-induced pCREB and BDNF expression, but only partially prevented the effects on PSD95 expression in the hippocampus. These findings suggest that Bay 60-7550 protects mice against PTSD-like stress induced traumatic injury by activation of cGMP- or cAMP-related neuroprotective molecules, such as synaptic proteins, pCREB and BDNF.

The receptor tyrosine kinase EPHB6 regulates catecholamine exocytosis in adrenal gland chromaffin cells.

The erythropoietin-producing human hepatocellular receptor EPH receptor B6 (EPHB6) is a receptor tyrosine kinase that has been shown previously to control catecholamine synthesis in the adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent fashion. EPHB6 also has a role in regulating blood pressure, but several facets of this regulation remain unclear. Using amperometry recordings, we now found that catecholamine secretion by AGCCs is compromised in the absence of EPHB6. AGCCs from male knockout (KO) mice displayed reduced cortical F-actin disassembly, accompanied by decreased catecholamine secretion through exocytosis. This phenotype was not observed in AGCCs from female KO mice, suggesting that testosterone, but not estrogen, contributes to this phenotype. Of note, reverse signaling from EPHB6 to ephrin B1 (EFNB1) and a 7-amino acid-long segment in the EFNB1 intracellular tail were essential for the regulation of catecholamine secretion. Further downstream, the Ras homolog family member A (RHOA) and FYN proto-oncogene Src family tyrosine kinase (FYN)-proto-oncogene c-ABL-microtubule-associated monooxygenase calponin and LIM domain containing 1 (MICAL-1) pathways mediated the signaling from EFNB1 to the defective F-actin disassembly. We discuss the implications of EPHB6's effect on catecholamine exocytosis and secretion for blood pressure regulation.

Alterations of estrous cycle, 3ß hydroxysteroid dehydrogenase activity and progesterone synthesis in female rats after exposure to hypobaric hypoxia.

The underlying mechanism regulating hypoxia induced alteration in female steroid hormones is first time explored in this study. To understand the mechanistic approach, female Sprague- Dawley rats were exposed to acute and chronic hypobaric hypoxia (282 mm-Hg, ~7620 m, 6 hours, 3 and 7 days). Estrous cycle, body weight, plasma progesterone and estradiol levels, morphology, histology and two key steroidogenic enzymes: 3ß hydroxysteroid dehydrogenase (HSD) and 17ß HSD activity of ovary and adrenal gland were studied. A persistent diestrous phase and a significant decrease in body weight were found in chronic hypoxia groups. Histological study suggested degenerative changes in ovarian corpus luteum of 7 days chronic hypobaric hypoxia (7CHH) group and a declined percentage of adrenocortical cells in 3 days chronic hypobaric hypoxia (3CHH) and 7CHH groups. Plasma estradiol level was unaltered, but progesterone level was decreased significantly in all hypoxic groups. Ovarian 3ß HSD activity was decreased significantly with increasing days of hypoxic treatment along with a significantly low adrenal 3ß HSD activity in 7CHH. In conclusion, hypobaric hypoxia causes a state of low circulatory progesterone level in females likely due to the degenerative changes in the female ovarian and adrenal tissues together with low steroidogenic 3ß HSD enzyme activity.

An integrated RNA-Seq and network study reveals the effect of nicotinamide on adrenal androgen synthesis.

Acne vulgaris is a chronic inflammatory disease of the skin resulting from androgen-induced increased sebum production and altered keratinization. Nicotinamide (NAM), an amide form of vitamin B3 with a well-established safety profile, has shown good therapeutic potential in treating acne and its complications. NAM has anti-inflammatory effects and reduces sebum but its function in androgen biosynthesis remains unknown. In this study, we used a widely used cell model, starved human adrenal NCI-H295R cells, to examine the effects of NAM in androgen production and its mediated network changes. By treating NCI-H295R cells with 1-25 mmol/L of NAM, we found that cell viability was only slightly inhibited at the highest dose (25 mmol/L). NAM reduced testosterone production in a dose-dependent manner. Transcriptomic analysis demonstrated that key enzymes of androgen biosynthesis were significantly decreased under NAM treatment. In addition, gene set enrichment analysis (GSEA) showed that gene sets of cell cycle, steroid biosynthesis, TGFß signalling, and targets of IGF1 or IGF2 were enriched in NAM-treated cells. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway and Gene ontology (GO) analysis of the differentially expressed genes also suggested that steroidogenesis and SMAD signalling were affected by NAM. Overall, these crucial genes and pathways might form a complex network in NAM-treated NCI-H295R cells and result in androgen reduction. These findings help explain the potential molecular actions of NAM in acne vulgaris, and position NAM as a candidate for the treatment of other hyperandrogenic disorders.

Fetal programming of adrenal PNMT and hypertension by glucocorticoids in WKY rats is dose and sex-dependent.

Biochemical changes in utero may alter normal fetal development, resulting in disease later in life, a phenomenon known as fetal programming. Recent epidemiological studies link fetal programming to negative health outcomes, such as low birth weight and hypertension in adulthood. Here, we used a WKY rat model and studied the molecular changes triggered by prenatal glucocorticoid (GC) exposure on the development of hypertension, and on the regulation of phenylethanolamine N-methyl transferase (PNMT), the enzyme responsible for biosynthesis of epinephrine, and a candidate gene linked to hypertension. Clinically, high doses of the synthetic GC dexamethasone (DEX) are used to treat infant respiratory distress syndrome. Elevated maternal GCs have been correlated with fetal programming of hypertension. The aim of this study was to determine if lower doses of DEX would not lead to detrimental fetal programming effects such as hypertension. Our data suggests that prenatal stress programs for increased expression of PNMT and altered regulation of PNMT in males and females. Importantly, we identified that DEX mediated programming was more apparent in the male rats, and the lower dose 10µg/kg/day of DEX did not lead to changes in blood pressure (BP) in female rats suggesting that this dose is below the threshold for programming of hypertension. Furthermore, sex-specific differences were observed in regards to programming mechanisms that may account for hypertension in males.

Adrenal Function in Adolescence is Related to Intrauterine and Postnatal Growth.

Background and objectives: Intrauterine growth restriction is thought to be implicated in long-term programming of hypothalamic-pituitary-adrenal axis activity. We investigated adrenal function in adolescents born small for gestational age (SGA) in relation to their postnatal growth and cardiovascular parameters. Materials and Methods: Anthropometric parameters, blood pressure, heart rate, dehydroepiandrosterone sulfate (DHEAS), and cortisol levels were assessed in 102 adolescents aged 11-14 years followed from birth (47 SGA and 55 born appropriate for gestational age (AGA)). Results: Mean DHEAS levels were higher in SGA adolescents with catch-up growth (SGACU+), compared with AGA. Second-year height velocity and body mass index (BMI) gain during preschool years were positively related to DHEAS levels. Morning cortisol levels and systolic and diastolic blood pressure were higher in SGA adolescents without catch-up growth (SGACU-) compared with AGA. Second-year BMI gain was inversely, and 2-12 years increase in subscapular skinfold thickness was directly associated with cortisol levels. Size at birth and postnatal growth explained 47.8% and 38.2% of variation in DHEAS and cortisol levels, respectively. Conclusion: Adrenal function in adolescence is affected by prenatal and postnatal growth: small size at birth with postnatal catch-up growth is related to higher DHEAS secretion, whereas increased cortisol levels and blood pressure are higher in short SGA adolescents.

Deficiency of T-type Ca2+ channels Cav3.1 and Cav3.2 has no effect on angiotensin II-induced hypertension but differential effect on plasma aldosterone in mice.

T-type Ca2+ channel Cav3.1 promotes microvessel contraction ex vivo. It was hypothesized that in vivo, functional deletion of Cav3.1, but not Cav3.2, protects mice against angiotensin II (ANG II)-induced hypertension. Mean arterial blood pressure (MAP) and heart rate were measured continuously with chronically indwelling catheters during infusion of ANG II (30 ng·kg-1·min-1, 7 days) in wild-type (WT), Cav3.1-/-, and Cav3.2-/- mice. Plasma aldosterone and renin concentrations were measured by radioimmunoassays. In a separate series, WT mice were infused with ANG II (100 ng·kg-1·min-1) with and without the mineralocorticoid receptor blocker canrenoate. Cav3.1-/- and Cav3.2-/- mice exhibited no baseline difference in MAP compared with WT mice, but day-night variation was blunted in both Cav3.1 and Cav3.2-/- mice. ANG II increased significantly MAP in WT, Cav3.1-/-, and Cav3.2-/- mice with no differences between genotypes. Heart rate was significantly lower in Cav3.1-/- and Cav3.2-/- mice compared with control mice. After ANG II infusion, plasma aldosterone concentration was significantly lower in Cav3.1-/- compared with Cav3.2-/- mice. In response to ANG II, fibrosis was observed in heart sections from both WT and Cav3.1-/- mice and while cardiac atrial natriuretic peptide mRNA was similar, the brain natriuretic peptide mRNA increase was mitigated in Cav3.1-/- mice ANG II at 100 ng/kg yielded elevated pressure and an increased heart weight-to-body weight ratio in WT mice. Cardiac hypertrophy, but not hypertension, was prevented by the mineralocorticoid receptor blocker canrenoate. In conclusion, T-type channels Cav3.1and Cav3.2 do not contribute to baseline blood pressure levels and ANG II-induced hypertension. Cav3.1, but not Cav3.2, contributes to aldosterone secretion. Aldosterone promotes cardiac hypertrophy during hypertension.

EPHB6 controls catecholamine biosynthesis by up-regulating tyrosine hydroxylase transcription in adrenal gland chromaffin cells.

EPHB6 is a member of the erythropoietin-producing hepatocellular kinase (EPH) family and a receptor tyrosine kinase with a dead kinase domain. It is involved in blood pressure regulation and adrenal gland catecholamine (CAT) secretion, but several facets of EPHB6-mediated CAT regulation are unclear. In this study, using biochemical, quantitative RT-PCR, immunoblotting, and gene microarray assays, we found that EPHB6 up-regulates CAT biosynthesis in adrenal gland chromaffin cells (AGCCs). We observed that epinephrine content is reduced in the AGCCs from male Ephb6-KO mice, caused by decreased expression of tyrosine hydroxylase, the rate-limiting enzyme in CAT biosynthesis. We demonstrate that the signaling pathway from EPHB6 to tyrosine hydroxylase expression in AGCCs involves Rac family small GTPase 1 (RAC1), MAP kinase kinase 7 (MKK7), c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, activator protein 1 (AP1), and early growth response 1 (EGR1). On the other hand, signaling via extracellular signal-regulated kinase (ERK1/2), p38 mitogen-activated protein kinase, and ELK1, ETS transcription factor (ELK1) was not affected by EPHB6 deletion. We further report that EPHB6's effect on AGCCs was via reverse signaling through ephrin B1 and that EPHB6 acted in concert with the nongenomic effect of testosterone to control CAT biosynthesis. Our findings elucidate the mechanisms by which EPHB6 modulates CAT biosynthesis and identify potential therapeutic targets for diseases, such as hypertension, caused by dysfunctional CAT biosynthesis.

Tyrosine-hydroxylase immunoreactivity in the mouse transparent brain and adrenal glands.

Working on catecholamine systems for years, the neuropharmacologist Arvid Carlsson has made a number of important and pioneering discoveries, which have highlighted the key role of these neuronal and peripheral neurotransmitters in brain functions and adrenal regulations. Since then, major advances have been made concerning the distribution of the catecholaminergic systems in particular by studying their rate-limiting enzyme, tyrosine hydroxylase (TH). Recently new methods of tissue transparency coupled with in toto immununostaining and three-dimensional (3D) imaging technologies allow to precisely map TH immunoreactive pathways in the mouse brain and adrenal glands. High magnification images and movies obtained with combined technologies (iDISCO+ and light-sheet microscopy) are presented in this review dedicated to the pioneer work of Arvid Carlsson and his collaborators.

Prenatal caffeine ingestion induces long-term alterations in scavenger receptor class B type I expression and glucocorticoid synthesis in adult male offspring rat adrenals.

Caffeine is contained within many drinks and food that are consumed daily. Prenatal caffeine ingestion (PCI) is a risk factor for intrauterine growth retardation (IUGR). We previously observed that PCI inhibits scavenger receptor class B type I (SR-BI)-mediated cholesterol uptake in fetal adrenals, subsequently decreasing glucocorticoid synthesis and inducing IUGR. In the present study, we aimed to investigate the long-term effects of PCI on adrenal glucocorticoid synthesis in adult male offspring rats. After establishing the PCI-induced IUGR, adult male offspring was injected intraperitoneally with 5 mg/kg·d lipopolysaccharide (LPS) for 2 days to induce acute stress. We observed persistent inhibition of SR-BI expression in PCI adrenals before and after stress. Compared with the controls, the PCI offspring had higher corticosterone concentrations after stress. The serum cholesterol concentration was stable without intergroup differences before and after stress. The cholesterol concentration in PCI adrenals showed a higher decrease rate than that of the control after stress. In summary, PCI induced long-term alterations in SR-BI expression and glucocorticoid synthesis in adult male offspring rat adrenals. Cholesterol has to be over-consumed in PCI adrenals against acute stress. This study provides an experimental basis to explain the susceptibility of IUGR offspring to metabolic diseases in adults.

Epigenetic Regulation of Aldosterone Synthase Gene by Sodium and Angiotensin II.

BACKGROUND: DNA methylation is believed to be maintained in adult somatic cells. Recent findings, however, suggest that all methylation patterns are not stable. We demonstrate that stimulatory signals can change the DNA methylation status around transcription factor binding sites and a transcription start site and activate expression of the aldosterone synthase gene (CYP11B2). METHODS AND RESULTS: DNA methylation of CYP11B2 was analyzed in aldosterone-producing adenomas, nonfunctioning adrenal adenomas, and adrenal glands and compared with the gene expression levels. CpG dinucleotides in the CYP11B2 promoter were found to be hypormethylated in tissues with high expression, but not in those with low expression, of CYP11B2. Methylation of the CYP11B2 promoter fused to a reporter gene decreased transcriptional activity. Methylation of recognition sequences of transcription factors, including CREB1, NGFIB (NR4A1), and NURR1 (NR4A2) diminished their DNA-binding activity. A methylated-CpG-binding protein MECP2 interacted directly with the methylated CYP11B2 promoter. In rats, low salt intake led to upregulation of CYP11B2 expression and DNA hypomethylation in the adrenal gland. Treatment with angiotensin II type 1 receptor antagonist decreased CYP11B2 expression and led to DNA hypermethylation. CONCLUSIONS: DNA demethylation may switch the phenotype of CYP11B2 expression from an inactive to an active state and regulate aldosterone biosynthesis.

Congenital Adrenal Hyperplasia (CAH) due to 21-Hydroxylase Deficiency: A Comprehensive Focus on 233 Pathogenic Variants of CYP21A2 Gene.

Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive disorders caused by complete or partial defects in one of the several steroidogenic enzymes involved in the synthesis of cortisol from cholesterol in the adrenal glands. More than 95-99% of all cases of CAH are caused by deficiency of steroid 21-hydroxylase, an enzyme encoded by the CYP21A2 gene. Currently, CYP21A2 genotyping is considered a valuable complement to biochemical investigations in the diagnosis of 21-hydroxylase deficiency. More than 200 mutations have been described in literature reports, and much energy is still focused on the clinical classification of new variants. In this review, we focus on molecular genetic features of 21-hydroxylase deficiency, performing an extensive survey of all clinical pathogenic variants modifying the whole sequence of the CYP21A2 gene. Our aim is to offer a very useful tool for clinical and genetic specialists in order to ease clinical diagnosis and genetic counseling.

Effects of body condition score (BCS) on steroid- and eicosanoid-metabolizing enzyme activity in various mare tissues during winter anoestrus.

The objective of this study was to determine the activity of steroid- and eicosanoid-metabolizing enzymes in horses with varying BCSs. The BCSs of twenty non-pregnant, anoestrous mares were determined prior to euthanasia, and tissue samples were collected from the liver, kidney, adrenal gland, ovary and endometrium. Cytochrome P450 1A (CYP1A), 2C (CYP2C), 3A (CYP3A) and uridine 5'-diphospho-glucuronosyltransferase (UGT) activities were determined using luminogenic substrates. The MIXED procedure of SAS was used to test the effect of BCS on enzyme activity and differences between tissues. Activity of CYP1A in adrenals was increased (p ≤ .05) in BCS 5 versus BCSs 4 and 6. Activity of CYP1A in the liver was increased (p = .05) in BCS 4 versus BCSs 5 and 6. Activity of CYP1A was 100-fold greater (p < .0001) in the liver than in the adrenal, ovary and kidney. Activity of CYP2C was 100-fold greater (p < .0001) in the liver than in the adrenal, ovary and endometrium. Activity of CYP3A was only detectable in the liver. Activity of UGT in the kidney was decreased (p = .02) in BCS 4 versus BCSs 5 and 6. Activity of UGT was threefold greater (p < .0001) in the liver than in the kidney, whereas activity of UGT was ninefold greater (p < .0001) in the kidney than in the ovary and endometrium. In general, BCS did not alter the activity of steroid- and eicosanoid-metabolizing enzymes in horses. However, tissue differences in these enzymes indicated abundant hepatic metabolism in horses, which is similar to other livestock species.

Molecular mechanisms of synthesis of noradrenaline as an inducer of development in the adrenal glands of rats in ontogenesis.

The level of gene expression and the protein content of tyrosine hydroxylase and dopamine ß-hydroxylase were determined. In the perinatal period of rats, when noradrenaline functions as a morphogenetic factor, the level of gene expression of these enzymes increased and the content of protein products of these genes was almost unchanged, indicating the difference in the regulatory mechanisms of their transcription and translation.

Sphingosine-1-phosphate lyase mutations cause primary adrenal insufficiency and steroid-resistant nephrotic syndrome.

Primary adrenal insufficiency is life threatening and can present alone or in combination with other comorbidities. Here, we have described a primary adrenal insufficiency syndrome and steroid-resistant nephrotic syndrome caused by loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1). SGPL1 executes the final decisive step of the sphingolipid breakdown pathway, mediating the irreversible cleavage of the lipid-signaling molecule sphingosine-1-phosphate (S1P). Mutations in other upstream components of the pathway lead to harmful accumulation of lysosomal sphingolipid species, which are associated with a series of conditions known as the sphingolipidoses. In this work, we have identified 4 different homozygous mutations, c.665G>A (p.R222Q), c.1633_1635delTTC (p.F545del), c.261+1G>A (p.S65Rfs*6), and c.7dupA (p.S3Kfs*11), in 5 families with the condition. In total, 8 patients were investigated, some of whom also manifested other features, including ichthyosis, primary hypothyroidism, neurological symptoms, and cryptorchidism. Sgpl1-/- mice recapitulated the main characteristics of the human disease with abnormal adrenal and renal morphology. Sgpl1-/- mice displayed disrupted adrenocortical zonation and defective expression of steroidogenic enzymes as well as renal histology in keeping with a glomerular phenotype. In summary, we have identified SGPL1 mutations in humans that perhaps represent a distinct multisystemic disorder of sphingolipid metabolism.

Disordered zonal and cellular CYP11B2 enzyme expression in familial hyperaldosteronism type 3.

Three forms of familial primary aldosteronism have been recognized. Familial Hyperaldosteronism type 1 (FH1) or dexamethasone suppressible hyperaldosteronism, FH2, the most common form of as yet unknown cause(s), and FH3. FH3 is due to activating mutations of the potassium channel gene KCNJ5 that increase constitutive and angiotensin II-induced aldosterone synthesis. In this study we examined the cellular distribution of CYP11B2, CYP11B1, CYP17A1 and KCNJ5 in adrenals from two FH3 siblings using immunohistochemistry and immunofluorescence and obtained unexpected results. The adrenals were markedly enlarged with loss of zonation. CYP11B2 was expressed sporadically throughout the adrenal cortex. CYP11B2 was most often expressed by itself, relatively frequently with CYP17A1, and less frequently with CYP11B1. KCNJ5 was co-expressed with CYP11B2 and in some cells with CYP11B1. This aberrant co-expression of enzymes likely explains the abnormally high secretion rate of the hybrid steroid, 18-oxocortisol.

A Homodimer Model Can Resolve the Conundrum as to How Cytochrome P450 Oxidoreductase and Cytochrome b5 Compete for the Same Binding Site on Cytochrome P450c17.

Cytochrome P450 17α-hydroxylase, 17,20-lyase (P450c17) is a key enzyme in the synthesis of cortisol in the zona fascicula of the adrenal cortex, and the synthesis of androgen precursors in the adrenal zona reticularis and the gonads. Each of these reactions require electrons transferred by the electron donor cytochrome P450 oxidoreductase. The 17α-hydroxylation of its substrate occurs in all cells where P450c17 is expressed. Remarkably, a second, subsequent reaction, namely the 17,20-lyase activity, only occurs in the zona reticularis and gonads. The specificity of the second reaction is due to the interaction with the haem-protein cytochrome b5. Surprisingly, cytochrome b5 and cytochrome P450 oxidoreductase have overlapping sites of interaction on the surface of P450c17. This poses the question as to how cytochrome b5 and cytochrome P450 oxidoreductase interact with P450c17 structurally, functionally and physiologically? This conundrum can be resolved based on the observation that P450c17 can homo-dimerise. A homodimer would allow cytochrome P450 oxidoreductase to bind to one P450c17 monomer of the P450c17 homodimer whilst cytochrome b5 could bind to the other P450c17 monomer simultaneously at the surfaces distal to the dimer interface. This structure is likely to be dynamic in vivo. Our modelling predicts that the proteins can assemble as a stable tetramer and is fully consistent with extensive experimental data that have been published over the last two decades. Predictions derived from this structural model are currently being tested by a range of in vitro and in vivo experimental approaches.