Adrenal Gland Chromaffin Cells Research Articles

SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas.

SUMMARYInherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation.

A Variant in the Nicotinic Acetylcholine Receptor Alpha 3 Subunit Gene Is Associated With Hypertension Risks in Hypogonadic Patients.

Ephb6 gene knockout causes hypertension in castrated mice. EPHB6 controls catecholamine secretion by adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent way. Nicotinic acetylcholine receptor (nAChR) is a ligand-gated Ca2+/Na+ channel, and its opening is the first signaling event leading to catecholamine secretion by AGCCs. There is a possibility that nAChR might be involved in EPHB6 signaling, and thus sequence variants of its subunit genes are associated with hypertension risks. CHRNA3 is the major subunit of nAChR used in human and mouse AGCCs. We conducted a human genetic study to assess the association of CHRNA3 variants with hypertension risks in hypogonadic males. The study cohort included 1,500 hypogonadic Chinese males with (750 patients) or without (750 patients) hypertension. The result revealed that SNV rs3743076 in the fourth intron of CHRNA3 was significantly associated with hypertension risks in the hypogonadic males. We further showed that EPHB6 physically interacted with CHRNA3 in AGCCs, providing a molecular basis for nAChR being in the EPHB6 signaling pathway.

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.

Резистентна артеріальна гіпертензія в пацієнта з феохромоцитомою/парагангліомою

Представлено клінічний випадок феохромоцитоми в поєднанні з парагангліомою в пацієнта із резистентною артеріальною гіпертензією (АГ). Феохромоцитома/парагангліома є катехоламінопродукувальною пухлиною з адреномедулярних хромафінних клітин наднирникових залоз або симпатичних і парасимпатичних гангліїв. Перебіг захворювання залежить від характеру та кількості катехоламінів, які виділяє пухлина, а також від того, наскільки стійким чи спорадичним є їх вивільнення в плазму крові. Тому спостерігається стійке підвищення артеріального тиску (АТ), або АГ має пароксизмальний (кризовий) перебіг. Особливістю представленого клінічного випадку є відсутність деяких характерних діагностичних критеріїв. Попри нормальний вміст катехоламінів у добовій сечі, відсутність змін наднирників при ультразвуковому обстеженні, проводився цілеспрямований пошук вторинних причин підвищення АТ. Клінічними виявами феохромоцитоми, окрім підвищеного АТ, були головний біль і серцебиття. Привертала увагу суттєва втрата маси тіла пацієнтом за останні 1,5 року, що обумовлено підвищеним метаболізмом на тлі активації симпатоадреналової системи. При виконанні дуплексного сканування брахіоцефальних артерій у ділянці біфуркації обох загальних сонних артерій виявлено утворення овальної форми з чіткими рівними контурами, що дало підстави запідозрити нетипове розташування феохромоцитоми. Виконано мультиспіральну комп’ютерну томографію з контрастуванням: черевної порожнини, наднирників, нирок та ниркових артерій. У ході дослідження в тілі медіальної ніжки правого наднирника виявлено округле гіперваскулярне утворення з чіткими рівними контурами. Для уточнення діагнозу в пацієнта було вивчено вміст адреналіну і норадреналіну в плазмі крові. Підвищення норадреналіну більш ніж у 4 рази вказувало на наявність катехоламінпродукувальної пухлини. Таким чином, підтверджено діагноз феохромоцитоми правого наднирника і виявлено каротидні парагангліоми шиї. Пацієнту виконана лапароскопічна адреналектомія справа, а також видалення каротидних парагангліом шиї. Дози антигіпертензивних препаратів були суттєво зменшені, АТ знизився до рівня 130–140/80–90 мм рт. ст. Цей клінічний випадок ілюструє необхідність акцентувати увагу лікарів на ймовірності наявності цієї патології за відсутності характерних лабораторно-інструментальних відхилень, що потребує наполегливого пошуку причин АГ.

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.

EPHB6 and testosterone in concert regulate epinephrine release by adrenal gland chromaffin cells

Erythropoietin-producing human hepatocellular receptor (EPH) B6 (EPHB6) is a member of the receptor tyrosine kinase family. We previously demonstrated that EPHB6 knockout reduces catecholamine secretion in male but not female mice, and castration reverses this phenotype. We showed here that male EPHB6 knockout adrenal gland chromaffin cells presented reduced acetylcholine-triggered Ca2+ influx. Such reduction depended on the non-genomic effect of testosterone. Increased large conductance calcium-activated potassium channel current densities were recorded in adrenal gland chromaffin cells from male EPHB6 knockout mice but not from castrated knockout or female knockout mice. Blocking of the large conductance calcium-activated potassium channel in adrenal gland chromaffin cells from male knockout mice corrected their reduced Ca2+ influx. We conclude that the absence of EPHB6 and the presence of testosterone would lead to augmented large conductance calcium-activated potassium channel currents, which limit voltage-gated calcium channel opening in adrenal gland chromaffin cells. Consequently, acetylcholine-triggered Ca2+ influx is reduced, leading to lower catecholamine release in adrenal gland chromaffin cells from male knockout mice. This explains the reduced resting-state blood catecholamine levels, and hence the blood pressure, in male but not female EPHB6 knock mice. These findings have certain clinical implications.

Rab3a ablation related changes in morphology of secretory vesicles in major endocrine pancreatic cells, pituitary melanotroph cells and adrenal gland chromaffin cells in mice

In this work we have compared the ultrastructural characteristics of major pancreatic endocrine cells, pituitary melanotrophs and adrenal chromaffin cells in the normal mouse strain (wild type, WT) and mice with a known secretory deficit, the Rab3a knockout strain (Rab3a KO). For this purpose, pancreata, pituitary glands and adrenal glands from the Rab3a KO and from the WT mice were analysed, using conventional transmission electron microscopy (TEM). In order to assess the significance of the presence of Rab3a proteins in the relevant cells, we focused primarily on their secretory vesicle morphology and distribution. Our results showed a comparable general morphology in Rab3a KO and WT in all assessed endocrine cell types. In all studied cell types, the distribution of secretory granules along the plasma membrane (number of docked and almost-docked vesicles) was comparable between Rab3a KO and WT mice. Specific differences were found in the diameters of their secretory vesicles, diameters of their electron-dense cores and the presence of autophagic structures in the cells of Rab3A KO mice only. Occasionally, individual electron-dense round vesicles were present inside autophagosome-like structures; these were possibly secretory vesicles or their remnants. The differences found in the diameters of the secretory vesicles confirm the key role of Rab3a proteins in controlling the balance between secretory vesicle biogenesis and degradation, and suggest that the ablation of this protein probably changes the nature of the reservoir of secretory vesicles available for regulated exocytosis.

PAC1 Receptor Activation by PACAP-38 Mediates Ca2+ Release from a cAMP-dependent Pool in Human Fetal Adrenal Gland Chromaffin Cells

Previous studies have shown that human fetal adrenal gland from 17- to 20-week-old fetuses expressed pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, which were localized on chromaffin cells. The aim of the present study was to identify PACAP receptor isoforms and to determine whether PACAP can affect intracellular calcium concentration ([Ca(2+)](i)) and catecholamine secretion. Using primary cultures and specific stimulation of chromaffin cells, we demonstrate that PACAP-38 induced an increase in [Ca(2+)](i) that was blocked by PACAP (6-38), was independent of external Ca(2+), and originated from thapsigargin-insensitive internal stores. The PACAP-triggered Ca(2+) increase was not affected by inhibition of PLC beta (preincubation with U-73122) or by pretreatment of cells with Xestospongin C, indicating that the inositol 1,4,5-triphosphate-sensitive stores were not mobilized. However, forskolin (FSK), which raises cytosolic cAMP, induced an increase in Ca(2+) similar to that recorded with PACAP-38. Blockage of PKA by H-89 or (R(p))-cAMPS suppressed both PACAP-38 and FSK calcium responses. The effect of PACAP-38 was also abolished by emptying the caffeine/ryanodine-sensitive Ca(2+) stores. Furthermore, treatment of cells with orthovanadate (100 microm) impaired Ca(2+) reloading of PACAP-sensitive stores indicating that PACAP-38 can mobilize Ca(2+) from secretory vesicles. Moreover, PACAP induced catecholamine secretion by chromaffin cells. It is concluded that PACAP-38, through the PAC(1) receptor, acts as a neurotransmitter in human fetal chromaffin cells inducing catecholamine secretion, through nonclassical, recently described, ryanodine/caffeine-sensitive pools, involving a cAMP- and PKA-dependent phosphorylation mechanism.