Adrenal Medullary Chromaffin Cells Research Articles

A comparative analysis of surgically excised hereditary and sporadic pheochromocytomas: Insights from a single-center experience.

Pheochromocytoma is a tumor that usually originating from adrenal medullary chromaffin cells and producing one or more catecholamines, can manifest as hereditary or sporadic. While the majority pheochromocytomas are sporadic, hereditary forms are often associated with genetic syndromes such as von Hippel-Lindau, multiple endocrine neoplasia type 2, and neurofibromatosis type 1. This study aims to analyze data from our series of surgically excited pheochromocytoma patients and compare the characteristics between hereditary and sporadic cases. We retrospectively evaluated 33 diagnosed pheochromocytoma patients, documenting clinical features, surgical complications, and tumor characteristics in both hereditary and sporadic cases. Among the patients, 21% (7 individuals) had hereditary pheochromocytoma, while 79% (26 individuals) had sporadic cases. During diagnosis, hereditary pheochromocytoma patients exhibited a significantly lower mean age compared to the sporadic group (26.4 ± 9.9 years vs. 50.4 ± 14.0 years; p < 0.001). The maximum tumor size was also lower in hereditary cases compared to sporadic cases (p = 0.004). Adrenal tumor localization analysis showed that 63.6% were right-sided, 24.2% were left-sided, and 12.1% were bilateral. Laboratory analysis revealed significantly higher urinary norepinephrine levels in hereditary pheochromocytoma patients (p = 0.021). Our findings suggest that hereditary pheochromocytoma cases are characterized by a younger age at diagnosis, smaller tumor size, and a higher prevalence of multiple bilateral adrenal adenomas. We recommend genetic testing for all pheochromocytoma patients, particularly those with early-onset disease and bilateral adrenal tumors.

Enhancement of muscarinic receptor-mediated excitation in spontaneously hypertensive rat adrenal medullary chromaffin cells.

One of the mechanisms for hypertension is an increase in blood catecholamines due to increased secretion from sympathetic nerve terminals and adrenal medullary chromaffin (AMC) cells. Spontaneously hypertensive rats (SHRs) are used as an animal model of hypertension. Catecholamine secretion in AMC cells occurs in response to humoral factors and neuronal inputs from the sympathetic nerve fibres. Acetylcholine (ACh) released from the nerve terminals activates nicotinic as well as muscarinic ACh receptors. The present experiment aimed to elucidate whether muscarinic receptor-mediated excitation is altered in SHR AMC cells and, if it is, how. Compared with normotensive rat AMC cells, muscarinic stimulation induced greater catecholamine secretion and larger depolarising inward currents in SHR AMC cells. In contrast to normotensive rat AMC cells, the muscarine-induced current consisted of quinine-sensitive and quinine-insensitive components. The former and the latter are possibly ascribed to nonselective cation channel activation and TWIK-related acid-sensitive K+ (TASK) channel inhibition, as noted in guinea pig AMC cells. In fact, immunoreactive material for TASK1 and several isoforms of transient receptor potential canonical (TRPC) channels was detected in SHR AMC cells. Stromal interaction molecule 1 (STIM1), which plays an essential role for heteromeric TRPC1-TRPC4 channel formation and is not expressed in normotensive rat AMC cells, was detected in the cytoplasm and co-localised with TRPC1. The expression of muscarinic M1 receptors was enhanced in SHR AMC cells compared with normotensive rats. The results indicate that muscarinic excitation is enhanced in SHR AMC cells, probably through facilitation of TRPC channel signalling.

Muscarinic Receptor Stimulation Does Not Inhibit Voltage-dependent Ca2+ Channels in Rat Adrenal Medullary Chromaffin Cells.

Adrenal medullary chromaffin (AMC) and sympathetic ganglion cells are derived from the neural crest and show a similar developmental path. Thus, these two cell types have many common properties in membrane excitability and signaling. However, AMC cells function as endocrine cells while sympathetic ganglion cells are neurons. In rat sympathetic ganglion cells, muscarinic M1 and M4 receptors mediate excitation and inhibition via suppression of M-type K+ channels and suppression of voltage-dependent Ca2+ channels, respectively. On the other hand, M1 receptor stimulation in rat AMC cells also produces excitation by suppressing TWIK-related acid sensitive K+ (TASK) channels. However, whether M4 receptors are coupled with voltage-dependent Ca2+ channel suppression is unclear. We explore this issue electrophysiologically and biochemically. Electrical stimulation of nerve fibers in rat adrenal glands trans-synaptically increased the Ca2+ signal in AMC cells. This electrically evoked increased Ca2+ signal was not altered during muscarine-induced increase in Ca2+ signal, whereas it decreased significantly during a GABA-induced increase, due to a shunt effect of increased Cl- conductance. The whole-cell current recordings revealed that voltage-dependent Ca2+ currents in AMC cells were suppressed by adenosine triphosphate, but not by muscarinic agonists. The fractionation analysis and immunocytochemistry indicated that CaV1.2 Ca2+ channels and M4 receptors are located in the raft and non-raft membrane domains, respectively. We concluded that muscarinic stimulation in rat AMC cells does not produce voltage-dependent Ca2+ channel inhibition. This lack of muscarinic inhibition is at least partly due to physical separation of voltage-dependent Ca2+ channels and M4 receptors in the plasma membrane.

Post-hatching developmental changes in the adrenal gland of the Japanese quail (Coturnix coturnix japonica): Histological, immunohistochemical, and electron microscopic studies.

The adrenal glands are paired abdominal endocrine organs vital to the bird's health. The present research aimed to provide a comprehensive examination of the histological, ultrastructural, and immunohistochemical investigations of the adrenal gland in Japanese quail during the post hatching period. The current study was performed on 21 healthy Japanese quail chicks at different post hatching periods. Our results showed the adrenal gland is surrounded by a connective tissue capsule, which consists of dense collagen fibers containing large blood vessels, chromaffin cells, autonomic ganglia, fibroblasts, and migrating Schwann cells. The zonation of the adrenal gland is composed of a subcapsular layer, a peripheral zone, and a central zone, which gets more pronounced with age. At the ultrastructural level, the interrenal cells take the steroid-secreting cells characters; they have varying amounts of lipid droplets and abundant mitochondria. Adrenal medullary chromaffin cells showed positive immunoreactivity to the NSE. With increasing age, the chromaffin tissue's Sox10 immunoreactivity increased. β-catenin is expressed within the plasmalemma and the cytoplasm of the interrenal and chromaffin cells and its reactivity increased with age, especially in the chromaffin cells. Our results indicate the adrenal gland undergoes significant morphological changes during the postnatal life. Overall, the postnatal period is an important time for the development and maturation of the adrenal glands.

Expression and function of mitochondrial inhibitor factor-1 and TASK channels in adrenal cells

Adrenal medullary chromaffin (AMC) cells in the perinatal period and carotid body glomus cells after birth respond to hypoxia with catecholamine secretion. The hypoxia detection mechanism in such O2-sensitive cells is still not well defined. One hypothesis is that a decrease in cellular ATP may be involved in the hypoxia detection. This idea is based on ATP dependence of TASK channel activity that regulates the resting membrane potential and is suppressed by hypoxia in glomus cells. Mitochondrial ATPase inhibitor factor-1 (IF1), a physiological regulator of ATP synthase, helps prevent ATP hydrolysis under hypoxic conditions. In cells where IF1 expression is high, exposure to hypoxia is expected to have no effect on TASK channel activity. This possibility was electrophysiologically and immunocytochemically explored. Single channel recordings revealed that 36-pS TASK3-like channels contribute to the resting membrane potential in young rat adrenal cortical (AC) cells. TASK3-like channel activity in a cell-attached patch was not affected by bath application of mitochondrial inhibitors. Consistent with this finding, IF1-like immunoreactive material was well expressed in rat AC cells. In further support of our hypothesis, IF1-like immunoreactive material was well expressed in adult rat AMC cells that are known to be hypoxia-insensitive and minimally expressed in newborn AMC cells that are hypoxia-sensitive. These results provide evidence for the functional relevance of IF1 expression in excitability in O2-sensitive cells in response to mitochondrial inhibition.

Nerve growth factor causes epinephrine release dysfunction by regulating phenotype alterations and the function of adrenal medullary chromaffin cells in mice with allergic rhinitis.

The presence of allergic rhinitis (AR) is an increased risk factor for the occurrence of bronchial asthma (BA). Nerve growth factor (NGF), in addition to its key role in the development and differentiation of neurons, may also be an important inflammatory factor in AR and BA. However, the pathogenesis of the progression of AR to BA remains to be elucidated. The present study aimed to investigate the ability of NGF to mediate nasobronchial interactions and explore possible underlying molecular mechanisms. In the present study, an AR mouse model was established and histology of nasal mucosa tissue injury was determined. The level of phenylethanolamine N‑methyl transferase in adrenal medulla was determined by immunofluorescence. Primary adrenal medullary chromaffin cells (AMCCs) were isolated and cultured from the adrenal medulla of mice. The expression levels of synaptophysin (SYP), STAT1, JAK1, p38 and ERK in NGF‑treated and untreated AMCCs were detected by reverse‑transcription‑quantitative PCR and western blotting. The epinephrine (EPI) and norepinephrine (NE) concentrations were measured by ELISA. It was found that the expression of SYP in AMCCs was enhanced in the presence of NGF, whereas, the concentration of EPI decreased significantly under the same conditions. Furthermore, NGF mediated the phenotypic and functional changes of AMCCs, resulting in decreased EPI secretion via JAK1/STAT1, p38 and ERK signaling. In conclusion, these findings could provide novel evidence for the role of NGF in regulating neuroendocrine mechanisms.

Pheochromocytoma with Type 2 Diabetes Mellitus and Subclinical Cushing's Syndrome

Pheochromocytoma is a catecholamine-producing tumor that originates from adrenal medullary chromaffin cells. It affects 1–8 per million people annually and accounts for 5% of adrenal incidentalomas. Here, we report a 62-year-old man with sudden excessive sweating, palpitations, and headaches. He has a history of type 2 diabetes mellitus, hypertension, and an adrenal mass found incidentally by ultrasonography. Clinical history and further imaging suggest a pheochromocytoma; however, the presence of subclinical Cushing's syndrome and a normal urine vanillylmandelic acid level have led to diagnostic challenges. Finally, the patient underwent a left adrenalectomy. Histopathology showed the tumor as thick nests of cells, positive chromogranin A staining, and negative inhibin staining that confirmed the diagnosis of pheochromocytoma. In conclusion, our patient had pheochromocytoma that was thought to produce adrenocorticotropic hormone ectopically, resulting in subclinical Cushing's syndrome.

Immunocytochemistry of Acutely Isolated Adrenal Medullary Chromaffin Cells.

Immunocytochemistry enables the detection and localization of proteins in cells that are acutely dissociated or in culture. There are advantages and disadvantages to the use of cultured cells for immunocytochemistry. One of the advantages is that cultured cells can be used for one or more weeks after the dissociation of cells, whereas one of the disadvantages is that the properties of cells in culture might change under artificial conditions. On the other hand, acutely dissociated cells are expected to have the original properties of cells because almost all procedures before fixation, except for enzymatic digestion, are carried out at low temperatures. Here, we describe how adrenal medullary cells of small animals are acutely dissociated for immunostaining.

La Esclerosis lateral amiotrófica no es solo una enfermedad de la motoneurona: impacto del eje simpatoadrenal

Amyotrophic lateral sclerosis (ALS), an adult onset, fatal neurodegenerative disease, has as a cardinal pathogenic feature the selective death of motor neurons (MNs) at the cortex, brainstem, and spinal cord. In this review we focus on four aspects: (i) the hypothesis of disease propagation through the cerebrospinal fluid (CSF); (ii) the distortion of the exocytotic release of neurotransmitters at the sympathoadrenal axis; (iii) the ultrastructural and functional alterations of mitochondria from adrenal medullary chromaffin cells (CCs); and (iv) the purinergic P2X7 receptor (P2X7R) as a potential target for neuroprotection. Concerning disease propagation from one to another area of the central nervous system (CNS), the pattern of clinical progression suggests that the disease spreads centrifugally. This indicates that a kind of toxin agent may be released and propagated through the CSF. In our laboratory we found that CSF from ALS patients exerted toxic effects on cultured cortical MNs. In CCs, we found deep distortions of the exocytotic kinetics and the exocytotic fusion pore in the process of catecholamine release, in the SOD1G93A mouse model of ALS. Furthermore, we found that these alterations could be related to the accumulation of mutated SOD1 into mitochondria; this resulted in mitochondrial depolarization, excess production of reactive oxygen species and deficiency in oxidative phosphorylation. Finally, we discuss recent data on the potential therapeutic effect of compound JNJ-47965567, a blocker of P2X7Rs known to be central-stage in neuroinflammation. Upon its chronic administration to SOD1G93A, we found that the compound delayed disease onset but only in females mice. In conclusion, why MN selectively die in ALS disease, remains a mystery; On the other hand, it seems that other cell types are also affected, particularly at the sympathoadrenal axis. As disease pathogenesis remains obscure, the search of therapeutic targets to slow disease progression in ALS, remains puzzling.

Mechanisms for establishment of GABA signaling in adrenal medullary chromaffin cells.

γ-Aminobutyric acid (GABA) is thought to play a paracrine role in adrenal medullary chromaffin (AMC) cells. Comparative physiological and immunocytochemical approaches were used to address the issue of how the paracrine function of GABA in AMC cells is established. GABAA receptor Cl- channel activities in AMC cells of rats and mice, where corticosterone is the major glucocorticoid, were much smaller than those in AMC cells of guinea-pigs and cattle, where cortisol is the major. The extent of enhancement of GABAA receptor α3 subunit expression in rat pheochromocytoma (PC12) cells by cortisol was larger than that by corticosterone in parallel with their glucocorticoid activities. Thus, the species difference in GABAA receptor expression may be ascribed to a difference in glucocorticoid activity between corticosterone and cortisol. GABAA receptor Cl- channel activity in mouse AMC cells was enhanced by allopregnanolone, as noted with that in guinea-pig AMC cells, and the enzymes involved in allopregnanolone production were immunohistochemically detected in the zona fasciculata in both mice and guinea pigs. The expression of glutamic acid decarboxylase 67 (GAD67), one of the GABA synthesizing enzymes, increased after birth, whereas GABAA receptors already developed at birth. Stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, but not nicotinic or muscarinic receptors, in PC12 cells, resulted in an increase in GAD67 expression in a protein-kinase A-dependent manner. The results indicate that glucocorticoid and PACAP are mainly responsible for the expressions of GABAA receptors and GAD67 involved in GABA signaling in AMC cells, respectively.

Gaseous transmitter regulation of hypoxia-evoked catecholamine secretion from murine adrenal chromaffin cells.

Emerging evidence suggests that gaseous molecules, carbon monoxide (CO), and hydrogen sulfide (H2S) generated by heme oxygenase (HO)-2 and cystathionine γ-lyase (CSE), respectively, function as transmitters in the nervous system. Present study examined the roles of CO and H2S in hypoxia-induced catecholamine (CA) release from adrenal medullary chromaffin cells (AMCs). Studies were performed on AMCs from adult (≥6 wk of age) wild-type (WT), HO-2 null, CSE null, and HO-2/CSE double null mice of either gender. CA secretion was determined by carbon fiber amperometry and [Ca2+]i by microflurometry using Fura-2. HO-2- and CSE immunoreactivities were seen in WT AMC, which were absent in HO-2 and CSE null mice. Hypoxia (medium Po2 30-38 mmHg) evoked CA release and elevated [Ca2+]i. The magnitude of hypoxic response was greater in HO-2 null mice and in HO inhibitor-treated WT AMC compared with controls. H2S levels were elevated in HO-2 null AMC. Either pharmacological inhibition or genetic deletion of CSE prevented the augmented hypoxic responses of HO-2 null AMC and H2S donor rescued AMC responses to hypoxia in HO-2/CSE double null mice. CORM3, a CO donor, prevented the augmented hypoxic responses in WT and HO-2 null AMC. CO donor reduced H2S levels in WT AMC. The effects of CO donor were blocked by either ODQ or 8pCT, inhibitors of soluble guanylyl cyclase (SGC) or protein kinase G, respectively. These results suggest that HO-2-derived CO inhibits hypoxia-evoked CA secretion from adult murine AMC involving soluble guanylyl cyclase (SGC)-protein kinase G (PKG)-dependent regulation of CSE-derived H2S.NEW & NOTEWORTHY Catecholamine secretion from adrenal chromaffin cells is an important physiological mechanism for maintaining homeostasis during hypoxia. Here, we delineate carbon monoxide (CO)-sensitive hydrogen sulfide (H2S) signaling as an important mediator of hypoxia-induced catecholamine secretion from murine adrenal chromaffin cells. Heme oxygenase-2 derived CO is a physiological inhibitor of catcholamince secretion by hypoxia and the effects of CO involve inhibition of cystathionine γ-lyase-derived H2S production through soluble guanylyl cyclase-protein kinase G signaling cascade.

52-Year-Old Woman With Fever, Diaphoresis, and Abdominal Pain

A 52-year-old woman with history of generalized anxiety disorder and perimenopausal hot flashes presented to the emergency department with 3 days of subjective fever, diaphoresis, and right upper quadrant abdominal pain. She described the pain as sharp, intermittent, and radiating across her upper abdomen with no relieving factors. She reported intermittent dyspnea and palpitations over the past several months, but denied any chest pain, nausea, or vomiting. She consumed alcohol rarely and never used tobacco or illicit drugs. Her medications included St John’s Wort for anxiety and clonidine 0.1 mg twice daily for hot flashes. Her family history was unremarkable. Upon arrival to the emergency department, she was afebrile with blood pressure 190/100 mm Hg, heart rate 86 beats/min, and oxygen saturation 97% on room air. Physical examination revealed a diaphoretic female with mild right upper quadrant abdominal tenderness to deep palpation with no rebound, guarding, masses, or organomegaly. Cardiac exam revealed a regular rhythm without murmurs and no jugular venous pressure elevation. Lung fields were clear. Laboratory testing demonstrated the following (reference ranges provided parenthetically): hemoglobin 14.0 g/dL (12.0 to 15.5 g/dL), leukocyte count 27.3 white blood cells/L (3.4 to 9.6×109), creatinine 0.95 mg/dL (0.84 to 1.21 mg/dL), aspartate aminotransferase 32 U/L (8 to 48 U/L), alanine aminotransferase 20 U/L (7 to 55), alkaline phosphatase 73 U/L (40 to 129 U/L), total bilirubin 0.6 mg/dL (≤1.2 mg/dL), sensitive thyroid stimulating hormone 0.9 mIU/L (0.3 to 4.2 mIU/L), lactate 4.8 mmol/L (0.5 to 2.2mmol/L), baseline troponin T 0.72 ng/mL (<0.01 ng/mL), and N-terminal pro–B-type natriuretic peptide 20,800 pg/mL (≤152). Chest x-ray revealed bilateral hazy opacities. Electrocardiogram showed sinus rhythm with nonspecific T-wave changes. 1. Which one of the following diagnoses is most consistent with the patient’s clinical picture?a.Acute cholecystitisb.Pulmonary embolismc.Community-acquired pneumoniad.Hypertensive emergencye.Acute coronary syndrome Acute cholecystitis is a reasonable consideration in this patient with right upper quadrant abdominal pain and tenderness, leukocytosis, and subjective fever episodes, but this diagnosis is unlikely in a patient with normal hepatic function tests and would not account for the elevated troponin T or N-terminal pro–B-type natriuretic peptide levels. Whereas pulmonary emboli can cause heart strain and lead to elevated cardiac biomarkers, this patient denies chest pain and lacks a clinical history that would fit with this diagnosis. Moreover, pulmonary emboli causing heart strain would be more likely to cause hypotension. The presence of subjective fevers, leukocytosis, lactic acidosis and infiltrates on chest x-ray could be consistent with a diagnosis of community-acquired pneumonia but would not adequately explain the cardiac biomarker abnormalities. Hypertensive emergency applies to patients with a systolic pressure greater than or equal to 180 mm Hg and/or diastolic pressure greater than or equal to 120 mm Hg with evidence of end-organ dysfunction, which in this case includes pulmonary edema and non–ST-elevation myocardial infarction (MI). Acute coronary syndrome is caused by acute atherosclerotic plaque rupture leading to myocardial ischemia and infarction (type 1 MI), whereas type 2 MI is caused by mismatch between oxygen supply and demand without acute atherosclerotic plaque rupture. Whereas coronary angiography may be needed to differentiate the two etiologies, this patient displays neither ischemic symptoms nor major atherosclerotic risk factors, making acute coronary syndrome less likely. Given her elevated troponin T in the setting of severe hypertension, type 2 MI secondary to hypertensive emergency is the more likely diagnosis. Transthoracic echocardiogram revealed a left ventricular ejection fraction (LVEF) of 31% (50% to 55%) with regional wall motion abnormalities and grade 3/4 diastolic dysfunction. Cardiac catheterization demonstrated nonobstructive atherosclerosis with a left ventricular end-diastolic pressure of 32 mm Hg (14 to 24 mm Hg) and findings consistent with dilated cardiomyopathy. The patient continued to be hypertensive and was administered intravenous metoprolol for blood pressure control. She subsequently experienced worsening hypertension and sinus tachycardia. Her respiratory status deteriorated, and bedside echocardiogram revealed a worsening LVEF. She was intubated due to progressive respiratory distress. Computed tomography (CT) scan of the chest revealed pulmonary edema with possible alveolar infiltrates. 2. Which one of the following is the most likely condition that is driving this patient’s worsening clinical status after administration of metoprolol?a.Essential hypertensionb.Coronary vasospasmc.Thyrotoxicosisd.Pheochromocytomae.Stimulant intoxication Essential hypertension can lead to hypertensive emergency but would be expected to respond to intravenous beta blockers with an improvement in blood pressure rather than worsening hypertension. Coronary vasospasm can be induced by administration of beta blockers, especially in the setting of stimulant intoxication, and could explain the intermittent epigastric pain, elevated cardiac biomarkers, and worsening systolic function in the setting of nonobstructive coronary angiography.1Robertson R.M. Wood A.J. Vaughn W.K. Robertson D. Exacerbation of vasotonic angina pectoris by propranolol.Circulation. 1982; 65: 281-285Crossref PubMed Scopus (210) Google Scholar However, coronary vasospasm often produces ST-elevation on electrocardiogram during symptoms and would not explain the worsening hypertension that this patient experienced. Thyrotoxicosis is a consideration for a patient with accelerated hypertension but would be unlikely given her normal thyroid stimulating hormone level on admission. Furthermore, beta blockers would block the sympathetic effects of thyroid hormone and alleviate hypertension. Pheochromocytoma is a rare catecholamine-secreting adrenal tumor that can cause paroxysmal hypertension and hypertensive emergency. It should be suspected in patients with worsening hypertension after administration of selective beta-adrenergic receptor blockers, as these lead to unopposed alpha-adrenergic activity by the elevated catecholamine levels. Use of stimulants, such as cocaine or methamphetamine, can similarly cause hypertension after administration of beta blockers mimicking pheochromocytoma. Although stimulant abuse may be a more common cause of this clinical presentation among the general population, this was thought to be less likely in our patient with no history of substance abuse and a urine toxicity screen was not obtained. Her history of anxiety, hot flashes, dyspnea, and paroxysmal palpitations was more consistent with a diagnosis of pheochromocytoma. Patients with persistently high levels of circulating catecholamines can develop a stress cardiomyopathy, which can acutely worsen as the systemic vascular resistance increases. Stress cardiomyopathy can present with regional wall motion abnormalities on echocardiogram, although they are not typically in the distribution of a single coronary artery. This patient was thought to have developed flash pulmonary edema as a result of the acute increase in afterload. 3. Which one of the following is the best initial test to evaluate a patient with suspected pheochromocytoma?a.Plasma or urine catecholaminesb.Plasma or urine metanephrinesc.Plasma or urine vanillylmandelic acid (VMA)d.Plasma or urine chromogranin Ae.CT scan of abdomen and pelvis Diagnosis of a suspected pheochromocytoma involves laboratory testing for evidence of excess catecholamines or imaging to identify the tumor. Laboratory testing can include concentrations of catecholamines (epinephrine, norepinephrine, or dopamine) or their metabolites (metanephrine, normetanephrine, VMA, or 3-methoxytyramine) in the plasma or urine. The Endocrine Society Clinical Practice Guidelines recommends initial screening with plasma free or 24-hour urinary fractionated metanephrines, as these appear to be the most sensitive tests.2Lenders J.W. Duh Q.Y. Eisenhofer G. et al.Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline.J Clin Endocrinol Metab. 2014; 99: 1915-1942Crossref PubMed Scopus (1124) Google Scholar Certain medications can interfere with laboratory measurements and/or the degradation of catecholamines and result in falsely elevated metanephrine levels, including acetaminophen, mesalamine, labetalol, buspirone, and tricyclic antidepressants. Chromogranin A is released with catecholamines in adrenal medullary and sympathetic neuronal vesicles and is sometimes used for patients with borderline or unclear results or as monitoring for recurrence. Imaging should not be obtained until there is biochemical evidence of a pheochromocytoma, as there is an increased likelihood of identifying asymptomatic, nonfunctioning incidentalomas. CT scan of the abdomen and pelvis is the preferred first-line imaging modality given its wide availability, low cost, and excellent visualization of the adrenal glands.2Lenders J.W. Duh Q.Y. Eisenhofer G. et al.Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline.J Clin Endocrinol Metab. 2014; 99: 1915-1942Crossref PubMed Scopus (1124) Google Scholar In this case, plasma free metanephrine was elevated to 55 nmol/L (<0.50 nmol/L). Subsequent testing for plasma catecholamines revealed levels that were above the maximum testing limit of the laboratory equipment, requiring dilution of the samples to obtain results. Diluted samples revealed elevated norepinephrine to 71,159 pg/mL (70 to 750 pg/mL), epinephrine to 48,015 pg/mL (≤111 pg/mL), and dopamine to 994 pg/mL (<30 pg/mL). Plasma chromogranin A was 6,246 ng/mL (<93 ng/mL). A CT scan of the abdomen and pelvis identified a large right adrenal heterogeneous mass with signs of recent hemorrhage.4.Which one of the following medications is the best initial choice for preoperative management?a.Alpha blockersb.Beta blockersc.Calcium channel blockers (CCBs)d.Angiotensin-converting enzyme inhibitorse.Tyrosine hydroxylase inhibitors The treatment of choice for pheochromocytoma is surgical resection, and an experienced endocrine surgeon should be involved once the diagnosis has been established. Anesthesia induction and surgical manipulation of the tumor can lead to dangerous abrupt increases in catecholamine levels, conferring a substantial perioperative risk to these patients. Preoperative medical management is essential, and surgery is typically delayed for 10 to 14 days for medical optimization to ensure adequate pharmacologic adrenergic receptor blockade in the event of intraoperative catecholamine surges.3Lenders J.W.M. Eisenhofer G. Update on modern management of pheochromocytoma and paraganglioma.Endocrinol Metab (Seoul). 2017; 32: 152-161Crossref PubMed Scopus (62) Google Scholar Several medication classes can diminish the effects of catecholamines and have been shown to be effective options in managing patients with functioning pheochromocytomas. The most widely accepted approach involves a combination of alpha and beta blockade. Surging catecholamines lead to activation of alpha-adrenergic receptors, resulting in vasoconstriction, hypertension, and end-organ damage. Phenoxybenzamine, an irreversible nonselective alpha-1 and alpha-2 blocker, is the most commonly administered alpha-adrenergic medication due to its long half-life. Other options include reversible nonselective alpha blockers, such as phentolamine, or reversible selective alpha-1 blockers, such as prazosin, terazosin, or doxazosin. Once adequate alpha-adrenergic blockade has been established, usually after 2 to 3 days, beta blockers should be added to reduce the tachycardia that is precipitated by the alpha blockade and prevent adverse cardiovascular effects of excessive beta-adrenergic stimulation by catecholamines. As seen in this case, beta blockers should not be used until alpha blockers have reached their full effects, otherwise the unopposed alpha-adrenergic stimulation will lead to worsening hypertension, vasoconstriction, and systolic dysfunction.4Garcia M. Palasi R. Gomez R.C. Marco J.L.P. Merino-Torres J.F. Surgical and Pharmacological Management of Functioning Pheochromocytoma and Paraganglioma.in: Mariani-Costantini R. Paraganglioma: A Multidisciplinary Approach. Codon Publications, Brisbane, Australia2019Crossref Google Scholar The modest degree of alpha blockade provided by mixed alpha/beta blockers such as labetalol or carvedilol may not be adequate to prevent this effect. CCBs inhibit calcium influx in vascular smooth muscle, thereby leading to direct arterial vasodilation. Both dihydropyridine and non-dihydropyridine calcium channel antagonists can be used when patients are persistently hypertensive or experiencing dose-limiting adverse effects from alpha blockers.4Garcia M. Palasi R. Gomez R.C. Marco J.L.P. Merino-Torres J.F. Surgical and Pharmacological Management of Functioning Pheochromocytoma and Paraganglioma.in: Mariani-Costantini R. Paraganglioma: A Multidisciplinary Approach. Codon Publications, Brisbane, Australia2019Crossref Google Scholar There is no current evidence that angiotensin-converting enzyme inhibitors are useful as primary or adjunctive medications in the preoperative management of pheochromocytomas. Metyrosine, a tyrosine hydroxylase inhibitor, prevents further production of catecholamines and can be used to augment blood pressure control. It is particularly useful for patients with high catecholamine levels but its use is limited by its cost.4Garcia M. Palasi R. Gomez R.C. Marco J.L.P. Merino-Torres J.F. Surgical and Pharmacological Management of Functioning Pheochromocytoma and Paraganglioma.in: Mariani-Costantini R. Paraganglioma: A Multidisciplinary Approach. Codon Publications, Brisbane, Australia2019Crossref Google Scholar In this case, the patient initially displayed hemodynamic evidence of normotensive cardiogenic shock, which responded to intravenous milrinone and clevidipine. Phenoxybenzamine was started the day after diagnosis and propranolol, a nonselective beta-adrenergic receptor blocker, was added 2 days later. Metyrosine was subsequently added to reduce catecholamine biosynthesis before surgery. Phenoxybenzamine was transitioned to doxazosin upon discharge. The patient underwent right adrenalectomy 20 days after diagnosis and pathology confirmed pheochromocytoma. She tolerated the surgery without any major complications and was discharged on postoperative day 3. 5. Which one of the following statements is true regarding post-discharge follow-up for this patient?a.Genetic testing is not indicatedb.She requires annual biochemical screening for recurrencec.She should have repeat abdominal CT or positron-emission tomography–CT scan in 1 yeard.Her cardiomyopathy is likely irreversiblee.She will likely require long-term anti-hypertensive therapy Recent advances in genetic testing have enhanced our ability to detect mutations in patients with pheochromocytomas. Contrary to the popular “10% rule” that is commonly taught to physicians (that pheochromocytomas are 10% familial, 10% malignant, and 10% extra-adrenal), approximately 30% of tumors have a known genetic basis.5Tischler A.S. Pheochromocytoma and extra-adrenal paraganglioma: updates.Arch Pathol Lab Med. 2008; 132: 1272-1284PubMed Google Scholar Identifying these mutations provides valuable prognostic information about the risk of recurrence and metastasis, as well as the need for screening in family members.3Lenders J.W.M. Eisenhofer G. Update on modern management of pheochromocytoma and paraganglioma.Endocrinol Metab (Seoul). 2017; 32: 152-161Crossref PubMed Scopus (62) Google Scholar All patients need to be monitored for recurrence following surgical resection. The current recommendations include repeat biomarker testing 2 to 6 weeks after surgery followed by yearly biomarker testing for 10 years. In high-risk patients, including those who are young, have large tumors, or high-risk genetic mutations, screening should be offered lifelong. Postoperative imaging is not typically recommended unless biomarkers remain elevated or there is concern for metastatic disease at the time of presentation.6Plouin P.F. Amar L. Dekkers O.M. et al.European Society of Endocrinology Clinical Practice Guideline for long-term follow-up of patients operated on for a phaeochromocytoma or a paraganglioma.Eur J Endocrinol. 2016; 174: G1-G10Crossref PubMed Scopus (188) Google Scholar Patients with pheochromocytomas are at an elevated risk of myocardial injury and some can develop a catecholamine-induced cardiomyopathy. The majority will experience improved cardiac function as the circulating catecholamine levels normalize.7Kassim T.A. Clarke D.D. Mai V.Q. Clyde P.W. Mohamed Shakir K.M. Catecholamine-induced cardiomyopathy.Endocr Pract. 2008; 14: 1137-1349Crossref PubMed Scopus (115) Google Scholar Repeat echocardiograms are necessary to ensure that cardiac function recovers appropriately. Hypertension typically resolves after removal of the pheochromocytoma, and some patients may develop hypotension secondary to adrenal insufficiency if adrenalectomy is performed.3Lenders J.W.M. Eisenhofer G. Update on modern management of pheochromocytoma and paraganglioma.Endocrinol Metab (Seoul). 2017; 32: 152-161Crossref PubMed Scopus (62) Google Scholar This patient was referred for genetic testing but declined. Repeat biomarker testing 8 weeks after surgery demonstrated normalization of serum metanephrines, 24-hour urinary metanephrines, and 24-hour urinary catecholamines. She will continue to have annual testing for the next 10 years. Repeat transthoracic echocardiogram 6 days after initiation of preoperative medical treatment demonstrated resolution of systolic and diastolic dysfunction with LVEF 55%, as well as resolution of previously seen regional wall motion abnormalities. She was weaned off antihypertensive therapy after surgery and was discharged on a propranolol taper. Pheochromocytomas are rare catecholamine-producing neuroendocrine tumors that arise from adrenal medullary chromaffin cells. The clinical presentation is highly variable and typically includes nonspecific symptoms secondary to direct effects from catecholamines. The most common symptoms include “spells” with episodic headaches (60% to 90%), palpitations (50% to 70%), and diaphoresis (55% to 75%).8Lenders J.W. Eisenhofer G. Mannelli M. Pacak K. Phaeochromocytoma. Lancet. 2005; 366: 665-675Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar When all three of these symptoms are present, the specificity has been shown to be upwards of 90%.9Plouin P.F. Degoulet P. Tugaye A. Ducrocq M.B. Menard J. Screening for phaeochromocytoma: in which hypertensive patients? A semiological study of 2585 patients, including 11 with phaeochromocytoma (author's transl) [in French].Nouv Presse Med. 1981; 10: 869-872PubMed Google Scholar Less common symptoms include anxiety (20% to 40%), hyperglycemia (40%), fatigue (25% to 40%), and paroxysmal hypertension (30%); hypertension may be sustained and resistant to therapy. The paroxysmal nature of these symptoms provides an important clue to this diagnosis.8Lenders J.W. Eisenhofer G. Mannelli M. Pacak K. Phaeochromocytoma. Lancet. 2005; 366: 665-675Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar As the mass enlarges, patients can develop pain from the mass compressing on nearby structures. Pheochromocytomas can be imminently life-threatening and all patients with a suspected diagnosis should undergo biochemical testing for this disease. The majority of pheochromocytomas produce at least one catecholamine and patients typically have elevated levels of epinephrine, norepinephrine, and/or dopamine. Epinephrine and norepinephrine are metabolized into metanephrine and normetanephrine, which are further metabolized into VMA. Dopamine is metabolized into 3-methoxytyramine. The Endocrine Society Clinical Practice Guidelines recommends initial testing for plasma free metanephrine or 24-hour urinary fractionated metanephrines, as these appear to have the greatest diagnostic sensitivity.2Lenders J.W. Duh Q.Y. Eisenhofer G. et al.Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline.J Clin Endocrinol Metab. 2014; 99: 1915-1942Crossref PubMed Scopus (1124) Google Scholar Patients with borderline results may benefit from a clonidine suppression test to distinguish patients with increased sympathetic activity from those with underlying pheochromocytomas. In this test, patients are given oral clonidine and have a repeat plasma normetanephrine drawn 3 hours later. Clonidine, an alpha-2 antagonist, will suppress neuronal norepinephrine release in patients without an underlying pheochromocytoma but will not have this effect on patients with a tumor. In patients with confirmed biochemical abnormalities, imaging should be obtained to localize the tumor. A CT scan of the abdomen and pelvis with and without intravenous contrast is the initial modality of choice, as the majority of pheochromocytomas are located within the abdomen and pelvis. In patients with adrenal tumors greater than 5 cm or concern for metastatic disease, further imaging with 123I-metaiodobenzylguanidine, magnetic resonance imaging, or positron-emission tomography–CT scans may be warranted. Patients with pheochromocytomas can develop cardiovascular complications secondary to toxic effects of circulating catecholamines and acute cardiac disease is often the initial presentation. Pheochromocytomas can cause hypertension, cardiac arrhythmias, acute MI, left apical ventricular hypertrophy, and catecholamine-induced dilated cardiomyopathy with heart failure. When excessive catecholamine release produces peripheral vasoconstriction and reduced myocardial systolic function, a low-output state progressing to cardiogenic shock can occur.10Sibal L. Jovanovic A. Agarwal S.C. et al.Phaeochromocytomas presenting as acute crises after beta blockade therapy.Clin Endocrinol (Oxf). 2006; 65: 186-190Crossref PubMed Scopus (66) Google Scholar Many of these cardiovascular complications are reversible after the effects of catecholamines have been blunted, although the damage can be irreversible and close monitoring is necessary.7Kassim T.A. Clarke D.D. Mai V.Q. Clyde P.W. Mohamed Shakir K.M. Catecholamine-induced cardiomyopathy.Endocr Pract. 2008; 14: 1137-1349Crossref PubMed Scopus (115) Google Scholar As exemplified in our case, the acute use of beta-adrenergic receptor blockers can be life-threatening in patients with pheochromocytomas. Even in severe cases, aggressive pharmacologic management should be prioritized over early surgical resection because of the high mortality associated with catecholamine surges in a patient without adequate preoperative adrenergic blockade.3Lenders J.W.M. Eisenhofer G. Update on modern management of pheochromocytoma and paraganglioma.Endocrinol Metab (Seoul). 2017; 32: 152-161Crossref PubMed Scopus (62) Google Scholar,4Garcia M. Palasi R. Gomez R.C. Marco J.L.P. Merino-Torres J.F. Surgical and Pharmacological Management of Functioning Pheochromocytoma and Paraganglioma.in: Mariani-Costantini R. Paraganglioma: A Multidisciplinary Approach. Codon Publications, Brisbane, Australia2019Crossref Google Scholar The most common regimen for preoperative management includes alpha blockade followed by beta blockade. CCBs and metyrosine have a role as adjunctive medications.4Garcia M. Palasi R. Gomez R.C. Marco J.L.P. Merino-Torres J.F. Surgical and Pharmacological Management of Functioning Pheochromocytoma and Paraganglioma.in: Mariani-Costantini R. Paraganglioma: A Multidisciplinary Approach. Codon Publications, Brisbane, Australia2019Crossref Google Scholar Patients with confirmed pheochromocytomas require yearly physical exams, blood pressure measurements, and biochemical screening for at least 10 years after surgery to assess for recurrence.3Lenders J.W.M. Eisenhofer G. Update on modern management of pheochromocytoma and paraganglioma.Endocrinol Metab (Seoul). 2017; 32: 152-161Crossref PubMed Scopus (62) Google Scholar Genetic testing should be considered for all patients, as identification of mutations provides prognostic information regarding the likelihood of recurrence, metastases, and hereditability in family members.11Pillai S. Gopalan V. Smith R.A. Lam A.K. Updates on the genetics and the clinical impacts on phaeochromocytoma and paraganglioma in the new era.Crit Rev Oncol Hematol. 2016; 100: 190-208Crossref PubMed Scopus (64) Google Scholar Familial syndromes with an increased risk of developing pheochromocytomas include multiple endocrine neoplasia (MEN) 2A syndrome, MEN 2B syndrome, von Hippel-Lindau disease, and neurofibromatosis type 1, among other rarer syndromes.8Lenders J.W. Eisenhofer G. Mannelli M. Pacak K. Phaeochromocytoma. Lancet. 2005; 366: 665-675Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar Pheochromocytomas are life-threatening catecholamine-secreting tumors that require prompt diagnosis and treatment. Patients often present with manifestations of acute cardiac disease, which can include catecholamine-induced cardiomyopathy. This case outlines the presentation, work-up, treatment, and follow-up of pheochromocytomas. It is imperative to avoid beta-blocker therapy until alpha blockade has been established in all suspected cases of pheochromocytoma.

MON-LB041 Pheochromocytoma - Illusive Myriad of Symptoms

Background: Pheochromocytoma is a rare neuroendocrine neoplasm arising from the adrenal medullary chromaffin cells. It contributes to 80-85% of catecholamine secreting tumors. The annual incidence of pheochromocytoma is 0.8 cases per 100,000 person-years. It can be both sporadic or familial. The classic triad of headache, palpitations and diaphoresis is seen in only 4% of cases. Rare presentations include cardiomyopathy, stroke, diabetes mellitus, ventricular arrhythmias and myocardial infarction. Our patient presented concurrently with dilated cardiomyopathy, hypertensive emergency and new-onset diabetes mellitus (DM) followed by ischemic stroke within a week. Heart failure can present as takotsubo or dilated cardiomyopathy with an incidence of 10%. The underlying pathophysiology is catecholamine mediated myocardial stunning, diffuse coronary vasospasm, microvasculature dysfunction and fibrosis. DM is seen in 23% of pheochromocytoma and is due to catecholamine-induced impaired glucose tolerance, and insulin resistance. Cerebral ischemia has an incidence of 3%, and is secondary to severe hypertension and cerebral vasospasm. Clinical Case: A 47-year-old African American woman presented with a 1-week duration of worsening dyspnea, orthopnea, dizziness, and palpitations. Past medical history includes HTN, non-ischemic cardiomyopathy on carvedilol and pravastatin. Physical exam: BP 182/119, HR 120, mild pulmonary crackles. Labs: proBNP 3533 (1-150 pg/ml), HbA1c 13.1%. Echocardiogram: moderate global hypokinesis with left ventricle ejection fraction (LVEF) of 30-35%. On imaging, CT of chest revealed an incidental finding of 4.7 cm right adrenal adenoma. MRI abdomen confirmed heterogeneously enhancing 4.3 cm right adrenal mass. Further testing showed high plasma catecholamines of 9963 (242-1125 pg/ml), 24-hour urine catecholamines of 2125 (50-100 mcg), thus confirming the diagnosis of pheochromocytoma. Further, her clinical course was complicated by left-sided weakness. MRI brain reported an acute infarct in right corona radiata. Pre-operative blockade was done with phenoxybenzamine and metoprolol. Subsequently, she underwent right adrenalectomy and tissue diagnosis confirmed pheochromocytoma. On follow up visit, her symptoms resolved. Also noted normalized catecholamine levels, HbA1c of 5.4% and LVEF of 40- 45%. Conclusion: Pheochromocytoma can rarely present with multi-organ failure. It warrants a high index of suspicion in non-ischemic cardiomyopathy. As per recent Mayo Clinic criteria, diagnosis of takotsubo cardiomyopathy mandates ruling out pheochromocytoma. As seen in our patient, it is a reversible cause of left ventricular dysfunction, focal weakness and DM. Based on our knowledge, this is the only contingently diagnosed pheochromocytoma with varied clinical presentations. It has been aptly described as “The Great Masquerader”.

Disruption of Exocytosis in Sympathoadrenal Chromaffin Cells from Mouse Models of Neurodegenerative Diseases.

Synaptic disruption and altered neurotransmitter release occurs in the brains of patients and in murine models of neurodegenerative diseases (NDDs). During the last few years, evidence has accumulated suggesting that the sympathoadrenal axis is also affected as disease progresses. Here, we review a few studies done in adrenal medullary chromaffin cells (CCs), that are considered as the amplifying arm of the sympathetic nervous system; the sudden fast exocytotic release of their catecholamines—stored in noradrenergic and adrenergic cells—plays a fundamental role in the stress fight-or-flight response. Bulk exocytosis and the fine kinetics of single-vesicle exocytotic events have been studied in mouse models carrying a mutation linked to NDDs. For instance, in R6/1 mouse models of Huntington’s disease (HD), mutated huntingtin is overexpressed in CCs; this causes decreased quantal secretion, smaller quantal size and faster kinetics of the exocytotic fusion pore, pore expansion, and closure. This was accompanied by decreased sodium current, decreased acetylcholine-evoked action potentials, and attenuated [Ca2+]c transients with faster Ca2+ clearance. In the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS), CCs exhibited secretory single-vesicle spikes with a slower release rate but higher exocytosis. Finally, in the APP/PS1 mouse model of Alzheimer’s disease (AD), the stabilization, expansion, and closure of the fusion pore was faster, but the secretion was attenuated. Additionally, α-synuclein that is associated with Parkinson’s disease (PD) decreases exocytosis and promotes fusion pore dilation in adrenal CCs. Furthermore, Huntington-associated protein 1 (HAP1) interacts with the huntingtin that, when mutated, causes Huntington’s disease (HD); HAP1 reduces full fusion exocytosis by affecting vesicle docking and controlling fusion pore stabilization. The alterations described here are consistent with the hypothesis that central alterations undergone in various NDDs are also manifested at the peripheral sympathoadrenal axis to impair the stress fight-or-flight response in patients suffering from those diseases. Such alterations may occur: (i) primarily by the expression of mutated disease proteins in CCs; (ii) secondarily to stress adaptation imposed by disease progression and the limitations of patient autonomy.

ATYPICAL TAKOTSUBO CARDIOMYOPATHY ASSOCIATED WITH PHEOCHROMOCYTOMA

Pheochromocytoma is an under-recognized, catecholamine-producing tumor arising from adrenal medullary chromaffin cells. Takotsubo cardiomyopathy is a rare complication of pheochromocytoma. Compared to primary Takotsubo cardiomyopathy, heart failure and cardiogenic shock may be more common in cases

Renin angiotensin system molecules and nitric oxide local interactions in the adrenal gland of Trypanosoma cruzi infected rats.

Chagas disease (CD) is a tropical zoonosis caused by the protozoan Trypanosoma cruzi. Severe autonomic dysfunction like reduced cardiac catecholamine-containing or acetylcholinesterase-positive innervation have been reported in CD. Renin-angiotensin system (RAS) seems to participate in the regulation of adrenal catecholamine secretion by adrenal medullary chromaffin cells, which might be dependent of nitric oxide (NO) pathways. To investigate the levels of RAS components in the adrenal gland during the acute infection with Y strain T. cruzi and in response to acute administration of an inhibitor of the enzyme NO synthase, L-NAME. Male Holtzman rats were inoculated intraperitoneally with Y strain T. cruzi and received L-NAME or tap water from one day before the infection until 13 or 17 days post-inoculation (dpi). The concentration of RAS molecules in the adrenal tissue was evaluated by ELISA immunoassay. Angiotensin converting enzyme 1 (ACE1) levels were significantly lower at 17 dpi when compared to 13 dpi. No significant differences were found compared with baseline, and no changes were detected in adrenal tissue levels of angiotensin converting enzyme 2 (ACE2), angiotensin II, or angiotensin-(1-7). Moreover, the treatment with L-NAME did not influence the levels of RAS components in adrenal tissue during the course of T. cruzi infection. We provided the first evidence that levels of RAS molecules change in the adrenal gland during acute phase of T. cruzi infection. Future studies are necessary to fully address the role of NO in RAS-associated adrenal gland function in CD.

Long-term facilitation of catecholamine secretion from adrenal chromaffin cells of neonatal rats by chronic intermittent hypoxia.

In neonates, catecholamine (CA) secretion from adrenal medullary chromaffin cells (AMC) is an important mechanism for maintaining homeostasis during hypoxia. Nearly 90% of premature infants experience chronic intermittent hypoxia (IH) because of high incidence of apnea of prematurity, which is characterized by periodic stoppage of breathing. The present study examined the effects of repetitive hypoxia, designed to mimic apnea of prematurity, on CA release from AMC of neonatal rats. Neonatal rats were exposed to either control conditions or chronic intermittent hypoxia (IH) from ages postnatal days 0-5 (P0-P5), and CA release from adrenal medullary slices was measured after challenge with repetitive hypoxia (5 episodes of 30-s hypoxia, Po2 ~35 mmHg). In response to repetitive hypoxia, chronic IH-treated AMC exhibited sustained CA release, and this phenotype was not seen in control AMC. The sustained CA release was associated with long-lasting elevation of intracellular Ca2+ concentration ([Ca2+]i), which was due to store-operated Ca2+ entry (SOCE). 2-Aminoethoxydiphenyl borate, an inhibitor of SOCE, prevented the long-lasting [Ca2+]i elevation and CA release. Repetitive hypoxia increased H2O2 abundance, and polyethylene glycol (PEG)-catalase, a scavenger of H2O2 blocked this effect. PEG-catalase also prevented repetitive hypoxia-induced SOCE activation, sustained [Ca2+]i elevation, and CA release. These results demonstrate that repetitive hypoxia induces long-term facilitation of CA release in chronic IH-treated neonatal rat AMC through sustained Ca2+ influx mediated by SOCE.NEW & NOTEWORTHY Apnea of prematurity and the resulting chronic intermittent hypoxia are major clinical problems in neonates born preterm. Catecholamine release from adrenal medullary chromaffin cells maintains homeostasis during hypoxia in neonates. Our results demonstrate that chronic intermittent hypoxia induces a hitherto uncharacterized long-term facilitation of catecholamine secretion from neonatal rat chromaffin cells in response to repetitive hypoxia, simulating hypoxic episodes encountered during apnea of prematurity. The sustained catecholamine secretion might contribute to cardiovascular morbidities in infants with apnea of prematurity.

Dopamine‐β‐monooxygenase inhibitors obtained by structure based methods exhibited anti‐hypertensive effect in L‐NAME induced hypertensive rats

IntroductionHypertension is the leading cause of mortality worldwide (Kwan G.F. et. al., Circulation, 2016). While classical anti‐hypertensives like ACE inhibitors, ARBs, calcium channel blockers, beta blockers and diuretics are widely used, they have their own limitations and no new type of drugs emerged in the last few years that use body's own blood pressure lowering mechanism. Dopamine β‐monooxygenase (DBM), expressed in noradrenergic nerve terminals as well as in adrenal medullary chromaffin cells, which convert dopamine into norepinephrine, is a novel target whose inhibition has been shown to help the treatment of hypertension with several advantages over the classical antihypertensives. However, DBM inhibitors are few in number, often result in side‐effects and are frequently non‐responsive to specific population, probably since the known inhibitors so far have been designed on ligand based methods. We hypothesize that rational and three dimensional structure based identification of novel inhibitors may overcome such problems.ObjectiveTo identify novel inhibitors of DBM and evaluate their anti‐hypertensive properties in L‐NAME induced hypertensive rat model.Design and methodAn experimentally validated computational model for human DBM, built in our lab, was used for structure‐based, rational drug design. The three‐dimensional model was used for virtual screening against various small molecule databases. Identified top hits were then tested in vitro against DBM with known inhibitors nepicastat and disulfiram as controls. Binding of the inhibitors to DBM were validated using fluorescence and CD spectroscopy as well as ITC. Pharmacokinetic analysis was performed computationally. Cyto‐ and hemo‐toxicities of the lead compounds were assessed ex vivo. Finally. their antihypertensive efficacies were evaluated in L‐NAME induced hypertensive rat model.ResultsVirtual screening of various compound libraries revealed 69 hits which were then assessed in vitro using a repertoire of biochemical and biophysical methods. UDSC‐171, 180 and 142 were discovered to be potent inhibitors of DBM with IC50s of 1μM, 5.5μM and 18μM, respectively. The inhibitors displayed KD values against DBM in the range of 100nm to 1μM. In silico pharmacokinetic analysis indicated that the molecules are unable to cross the BBB. High doses (up to 50μM) of the lead compounds showed acceptable cellular tolerance against HEK293 cell line and insignificant hemo‐toxicities against RBCs. These three leads were successful in preventing elevated systolic blood pressure in L‐NAME induced hypertensive rat models.ConclusionsPresent study successfully developed rapid, systematic and non‐expensive biophysical, biochemical and computational tools for the identification, characterization and validation of DBM inhibitors. Moreover, UDSC‐171, 180 and 142 have been discovered and validated as novel antihypertensives against L‐NAME induced hypertensive rats. (Indian Patent filed for the present study with app. no. 201711036983)Support or Funding InformationDepartment of Biotechnology, Govt. of India

Phosphopeptidomics Reveals Differential Phosphorylation States and Novel SxE Phosphosite Motifs of Neuropeptides in Dense Core Secretory Vesicles.

Neuropeptides are vital for cell-cell communication and function in the regulation of the nervous and endocrine systems. They are generated by post-translational modification (PTM) steps resulting in small active peptides generated from prohormone precursors. Phosphorylation is a significant PTM for the bioactivity of neuropeptides. From the known diversity of distinct neuropeptide functions, it is hypothesized that the extent of phosphorylation varies among different neuropeptides. To assess this hypothesis, neuropeptide-containing dense core secretory vesicles from bovine adrenal medullary chromaffin cells were subjected to global phosphopeptidomics analyses by liquid chromatography (LC)-mass spectrometry (MS/MS). Phosphopeptides were identified directly by LC-MS/MS and indirectly by phosphatase treatment followed by LC-MS/MS. The data identified numerous phosphorylated peptides derived from neuropeptide precursors such as chromogranins, secretogranins, proenkephalin and pro-NPY. Phosphosite occupancies were observed at high and low levels among identified peptides and many of the high occupancy phosphopeptides represent prohormone-derived peptides with currently unknown bioactivities. Peptide sequence analyses demonstrated SxE as the most prevalent phosphorylation site motif, corresponding to phosphorylation sites of the Fam20C protein kinase known to be present in the secretory pathway. The range of high to low phosphosite occupancies for neuropeptides demonstrates cellular regulation of neuropeptide phosphorylation. Graphical Abstract ᅟ.

Muscarinic receptors in adrenal chromaffin cells: physiological role and regulation of ion channels

Adrenal medullary chromaffin cells in mammals are innervated by sympathetic preganglionic nerve fibers, as are sympathetic ganglion neurons. Acetylcholine in the ganglion neurons is well established as mediating fast and slow excitatory postsynaptic potentials through nicotinic and muscarinic acetylcholine receptors (AChRs), respectively. The role of muscarinic AChRs during neuronal transmission in chromaffin cells varies among different mammals. Furthermore, the ion channel mechanisms associated with the muscarinic AChR-mediated increase in excitability of chromaffin cells are complicated and different from the excitation of ganglion neurons, which has been ascribed to the inhibition of M-type K+ channels. In this review, we focus on muscarinic receptor-mediated excitation in rodent and guinea pig chromaffin cells, in particular, on the role of muscarinic receptors in neuronal transmission, the muscarinic receptor subtypes involved in excitation and secretion, and the muscarinic regulation of ion channels including TWIK-related acid-sensitive K+ channels. Finally, we discuss prospectively the future of muscarinic receptor research in adrenal chromaffin cells.