Adrenal Medulla Cells Research Articles

CMOS-Based Amperometric Sensor Utilizing Redox-Cycling Technique for Selective Detection of Dopamine from PC12 Spheroids

Monitoring neurotransmitters, such as dopamine, released from neural cells in the brain is crucial to reveal the mechanism of intercellular communication. Since a released amount of dopamine is closely related to Alzheimer’s disease and Parkinson’s disease, quantitative estimation method of the released amount of dopamine from the cells is required. Among various methods, the electrochemical technique is a major method to monitor dopamine. The conventional electrochemical monitoring of dopamine is basically relay on a single microelectrode setting near a cell. However, this method can only obtain very local information. To detect wider area, the scanning electrochemical monitoring of dopamine is used but it cannot simultaneously obtain information from multiple points. To make an image of dopamine released from relatively big tissue-like samples, we previously developed an CMOS-based amperometric electrode array called Bio-LSI device [1]. The Bio-LSI has 400 micro-disk electrodes with 250 µm pitch. In the report, we successfully obtained the real-time images of dopamine released from the aggregates (ca. 500 µm in diameter) of pheochromocytomas of the rat adrenal medulla (PC12) cells, which is often used as a model of neural cells [2]. To detect the dopamine from the actual brain tissue, it is known that the ascorbic acid interfere the detection of dopamine, because the ascorbic acid is secreted from brain tissues much higher concentration than dopamine. In this study, we developed a novel electrochemical device incorporating the Bio-LSI and an IDA for selective imaging of dopamine release from tissues in the presence of ascorbic acid. We used Bio-LSI as a substrate of the device. We made 20×18 interdigitated electrode arrays (IDAs) by patterning the electrodes on the LSI surface. According to the report described by Niwa et al. [3] the IDAs amplify the amperometric signal of dopamine but does not amplify the signal of ascorbic acid, because dopamine is a reversible redox species which induces the redox cycling at IDA, but ascorbic acid is an irreversible redox species which does not induce the redox cycling at IDA (Fig. 1A). The device was fabricated by photolithography technique (Fig. 1B). The electrodes were made by Pt spattering. The width and the interval of the IDA were 5 µm. The 20×18 IDAs were arrayed with 250 µm pitch. The oxidation electrodes of IDAs on a same low were connected each other with a lead line connected to the oxidation voltage application pad (Fig. 1C). The reduction electrodes of IDAs were independently connected to the pad nearest to the each detection point. PC12 spheroids with 450 ~ 500 µm in diameter were obtained by the hanging drop method. A PC12 spheroid was simulated by accommodating on a device filled with a high K+solution (Fig. 1B). By applying an oxidation (+0.60 V vs. Ag/AgCl) and reduction (0.00 V vs. Ag/AgCl) potential to the each electrode, we successfully obtained the dopamine released image from the PC12 spheroid (Fig. 1D). This result shows that we can detect dopamine using our device incorporating the Bio-LSI and an IDA. We are now trying to the imaging of dopamine in the presence of ascorbic acid. The detail of the results with the advantage of the use of the IDA electrodes will be discussed in the presentation. Our device will be a promising tool for imaging a dopamine released from a brain tissue to reveal the mechanism of intercellular communication.

Pheochromocytoma with Ventricular Tachycardia as the Presenting Symptom

To the Editor: Although the triad of palpitations, headaches, and sweating is generally recognized as the classic presentation for pheochromocytoma, we reported the case of a patient with pheochromocytoma who first presented with ventricular tachycardia (VT). This case shows the importance of a broad differential diagnosis when the clinical presentation is unusual. A 17-year-old woman presented with episodic palpitation and headaches during physical activities for 4 years. The symptoms resolved after 3–5 min of rest. Two days before admission, she was admitted to the emergency department with persistent palpitations and headaches, and the blood pressure (BP) was 180/90 mmHg. The admission echocardiography (ECG) revealed a sinus rate of 92 beats/min. There was no family or personal history of syncope, cardiac arrest, and sudden death. The physical examination was unremarkable. The admission and peak creatine kinase-MB was 153 U/L and 157 U/L, respectively. During follow-up, the patient presented with recurrent palpitation and headaches when sitting or standing. During these episodes the BP was 160–200/90–100 mmHg and the pulse rate was elevated at 148 beats/min. Cardiac monitoring showed VT with a heart rate (HR) of 150 beats/min [Figure 1a]. Three episodes of VT were recorded during persistent hypertension. The symptoms were relieved after maintaining a recumbent position for 1–2 min. The 24 h urinary catecholamines and urinary vanillylmandelic acid levels were elevated. Abdominal ultrasound revealed a right adrenal mass with a diameter of 6 cm which was confirmed on abdominal computed tomography [Figure 1b]. A pheochromocytoma was diagnosed. Figure 1 (a) Echocardiography showing ventricular tachycardia. (b) Contrast-enhanced computed tomography scan of the upper abdomen revealing a tumor of the right adrenal gland. The patient was hemodynamically stabilized in the supine position with the administration of phentolamine (a nonspecific α-adrenergic blocker) and metoprolol (a selective β-antagonist). A right adrenalectomy was performed, yielding a 6.0 cm nodule confirmed to be a pheochromocytoma by histology. The hypertension was controlled and no arrhythmias were documented during 12 months of follow-up. Pheochromocytoma is a catecholamine-producing tumor that originates from the adrenal medulla or extra-adrenal chromaffin cells. The main clinical manifestation of pheochromocytoma is paroxysmal or persistent hypertension, accompanied by arrhythmias, such as sinus tachycardia, paroxysmal supra VT, atrial fibrillation, or ventricular premature contractions. We have reported a rare case of pheochromocytoma with VT as the presenting symptom. Frequent episodes of VT are almost always preceded by vital sign fluctuation (elevated HR and BP). Paulin et al.[1] reported that the sudden release of catecholamine from the pheochromocytoma is associated with the mechanism underlying VT. Excessively elevated catecholamine levels could lead to abnormal myocardial electrical activity, such as over opening of ion channels, enhanced ion exchange pump function, thus causing extremely large amounts of sodium, potassium, and calcium ion flow through the membranes, resulting in intensive myocardial autorhythmicity, conductivity, and triggered activities. Furthermore, increased catecholamine levels can reduce the cardiac ventricular fibrillation threshold, predispose to ventricular fibrillation, lead to sudden death,[2] and contribute to abnormal repolarization, thus causing all types of rapid arrhythmia. Ceremuzynski et al.[3] have shown that plasma catecholamine concentrations are increased in dogs during acute myocardial ischemia and a relationship between the blood catecholamine concentration and the severity of ventricular arrhythmias. Cardiac abnormalities caused by pheochromocytoma includes myocarditis, acute myocardial infarction, transient left ventricular systolic dysfunction, and cardiomyopathy. Bravo et al.[4] reported that catecholamines cause myocardial necrosis, focal myofibrillar degeneration, and subsequent fibrous scar formation. Left ventricular systolic dysfunction may resemble stress-induced cardiomyopathy, also known as takotsubo cardiomyopathy and is reversible in patients with pheochromocytoma.[5] Moreover, long-term high catecholamine levels can lead to myocardial hypertrophy and heart failure. The presence of sustained VT can be treated with radio-frequency ablation but such treatment would not abolish the systemic arrhythmogenic mechanism and would not resolve the symptoms or prevented the potential onset of other tachyarrhythmias. This case illustrates the importance of a complete differential diagnosis when the clinical presentation is unusual. In a hypertensive patient with a typical presentation consisting of palpitations and headache, and an ECG demonstrates VT or other tachyarrhythmias, a pheochromocytoma should be included in the differential diagnosis. In addition, biochemical testing and imaging studies will guide the diagnosis and allow complete resolution of symptoms following resection of the pheochromocytoma. The preoperative use of phentolamine, a nonspecific α-adrenergic blocker has a theoretical pharmacologic basis. The β-blocker propranolol is used when tachycardia or catecholamine-induced arrhythmias are present and should only be started after α-blocker has been established to avoid life-threatening hypertension. In conclusion, this case shows that VT is an unusual and initial cardiovascular presenting symptom in patients with a pheochromocytoma. Pheochromocytoma must be considered in patients with frequent VT plus sudden changes in BP. Resection of chromaffin cell tumor will completely relieve the symptoms. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

COMPARATIVE MORPHOFUNCTIONAL CHARACTERISTIC OF ADRENAL GLANDS IN ARTERIAL HYPERTENSION

The article is devoted to the study of morphological changes of the adrenal glands in arterial hyperten-sion. Adrenals investigated persons who do not suffer in life and suffering from hypertension during the life of hypertension and died from various causes – is incompatible with the life of a mechanical trauma, cerebral hemorrhage, and acute left ventricular failure. In each case, each of the adrenal glands were assessed: the presence and severity of focal and diffuse mononuclear infiltration, the number of lympho-cytes, monocytes, plasma cells and fibroblasts arranged in the respective zones of the cortex and medulla, the severity of hyperemia and edema of various structural parts, the number of zones cortex and medulla endocrinocytes with pycnotic nuclei in the reticular zone evaluated the distribution of secretory cells with lipofuscin and the degree of saturation of the cytoplasm of the secretory cells, in secretory cells of medul-la evaluated nuclear-cytoplasmic ratio, the severity of vacuolization and basophils cytoplasm revealed a number of regularities of morphological changes depending from the various clinical manifestations of hypertension, defining the ultimate option of dying. At the heart tanatogenesis version are more pro-nounced morphological features hyperfunctions glomerular zone – namely delipidization left and right glands. The beam and netted areas contralateral glands embodiment of dying of a heart compared to the brain, is more pronounced hyperemia. When cardiac variant tanatogenesis more pronounced focal lym-phoid infiltration and vacuolization in secretory cells of left adrenal medulla.

The role of miRNAs in the pheochromocytomas

Pheochromocytoma (PCC) is a catecholamine-producing and neuroendocrine tumor, which originates from chromaffin cells in adrenal medulla or at extra-adrenal sites. The clinical symptoms of PCC patients were including paroxysmal hypertension and cardiovascular crises. Despite the identification of many molecular-level basics involved in these pathogenesis processes, the 5-year survival of advanced stage PCC is <40%. The 5-year survival of advanced stage PCC is <40%. microRNAs (miRNAs) are a class of single-strand, highly conserved, small, non-coding RNAs which inhibit gene expression by binding to the target messenger RNA (mRNA) 3'UTR to induce translational repression or their degradation. Increasing data have shown that the deregulated expression of miRNAs has been implicated in the diagnosis and treatment of tumors including PCC. In this review, we summarized the expressions and roles of miRNAs in PCC and comment on the potential role of miRNAs in improving diagnosis, prognosis, and treatment of PCC.

Abstract P067: Novel Antagonists of Dopamine-β-hydroxylase Identified and Validated Through Structure Based Approach to Combat Hypertension

Human dopamine β-hydroxylase (hDBH), expressed in noradrenergic nerve terminals of nervous system and in chromaffin cells of adrenal medulla, is a key constituent of catecholamine biosynthetic pathway. DBH inhibition has been shown to help the treatment of hypertension, cardiac hypertrophy and cardiac heart failure, which are major causes of mortality and morbidity worldwide. Existing hDBH inhibitors are too few, often result in side effects and are frequently non-responsive to specific population. Since no three-dimensional structure existed for full-length hDBH, structure based rational drug design was elusive till date, an issue to which we provided solution lately by building an experimentally validated in silico model for hDBH. The model was used in Autodock, Glide SP and XP software for structure based virtual screening against small molecule databases from NCI, USA. The docked structures were scored using Autodock, X-Score and Prime MMGBSA. Thus, 69 compounds were identified as prospective inhibitors of DBH, which were then tested in vitro against human serum DBH and its nearly identical homologue, bovine DBH (586 of 617 amino acids homologous), with known inhibitors nepicastat and disulfiram as positive controls. Three lead molecules UDSC171, UDSC180 and UDSC142 were discovered in the process as potent inhibitors of DBH with IC50s of 1μM, 5.5μM and 18μM, respectively. The binding of the inhibitors to the enzyme were validated using fluorescence and CD spectroscopy as well as ITC, revealing KD values in the range of 100nm to 1μM. In silico pharmacokinetic analysis indicated the molecules to be latest generation of DBH inhibitors having very high cell permeability and inability to cross the blood brain barrier. High doses (up to 50μM) of the lead compounds showed acceptable cellular tolerance against HEK 293 cell line and insignificant hemo-toxicities against human RBCs. Hence, in vivo evaluation of the lead molecules were done in small model systems like C. elegans and D. melanogaster reconfirming their nontoxic properties up to 15μM doses. These three leads are now being tested in cardiac hypertrophy and hypertension rat models with exciting preliminary results.

Differences in CART expression and cell cycle behavior discriminate sympathetic neuroblast from chromaffin cell lineages in mouse sympathoadrenal cells.

Adrenal medullary chromaffin cells and peripheral sympathetic neurons originate from a common sympathoadrenal (SA) progenitor cell. The timing and phenotypic changes that mark this lineage diversification are not fully understood. The present study investigated the expression patterns of phenotypic markers, and cell cycle dynamics, in the adrenal medulla and the neighboring suprarenal ganglion of embryonic mice. The noradrenergic marker, tyrosine hydroxylase (TH), was detected in both presumptive adrenal medulla and sympathetic ganglion cells, but with significantly stronger immunostaining in the former. There was intense cocaine and amphetamine-regulated transcript (CART) peptide immunostaining in most neuroblasts, whereas very few adrenal chromaffin cells showed detectable CART immunostaining. This phenotypic segregation appeared as early as E12.5, before anatomical segregation of the two cell types. Cell cycle dynamics were also examined. Initially, 88% of Sox10 positive (+) neural crest progenitors were proliferating at E10.5. Many SA progenitor cells withdrew from the cell cycle at E11.5 as they started to express TH. Whereas 70% of neuroblasts (TH+/CART+ cells) were back in the cell cycle at E12.5, only around 20% of chromaffin (CART negative) cells were in the cell cycle at E12.5 and subsequent days. Thus, chromaffin cell and neuroblast lineages showed differences in proliferative behavior from their earliest appearance. We conclude that the intensity of TH immunostaining and the expression of CART permit early discrimination of chromaffin cells and sympathetic neuroblasts, and that developing chromaffin cells exhibit significantly lower proliferative activity relative to sympathetic neuroblasts.

Multipotent glia-like stem cells mediate stress adaptation.

The neural crest-derived adrenal medulla is closely related to the sympathetic nervous system; however, unlike neural tissue, it is characterized by high plasticity which suggests the involvement of stem cells. Here, we show that a defined pool of glia-like nestin-expressing progenitor cells in the adult adrenal medulla contributes to this plasticity. These glia-like cells have features of adrenomedullary sustentacular cells, are multipotent, and are able to differentiate into chromaffin cells and neurons. The adrenal is central to the body's response to stress making its proper adaptation critical to maintaining homeostasis. Our results from stress experiments in vivo show the activation and differentiation of these progenitors into new chromaffin cells. In summary, we demonstrate the involvement of a new glia-like multipotent stem cell population in adrenal tissue adaptation. Our data also suggest the contribution of stem and progenitor cells in the adaptation of neuroendocrine tissue function in general.

A multipotent stem cell population in the adult adrenal medulla mediates stress response

The neural crest derived adrenal medulla is closely related to the sympathetic nervous system; however, unlike neural tissue, it is characterized by high plasticity which suggests the involvement of stem cells. In this study, by using two transgenic mouse models, Nestin-GFP and the inducible Nestin-CreER:Rosa26YFP, we define a pool of multipotent glia-like nestin-GFP expressing progenitor cells in the adult adrenal medulla, which contribute to its plasticity. The adrenal plays a central role in response to stress. However, the role of adrenomedullary progenitor cells in stress adaptation has not been studied so far. Our results from stress experiments in vivo show the activation and differentiation of these progenitors into new chromaffin cells. In summary, we demonstrate the involvement of a new glia-like multipotent stem cell population in adrenal tissue adaptation. Our data also suggest the contribution of stem and progenitor cells in the adaptation of neuroendocrine tissue function in general.

Metachronous metastatic paraganglioma in jejunum as a rare entity: A case report.

Pheochromocytomas and paragangliomas are neuroendocrine tumors that arise from chromaffin cells of adrenal medulla and extra-adrenal paraganglia, respectively. The recurrence of these neuroendocrine tumors as a jejunal mass causing obstruction in the small intestine is an exceptional entity. The present study reports the case of a 70-year-old male who presented to the Emergency Department of Bezmialem Vakif University Hospital with abdominal pain and vomiting. The patient possessed a history of left nephrectomy due to malignant pheochromocytoma that had invaded into the left kidney eight months prior to presentation. Bowel obstruction was diagnosed and the patient underwent a laparoscopic procedure. Partial resection of the jejunum was performed and immunohistochemical studies revealed the lesion to be malignant paraganglioma. The majority of paragangliomas are chemo- and radioresistant. Surgical excision remains the primary treatment. Metachronous paraganglioma arising from the small intestine is an extremely rare entity and may be a relevant consideration in patients presenting with bowel obstruction.

Angiotensin II acting on brain AT1 receptors induces adrenaline secretion and pressor responses in the rat.

Angiotensin II (AngII) plays important roles in the regulation of cardiovascular function. Both peripheral and central actions of AngII are involved in this regulation, but mechanisms of the latter actions as a neurotransmitter/neuromodulator within the brain are still unclear. Here we show that (1) intracerebroventricularly (i.c.v.) administered AngII in urethane-anesthetized male rats elevates plasma adrenaline derived from the adrenal medulla but not noradrenaline with valsartan- (AT1 receptor blocker) sensitive brain mechanisms, (2) peripheral AT1 receptors are not involved in the AngII-induced elevation of plasma adrenaline, although AngII induces both noradrenaline and adrenaline secretion from bovine adrenal medulla cells, and (3) i.c.v. administered AngII elevates blood pressure but not heart rate with the valsartan-sensitive mechanisms. From these results, i.c.v. administered AngII acts on brain AT1 receptors, thereby inducing the secretion of adrenaline and pressor responses. We propose that the central angiotensinergic system can activate central adrenomedullary outflow and modulate blood pressure.

Abstract 267: Identification And Validation Of New Inhibitors Based On Rational Design Against Dopamine-β-hydroxylase To Combat Hypertension

Human dopamine β-hydroxylase (hDBH), expressed in noradrenergic nerve terminals of nervous system and in chromaffin cells of adrenal medulla, is a key constituent of catecholamine biosynthetic pathway. DBH inhibition has been shown to help the treatment of hypertension and cardiac heart failure, which are major causes of mortality and morbidity worldwide. Existing hDBH inhibitors are too few, often result in side effects and are frequently non-responsive to specific population. Since no three-dimensional structure existed for full-length hDBH, structure based rational drug design has been elusive till date, an issue to which we provided solution lately by building an experimentally validated in silico model for hDBH. The model was used in Autodock, Glide SP and XP software for structure based virtual screening against small molecule databases from NCI, USA. The docked structures were scored using Autodock, X-Score and Prime MMGBSA. Thus, 69 compounds were identified as prospective inhibitors of DBH, which were then tested in vitro against human serum DBH and its nearly identical homologue, bovine DBH (586 of 617 amino acids homologous), with known inhibitors nepicastat and disulfiram as positive controls. Three lead molecules (NSC637578, NSC99657 and NSC379555) were discovered in the process as potent inhibitors of DBH with IC50s of 1μM, 5.5μM and 18μM, respectively. The binding of the inhibitors to the enzyme were validated using fluorescence and CD spectroscopy as well as ITC, revealing KD values in the range of 100nm to 1μM. In silico pharmacokinetic analysis indicated the molecules to be latest generation of DBH inhibitors having very high cell permeability and inability to cross the blood brain barrier. High doses (up to 50μM) of the lead compounds showed acceptable cellular tolerance against HEK 293 cell line and insignificant hemo-toxicities against human RBCs. Hence, in vivo evaluation of the lead molecules has been initiated in model systems like C. elegans and D. melanogaster. These studies reconfirmed their nontoxic properties up to 15μM doses. CombiGlide and Ligand Based Core Hopping methods are being employed to optimize the lead compounds computationally to improve their pharmacokinetic properties.

Effects of a novel pesticide-particle conjugate on viability and reactive oxygen species generation in neuronal (PC12) cells

Development of new methods and compounds to eradicate insect vectors are desperately needed. To that end, our team has previously described the synthesis and characterization of a conjugate comprised of a silver nanoparticle core encapsulated by the pyrethroid pesticide, deltamethrin (pesticide encapsulated silver nanoparticle termed “PENS”). For this current work, the PENS conjugate was tested in neuronal cultured cells to compare the cytotoxic responses to the unconjugated pesticide deltamethrin – a known neurotoxic agent and pristine silver nanoparticles. The PC12 (pheochromocytoma of the rat adrenal medulla) cell line was chosen as a model neuronal culture system. Cells were exposed to known concentrations of PENS, deltamethrin or silver nanoparticle suspensions to assess the degree of toxicity in vitro. After 24 hours of incubation, cell viability and intracellular reactive oxygen species (ROS) were measured. Bright field images of high dose exposures to dosing solutions were also acquired to evaluate cell morphology. Exposure to PENS resulted in a 17% decline in viability at the highest concentration of 45 µM while exposure to deltamethrin caused a 47% decrease. These results suggest that cellular viability was less adversely affected by PENS than by the deltamethrin. Also, ROS production following PENS exposure indicated that the newly developed conjugate was responding in a similar manner as that of cells treated with deltamethrin only.

Bradykinin and histamine-induced cytosolic calcium increase in capillary endothelial cells of bovine adrenal medulla.

We have assessed the effect of bradykinin and histamine on the cytosolic free calcium concentration ([Ca(2+)]i ) of bovine adrenal medulla capillary endothelial cells (BAMCECs). To measure [Ca(2+)]i changes in BAMCECs the intracellular fluorescent probe, fluo-3 AM, was used. Bradykinin (3 µM) produced a transient monophasic increase in [Ca(2+)]i , which was depressed by B1650 (0.1 µM), a B2-bradykinin receptor antagonist (D-Arg-[Hyp(3), Thi(5,8) , D-Phe(7)]-Bradykinin). Similarly, increase in [Ca(2+)]i induced by histamine was also depressed by tripolidine (0.1 µM), an H1-histamine receptor antagonist. [Ca(2+)]i increase induced by both agonists was unaffected in the absence of extracellular Ca(2+) or presence of antagonists of voltage operated Ca(2+) channels (VOCCs). Thapsigargin (1 µM) did not abolish the increase of [Ca(2+)]i produced by bradykinin, but abolished that of histamine. In contrast, caffeine (100 µM), abolished the [Ca(2+)]i response induced by bradykinin (3 µM), but did not affect the [Ca(2+)]i increase induced by histamine (100 µM). The results indicate the presence of B2 bradykinin- and H1 histamine-receptors in BAMCECs. Liberation of Ca(2+) induced by both agonists occurs through 2 different intracellular mechanisms. While bradykinin activates a sarco(endo) plasmic reticulum (SER) containing a SER Ca(2+) -ATPase (SERCA) thapsigargin-insensitive, histamine activates a SER containing a SERCA thapsigargin-sensitive. We suggest that the increase in [Ca(2+)]i induced by bradykinin and histamine could be of physiological relevance, modulating adrenal gland microcirculation.

Identification and characterization of chromaffin progenitor cells in murine adrenal medulla

Background: The adrenal gland exhibits a high plasticity and chromaffin cell number can be regulated, in contrast to the closely related neurons, to meet physiological needs. It therefore has been suggested that a subpopulation of proliferation and differentiation competent progenitor cells continues to exist in the adult adrenal medulla. In our previous studies, we established protocols to in vitro enrich these cells from bovine and human adrenals in sphere cultures. These cells are characterized by the expression of neural crest and neural progenitor markers and are able to differentiate to the chromaffin and neural lineage.

Progenitor Cells in Chromospheres: In Response to Arthur S. Tischler

This is in response to the letter “What Happens in ‘Chromospheres’?” by Arthur S. Tischler, commenting on our article titled “Isolation, Characterization, and Differentiation of Progenitor Cells From Human Adult Adrenal Medulla” [1]. We would like to acknowledge the comments, the interesting discussion, and potential other point of view on our results. We agree with Dr. Tischler about the fact that human chromaffin cells have the capacity to acquire a neuron-like phenotype in monolayer cultures without a “chromosphere” stage. We also observed this phenomenon in a previous study using human adrenal chromaffin cells in monolayer cultures [2]. Since the cell culture conditions of human chromaffin cells in monolayer cultures and suspension chromosphere cultures are different, careful investigation should be made before it is assumed that the same changes are occurring. Therefore, as suggested by Dr. Tischler, it will be important to compare the two cell culture types in terms of cell proliferation capacity, expression of progenitor cell markers, duration of cell culture, regulatory peptide expression, and catecholamine content. This new set of experiments will provide information on whether the mechanisms required for chromaffin cell transdifferentiation will involve a dedifferentiation or an unnatural intermediate step [3]. Even if we do not know the mechanism, and even if we did not directly evaluate the proliferation capacity, human chromospheres, like bovine chromospheres [4], express nine markers of progenitor cells (nestin, CD133, Notch1, nerve growth factor receptor, Snai2, Sox 9, Sox 10, Phox2b, and Ascl1), and more importantly, nestin-positive cells are present in human adult adrenal medulla, suggesting the presence of progenitor cells in human adult adrenal medulla. The possibility that a “priming” process, with an upregulation of genes required for transdifferentiation, also occurs during chromosphere culture cannot be discarded. Nevertheless, we think that this could also represent an advantage of the use of chromospheres as a source of cells for clinical purposes. The survival rate of chromosphere cells after grafting is currently being investigated in animal experiments. From data available on the transplantation of neural progenitor cells, we expect that the number of spheres isolated from one adrenal gland (27,000–280,000) should give rise to enough cells for grafting. Moreover, one of the major problems of using embryonic stem cells and induced pluripotent stem cells still is their tumorigenicity. Therefore, if future data demonstrate that human chromospheres have a low cell proliferation capacity, this should emphasize the interest in their use for regenerative medicine.

Gamma‐aminobutyric acid B Receptor Immunoreactivity in the Mouse Adrenal Medulla

The present study examined gamma-aminobutyric acid B (GABAB ) receptor, GABA, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) immunoreactivities in the mouse adrenal medulla. GABAB receptor immunoreactivity was seen in numerous chromaffin cells and in a few ganglion cells of the adrenal medulla. By using a formaldehyde-induced fluorescence (FIF) method, GABAB receptor immunoreactivity was observed in numerous adrenaline (A) cells, but not in noradrenaline (NA) cells showing blue-white fluorescence. This suggests that GABAB receptors may be present in the A cells and be related to the secretory activity of A cells but not NA cells in the mouse adrenal medulla. GABAB receptor immunoreactive ganglion cells were shown to be nNOS immunopositive by using a double immunostaining method. Weak GABA immunoreactivity was visible in some chromaffin cells and in the numerous nerve fibers of the medulla. By using the FIF method, weak GABA-immunoreactive chromaffin cells were shown to be in the NA cells showing blue-white fluorescence. GABA-immunoreactive nerve fibers were in dense contact in A cells, but not NA cells. GABA-immunoreactive nerve fibers closely contacted a few ganglion cells. Numerous GABA-immunoreactive nerve fibers in the medulla showed ChAT immunoreactive. This result suggests that GABA and acetylcholine may be released from the same nerve fibers and may have a secretory effect on the A cells of the medulla.

Antagonism of Cortex Periplocae extract-induced catecholamines secretion by Panax notoginseng saponins in cultured bovine adrenal medullary cells by drug combinations

Ethnopharmacological relevanceTraditional Chinese Medicine (TCM) operates on the general principle that compatible components of different herbal decoction may work together to synergistically enhance therapeutic efficacy or reduce adverse effects. Cortex Periplocae is an herb that has been used in TCM clinics for a long time in the treatment of chronic heart failure. However, recently, the use of this herb has been restricted because of widespread abuse and misapplications. Radix Notoginseng is another herb that is used in TCM because of its protective role on cardiomyocytes. From our previous studies on these two herbs in a mouse model, we observed an increased LD50 after oral administration of Cortex Periplocae extract (CPE) and Panax notoginseng saponins (PNS) in a ratio of 1:1 compared with Cortex Periplocae extract used alone. Aim of the studyThis study aimed to investigate whether there are mutual synergistic effects of the two herbal extracts, CPE and PNS, on catecholamines (CAs) secretion, and their possible underlying mechanism(s) for such effects. Materials and methodsCPE and PNS were quantified by the LC-MS/MS method. HPLC-ECD was used to determine the CAs secreted into the medium by bovine adrenal medulla cells (BAMCs) and calcium influx was measured using a Calcium 4 reagent kit. ResultsWe found that the stimulatory effect of CPE on CAs secretion was inhibited when used together with PNS. For a better clarification of the different constituents of the extracts, a quantitative analysis was carried out. Periplocin was found to be the main active component of CPE valued as 0.99% and saponins were the principal constituents of PNS. These results also showed that CPE increased the secretion of CAs in a dose-dependent manner while the actions of PNS were seen to be inhibitory. Periplocin monomer of CPE could be implicated for the actions of CPE since it plays the role of increasing the ACh-induced CAs secretion in a calcium-dependent manner. We therefore conclude that; CPE and PNS exert antagonistic effects in regulating the concentration of intracellular calcium. ConclusionsPNS inhibits CPE-induced CAs secretion by suppressing calcium influx in bovine adrenal medulla cells while periplocin, one of the main components of CPE has the same secretagogue effect as CPE.

A Case of Pheochromocytoma Associated with Diabetic Ketoacidosis and Infective Endocarditis

Pheochromocytoma is a rare neuroendocrine tumor that is usually derived from adrenal medulla or chromaffin cells along with sympathetic ganglia. In Western countries, the prevalence of pheochromocytoma is estimated to be between 1:6,500 and 1:2,500, compared with an incidence in the United States of 500 to 1,100 cases per year. Despite this low incidence, pheochromocytoma should always be considered for differential diagnoses because previous studies have shown that this condition can be cured in approximately 90% of cases. However, an untreated tumor is likely to be fatal due to catecholamine-induced malignant hypertension, heart failure, myocardial infarction, stroke, ventricular arrhythmias or metastatic disease. Symptoms that result primarily from excess circulating catecholamines and hypertension include severe headaches, generalized inappropriate sweating and palpitations (with tachycardia or occasionally bradycardia). Pheochromocytoma, however, has highly variable and heterogeneous clinical manifestations, including fever, general weakness and dyspepsia, and can be observed in patients who are suffering from infectious diseases. Several of such case reports have been presented, but most of these included infectious patients with high blood pressure and severe fluctuations. In this study, we presented the case of a 53-year-old male who showed normal blood pressure, but had a sustained fever. He was diagnosed with diabetic ketoacidosis, infective endocarditis and asymptomatic adrenal incidentaloma. Despite treatment with antibiotics and valve replacement, the fever persisted. After the patient underwent evaluation for the fever, adrenal incidentaloma was identified as pheochromocytoma. After removal of the abdominal mass, his fever improved. (J Korean Diabetes 2013;14:156-161)

Isolation, Characterization, and Differentiation of Progenitor Cells from Human Adult Adrenal Medulla

Chromaffin cells, sympathetic neurons of the dorsal ganglia, and the intermediate small intensely fluorescent cells derive from a common neural crest progenitor cell. Contrary to the closely related sympathetic nervous system, within the adult adrenal medulla a subpopulation of undifferentiated progenitor cells persists, and recently, we established a method to isolate and differentiate these progenitor cells from adult bovine adrenals. However, no studies have elucidated the existence of adrenal progenitor cells within the human adrenal medulla. Here we describe the isolation, characterization, and differentiation of chromaffin progenitor cells obtained from adult human adrenals. Human chromaffin progenitor cells were cultured in low-attachment conditions for 10-12 days as free-floating spheres in the presence of fibroblast growth factor-2 (FGF-2) and epidermal growth factor. These primary human chromosphere cultures were characterized by the expression of several progenitor markers, including nestin, CD133, Notch1, nerve growth factor receptor, Snai2, Sox9, Sox10, Phox2b, and Ascl1 on the molecular level and of Sox9 on the immunohistochemical level. In opposition, phenylethanolamine N-methyltransferase (PNMT), a marker for differentiated chromaffin cells, significantly decreased after 12 days in culture. Moreover, when plated on poly-l-lysine/laminin-coated slides in the presence of FGF-2, human chromaffin progenitor cells were able to differentiate into two distinct neuron-like cell types, tyrosine hydroxylase (TH)(+)/β-3-tubulin(+) cells and TH(-)/β-3-tubulin(+) cells, and into chromaffin cells (TH(+)/PNMT(+)). This study demonstrates the presence of progenitor cells in the human adrenal medulla and reveals their potential use in regenerative medicine, especially in the treatment of neuroendocrine and neurodegenerative diseases.

Methyl Isocyanate and Carcinogenesis: Bridgeable Gaps in Scientific Knowledge

Methyl isocyanate may have a role in cancer etiology, although the link is unclear. There is evidence in the literature that it can induce cancer in animals but the carcinogenic potency is weak. Pheochromocytoma of adrenal medulla and acinar cell tumors of pancreas have been observed in methyl isocyanate exposed animals. Conversely, emerging data from population-based epidemiological studies are contradictory since there is no evidence of such cancers in methyl isocyanate exposed humans. Recently, we reported a high prevalence of breast and lung cancers in such a population in Bhopal. In vitro findings appearing in the latest scientific literature suggest that genomic instability is caused by methyl isocyanate analogs in lung, colon, kidney, ovary epithelial cells, and that hepatocytes may undergo oncogenic transformation, have obvious implications. The conflicting information prompted us to present this update over the last three decades on methyl isocyanate-induced cancers after an extensive literature search using PubMed. While the pertinent literature remains limited, with a scarcity of strong laboratory analyses and field-epidemiological investigations, our succinct review of animal and human epidemiological data including in vitro evidences, should hopefully provide more insight to researchers, toxicologists, and public health professionals concerned with validation of the carcinogenicity of methyl isocyanate in humans.