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chromaffin tissue [keywords]
- Lumenal Protein within Secretory Granules Affects Fusion Pore Expansion. [Journal Article]
- Biophys J 2014 Jul 1; 107(1):26-33.
It is often assumed that upon fusion of the secretory granule membrane with the plasma membrane, lumenal contents are rapidly discharged and dispersed into the extracellular medium. Although this is the case for low-molecular-weight neurotransmitters and some proteins, there are numerous examples of the dispersal of a protein being delayed for many seconds after fusion. We have investigated the role of fusion-pore expansion in determining the contrasting discharge rates of fluorescent-tagged neuropeptide-Y (NPY) (within 200 ms) and tissue plasminogen activator (tPA) (over many seconds) in adrenal chromaffin cells. The endogenous proteins are expressed in separate chromaffin cell subpopulations. Fusion pore expansion was measured by two independent methods, orientation of a fluorescent probe within the plasma membrane using polarized total internal reflection fluorescence microscopy and amperometry of released catecholamine. Together, they probe the continuum of the fusion-pore duration, from milliseconds to many seconds after fusion. Polarized total internal reflection fluorescence microscopy revealed that 71% of the fusion events of tPA-cer-containing granules maintained curvature for >10 s, with approximately half of the structures likely connected to the plasma membrane by a short narrow neck. Such events were not commonly observed upon fusion of NPY-cer-containing granules. Amperometry revealed that the expression of tPA-green fluorescent protein (GFP) prolonged the duration of the prespike foot ∼2.5-fold compared to NPY-GFP-expressing cells and nontransfected cells, indicating that expansion of the initial fusion pore in tPA granules was delayed. The t1/2 of the main catecholamine spike was also increased, consistent with a prolonged delay of fusion-pore expansion. tPA added extracellularly bound to the lumenal surface of fused granules. We propose that tPA within the granule lumen controls its own discharge. Its intrinsic biochemistry determines not only its extracellular action but also the characteristics of its presentation to the extracellular milieu.
- Protein Mobility within Secretory Granules. [Journal Article]
- Biophys J 2014 Jul 1; 107(1):16-25.
We investigated the basis for previous observations that fluorescent-labeled neuropeptide Y (NPY) is usually released within 200 ms after fusion, whereas labeled tissue plasminogen activator (tPA) is often discharged over many seconds. We found that tPA and NPY are endogenously expressed in small and different subpopulations of bovine chromaffin cells in culture. We measured the mobility of these proteins (tagged with fluorophore) within the lumen of individual secretory granules in living chromaffin cells, and related their mobilities to postfusion release kinetics. A method was developed that is not limited by standard optical resolution, in which a bright flash of strongly decaying evanescent field (∼64 nm exponential decay constant) produced by total internal reflection (TIR) selectively bleaches cerulean-labeled protein proximal to the glass coverslip within individual granules. Fluorescence recovery occurred as unbleached protein from distal regions within the 300 nm granule diffused into the bleached proximal regions. The fractional bleaching of tPA-cerulean (tPA-cer) was greater when subsequently probed with TIR excitation than with epifluorescence, indicating that tPA-cer mobility was low. The almost equal NPY-cer bleaching when probed with TIR and epifluorescence indicated that NPY-cer equilibrated within the 300 ms bleach pulse, and therefore had a greater mobility than tPA-cer. TIR-fluorescence recovery after photobleaching revealed a significant recovery of tPA-cer (but not NPY-cer) fluorescence within several hundred milliseconds after bleaching. Numerical simulations, which take into account bleach duration, granule diameter, and the limited number of fluorophores in a granule, are consistent with tPA-cer being 100% mobile, with a diffusion coefficient of 2 × 10(-10) cm(2)/s (∼1/3000 of that for a protein of similar size in aqueous solution). However, the low diffusive mobility of tPA cannot alone explain its slow postfusion release. In the accompanying study, we suggest that, additionally, tPA itself stabilizes the fusion pore with dimensions that restrict its own exit.
- HIF signaling pathway in pheochromocytoma and other neuroendocrine tumors. [JOURNAL ARTICLE]
- Physiol Res 2014 Jun 6; 63(Supplementum 2):S251-S262.
Hypoxia-inducible factors (HIFs) are transcription factors controlling energy, iron metabolism, erythropoiesis, and development. Dysregulation of these proteins contributes to tumorigenesis and cancer progression. Recent findings revealed the important role of HIFs in the pathogenesis of neuroendocrine tumors, especially pheochromocytoma (PHEO) and paraganglioma (PGL). PHEOs and PGLs are catecholamine-producing tumors arising from sympathetic- or parasympathetic-derived chromaffin tissue. To date, eighteen PHEO/PGL susceptibility genes have been identified. Based on the main signaling pathways, PHEOs/PGLs have been divided into two clusters, pseudohypoxic cluster 1 and cluster 2, rich in kinase receptor signaling and protein translation pathways. Recent data suggest that both clusters are interconnected via the HIF signaling and its role in tumorigenesis is supported by newly described somatic and germline mutations in HIF2A gene in patients with PHEOs/PGLs associated with polycythemia, and in some of them also with somatostatinoma. Moreover, HIFalpha signaling has also been shown to be upregulated in neuroendocrine tumors other than PHEO/PGL. Some of these tumors are components of hereditary tumor syndromes which can be associated with PHEO/PGL, but also in ileal carcinoids or melanoma. HIF signaling appears to be one of the crucial players in tumorigenesis, which could suggest new therapeutic approaches for treatment of neuroendocrine tumors.
- Chronic opioids regulate KATP channel subunit Kir6.2 and carbonic anhydrase I & II expression in rat adrenal chromaffin cells via HIF-2α and protein kinase A. [JOURNAL ARTICLE]
- Am J Physiol Cell Physiol 2014 Jun 4.
At birth, asphyxial stressors such as hypoxia and hypercapnia are important physiological stimuli for adrenal catecholamine release that is critical for the proper transition to extra-uterine life. We recently showed that chronic opioids blunt chemosensitivity of neonatal rat adrenomedullary chromaffin cells (AMCs) to hypoxia and hypercapnia. This blunting was attributable to increased KATP channel and decreased carbonic anhydrase (CA) I, II expression respectively, and involved μ- and δ-opioid receptor signaling pathways. To address underlying molecular mechanisms, we first exposed an O2- and CO2-sensitive, immortalized rat chromaffin cell line (MAH cells) to combined μ- (DALDA) and δ- (DPDPE) opioid agonists (2 μM) for ~7 days. Western blot and QPCR analysis revealed that chronic opioids increased KATP channel subunit Kir6.2, and decreased CAII expression; both effects were blocked by naloxone and were absent in hypoxia inducible factor (HIF)-2α-deficient MAH cells. Chronic opioids also stimulated HIF-2α accumulation along a time-course similar to Kir6.2. Chromatin immunoprecipitation assays on opioid-treated cells revealed the binding of HIF-2α to a hypoxia response element in the promoter region of the Kir6.2 gene. The opioid-induced regulation of Kir6.2 and CAII was dependent on PKA, but not PKC or CaM kinase, activity. Interestingly, a similar pattern of HIF-2α, Kir6.2, and CAII regulation (including downregulation of CAI) was replicated in chromaffin tissue obtained from rat pups, born to dams exposed to morphine throughout gestation. Collectively, these data reveal novel mechanisms by which chronic opioids blunt asphyxial chemosensitivity in AMCs, thereby contributing to abnormal arousal responses in the offspring of opiate-addicted mothers.
- Robot assisted laparoscopic excision of a paraganglioma: new therapeutic approach. [JOURNAL ARTICLE]
- Int Braz J Urol 2014 march-april; 40(2):279-280.
The Paraganglioma is the most common extra-adrenal pheochromocytoma arising from neural crest (1) (It will better to write: The paraganglioma is an extra-adrenal pheocromocytoma arising from the neural crest. 10% of pheocromocytomas are extra-adrenal and can arise form chromaffin tissue derived from primitive neuroectoderm). Minimally invasive techniques allow surgeons to perform the procedure without wide exposure and mobilization of intra abdominal organs. To our knowledge we present the third case of robotic excision of a retroperitoneal paraganglioma (2,3).
- A Defined, Controlled Culture System for Primary Bovine Chromaffin Progenitors Reveals Novel Biomarkers and Modulators. [JOURNAL ARTICLE]
- Stem Cells Transl Med 2014 May 22.
We present a method to efficiently culture primary chromaffin progenitors from the adult bovine adrenal medulla in a defined, serum-free monolayer system. Tissue is dissociated and plated for expansion under support by the mitogen basic fibroblast growth factor (bFGF). The cultures, although not homogenous, contain a subpopulation of cells expressing the neural stem cell marker Hes3 that also propagate. In addition, Hes3 is also expressed in the adult adrenal medulla from where the tissue is taken. Differentiation is induced by bFGF withdrawal and switching to Neurobasal medium containing B27. Following differentiation, Hes3 expression is lost, and cells acquire morphologies and biomarker expression patterns of chromaffin cells and dopaminergic neurons. We tested the effect of different treatments that we previously showed regulate Hes3 expression and cell number in cultures of fetal and adult rodent neural stem cells. Treatment of the cultures with a combination of Delta4, Angiopoietin2, and a Janus kinase inhibitor increases cell number during the expansion phase without significantly affecting catecholamine content levels. Treatment with cholera toxin does not significantly affect cell number but reduces the ratio of epinephrine to norepinephrine content and increases the dopamine content relative to total catecholamines. These data suggest that this defined culture system can be used for target identification in drug discovery programs and that the transcription factor Hes3 may serve as a new biomarker of putative adrenomedullary chromaffin progenitor cells.
- Succinate Dehydrogenase Subunit D and Succinate Dehydrogenase Subunit B Mutation Analysis in Canine Phaeochromocytoma and Paraganglioma. [JOURNAL ARTICLE]
- J Comp Pathol 2014 Mar 27.
Phaeochromocytomas (PCs) are tumours of the adrenal medulla chromaffin cells. Paragangliomas (PGLs) arise in sympathetic ganglia (previously called extra-adrenal PCs) or in non-chromaffin parasympathetic ganglia cells that are usually non-secretory. Parenchymal cells from these tumours have a common embryological origin from neural crest ectoderm. Several case series of canine PCs and PGLs have been published and a link between the increased incidence of chemoreceptor neoplasia in brachycephalic dog breeds and chronic hypoxia has been postulated. A similar link to hypoxia in man led to the identification of germline heterozygous mutations in the gene encoding succinate dehydrogenase subunit D (SDHD) and subsequently SDHA, SDHB and SDHC in similar tumours. We investigated canine PCs (n = 6) and PGLs (n = 2) for SDHD and SDHB mutations and in one PGL found a somatic SDHD mutation c.365A>G (p.Lys122Arg) in exon 4, which was not present in normal tissue from this brachycephalic dog. Two PCs were heterozygous for both c.365A>G (p.Lys122Arg) mutation and an exon 3 silent variant c.291G>A. We also identified the heterozygous SDHB exon 2 mutation c.113G>A (p.Arg38Gln) in a PC. These results illustrate that genetic mutations may underlie tumourigenesis in canine PCs and PGLs. The spontaneous nature of these canine diseases and possible association of PGLs with hypoxia in brachycephalic breeds may make them an attractive model for studying the corresponding human tumours.
- Signaling molecules and transcription factors involved in the development of the sympathetic nervous system, with special emphasis on the superior cervical ganglion. [JOURNAL ARTICLE]
- Cell Tissue Res 2014 Apr 26.
The cells that constitute the sympathetic nervous system originate from the neural crest. This review addresses the current understanding of sympathetic ganglion development viewed from molecular and morphological perspectives. Development of the sympathetic nervous system is categorized into three main steps, as follows: (1) differentiation and migration of cells in the neural crest lineage for formation of the primary sympathetic chain, (2) differentiation of sympathetic progenitors, and (3) growth and survival of sympathetic ganglia. The signaling molecules and transcription factors involved in each of these developmental stages are elaborated mostly on the basis of the results of targeted mutation of respective genes. Analyses in mutant mice revealed differences between the superior cervical ganglion (SCG) and the other posterior sympathetic ganglia. This review provides a summary of the similarities and differences in the development of the SCG and other posterior sympathetic ganglia. Relevant to the development of sympathetic ganglia is the demonstration that neuroendocrine cells, such as adrenal chromaffin cells and carotid body glomus cells, share a common origin with the sympathetic ganglia. Neural crest cells at the trunk level give rise to common sympathoadrenal progenitors of sympathetic neurons and chromaffin cells, while progenitors segregated from the SCG give rise to glomus cells. After separation from the sympathetic primordium, the progenitors of both chromaffin cells and glomus cells colonize the anlage of the adrenal gland and carotid body, respectively. This review highlights the biological properties of chromaffin cells and glomus cells, because, although both cell types are derivatives of sympathetic primordium, they are distinct in many respects.
- Combination chemotherapy regimen in a patient with metastatic malignant pheochromocytoma and neurofibromatosis type 1. [Journal Article]
- Am J Case Rep 2014.:123-7.
Patient: Female, 55 Final Diagnosis: Metastatic malignant pheochromocytoma Symptoms: Chest pain • tachycardia • tachypnea Medication: - Clinical Procedure: - Specialty: Oncology.Rare disease.Malignant pheochromocytoma is defined as the occurrence of the tumor in an area that is normally devoid of chromaffin tissue, direct tumor invasion, and/or metastasis. Metastatic malignant pheochromocytoma is very rare. Malignant pheochromocytoma carries a poor prognosis with a 5-year survival rate of 44%. The majority of pheochromocytoma cases are sporadic, but a small portion (10%) can be associated with hereditary syndromes such as neurofibromatosis type 1 (NF1).A 55-year-old Hispanic woman presented to our care with chest pain. Her past medical problems included hypertension, neurofibromatosis type 1 (NF1), and pheochromocytoma status after right laparoscopic adrenalectomy, which was converted to open procedure about 19 months ago. Initial vital signs were significant for tachycardia, tachypnea, and hypertension. Computed tomography (CT) angiography of the chest was performed to rule out a pulmonary embolism, but instead revealed multiple bilateral lung nodules measuring up to 8 mm, consistent with metastasis. CT of the abdomen and pelvis revealed a lytic lesion in the posterior aspect of the left pedicle and transverse process of L4, and the neck/greater trochanter of the left femur. MIBG scan revealed widespread metastatic disease. She received an outpatient chemotherapy regimen of cyclophosphamide, dacarbazine, and vincristine.Metaiodobenzylguanidine (MIBG) is an alkyl-guanidine derivative similar to noradrenaline, which accumulates in tissue derived from neural crest cells. Current medical literature suggests that therapeutic 131I MIBG has a response rate of 50-75%. Combination chemotherapy using cyclophosphamide, vincristine, and dacarbazine has been the most widely used regimen for malignant pheochromocytoma. This combination of chemotherapy has been shown to have a high response rate and symptomatic improvement. Numerous therapeutic regimens exist for metastatic malignant pheochromocytoma; however, no regimen has been shown to have a benefit significantly superior to the others.
- Complexin synchronizes primed vesicle exocytosis and regulates fusion pore dynamics. [Journal Article]
- J Cell Biol 2014 Mar 31; 204(7):1123-40.
ComplexinII (CpxII) and SynaptotagminI (SytI) have been implicated in regulating the function of SNARE proteins in exocytosis, but their precise mode of action and potential interplay have remained unknown. In this paper, we show that CpxII increases Ca(2+)-triggered vesicle exocytosis and accelerates its secretory rates, providing two independent, but synergistic, functions to enhance synchronous secretion. Specifically, we demonstrate that the C-terminal domain of CpxII increases the pool of primed vesicles by hindering premature exocytosis at submicromolar Ca(2+) concentrations, whereas the N-terminal domain shortens the secretory delay and accelerates the kinetics of Ca(2+)-triggered exocytosis by increasing the Ca(2+) affinity of synchronous secretion. With its C terminus, CpxII attenuates fluctuations of the early fusion pore and slows its expansion but is functionally antagonized by SytI, enabling rapid transmitter discharge from single vesicles. Thus, our results illustrate how key features of CpxII, SytI, and their interplay transform the constitutively active SNARE-mediated fusion mechanism into a highly synchronized, Ca(2+)-triggered release apparatus.