Expression of chromogranins A, B, and C (secretogranin II) in human adrenal medulla and in benign and malignant pheochromocytomas An immunohistochemical study with region-specific antibodies

In a recent immunohistochemical study of pheochromocytomas, a difference was observed between benign and malignant pheochromocytomas in their expression in different parts of the chromogranin (Cg) A molecule. The purpose of the present study was to extend the investigations by including two other members of this Cg family, CgB and C. Twenty-five patients operated on for clinicopathologically benign pheochromocytomas, and four for metastasizing pheochromocytomas, were studied. Expression of the different Cg regions was studied immunohistochemically by means of region-specific antibodies: four raised against CgA epitopes, five against CgB, and two against CgC. Adrenal medulla parenchyma from three surgical adrenalectomy specimens was used as non-neoplastic control. All cells of normal adrenal medulla were immunoreactive to all 11 region-specific Cg antibodies. In the pheochromocytomas, variations in the expression pattern occurred, but no significant quantitative differences were noted between benign and malignant tumours. Nevertheless, in all four malignant pheochromocytomas, the antibodies raised against the C-terminal regions of both CgB and CgC visualised a noticeable population of large spindle-shaped tumour cells, characterised by elongated processes. This cell type occurred in all four malignant pheochromocytomas but only in one benign tumour. Their structure and immunoreactivity differed from those of the sustentacular cells in the pheochromocytoma parenchyma. The use of region-specific antibodies raised against epitopes in the C-terminal region of CgB and CgC can facilitate the diagnosis of malignant pheochromocytoma.     

Sporadic type composite pheochromocytoma with neuroblastoma: Clinicomorphologic, DNA content, and ret gene analysis

Composite pheochromocytomas (CP) account for only 3% of all pheochromocytomas. We analyzed the clinical, immunohistochemical, ultrastructural, DNA content, and a 634 ret mutation feature in a 56-yr-old Mexican woman with a CP localized in the right adrenal gland and associated with a blood pressure of 140/90 mmHg. Clinical symptoms were absent after surgery. The tumor showed pheochromocytoma and neuroblastoma components. This dual phenotype was supported by light microscopy and corroborated by immunohistochemistry and ultrastructural findings. Flow cytometric analysis showed that both components were diploid. A genetic mutational analysis of the ret oncogene in exon 11 showed no 634 mutation. This case demonstrates the indolent behavior of neuroblastoma associated with a sporadic-type CP in an adult patient.     

Age-related structural characteristics of the adrenal medulla in hypertensive NISAG rats

Structural characteristics of the adrenal medulla in hypertensive NISAG rats (hereditary stress-induced arterial hypertension) were studied during various periods of postnatal ontogeny. Signs of hyperplasia of the adrenal medulla were most pronounced in adult hypertensive animals with persistent arterial hypertension, as well as during the period of late ontogeny. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 12, pp. 604–606, December, 2006     

Efflux of 45Ca from bovine adrenal medulla cells in culture caused by nicotinic receptor stimulation

Efflux of 45Ca was investigated in cultured bovine adrenal medulla cells with its relation to Ca influx. Carbachol caused a rapid and transient efflux of 45Ca from 45C preloaded cells which proceeded with a similar time course to 45Ca influx. Nicotine also caused efflux of 45Ca that is completely antagonized by hexamethonium, while muscarine and pilocarpine did not cause 45Ca efflux. Nicotine-induced efflux of 45Ca was abolished by the Ca-antagonist, diltiazem, and was not observed in Ca-free medium. These observations show that in bovine adrenal medulla, not only the influx of Ca but also the efflux of Ca are caused by nicotinic receptor stimulation and that the efflux of Ca is dependent on the Ca in the medium.     

肾上腺肿瘤诊治的分子生物学进展

目前已经报道约18种肾上腺髓质肿瘤相关基因突变,基于基因突变类型的靶向治疗前景广阔。肾上腺皮质腺瘤中,原发性醛固酮腺瘤的体细胞突变携带率约40%,但真正的致病基因还需进一步的研究。基因芯片、蛋白组学帮助快速发现更多致病候选基因。米托坦为目前唯一有效的治疗肾上腺皮质癌的药物,国际临床随机双盲实验已证实米托坦联合依托泊苷、阿霉素和顺铂可延长疾病无进展生存时间。     

自发性高血压大鼠交感神经节与肾上腺髓质内NPY的表达及超微结构的变化

目的:通过对自发性高血压大鼠(SHR)颈上神经节、肾上腺髓质内神经肽酪氨酸(NPY)表达及超微结构变化的研究,探讨NPY在高血压发生和发展中的作用。方法:随机选取成年大鼠60只,分成SHR组和正常对照组,每组各30只,采用Real-Time PCR技术和免疫组织化学法,检测两组大鼠颈上神经节、肾上腺髓质内NPYmRNA和蛋白的表达;应用透射电镜技术观察上述两种组织内超微结构的改变。结果:与同周龄的正常组大鼠相比,SHR大鼠的血压明显增高(P<0.05),RT-PCR和免疫组织化学方法检测到颈上神经节、肾上腺髓质内NPYmRNA和蛋白质的表达均增高(P<0.05),NPY免疫阳性神经元的数量和阳性细胞的光密度亦增加(P<0.05)。电镜结果示SHR大鼠两种组织内异染色质、线粒体、粗面内质网等数量均明显增加(P<0.05)。结论:SHR颈上神经节、肾上腺髓质内的NPY通过复杂的机制可能参与了高血压的形成。     

Expression of human GIPC1 in normal tissues, cancer cell lines, and primary tumors

GIPC1/RGS19IP1/GIPC, GIPC2, and GIPC3 are a family of central PDZ-domain proteins with GH1 and GH2 domains. GIPC1 interacts with GTPase-activating protein RGS19/RGS-GAIP, TGFbeta type III receptor, receptor tyrosine kinase TrkA, and integrin alpha6A subunit. Xenopus homologue of human GIPCs interacts with Frizzled-3 class of WNT receptor. We investigated expression of human GIPC1 mRNA in normal tissues, cancer cell lines, and primary tumors. GIP1A probe (nucleotide position 1075-1483 of GIPC1 cDNA) hybridized to GIPC1 mRNA of 1.8 kb in size. GIPC1 mRNA was almost ubiquitously expressed in various normal tissues. Expression level of GIPC1 mRNA was relatively lower in bone marrow and peripheral blood leukocytes. GIPC1 mRNA was relatively highly expressed in gastric cancer cell lines OKAJIMA, TMK1, MKN28, MKN45, MKN74, KATO-III, pancreatic cancer cell line AsPC-1, colorectal cancer cell line SW480, and lung cancer cell line A549. On the other hand, GIPC1 mRNA was almost undetectable in leukemia/lymphoma cell lines HL-60, Raji, and Daudi. Expression of GIPC1 mRNA was down-regulated in 12 out of 14 cases of primary kidney tumors, 10 out of 18 cases of primary colorectal tumors, 3 out of 8 cases of primary gastric cancer, 3 out of 3 cases of primary prostate cancer. Because GIPC1 induces increased expression of TGFbeta type III receptor at the cell surface and enhanced responsiveness to TGFbeta, down-regulation of GIPC1 mRNA in tumors might promote cellular proliferation through interference of TGFbeta signaling.     

Weight and shape of the human adrenal medulla in various age groups

Summary The weight and shape of the adrenal medulla were studied in 118 adrenal glands obtained at autopsies of 62 patients (38 males and 24 females) between 0 and 52 years of age. In adolescents 15 years and older and adults cases of sudden death only were entered in this study. The weight was calculated using morphometric measurements done on serial sections of the glands. In the new-born, the medulla accounts for less than 1% of the total volume of the adrenal gland. Though there is a rapid growth of the adrenal medulla after birth, the percentage of adrenal medullary volume at all age levels during childhood and adolescence is lower than in adults where it constitutes 9% of the total adrenal volume on the average. This corresponds to an average medullary weight of 0.43 g. In the new-born, the medulla consists of a thin plate made up of immature medulloblasts. Within a few months these are transformed into mature medullary cells; the shape of the medulla soon approaches that of the adult gland: an increase in thickness around the central vein and flat processes into the alae. With increasing age the cortico-medullary border becomes irregular and ragged. Especially in the vicinity of the central vein an intermingling of medullary and cortical cell complexes is found. An unequivocal diagnosis of genuine adrenal medullary hyperplasia can be established only by weighing the dissected medulla or by applying morphometric methods.     

A physiological view of the central and peripheral mechanisms that regulate the release of catecholamines at the adrenal medulla

Here we review the tight neural control of the differential secretion into the circulation, of the adrenal medullary hormones adrenaline and noradrenaline. One or the other catecholamines are differentially released on various stress conditions. This is specifically controlled by central nervous system nuclei at the cortex, hypothalamus and spinal cord. Different firing patterns of splanchnic nerves and nicotinic or muscarinic receptors cause the selective release of noradrenaline or adrenaline, to adapt the body to the 'fight or flight' reaction, or during severe hypoglycaemia, haemorrhage, cold, acute myocardial infarction or other severe stressful conflicts. Endogenously acetylcholine (ACh) released at the splanchnic nerve-chromaffin cell synapse, acting on muscarinic and nicotinic receptors, causes membrane depolarization and action potentials (AP) in chromaffin cells. These changes vary with the animal species, the cell preparation (intact bisected adrenal, adrenal slices, or isolated fresh or cultured cells) or the recording technique (intracellular microelectrodes, patch-clamp, perforated-patch, cell-attached). Conflicting results leave many open questions concerning the actions of ACh on chromaffin cell excitability. The use of adrenal slices and field electrical stimulation will surely provide new insights into these mechanisms. Chromaffin cells have been thoroughly used as models to study the relationship between Ca2+ entry, cytosolic Ca2+ signals, exocytosis and endocytosis, using patch-clamp and amperometric techniques. Cells have been stimulated with single depolarizing pulses (DPs), DP trains and with simulated AP waveforms. These approaches have provided useful information but we have no data on APs generated by pulsatile secretory quanta of ACh, trying to mimic the intermittent and repetitive splanchnic nerve discharge of the neurotransmitter. We present some recent experiments using ultrashort ACh pulses (25 ms), that cause non-desensitizing repetitive APs with each ACh pulse, at low ACh concentrations (30 microM). Ultrashort pulses of a high ACh concentration (1000 microM) causes a single AP followed by a prolonged depolarization. It could be interesting trying to correlate these 'patterns of splanchnic nerve discharge' with Ca2+ signals and exocytosis. This, together with the use of adrenal slices and transmural electrical stimulation of splanchnic nerves will provide new physiologically sound data on the regulation of adrenal medullary secretion.     

In vitro studies on a two-pool storage of adrenaline and noradrenaline in granule material from bovine adrenal medulla

Adrenal medullary granules were isolated by millipore filtration according to Poisner & Trifaró (1967) and then lysed in deionized water. In a perfusion system the influence of concentration and pH was studied on the uptake of biogenic amines (PhEA, TA, DA, NA, A, Ach and Hi) and sodium ions by lysed and by dialyzed material. The results suggest a two-pool storage of A and NA in the granules. A minor pool with cation exchanger properties binds unselectively organic (biogenic amines) and inorganic cations with a U_max of 400–500 nmol/mg granules dry-weight. This minor pool (pool 1) was fully charged at amine concentrations >10 mM. A larger pool binds selectively A and NA-possibly stereospecifically L-NA and L-A—with a U_max?1500 nmol/mg granules. This larger pool (pool 2) required A and NA concentrations ?200–300 mM for maximal filling. In pool 2 CA⁺ ions are assumed to be electrostatically linked to carboxyl groups, which become available as hypothetical COO^-—⁺H₃N salt bridges are successively forced open by increasing CA concentrations (>10–30 mM). ATP^- ions become attached to the concomitantly unmasked ⁺NH₃ groups.     

Brief and repeated noise exposure produces different morphological and biochemical effects in noradrenaline and adrenaline cells of adrenal medulla

Exposure to stressful stimuli is known to activate the peripheral sympathetic nervous system and the adrenal gland. In this study, we evaluated the effects of single or repeated bouts of exposure to a readily measurable stressful stimulus (loud noise) on the catecholamine content and ultrastructure of the rat adrenal medulla. In particular, we measured tissue levels of dopamine, noradrenaline, adrenaline and metabolites. In parallel studies, we evaluated the fine ultrastructure of catecholamine cells, including a detailed study of catecholamine granules and a morphometric analysis of adrenaline and noradrenaline medullary cells. Animals were exposed either to a single (6 h) session of loud (100 dBA) noise, or to this noise stimulus repeated every day for 21 consecutive days. There was a marked correlation between biochemical indexes of catecholamine activity and the ultrastructural morphometry of specific catecholamine granules. Exposure to loud noise for 6 h induced a parallel increase in dopamine, noradrenaline, adrenaline and their metabolites, a polarization and an increased numerical density of noradrenaline and adrenaline granules in the cells. After repeated noise exposure, noradrenaline levels were significantly higher than in controls, and adrenaline decreased significantly. In addition, adrenaline cells also exhibited ultrastructural alterations consisting of wide homogeneous cytoplasmic areas and large, pale vesicles.     

肾上腺肿瘤光镜,电镜鉴别诊断价值

收集２２例肾上腺皮髓质肿瘤电镜材料，根据电镜结果，明确皮髓质来源后，重新认识光镜特点。结果发现１６例单凭光镜即可诊断，另６例单凭光镜则难以明确。根据电镜表现，对以嗜酸细胞瘤样表现的肿瘤和以实性片块为主体的肿瘤，则应行电镜检查才能获得可靠的诊断，而临床表现、实验室检查、肿瘤大小、有无包膜等指标在肾上腺皮髓质肿瘤鉴别上帮助不大。     

大鼠实验性胃溃疡自愈期间肾上腺髓质神经肽Y、5羟色胺及嗜铬颗粒素A免疫阳性细胞的变化

目的:探讨大鼠实验性胃溃疡自愈期间肾上腺髓质神经肽Y(NPY)、5羟色胺(5-HT)及嗜铬颗粒素A(CgA)免疫阳性细胞的变化。方法:免疫组织化学方法显示阳性细胞并用病理图像分析系统软件做面密度分析。结果:溃疡组NPY阳性细胞面密度于术后4d升高,术后6、10d降低;溃疡组5-HT阳性细胞面密度于术后4d增高,6d达高峰;溃疡组CgA阳性细胞面密度于术后4、6、10、14d低于正常组。结论:肾上腺髓质NPY、5-HT及CgA阳性细胞可能直接或间接参与大鼠实验性胃溃疡修复的调节。     

Preganglionic fibres of the adrenal medulla and cervical sympathetic ganglia: differential involvement during experimental American trypanosomiasis in rats

The acute phase of experimental Chagas' disease in rats is associated with early lesions of the post-ganglionic sympathetic nerve terminals of the heart, the varicosities being the target. In the superior cervical and stellate ganglia the preganglionic fibres showed no signs of lesion in the course of experimental Chagas' disease. In the adrenal medulla, however, the acute phase of the Trypanosoma cruzi infection induced a clear rarefaction of the acetylcholinesterase-positive nerve fibres (20 and 32 days after infection). Recovery of the normal pattern occurred in most animals by day 125 after infection. At days 20, 32 and 46 after infection, electron-microscopic studies demonstrated the occurrence of damage in cholinergic nerve terminals contacting the chromaffin cells. The signs of damage included dense bodies, clumps of synaptic vesicles and filaments, rarefaction of all organelles, vacuoles and irregular contour. The ultrastructural peculiarities of the sympathetic ganglia may explain the ganglionar microenvironment protective against the hazardous factors elicited by acute Chagas' disease.     

Fine structure and morphogenesis of spironolactone bodies in the zona glomerulosa of the human adrenal cortex

Numerous spironolactone bodies have been detected in the zona glomerulosa cells of the adrenal cortex of a 36-year-old spironolactone-treated woman whose non-tumorous right adrenal gland was removed surgically because of primary hyperaldosteronism. Electron microscopy revealed spherical laminated whorls which consisted of a central core composed of an amorphous electron-dense material surrounded by numerous smooth-walled concentric membranes. Continuous with and deriving from the endoplasmic reticulum, they were present in viable cells and were not associated with ultrastructural features indicating cellular injury. Cytoplasmic inclusions similar to spironolactone bodies can be detected in other organs after the administration of various compounds. Thus, they can be regarded as neither specific to spironolactone treatment nor exclusively inducible in the zona glomerulosa of the adrenal cortex.     

The role of the adrenal medulla in cardiovascular responses to hypertonic saline in haemorrhaged conscious rats

The aim of this study was to determine if the adrenal medulla plays a role in mediating haemodynamic effects of hypertonic saline (HTS) resuscitation during haemorrhagic hypotension in conscious rats. Wistar-Kyoto rats were either adrenomedullectomized (ADMX, n = 11) or sham-operated (SHAM, n = 10) and implanted with intravascular catheters. Pre-haemorrhage resting mean arterial pressure (MAP) was lower in the ADMX than in the SHAM group. Haemorrhage was performed by withdrawal of blood through the venous catheter, and a MAP of 50 mmHg was maintained for 1 h by further withdrawal when necessary. Both groups responded to haemorrhage with marked bradycardia. Plasma adrenaline (A) rose 10-fold in response to hypotension in the SHAM group, while remaining at pre-haemorrhage levels in the ADMX group, indicating successful adrenal demedullation. Infusion of 2.0 ml kg^-1 HTS (NaCl 8.0 mg ml^-1 i.v.) produced an immediate increase in MAP and heart rate (HR) in both groups, to pre-haemorrhage values or higher. Plasma noradrenaline (NA) increased in both groups after HTS, while the high levels of A in the SHAM group slowly returned toward baseline. Plasma glucose and rate of haemodilution was higher in the SHAM group during and after hypotension. The maintenance of MAP above 60 mmHg was less effective in the SHAM group during the first 2 h after HTS, but after 24 h, pre-haemorrhage MAP was established in both groups. In conclusion, the adrenal medulla does not play a major role in the response to HTS resuscitation.     

Transient Ca2+-channel current characterized by a low-threshold voltage in zona glomerulosa cells of rat adrenal cortex

Voltage-gated Ca²⁺-current was identified in single isolated cells of the zona glomerulosa of adrenal cortex and its properties were studied by the tight-seal whole cell recording technique. The Ca²⁺-channel current was dissected from the net current by dialyzing the cells with CsCl. The identified Ca²⁺-current was found to be activated by a relatively small depolarization only when the cells were held at a large negative holding potential, but it was inactivated within 10–30 ms. The time course of activation and inactivation was voltage-dependent and became faster when the amplitude of depolarization was increased. The transmembrane potential of the glomerulosa cells was highly sensitive to [K⁺]_e, the slope of the potential change per tenfold change in [K⁺]_e being 48 mV. An increase in [K⁺]_e from 4.7 to 10 mM induce a membrane depolarization by 15 mV, which was sufficient to cause the membrane to reach the threshold potential (–60 mV) for activation of the Ca²⁺-current at physiological concentration of [Ca²⁺]_e (2.5 mM –CaCl₂). The observed properties of the Ca²⁺-current and K⁺-dependence of the membrane potential may give reasonable explanation for the mechanism of Ca²⁺-uptake and consequent aldosterone secretion induced by a small increase in [K⁺]_e, which is known to be one of the major stimulations for aldosterone secretion.     

Stathmin Expression in Pheochromocytomas,Paragangliomas, and in other Endocrine Tumors

Pheochromocytomas are neuroendocrine tumors confined to the adrenal glands, and malignant pheochromocytomas can spread to various sites including the liver, lung, and bones. Paragangliomas occur in numerous locations in the body, so assessment of metastatic disease is more challenging, as patients with familial syndromes often have multiple, possibly independent paragangliomas. The most reliable criterion for malignancy in pheochromocytomas and paragangliomas is metastatic disease. Because there are few immunohistochemical markers that are useful in the diagnosis of malignancy in pheochromocytomas and paragangliomas before they metastasize, more markers are needed to characterize these tumors. Stathmin is a widely expressed 17-kDa cytoplasmic, microtubule destabilizing and sequestering phosphoprotein that is important in cell motility and cancer cell metastasis. It is upregulated in various malignancies. We examined stathmin expression in tissues from patients with pheochromocytomas (n = 48), malignant pheochromocytomas (n = 28), paragangliomas (n = 42), and malignant paragangliomas (n = 21) by immunohistochemistry using tissue microarrays (TMA) with a polyclonal antibody against stathmin. A series of other endocrine tissues and tumors (n = 70) were also examined for stathmin expression. Stathmin was more highly expressed in pheochromocytomas compared to normal adrenals, a finding confirmed by Western blot. There was higher expression of stathmin by immunohistochemical staining in malignant pheochromocytomas compared to pheochromocytomas without metastasis when analyzed by maximal staining (p = 0.012). Stathmin was present in a wide variety of endocrine tumors and was most highly expressed in rapidly proliferating tumors including anaplastic thyroid carcinomas, Merkel cell carcinomas of the skin and small cell carcinomas of the lung. These results show that stathmin is expressed at higher levels in more rapidly proliferating endocrine tumors. However, it is probably not useful as a stand-alone marker to determine malignancy in pheochromocytomas for individual tumors.