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.     

Possible role of nerve impulse induced sodium ion flux in a proposed multivesicular fractional release of adrenaline and noradrenaline from the chromaffin cell

Based on own observations concerning a two-compartment storage of CA in the adrenal medulla and a cation exchange dependent release of CA from perfused chromaffin granules in vitro, and encouraged by recent reports from other laboratories about the importance of sodium ions for the CA release from the adrenal gland, we propose a modification of the current quantal theory of CA secretion. Instead of secretion of quanta, each quantum corresponding to the content of one vesicle, we envisage a concomitant fractional release of CA from multiple vesicles adjacent to the chromaffin cell membrane. The CA secretion should be the result of a cation exchange across the contact area between the plasma membrane and the granule membrane during the period of depolarization caused by the nerve impulse. The size of the released quanta should be determined by the nerve impulse induced sodium ion flux and the number of such ions which reach the CA binding ionic sites in the cation exchanger pool (the release pool) of the granules.     

Catecholamines (CA) and adenosine triphosphate (ATP) are separately stored in bovine adrenal medulla,both in ionic linkage to granule sites,and not as a non-diffusible CA-ATP-protein complex*

On superfusion of chromaffin granules from bovine adrenals with isotonic sodium and potassium salts, catecholamines and ATP were released in parallel and both in accordance with ion exchange kinetics. An artificial model was prepared by mixing a cationic (IRC-50) and an anionic (IR-4B) ion exchanger with COO and NH⁺₃ groups, respectively, as binding sites. This mixed ion exchanger showed in its storage and release of CA⁺ and ATP^- striking similarities to the chromaffin granules. Within the pH range given for the interior of the granules - 5.5–6 - the artificial model even stored and released CA⁺ and ATP^- within the same molar ratio as observed for the granules. We hypothesize that the chromaffin granule matrix in its storage and release functions operates as an amphoteric ion exchanger with COO^- and NH⁺₃, groups as the binding sites.     

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.     

Galanin and neuropeptide Y in chromaffin granules from the guinea-pig

We have examined the subcellular distribution of galanin-like immunoreactivity, neuropeptide Y-like immunoreactivity and the catecholamines noradrenaline and adrenaline in the adrenal medulla from guinea-pigs. By differential centrifugation of the adrenal medulla homogenate the neuropeptides as well as the catecholamines sedimented in a 10000 g pellet. This pellet was resuspended and further examined in discontinuous and continuous density gradients. In the discontinuous gradient the catecholamines peaked in the heavy bottom fraction, assumed to contain chromaffin granules. Galanin-like immunoreactivity and neuropeptide Y-like immunoreactivity were also enriched in this fraction. However, both neuropeptides showed high levels of sedimentable material also in a fraction of intermediate density. In the continuous density gradient, the sum of sedimentable and soluble catecholamines showed peak values in two fractions corresponding to 1.07 and 1.47 m sucrose, respectively. The NA peak in the denser fraction was more pronounced than the corresponding A peak. Galanin-like immunoreactivity showed only one peak, in the fraction corresponding to 1.07 M sucrose. The data suggest that galanin-like immunoreactivity and neuropeptide Y-like immunoreactivity are partly stored with catecholamines in chromaffin granules. However, galanin-like immunoreactivity and neuropeptide Y-like immunoreactivity was also found in fractions lighter than those containing the bulk of the catecholamines.     

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.     

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.     

Co-storage of enkephalins and adrenaline in the bovine adrenal medulla

Fluorescence histochemical and immunohistochemical techniques have been used to examine the distribution of catecholamine-containing and enkephalin-containing cells in sections of adult bovine adrenal medulla. Noradrenaline-containing cells were identified by fluorescence microscopy following perfusion fixation with 4% paraformaldehyde (formaldehyde-induced fluorescence, technique of Era¨nko¨⁸). Adrenaline-containing cells did not fluoresce under these conditions. Adrenaline-synthesizing cells were identified by immunofluorescence with an antiserum to bovinephenyl-N-methyl transferase. An antiserum to bovine dopamine-β-hydroxylase was used to identify noradrenaline plus adrenaline cells in the same section. Leu- and met-enkephalin-containing cells were identified immunohistochemically with their respective antisera. To determine whether there was a preferential association of leu- or met-enkephalin with adrenaline or noradrenaline cells, these various antisera were used singly or sequentially on sections treated with formaldehyde in which the localization of endogenous noradrenaline fluorescence had been recorded and then the fluorescence removed by washing overnight.Immunoreactive leu- and met-enkephalin were found to be associated exclusively with adrenaline-synthesizing cells. The finding that both enkephalins are localized in the one cell type (adrenaline cells) in the bovine adrenal medulla is consistent with the proposed common precursor model for synthesis of the two opioid pentapeptides. These findings on co-storage of enkephalins with adrenaline in the adrenal medulla may have implications for other areas of the peripheral and central nervous system where co-storage of catecholamines and enkephalins is known to occur.     

The ability of ATP-free granule material from bovine adrenal medulla to bind inorganic cations and biogenic amines.

Bovine adrenal medullary granules isolated by millipore filtration were depleted of CA and ATP by dialysis. The resulting material showed an ability to bind inorganic cations and biogenic amines in a concentration-dependent manner. The similarity of the uptake curves, the identical uptake maxima and the narrow pH range (between 4-7) over which the uptake of the inorganic and organic cations took place indicated a binding of these ions to common sites. In addition, the fact that all the uptake curves fitted the Rothmund-Kornfeld equation for cation exchangers corroborated the cation exchanger properties of the dialyzed granule material. The CA binding capacity corresponded to 20-30% of the normal CA content of bovine medullary granules.     

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。     

Further studies on renal nerve stimulation induced release of noradrenaline and dopamine from the canine kidney in situ

The renal venous outflow of dopamine and noradrenaline were studied in the canine kidney in situ in connection with renal nerve stimulation (RNS). RNS (0.5-4 Hz) caused frequency-dependent increases in the outflow of both catecholamines, which could be detected already at 0.5 Hz. The ratio dopamine/noradrenaline in renal venous plasma (approximately 0.15) was not influenced by varying the RNS parameters but was significantly enhanced (to about 0.25) by pretreatment with guanethidine according to a procedure previously used to demonstrate renal dopaminergic vasodilatation. The unstimulated kidney removed conjugated dopamine (which represents 98–99% of the total dopamine in plasma). During RNS the conjugated dopamine outflow to renal venous blood increased, but measurements of conjugated dopamine were less reliable than measurements of free dopamine to assess dopamine release from the kidney. When studying the renal nerve contributions to the renal venous outflow of dopamine and noradrenaline more accurate estimates may be obtained by correcting for the removal of catecholamines delivered to the kidney in arterial plasma. Such corrections were performed with endogenous adrenaline or radiolabelled noradrenaline. The two methods of correction yielded similar results and showed that RNS reduced catecholamine extraction in the kidney. The high ratio of dopamine/noradrenaline in kidney tissue (with a preferential distribution of dopamine to the cortex) and the dopamine outflow to renal venous plasma during RNS support the existence of specific dopaminergic nerves in the dog kidney.     

寒冷应激对大鼠肾上腺髓质亨廷顿蛋白相关蛋白1表达的影响

目的 探讨亨廷顿蛋白相关蛋白1(HAP1)在大鼠肾上腺髓质的超微结构定位,以及寒冷应激对大鼠肾上腺髓质HAP1表达的影响. 方法 成年雄性Wistar大鼠14只,2只用于免疫电镜研究,12只用于寒冷实验研究.寒冷实验中,将动物随机分为对照组和寒冷组,每组6只,寒冷组动物放置4℃环境下,12h后用免疫组织化学和Western blotting方法 检测大鼠肾上腺髓质HAP1表达的变化. 结果 免疫电镜结果 显示,HAP1免疫反应产物分布在肾上腺髓质细胞分泌颗粒外膜及分泌颗粒间的膜性细胞器上.寒冷组大鼠肾上腺髓质HAP1的表达明显减少,和对照组比较有显著性差异( P <0.01). 结论 HAP1可能与肾上腺髓质细胞内分泌颗粒及位于分泌颗粒内的肾上腺素/去甲肾上腺素的运输和释放有关.     

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.     

Clonality studies in the analysis of adrenal medullary proliferations: Application principles and limitations

Clonality remains as the hallmark of neoplasms. A dual genetic approach using markers nonrelated (e.g., X-chromosome inactivation assays) and related to the malignant transformation (such as loss of heterozygosity analyses of tumor-suppressor genes) would provide useful clonality information from early and advanced tumor stages, respectively. Tumor progression and clonal selection would result in genetic instability and heterogeneous expression of those molecular markers related to the malignant pathway. Therefore, only the coexistence of multiple genetic abnormalities would support the clonal nature as an expression of convergent cell selection. Considering those facts, the currently available evidence on tumorigenesis and clonality in the adrenal medulla can be summarized as follows:

A quantitative electron microscopic study of the effect of glucocorticoids in vivo on the early postnatal differentiation of paraneuronal cells in the carotid body and the adrenal medulla of the rat

Summary The postnatal differentiation of carotid body chief cells and endocrine adrenal medullary cells was comparatively examined during ontogenesis and in rats which were treated with dexamethasone for 7 days after birth. Ultrastructure and innervation of carotid body chief cells are mature in neonates according to the functional requirements of chemoreception. By the end of the first postnatal week, only an increase in number of dense core vesicles can be noticed, the concentration of which then will reach the adult level. Under the effect of dexamethasone most of the heterochromatin is transformed into finely dispersed euchromatin within the nuclei of carotid body chief cells. In the cytoplasm, the Golgi apparatus becomes larger and the granular endoplasmic reticulum hypertrophic. The number of catecholamines storing dense core vesicles increases considerably. The innervation density remains constant. In contrast to the carotid body chief cells, the adrenal medullary cells have not reached their definitive maturity at the time of birth. Besides phenotypes of adrenaline-cells, noradrenaline-cells and small granules containing cells, pheochromoblasts and intermediary cells can be seen as well. Their cytoplasm is sparse, the concentration of dense core vesicles and the innervation density very low. After 8 days of postnatal ontogenesis, pheochromoblasts and intermediary cells are no longer present in the adrenal medulla. In adrenaline-cells and noradrenaline-cells, important processes of growth can be noticed, the cytoplasm has grown in extent, the number of dense core vesicles doubled and the innervation density of single cells triplicated. Only the few small granules containing cells remain small. Under the effect of dexamethasone also in the nuclei of chromaffin cells a transformation of heterochromatin into euchromatin occurs. The increase in number of dense core vesicles is relatively lower than in carotid body chief cells. The significant growth of innervation density during the first postnatal week was inhibited. Our observations suggest that dexamethasone stimulates the synthesis of catecholamines in adrenal medullary cells of newborn rats less pronouncedly than in carotid body chief cells. This could be attributed to the inhibited formation of synapses of growing chromaffin cells and to the in vivo active endocrine counter-regulation.     

Effects of 2-deoxyglucose on plasma catecholamines in adult and aged rats

To examine the effects of aging on the responsiveness of the sympathetic-adrenal medullary system, I have measured plasma levels of norepinephrine (NE) and epinephrine (EPI) in adult (6 months old) and aged (24 months old) Fischer 344 male rats. Two days prior to testing, rats were surgically prepared with chronic tail artery catheters to permit remote sampling of blood in conscious, unrestrained animals. Following collection of basal blood samples, each rat received a single injection of 2-deoxyglucose (2-DG, 250 or 500 mg/kg, IP) and additional blood samples were collected 1, 2 and 4 hours later. 2-DG, a glucose analogue, stimulates a centrally mediated activation of the adrenal medulla and to a lesser extent the postganglionic sympathetic neurons. For purposes of analysis, data were excluded from animals which died within 4 hours after injection. Basal plasma levels of both catecholamines were similar in adult and aged rats. Administration of 2-DG was attended by significant and sustained increases in plasma NE and EPI in rats of both ages. A greater proportion of aged rats died following administration of 2-DG compared to adult rats. At the higher dose of 2-DG, plasma levels of NE were significantly higher in 6 month old rats at 1 and 2 hours post-injection. In contrast, plasma levels of EPI were significantly higher in 24 month old rats at 1 and 2 hours after administration of 250 mg/kg 2-DG and at 1 hour after administration of 500 mg/kg 2-DG. These findings indicated that the adrenal medullary response of aged rats to an intense and prolonged stressor is greater than that of adult rats. The attenuated plasma NE response of aged rats to 2-DG may be due to a lessor release of NE from the adrenal medulla and/or a diminished activation of postganglionic sympathetic nerves.     

Guanylate cyclase from bovine adrenal medulla: Subcellular distribution and studies on the effect of lysolecithin on enzyme activity

The localization of guanylate cyclase in bovine adrenal medulla was explored by preparation of subcellular organelles. Forty per cent of the activity was recovered in the soluble fraction (cytosol), and 46% in the low-speed (800g) pellet as particulate. In order to determine the exact localization of the particulate enzyme, continuous sucrose density gradient centrifugation was used. The results indicate that guanylate cyclase activity sedimented together with acetylcholinesterase and adenylate cyclase, two plasma membrane markers. Enrichment of specific activity over that present in the crude chromaffin granule fraction was identical for these three enzymes (from 4.4 to 6.2-fold). Particulate guanylate cyclase appears therefore to be a constituent of plasma membranes.The non-ionic detergent Triton X-100 and the natural phospholipid, lysolecithin, stimulated particulate guanylate cyclase activity 20-fold, but that of the soluble enzyme only 2- to 3-fold. The kinetic behaviour of particulate guanylate cyclase was not altered by treatment with lysolecithin: the Hill n coefficient for guanosine 5′-triphosphate remained close to 1.35 and the S_0.5 value to 320 μM. Among lecithin, lysophosphatidyl ethanolamine and phosphatidyl serine, lysolecithin was the only phospholipid to induce activation of particulate guanylate cyclase. Incubation of membranes with phospholipase A₂ led to a 5-fold stimulation of particulate guanylate cyclase activity, while the soluble enzyme activity was not affected. These results suggest a possible role for lysolecithin in the regulation of the intracellular levels of cyclic guanosine monophosphate during or following the excitation-secretion coupling process in the medullary cell.     

Effects of 2-deoxyglucose on plasma catecholamines in adult and aged rats

To examine the effects of aging on the responsiveness of the sympathetic-adrenal medullary system, I have measured plasma levels of norepinephrine (NE) and epinephrine (EPI) in adult (6 months old) and aged (24 months old) Fischer 344 male rats. Two days prior to testing, rats were surgically prepared with chronic tail artery catheters to permit remote sampling of blood in conscious, unrestrained animals. Following collection of basal blood samples, each rat received a single injection of 2-deoxyglucose (2-DG, 250 or 500 mg/kg, IP) and additional blood samples were collected 1, 2 and 4 hours later. 2-DG, a glucose analogue, stimulates a centrally mediated activation of the adrenal medulla and to a lesser extent the postganglionic sympathetic neurons. For purposes of analysis, data were excluded from animals which died within 4 hours after injection. Basal plasma levels of both catecholamines were similar in adult and aged rats. Administration of 2-DG was attended by significant and sustained increases in plasma NE and EPI in rats of both ages. A greater proportion of aged rats died following administration of 2-DG compared to adult rats. At the higher dose of 2-DG, plasma levels of NE were significantly higher in 6 month old rats at 1 and 2 hours post-injection. In contrast, plasma levels of EPI were significantly higher in 24 month old rats at 1 and 2 hours after administration of 250 mg/kg 2-DG and at 1 hour after administration of 500 mg/kg 2-DG. These findings indicated that the adrenal medullary response of aged rats to an intense and prolonged stressor is greater than that of adult rats. The attenuated plasma NE response of aged rats to 2-DG may be due to a lessor release of NE from the adrenal medulla and/or a diminished activation of postganglionic sympathetic nerves.