Ret protein expression in adrenal medullary hyperplasia and pheochromocytoma

Ret is a developmentally regulated tyrosine kinase involved in formation and maintenance of the nervous system. Ret mutations predisposing to pheochromocytomas and medullary thyroid carcinomas occur in multiple endocrine neoplasia (MEN) syndromes 2A and 2B. Biochemical studies have demonstrated overexpression of Ret mRNA and protein in pheochromocytomas compared to normal adrenal medulla. However, the cellular distribution of Ret in the normal human adrenal and in hyperplastic lesions that antecede pheochromocytomas are unclear. The present investigation was undertaken to resolve the histological distribution of Ret in the normal human adrenal, in pheochromocytomas evolving from adrenal medullary hyperplasia in MEN2A and in sporadic pheochromocytomas. Ret expression was studied by immunohistochemistry using both a polyclonal and a monoclonal antibody, with confirmation by immunoblotting of representative cases. Only occasional cells stained for Ret in the normal adrenal, consistent with the distribution in adult adrenals of other species. Heterogeneous, progressively increased Ret expression was observed during the evolution of pheochromocytomas. In both normal and neoplastic adrenal, the most intense immunoreactivity was observed in cells with neuron-like features. Our finding that Ret is not expressed at high levels in the early stages of disease suggests that elucidation of mechanisms that regulate Ret expression is required for understanding the pathobiology of MEN2A. The association of high-level Ret expression with neuronal morphology suggests that the variable overexpression of Ret in pheochromocytomas might in part be an epiphenomenon, reflecting the known phenotypic plasticity of these tumors.     

Effects of elevated plasma adrenaline levels on substrate metabolism, effort perception and muscle activation during low-to-moderate intensity exercise

The aim of this study was to differentiate the role of raised plasma adrenaline (Adr) concentrations from sympathoadrenal activation associated with moderate-intensity exercise, on muscle activation, cardiopulmonary responses, fuel metabolism, and ratings of perceived exertion (RPE) during low-intensity exercise. Two groups of subjects (MOD, n=6; LOW, n=7) cycled on two occasions for 90 min. MOD cycled at 68% VO_2max with saline infusion, and at 34% VO_2max with Adr infusion. LOW cycled twice at 34% VO_2max, with either Adr or saline infusion. Infusions (0.015 g Adr/kg/min) started at 15 min and increased plasma [Adr] somewhat higher than during exercise at 68% VO_2max (~1.9 vs. 1.4 nM, at 75 min). Mean plasma glucose and lactate concentrations during LOW were significantly higher with Adr than saline infusion (5.1±0.6 vs. 4.4±0.3 mmol/l, P<0.01 and 2.1±0.8 vs. 1.3±0.5 mmol/l, P<0.01, respectively). Elevated [Adr], without increased exercise intensity, did not alter glycogenolysis. There were also no effects of Adr infusion at 34% VO_2max on heart rate, oxygen consumption, [FFA], respiratory exchange ratio, intramuscular triglyceride utilization, muscle activation or RPE. In conclusion, elevated [Adr] similar to those found during moderate-intensity exercise increased plasma glucose and lactate availability, but did not alter intramuscular fuel utilization, effort perception or muscle activation.     

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.     

Phospholipase C isozymes are differentially distributed in the rat adrenal medulla

Immunohistochemistry has been used to examine the distribution of selected phospholipase C (PLC) isozymes within the adrenal medulla of the rat. PLCbeta isozymes were expressed at moderate levels in the chromaffin cells but more strongly in association with ganglion cell clusters. PLCbeta2 and PLCbeta3 staining of clusters did not overlap suggesting selective PLC isozyme expression in two distinct ganglionic types. The distribution of PLCbeta4 immunoreactivity was very similar to PLCbeta3 with the strongest staining observed in the same cell clusters. Antibodies to PLCbeta1 labelled multiple bands on Western blots and were not therefore used for immunohistochemistry. The chromaffin cells were also immunoreactive for PLCgamma1, although the strongest staining with this antibody was seen in cells surrounding large sinus vessels. PLCdelta1 and PLCdelta2 had quite distinct distributions, with the former selectively localized to an endothelial cell population surrounding the chromaffin cells. This observation was supported by experiments on isolated bovine adrenal medullary cells where PLCdelta1 expression was lost when the cell preparation was enriched for chromaffin cells. Antibodies to PLCdelta2 labelled a network of nerve fibres throughout the medulla and clusters of ganglion cells located primarily at the medullary-cortical boundary. PLCdelta2 immunoreactivity was also present in nerve fibres within the adrenal capsule where it appeared to be co-localized with PLCbeta4 staining.     

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.     

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.     

Plasma catecholamines in rats: Daily variations in basal levels and increments in response to stress

Chronic tail artery catheters were surgically implanted into male Sprague-Dawley rats to allow for repeated sampling of blood. Animals were housed individually and maintained on a controlled photoperiod (lights on 0600–1800 hours). In the first experiment, blood samples were obtained 2 days after surgery from animals at 0800, 1600, and 2400 hours and again at 0800 hours on the next day. Plasma levels of the sympathetic transmitter norepinephrine (NE) were higher at 2400 hours compared to all other sampling times. In contrast, plasma levels of the adrenal medullary hormone, epinephrine (EPI), were similar across sampling times. In a second experiment, individual rats were transferred to a shock chamber and after 3 minutes received 10 footshocks (2.0 mA, 0.5 sec duration, every 6 sec). Separate groups of rats were stressed in this manner at 0800, 1600, or 2400 hours. Basal and transfer-induced increments in plasma levels of NE and EPI did not vary with time of day. However, footshock-induced increments in plasma levels of EPI were greater at 0800 hours than at 1600 or 2400 hours. Footshock-induced alterations in plasma NE and behavior of rats did not vary with time of day. These results suggest that basal plasma levels of NE are elevated at 2400 hours due to postural changes associated with increased locomotor activity and not to an endogenous circadian rhythm. Furthermore, this effect is consistent within but not across groups of rats. The responsiveness of the adrenal medulla to the intense stress of footshock appears to be greatest in early morning compared to later afternoon and midnight.     

肺心病患者肾上腺髓质素测定的临床意义及其相关因素分析

为探讨肾上腺髓质素(ADM)在慢性肺心病发生发展中的作用, 以及吸烟、缺氧、肺功能等因素对肾上腺髓质素的影响, 测定63例慢性肺心病患者急性发作期和缓解期的ADM, 并用多元回归分析方法分析患者年龄、病程、吸烟和肺功能等因素对ADM的影响. 发现慢性肺心病患者急性发作期及缓解期ADM比正常人显著升高(P＜0.001).一秒钟用力呼气容积(FEV1)与动脉氧分压PaO2、吸烟、阻塞性通气功能障碍、缺氧是影响ADM水平的因素. 结果表明, 慢性肺心病患者ADM高于正常人, 且ADM升高与病情的严重程度有关.     

Sympatho-adrenal medullary activity and behavior during exposure to footshock stress: A comparison of seven rat strains

The effects of footshock stress on circulating levels of norepinephrine (NE) and epinephrine (EPI) and behavioral activation were examined in male rats of seven strains: spontaneously hypertensive (SHR), stroke-prone hypertensive (SP-SHR), Wistar-Kyoto (WKY), Sprague-Dawley (SD), Osborne-Mendel (OM), Brown Norway (BN), and Charles River Wistar (CRW). A catheter was inserted into the ventral caudal artery of each rat to allow for direct measurement of blood pressure and heart rate and for repeated sampling of blood in conscious, undisturbed animals. Two days after insertion of the catheter, resting levels of plasma NE and EPI were higher in SHRs than in CRWs; no other comparisons among strains were significant. No strain differences were noted in the increments in plasma NE and EPI following transfer of rats from the home cage to a shock chamber. There were, however, significant strain differences in the responses of rats to 5 min of footshock (2.5 mA, 0.4 sec duration every 5 sec). Shock-induced increments in plasma NE and EPI were greatest in SHRs and lowest in S-D, O-M, B-N and CRW strains. In addition, there was a significant positive relationship between activity and shock-induced increments in plasma NE (r=0.89, p<0.01) and EPI (r=0.93, p<0.01). These findings indicate an association between activity of the sympatho-adrenal medullary system and behavioral activation during footshock stress. In addition, responsivity to stress was greatest in those strains (SHR and SP-SHR) which are genetically predisposed to increases in blood pressure.     

Fine structure of adrenal medullary grafts in the pain modulatory regions of the rat periaqueductal gray

Summary Recent findings in our laboratory indicate that adrenal medullary grafts produce significant alterations in pain sensitivity. Electron microscopic studies were undertaken to correlate these behavioral changes with the neural interactions of the host and graft tissue in the periaqueductal gray. A striking change found 8 weeks after transplantation is that pronounced myelination has taken place both in the graft and in the host tissue. The new myelin formation in the graft has the typical appearance of PNS myelination and, in the host the appearance of CNS myelination. The endothelial cells of the capillaries in the grafted tissue are attenuated and fenestrated in contrast to those of the surrounding parenchymal tissue of the host. By 8 weeks, the graft becomes heavily encapsulated with collagen, while the host CNS tissue develops layers of glial processes outlining the graft. However, collagen and glial layers apparently do not form an absolute barrier to either cellular or humoral interaction between the host and graft tissue. Chromaffin cells can be found protruding into the host CNS tissue and sometimes forming synapses with presumably the host neuronal processes. Grafted chromaffin cells may participate as both postsynaptic and, less often, as presynaptic components of synaptic junctions. The behavioral relevance of these synaptic contacts is unclear, since similar implants of adrenal medullary tissue into the dorsal spinal cord subarachnoid space, which also induce potent analgesia, do not contain synapses. Thus, it is more likely that behavioral changes are brought about by diffusion of neuroactive substances from grafted chromaffin cells to host receptors.     

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.     

Stimulation of a bulbospinal 5-HT pathway in the rat brain produces hyperglycaemia

The glucoregulatory role of spinally projecting serotonin (5-HT) neurones near the ventrolateral surface of the medulla oblongata was investigated by stimulating these nerve cells in normal rats and in rats with selective chemical ablation of 5-HT nerves in the spinal cord. Electrical stimulation of the lateral medulla produced hyperglycaemia in normal rats; the increase in blood glucose was proportional to the intensity and frequency of stimulation. Furthermore, microinjection of kainic acid or L-glutamate at the same sites also produced hyperglycaemia. This stimulation-induced hyperglycaemia was significantly reduced by spinal transection or adrenalectomy. Selective destruction of spinal 5-HT nerves produced by intraspinal injection of 5,7-dihydroxytryptamine also reduced the magnitude of the hyperglycaemia response to electrical stimulation of the lateral medulla. This indicates that stimulation of 5-HT nerve cells adjacent to the ventrolateral surface of the medulla oblongata and projecting to the spinal cord increases the adrenal-sympathetic efferent activity and leads to hyperglycaemia in rats.     

Histomorphometric and sympathetic innervation of the human internal thoracic artery

INTRODUCTION:

Internal thoracic artery (ITA) is an established arterial graft for the coronary artery by‐pass surgery. Special micro‐anatomical features of the ITA wall may protect it from age related pathological changes. One of the complications seen after coronary artery bypass grafting is vasospasm. Sympathetic nerves may be involved in vasospasm.

OBJECTIVE:

To ascertain the sympathetic innervation of the internal thoracic artery and to assess the effect of aging on this artery by histomorphometry.

METHOD:

Fifty‐four human internal thoracic artery samples were collected from 27 cadavers (19 male and 8 female) with ages of 19 to 83 years. Samples were divided into three age groups: G1, 19–40 years; G2, 41–60 years; G3, ≥61 years. Sections (thickness 5 µm) of each sample were taken and stained with hematoxylin–eosin and Verhoeff–Van Gieson stains. Five of fifty‐four samples were processed for tyrosine hydroxylase immunostaining.

RESULTS:

The thickness of the tunica intima was found to be constant in all age groups, whereas the thickness of the tunica media decreased in proportion to age. Verhoeff–Van Gieson staining showed numerous elastic laminae in the tunica media. Significant differences (p<0.0001) in the number of elastic laminae were found between G1 with G2 cadavers, between G2 and G3 cadavers and between G3 and G1 cadavers. Tyrosine hydroxylase immunostaining demonstrated sympathetic fibers, located mainly in the tunica adventitia and the adventitia–media border. The sympathetic nerve fiber area and sympathetic index were found to be 0.0016 mm² and 0.012, respectively.

DISCUSSION:

Histology of the ITA showed features of the elastic artery. This may be associated with lower incidence of Atherosclerosis or intimal hyperplasia in ITA samples even in elderly cases. Low sympathetic index (0.012) of ITA may be associated with fewer incidences of sympathetic nervous systems problems (vasospasm) of the ITA.

CONCLUSION:

Sympathetic nerve fibers are present in the adventitia of the internal thoracic artery. This is an elastic artery, although anatomically it is considered to be medium‐sized. The sympathetic index may be used for analysis of sympathetic nerve fiber‐related problems of the internal thoracic artery.     

Physiological and behavioral responses of New Zealand hypertensive and normotensive rats to stress

A chronic catheter was inserted into the ventral tail artery of adult male New Zealand hypertensive (NZH) and normotensive (NZN) rats to allow for repeated sampling of blood and measurement of blood pressure and heart rate in conscious animals without handling. Two days after surgery, plasma levels of norepinephrine (NE) and epinephrine (EPI) were similar in NZH and NZN rats while resting and undisturbed in their home cages. Mean arterial blood pressure was significantly higher in NZH rats (166±9 mm Hg) than in NZN rats (124±4 mm Hg) but basal heart rates did not differ (345±8 and 342±14 beats/min, respectively). Increments in plasma levels of NE and EPI and in mean arterial blood pressure and heart rate were similar in NZH and NZN rats following transfer to a shock box and immediately and 10 minutes after exposure to 1 minute of intermittent footshock. Male rats of the two strains also did not differ in their behaviors during tests in an open field arena. These results indicated that NZH and NZN rats do not differ with respect to basal or stress-induced increments in sympathetic-adrenal medullary activity or in several behavioral measures. These results are in striking contrast to previous studies with the Okamoto strain of spontaneously hypertensive (SHR) rats and indicate that genetically determined increases in arterial blood pressure are not necessarily associated with sympathetic-adrenal medullary and behavioral hyperresponsivity to stress.     

Differential occurrence and distribution of galanin in adrenal nerve fibres and medullary cells in rodent and avian species

The presence and distribution of galanin (GAL) in adrenal glands of rodent and avian species was investigated by light microscopic immunohistochemistry. GAL immunoreactivity was found in all medullary cells of guinea pig, duck and chicken adrenals. In contrast, only a subpopulation of medullary cells stained for GAL in Phodopus (Djungarian hamster) while the neuropeptide was completely missing in chromaffin cells of rat and pigeon. In rat, guinea pig and pigeon, GAL-immunoreactive nerve fibres were frequent in subcapsular regions and sparse in deeper cortical layers and in the chromaffin tissue. In contrast, only very few GAL fibres were found in Phodopus and no GAL fibres were observed in the adrenal glands of duck. In the chicken adrenal gland, fibres containing GAL were numerous throughout the organ and occurred in close vicinity to both steroidogenic as well as catecholaminogenic cells. The striking differences in the presence of GAL-positive cells and fibres are more pronounced between species within the rodent or avian group, respectively, than between the different vertebrate orders. The hitherto unknown and surprising variability of GAL expression and distribution in adrenal glands of various species suggests species-dependent functional (autocrine, paracrine and/or endocrine) roles of GAL in the neuroadrenal axis.     

Sympatho-adrenal activity of SHR and WKY rats during recovery from forced immobilization

Circulating levels of norepinephrine (NE), epinephrine (EPI), dopamine-β-hydroxylase (DBH), and corticosterone were compared in spontaneously hypertensive (SHR) and Wistar-Kyoto normotensive (WKY) rats during recovery from a 2-hr period of immobilization stress. On the day before the experiments, a catheter was inserted into the ventral caudal artery of each rat to measure directly blood pressure and heart rate and to obtain samples of blood from conscious, unhandled rats. Immediately prior to the period of forced immobilization, while animals were at rest in their home cages, plasma levels of NE and EPI did not differ between rats of the 2 strains. After 2 hr of immobilization, circulating levels of NE, EPI, and DBH were significantly higher in SHR rats than in WKY rats. In contrast, increments in mean blood pressure after 2 hr of stress were greater in WKY rats. When compared to WKY rats, SHR rats had significantly higher circulating levels of NE, EPI, and DBH for up to 4 hr after the period of immobilization. These results are consistent with the view that the sympatho-adrenal medullary system of SHR rats is more responsive than WKY rats to immobilization stress. In addition, in SHR rats, the sympatho-adrenal system remains in a heightened state of activity for a longer period of time following stressful stimulation. The excessive and prolonged discharge of catecholamines into the circulation during stress may be an important factor in the etiology of this form of experimental hypertension.