Effects of leptin and leptin fragments on steroid secretion and proliferative activity of regenerating rat adrenal cortex

Leptin, an adipose tissue-secreted hormone, acts via several isoforms of specific receptors (Ob-Rs), which may variously interact with the native leptin molecule and its fragments. Evidence has been provided that leptin affects rat adrenal functions, but the results were rather conflicting depending on the experimental condition examined (e.g. regenerating vs. mature or immature adrenal gland). Hence, we investigated the effects of three subcutaneous injections of murine leptin(1-147) and several leptin fragments (3 nmol/100 g body weight; 28, 16 and 4 h before the sacrifice) on the secretory activity and growth of regenerating rat adrenal cortex. The following leptin fragments were tested: murine leptin(116-130), and human leptin fragments 150-167, 138-167, 93-105, 22-56 and [Tyr]26-39. Leptin(1-147) enhanced plasma concentration of both aldosterone and corticosterone. The blood level of aldosterone was raised by leptin(116-130), leptin(138-167) and leptin(93-105), and that of corticosterone by leptin(93-105) and Tyr-leptin(26-39). Metaphase index (stachmokinetic method with vincristine) was unaffected by leptin(1-147), and lowered by leptin(116-130), leptin(150-167) and leptin(138-167). Collectively, our findings allow us to conclude that leptin and leptin fragments enhance the secretory activity and inhibit the growth of regenerating rat adrenal cortex, the biological activity of leptin being located in the C-terminal segment of its molecule.     

Effects of adrenomedullin and its fragment 22-52 on basal and ACTH-stimulated secretion of cultured rat adrenocortical cells

Adrenomedullin (ADM) and its receptors are expressed in the adrenal cortex, where ADM is currently known to inhibit agonist-stimulated aldosterone secretion from zona glomerulosa (ZG), without affecting either basal aldosterone release or the secretory activity of zona fasciculata-reticularis (ZF/R) cells. These functional findings have been obtained using freshly dispersed adrenocortical cells, but evidence has been provided that ADM is able to enhance basal aldosterone secretion from rat capsule-ZG preparations. Hence, we investigated the effect of ADM and ADM22-52, a putative antagonist of ADM receptors, on the secretory activity of rat adrenal cell cultured in vitro for 72 h. Cultures were exposed for 3 or 24 h to 10(-7) M ADM and/or ADM22-52, in the absence or the presence of 10(-8) M ACTH, and the concentration of aldosterone and corticosterone in the culture medium was measured by radioimmune assay. ADM and/or ADM22-52 raised basal aldosterone secretion at 3 h, but not 24 h exposure, and did not affect ACTH-stimulated aldosterone production. Corticosterone secretion was not changed at 3 h. In contrast, at 24 h exposure ADM22-52 alone or with ADM decreased basal corticosterone secretion; ADM evoked a small rise in ACTH-stimulated corticosterone production, and the effect was annulled by ADM22-52. These puzzling findings are interpreted in light of the fact that i) our cultures were actually a mixture of ZG, ZF/R and medullary chromaffin cells; ii) ADM stimulates adrenomedullary cells to release catecholamines, which are able to enhance aldosterone secretion from ZG cells; and iii) the prolonged exposure to ADM may modify, under in vitro culture conditions, ZF/R cells, switching their phenotype from an ADM-unresponsive to an ADM-responsive one. Our study casts doubts on the selectivity of ADM22-52 as ADM receptor antagonist, and stresses that great caution must be used in comparing adrenal-secretion findings obtained with different in vitro techniques.     

Effect of histamine on corticosteroid secretion of isolated human and rat adrenocortical cells

Inflammation Research -     

Leptin对缺氧诱导胎肺Ⅱ型上皮细胞凋亡的影响及机制

目的： 观察瘦素(LEP)对缺氧诱导胎鼠肺Ⅱ型上皮细胞（AECⅡ）凋亡的拮抗作用，并探讨其机制。方法： 采用改良免疫黏附法原代培养胎鼠AECⅡ细胞，并用SP-A免疫细胞化学法和透射电镜进行鉴定；用含5 mmol/L连二亚硫酸钠（Na₂S₂O₄）培养液培养AECⅡ细胞12 h建立缺氧诱导细胞凋亡模型，处理组含不同浓度LEP（100-1 600 μg/L）；噻唑兰（MTT）比色法检测细胞存活情况；流式细胞术分析细胞凋亡和细胞周期；蛋白质免疫印迹法（Western blotting）检测凋亡蛋白caspase 3的表达。结果： 采用免疫黏附法获得高纯度原代培养的AECⅡ细胞，免疫细胞化学法示SP-A阳性表达，电镜下可见细胞内特征性板层小体；5 mmol/L Na₂S₂O₄能诱导AECⅡ细胞凋亡和caspase 3活化，LEP（100-1 600 μg/L） 能减轻Na₂S₂O₄所致的细胞损伤，表现为AECⅡ存活率提高、增殖指数（PI）增高及凋亡峰下降、细胞形态恢复和caspase 3活化受抑制。结论： LEP可拮抗缺氧所致AECⅡ细胞凋亡，这可能与其促使细胞周期从G₁期进入S期及抑制凋亡蛋白caspase 3活化有关。     

Endocrine disruptors and rat adrenocortical function: studies on freshly dispersed and cultured cells

The effects of four endocrine disruptors: resveratrol, diphenylolpropane (bisphenol-A; BSP), benzophenone-3 (BP3) and silymarin on the secretory and proliferative activity of rat adrenocortical cells were investigated in vitro. Resveratrol and BP3 acutely increased basal corticosterone secretion from freshly dispersed adrenocortical cells, and resveratrol and BSP enhanced ACTH-stimulated cells. The 24-h exposure to resveratrol and BP3 increased basal corticosterone production from cultured adrenocortical cells, while ACTH-stimulated secretion was increased only by resveratrol. BSP was ineffective, while silymarin decreased basal, but not ACTH-stimulated secretion. The proliferative activity of the cultured adrenocortical cells was unaffected by the tested disruptors. In conclusion, the in vitro direct effect of endocrine disruptors on adrenocortical steroidogenesis displays a great variability, which seems to depend not only on their chemical nature, but also on their dose and the duration of the exposure of the studied cells.     

LePtin对缺氧诱导胎肺H型上皮细胞凋亡的影响及机制

目的观察瘦素(LEP)对缺氧诱导胎鼠肺Ⅱ型上皮细胞(AECⅡ)凋亡的拮抗作用,并探讨其机制.方法采用改良免疫黏附法原代培养胎鼠AECⅡ细胞,并用SP-A免疫细胞化学法和透射电镜进行鉴定;用含5 mmol/L连二亚硫酸钠(Na2S2O4)培养液培养AECⅡ细胞12 h建立缺氧诱导细胞凋亡模型,处理组含不同浓度LEP(100-1 600 μg/L);噻唑兰(MTT)比色法检测细胞存活情况;流式细胞术分析细胞凋亡和细胞周期;蛋白质免疫印迹法(Western blotting)检测凋亡蛋白caspase 3的表达.结果采用免疫黏附法获得高纯度原代培养的AECⅡ细胞,免疫细胞化学法示SP-A阳性表达,电镜下可见细胞内特征性板层小体;5 mmol/L Na2S2O4能诱导AECⅡ细胞凋亡和caspase 3活化,LEP(100-1 600 μg/L)能减轻Na2S2O4所致的细胞损伤,表现为AECⅡ存活率提高、增殖指数(PI)增高及凋亡峰下降、细胞形态恢复和caspase 3活化受抑制.结论LEP可拮抗缺氧所致AECⅡ细胞凋亡,这可能与其促使细胞周期从G1期进入S期及抑制凋亡蛋白caspase 3活化有关.     

Cholinergic-nicotinic control of growth and secretion of cultured pulmonary neuroendocrine cells

Dispersed newborn hamster lung cells were established in vitro in a defined, low-serum growth medium. Neuroendocrine markers (immunohistochemistry for bombesin/gastrin-releasing peptide and calcitonin) revealed a cellular predominance of pulmonary neuroendocrine (PNE) cells. While the supernatant concentration remained stable, the concentration of PNE cell immunoreactive calcitonin (iCT) gradually declined over 4 weeks. Supplementation of the medium with nicotine for 3 weeks prevented this decline in cellular iCT. Concurrently, the number of cells and [³H]thymidine incorporation were significantly increased. The stimulatory effect of chronic nicotine was reversed by the coadministration of the nicotinic antagonist hexamethonium. In another set of experiments, prior multiple transplacental nicotine pretreatments resulted in a significant increase in iCT in the lungs of newborns; when these lungs were subsequently placed in cell culture without nicotine, despite the higher concentration of iCT, there was a drop in iCT similar to that observed in the control culture. In contrast, in vivo, the lung iCT remained significantly elevated at 1 week postparturition. Cell culture supernatants were analyzed at week 4 for the evoked release of iCT; cholinergic-nicotinic agonists promptly increased the supernatant iCT, which was blocked by nicotinic but not by muscarinic antagonists. We suggest that this in vitro system provides a useful tool to study directly the PNE cell. The acute and chronic effects of nicotine are most likely related to stimulation of cholinergic-nicotinic receptors on iCT-containing PNE cells. © 1993 Wiley-Liss, Inc.     

Calcium action potentials in cultured adrenocortical cells

Membrane potential has been recorded in Y-1 adrenocortical cells with intracellular microelectrodes. Resting potential averaged –68±13 mV (n=63). Cells were silent at rest but depolarization by current pulses evoked repetitive action potentials of 80–120 mV amplitude and variable duration (between 10–300 ms). Action potentials were unaffected by removal of external Na or addition of TTX, however they were completely abolished by substitution of external Ca by Co. In Ca-free solutions with 2.4 mM Ba, action potentials had a lower threshold and lasted for the whole duration of the current pulse.     

Effect of compound 48/80 on mast cells and histamine levels in rat brain and on corticosterone secretion

Inflammation Research -     

血管紧张素Ⅳ促新生大鼠心肌细胞生长的实验研究

目的： 研究血管紧张素Ⅳ（Ang Ⅳ）对心肌细胞生长的影响及特点。 方法： 体外培养新生大鼠心肌细胞，将培养的心肌细胞分为对照组、不同浓度和不同干预时间组。分别用改良的Bradford法、流式细胞仪检测Ang Ⅳ对新生大鼠心肌细胞的蛋白表达及细胞周期的影响。 结果： Ang Ⅳ使心肌细胞的蛋白量明显大于对照组（P<0.05），并且存在着时间依赖性(36、48 h时，P<0.01)；而且，不同浓度的Ang Ⅳ均加速心肌细胞由静止期、 DNA合成前期向DNA合成期的转换，使DNA合成期及DNA合成后期的细胞数增加，促进心肌细胞的生长；且10-6mol/L的AngⅣ比10-5mol/L的AngⅣ使更多的细胞从G1期向S期转化。 结论： AngⅣ可能是通过AT4介导，对新生大鼠心肌细胞具有直接促蛋白合成及促进心肌细胞生长，并各自存在着时间及浓度依赖性。