Cholecystokinin octapeptide: Vesicular localization and calcium dependent release from rat brain in vitro

Sub-cellular fractionation tissue from rat hypothalamus and cerebral cortex in sucrose gradients indicated a concentration of cholecystokinin-like peptides in synaptosomal fractions. Lysis of synaptosomes yielded a vesicle rich fraction which was further enriched in cholecystokinin-like peptides, particularly the octapeptide (CCK-8). In vitro release experiments carried out using rat cerebral cortex tissue slices showed a calcium dependent release of cholecystokinin (primarily as CCK-8). The demonstration of a vesicular localization and calcium evoked release of cholecystokinin is consistent with a role for cholecystokinin as a neurotransmitter/neuromodulator in the CNS.     

Epidermal overexpression of granulocyte-macrophage colony-stimulating factor induces both keratinocyte proliferation and apoptosis.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is released by keratinocytes in sizeable amounts only under pathological conditions, e.g., after topical application of a tumor promoter, in atopic dermatitis (AD), and after wounding. To study the biological function of this cytokine release, we generated transgenic mice that constitutively overexpress GM-CSF in the epidermis. An increase in the numbers of mast cells and Langerhans cells (LCs) in transgenics versus nontransgenic controls was observed but no severe inflammation. This is consistent with a central role of this cytokine in the development and maturation of LCs. Mitotic activity in the epidemnis of transgenic mice was elevated, but epidermal thickness and differentiation were normal. Homeostasis is maintained by an increase of apoptosis in the epidermis. We describe the differential expression of regulators of apoptosis and discuss a potential mechanism for this novel proapoptotic activity of GM-CSF on keratinocytes. Both stimulation of proliferation and promotion of apoptosis are of great relevance to tumorigenesis. The latter may be a means of removing damaged cells after genotoxic stress or injury.     

Cyclic stretch induces both apoptosis and secretion in rat alveolar type II cells

We examined the effects of short-term cyclic stretch on both phosphatidylcholine (PC) secretion and apoptosis in primary cultures of rat alveolar type II cells. A 22% cyclic stretch (3 cycles/min) was applied to type II cells cultured on silastic membranes using a Flexercell strain unit. This induced, after a lag period of about 1 h, a small, but significant release of [3H]PC from prelabelled cells. In addition, stretch increased nuclear condensation, the generation of oligosomal DNA fragments and the activation of caspases. Similar responses were triggered by sorbitol-induced osmotic shock, but not by the secretagogue ATP. We conclude that stretch can induce both apoptosis and PC secretion in alveolar type II cells and propose that these diverse responses occur within the lung as a consequence of normal respiratory distortion of the alveolar epithelium.     

Cholecystokinin octapeptide releases growth hormone from the pituitary in vitro

Cholecystokinin-octapeptide (CCK-8)(10^?6 to 10^?8M) produced a marked increase in growth hormone (GH) release from incubated rat anterior pituitary quarters and from cultured GH₃ pituitary tumor cells. Although several CCK-8 analogues also caused GH release, bombesin, secretin and pancreatic polypeptide had no effect on GH secretion $\text{in vitro}$. In the GH₃ cell line, CCK-8 (10^?7M) reversed the inhibitory effect of somatostatin (10^?5M) on GH release. As CCK immunoreactivity has been demonstrated to be present in the hypothalamus, these results suggest that CCK-8 may be a physiologically important growth hormone releasing factor.     

Cholecystokinin octapeptide, proglumide, and passive avoidance in rats

Rats were conditioned to avoid a darkened chamber using electric footshock (0.25 mA for 2 sec). Cholecystokinin octapeptide (CCK-8), a CCK-8 antagonist proglumide, or 0.9% NaCl solution was injected immediately following the footshock to study the effect upon passive avoidance behavior. The passive avoidance behavior was observed one day following the conditioning footshock and treatment. CCK-8 produced a reduction of the passive avoidance latency of rats at doses ranging from 30 micrograms/kg to 500 micrograms/kg. Proglumide (5 mg/kg) was able to block the CCK-8 effect on rat passive avoidance conditioning. Proglumide by itself at a dose of 2 mg/kg decreased the latency to enter the darkened chamber. Endogenous CCK-8 activity may be involved in passive avoidance conditioning in rats.     

Cholecystokinin octapeptide immunoreactivity in the nucleus tractus solitarius of the cat

The nucleus tractus solitarius possessed distinct patterns of cholecystokinin immunoreactive fibers and cell bodies within its various subdivisions. The commissural, medial, intermediate, parvocellular, dorsolateral and interstitial subdivisions contained relatively dense amounts of CCK immunolabelled fibers. In contrast, CCK immunoreactivity within the ventrolateral subdivision consisted of a few scattered fibers and small neurons. The commissural, intermediate, medial, dorsolateral and parvocellular subdivisions contained CCK immunoreactive neurons following colchicine treatment. The presence of CCK in the NTS suggest that it may be involved as a neuromodulator and/or neurotransmitter in circuitry that mediate cardiovascular, respiratory, gastrointestinal and taste functions.     

八肽胆囊收缩素对大鼠颈动脉窦压力感受器反射的抑制作用

在36只隔离灌流颈动脉窦区的麻醉大鼠上,观察了八肽胆囊收缩素(cholecystokinin octapepide,CCK-8)对颈动脉窦压力感受器反射的影响。其结果如下：(1)以CCK-8(0．1、0．5、1．0μmol／L)隔离灌流颈动脉窦区时,压力感受器机能曲线向右上方移位,曲线最大斜率(peak slope,PS)减小,反射性血压下降幅度(reflex decrease,RD)减少,阈压(threshold pressure,TP)和饱和压(saturation pressure,SP)均增高。其中RD、PS和TP呈明显的剂量依赖性;(2)用CCK-8的非特异性受体拮抗剂丙谷胺(100μmol／L)预处理后,能明显减弱CCK-8(0．50mol/L)对压力感受器反射的抑制;(3)预先灌流一氧化氮合酶(nitric oxide synthase,NOS)阻断剂(L-NAME,100μmol／L),不能阻断CCK-8(0．5μmol/L)对压力感受器反射的影响;(4)用Ca^2 通道激动剂Bay K 8644(500nmol／L)预处理后,也能明显减弱CCK-8(0．5μmol／L)对压力感受器反射的抑制作用。以上结果提示,CCK-8是通过作用于颈动脉窦压力感受器神经元末梢上的受体而起到抑制作用的,其机制可能为抑制了牵张敏感性通道,致使Ca^2 离子内流减少,而与内皮细胞释放NO无关。     

Cholecystokinin octapeptide decreases intake of solid food in man

Cholecystokinin octapeptide (CCK-OP) was reported to decrease the intake of liquid food in lean and in obese man. This study investigated the effect of CCK-OP on the consumption of real life food, i.e., of standardized sandwiches. Sixteen young non-obese females and males participated, after an overnight fast, each in two experiments. After a basal 30 min, saline or CCK-OP, 1.5 or 3.0 Ivy Dog Units/kg body weight/15 min, was infused in random double blind fashion, while sandwiches were placed in front of the subjects. For the next three 15-min periods, the subjects were instructed to eat as much as they liked. In the first 15 min after 3.0 as well as 1.5 U CCK-OP/kg/15 min significantly fewer sandwiches (50 and 17 percent) were eaten than after saline (p less than 0.01 and p less than 0.05) and less hunger was reported (p less than 0.02 and p less than 0.05). Self-reported activation decreased only with 3.0 U CCK-OP (p less than 0.005). Reports of well-bring , electroencephalogram, heart rate, and respiration were not altered. The results support the notion that CCK is involved in the regulation of food intake.     

Cyclic mechanical stretch inhibits cell proliferation and induces apoptosis in fetal rat lung fibroblasts

Development of the pulmonary air sacs is crucial for extrauterine survival. Late fetal lung development is characterized by a thinning of the mesenchyme, which brings pneumocytes and endothelial cells into apposition. We hypothesized that mechanical stretch, simulating fetal breathing movements, plays an important role in this remodeling process. Using a Flexercell Strain Unit, we analyzed the effects of intermittent stretch on cell proliferation and apoptosis activation in fibroblasts isolated from fetal rat lungs during late development. On day 19, intermittent stretch increased cells in G(0)/G(1) by 22% (P = 0.001) and decreased in S phase by 50% (P = 0.003) compared with unstretched controls. Cell proliferation analyzed by 5-bromo-2'-deoxyuridine incorporation showed a similar magnitude of cell cycle arrest (P = 0.04). At this same gestational age, stretch induced apoptosis by two- to threefold over controls, assayed by DNA flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick-end labeling, and caspase-3 activation. These results indicate that mechanical stretch of fibroblasts isolated during the canalicular stage inhibits cell cycle progression and activates apoptosis. These findings are cotemporal with the mesenchymal thinning that normally occurs in situ.     

The influence of nitric oxide on basal and cholecystokinin-8-induced proliferation and apoptosis in the rat pancreas

Nitric oxide (NO) is formed by different cell types in the pancreas. In this study, inhibition of endogenous nitric oxide by N(omega)-nitro-L-arginine (L-NNA) reduced the urinary excretion of NO(2)/NO(3) and raised serum L-arginine and the NO donator S-nitroso-N-acetylpenicillamine (SNAP) increased the urinary excretion of NO(2)/NO(3). The peptide cholecystokinin-8 (CCK-8) has a strong influence on exocrine pancreatic proliferation. Rat pancreas was excised and studied with regard to tissue weight, protein and DNA contents after 3 days of treatment with saline, L-NNA or SNAP given separately or combined with CCK-8. Further, proliferation of different pancreatic cells was studied with [3H]-thymidine incorporation and apoptotic activity was studied by analysing caspase-3 activity and histone-associated DNA fragments. The effects of L-NNA indicate that endogenous nitric oxide formation has a tonic inhibition on apoptosis in the pancreas during both basal condition and growth stimulation by CCK-8. In CCK-induced hyperplasia, NO inhibits the proliferation of acinar cells but stimulates ductal cells. Endogenous NO may regulate the balance between proliferation and apoptosis and in a situation of growth stimulation by CCK-8, it has a tonic inhibition on both mitogenesis and apoptosis thus slowing down the acinar cell turnover in the pancreas.     

Cholecystokinin/pancreozymin induces the parallel discharge of digestive enzymes from the in vitro rabbit pancreas.

Considerable controversy has surrounded the question of whether the exocrine pancreas discharges digestive enzymes in a parallel or nonparallel fashion. A recent report (Rothman, S.S., and Wilking, H. (1978) J. Biol. Chem. 253, 3543-3549) claimed that the in vitro rabbit pancreas demonstrated nonparallel enzyme discharge after stimulation with cholecystokinin/pancreozymin, but that parallel discharge followed stimulation with the COOH-terminal octapeptide of cholecystokinin/pancreozymin. It was suggested that the full hormone acted to inhibit chymotrypsinogen secretion while stimulating trypsinogen secretion. Because of the fundamental importance of this question to our understanding of the exocrine secretion of exportable proteins, we have repeated these experiments using the same preparation and stimulant but have observed only parallel enzyme discharge. We conclude that it is unlikely that cholecystokinin/pancreozymin causes the selective inhibition of chymotrypsinogen secretion.     

Cholecystokinin octapeptide: effects on the excitability of cultured spinal neurons

The actions of cholecystokinin octapeptide (CCK) on the membrane properties of mouse spinal neurons grown in monolayer culture were examined using intracellular recording techniques. In a subpopulation of cells, application of CCK (0.2-100 micron) by pressure ejection from micropipettes produced a small (approximately 2 mV) membrane depolarization that was accompanied by a decrease in membrane conductance (approximately 11 percent). These effects were associated with an enhanced tendency of the cells to generate action potentials when stimulated with intracellular depolarizing current. The unsulfated analog of CCK, which possesses weak biological activity in the gut, had little or no effect on cultured spinal neurons. A number of differences were noted between the responses to CCK and the excitatory amino acid glutamate. First, the effects of CCK were more delayed in onset (approximately 17 sec) and prolonged in duration (approximately 124 sec). Second, the depolarizations produced by glutamate were of larger magnitude and associated with variable effects on membrane conductance. Third, the response to CCK showed tachyphylaxis with repeated applications whereas glutamate remained effective as often as it was applied. It is concluded that CCK facilitates the excitability of spinal neurons in a manner distinct from that of the conventional excitant glutamate.     

Anti-immunoglobulin M induces both B-lymphocyte proliferation and differentiation in Xenopus laevis

Anti-IgM induced the proliferation of spleen lymphocytes of the amphibian Xenopus laevis as determined by 3H-thymidine uptake. The responding cells were B lymphocytes, since lymphocyte populations enriched in surface-Ig-positive cells exhibited an increased proliferative response, and spleen cells from larvally thymectomized animals still responded to anti-IgM. Immunofluorescence analysis and gel electrophoresis of biosynthetically labeled Ig polypeptides revealed that lymphoblasts induced by anti-IgM differentiated into plasmablasts that synthesized and secreted mainly IgM and small amounts of IgY. The in vitro differentiation of B lymphocytes also occurred in spleen cells obtained from thymectomized animals. These findings are in contrast with those obtained in mammals and suggest that the differentiation of B lymphocytes in X. laevis is subject to different regulatory mechanisms.     

Fat-rich diet induces inflammatory changes in the intact rat pancreas

The effects of chronic ethanol ingestion combined with fat-rich, protein-rich or carbohydrate-rich diets on the histology of the intact rat pancreas were studied. 192 male Wistar rats were randomly divided into four different dietary groups. Half of each group received 15% (v/v) ethanol in their drinking solution and the rest were used as controls and given tap water. After a 12-week diet period the pancreas were removed and histological specimens were stained with hematoxylin and eosin. No significant difference was observed between the groups in occurrence of edema, but inflammatory cells were found in (9/24) rats in the fat-rich group receiving water (p less than 0.01. In the fat-rich diet group receiving ethanol this finding was observed in 5 of 24 rats. In these groups slight parenchymal cell necrosis was also observed in conjunction with the inflammatory cells. All specimens in the other groups were normal. It is suggested that inflammatory changes caused by a fat-rich diet may be due to unknown toxic effects of this diet.     

Escherichia coli induces apoptosis and proliferation of mammary cells

Mammary cell apoptosis and proliferation were assessed after injection of Escherichia coli into the left mammary quarters of six cows. Bacteriological analysis of foremilk samples revealed coliform infection in the injected quarters of four cows. Milk somatic cell counts increased in these quarters and peaked at 24 h after bacterial injection. Body temperature also increased, peaking at 12 h postinjection. The number of apoptotic cells was significantly higher in the mastitic tissue than in the uninfected control. Expression of Bax and interleukin-1beta converting enzyme increased in the mastitic tissue at 24 h and 72 h postinfection, whereas Bcl-2 expression decreased at 24 h but did not differ significantly from the control at 72 h postinfection. Induction of matrix metalloproteinase-9, stromelysin-1 and urokinase-type plasminogen activator was also observed in the mastitic tissue. Moreover, cell proliferation increased in the infected tissue. These results demonstrate that Escherichia coli-induced mastitis promotes apoptosis and cell proliferation.     

Acetaminophen induces apoptosis in rat cortical neurons

Background

Acetaminophen (AAP) is widely prescribed for treatment of mild pain and fever in western countries. It is generally considered a safe drug and the most frequently reported adverse effect associated with acetaminophen is hepatotoxicity, which generally occurs after acute overdose. During AAP overdose, encephalopathy might develop and contribute to morbidity and mortality. Our hypothesis is that AAP causes direct neuronal toxicity contributing to the general AAP toxicity syndrome.

Methodology/Principal Findings

We report that AAP causes direct toxicity on rat cortical neurons both in vitro and in vivo as measured by LDH release. We have found that AAP causes concentration-dependent neuronal death in vitro at concentrations (1 and 2 mM) that are reached in human plasma during AAP overdose, and that are also reached in the cerebrospinal fluid of rats for 3 hours following i.p injection of AAP doses (250 and 500 mg/Kg) that are below those required to induce acute hepatic failure in rats. AAP also increases both neuronal cytochrome P450 isoform CYP2E1 enzymatic activity and protein levels as determined by Western blot, leading to neuronal death through mitochondrial–mediated mechanisms that involve cytochrome c release and caspase 3 activation. In addition, in vivo experiments show that i.p. AAP (250 and 500 mg/Kg) injection induces neuronal death in the rat cortex as measured by TUNEL, validating the in vitro data.

Conclusions/Significance

The data presented here establish, for the first time, a direct neurotoxic action by AAP both in vivo and in vitro in rats at doses below those required to produce hepatotoxicity and suggest that this neurotoxicity might be involved in the general toxic syndrome observed during patient APP overdose and, possibly, also when AAP doses in the upper dosing schedule are used, especially if other risk factors (moderate drinking, fasting, nutritional impairment) are present.     

miR-376a suppresses proliferation and induces apoptosis in hepatocellular carcinoma

MicroRNAs are known to be involved in the pathogenesis of hepatocellular carcinoma (HCC). This study aims to explore the potential biological function of miR-376a, which was found to be inhibited after partial hepatectomy, in HCC. We discovered that miR-376a was frequently down-regulated in HCC cell lines and HCC tissues, while higher relative level of miR-376a was significantly associated with high serum AFP level. Over-expression of miR-376a not only inhibited proliferation but induced apoptosis in Huh7 cells. Additionally, p85α (PIK3R1) was identified as a direct and functional target of miR-376a in Huh7 cells. Moreover, we confirmed that p85α and miR-376a were inversely correlated in HCC. These findings suggest that down-regulation of miR-376a may contribute to the development of HCC by targeting p85α.