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1.
The amounts and time courses of dopamine and ATP released from perfused PC12 cells were examined using a simultaneous on-line recording system. High KCl (60 mM) caused dopamine and ATP release with similar time courses. The relative amount of dopamine to ATP in the effluent was 9.5. In PC12 cells cultured with dexamethasone, reserpine or bafilomycin A1 for 2 days, these drugs did not affect increases of intracellular Ca2+ in response to high KCl. Dexamethasone doubled the amount of dopamine release induced by high KCl without changing the amount of ATP release. High KCl failed to cause dopamine release in reserpine-treated cells but evoked ATP release. Bafilomycin A1 decreased both high KCl-induced dopamine and ATP release. The ratio of released ATP to total adenine nucleotides and adenosine in response to high KCl was not changed by treatment with the drugs. These results suggest that dopamine and ATP are simultaneously released from secretory vesicles of PC12 cells, in which they are stored via different pathways. Similar to dopamine uptake into secretory vesicles, the H+-gradient across the vesicular membrane developed by vacuolar ATPase may play an important role in the vesicular uptake of ATP.  相似文献   

2.
Rotenone is an inhibitor of mitochondrial complex I that produces a model of Parkinson’s disease (PD), in which neurons undergo dopamine release dysfunction and other features. In neurons, exocytosis is one of the processes associated with dopamine release and is dependent on Ca2+ dynamic changes of the cell. In the present study, we have investigated the exocytosis of dopamine and the involvement of Ca2+ in dopamine release in PC12 cells administrated with rotenone. Results demonstrated that rotenone led to an elevation of intracellular Ca2+ through Ca2+ influx by opening of the voltage-gated Ca2+ channel and influenced the soluble N-ethylmaleimide attachment protein receptor (SNARE) proteins expression (including syntaxin, vesicle-associated membrane protein 2 (VAMP2) and synaptosome-associated protein 25 (SNAP-25)); pretreatment with a blocker of L-type voltage-activated Ca2+ channels (nifedipine) decreased the intracellular dopamine levels and ROS formation, increased the cell viability and enhanced the neurite outgrowth and exocytosis of synaptic vesicles. These results indicated that the involvement of intracellular Ca2+ was one of the factors resulting in suppression of dopamine release suppression in PC12 cells intoxicated with rotenone, which was associated with the rotenone-induced dopamine neurotoxicity.  相似文献   

3.
Cell secretion and membrane fusion   总被引：4，自引：0，他引：4  
Secretion occurs in all cells of multicellular organisms and involves the delivery of secretory products packaged in membrane-bound vesicles to the cell exterior. Specialized cells for neurotransmission, enzyme secretion or hormone release utilize a highly regulated secretory process. Secretory vesicles are transported to specific sites at the plasma membrane, where they dock and fuse to release their contents. Similar to other cellular processes, cell secretion is found to be highly regulated and a precisely orchestrated event. It has been demonstrated that membrane-bound secretory vesicles dock and fuse at porosomes, which are specialized supramolecular structures at the cell plasma membrane. Swelling of secretory vesicles results in a build-up of pressure, allowing expulsion of intravesicular contents. The extent of secretory vesicle swelling dictates the amount of intravesicular contents expelled during secretion. The discovery of the porosome, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution into artificial lipid membrane, have been determined. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and vesicle swelling, has also been resolved. These findings reveal the molecular mechanism of cell secretion.  相似文献   

4.
Most species of Corydoras exhibited a reproductive behaviour called 'T-position', and exhibited an accessory gland in the male genital tract, called the seminal vesicle. It appeared that both the structure and the composition of the fluid varied considerably between the species investigated. Consequently, different opinions were proposed regarding the possible role of seminal vesicle on this particular reproductive behaviour. Male adults of Corydoras aeneus were collected, anaesthetized, and samples of seminal vesicle were fixed in Bouin's solution. The sections were stained with haematoxylin-eosin and periodic acid Schiff. The seminal vesicle showed a system of anastomosed secretory tubules, forming a vesicular collective network, which gave rise to the vesicular ducts. The latter fused with the testicular efferent ducts and formed the spermatic ducts. Considering this fusion, when the sperm cells reached the spermatic ducts, the fluid produced at the seminal vesicle covered them. Histochemical studies evidenced the presence of neutral and acid glycosaminoglycans in the seminal fluid. Considering the reproductive behaviour of C. aeneus, it is believed that the protection associated with the immobilization of the sperm cells assures the sperm integrity during the passage through female's intestine until fertilization.  相似文献   

5.
To define the role of activated neutrophils in lung injury during bovine respiratory tract infections (BRTI) their in vitro function was investigated. As a means to achieve this goal the comparison of secretory action between neutrophils from the BRTI group and control was made on the basis of elastase, myeloperoxidase (MPO), alkaline phosphatase (ALK-P) release, and nitric oxide production. We noted that there is an interdependence between secretory response of neutrophils and clinical severity of BRTI. The release of elastase was greater in the BRTI group than in the control group (49.17+/-4.41 versus 46.43+/-4.95% of the total content). Neutrophils from infected heifers exhibited a significantly (p<0.05) higher value of MPO release than from healthy heifers and reached 39.23+/-10.18 versus 25.54+/-8.41% of the total content. ALK-P containing granules released significantly (p<0.001) more enzyme in the group with BRTI than in the control group (22.42+/-6.27 versus 13.74+/-2.01% of the total enzyme content). The level of nitrite accumulation rose in the culture of cells isolated from heifers with BRTI from 4+/-0.53 microM after 0.5h to 6.9+/-0.52 microM after 72 h. Our data suggest that during BRTI the increase of neutrophil secretory action results in augmentation of enzyme release including elastase, MPO and ALK-P, and the nitrite production. During an excessive secretory response of neutrophils all these factors contribute to lung injury and worsen the course of a disease and might be recognised as markers of lung injury. Moreover, such a destructive action of neutrophils must be taken into account during the introduction of new methods of BRTI treatment.  相似文献   

6.
The effects of K+ channel blockers and P2Y receptor agonist/antagonist on the vasorelaxation mediated by endothelium-derived hyperpolarizing factor (EDHF) were investigated in the rabbit renal artery. Acetylcholine (ACh, 1 nM-10 microM) induced endothelium-dependent relaxation of arterial rings precontracted with norepinephrine (NE, 1 microM) in a concentration-dependent manner. NG-nitro-L-arginine (L-NAME. 0.1 mM), an inhibitor of NO synthase, partially inhibited the ACh-induced endothelium-dependent relaxation. The ACh-induced relaxation was only partially inhibited by L-NAME whereas combined addition of L-NAME and 30 mM KCl completely inhibited the relaxation. The ACh-induced relaxation observed in the presence of L-NAME was significantly reduced by a combination of iberiotoxin (0.1 microM) and apamin (1 microM), and almost completely blocked by 4-aminopyridine (5 mM). The ACh-induced relaxation was antagonized by P2Y receptor antagonist, cibacron blue (10 and 100 microM) in a concentration-dependent manner. Furthermore, ADPbetaS, a potent P2Y agonist, induced the endothelium-dependent relaxation, and this relaxation was markedly reduced by either the combination of iberiotoxin and apamin or by cibacron blue alone. In conclusion, ACh may activate the release of ATP from endothelial cells which in turn activates a P2Y receptor on the endothelial cells followed by a release of EDHF, resulting in a vasorelaxation via a mechanism that involves activation of both the voltage-gated K+ channels and the Ca2+-activated K+ channels. EY WORDS: ATP, K+ channel, rabbit renal artery.  相似文献   

7.
Loop diuretics are derivatives of 4-sulfamoylbenzoic acid, which derived originally from sulfonamides. Their diuretic effect is due to the inhibition of the Na-K-Cl-cotransport system in the distal part of Henle's loop. The compounds react with different affinity with the chloride binding site of the transporter. Bumetanide is among the most potent blockers with an IC50 of 0.2 microM. The compound was developed by P. W. FEIT, 1971; the saluretic response was described first by H. H. FREY 1972. Bumetanide has outgrown to become a tool for physiologists and pharmacologists in renal transport research. The compound has essentially contributed to the elucidation of the mechanisms of volume regulation of cells. Bumetanide is taken up into tubule cells and hepatocytes by active transport. The uptake in tubule cells ist mediated by an organic anion transporter, which is involved in the renal secretion of drugs. In the liver bumetanide is transporter by the bile acid carrier. The carrier is a multispecific drug transporter, too. It is not yet known, whether both drug transport systems contain identical membrane proteins. For this purpose bumetanide is currently used to investigate by photoaffinity labeling and functional expression cloning molecular principles of the drug elimination in kidney and liver.  相似文献   

8.
Glutamate (Glu), the major excitatory neurotransmitter in vertebrate central nervous system, is actively taken up and stored in synaptic vesicles. On the arrival of an action potential to the pre‐synaptic membrane and the subsequent opening of the voltage‐gated calcium channels and movement of Ca2+ into the neuron, these small vesicles fuse with the pre‐synaptic membrane to release the neurotransmitter content into the synaptic cleft. Because it has previously been shown that intracerebroventricular (ICV) glutamate plays a role in feed intake in broilers, the manipulation of its vesicular concentration could affect feeding behaviour. On the contrary, research on vesicular glutamate transporters has, so far, been carried out on mammalian species. In the present study, we aim to examine the effect of Chicago sky blue 6B (CSB6B), a glutamate vesicular uptake inhibitor, on feed intake and latency to start feeding in a commercial strain of meat type chickens. To do this, four experiments have been designed to investigate the effect of ICV injection of saline, glutamate, as a general agonist for glutamate receptors, CSB6B and the combination of Glu and CSB6B. The findings indicate that CSB6B increases feed intake and decreases the latency to start feeding in 24‐h‐feed‐deprived Ross 208 broilers.  相似文献   

9.
Immunohistochemistry was applied to determine the distribution patterns of nerve fibres containing tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and vesicular acetylcholine transporter (VAChT) in the prostate, seminal vesicle (SV) and bulbourethral glands (BU) of male sheep. In all organs studied, cholinergic innervation was more developed than noradrenergic innervation. Numerous VAChT-immunoreactive (IR) nerve fibres were found in the muscular layer and mucosa of the SV and BU as well as in the prostate. A similar abundance of noradrenergic nerve fibres (showing immunoreactivity both to TH and DbetaH) was also found in both layers of the SV and BU (but not in the prostate). In the prostate a moderate density of VIP-IR nerve fibres was present but only very scarce NPY-IR nerve fibres were shown. All the studied accessory sexual glands (ASG) of male sheep contained VIP-IR nerve fibres in a similar frequency. Double immunohistochemistry revealed that the vast majority of noradrenergic nerve fibres also contained NPY. None of the noradrenergic nerve fibres showed the presence of VAChT or VIP. The possible functional significance of these findings is discussed.  相似文献   

10.
The ultrastructure of Sertoli cells in the seminiferous tubules of water buffaloes before and during sexual maturity was studied by transmission electron microscopy, with emphasis on the intranucleolar vesicular elements. Sertoli cells of animals under 12 months of age were distinguished from the germ cells by the presence of electron dense membrane bound bodies within their cytoplasm. These cells, referred to as basal indifferent supporting cells, were probably involved in the phagocytosis and elimination of degenerating spermatocytes, which failed to differentiate into spermatids and spermatozoa in animals under one year of age. In 12 month old animals, a few Sertoli cells exhibiting the vesicular elements appeared in the nucleolar region while in animals over 15 months of age Sertoli cells could be positively identified by the characteristic cytoplasm containing microtubules, elongated and electron dense mitochondria, extensive granular endoplasmic reticulum and the presence of spermatids in various stages of spermiogenesis. The vesicular elements in the nucleolar region of the Sertoli cells were most prominent at this stage. Ultrastructural features of the Sertoli cells revealed an abundance of ribosome-like particles surrounding the vesicles of varying size. Some of these vesicular elements contained amorphous material suggesting that they represent the products sequestered in the nuclear region for transport to the cytoplasm and that the process of spermiogenesis may be dependent on the ability of Sertoli cells to generate these products at sexual maturity.  相似文献   

11.
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12.
A study of the crista ampullaris of the vestibular apparatus was carried out in chicken embryos. The study group included embryos between stages 24 and 39 of Hamburger-Hamilton. This study elucidates the relationship of the cupula with respect to the epithelium of the crista ampullaris. With electron microscopic examination, the rough endoplasmic reticulum of the crista epithelial sustentacular cells at developmental stage of 31 H-H, demonstrated dilatations containing secretory material. Vesicles, with adhering ribosomes appear to be formed from these dilatations. At later stages of development, the vesicular material took on the characteristics of the fibrillary material composing the cupula. In some cells, secretory vesicles are seen near the apical border of these cells, where they apparently secrete vesicular contents into the endolymphatic space, contributing to the formation of the cupula.  相似文献   

13.
Ergocryptine is an ergot alkaloid that affects dopaminergic activity principally by interacting with D2-type receptors. In this study the ability of ergocryptine and several other ergot alkaloids to release [3H]dopamine from isolated nerve endings was demonstrated using in vitro superfusion of rat striatal synaptosomes. Ergocryptine, ergocristine, and bromocryptine produced an elevation in baseline dopamine release of approximately 400% with effective concentrations (EC50) of approximately 30 microM. Ergotamine, ergonovine, ergovaline, and ergocornine were devoid of activity. The time-course of the ergocryptine-stimulated release was relatively slow compared with amphetamine, nicotine, or K+-stimulated [3H]dopamine release; the maximal increase in release required a 5-min treatment. A number of receptor antagonists were examined for their ability to block ergocryptine-stimulated release. Of the dopaminergic, adrenergic, serotonergic, GABA-ergic, and cholinergic antagonists examined, only phentolamine produced a moderate attenuation in evoked release. Omission of Ca++ from the medium did not affect ergocryptine-evoked release. Following ergocryptine treatment, the synaptosomes were fully responsive to other stimulant. The results indicate that, in addition to interacting with dopamine receptors, several ergot alkaloids may produce dopaminergic effects by increasing the release of dopamine from central nerve endings. Several mechanisms to account for the evoked neurotransmitter release are discussed.  相似文献   

14.
In the present study, we investigated expressions of vesicular glutamate transporter (VGLUT) and of the plasma membrane glutamate transporters [glutamate transporter 1 (GLT-1), glutamate/aspartate transporter (GLAST) and excitatory amino acid carrier 1 (EAAC-1)] in the gerbil hippocampus following transient ischaemia. The expressional levels and distribution patterns of VGLUT immunoreactivities were unaltered until 3 days after ischaemic-insults. However, VGLUT-2 immunoreactivity in the CA1 region was reduced at 4 days after ischaemia due to delayed neuronal death. In addition, both GLT-1 and GLAST immunoreactivities in the CA1 region were enhanced at 30 min - 12 h after ischaemia-reperfusion and their expression began to reduce at 24 h after ischaemia-reperfusion. In contrast, EAAC-1 immunoreactivity was transiently reduced in the CA1 region at 30 min after ischaemia, re-enhanced at 3-12 h after ischaemia, and re-reduced at 24 h after ischaemia. These findings suggest that malfunctions of plasma membrane glutamate transporters, not of VGLUT, may play an important role in the elevation of extracellular glutamate concentration following ischaemic insults.  相似文献   

15.
Gossypol, a male anti-fertility agent, was found to be an inhibitor of cyclic nucleotide-independent phosvitin kinase from pig testis. The IC50 value (concentration causing 50% inhibition) was 42 microM using phosvitin as substrate. Kinetic analysis indicated that the compound inhibited the enzyme noncompetitively with respect to ATP (Ki = 17 microM) and competitively with respect to phosvitin (Ki = 11 microM). The gossypol inhibition was reversed by the addition of polyamines. The compound also inhibited phosphorylation of high mobility group (HMG) proteins HMG 14 and 17 (the endogenous substrates in the testis located in nucleosome), with an IC50 value of 48 microM.  相似文献   

16.
Mannheimia haemolytica, one of the agents associated with bovine respiratory disease complex, can cause severe lung pathology including the leakage of vascular products into the airways and alveoli. Previous work by this laboratory has demonstrated that bovine lung endothelial and epithelial cells undergo dramatic permeability increases when exposed to adenosine-5'-triphosphate (ATP). Therefore, we wanted to determine if ATP levels were elevated in bronchoalveolar lavage (BAL) samples from calves experimentally infected with M. haemolytica. In addition, cultured bovine pulmonary epithelial (BPE) cells were stimulated with heat-killed and live M. haemolytica bacteria, lipopolysaccharide (LPS), lipoteichoic acid (LTA), interleukin-1 (IL-1), and zymosan activated plasma (ZAP) to determine whether they might release extracellular ATP during in vitro infection. Calves experimentally exposed to M. haemolytica had an approximately 2-fold higher level of ATP in their BAL samples compared to control. BPE cells exposed to increasing numbers of heat-killed or live M. haemolytica had significantly increased levels of ATP release as compared to time-matched controls. Finally, BPE cells treated with several concentrations of LPS and IL-1 had increases in ATP release as compared to time-matched controls. This increase appeared to be a result of active ATP secretion by the cells, as cell viability was similar between treated and non-treated cells. Neither ZAP nor LTA induced any ATP release by the cells. In conclusion, ATP levels are elevated in lung secretions from calves infected with M. haemolytica. In addition, lung epithelial cells can actively release ATP when exposed to heat-killed or live M. haemolytica, LPS or IL-1.  相似文献   

17.
旨在研究Fas/FasL通路对镉激活PC12细胞MAPK通路的影响,用10 μmol L-1醋酸镉（CdAc2）分别处理插入非特异性序列PC12细胞与Fas基因沉默PC12细胞12 h;用10 μmol·L-1CdAc2与40 μmol·L-1 Z-IETD-FMK （caspase-8特异性抑制剂）单独或联合处理PC12细胞12 h。通过Western blot检测Fas/FasL通路相关蛋白Fas、FasL、Fas相关死亡域蛋白（FADD）、Cleaved caspase-8、死亡结构域相关蛋白（Daxx）、凋亡信号调节激酶1（ASK1）的表达与MAPK通路相关蛋白ERK1/2、JNK1/2、p-ERK1/2、p-JNK1/2的表达;Hoechst33258荧光染色检测细胞凋亡形态学变化,流式细胞术检测细胞凋亡率。结果显示,Fas shRNA慢病毒极显著抑制镉引起的PC12细胞Fas/FasL通路相关蛋白表达量和凋亡率的升高（P<0.01）,缓解镉引起的PC12细胞凋亡形态学变化;Fas shRNA与Z-IETD-FMK均能够极显著抑制镉引起的PC12细胞MAPK通路相关蛋白ERK1/2与JNK1/2磷酸化水平升高（P<0.01）。综上表明,Fas/FasL通路调控MAPK通路参与镉致PC12细胞凋亡。  相似文献   

18.
Macrophages are essential for controlling the majority of infections, and are mediators of natural immunity. During infection, lipopolysaccharide (LPS) stimulates macrophages to produce pro-inflammatory cytokines. Adenosine and ATP released into the extracellular space by immunological stimuli have been shown to regulate various immune functions. More recently, it has been shown adenosine and ATP have a critical role on the physiological negative feedback mechanism for limitation and termination of tissue-specific and systemic inflammatory responses. It was useful and meaningful to gain information about interaction between LPS, which generates the inflammation, and adenosine and ATP, which terminate the inflammation. We evaluate effects of adenosine and ATP on the production of cytokines related to inflammation in canine macrophage cell line DH82 cells. Adenosine and ATP respectively increased the production of IL-10 without affecting the production of IL-6, TNF-α and IL-12 in DH82 cells. In addition, adenosine and ATP prevented the production of LPS-induced IL-6, TNF-α and IL-12 in DH82 cells. In contrast, adenosine and ATP potentiated LPS-induced IL-10 production in DH82 cells. Moreover, adenosine, but not ATP inhibited LPS-induced expression of TLR4 in DH82 cells. These results suggest that conditions related to increased adenosine and/or ATP may play an important role in the inflammatory reactions.  相似文献   

19.
旨在探究死亡受体Fas在镉致大鼠肾上腺嗜铬细胞瘤细胞（PC12）凋亡中的作用及其对线粒体通路的调控机制,用10 μmol·L-1镉处理Fas基因沉默的PC12细胞株12 h,通过Western blot检测BH3相互作用域死亡激动剂（BID）、半胱氨酸蛋白酶-9（caspase-9）、半胱氨酸蛋白酶-3（caspase-3）、多聚二磷酸腺苷核糖聚合酶（PARP）的活化情况,Bcl-2相关X蛋白（Bax）、B细胞淋巴瘤/白血病-2基因（Bcl-2）、凋亡诱导因子（AIF）、核酸内切酶G（Endo G）的表达情况,以及细胞色素C（Cyt C）在细胞内的分布情况,免疫荧光染色检测AIF核转位。结果显示,镉极显著上调tBID/BID比值和Bax/Bcl-2比值,诱导Cyt C从线粒体释放到细胞质,激活caspase-9、caspase-3和PARP,增加AIF和Endo G蛋白表达水平（P<0.01）,并诱导AIF核转位;沉默Fas极显著抑制镉引起的tBID/BID比值和Bax/Bcl-2比值升高,Cyt C从线粒体释放到胞浆,caspase-3、PARP蛋白活化和AIF、Endo G蛋白表达水平极显著升高（P<0.01）,显著抑制镉激活的caspase-9（P<0.05）,并抑制AIF核转位。综上表明,Fas通过调控线粒体通路参与镉致PC12细胞凋亡。  相似文献   

20.
The Chinese Meishan pig is prolific breed and it is considered that this pig has a capacity with higher rates of embryonic survival. The oviductal secretory cells may affect the embryonic development and survival. The aim of the present study was to investigate the ultrastructural features of secretory cells in the various regions of the Chinese Meishan pig oviduct during the follicular and luteal phases of the oestrous cycle. In the ampullar secretory cells, numerous secretory granules with moderately electron-dense matrices were present in the supranuclear cytoplasm and exocytosis of secretory granules was observed. The number of secretory granules was dramatically reduced in the ampullar secretory cells in the luteal phase. During the follicular phase in the fimbrial epithelium, the secretory cells contained rough endoplasmic reticulum and Golgi apparatus, but most cells had few small granules. In the luteal phase, the secretory cells in the ampullar and fimbrial epithelia extended beyond the luminal border of the ciliated cells. In the isthmus, many granules were present in the cytoplasm of secretory cells throughout the oestrous cycle, but the number of secretory granules was reduced in the luteal phase. The cytomorphometric data revealed that the height of ciliated cells decreased substantially in the fimbriae and ampulla at the luteal phase, while that of non-ciliated cells was less affected. These results suggest that the drastic reduction of cell height of ciliated cells cause the extrusion of most secretory cells beyond the ciliated cells in the fimbriae and ampulla during the luteal phase. In summary, our ultrastructural observations of Chinese Meishan pig oviduct revealed marked cyclic changes in the ultrastructural features of secretory cells. In particular, the ultrastructural features and the numbers of secretory granules were distinctive for each particular segment. These findings should provide insight into the regional and cellular differences in functions of secretory cells of the Chinese Meishan pig oviduct.  相似文献   

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