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1.
ABSTRACT. The potencies of seventeen analogues of ATP as gorging inducers for Glossina palpalis palpalis were evaluated. The ranking for effective dose that induced half the flies to gorge (ED50) was: A tetra P 5 ATP=2'd ATP ADP=2'd ADP > AMP-PNP > 3'd ATP 2'3'dd ATP > AMP-PCP > adenosine 5' triphosphate 2',3'dialdehyde AMP-CPP >> AMP. Females detect ATP and its analogues better than males. The ED50 of ATP was 5 × 10-7 M for teneral females and 1.5 × 10-6 M for males. According to the potency order of the ATP analogues, the G.p.palpalis gustatory receptors recognizing ATP can be classified as P2y purinoceptors.  相似文献   

2.
Abstract: ATP, an established neurotransmitter, causes elevation of cytosolic Ca2+ and catecholamine secretion when applied to chromaffin cells in the intact adrenal gland. The ATP-induced rise in Ca2+ is due both to release from internal stores and to entry across the plasma membrane. The latter source of Ca2+ causes secretion; the primary role of Ca2+ released from internal stores remains undetermined. In this article, we have studied the nucleotide specificity for activating the two types of Ca2+ increases. The agonist potency order for the increase in fluorescence from fura-2-loaded chromaffin cells due to release of Ca2+ from internal stores is ATP = UTP > ADP > 2-methylthio-ATP, α,β-methylene ATP, identifying the receptor as a P2U purinoceptor. The potency order for secretion is 2-methylthio-ATP > ATP > α,β-methylene ATP, ADP, UTP, placing the receptor in the P2Y subtype. Thus, two distinct receptors are responsible for Ca2+ release and secretion. Agonists were more effective in the absence of extracellular Mg2+, suggesting that ATP uncomplexed with divalent cations binds preferentially to both receptors. The low response of both receptors to ADP distinguishes them from the ATP receptor on these cells that inhibits voltage-dependent Ca2+ current and secretion.  相似文献   

3.
Abstract: Cultured astroglia express both adenosine and ATP purinergic receptors that are coupled to increases in intracellular calcium concentration ([Ca2+]i). Currently, there is little evidence that such purinergic receptors exist on astrocytes in vivo. To address this issue, calcium-sensitive fluorescent dyes were used in conjunction with confocal microscopy and immunocytochemistry to examine the responsiveness of astrocytes in acutely isolated hippocampal slices to purinergic neuroligands. Both ATP and adenosine induced dynamic increases in astrocytic [Ca2+]i that were blocked by the adenosine receptor antagonist 8-( p -sulfophenyl)theophylline. The responses to adenosine were not blocked by tetrodotoxin, 8-cyclopentyltheophylline, 8-(3-chlorostyryl)caffeine, dipyridamole, or removal of extracellular calcium. The P2Y-selective agonist 2-methylthioadenosine triphosphate was unable to induce increases in astrocytic [Ca2+]i, whereas the P2 agonist adenosine 5'- O -(2-thiodiphosphate) induced astrocytic responses in a low percentage of astrocytes. These results indicate that the majority of hippocampal astrocytes in situ contain P1 purinergic receptors coupled to increases in [Ca2+]i, whereas a small minority appear to contain P2 purinergic receptors. Furthermore, individual hippocampal astrocytes responded to adenosine, glutamate, and depolarization with increases in [Ca2+]i. The existence of both purinergic and glutamatergic receptors on individual astrocytes in situ suggests that astrocytes in vivo are able to integrate information derived from glutamate and adenosine receptor stimulation.  相似文献   

4.
Abstract: Ischemia-induced changes in 31P NMR relaxation were examined in 16 piglets. NMR spectra were acquired under control conditions and during complete cerebral ischemia induced via cardiac arrest. Changes in T 1 were assessed directly in six animals during control conditions and after 30–45 min of complete ischemia when changes in brain P1 levels had reached a plateau. The T 1 for P1 did not change, i.e., 2.3 ± 0.5 s during control conditions versus 2.4 ± 1.0 s during ischemia. To evaluate phosphocreatine and ATP, two types of spectra, with a long (25-s) or short (1-s) interpulse delay time, were collected during the first 10 min of ischemia (n = 10). Both types of spectra showed the same time course of changes in phosphocreatine and ATP levels, implying that the T 1 relaxation times do not change during ischemia. There were no changes in the linewidths of phosphocreatine, ATP, or P1 during ischemia, implying that the T *2 values remain constant. Our results suggest that the 31P T 1 and T *2 for phosphocreatine, Pi, and ATP do not change during ischemia, and therefore changes in 31P NMR peak intensity accurately reflect changes in metabolite concentrations.  相似文献   

5.
PROTEIN COMPOSITION OF MYELIN OF THE PERIPHERAL NERVOUS SYSTEM   总被引:33,自引:15,他引:18  
Abstract— Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P0), two uncharacterized proteins, and two basic proteins (P1 and P2). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P1 and P2 proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P2 protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P0, P1 and P2 proteins from rabbit sciatic nerve and P0 and P2 proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P1 protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P0 and P2 proteins are unique to the PNS.  相似文献   

6.
An increase in phosphatidylcholine (PC), phosphatidyl-ethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI) occurred during bud break induced by decapitation. Inositol-1-phosphate [Ins(l)P1], inositol-1,4-bisphosphate [Ins(1,4)P2], and inositol-1,4,5-triphosphate [Ins(1,4,5)P3] were found in apple buds and increased progressively following decapitation. Ins(1)P1 and Ins(1,4)P2 peaked 48 h after decapitation and Ins(1,4,5)P3 peaked 72 h after decapitation during the metabolic transition when buds emerged from dormancy. Ins(1,4)P2 and Ins(1,4,5)P3 levels declined there after. The lateral buds on shoots with intact terminals and decapitated shoots treated with indole-3-acetic acid (IAA) in the terminals tip remained dormant and there were no significant changes in phospholipid and inositol phosphate contents.  相似文献   

7.
Abstract: The myelin specific protein, P2, was localized immunocytochemically in electron micrographs of 4-day-old rat peripheral nerve by a preembedding technique. P2 staining was restricted to Schwann cells that had established a one-to-one relationship with an axon. P2 antiserum produced a diffuse staining throughout the entire cytosol of myelinating Schwann cells. In addition, the cytoplasmic side of Schwann cell plasma membranes and the membranes of cytoplasmic organelles that were exposed to cytosol were stained by P2 antiserum. This cytoplasmic localization of P2 protein is similar to that described for soluble or peripheral membrane proteins that are synthesized on free ribosomes. P2 antiserum stained the cytoplasmic side of Schwann cell membranes that formed single or multiple loose myelin spirals around an axon. In the region of the outer mesaxon, P2 antiserum stained the major dense line of compact myelin. These results demonstrate that P2 protein is located on the cytoplasmic side of compact myelin membranes and are consistent with biochemical studies demonstrating P2 to be a peripheral membrane protein.  相似文献   

8.
Abstract: Calcium signaling in fura-2 acetoxymethyl ester-loaded enteric glia was investigated in response to neuroligands; responses to ATP were studied in detail. Carbachol (1 m M ), glutamate (100 µ M ), norepinephrine (10 µ M ), and substance P (1 µ M ) did not increase the intracellular calcium concentration ([Ca2+]i) in cultured enteric glia. An increasing percentage of glia responded to serotonin (4%; 100 µ M ), bradykinin (11%; 10 µ M ), and histamine (31%; 100 µ M ), whereas 100% of glia responded to ATP (100 µ M ). ATP-evoked calcium signaling was concentration dependent in terms of the percentage of glia responding and the peak [Ca2+]i achieved; responses were pertussis toxin insensitive. Based on responsiveness of enteric glia to purinergic agonists and peak [Ca2+]i evoked, ATP = UTP > ADP > β,γ-methyleneadenosine 5'-triphosphate ≫ 2-methylthioadenosine 5'-triphosphate = α,β-methyleneadenosine 5'-triphosphate = AMP = adenosine, suggesting a glial P2U receptor. Depletion of d - myo -inositol 1,4,5-trisphosphate-sensitive calcium stores by thapsigargin (10 µ M ) abolished glial responses to ATP. Similarly, calcium responses were decreased 92% by U-73122 (10 µ M ), an inhibitor of phospholipase C, and 93% by the phorbol ester phorbol 12-myristate 13-acetate (100 n M ), an activator of protein kinase C. Thus, cultured enteric glia can respond to neurotransmitters with increases in [Ca2+]i. Our data suggest that glial responses to ATP are mediated by a P2U receptor coupled to activation of phospholipase C and release of intracellular calcium stores.  相似文献   

9.
Process pasteurization values for reference temperature 70°C (P70) were calculated from the temperature profiles of 250 g luncheon meat chubs cooked under experimental conditions. A simple equation relating Process P70-value and the time and temperature of cooking was derived. With minimal cooking (P70= 40) the surviving microflora (103/g) was dominated by species of Lactobacillus, Brochothrix and Micrococcus. These organisms were destroyed by more intensive cooking (P70= 105), leaving a flora (102/g) composed of Bacillus and Micrococcus species. The spoilage that developed after 14 d storage at 25°C reflected the severity of the heat treatment received by each chub: with P70 between 40 and 90, a Streptococcus spoilage sequence occurred; with P70 between 105 and 120, a Bacillus/Streptococcus spoilage sequence occurred; with P70 of 135 and above, a Bacillus spoilage sequence occurred. Cooking to a P70= 75 was adequate to reduce the surviving microflora to the 102/g level associated with current good manufacturing practice.  相似文献   

10.
Abstract: The effects of extracellular ATP and polypeptide growth factors on DNA synthesis in primary cultures of rat astrocytes have been examined. It was found that ATP acts synergistically with either acidic or basic fibroblast growth factor to stimulate DNA synthesis. The specificity of this effect was demonstrated by the inability of ATP to potentiate DNA synthesis induced by platelet-derived growth factor or epidermal growth factor. ATP appears to act via P2 purinergic receptors, because (a) it was more effective than adenosine and (b) the synergistic effect was observed with the hydrolysis-resistant P2 agonists, ADPβS and ATPγS. The evidence suggests that extracellular ATP may be an important factor in regulating the extent of gliosis and, as such, may be involved in mechanisms of neural injury and repair.  相似文献   

11.
We evaluated the effect of haloperidol (HP) and its metabolites on [3H](+)-pentazocine binding to σ1 receptors in SH-SY5Y human neuroblastoma cells and guinea pig brain P1, P2 and P3 subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other σ1 antagonists or (−)-sulpiride), [3H](+)-pentazocine binding to brain membranes was markedly decreased. Recovery of σ1 receptor density to steady state after HP-induced inactivation required more than 30 days. HP-metabolite II (reduced HP, 4-(4-chlorophenyl)-α-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol), but not HP-metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), irreversibly blocked σ1 receptors in guinea pig brain homogenate and P2 fraction in vitro . We found similar results in SH-SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated σ1 receptors when it was incubated with brain homogenate and SH-SY5Y cells, but not when incubated with P2 fraction membranes, which suggests that HP is metabolized to inactivate σ1 receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP-metabolite II, completely prevented HP-induced inactivation of σ1 receptors in brain homogenates. These results suggest that HP may irreversibly inactivate σ1 receptors in guinea pig and human cells, probably after metabolism to reduced HP.  相似文献   

12.
Abstract: Circular dichroism (CD) was used to study the conformations of bovine nerve root P2 basic protein, its reduced and carboxymethylated derivative (RCM-P2), and its large cyanogen bromide fragment (CN1). Data in the far UV show that both the parent protein and RCM-P2 have conformations dominated by a large amount of β structure. However, the CN1 peptide appears to exist in a largely unordered conformation. Since CN1 lacks short (20 residue) amino- and carboxy-terminal segments of the P2 protein, the spectral data suggest that these regions are important for determining and/or maintaining folding of the P2 protein in aqueous solutions. The P2 protein was found to have a distinctive CD spectrum in the near UV. The characteristics of molar ellipticities indicate that the spectrum contains significant contributions from tyrosine residues, and that these contributions suggest different environments for the two tyrosines in P2 protein. Both environments depend on protein conformation, since CD side chain absorptions are lost when P2 protein is denatured with 5 M urea.  相似文献   

13.
Abstract: We examined the mechanism underlying the ATP-induced increase in the cytosolic Ca2+ concentration ([Ca]in) in acutely isolated chick ciliary ganglion neurons, using fura-2 microfluorometry. The ATP-induced increase in [Ca]in was dependent on external Ca2+, was blocked in a dose-dependent manner by reactive blue 2, and was substantially inhibited by both L- and N-type Ca2+ channel blockers. ATP was effective in increasing [Ca]in in the presence of a desensitizing concentration of nicotine (100 µ M ), and simultaneous addition of maximal doses of ATP and nicotine caused an additive increase in [Ca]in, suggesting that ATP acts on a site distinct from nicotinic acetylcholine receptors. ATP also increased the cytosolic Na+ concentration as determined by sodium-binding benzofuran isophthalate microfluorometry. These results suggest that ATP increases Na+ influx through P2 purinoceptor-associated channels resulting in membrane depolarization, which in turn increases Ca2+ influx through voltage-dependent Ca2+ channels. However, ATP still caused a small increase in [Ca]in under Na+-free conditions, and this [Ca]in increase was little affected by Ca2+ channel blockers. ATP also increased Mn2+ influx under Na+-free conditions, as indicated by quenching of fura-2 fluorescence. These results suggest that nonselective cationic channels activated by ATP are permeable not only to Ca2+ but also to Mn2+, in addition to monovalent cations.  相似文献   

14.
Fibroblast growth factor 2 (FGF-2) is a mitogen that is exported from cells by an endoplasmic reticulum/Golgi-independent secretory pathway. Recent findings have shown that FGF-2 export occurs by direct translocation from the cytoplasm across the plasma membrane into the extracellular space. Here, we report that FGF-2 contains a binding site for phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], the principal phosphoinositide species associated with plasma membranes. Intriguingly, in the context of a lipid bilayer, the interaction between FGF-2 and PI(4,5)P2 is shown to depend on a lipid background that resembles plasma membranes. We show that the interaction with PI(4,5)P2 is critically important for FGF-2 secretion as experimental conditions reducing cellular levels of PI(4,5)P2 resulted in a substantial drop in FGF-2 export efficiency. Likewise, we have identified FGF-2 variant forms deficient for binding to PI(4,5)P2 that were found to be severely impaired with regard to export efficiency. These data show that a transient interaction with PI(4,5)P2 associated with the inner leaflet of plasma membranes represents the initial step of the unconventional secretory pathway of FGF-2.  相似文献   

15.
Abstract: Extracellular ATP is known to cause a variety of changes, including the alteration of ion fluxes, cell growth, and other physiological activities. Recently, it has been suggested that ATP acts as an excitatory synaptic transmitter, which may produce a Ca2+ influx via the activation of a P2y purinoceptor. Rat pheochromocytoma (PC-12) cells are known to resemble rat sensory neurons and to possess a P2y purinoceptor. In this study, we demonstrated that extracellular ATP dose-dependently increased PC-12 cell death in the presence of ferrous ions. Voltage-sensitive calcium channel blockers and calpain and xanthine oxidase inhibitors were found to be effective at protecting PC-12 cells from Fe2+/ATP-induced lipid peroxidation and cell death. These results suggest that xanthine oxidase activation induced by calpains and subsequent free radical formation may be responsible for Fe2+/ATP-induced neuronal cell death.  相似文献   

16.
Abstract: P0 glycoprotein, the major protein of PNS myelin, contains approximately 1 mol of covalently bound long-chain fatty acids. To determine the chemical nature of the fatty acid-protein linkage, P0 was labeled in rat sciatic nerve slices with [3H]palmitic acid and subsequently treated with various reagents. The protein-bound palmi-tate was released by incubation with the reducing agents dithiothreitol and 2-mercaptoethanol, and with 1 M hydrox-ylamine at pH 7.5. In addition, P0 was deacylated by treatment with 10 m M NaBH4 with the concomitant production of [3H]hexadecanol, indicating that the fatty acid is bound in a thioester linkage. This conclusion was supported further by the fact that deacylation with hydroxylamine generated free thiol groups, which were titrated with [14C]-iodoacetamide. To identify the cysteine residue involved in the thioester linkage, [14C]carboxyamidomethylated P0was digested with trypsin and the resulting peptides analyzed by reversed-phase HPLC. Identification of the radioactive protein fragments by amino acid analysis and amino-terminal peptide sequencing revealed that Cys153 in rat P0 glycoprotein is the acylation site. The acylated cysteine is located at the junction of the putative transmem-brane and cytoplasmic domains. This residue is also present in the P0 glycoprotein of other species, including human, bovine, mice, and chicken.  相似文献   

17.
Abstract— A subfraction, derived from the microsomal fraction of rat cerebral cortex, with a buoyant density of 1.112 g μ ml−1 appears to be enriched in receptor sites for a number of potential neurotrans-mitters. These include the cholinergic (nicotinic and muscarinic) and ß-adrenergic receptors. This microsomal subfraction (P3B2) has been isolated on a preparative scale by two sequential isopycnic sedimentations in discontinuous sucrose gradients.
We have studied the morphology, enzymatic markers and protein composition of this fraction and have compared them with the properties of other subcellular fractions from the same source. Synaptic plasma membranes resembled P3B2 by exhibiting the same high extent of enrichment in receptors. However, the synaptic membranes appear to contain more mitochondrial and presynaptic (axonal and cell surface) membranes than does P3B2, and the postsynaptic membranes in the two fractions appear morphologically distinct since P3B2 does not contain the characteristic postsynaptic densities. Thus these membranes may be derived from Gray's type II synapses.  相似文献   

18.
Abstract: The effect of an inhibitor of N -glycosylation of glycoproteins, tunicamycin, on synthesis of PNS myelin proteins was investigated in vitro by using chopped sciatic nerves or spinal roots of 21-day-old Wistar rats. Tunicamycin when incubated with these nerves in the presence of 3H-labeled fucose, mannose, or glucosamine inhibited the uptake of radioactivity into myelin proteins including some high-molecular-weight proteins, P0, 23K protein, and 19K protein by amounts ranging from 42 to 79%. Uptake of 14Camino acid mixture was inhibited much less by tunicamycin, but a new radioactive protein peak appeared when the protein mixtures had been separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This protein ran directly in front of the P0 peak, did not correspond to any bands stained by Fast green, and was not labeled by fucose. This peak appeared in increasing larger proportions with progressive time of incubation of nerves with 3H amino acids in the presence of tunicamycin. The new protein, which cross-reacts with P0 antiserum, was tentatively identified as a nonglycosylated P0 protein that appears to be almost as well incorporated as P0 into the subcellular fraction containing myelin. At this time it is not possible to determine whether the unglycosylated P0 is actually assembled into a site and configuration like that of P0.  相似文献   

19.
Abstract: Uptake and release of cysteine sulfinic acid by synaptosomal fractions (P2) and slices of rat cerebral cortex were investigated. The P2 fraction had a Na+-dependent high-affinity uptake system for cysteine sulfinic acid (Km, 12μM), which was restricted to the synaptosomes. High-affinity uptake of cysteine sulfinic acid was competitively inhibited by glutamate, aspartate, and cysteic acid. None of the various centrally acting drugs tested specifically inhibited this transport system. Release of [14C]cysteine sulfinic acid from preloaded cortical slices or P2 fractions was examined by a superfusion method, which avoided reuptake of released [14C]cysteine sulfinic acid. High K+ (56 m M ) and veratridine (10μM) stimulated the release of cysteine sulfinic acid from slices and the P2 fraction in a partly Ca2+-dependent manner. Diazepam at concentrations of 10 and 100 μM markedly inhibited the stimulated release, but not the spontaneous release, by cortical slices. On the contrary, it had no effect on the stimulated release of cysteine sulfinic acid from the P2 fraction.  相似文献   

20.
Abstract: The cellular localization of two Ca2+-dependent protein phosphorylation systems was investigated using the kainic acid lesioning technique for the selective destruction of neurons. In one of these systems, a crude synaptosomal (P2) fraction was preincubated with 32Pj for 30 min; the phosphorylation of several proteins was increased during a short subsequent incubation with veratridine plus Ca2+. In the second system, crude synaptosomal membranes isolated from the P2 fraction were incubated with [γ-32P]ATP; in this system, the phosphorylation of several proteins was increased in the presence of a "calcium-dependent regulator" plus Ca2+. Kainic acid lesioning greatly reduced the amount of Ca-+-dependent protein phosphorylation in both systems. The results indicate a predominantly neuronal localization for both Ca2+-dependent protein phosphorylation systems.  相似文献   

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