Neuropeptides modulate the beta-adrenergic response of purified astrocytes in vitro

Neuropeptides may have functions in the central nervous system (CNS) other than altering neuronal excitability. For example, they may act as regulators of brain metabolism by affecting glycogenolysis. Since it has been suggested that glial cells might provide metabolic support for neuronal activity, they may well be one of the targets for neuropeptide regulation of metabolism. Consistent with this view are reports that peptide-containing nerve terminals have been seen apposed to astrocytes, but it is also quite possible that peptides could act at sites lacking morphological specialization. Primary cultures containing CNS glial cells have been shown to respond to beta-adrenergic agonists with an increase in cyclic AMP and, as a result, with an increase in glycogenolysis and have also been shown to respond to a variety of peptides with changes in cyclic AMP. In the study reported here, we have examined the effects of several peptides on relatively pure cultures of rat astrocytes. We demonstrate that the increase in intracellular cyclic AMP induced by noradrenaline is markedly enhanced by somatostatin and substance P and is inhibited by enkephalin, even though these peptides on their own have little or no effect on the basal levels of cyclic AMP. Vasoactive intestinal peptide (VIP) on the other hand increases cyclic AMP in the absence of noradrenaline. These results suggest that neuropeptides influence glial cells as well as neurones in the CNS and, in the case of somatostatin and substance P, provide further examples of neuropeptides modulating the response to another chemical signal without having a detectable action on their own.     

Chronic ethanol causes heterologous desensitization of receptors by reducing alpha s messenger RNA

One of the biochemical results of ethanol exposure is a change in the amount of the intracellular second messenger cyclic AMP (cAMP) produced in response to receptor stimulation. In general, acute ethanol exposure increases the amount of cAMP produced on stimulation of receptors coupled to the enzyme adenylyl cyclase via the GTP-binding protein Gs, whereas chronic ethanol exposure has the opposite effect (results for receptors coupled via Gi have been more variable). We previously reported that adaptation to continuous ethanol exposure reduces receptor-stimulated cAMP production by 25-35% in a neuroblastoma cell line (NG108-15), and an even greater reduction of 75% was observed in lymphocytes taken from actively-drinking alcoholics. This reduction in receptor-stimulated cAMP levels was recently confirmed in platelets from alcoholics. None of these studies, however, determined whether more than one receptor coupled to adenylyl cyclase activity was affected in the same cell. Here we report that chronic ethanol exposure causes desensitization of heterologous receptors coupled to Gs as cAMP production mediated by prostaglandin E1 as well as by adenosine is reduced by approximately 30% in NG108-15 cells. We show that, after chronic ethanol exposure, the activity of the alpha subunit of Gs is decreased by 29%, the amount of alpha s protein is decreased by 38.5%, and alpha s messenger RNA is decreased by 30%. Thus, cellular adaptation to ethanol involves a reduction in alpha s mRNA and, as a consequence, reduced cAMP production by heterologous receptors coupled to Gs. Such changes in cAMP production may account for the tolerance and physical dependence on ethanol in alcoholism.     

Defect in cyclic AMP phosphodiesterase due to the dunce mutation of learning in Drosophila melanogaster

Cyclic AMP is an intracellular mediator ('second messenger') in the nervous and endocrine control of cellular function, regulating different processes in different cell types. Although evidence is incomplete, it seems that cyclic AMP enhances the calcium-mediated release of neurotransmitter in some neurones. A simple form of memory in the mollusc Aplysia is probably encoded as a cyclic AMP-induced enhancement of neurotransmission at certain synapses of the central nervous system. The possibility that cyclic AMP participates in learning mechanisms may be explored using genetic mutants. For this purpose the fruitfly Drosophila is suitable as it is genetically well characterized and can learn through olfaction, vision or taste. We show here that independent searches for mutations of olfactory learning and of cyclic AMP metabolism, and for mutations causing female infertility have each led to the same gene--the dunce gene. Our evidence indicates that the normal dunce gene may specify a cyclic AMP phosphodiesterase.     

Selective reconstitution of nude mice with long-term cultured and cloned specific helper T cells

An antibody response is the end result of complex interactions among T cells, adherent cells and B cells. An understanding of the interactions involved has proved difficult as pure populations of these cells have not been available. By making use of T-cell growth factor, we were able to grow normal helper T cells specific for heterologous erythrocytes. Because specificity and mechanism of action of these cells had been demonstrated solely in culture, we sought to establish their competence in the whole animal. We have therefore examined here whether antigen-specific helper T cells, maintained in culture over long periods, would enable syngeneic nude mice to respond to T-cell dependent antigens. The results show that specific helper T cells, propagated in serum-free medium in vitro for up to 15 months, can selectively and specifically reconstitute syngeneic C57BL/6J nu/nu mice. Depending on the specificity of the injected helper T cells, such nude mice could respond to sheep red blood cells (SRC) but not to horse red blood cells (HRC) and vice versa. The magnitude of the response was comparable to that of normal mice and could exceed it by almost 10-fold, depending on the source and number of injected helper T cells.     

Cyclic nucleotides may mediate taste transduction

Taste stimulus adsorption is believed to occur at the taste cell microvillous membrane. But due to technical difficulties of inserting glass electrodes into the mammalian taste cell, little is known about the mechanisms of taste transduction. Reliable intracellular recordings are necessary to determine the characteristics of taste cells. This has been accomplished previously in the mouse and is reported here. Recent experiments indicated that cyclic nucleotides can act on the inner surface of the membranes of a variety of cells to alter their ion-channel activity, and these substances might act as intracellular transmitters in taste cells. But tight junctions found at the apical membrane of mammalian taste cells do not allow stimuli to enter the taste bud, making it difficult to alter the environment of the taste cell by perfusing with chemical solutions. Here we report that cyclic AMP, cyclic GMP, EGTA or tetraethyl-ammonium electrophoretically injected into the mouse taste cell induce membrane depolarization and increased membrane resistance. These results suggest that a cyclic nucleotide enzymatic cascade, modulated by calcium ions, may mediate the potassium permeability that controls taste, in a way analogous to visual and olfactory transduction.     

IgE-mediated degranulation of mast cells does not require opening of ion channels

Rat peritoneal mast cells respond to antigenic stimulation by releasing histamine through exocytosis. The dynamics of exocytosis can be investigated by dialysing single cells with patch pipettes using the whole-cell recording configuration of the patch-clamp technique. However, dialysed cells fail to respond to external stimuli such as compound 48/80 or antigens, suggesting that essential cytoplasmic components have been washed out. We have developed a new patch-clamp configuration in which the patch under the pipette tip is not disrupted but instead permeabilized, preventing the diffusion of large molecules out of the cell. In this configuration the cell responds to external stimulation, and the capacitance as well as the conductance of the cell membrane can be recorded during degranulation. On antigenic stimulation, the cell capacitance (proportional to plasma membrane area), after an initial delay, increases by a factor of about 3. This increase in capacitance is often preceded by a transient increase in conductance. Agents that block Ca-activated channels inhibit this conductance change without affecting the amplitude and time course of degranulation. We therefore conclude that, in contrast to excitable secretory cells such as chromaffin cells, mast cells do not use ion channels in stimulus-secretion coupling.     

Nerve growth factor induces adrenergic neuronal differentiation in F9 teratocarcinoma cells

Teratocarcinoma cells have been used as a model to study differentiation and development in vertebrates. Treatment with retinoic acid (RA) and dibutyryl cyclic AMP can in some embryonal carcinoma (EC) cell lines lead to neural differentiation, as judged by neurofilament expression and by the induction of enzymes involved in cholinergic transmission. Short-term culture of F9 line cells with RA and dibutyryl cyclic AMP results in a biochemically demonstrable rise in acetylcholinesterase (AChE) activity. We now report that long-term culture of F9 cells with RA and dibutyryl cyclic AMP induces neurofilament expression, demonstrated by immunofluorescence with specific antibodies. Furthermore, if nerve growth factor (NGF) is also added, the developing neurone-like cells exhibit immunoreactivity to tyrosine hydroxylase, a rate-limiting enzyme of catecholamine synthesis specific for adrenergic neurones. Immunoreactivity for Leu-enkephalin-like peptides is also induced. These results suggest that F9 cells can differentiate into cells with adrenergic characteristics.     

Cytokine-induced pseudopodial protrusion is coupled to tumour cell migration

Pseudopodia protrusion is a prominent feature of actively motile cells in vitro and invading tumour cells in vivo; however, the function and regulation of pseudopodia are poorly understood. Tumour autocrine motility factor (AMF) represents a new class of cytokines which are secreted by tumour cells and embryonic cells and induce random motility in the producer cells or in heterologous cells with appropriate receptors. Here we report that a major effect of this factor is to induce the extension of cell pseudopodia before cell translocation. Using a new method to quantify and isolate pseudopodia, we find that human breast carcinoma cell AMF (at concentrations of 1 nM or below) stimulates random pseudopodia formation in a dose-dependent and time-dependent manner. Anti-AMF antibodies inhibit pseudopodia protrusion and cell motility, showing the importance of pseudopodia formation during locomotion. AMF-stimulated motility and pseudopodia formation occur on a wide variety of adhesive substrata which suggests that certain intrinsic motility events are independent of the attachment mechanism. Induced pseudopodia show a prominent axial actin network in the electron microscope. The number of laminin receptor and fibronectin RGD recognition sites is increased by a factor of 20 in the induced pseudopodia when compared to the average distribution in unstimulated cells. Exploratory pseudopodia regulated by cell-derived motility factors contain receptors for matrix proteins and could serve as 'senseorgans' essential to the process of cell locomotion.     

半乳糖氧化酶的生物电化学特性

采用循环伏安法对半乳糖氧化酶(GAO)进行了生物电化学特性的研究。实验结果表明,由于半乳糖氧化酶的酶活性中心深埋在酶内部,所以该酶在铂电极上的直接电化学行为较难观察到,而二茂铁甲醇作为介体能明显地改善酶活性中心的电子传递,加快电子传递速率。且测定了半乳糖氧化酶与二茂铁甲醇之间的反应速度常数Ks为1.2×10~5L·mol~(-1)·s~(-1)。     

A membrane form of guanylate cyclase is an atrial natriuretic peptide receptor

Atrial natriuretic peptide (ANP) is a polypeptide hormone whose effects include the induction of diuresis, natriuresis and vasorelaxation. One of the earliest events following binding of ANP to receptors on target cells is an increase in cyclic GMP concentration, indicating that this nucleotide might act as a mediator of the physiological effects of the hormone. Guanylate cyclase exists in at least two different molecular forms: a soluble haem-containing enzyme consisting of two subunits and a non-haem-containing transmembrane protein having a single subunit. It is the membrane form of guanylate cyclase that is activated following binding of ANP to target cells. We report here the isolation, sequence and expression of a complementary DNA clone encoding the membrane form of guanylate cyclase from rat brain. Transfection of this cDNA into cultured mammalian cells results in expression of guanylate cyclase activity and ANP-binding activity. The ANP receptor/guanylate cyclase represents a new class of mammalian cell-surface receptors which contain an extracellular ligand-binding domain and an intracellular guanylate cyclase catalytic domain.     

mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake

How adult tissue stem and niche cells respond to the nutritional state of an organism is not well understood. Here we find that Paneth cells, a key constituent of the mammalian intestinal stem-cell (ISC) niche, augment stem-cell function in response to calorie restriction. Calorie restriction acts by reducing mechanistic target of rapamycin complex 1 (mTORC1) signalling in Paneth cells, and the ISC-enhancing effects of calorie restriction can be mimicked by rapamycin. Calorie intake regulates mTORC1 in Paneth cells, but not ISCs, and forced activation of mTORC1 in Paneth cells during calorie restriction abolishes the ISC-augmenting effects of the niche. Finally, increased expression of bone stromal antigen 1 (Bst1) in Paneth cells—an ectoenzyme that produces the paracrine factor cyclic ADP ribose—mediates the effects of calorie restriction and rapamycin on ISC function. Our findings establish that mTORC1 non-cell-autonomously regulates stem-cell self-renewal, and highlight a significant role of the mammalian intestinal niche in coupling stem-cell function to organismal physiology.     

An autoregulatory circuit for long-range self-organization in Dictyostelium cell populations

Nutrient-deprived Dictyostelium amoebae aggregate to form a multicellular structure by chemotaxis, moving towards propagating waves of cyclic AMP that are relayed from cell to cell. Organizing centres are not formed by founder cells, but are dynamic entities consisting of cores of outwardly rotating spiral waves that self-organize in a homogeneous cell population. Spiral waves are ubiquitously observed in chemical reactions as well as in biological systems. Although feedback control of spiral waves in spatially extended chemical reactions has been demonstrated in recent years, the mechanism by which control is achieved in living systems is unknown. Here we show that mutants of the cyclic AMP/protein kinase A pathway show periodic signalling, but fail to organize coherent long-range wave territories, owing to the appearance of numerous spiral cores. A theoretical model suggests that autoregulation of cell excitability mediated by protein kinase A acts to optimize the number of signalling centres.     

Release of chromatin protein HMGB1 by necrotic cells triggers inflammation

High mobility group 1 (HMGB1) protein is both a nuclear factor and a secreted protein. In the cell nucleus it acts as an architectural chromatin-binding factor that bends DNA and promotes protein assembly on specific DNA targets. Outside the cell, it binds with high affinity to RAGE (the receptor for advanced glycation end products) and is a potent mediator of inflammation. HMGB1 is secreted by activated monocytes and macrophages, and is passively released by necrotic or damaged cells. Here we report that Hmgb1(-/-) necrotic cells have a greatly reduced ability to promote inflammation, which proves that the release of HMGB1 can signal the demise of a cell to its neighbours. Apoptotic cells do not release HMGB1 even after undergoing secondary necrosis and partial autolysis, and thus fail to promote inflammation even if not cleared promptly by phagocytic cells. In apoptotic cells, HMGB1 is bound firmly to chromatin because of generalized underacetylation of histone and is released in the extracellular medium (promoting inflammation) if chromatin deacetylation is prevented. Thus, cells undergoing apoptosis are programmed to withhold the signal that is broadcast by cells that have been damaged or killed by trauma.     

Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides

Murine embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo which can contribute differentiated progeny to all adult tissues, including the germ-cell lineage, after re-incorporation into the normal embryo. They provide both a cellular vector for the generation of transgenic animals and a useful system for the identification of polypeptide factors controlling differentiation processes in early development. In particular, medium conditioned by Buffalo rat liver cells contains a polypeptide factor, ES cell differentiation inhibitory activity (DIA), which specifically suppresses the spontaneous differentiation of ES cells in vitro, thereby permitting their growth as homogeneous stem cell populations in the absence of heterologous feeder cells. ES cell pluripotentiality, including the ability to give rise to functional gametes, is preserved after prolonged culture in Buffalo rat liver media as a source of DIA. Here, we report that purified DIA is related in structure and function to the recently identified hematopoietic regulatory factors human interleukin for DA cells and leukaemia inhibitory factor. DIA and human interleukin DA/leukaemia inhibitory factor have thus been identified as related multifunctional regulatory factors with distinct biological activities in both early embryonic and hematopoietic stem cell systems.     

Correction of cell-cell communication defect by introduction of a protein kinase into mutant cells

The cell-to-cell permeability of the junctions of various cultured mammalian cell types depends on the concentration of intracellular cyclic AMP [( cAMP]i). The permeability rises when the cells are supplied with exogenous cyclic AMP or when their cyclic AMP synthesis is stimulated with choleragen or hormones; it falls when [cAMP]i is lowered by application of serum or due to increase in cell density. The rise and fall in permeability take several hours to develop (the rise is protein synthesis-dependent) and they occur concurrently with the rise and fall in the number of intramembrane particles of the gap junctions, which probably embody the cell-to-cell channels. Is this permeability regulation mediated by phosphorylating protein kinase? In many eukaryotes, the cyclic AMP receptor is a protein kinase consisting of a pair of regulatory subunits and a pair of catalytic subunits. The latter dissociate from the holoenzyme as the cyclic AMP binds to the regulatory subunits and, in this dissociated form, catalyse the phosphorylation of the target. The regulatory subunit occurs in two isoenzyme forms, I and II. The catalytic subunit seems invariant; subunits from different isoenzymes can substitute for each other. We show here that a mutant cell lacking the isoenzyme I is deficient in permeable junctions, and that this junctional defect is corrected when the mutant is supplied with exogenous catalytic subunit.     

Insulin trigger, cyclic AMP-dependent activation and phosphorylation of a plasma membrane cyclic AMP phosphodiesterase

Regulation of blood glucose levels by the liver is primarily achieved by the action of two peptide hormones, insulin and glucagon, which bind to specific receptors associated with the hepatocyte plasma membrane. Whilst the molecular action of glucagon at the level of the cell plasma membrane in activating adenylate cyclase is relatively well understood, we know little, if anything, of the molecular consequences of insulin occupying its receptor. We demonstrate here that insulin, at physiologically relevant concentrations, can trigger the cyclic AMP-dependent activation and phosphorylation of a low Km cyclic AMP phosphodiesterase attached to the liver plasma membrane. Such an effect may in part explain the ability of insulin to inhibit the increase in cellular cyclic AMP content that glucagon alone produces by activation of adenylate cyclase. Our observation that basal, intracellular cyclic AMP levels are insufficient to allow insulin to activate the cyclic AMP phosphodiesterase, yet those cyclic AMP levels achieved after exposure of the cells to glucagon are sufficient, gives a molecular rationale to Butcher and Sutherland's proposal that it is necessary to first elevate cellular cyclic AMP levels before they can be depressed by insulin.     

Differentiation of PC12 phaeochromocytoma cells induced by v-src oncogene

PC12 rat phaeochromocytoma cells are a model system that can be used to study both neuronal differentiation and the mechanism of action of nerve growth factor (NGF). PC12 cells respond to NGF protein by shifting from a chromaffin-cell-like phenotype to a neurite-bearing sympathetic neurone-like phenotype. Here we present data on the effect of infection of PC12 cells with retroviruses carrying the src oncogene of Rous sarcoma virus. Previous studies have demonstrated that the expression of src severely affects the synthesis and accumulation of differentiated cell products in a variety of cell types. We show that in the PC12 cell system, expression of v-src appears to have an inductive effect on differentiation that resembles the action of a 'physiological' growth factor.     

新亚甲蓝修饰玻碳电极测定过氧化氢

利用新亚甲蓝(NMB)为中间媒介体,以对苯二甲醛为桥联试剂将其与辣根过氧化物酶(HRP)修饰于玻碳电极表面,制备成过氧化氢电化学传感器。实验表明,NMB作为中间介体与对苯二甲醛键合后能有效地传输电子,可求得电子转移系数(α)为0.84,表观反应速率常数Ks为1.09 s-1。该传感器能快速地对过氧化氢响应,线性范围为0.0~75.0mol L-1,检出限低至0.278 mol.L-1。该方法用于检测过氧化氢简单快速而又灵敏,性能稳定。