Pancreatic stellate cell migration: role of the phosphatidylinositol 3-kinase(PI3-kinase) pathway

Pancreatic stellate cells (PSCs) are implicated as key mediators of pancreatic fibrogenesis and are found in increased numbers in areas of pancreatic injury. This increase in number may be due to increased local proliferation and/or migration of PSCs to affected areas from surrounding tissue. We have recently shown that PSCs can migrate and that this migration is stimulated by PDGF in a predominantly chemotactic manner [Gut 52 (2003) 677]. However, the signalling mechanisms responsible for PDGF-induced PSC migration are not known. Aims: (i) To determine whether PDGF-induced PSC migration is mediated by the PI3-kinase pathway. (ii) To investigate whether cell migration is influenced by cell proliferation and whether an interaction exists between the PI3-kinase pathway and the ERK1/2 pathway (known to mediate cell proliferation) in PSCs exposed to PDGF. Methods: (i) PI3-kinase activity was assessed by measuring the activation (phosphorylation) of its downstream substrate Akt in rat PSCs incubated with PDGF (10ng/mL) for 5min, 15min, 60min, and 24hr in the presence or absence of the specific PI3-kinase inhibitor wortmannin. (ii) The role of the PI3-kinase pathway in PSC migration was examined by assessing PSC migration through a porous membrane after exposure to PDGF in the presence and absence of wortmannin for 24hr. (iii) The relationship between migration and proliferation was assessed by examining migration of PSCs exposed to PDGF in the presence and absence of mitomycin C, an inhibitor of cell proliferation. (iv) The interaction between PI3-kinase and ERK1/2 was examined by incubation of PSCs with PDGF in the presence and absence of wortmannin, followed by assessment of ERK1/2 activation by western blot. Results: PDGF increased Akt activation in PSCs as early as at 5min of incubation and this increase was sustained for 24hr. Inhibition of PI3-kinase by wortmannin decreased basal as well as PDGF-induced migration and also inhibited ERK1/2 activation. Inhibition of PSC proliferation with mitomycin C significantly reduced (but did not abolish) basal and PDGF-induced PSC migration. Conclusions: (i) The PI3-kinase pathway is induced in PSCs after exposure to PDGF and this induction is sustained for at least 24hr. (ii) The PI3-kinase pathway plays a role in PDGF-induced PSC migration and is partially involved in mediating ERK1/2 activation. (iii) PSC migration is dependent, at least in part, on cell proliferation.     

Inhibition of pancreatic stellate cell activation by the hydroxymethylglutaryl coenzyme A reductase inhibitor lovastatin

Pancreatic stellate cells (PSCs) play a key role in pancreatic fibrosis, a constant feature of chronic pancreatitis. PSC activation occurs in response to profibrogenic mediators such as cytokines and involves proliferation, transition towards a myofibroblastic phenotype and enhanced production of extracellular matrix proteins. Previously, we have shown that PSC activation correlates with the activity of the Ras-Raf-ERK (extracellular signal-regulated kinase) signalling cascade [Gut 51 (2002) 579]. Using a rat culture model of PSCs, we have now evaluated the effects of lovastatin, a hydroxymethylglutaryl coenzyme A reductase inhibitor that interferes with protein isoprenylation, on PSC viability and activation as well as on signalling through Ras proteins. Apoptotic cells were detected applying the TUNEL assay. Proliferation of PSCs was quantitated using the bromodeoxyuridine DNA incorporation assay. Expression of alpha-smooth muscle actin (an indicator of the myofibroblastic phenotype), ERK activation and membrane translocation of the Ras superfamily member RhoA were analysed by immunoblotting. Lovastatin inhibited serum- and platelet-derived growth factor-stimulated PSC proliferation in a dose-dependent manner. At drug concentrations above the level required for growth inhibition, a strong increase of apoptotic cells was observed. Furthermore, lovastatin inhibited induction of alpha-smooth muscle actin expression in the course of primary culture. Immunoblot experiments indicated that lovastatin suppressed both Ras-mediated ERK 1/2 activation and platelet-derived growth factor-induced membrane translocation of RhoA. Together, our data suggest that lovastatin, through the interruption of Ras signalling, interferes with PSC activation. The antifibrotic efficiency of statins should be tested in animal models of chronic pancreatitis.     

Pluripotent stem cells: An in vitro model for nanotoxicity assessments

The advent of technology has led to an established range of engineered nanoparticles that are used in diverse applications, such as cell–cell interactions, cell–material interactions, medical therapies and the target modulation of cellular processes. The exponential increase in the utilization of nanomaterials and the growing number of associated criticisms has highlighted the potential risks of nanomaterials to human health and the ecosystem. The existing in vivo and in vitro platforms show limitations, with fluctuations being observed in the results of toxicity assessments. Pluripotent stem cells (PSCs) are viable source of cells that are capable of developing into specialized cells of the human body. PSCs can be efficiently used to screen new biomaterials/drugs and are potential candidates for studying impairments of biophysical morphology at both the cellular and tissue levels during interactions with nanomaterials and for diagnosing toxicity. Three‐dimensional in vitro models obtained using PSC‐derived cells would provide a realistic, patient‐specific platform for toxicity assessments and in drug screening applications. The current review focuses on PSCs as an alternative in vitro platform for assessing the hazardous effects of nanomaterials on health systems and highlights the importance of PSC‐derived in vitro platforms. Copyright © 2016 John Wiley & Sons, Ltd.     

PCB-induced inhibition of the vesicular monoamine transporter predicts reductions in synaptosomal dopamine content.

Both Aroclor mixtures and individual non-coplanar polychlorinated biphenyl (PCB) congeners reduce dopamine (DA) concentrations in cells in culture and in the brains of developing and adult laboratory animals. These reductions may involve inhibition of the dopamine transporter (DAT) and the vesicular monoamine transporter (VMAT) responsible, respectively, for the uptake of extracellular DA and the packaging of nerve terminal cytosolic DA into synaptic vesicles. However, the relative contribution of each monoamine transporter to the PCB-induced reductions in tissue DA has not been determined. Accordingly, we exposed striatal synaptosomes from adult rats to individual PCB congeners, a commercial mixture of PCBs or known monoamine transporter inhibitors; measured synaptosomal DA; and related these changes to media DA and concentrations of 3,4-dihydroxyphenylacetic (DOPAC). PCB-induced elevations in media DA concentrations are not sufficient to explain the reductions in tissue DA because known DAT inhibitors elevate media DA to a much greater extent than PCBs and yet induce similar decreases in tissue DA concentrations. On the other hand, PCB-induced elevations in DOPAC, reflective of increases in nerve terminal cytosolic DA, are sufficient to explain the reductions in tissue DA, because a known VMAT inhibitor elevates DOPAC and reduces tissue DA to an extent similar to that seen with PCBs. Taken together, these results suggest that elevations in DOPAC, reflective of increases in nerve terminal cytosolic DA due to VMAT inhibition, rather than elevations in media DA due to DAT inhibition, are largely responsible for the observed decreases in tissue DA content.     

Muscarinic acetylcholine receptors potentiate the GABAergic transmission in the developing rat inferior colliculus

Postsynaptic currents (PSCs) were recorded using the patch-clamp technique in neurons of the rat inferior colliculus (IC) to investigate the muscarinic modulation of the GABAergic transmission. In the presence of strychnine (0.5 microM) and kynurenic acid (1 mM), spontaneous GABAergic PSCs were observed in all IC neurons investigated. Muscarine (10 microM) greatly potentiated the frequency of these GABAergic PSCs (618% of the control). 4-DAMP (50 nM), a M3 receptor preferring antagonist, greatly blocked the muscarine-evoked PSC frequency increase. The muscarinic antagonists telenzepine (50 nM; M1 preferring), methoctramine (10 microM; M2 preferring), and himbazine (10 microM; M4 preferring), and the nicotinic antagonist mecamylamine (10 microM) did not significantly affect the muscarine effect. These findings indicate that the muscarinic modulation of the GABAergic transmission is primarily mediated by M3 receptors, while M1-, M2- and M4- and nicotinic receptors do not participate substantially. In the presence of tetrodotoxin (0.5 microM), muscarine failed to increase the PSC frequency indicating that its effect depended on the generation of spikes. We suggest that GABAergic interneurones express M3 receptors at some distance from the terminal. Their activation excites GABAergic interneurones, thereby enhancing GABA release in the IC. The muscarinic modulation of the GABAergic transmission may play an important role in the maturation of inhibitory synapses in the developing IC.     

Stem cells as a novel tool for drug screening and treatment of degenerative diseases

Degenerative diseases similarly as acute tissue injuries lead to massive cell loss and may cause organ failure of vital organs (e.g., heart, central nervous system). Therefore, they belong to a group of disorders that may significantly benefit from stem cells (SCs)-based therapies. Several stem and progenitor cell populations have already been described as valuable tools for developing therapeutic strategies in regenerative medicine. In particular, pluripotent stem cells (PSCs), including adult-tissue-derived PSCs, neonatal-tissue-derived SCs, embryonic stem cells (ESCs), and recently described induced pluripotent stem cells (iPSCs), are the focus of particular attention because of their capacity to differentiate into all the cell lineages. Although PSCs are predominantly envisioned to be applied for organ regeneration, they may be also successfully employed in drug screening and disease modeling. In particular, adult PSCs and iPSCs derived from patient tissues may not only be a source of cells for autologous therapies but also for individual customized in vitro drug testing and studies on the molecular mechanisms of disease. In this review, we will focus on the potential applications of SCs, especially PSCs i) in regenerative medicine therapies, ii) in studying mechanisms of disease, as well as iii) in drug screening and toxicology tests that are crucial in new drug development. In particular, we will discuss the application of SCs in developing new therapeutic approaches to treat degenerative diseases of the neural system and heart. The advantage of adult PSCs in all the above-mentioned settings is that they can be directly harvested from patient tissues and used not only as a safe non-immunogenic source of cells for therapy but also as tools for personalized drug screening and pharmacological therapies.     

Standardization of pluripotent stem cell cultures for toxicity testing

INTRODUCTION: Pluripotent stem cell (PSC) lines offer a unique opportunity to derive various human cell types that can be exploited for human safety assessments in vitro and as such contribute to modern mechanistically oriented toxicity testing. AREAS COVERED: This article reviews the two major types of PSC cultures that are currently most promising for toxicological applications: human embryonic stem cell lines and human induced PSC lines. Through the review, the article explains how these cell types will improve the current safety evaluations of chemicals and will allow a more efficient selection of drug candidates. Additionally, the article discusses the important issues of maintaining PSCs as well as their differentiation efficiency. EXPERT OPINION: The demonstration of the reliability and relevance of in vitro toxicity tests for a given purpose is mandatory for their use in regulatory toxicity testing. Given the peculiar nature of PSCs, a high level of standardization of undifferentiated cell cultures as well as of the differentiation process is required in order to ensure the establishment of robust test systems. It is, therefore, of pivotal importance to define and internationally agree on crucial parameters to judge the quality of the cellular models before enrolling them for toxicity testing.     

Nitric oxide release and long term potentiation at synapses in autonomic ganglia

• 1.1. Long-term potentiation (LTP) of synaptic transmission in autonomic ganglia is reviewed, together with the possible role of nitric oxide (NO) in this process.
• 2.2. Calcium levels in preganglionic nerve terminals are elevated during at least the induction phase of LTP following a tetanus as well as during LTP induced by transmitter substances acting on the nerve terminals. Of the large number of calcium-dependent processes in the nerve terminal that might affect transmitter release, only calcium-calmodulin has been shown to be important in both the induction and maintenance of LTP.
• 3.3. The possibility that there is a decrease in the open time of nerve-terminal potassium channels following a tetanus, leading to an increase in duration of the terminal action potential and hence an increase in calcium influx and transmitter release is considered. There is little evidence for such an effect as yet for preganglionic nerve terminals.
• 4.4. Phosphorylation of potassium channels by cAMP-dependent protein kinase can lead to their inactivation with consequent action potential broadening in some systems. Exogenous cAMP enhances synaptic efficacy at preganglionic nerve terminals. Whether this occurs through an inactivation of potassium channels is not known.
• 5.5. Nitric oxide (NO) synthase is present in both sympathetic ganglia and the ciliary ganglia. NO increases synaptic efficacy in both ganglia. In at least the case of ciliary ganglion this is due to elevation of quantal secretion.
• 6.6. NO can in some conditions increase the terminal action potential duration in ciliary ganglia, probably through decrease in the Ic potassium current. There is evidence that this happens through cGMP modulating cAMP phosphodiesterases, thereby affecting cAMP phosphorylation of the Ic channel.
• 7.7. Blocking NO synthase markedly decreases LTP following a tetanus in the ciliary ganglion. The possibility is considered that NO is released from the terminal during a tetanus and through altering cAMP phosphorylation of Ic enhances transmitter release.

     

Calcium overload in nerve terminals of cultured neurons intoxicated by alpha-latrotoxin and snake PLA2 neurotoxins

Snake presynaptic neurotoxins with phospholipase A2 (PLA2) activity cause degeneration of the neuromuscular junction. They induce depletion of synaptic vesicles and increase the membrane permeability to Ca²⁺ which fluxes from the outside into the nerve terminal. Moreover, several toxins were shown to enter the nerve terminals of cultured neurons, where they may display their PLA2 activity on internal membranes. The relative contribution of these different actions in nerve terminal degeneration remains to be established. To gather information on this point, we have compared the effects of β-bungarotoxin, taipoxin, notexin and textilotoxin with those of alpha-latrotoxin on the basis of the notion that this latter toxin is well known to cause massive Ca²⁺ influx and exocytosis of synaptic vesicles. All the parameters analysed here, including calcium imaging, are very similar for the two classes of neurotoxins. This indicates that Ca²⁺ overloading plays a major role in the degeneration of nerve terminals induced by the snake presynaptic neurotoxins.     

Selective phytochemicals targeting pancreatic stellate cells as new anti-fibrotic agents for chronic pancreatitis and pancreatic cancer

Activated pancreatic stellate cells (PSCs) have been widely accepted as a key precursor of excessive pancreatic fibrosis, which is a crucial hallmark of chronic pancreatitis (CP) and its formidable associated disease, pancreatic cancer (PC). Hence, anti-fibrotic therapy has been identified as a novel therapeutic strategy for treating CP and PC by targeting PSCs. Most of the anti-fibrotic agents have been limited to phase I/II clinical trials involving vitamin analogs, which are abundant in medicinal plants and have proved to be promising for clinical application. The use of phytomedicines, as new anti-fibrotic agents, has been applied to a variety of complementary and alternative approaches. The aim of this review was to present a focused update on the selective new potential anti-fibrotic agents, including curcumin, resveratrol, rhein, emodin, green tea catechin derivatives, metformin, eruberin A, and ellagic acid, in combating PSC in CP and PC models. It aimed to describe the mechanism(s) of the phytochemicals used, either alone or in combination, and the associated molecular targets. Most of them were tested in PC models with similar mechanism of actions, and curcumin was tested intensively. Future research may explore the issues of bioavailability, drug design, and nano-formulation, in order to achieve successful clinical outcomes with promising activity and tolerability.     

Effects of synthetic omega-conotoxin on synaptic transmission

The effects of chemically synthesized omega-conotoxin GVIA (a neurotoxic peptide from Conus geographus) on synaptic transmission at the bullfrog sympathetic ganglion, frog neuromuscular junction and electric organ of the ray, Narke japonica, were studied. The synthetic toxin irreversibly suppressed synaptic transmission at these synapses by arresting the release of transmission from the nerve terminals without showing postsynaptic effects. This action of the toxin was effectively antagonized by high concentrations of extracellular Ca2+. The synthetic toxin irreversibly blocked the Ca2+-dependent action potential of bullfrog sympathetic ganglion cells. These results suggest that omega-conotoxin GVIA blocks synaptic transmission by interfering with the Ca2+ influx through the voltage-sensitive Ca2+ channel of the nerve terminal. These results indicate that the chemically synthesized omega-conotoxin GVIA acts exactly like the natural omega-conotoxin GVIA. Thus, the synthetic toxin can be used in place of the natural toxin as a useful probe for the voltage-sensitive Ca2+ channel in the nervous system.     

Characterization of rabphilin phosphorylation using phospho-specific antibodies.

Rab3A is a GTP-binding protein of synaptic vesicles that regulates neurotransmitter release and cycles on and off synaptic vesicles as a function of exocytosis. Rab3A presumably functions via GTP-dependent interactions with effectors. Two putative rab3A effectors have been described in neurons, rabphilin which is a soluble protein that moves onto and off synaptic vesicles in concert with rab3A, and RIM which is an active zone protein that only binds to rab3A on docked vesicles. Rabphilin is an abundant, evolutionarily conserved protein whose function has remained enigmatic since a knockout of rabphilin does not display the functional deficiencies observed in the rab3A knockout. However, previous studies have shown that rabphilin is phosphorylated by protein kinase A and CaM Kinase II, suggesting that it may have a regulatory role. In the present study, we have examined the site and regulation of rabphilin phosphorylation in living nerve terminals using phospho-specific antibodies raised against phospho-serine234 of rabphilin. With these antibodies, we demonstrate that rabphilin is physiologically phosphorylated on serine234, and that soluble rabphilin which is not bound to rab3A on synaptic vesicles is the primary target. However, different from synapsins which are induced to dissociate from synaptic vesicles by PKA phosphorylation, phosphorylation of rabphilin is not instrumental for dissociating rabphilin from synaptic vesicles. Our data support the notion that dissociated rabphilin is a synaptic phosphoprotein in vivo that may play a role in the regulation of nerve terminal protein-protein interactions.     

An electrophysiological analysis of the effects of reserpine on adrenergic neuromuscular transmission.

1 An electrophysiological study has been made of the effects of depleting synaptic vesicles (i.e. small vesicles less than 60 nm diameter) of their transmitter with reserpine on the quantity of transmitter released by nerve impulses, using the amplitude of the synaptic potential as a measure of transmitter release. 2 Pretreatment of adrenergic nerve terminals with reserpine sufficient to deplete the terminals of 70% of their noradrenaline (NA) did not change the total number of synaptic vesicles in the terminals, but did reduce the number with a large granular core as well as the quantity of NA released by a single nerve impulse by 80%. 3 Pretreatment of adrenergic nerve terminals with reserpine and iproniazid, to decrease vesicular NA but enhance cytoplasmic NA, had the same effect on synaptic vesicles and on the NA released by a single nerve impulse as did reserpine alone. 4 During a short train of impulses at high frequencies in reserpine pretreated terminals, the quantity of NA released by successive impulses increased until a steady-state release was reached comparable to that in untreated preparations. This facilitated release could be quantitatively predicted in terms of the addition of the individual potentiations introduced by each impulse in the train. 5 These results are consistent with the idea that each quantum of transmitter is stored in a synaptic vesicle, and that these may be released by nerve impulses directly from the terminal by a process of exocytosis.     

Synaptic terminal degeneration and remodeling at the rat neuromuscular junction resulting from a single exposure to acrylamide

Repetitive exposure to low doses of acrylamide results in extensive pathological changes at the neuromuscular junction (NMJ), but it remains undetermined if a single exposure to a larger dose will produce a similar neuropathological outcome. In the present study, morphometric and ultrastructural analyses of rat soleus NMJ were performed to determine early pathological effects of an intraperitoneal injection of 100 mg/kg acrylamide. Widespread nerve terminal degeneration, terminal sprouting, and endplate lengthening were evident as early as 4 days after injection. Degenerating terminal branches were swollen and exhibited enhanced argyrophilia. Ultrastructurally, the majority of terminals exhibited axolemmal abnormalities, neurofilament accumulations, and a paucity of synaptic vesicles; occasional swollen terminals lacked neurofilaments but contained increased numbers of tubulovesicular profiles. This early morphological pattern of nerve terminal changes suggests that acrylamide may disrupt both synaptic vesicle recycling and neurofilament degradation. These findings indicate that a single high dose of acrylamide triggers pathological lesions and remodeling in motor nerve terminals virtually identical to those resulting from multiple low doses.     

Oral dyskinesias and morphological changes in rat striatum during long-term haloperidol administration

RATIONALE: Neuroleptic-induced oral dyskinesias in rats, a putative analogue to human tardive dyskinesia, may be due to increased glutamate release within the striatum. This may lead to excitotoxic degeneration and, as a consequence, persistent motor side effects. OBJECTIVES: To investigate whether alterations in glutamatergic synapses within the striatum are associated with the development of neuroleptic-induced oral dyskinesia. METHODS: Haloperidol was administered for 20 weeks, and rats with high and low levels of vacuous chewing movements (VCM) were analyzed for morphological changes with electron microscopy at three time points. RESULTS: At week 8, the high VCM rats had a larger nerve terminal area and lower density of nerve terminal glutamate immunoreactivity than the other groups. After 18 weeks of treatment, the nerve terminal area was increased relative to controls in both the high and low VCM groups. After discontinuation of treatment, there were no significant morphological differences between the groups, but the level of VCM was still significantly increased in the high VCM group. CONCLUSIONS: These results show that striatal glutamatergic transmission is affected during haloperidol treatment and the nerve terminal area and the density of nerve terminal glutamate immunoreactivity are important in determining the VCM response to haloperidol treatment. This indicates that increased glutamatergic synaptic activity in the striatum contributes to the development of human tardive dyskinesia.     

Screening of Bioactive Peptides Using an Embryonic Stem Cell-Based Neurodifferentiation Assay

Differentiation of pluripotent stem cells, PSCs, towards neural lineages has attracted significant attention, given the potential use of such cells for in vitro studies and for regenerative medicine. The present experiments were designed to identify bioactive peptides which direct PSC differentiation towards neural cells. Fifteen peptides were designed based on NCAM, FGFR, and growth factors sequences. The effect of peptides was screened using a mouse embryonic stem cell line expressing luciferase dual reporter construct driven by promoters for neural tubulin and for elongation factor 1. Cell number was estimated by measuring total cellular DNA. We identified five peptides which enhanced activities of both promoters without relevant changes in cell number. We selected the two most potent peptides for further analysis: the NCAM-derived mimetic FGL_L and the synthetic NCAM ligand, Plannexin. Both compounds induced phenotypic neuronal differentiation, as evidenced by increased neurite outgrowth. In summary, we used a simple, but sensitive screening approach to identify the neurogenic peptides. These peptides will not only provide new clues concerning pathways of neurogenesis, but they may also be interesting biotechnology tools for in vitro generation of neurons.     

An electrophysiological analysis of the effects of morphine on the calcium dependence of neuromuscular transmission in the mouse vas deferens.

1 The effects of morphine on the Ca-dependence of the synaptic potential amplitude in the mouse vas deferens have been determined. 2 The synaptic potential increased with a power factor of 2.4 for [Ca]o between 0.7 mM and 1.8 mM. Morphine (40 nM) decreased the synaptic potential, without altering the second power relationship between the synaptic potential and [Ca]o. 3 Morphine reversed the depression in the synaptic potential which develops during a short high-frequency (10 Hz) train of impulses to facilitation. Consequently the synaptic potential beyond the tenth impulse was unaffected by morphine. 4 Morphine did not alter the facilitation of the synaptic potential which develops during a short low-frequency (less than or equal to 2 Hz) train of impulses in normal [Ca]o. Consequently morphine decreased the synaptic potential for each impulse by about the same percentage amount. 5 Morphine increased the small facilitation in the synaptic potential which occurs during a short low-frequency (less than or equal to 2 Hz) train of impulses in high [Ca]o. This facilitation approximated the predictions based on the assumption that each impulse leaves residual Ca ions bound to receptors involved in transmitter release from the nerve terminal.