Sphingosine-1-phosphate, a novel second messenger involved in cell growth regulation and signal transduction, affects growth and invasiveness of human breast cancer cells

This review will focus on the role of sphingosine and its phosphorylated derivative sphingosine-1-phosphate (SPP) in cell growth regulation and signal transduction. We will show that many of the effects attributed to sphingosine in quiescent Swiss 3T3 fibroblasts are mediated via its conversion to SPP. We propose that SPP has appropriate properties to function as an intracellular second messenger based on the following: it elicits diverse cellular responses; it is rapidly produced from sphingosine by a specific kinase and rapidly degraded by a specific lyase; its concentration is low in quiescent cells but increases rapidly and transiently in response to the growth factors, fetal calf serum (FCS) and platelet derived growth factor (PDGF); it releases Ca2+ from internal sources in an InsP3-independent manner; and finally, it may link sphingolipid signaling pathways to cellular ras-mediated signaling pathways by elevating phosphatidic acid levels. The effects of this novel second messenger on growth, differentiation and invasion of human breast cancer cells will be discussed. © 1994 Kluwer Academic Publishers.

Cyclic AMP in Mycobacteria: the second messenger comes first

Cyclic AMP (cAMP) has emerged as a pivotal molecule for signalling in all life forms. Mycobacterial genomes have been found to encode for numerous proteins that are involved in cAMP generation, degradation and utilization. Many of these proteins have domain organizations unique to mycobacteria. This review summarizes recent advances in mechanisms of cAMP synthesis and degradation, focusing on the processes by which cAMP modulates mycobacterial signalling. We explore its impact on the physiology of the organism and on the discourse between M. tuberculosis and its host.

Quorum Sensing and Biofilm Formation in Mycobacteria: Role of c-di-GMP and Methods to Study This Second Messenger

Bacteria have evolved to survive the ever-changing environment using intriguing mechanisms of quorum sensing (QS). Very often, QS facilitates formation of biofilm to help bacteria to persist longer and the formation of such biofilms is regulated by c-di-GMP. It is a well-known second messenger also found in mycobacteria. Several methods have been developed to study c-di-GMP signaling pathways in a variety of bacteria. In this review, we have attempted to highlight a connection between c-di-GMP and biofilm formation and QS in mycobacteria and several methods that have helped in better understanding of c-di-GMP signaling. (c) 2014 IUBMB Life, 66(12):823-834, 2014

Altered endothelin-1 induced contraction and second messenger generation in bovine retinal microvascular pericytes cultured in high glucose medium.

The effect of simulated hyperglycaemia on bovine retinal pericytes was studied following culture of these cells for 10 days under normal (5 mmol/l) and elevated (25 mmol/l) glucose conditions in the absence of endothelial cells. Pericytes cultured under high ambient glucose exhibited both a delayed and reduced contractile response following stimulation with endothelin-1. Stimulation with 10(-7) mol/l endothelin-1 for 30 s caused significant contraction in cells grown in both 5 mmol/l and 25 mmol/l glucose. The former also contracted significantly with 10(-8) mol/l endothelin-1. Further, at all concentrations tested, statistical comparison of the time course of contraction showed a significant difference (p 0.1) between bovine retinal pericytes grown for 10 days under normo- or hyperglycaemic conditions, it became apparent that the altered contractility in bovine retinal pericytes following culture in high glucose must be due to post-binding intracellular disturbance(s). Indeed, both basal and 15 s post-stimulation with 10(-8) mol/l endothelin-1, levels of inositol trisphosphate were significantly reduced (p

Analysis of second messenger pathways stimulated by different chemokines acting at the chemokine receptor

The chemokine receptor, CCR5, responds to several chemokines leading to changes in activity in several signalling pathways. Here, we investigated the ability of different chemokines to provide differential activation of pathways. The effects of five CC chemokines acting at CCR5 were investigated for their ability to inhibit forskolin- stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation and to stimulate Ca2+ mobilisation. in Chinese hamster ovary (CHO) cells expressing CCR5. Macrophage inflammatory protein 1 alpha (D26A) (MIP-1 alpha (D26A), CCL3 (D26A)), regulated on activation, normal T-cell expressed and secreted (RANTES, CCLS), MIP-1 beta (CCL4) and monocyte chemoattractant protein 2 (MCP-2, CCL8) were able to inhibit forskolin -stimulated CAMP accumulation, whilst MCP-4 (CCL13) could not elicit a response. CCL3 (D26A), CCL4, CCLS, CCL8 and CCL13 were able to stimulate Ca2+ mobilisation. through CCRS, although CCL3 (D26A) and CCL5 exhibited biphasic concentration-response curves. The Ca2+ responses induced by CCL4, CCL5, CCL8 and CCL13 were abolished by pertussis toxin, whereas the response to CCL3 (D26A) was only partially inhibited by pertussis toxin, indicating G(i/o)-independent signalling induced by this chemokine. Although the rank order of potency of chemokines was similar between the two assays, certain chemokines displayed different pharmacological profiles in cAMP inhibition and Ca2+ mobilisation assays. For instance, whilst CCL13 could not inhibit forskolin-stimulated cAMP accumulation, this chemokine was able to induce Ca2+ mobilisation via CCR5. It is concluded that different chemokines acting at CCR5 can induce different pharmacological responses, which may account for the broad spectrum of chemokines that can act at CCRS. (C) 2007 Elsevier Inc. All rights reserved.

The role of the intracellular second messenger cAMP in the induction of long-term morphological changes in sensory neurons of {\it Aplysia\/}

The present work examines the role of cAMP in the induction of the type of long-term morphological changes that have been shown to be correlated with long-term sensitization in Aplysia.^ To examine this issue, cAMP was injected into individual tail sensory neurons in the pleural ganglion to mimic, at the single cell level, the effects of behavioral training. After a 22 hr incubation period, the same cells were filled with horseradish peroxidase and 2 hours later the tissue was fixed and processed. Morphological analysis revealed that cAMP induced an increase in two morphological features of the neurons, varicosities and branch points. These structural alterations, which are similar to those seen in siphon sensory neurons of the abdominal ganglion following long-term sensitization training of the siphon-gill withdrawal reflex, could subserve the altered behavioral response of the animal. These results expose another role played by cAMP in the induction of learning, the initiation of a structural substrate, which, in concert with other correlates, underlies learning.^ cAMP was injected into sensory neurons in the presence of the reversible protein synthesis inhibitor, anisomycin. The presence of anisomycin during and immediately following the nucleotide injection completely blocked the structural remodeling. These results indicate that the induction of morphological changes by cAMP is a process dependent on protein synthesis.^ To further examine the temporal requirement for protein synthesis in the induction of these changes, the time of anisomycin exposure was varied. The results indicate that the cellular processes triggered by cAMP are sensitive to the inhibition of protein synthesis for at least 7 hours after the nucleotide injection. This is a longer period of sensitivity than that for the induction of another correlate of long-term sensitization, facilitation of the sensory to motor neuron synaptic connection. Thus, these findings demonstrate that the period of sensitivity to protein synthesis inhibition is not identical for all correlates of learning. In addition, since the induction of the morphological changes can be blocked by anisomycin pulses administered at different times during and following the cAMP injection, this suggests that cAMP is triggering a cascade of protein synthesis, with successive rounds of synthesis being dependent on successful completion of preceding rounds. Inhibition at any time during this cascade can block the entire process and so prevent the development of the structural changes.^ The extent to which cAMP can mimic the structural remodeling induced by long-term training was also examined. Animals were subjected to unilateral sensitization training and the morphology of the sensory neurons was examined twenty-four hours later. Both cAMP injection and long-term training produced a twofold increase in varicosities and approximately a fifty percent increase in the number of branch points in the sensory neuron arborization within the pleural ganglion. (Abstract shortened by UMI.) ^

Second messenger pathways in the action of cachectic factors

Cachexia in cancer is characterised by progressive depletion of both adipose tissue stores and skeletal muscle mass. Two catabolic factors produced by cachexia-inducing tumours have the potential for inducing these changes in body composition: (i) proteolysis-inducing factor (PIF) which acts on skeletal muscle to induce both protein degradation and inhibit protein synthesis, (ii) lipid-mobilising factor (LMF), which has been shown to directly induce lipolysis in isolated epididymal murine white adipocytes. Administration of lipid-mobilising factor (LMF) to mice produced a specific reduction in carcass lipid with a tendency to increase non-fat carcass mass. Treatment of murine myoblasts, myotubes and tumour cells with tumour-produced LMF, caused concentration dependent stimulation of protein synthesis, within a 24hr period. It produced an increase in intracellular cyclic AMP levels, which was linearly related to the increase in protein synthesis. The observed effect was attenuated by pretreating cells with the adenylate cyclase inhibitor, MDL12330A and was additive with stimulation produced by forskolin. Both propranolol and a specific 3 adrenergic antagonist SR59230A, significantly reduced the stimulation of protein synthesis induced by LMF. LMF also affected protein degradation in vitro, as demonstrated by a reduction in proteasome activity, a key component of the ubiquitin-dependent proteolytic pathway. These effects were opposite to those produced by PIF which caused both a decrease in the rate of protein synthesis and an elevation on protein breakdown when incubated in vitro.Incubation of LMF with a fat cell line produced alterations in the levels of guanine-nucleotide binding proteins (G proteins). This was also evident in adipocyte plasma membranes isolated from mice bearing the tumour model of cachexia, MAC16 adenocarcinoma and from patients with cancer cachexia. Progression through the cachectic state induced an upregulation of stimulatory G proteins paralleled with a downregulation of inhibitory G proteins. These changes would contribute to the increased lipid mobilisation seen in cancer cachexia.

Localization of Mineralocorticoid Receptors at Mammalian Synapses

In the brain, membrane associated nongenomic steroid receptors can induce fast-acting responses to ion conductance and second messenger systems of neurons. Emerging data suggest that membrane associated glucocorticoid and mineralocorticoid receptors may directly regulate synaptic excitability during times of stress when adrenal hormones are elevated. As the key neuron signaling interface, the synapse is involved in learning and memory, including traumatic memories during times of stress. The lateral amygdala is a key site for synaptic plasticity underlying conditioned fear, which can both trigger and be coincident with the stress response. A large body of electrophysiological data shows rapid regulation of neuronal excitability by steroid hormone receptors. Despite the importance of these receptors, to date, only the glucocorticoid receptor has been anatomically localized to the membrane. We investigated the subcellular sites of mineralocorticoid receptors in the lateral amygdala of the Sprague-Dawley rat. Immunoblot analysis revealed the presence of mineralocorticoid receptors in the amygdala. Using electron microscopy, we found mineralocorticoid receptors expressed at both nuclear including: glutamatergic and GABAergic neurons and extra nuclear sites including: presynaptic terminals, neuronal dendrites, and dendritic spines. Importantly we also observed mineralocorticoid receptors at postsynaptic membrane densities of excitatory synapses. These data provide direct anatomical evidence supporting the concept that, at some synapses, synaptic transmission is regulated by mineralocorticoid receptors. Thus part of the stress signaling response in the brain is a direct modulation of the synapse itself by adrenal steroids.

In vivo inhibition of elevated myocardial beta-adrenergic receptor kinase activity in hybrid transgenic mice restores normal beta-adrenergic signaling and function.

BACKGROUND: The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac beta-adrenergic receptor (betaAR) system, which is critical in the regulation of myocardial function. Expression of the betaAR kinase (betaARK1), which phosphorylates and uncouples betaARs, is elevated in human HF; this likely contributes to the abnormal betaAR responsiveness that occurs with beta-agonist administration. We previously showed that transgenic mice with increased myocardial betaARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous betaARK1 activity in the heart led to enhanced myocardial function. METHODS AND RESULTS: We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both betaARK1 and an inhibitor of betaARK1 activity to study the feasibility and functional consequences of the inhibition of elevated betaARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of betaARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial betaARK1 levels remained elevated due to transgene expression, in vitro betaARK1 activity returned to control levels and the percentage of betaARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to betaAR stimulation was restored to normal in the hybrid double-transgenic mice. CONCLUSIONS: Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial betaARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac betaARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function.

The effect of endothelin 1 on the retinal microvascular pericyte

The effect of the highly vasoactive peptide endothelin 1 (ET1) was tested on bovine retinal microvascular pericytes propagated in vitro. Specific binding of 125I-ET1 to retinal pericytes was documented by autoradiography. ET1 caused contraction of pericytes at a concentration of 0.1 nM which was accompanied by increases in inositol phosphates. Exposure of pericytes to 10 nM ET1 resulted in the aggregation and realignment of muscle-specific actins into bundles which were oriented parallel to the long axis of the cell, and ET1 was also mitogenic to pericytes in the presence of low levels of fetal calf serum. These observations suggest that ET1 may play an important role in endothelial cell-pericyte interactions within the microvasculature of the retina and that it may be involved in the autoregulation of retinal blood flow.