Characterization of a functional V1B vasopressin receptor in the male rat kidney: evidence for cross talk between V1B and V2 receptor signaling pathways.

Although vasopressin V1B receptor (V1BR) mRNA has been detected in the kidney, the precise renal localization as well as pharmacological and physiological properties of this receptor remain unknown. Using the selective V1B agonist d[Leu4, Lys8]VP, either fluorescent or radioactive, we showed that V1BR is mainly present in principal cells of the inner medullary collecting duct (IMCD) in the male rat kidney. Protein and mRNA expression of V1BR were very low compared with the V2 receptor (V2R). On the microdissected IMCD, d[Leu4, Lys8]VP had no effect on cAMP production but induced a dose-dependent and saturable intracellular Ca2+ concentration increase mobilization with an EC50 value in the nanomolar range. This effect involved both intracellular Ca2+ mobilization and extracellular Ca2+ influx. The selective V1B antagonist SSR149415 strongly reduced the ability of vasopressin to increase intracellular Ca2+ concentration but also cAMP, suggesting a cooperation between V1BR and V2R in IMCD cells expressing both receptors. This cooperation arises from a cross talk between second messenger cascade involving PKC rather than receptor heterodimerization, as supported by potentiation of arginine vasopressin-stimulated cAMP production in human embryonic kidney-293 cells coexpressing the two receptor isoforms and negative results obtained by bioluminescence resonance energy transfer experiments. In vivo, only acute administration of high doses of V1B agonist triggered significant diuretic effects, in contrast with injection of selective V2 agonist. This study brings new data on the localization and signaling pathways of V1BR in the kidney, highlights a cross talk between V1BR and V2R in the IMCD, and suggests that V1BR may counterbalance in some pathophysiological conditions the antidiuretic effect triggered by V2R activation.NEW & NOTEWORTHY Although V1BR mRNA has been detected in the kidney, the precise renal localization as well as pharmacological and physiological properties of this receptor remain unknown. Using original pharmaceutical tools, this study brings new data on the localization and signaling pathways of V1BR, highlights a cross talk between V1BR and V2 receptor (V2R) in the inner medullary collecting duct, and suggests that V1BR may counterbalance in some pathophysiological conditions the antidiuretic effect triggered by V2R activation.

Regulation of cell growth, survival and migration by ceramide 1-phosphate - implications in lung cancer progression and inflammation.

Ceramide 1-phosphate (C1P) is a bioactive sphingolipid that is implicated in the regulation of vital cellular functions and plays key roles in a number of inflammation-associated pathologies. C1P was first described as mitogenic for fibroblasts and macrophages and was later found to promote cell survival in different cell types. The mechanisms involved in the mitogenic actions of C1P include activation of MEK/ERK1-2, PI3K/Akt/mTOR, or PKC-α, whereas promotion of cell survival required a substantial reduction of ceramide levels through inhibition of serine palmitoyl transferase or sphingomyelinase activities. C1P and ceramide kinase (CerK), the enzyme responsible for its biosynthesis in mammalian cells, play key roles in tumor promotion and dissemination. CerK-derived C1P can be secreted to the extracellular milieu by different cell types and is also present in extracellular vesicles. In this context, whilst cell proliferation is regulated by intracellularly generated C1P, stimulation of cell migration/invasion requires the intervention of exogenous C1P. Regarding inflammation, C1P was first described as pro-inflammatory in a variety of cell types. However, cigarette smoke- or lipopolysaccharide-induced lung inflammation in mouse or human cells was overcome by pretreatment with natural or synthetic C1P analogs. Both acute and chronic lung inflammation, and the development of lung emphysema were substantially reduced by exogenous C1P applications, pointing to an anti-inflammatory action of C1P in the lungs. The molecular mechanisms involved in the regulation of cell growth, survival and migration with especial emphasis in the control of lung cancer biology are discussed.

Phosphatidic Acid Stimulates Myoblast Proliferation through Interaction with LPA1 and LPA2 Receptors.

Phosphatidic acid (PA) is a bioactive phospholipid capable of regulating key biological functions, including neutrophil respiratory burst, chemotaxis, or cell growth and differentiation. However, the mechanisms whereby PA exerts these actions are not completely understood. In this work, we show that PA stimulates myoblast proliferation, as determined by measuring the incorporation of [3H]thymidine into DNA and by staining the cells with crystal violet. PA induced the rapid phosphorylation of Akt and ERK1/2, and pretreatment of the cells with specific small interferin RNA (siRNA) to silence the genes encoding these kinases, or with selective pharmacologic inhibitors, blocked PA-stimulated myoblast proliferation. The mitogenic effects of PA were abolished by the preincubation of the myoblasts with pertussis toxin, a Gi protein inhibitor, suggesting the implication of Gi protein-coupled receptors in this action. Although some of the effects of PA have been associated with its possible conversion to lysoPA (LPA), treatment of the myoblasts with PA for up to 60 min did not produce any significant amount of LPA in these cells. Of interest, pharmacological blockade of the LPA receptors 1 and 2, or specific siRNA to silence the genes encoding these receptors, abolished PA-stimulated myoblast proliferation. Moreover, PA was able to compete with LPA for binding to LPA receptors, suggesting that PA can act as a ligand of LPA receptors. It can be concluded that PA stimulates myoblast proliferation through interaction with LPA1 and LPA2 receptors and the subsequent activation of the PI3K/Akt and MEK/ERK1-2 pathways, independently of LPA formation.

Role of bioactive sphingolipids in physiology and pathology.

Sphingolipids are a class of complex lipids containing a backbone of sphingoid bases, namely the organic aliphatic amino alcohol sphingosine (Sph), that are essential constituents of eukaryotic cells. They were first described as major components of cell membrane architecture, but it is now well established that some sphingolipids are bioactive and can regulate key biological functions. These include cell growth and survival, cell differentiation, angiogenesis, autophagy, cell migration, or organogenesis. Furthermore, some bioactive sphingolipids are implicated in pathological processes including inflammation-associated illnesses such as atherosclerosis, rheumatoid arthritis, inflammatory bowel disease (namely Crohn's disease and ulcerative colitis), type II diabetes, obesity, and cancer. A major sphingolipid metabolite is ceramide, which is the core of sphingolipid metabolism and can act as second messenger, especially when it is produced at the plasma membrane of cells. Ceramides promote cell cycle arrest and apoptosis. However, ceramide 1-phosphate (C1P), the product of ceramide kinase (CerK), and Sph 1-phosphate (S1P), which is generated by the action of Sph kinases (SphK), stimulate cell proliferation and inhibit apoptosis. Recently, C1P has been implicated in the spontaneous migration of cells from some types of cancer, and can enhance cell migration/invasion of malignant cells through interaction with a Gi protein-coupled receptor. In addition, CerK and SphK are implicated in inflammatory responses, some of which are associated with cancer progression and metastasis. Hence, targeting these sphingolipid kinases to inhibit C1P or S1P production, or blockade of their receptors might contribute to the development of novel therapeutic strategies to reduce metabolic alterations and disease.

Novel signaling aspects of ceramide 1-phosphate.

The bioactive sphingolipid ceramide 1-phosphate (C1P) regulates key physiologic cell functions and is implicated in a number of metabolic alterations and pathological processes. Initial studies using different types of fibroblasts and monocytes/macrophages revealed that C1P was mitogenic and that it promoted cell survival through inhibition of apoptosis. Subsequent studies implicated C1P in inflammatory responses with a specific role as pro-inflammatory agent. Specifically, C1P potently stimulated cytosolic phospholipase A2 (cPLA2) resulting in elevation of arachidonic acid and pro-inflammatory eicosanoid levels. However, increasing experimental evidence suggests that C1P can also exert anti-inflammatory actions in some cell types and tissues. Specifically, it has been demonstrated that C1P inhibits the release of pro-inflammatory cytokines and blocks activation of the pro-inflammatory transcription factor NF-κB in some cell types. Moreover, C1P was shown to increase the release of anti-inflammatory interleukin-10 in macrophages, and to overcome airway inflammation and reduce lung emphysema in vivo. Noteworthy, C1P stimulated cell migration, an action that is associated with diverse physiological cell functions, as well as with inflammatory responses and tumor dissemination. More recently, ceramide kinase (CerK), the enzyme that produces C1P in mammalian cells, has been shown to be upregulated during differentiation of pre-adipocytes into mature adipocytes, and that exogenous C1P, acting through a putative Gi protein-coupled receptor, negatively regulates adipogenesis. Although the latter actions seem to be contradictory, it is plausible that exogenous C1P may balance the adipogenic effects of intracellularly generated (CerK-derived) C1P in adipose tissue. The present review highlights novel signaling aspects of C1P and its impact in the regulation of cell growth and survival, inflammation and tumor dissemination.

Regulation of adipogenesis by ceramide 1-phosphate.

We showed previously that ceramide kinase (CerK) expression increases during adipogenesis pointing to a relevant role of intracellular C1P in this process. In the present work we demonstrate that administration of exogenous C1P inhibits the differentiation of 3T3-L1 pre-adipocytes into mature adipocytes through a mechanism involving activation of extracellularly regulated kinases (ERK) 1-2. Exogenous C1P reduced the accumulation of lipid droplets and the content of triacylglycerol in these cells, and potently inhibited the expression of the early and late adipogenic markers C/EBPß and PPARγ, respectively. C1P also reduced the secretion of leptin, which is a crucial regulator of energy balance and appetite in the organism, and is considered to be a late marker of adipogenesis. Interestingly, all of these C1P actions were reversed by pertussis toxin, suggesting the intervention of a Gi protein-coupled receptor previously identified for C1P, in this process. Also, exogenous C1P significantly reduced CerK activity. Altogether, the data presented in this work suggest that exogenous C1P may balance adipogenesis, and that targeting CerK may be a novel way for potential applications in the treatment of obesity or other inflammation-associated diseases.

Neuroanatomical distribution and function of the vasopressin V1B receptor in the rat brain deciphered using specific fluorescent ligands.

It is now accepted that vasopressin, through V1A/V1B receptors, centrally regulates cognitive functions such as memory, affiliation, stress, fear and depression. However, the respective roles of these receptor isoforms and their contribution to stress-related pathologies remain uncertain. The development of new therapeutic treatments requires a precise knowledge of the distribution of these receptors within the brain, which has been so far hampered by the lack of selective V1B markers. In the present study, we have determined the pharmacological properties of three new potent rat V1B fluorescent ligands and demonstrated that they constitute valuable tools for simultaneous visualization and activation of native V1B receptors in living rat brain tissue. Thus, d[Leu4,Lys-Alexa 647)8]VP (analogue 3), the compound with the best affinity-selectivity/fluorescence ratio for the V1B receptor emerged as the most promising. The rat brain regions most concerned by stress such as hippocampus, olfactory bulbs, cortex and amygdala display the highest V1B fluorescent labelling with analogue 3. In the hippocampus CA2, V1B receptors are located on glutamatergic, not GABAergic neurones, and are absent from astrocytes. Using AVP-EGFP rats, we demonstrate the presence of V1B autoreceptors on AVP-secreting neurones not only in the hypothalamus, but also sparsely in the hippocampus. Finally, using both electrophysiology and visualization of ERK phosphorylation, we show analogue 3-induced activation of the V1B receptor in situ. This will help to analyse expression and functionality of V1B receptors in the brain and contribute to further explore the AVPergic circuitry in normal and pathological conditions.

Implication of Ceramide Kinase in Adipogenesis.

Ceramide kinase (CerK) plays a critical role in the regulation of cell growth and survival and has been implicated in proinflammatory responses. In this work, we demonstrate that CerK regulates adipocyte differentiation, a process associated with obesity, which causes chronic low-grade inflammation. CerK was upregulated during differentiation of 3T3-L1 preadipocytes into mature adipocytes. Noteworthy, knockdown of CerK using specific siRNA to silence the gene encoding this kinase resulted in substantial decrease of lipid droplet formation and potent depletion in the content of triacylglycerols in the adipocytes. Additionally, CerK knockdown caused blockade of leptin secretion, an adipokine that is crucial for regulation of energy balance in the organism and that is increased in the obese state. Moreover, CerK gene silencing decreased the expression of peroxisome proliferator-activated receptor gamma (PPARγ), which is considered the master regulator of adipogenesis. It can be concluded that CerK is a novel regulator of adipogenesis, an action that may have potential implications in the development of obesity, and that targeting this kinase may be beneficial for treatment of obesity-associated diseases.

Regulation of cell migration and inflammation by ceramide 1-phosphate.

Ceramide 1-phosphate (C1P) is a bioactive sphingolipid metabolite first shown to regulate cell growth and death. Subsequent studies revealed that C1P was a potent stimulator of cytosolic phospholipase A2 (cPLA2) with ensuing release of arachidonic acid and prostaglandin biosynthesis. The latter findings placed C1P on the list of pro-inflammatory metabolites. More recently, C1P was found to potently stimulate cell migration, an action that is associated to diverse physiological effects, as well as to inflammatory responses and tumor dissemination. The implication of C1P in inflammation has gained further interest in the last few years due to the discovery that it can exert anti-inflammatory actions in some cell types and tissues. In particular, C1P has been demonstrated to inhibit pro-inflammatory cytokine release and blockade of the pro-inflammatory transcription factor NF-κB in some cell types, as well as to reduce airway inflammation and lung emphysema. The present review is focused on novel aspects of C1P regulation of cell migration and the impact of C1P as novel anti-inflammatory agent. GLOSS: Ceramide 1-phosphate (C1P) is a phosphosphingolipid with potent biological activities. It promotes cell growth and survival, and is a key regulator of cell migration. Both C1P and the enzyme that catalyzes its biosynthesis, ceramide kinase, are implicated in inflammatory responses. Although C1P has pro-inflammatory properties, it reduces pulmonary emphysema and exerts anti-inflammatory actions in the lung. Synthetic C1P analogs may be promising tools to treat lung inflammation.

Ceramide 1-phosphate regulates cell migration and invasion of human pancreatic cancer cells.

Pancreatic cancer is an aggressive and devastating disease characterized by invasiveness, rapid progression and profound resistance to treatment. Despite years of intense investigation, the prognosis of this type of cancer is poor and there is no efficacious treatment to overcome the disease. Using human PANC-1 and MIA PaCa-2 cells, we demonstrate that the bioactive sphingolipid ceramide 1-phosphate (C1P) increases pancreatic cancer cell migration and invasion. Treatment of these cells with selective inhibitors of phosphatidylinositol 3-kinase (PI3K), Akt1, or mammalian target of rapamycin 1 (mTOR1), or with specific siRNAs to silence the genes encoding these kinases, resulted in potent inhibition of C1P-induced cell migration and invasion. Likewise, the extracellularly regulated kinases 1 and 2 (ERK1-2), and the small GTPase RhoA, which regulates cytoskeleton reorganization, were also found to be implicated in C1P-stimulated ROCK1-dependent cancer cell migration and invasion. In addition, pre-treatment of the cancer cells with pertussis toxin abrogated C1P-induced cell migration, suggesting the intervention of a Gi protein-coupled receptor in this process. Pancreatic cancer cells engineered to overexpress ceramide kinase (CerK), the enzyme responsible for C1P biosynthesis in mammalian cells, showed enhanced spontaneous cell migration that was potently blocked by treatment with the selective CerK inhibitor NVP-231, or by treatment with specific CerK siRNA. Moreover, overexpression of CerK with concomitant elevations in C1P enhanced migration of pancreatic cancer cells. Collectively, these data demonstrate that C1P is a key regulator of pancreatic cancer cell motility, and suggest that targeting CerK expression/activity and C1P may be relevant factors for controlling pancreatic cancer cell dissemination.

Control of inflammatory responses by ceramide, sphingosine 1-phosphate and ceramide 1-phosphate.

Inflammation is a network of complex processes involving a variety of metabolic and signaling pathways aiming at healing and repairing damage tissue, or fighting infection. However, inflammation can be detrimental when it becomes out of control. Inflammatory mediators involve cytokines, bioactive lipids and lipid-derived metabolites. In particular, the simple sphingolipids ceramides, sphingosine 1-phosphate, and ceramide 1-phosphate have been widely implicated in inflammation. However, although ceramide 1-phosphate was first described as pro-inflammatory, recent studies show that it has anti-inflammatory properties when produced in specific cell types or tissues. The biological functions of ceramides and sphingosine 1-phosphate have been extensively studied. These sphingolipids have opposing effects with ceramides being potent inducers of cell cycle arrest and apoptosis, and sphingosine 1-phosphate promoting cell growth and survival. However, the biological actions of ceramide 1-phosphate have only been partially described. Ceramide 1-phosphate is mitogenic and anti-apoptotic, and more recently, it has been demonstrated to be key regulator of cell migration. Both sphingosine 1-phosphate and ceramide 1-phosphate are also implicated in tumor growth and dissemination. The present review highlights new aspects on the control of inflammation and cell migration by simple sphingolipids, with special emphasis to the role played by ceramide 1-phosphate in controlling these actions.

Sphingomyelinase D/ceramide 1-phosphate in cell survival and inflammation.

Sphingolipids are major constituents of biological membranes of eukaryotic cells. Many studies have shown that sphingomyelin (SM) is a major phospholipid in cell bilayers and is mainly localized to the plasma membrane of cells, where it serves both as a building block for cell architecture and as a precursor of bioactive sphingolipids. In particular, upregulation of (C-type) sphingomyelinases will produce ceramide, which regulates many physiological functions including apoptosis, senescence, or cell differentiation. Interestingly, the venom of some arthropodes including spiders of the genus Loxosceles, or the toxins of some bacteria such as Corynebacterium tuberculosis, or Vibrio damsela possess high levels of D-type sphingomyelinase (SMase D). This enzyme catalyzes the hydrolysis of SM to yield ceramide 1-phosphate (C1P), which promotes cell growth and survival and is a potent pro-inflammatory agent in different cell types. In particular, C1P stimulates cytosolic phospholipase A2 leading to arachidonic acid release and the subsequent formation of eicosanoids, actions that are all associated to the promotion of inflammation. In addition, C1P potently stimulates macrophage migration, which has also been associated to inflammatory responses. Interestingly, this action required the interaction of C1P with a specific plasma membrane receptor, whereas accumulation of intracellular C1P failed to stimulate chemotaxis. The C1P receptor is coupled to Gi proteins and activates of the PI3K/Akt and MEK/ERK1-2 pathways upon ligation with C1P. The proposed review will address novel aspects on the control of inflammatory responses by C1P and will highlight the molecular mechanisms whereby C1P exerts these actions.

Phosphatidic acid inhibits ceramide 1-phosphate-stimulated macrophage migration.

Ceramide 1-phosphate (C1P) was recently demonstrated to potently induce cell migration. This action could only be observed when C1P was applied exogenously to cells in culture, and was inhibited by pertussis toxin. However, the mechanisms involved in this process are poorly understood. In this work, we found that phosphatidic acid (PA), which is structurally related to C1P, displaced radiolabeled C1P from its membrane-binding site and inhibited C1P-stimulated macrophage migration. This effect was independent of the saturated fatty acid chain length or the presence of a double bond in each of the fatty acyl chains of PA. Treatment of RAW264.7 macrophages with exogenous phospholipase D (PLD), an enzyme that produces PA from membrane phospholipids, also inhibited C1P-stimulated cell migration. Likewise, PA or exogenous PLD inhibited C1P-stimulated extracellularly regulated kinases (ERK) 1 and 2 phosphorylation, leading to inhibition of cell migration. However, PA did not inhibit C1P-stimulated Akt phosphorylation. It is concluded that PA is a physiological regulator of C1P-stimulated macrophage migration. These actions of PA may have important implications in the control of pathophysiological functions that are regulated by C1P, including inflammation and various cellular processes associated with cell migration such as organogenesis or tumor metastasis.

Ceramide 1-phosphate induces macrophage chemoattractant protein-1 release: involvement in ceramide 1-phosphate-stimulated cell migration.

The bioactive sphingolipid ceramide 1-phosphate (C1P) is implicated in inflammatory responses and was recently shown to promote cell migration. However, the mechanisms involved in these actions are poorly described. Using J774A.1 macrophages, we have now discovered a new biological activity of C1P: stimulation of monocyte chemoattractant protein-1 (MCP-1) release. This novel effect of C1P was pertussis toxin (PTX) sensitive, suggesting the intervention of Gi protein-coupled receptors. Treatment of the macrophages with C1P caused activation of the phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase kinase (MEK)/extracellularly regulated kinases (ERK), and p38 pathways. Inhibition of these kinases using selective inhibitors or specific siRNA blocked the stimulation of MCP-1 release by C1P. C1P stimulated nuclear factor-κB activity, and blockade of this transcription factor also resulted in complete inhibition of MCP-1 release. Also, C1P stimulated MCP-1 release and cell migration in human THP-1 monocytes and 3T3-L1 preadipocytes. A key observation was that sequestration of MCP-1 with a neutralizing antibody or treatment with MCP-1 siRNA abolished C1P-stimulated cell migration. Also, inhibition of the pathways involved in C1P-stimulated MCP-1 release completely blocked the stimulation of cell migration by C1P. It can be concluded that C1P promotes MCP-1 release in different cell types and that this chemokine is a major mediator of C1P-stimulated cell migration. The PI3K/Akt, MEK/ERK, and p38 pathways are important downstream effectors in this action.

New insights on the role of ceramide 1-phosphate in inflammation.

Inflammation is a complex biological process involving a variety of locally produced molecules, as well as different types of white blood cells. Some of the so-called inflammatory mediators include cytokines, chemokines, interleukins, prostaglandins, or bioactive lipids, all of which provide protection from infection and foreign substances, such as bacteria, yeast, viruses or some chemicals. Under some circumstances, however, the organism inappropriately activates the immune system triggering an inflammatory response in the absence of foreign insults thereby leading to the establishment of autoimmune diseases. Therefore, inflammation must be tightly regulated in order to ensure sufficient protection to the organism in the absence of unwanted, and at times dangerous, side effects. Increasing experimental evidence implicates sphingolipids as major inducers of inflammatory responses and regulators of immune cell functions. In particular, ceramides and sphingosine 1-phosphate have been extensively implicated in inflammation, and ceramide 1-phosphate has also been shown to participate in these processes. The present review highlights novel aspects on the regulation of inflammation by sphingolipids, with special emphasis to the role played by ceramide 1-phosphate and ceramide kinase, the enzyme responsible for its biosynthesis, in inflammatory responses.

Ceramide 1-phosphate stimulates glucose uptake in macrophages.

It is well established that ceramide 1-phosphate (C1P) is mitogenic and antiapoptotic, and that it is implicated in the regulation of macrophage migration. These activities require high energy levels to be available in cells. Macrophages obtain most of their energy from glucose. In this work, we demonstrate that C1P enhances glucose uptake in RAW264.7 macrophages. The major glucose transporter involved in this action was found to be GLUT 3, as determined by measuring its translocation from the cytosol to the plasma membrane. C1P-stimulated glucose uptake was blocked by selective inhibitors of phosphatidylinositol 3-kinase (PI3K) or Akt, also known as protein kinase B (PKB), and by specific siRNAs to silence the genes encoding for these kinases. C1P-stimulated glucose uptake was also inhibited by pertussis toxin (PTX) and by the siRNA that inhibited GLUT 3 expression. C1P increased the affinity of the glucose transporter for its substrate, and enhanced glucose metabolism to produce ATP. The latter action was also inhibited by PI3K- and Akt-selective inhibitors, PTX, or by specific siRNAs to inhibit GLUT 3 expression.

Generation of reactive oxygen species (ROS) is a key factor for stimulation of macrophage proliferation by ceramide 1-phosphate.

We previously demonstrated that ceramide 1-phosphate (C1P) is mitogenic for fibroblasts and macrophages. However, the mechanisms involved in this action were only partially described. Here, we demonstrate that C1P stimulates reactive oxygen species (ROS) formation in primary bone marrow-derived macrophages, and that ROS are required for the mitogenic effect of C1P. ROS production was dependent upon prior activation of NADPH oxidase by C1P, which was determined by measuring phosphorylation of the p40phox subunit and translocation of p47phox from the cytosol to the plasma membrane. In addition, C1P activated cytosolic calcium-dependent phospholipase A(2) and protein kinase C-α, and NADPH oxidase activation was blocked by selective inhibitors of these enzymes. These inhibitors, and inhibitors of ROS production, blocked the mitogenic effect of C1P. By using BHNB-C1P (a photolabile caged-C1P analog), we demonstrate that all of these C1P actions are caused by intracellular C1P. It can be concluded that the enzyme responsible for C1P-stimulated ROS generation in bone marrow-derived macrophages is NADPH oxidase, and that this enzyme is downstream of PKC-α and cPLA(2)-α in this pathway.

Design, synthesis, and pharmacological characterization of fluorescent peptides for imaging human V1b vasopressin or oxytocin receptors.

Among the four known vasopressin and oxytocin receptors, the specific localization of the V1b isoform is poorly described because of the lack of selective pharmacological tools. In an attempt to address this need, we decided to design, synthesize, and characterize fluorescent selective V1b analogues. Starting with the selective V1b agonist [deamino-Cys(1),Leu(4),Lys(8)]vasopressin (d[Leu(4),Lys(8)]VP) synthesized earlier, we added blue, green, or red fluorophores to the lysine residue at position 8 either directly or by the use of linkers of different lengths. Among the nine analogues synthesized, two exhibited very promising properties. These are d[Leu(4),Lys(Alexa 647)(8)]VP (3) and d[Leu(4),Lys(11-aminoundecanoyl-Alexa 647)(8)]VP (9). They remained full V1b agonists with nanomolar affinity and specifically decorated the plasma membrane of CHO cells stably transfected with the human V1b receptor. These new selective fluorescent peptides will allow the cellular localization of V1b or OT receptor isoforms in native tissues.

Activation of mTOR and RhoA is a major mechanism by which Ceramide 1-phosphate stimulates macrophage proliferation.

This study tested the hypothesis that Ceramide 1-phosphate (C1P) stimulates macrophage proliferation through activation of the mammalian target of rapamycin (mTOR). We first reported that C1P is mitogenic for fibroblasts and macrophages, but the mechanisms whereby it stimulates cell proliferation are incompletely understood. Here we demonstrate that C1P causes phosphorylation of mTOR in primary (bone marrow-derived) macrophages. Activation of this kinase was tested my measuring the phosphorylation state of its downstream target p70S6K after treatment with C1P. These actions were dependent upon prior activation of phosphoinositide 3 kinase (PI3-K), as selective inhibition of this kinase blocked mTOR phosphorylation and activation. In addition, C1P caused phosphorylation of PRAS40, a component of the mTOR complex 1 (mTORC1) that is absent in mTORC2. Furthermore, inhibition of the small G protein Ras homolog enriched in brain (Rheb), which is also a specific component of mTORC1, with FTI277, completely blocked C1P-stimulated mTOR phosphorylation, DNA synthesis and macrophage growth. In addition, C1P caused phosphorylation of another Ras homolog gene family member, RhoA, which is also involved in cell proliferation. Interestingly, inhibition of the RhoA downstream effector RhoA-associated kinase (ROCK) also blocked C1P-stimulated mTOR and cell proliferation. It can be concluded that mTORC1, and RhoA/ROCK are essential components of the mechanism whereby C1P stimulates macrophage proliferation.