Profiling of drugs and environmental chemicals for functional impairment of neural crest migration in a novel stem cell-based test battery.

Developmental toxicity in vitro assays have hitherto been established as stand-alone systems, based on a limited number of toxicants. Within the embryonic stem cell-based novel alternative tests project, we developed a test battery framework that allows inclusion of any developmental toxicity assay and that explores the responses of such test systems to a wide range of drug-like compounds. We selected 28 compounds, including several biologics (e.g., erythropoietin), classical pharmaceuticals (e.g., roflumilast) and also six environmental toxicants. The chemical, toxicological and clinical data of this screen library were compiled. In order to determine a non-cytotoxic concentration range, cytotoxicity data were obtained for all compounds from HEK293 cells and from murine embryonic stem cells. Moreover, an estimate of relevant exposures was provided by literature data mining. To evaluate feasibility of the suggested test framework, we selected a well-characterized assay that evaluates 'migration inhibition of neural crest cells.' Screening at the highest non-cytotoxic concentration resulted in 11 hits (e.g., geldanamycin, abiraterone, gefitinib, chlorpromazine, cyproconazole, arsenite). These were confirmed in concentration-response studies. Subsequent pharmacokinetic modeling indicated that triadimefon exerted its effects at concentrations relevant to the in vivo situation, and also interferon-ß and polybrominated diphenyl ether showed effects within the same order of magnitude of concentrations that may be reached in humans. In conclusion, the test battery framework can identify compounds that disturb processes relevant for human development and therefore may represent developmental toxicants. The open structure of the strategy allows rich information to be generated on both the underlying library, and on any contributing assay.

[Cell transplantation: current treatments and future prospects]. / Transplantation cellulaire: traitements actuels et perspectives d'avenir.

Regenerative medicine aims to replace a body function or specific cell loss. It includes therapies at the forefront of modem medicine, issuing from translational biomedical research. Transplantation of organs and cells has revolutionized the management of patients for whom medical treatment is a failure. Unfortunately, organ shortage is limiting treatment possibility. As an example, among the 15,000 patients with type I diabetes in Switzerland, only approximately 30 can receive a pancreas or an islet transplant per year. Second example, 500 patients die each year in Switzerland from alcoholic cirrhosis because no treatment is available. Transplantation of islet cells, hepatocytes, mesenchymal stem cells or dopaminergic neurons represents hope fora therapy available for large populations of patients.

Deficiency in the NADPH oxidase 4 predisposes towards diet-induced obesity.

OBJECTIVE: NADPH oxidase 4 (NOX4) is a reactive oxygen species (ROS) producing NADPH oxidase that regulates redox homeostasis in diverse insulin-sensitive cell types. In particular, NOX4-derived ROS is a key modulator of adipocyte differentiation and mediates insulin receptor signaling in mature adipocytes in vitro. Our study was aimed at investigating the role of NOX4 in adipose tissue differentiation, whole body metabolic homeostasis and insulin sensitivity in vivo. DESIGN: Mice with genetic ablation of NOX4 (NOX4-deficient mice) were subjected to chow or high-fat-containing diet for 12 weeks. Body weight gain, adiposity, insulin sensitivity, and adipose tissue and liver gene and protein expression were analyzed and compared with similarly treated wild-type mice. RESULTS: Here, we report that NOX4-deficient mice display latent adipose tissue accumulation and are susceptible to diet-induced obesity and early onset insulin resistance. Obesity results from accelerated adipocyte differentiation and hypertrophy, and an increase in whole body energy efficiency. Insulin resistance is associated with increased adipose tissue hypoxia, inflammation and adipocyte apoptosis. In the liver, more severe diet-induced steatosis was observed due to the lack of proper upregulation of mitochondrial fatty acid ß-oxidation. CONCLUSION: These findings identify NOX4 as a regulator of metabolic homeostasis. Moreover, they indicate an anti-adipogenic role for NOX4 in vivo and reveal its function as a protector against the development of diet-induced obesity, insulin resistance and hepatosteatosis.

The calcium-binding protein calreticulin is a major constituent of lytic granules in cytolytic T lymphocytes.

Cytolytic T lymphocytes (CTL), natural killer cells, and lymphokine-activated killer (LAK) cells are cytolytic cells known to release the cytolytic protein perforin and a family of proteases, named granzymes, from cytoplasmic stores upon interaction with target cells. We now report the purification of an additional major 60-kD granule-associated protein (grp 60) from human LAK cells and from mouse cytolytic T cells. The NH2-terminal amino acid sequence of the polypeptide was found to be identical to calreticulin. Calreticulin is a calcium storage protein and carries a COOH-terminal KDEL sequence, known to act as a retention signal for proteins destined to the lumen of the endoplasmic reticulum. In CTLs, however, calreticulin colocalizes with the lytic perforin to the lysosome-like secretory granules, as confirmed by double label immunofluorescence confocal microscopy. Moreover, when the release of granule-associated proteins was triggered by stimulation of the T cell receptor complex, calreticulin was released along with granzymes A and D. Since perforin is activated and becomes lytic in the presence of calcium, we propose that the role of calreticulin is to prevent organelle autolysis due to the protein's calcium chelator capacity.

A novel H(+) conductance in eosinophils: unique characteristics and absence in chronic granulomatous disease.

Efficient mechanisms of H(+) ion extrusion are crucial for normal NADPH oxidase function. However, whether the NADPH oxidase-in analogy with mitochondrial cytochromes-has an inherent H(+) channel activity remains uncertain: electrophysiological studies did not find altered H(+) currents in cells from patients with chronic granulomatous disease (CGD), challenging earlier reports in intact cells. In this study, we describe the presence of two different types of H(+) currents in human eosinophils. The "classical" H(+) current had properties similar to previously described H(+) conductances and was present in CGD cells. In contrast, the "novel" type of H(+) current had not been described previously and displayed unique properties: (a) it was absent in cells from gp91- or p47-deficient CGD patients; (b) it was only observed under experimental conditions that allowed NADPH oxidase activation; (c) because of its low threshold of voltage activation, it allowed proton influx and cytosolic acidification; (d) it activated faster and deactivated with slower and distinct kinetics than the classical H(+) currents; and (e) it was approximately 20-fold more sensitive to Zn(2+) and was blocked by the histidine-reactive agent, diethylpyrocarbonate (DEPC). In summary, our results demonstrate that the NADPH oxidase or a closely associated protein provides a novel type of H(+) conductance during phagocyte activation. The unique properties of this conductance suggest that its physiological function is not restricted to H(+) extrusion and repolarization, but might include depolarization, pH-dependent signal termination, and determination of the phagosomal pH set point.

s-cyclophilin is retained intracellularly via a unique COOH-terminal sequence and colocalizes with the calcium storage protein calreticulin.

Cyclophilins (cyclosporin A-binding proteins) are conserved, ubiquitous, and abundant proteins that accelerate the isomerization of XaaPro peptide bonds and the refolding of proteins in vitro. s-Cyclophilin is a member of the cyclophilin family with unique NH2- and COOH-terminal extensions, and with a signal sequence. We now report that s-cyclophilin is retained in the cell, and that the conserved s-cyclophilin-specific COOH-terminal extension VEKPFAIAKE is sufficient to direct a secretory protein to s-cyclophilin containing structures. Antibodies to s-cyclophilin-specific peptides were produced and the location of the protein was determined by an immunocytochemical study at the light microscopic level. s-Cyclophilin colocalized with the Ca(2+)-binding protein calreticulin and, to a lesser extent, with the microsomal Ca(2+)-ATPase in the myogenic cell line L6, and with the Ca(2+)-binding protein calsequestrin in skeletal muscle. In activated platelets, s-cyclophilin immunoreactivity was detected in a ring-like structure that might correspond to the Ca(2+)-storing and -releasing dense tubular network. In spreading cells, s-cyclophilin containing vesicular structures accumulated at actin-rich protrusion sites. While s-cyclophilin consistently codistributed with Ca2+ storage site markers, the distribution of s-cyclophilin immunoreactivity was not identical to that of ER markers. To determine whether the COOH-terminal extension of s-cyclophilin was involved in its intracellular transport we added this sequence to the COOH-terminus of the secretory protein glia-derived nexin. Appropriate constructs were expressed transiently in cultured cells and proteins were detected with specific antibodies. We found that glia-derived nexin with the COOH-terminal sequence VEKPFAIAKE (but not with the control sequence GLVVMNIT) colocalized with endogenous s-cyclophilin, indicating that the sequence contained retention information. These results indicate that s-cyclophilin is a retained component of an intracellular organelle and that it may accumulate in specialized portions of the ER, and possibly in calciosomes. Because of its conserved structure, widespread distribution, and abundance s-cyclophilin may be a useful marker to study the biogenesis and distribution of ER subcompartments.

Nef-mediated clathrin-coated pit formation.

The sequence of events leading to clathrin-coated pit (CCP) nucleation on the cell surface and to the incorporation of receptors into these endocytic structures is still imperfectly understood. In particular, the question remains as to whether receptor tails initiate the assembly of the coat proteins or whether receptors migrate into preformed CCP. This question was approached through a dissection of the mechanisms implemented by Nef, an early protein of human and simian immunodeficiency virus (HIV and SIV, respectively), to accelerate the endocytosis of cluster of differentiation antigen type 4 (CD4), the major receptor for these viruses. Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation. Taken together, these observations lead us to propose that CD4 can promote CCP generation via the connector molecule Nef. In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins). These Nef-generated complexes would then initiate the nucleation of CCP.

Calreticulin is essential for cardiac development.

Calreticulin is a ubiquitous Ca2+ binding protein, located in the endoplasmic reticulum lumen, which has been implicated in many diverse functions including: regulation of intracellular Ca2+ homeostasis, chaperone activity, steroid-mediated gene regulation, and cell adhesion. To understand the physiological function of calreticulin we used gene targeting to create a knockout mouse for calreticulin. Mice homozygous for the calreticulin gene disruption developed omphalocele (failure of absorption of the umbilical hernia) and showed a marked decrease in ventricular wall thickness and deep intertrabecular recesses in the ventricular walls. Transgenic mice expressing a green fluorescent protein reporter gene under the control of the calreticulin promoter were used to show that the calreticulin gene is highly activated in the cardiovascular system during the early stages of cardiac development. Calreticulin protein is also highly expressed in the developing heart, but it is only a minor component of the mature heart. Bradykinin-induced Ca2+ release by the InsP3-dependent pathway was inhibited in crt-/- cells, suggesting that calreticulin plays a role in Ca2+ homeostasis. Calreticulin-deficient cells also exhibited impaired nuclear import of nuclear factor of activated T cell (NF-AT3) transcription factor indicating that calreticulin plays a role in cardiac development as a component of the Ca2+/calcineurin/NF-AT/GATA-4 transcription pathway.

Functional specialization of calreticulin domains.

Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.

A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1.

Voltage-gated proton (H+) channels are found in many human and animal tissues and play an important role in cellular defense against acidic stress. However, a molecular identification of these unique ion conductances has so far not been achieved. A 191-amino acid protein is described that, upon heterologous expression, has properties indistinguishable from those of native H+ channels. This protein is generated through alternative splicing of messenger RNA derived from the gene NOH-1 (NADPH oxidase homolog 1, where NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate).

Redistribution of intracellular Ca2+ stores during phagocytosis in human neutrophils.

Subcellular gradients of cytosolic free Ca2+ concentration, [Ca2+]i, are thought to be critical for the localization of functional responses within a cell. A potential but previously unexplored mechanism for the generation of gradients of [Ca2+]i is the accumulation of Ca2+ stores at the site of Ca2+ action. The distribution of the Ca2+ store markers Ca(2+)-dependent adenosine triphosphatase and calreticulin was investigated in resting and phagocytosing human neutrophils. Both proteins showed an evenly distributed fine granular pattern in nonphagocytosing cells, but became markedly concentrated in the filamentous actin-rich cytoplasmic area around the ingested particle during phagocytosis. This redistribution began at early stages of phagocytosis and did not depend on an increase in [Ca2+]i. Thus, accumulation of Ca2+ stores in a restricted area of the cell may contribute to the generation of localized increases in [Ca2+]i.

Transplantation of gene-modified haematopoietic stem cells: Application and clinical considerations.

Autologous and allogeneic haematopoietic stem cell (HSC) transplantation has been performed in patients with various malignant and non-malignant haematological disorders for more than 50 years. Ex vivo gene modification of HSCs for autologous transplantation opens up new therapeutic avenues for genetic and infectious diseases. Major advances have been made over the last three decades with respect to gene modification of HSCs and transplantation strategies, ultimately culminating in the approval of two such therapies in Europe (Strimvelis for a rare primary immune deficiency, and LentiGlobin for beta-thalassaemia). Newer gene-modifying technologies and treatment regimens have also recently come to the fore, which hold great promise for the development of safer and more effective treatments. We provide an overview of the current state of gene-modified HSC therapies, highlighting success stories, limitations and important considerations for achieving successful translation of these therapies to the clinic.

Embryonic and adult stem cell-derived cardiomyocytes: lessons from in vitro models.

For years, research has focused on how to treat heart failure by sustaining the overloaded remaining cardiomyocytes. Recently, the concept of cell replacement therapy as a treatment of heart diseases has opened a new area of investigation. In vitro-generated cardiomyocytes could be injected into the heart to rescue the function of a damaged myocardium. Embryonic and/or adult stem cells could provide cardiac cells for this purpose. Knowledge of fundamental cardiac differentiation mechanisms unraveled by studies on animal models has been improved using in vitro models of cardiogenesis such as mouse embryonal carcinoma cells, mouse embryonic stem cells and, recently, human embryonic stem cells. On the other hand, studies suggesting the existence of cardiac stem cells and the potential of adult stem cells from bone marrow or skeletal muscle to differentiate toward unexpected phenotypes raise hope and questions about their potential use for cardiac cell therapy. In this review, we compare the specificities of embryonic vs adult stem cell populations regarding their cardiac differentiation potential, and we give an overview of what in vitro models have taught us about cardiogenesis.

Scapuloperoneal syndrome type Kaeser and a wide phenotypic spectrum of adult-onset, dominant myopathies are associated with the desmin mutation R350P.

In 1965, an adult-onset, autosomal dominant disorder with a peculiar scapuloperoneal distribution of weakness and atrophy was described in a large, multi-generation kindred and named 'scapuloperoneal syndrome type Kaeser' (OMIM #181400). By genetic analysis of the original kindred, we discovered a heterozygous missense mutation of the desmin gene (R350P) cosegregating with the disorder. Moreover, we detected DES R350P in four unrelated German families allowing for genotype-phenotype correlations in a total of 15 patients carrying the same mutation. Large clinical variability was recognized, even within the same family, ranging from scapuloperoneal (n = 2, 12%), limb girdle (n = 10, 60%) and distal phenotypes (n = 3, 18%) with variable cardiac (n = 7, 41%) or respiratory involvement (n = 7, 41%). Facial weakness, dysphagia and gynaecomastia were frequent additional symptoms. Overall and within each family, affected men seemingly bear a higher risk of sudden, cardiac death as compared to affected women. Moreover, histological and immunohistochemical examination of muscle biopsy specimens revealed a wide spectrum of findings ranging from near normal or unspecific pathology to typical, myofibrillar changes with accumulation of desmin. This study reveals that the clinical and pathological variability generally observed in desminopathies may not be attributed to the nature of the DES mutation alone, but may be influenced by additional genetic and epigenetic factors such as gender. In addition, mutations of the desmin gene should be considered early in the diagnostic work-up of any adult-onset, dominant myopathy, even if specific myofibrillar pathology is absent.

GROW: a model for mentorship to advance women's leadership in global health.

In this essay, we discuss the under-representation of women in leadership positions in global health (GH) and the importance of mentorship to advance women's standing in the field. We then describe the mentorship model of GROW, Global Research for Women. We describe the theoretical origins of the model and an adapted theory of change explaining how the GROW model for mentorship advances women's careers in GH. We present testimonials from a range of mentees who participated in a pilot of the GROW model since 2015. These mentees describe the capability-enhancing benefits of their mentorship experience with GROW. Thus, preliminary findings suggest that the GROW mentorship model is a promising strategy to build women's leadership in GH. We discuss supplemental strategies under consideration and next steps to assess the impact of GROW, providing the evidence to inform best practices for curricula elsewhere to build women's leadership in GH.

Subcellular distribution of Ca2+ pumping sites in human neutrophils.

The distribution of nonmitochondrial Ca2+ pumping sites and the site of action of inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) were studied in subcellular fractions of human neutrophils. In homogenates, two different Ca2+ pools could be observed: a mitochondrial Ca2+ pool and a nonmitochondrial, ATP-dependent, Ins 1,4,5-P3-responsive Ca2+ pool. When the homogenate was separated into microsomes, primary granules, and secondary granules, the nonmitochondrial Ca2+ pumping and the Ins 1,4,5-P3-induced Ca2+ release occurred only in the microsomal fraction. In a gradient developed to separate different microsomal organelles, maximal Ca2+ pumping activity occurred in fractions of low densities. Correlations between Ca2+ uptake and organelle markers were negative for the endoplasmic reticulum (r = -0.49) and positive for plasma membrane (r = 0.47), Golgi (r = 0.62), and endosomes (r = 0.96). Because the Ca2+ pumping organelles in these fractions were insensitive to micromolar vanadate and digitonin treatment, they are unlikely to be plasma membrane vesicles. We conclude first that microsomal fractions of human neutrophils contain organelles that lower the ambient free Ca2+ concentration and respond to Ins 1,4,5-P3. Second, granules are not involved in intracellular Ca2+ regulation in neutrophils. Third, nonendoplasmic reticulum organelles, such as endosomes, Golgi elements, or yet undefined specialized structures, play a major role in intracellular Ca2+ homeostasis in human neutrophils.

Voltage-dependent and Ca2(+)-activated ion channels in human neutrophils.

To investigate the regulation of membrane voltage and transmembrane ion fluxes in human neutrophils, we studied plasma membrane currents using the whole-cell patch-clamp method. We observed three distinct ion channel currents: (a) a voltage-dependent K+ current, (b) a Ca2(+)-activated K+ current, and (c) a Ca2(+)-activated Cl- current. The voltage-dependent K+ current was found in cells at rest. Its conductive properties suggested an inwardly rectifying channel. The channel was activated at membrane potentials more positive than -60 mV, suggesting that it may determine the resting membrane potential of neutrophils. Activation of neutrophils by the Ca2+ ionophore ionomycin led to an increase in whole-cell K+ and Cl- currents. The Ca2(+)-activated K+ channel differed from the voltage-dependent K+ channel because it was insensitive to voltage, because it rectified outwardly, and because the voltage-sensitive K+ channel was Ca2(+)-independent. The Ca2(+)-activated Cl- channel showed outward rectification and no apparent voltage dependency. The Ca2(+)-activated K+ and Cl- channels may play a role in cell volume homeostasis and/or cellular activation.

Regulation of Ca2+ influx in myeloid cells. Role of plasma membrane potential, inositol phosphates, cytosolic free [Ca2+], and filling state of intracellular Ca2+ stores.

To study the mediation of Ca2+ influx by second messengers in myeloid cells, we have combined the whole-cell patch clamp technique with microfluorimetric measurements of [Ca2+]i. Me2SO-differentiated HL-60 cells were loaded with the fluorescent Ca2+ indicator Indo-1, allowed to adhere to glass slides, and patch-clamped. Receptor agonists and Ca(2+)-ATPase inhibitors were applied by superfusion and inositol phosphates by microperfusion through the patch pipette. In voltage-clamped cells, [Ca2+]i elevations with a sustained phase could be induced by (a) the chemoattractant receptor agonist FMLP, (b) the Ca(2+)-releasing second messenger myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P3], as well as its nonmetabolizable analogues, and (c) the Ca(2+)-ATPase inhibitor cyclopiazonic acid, which depletes intracellular Ca2+ stores. In the absence of extracellular Ca2+, responses to all stimuli were short-lasting, monophasic transients; however, subsequent addition of Ca2+ to the extracellular medium led to an immediate [Ca2+]i increase. In all cases, the sustained phase of the [Ca2+]i elevations could be inhibited by millimolar concentrations of extracellular Ni2+, and its amplitude could be decreased by depolarization of the plasma membrane. Thus, the sustained phase of the Ca2+ elevations was due to Ca2+ influx through a pathway sensitive to the electrical driving force and to Ni2+. No Ca2+ influx could be observed after (a) plasma membrane depolarization in resting cells, (b) an imposed [Ca2+]i transient independent of receptor activation, or (c) microperfusion of myo-inositol(1,3,4,5)tetrahisphosphate (Ins(1,3,4,5)P4). Also, Ins(1,3,4,5)P4 did not have additive effects when co-perfused with a submaximal concentration of Ins(1,4,5)P3. Our results suggest that, in myeloid cells, activation of chemoattractant receptors induces an electrogenic, Ni(2+)-sensitive Ca2+ influx via generation of Ins(1,4,5)P3. Ins(1,4,5)P3 might activate Ca2+ influx directly, or by depletion of intracellular Ca2+ stores, but not via [Ca2+]i increase or Ins(1,3,4,5)P4 generation.

Chemotactic peptide activation of human neutrophils and HL-60 cells. Pertussis toxin reveals correlation between inositol trisphosphate generation, calcium ion transients, and cellular activation.

The mechanism of neutrophil activation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) has been studied by pretreatment of human neutrophils with pertussis toxin. Upon stimulation with FMLP, the cytosolic-free calcium concentration, [Ca2+]i, is increased both by stimulation of calcium influx and mobilization of cellular calcium. We have measured [Ca2+]i as well as the generation of the phospholipid breakdown product inositol trisphosphate (IP3), which is thought to mediate Ca2+ mobilization. As the phosphoinositide pool in human neutrophils is difficult to prelabel with [3H]myoinositol, experiments were also carried out in the cultured human promyelocytic leukemia cell line HL-60 after differentiation with dimethylsulfoxide. Pertussis toxin pretreatment of both cell types inhibited FMLP stimulated membrane depolarization, exocytosis, and superoxide production in a dose-dependent manner. This toxin effect was selective for the receptor agonist, since stimulation of these parameters by two substances bypassing the transduction mechanism, the calcium ionophore ionomycin and the phorbolester phorbol myristate acetate, were unaffected. Rises in [Ca2+]i, as well as generation of IP3 in response to FMLP, were inhibited in parallel; for the inhibition of functional responses, slightly lower toxin concentrations were required. The attentuation of the [Ca2+]i rise was more marked in the absence of extracellular calcium, i.e., when the rise is due only to calcium mobilization. The results provide evidence that phospholipase C stimulation by FMLP resulting in IP3 generation is involved in the signal transduction mechanism. Coupling of FMLP receptor occupancy to phospholipase C activation is sensitive to pertussis toxin, suggesting the involvement of a GTP binding protein (N protein), which has been shown to be a pertussis toxin substrate. The parallel changes in [Ca2+]i and IP3 further support the hypothesis that IP3 is the calcium-mobilizing mediator in FMLP-activated cells.

Second-messenger regulation of receptor association with clathrin-coated pits: a novel and selective mechanism in the control of CD4 endocytosis.

CD4, a member of the immunoglobulin superfamily, is not only expressed in T4 helper lymphocytes but also in myeloid cells. Receptor-mediated endocytosis plays a crucial role in the regulation of surface expression of adhesion molecules such as CD4. In T lymphocytes p56lck, a CD4-associated tyrosine kinase, prevents CD4 internalization, but in myeloid cells p56lck is not expressed and CD4 is constitutively internalized. In this study, we have investigated the role of cyclic AMP (cAMP) in the regulation of CD4 endocytosis in the myeloid cell line HL-60. Elevations of cellular cAMP were elicited by 1) cholera toxin, 2) pertussis toxin, 3) forskolin and IBMX, 4) NaF, or 5) the physiological receptor agonist prostaglandin E1. All five interventions led to an inhibition of CD4 internalization. Increased cAMP levels did not inhibit endocytosis per se, because internalization of insulin receptors and transferrin receptors and fluid phase endocytosis were either unchanged or slightly enhanced. The mechanism of cAMP inhibition was further analyzed at the ultrastructural level. CD4 internalization, followed either by quantitative electron microscopy autoradiography or by immunogold labeling, showed a rapid and temperature-dependent association of CD4 with clathrin-coated pits in control cells. This association was markedly inhibited in cells with elevated cAMP levels. Thus these findings suggest a second-messenger regulation of CD4 internalization through an inhibition of CD4 association with clathrin-coated pits in p56lck-negative cells.