Mobilization Of Intracellular Ca2 Research Articles

Differential effects of β-arrestins on the internalization, desensitization and ERK1/2 activation downstream of protease activated receptor-2

Beta-arrestins-1 and 2 are known to play important roles in desensitization of membrane receptors and facilitation of signal transduction pathways. It has been previously shown that beta-arrestins are required for signal termination, internalization, and ERK1/2 activation downstream of protease-activated-receptor-2 (PAR-2), but it is unclear whether they are functionally redundant or mediate specific events. Here, we demonstrate that in mouse embryonic fibroblasts (MEFs) from beta-arrestin-1/2 knockout mice, G alpha q signaling by PAR-2, as measured by mobilization of intracellular Ca(2+), is prolonged. Only expression of beta-arrestin-1 shortened the signal duration, whereas either beta-arrestin-1 or 2 was able to restore PKC-induced receptor desensitization. Beta-arrestin-1 also mediated early, while beta-arrestin-2 mediated delayed, receptor internalization and membrane-associated ERK1/2 activation. While beta-arrestin-1 colocalized with a lysosomal marker (LAMP-1), beta-arrestin-2 did not, suggesting a specific role for beta-arrestin-1 in lysosomal receptor degradation. Together, these data suggest distinct temporal and functional roles for beta-arrestins in PAR-2 signaling, desensitization, and internalization.

CXCR1 and CXCR2 Activation and Regulation: ROLE OF ASPARTATE 199 OF THE SECOND EXTRACELLULAR LOOP OF CXCR2 IN CXCL8-MEDIATED RAPID RECEPTOR INTERNALIZATION

CXCL8 (interleukin-8) interacts with two receptors, CXCR1 and CXCR2, to activate leukocytes. Upon activation, CXCR2 internalizes very rapidly relative to CXCR1 ( approximately 90% versus approximately 10% after 5 min). The C termini of the receptors have been shown to be necessary for internalization but are not sufficient to explain the distinct kinetics of down-regulation. To determine the structural determinant(s) that modulate receptor internalization, various chimeric and point mutant receptors were generated by progressively exchanging specific domains or amino acids between CXCR1 and CXCR2. The receptors were stably expressed in rat basophilic leukemia 2H3 cells and characterized for receptor binding, intracellular Ca(2+) mobilization, phosphoinositide hydrolysis, phosphorylation, internalization, and MAPK activation. The data herein indicate that the second extracellular loop (2ECL) of the receptors is critical for the distinct rate of internalization. Replacing the 2ECL of CXCR2 with that of CXCR1 (B(2ECL)A) or Asp(199) with its CXCR1 valine counterpart (B(D199V)A) delayed CXCR2 internalization similarly to CXCR1. Replacing Asp(199) with Asn (B(D199N)) restored CXCR2 rapid internalization. Structure modeling of the 2ECL of the receptors also suggested that Asp(199) plays a critical role in stabilizing and modulating CXCR2 rapid internalization relative to CXCR1. B(D199N) internalized rapidly but migrated as a single phosphorylated form like CXCR1 ( approximately 75 kDa), whereas B(2ECL)A and B(D199V)A showed slow and fast migrating forms like CXCR2 ( approximately 45 and approximately 65 kDa, respectively) but internalized like CXCR1. These data further undermine the role of receptor oligomerization in CXCL8 receptor internalization. Like CXCR1, B(D199V)A also induced sustained ERK activation and cross-desensitized Ca(2+) mobilization to CCR5 relative to B(D199N) and CXCR2. Altogether, the data suggest that the 2ECL of the CXCL8 receptors is important in modulating their distinct rate of down-regulation and thereby signal length and post-internalization activities.

Ginkgolide C Inhibits Platelet Aggregation in cAMP- and cGMP-Dependent Manner by Activating MMP-9

In this report, we investigated the effect of ginkgolide C (GC) from Ginkgo biloba leaves in collagen (10 mug/ml)-stimulated platelet aggregation. It has been known that matrix metalloproteinase-9 (MMP-9) is released from human platelets, and that it significantly inhibited platelet aggregation stimulated by collagen. Zymographic analysis confirmed that pro-MMP-9 (92-kDa) was activated by GC to form an activated MMP-9 (86-kDa) on gelatinolytic activities. And then, GC dose-dependently inhibited platelet aggregation, intracellular Ca(2+) mobilization, and thromboxane A(2) (TXA(2)) formation in collagen-stimulated platelets. In addition, GC significantly increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have an anti-platelet function in both resting and collagen-stimulated platelets. Therefore, we demonstrate that the inhibitory effect of GC on platelet aggregation might be involved into the following pathways. GC may increase intracellular cAMP and cGMP production and MMP-9 activity, inhibit intracellular Ca(2+) mobilization and TXA(2) production, thereby leading to inhibition of platelet aggregation. These results strongly indicate that GC is a potent inhibitor of collagen-stimulated platelet aggregation. It may be a suitable tool for a negative regulator during platelet activation.

Paroxetine-induced apoptosis in human osteosarcoma cells: Activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca2+]i elevation

Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are generally used for treatment of various mood and anxiety disorders. There has been much research showing the anti-tumor and cytotoxic activities of some antidepressants; but the detailed mechanisms were unclear. In cultured human osteosarcoma cells (MG63), paroxetine reduced cell viability in a concentration- and time-dependent manner. Paroxetine caused apoptosis as assessed by propidium iodide-stained cells and increased caspase-3 activation. Although immunoblotting data revealed that paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine also induced [Ca(2+)](i) increases which involved the mobilization of intracellular Ca(2+) stored in the endoplasmic reticulum and Ca(2+) influx from extracellular medium. However, pretreatment with BAPTA/AM, a Ca(2+) chelator, to prevent paroxetine-induced [Ca(2+)](i) increases did not protect cells from death. The results suggest that in MG63 cells, paroxetine caused Ca(2+)-independent apoptosis via inducing p38 MAPK-associated caspase-3 activation.

Role of Sustained Overexpression of Central Nervous System IGF-I in the Age-Dependent Decline of Mouse Excitation-Contraction Coupling

We investigated the effects of exclusive and sustained transgenic overexpression of insulin-like growth factor (IGF)-I in the central nervous system (CNS) on the age-dependent decline in muscle strength, excitation-contraction coupling, muscle innervation and neuromuscular junction postterminal architecture. We found that (1) transgenic IGF-I overexpression in the CNS does not modify the decline in extensor digitorum longus (EDL) and soleus muscle weight with aging and (2) strength significantly decreases in transgenic (Tg) compared to wild-type mice. The latter finding is consistent with (3) the decreased absolute and specific force measured in the EDL muscle in vitro and (4) the decreased charge movement and peak intracellular Ca(2+) mobilization in individual muscle fibers from old IGF-I Tg mice compared to young wild-type mice, which also is associated with (5) decreased dihydropyridine receptor alpha(1)-subunit expression in old compared to young IGF-I Tg mice. (6) Tg IGF-I prevents a change in muscle fiber type that is associated with (7) improved muscle innervation and postterminal neuromuscular structure. (8) IGF-I is expressed extensively across the spinal cord gray matter and the lateral motor column. Our results raise questions about the timing and cell location of CNS IGF-I overexpression necessary to prevent or to ameliorate age-dependent alterations in the structure and function of skeletal muscle.

Muscarinic Acetylcholine Receptors

Muscarinic acetylcholine receptors mediate diverse physiological functions. At present, five receptor subtypes (M(1) - M(5)) have been identified. The odd-numbered receptors (M(1), M(3), and M(5)) are preferentially coupled to G(q/11) and activate phospholipase C, which initiates the phosphatidylinositol trisphosphate cascade leading to intracellular Ca(2+) mobilization and activation of protein kinase C. On the other hand, the even-numbered receptors (M(2) and M(4)) are coupled to G(i/o), and inhibit adenylyl cyclase activity. They also activate G protein-gated potassium channels, which leads to hyperpolarization of the plasma membrane in different excitable cells. Individual members of the family are expressed in an overlapping fashion in various tissues and cell types. Recent gene targeting approaches have unraveled the specific function of these muscarinic receptor subtypes, which were not able to be fully elucidated with pharmacological approaches because of the non-selective effects of the available ligands. Based on these findings, muscarinic receptors have been emerging as an important therapeutic target for various diseases, including dry mouth, incontinence and chronic obstructive pulmonary disease. Here we review the latest advances in the structural and functional characterization of muscarinic acetylcholine receptors and the pharmaceutical development of muscarinic receptor ligands.

Preclinical Pharmacology of AL-12182, a New Ocular Hypotensive 11-Oxa Prostaglandin Analog

The aim of this study was to determine selected in vivo ocular properties of AL-12182 (5,6-dihydro-4,5-didehydro-11-deoxy-11-oxa-16-(3-chlorophenoxy)-omega-tetranor-PGF(2alpha) isopropyl ester) and the in vitro profile of its free acid, AL-12180. Previously documented radioligand binding and functional assays involving human ciliary muscle cells (h-CM), human trabecular meshwork (h-TM) and other cells, and porcine ocular arteries were utilized. For in vivo procedures, we utilized rabbits, cats, and nonhuman primates to measure hyperemia, pupil diameter, and intraocular pressure (IOP), respectively. AL-12180 exhibited the highest affinity for the FP-receptor (K(i) = 143 +/- 36 nM) and much lower affinity for DP-, EP(3)-, IP-, and TP-receptors, and for several nonprostanoid receptors, enzymes, neurotransmitter uptake sites, ion channels, and other regulatory sites. AL-12180 activated phospholipase C-mediated phosphoinositide hydrolysis (potency, EC(50) = 13.7-42.7 nM) through the FP-receptor in a variety of cells, such as h-CM, h-TM cells, human embryonic kidney cells expressing the cloned human ciliary body FP-receptor (HEK-FP), mouse 3T3 cells, and rat vascular smooth muscle cells. AL-8810, an FP-antagonist, blocked the effects of AL-12180 in h-CM cells (IC(50) = 8.7 microM). AL-12180 also stimulated the mobilization of intracellular Ca(2+) ([Ca(2+)](i)) in h-TM cells (EC(50) = 111 +/- 36 nM), h-CM cells (EC(50) = 11 nM), and in host cells expressing the cloned human ciliary body FP-receptor (EC(50) = 5.9 +/- 3.1 nM). AL-12180 lacked significant agonist activity at DP-, EP(2)-, EP(4)-, IP-, and TP-receptors in cell-based assays. However, AL-12180 contracted porcine central retinal and short posterior ciliary arteries in vitro with micromolar potencies that appeared to involve TP-receptor activation. in vivo, AL-12182 elicited dose-related hyperemia in the rabbit eye, miosis in the cat eye, and ocular hypotension in the nonhuman primate eye. AL-12180 is a relatively potent and selective FP-receptor agonist whose isopropyl ester prodrug (AL-12182) lowers IOP by as much as 40% following topical ocular dosing in a laser-induced nonhuman primate model of ocular hypertension.

Ontogeny and mechanisms of action for the stimulatory effect of kisspeptin on gonadotropin-releasing hormone system of the rat

Kisspeptins have recently emerged as essential regulators of gonadotropin secretion and puberty onset. These functions are primarily conducted by stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) secretion. However, relevant aspects of KiSS-1 physiology, including the ontogeny and major signaling systems of its stimulatory action, remain to be fully elucidated. To cover these issues, the effects of kisspeptin-10 on GnRH and LH secretion were monitored at early stages of postnatal maturation, and potential changes in the sensitivity to kisspeptin were assessed along the pubertal transition in the rat. In addition, the signaling cascades involved in kisspeptin-induced GnRH secretion were explored by means of pharmacological blockade using rat hypothalamic explants. Despite sexual immaturity, kisspeptin-10 potently elicited GnRH release ex vivo and LH secretion in vivo at early stages (neonatal to juvenile) of postnatal development. Yet, LH responsiveness to low doses of kisspeptin was enhanced in peri-pubertal animals. Concerning GnRH secretion, the stimulatory action of kisspeptin-10 required activation of phospholipase-C, mobilization of intracellular Ca 2+ and recruitment of ERK1/2 and p38 kinases, but was preserved after blockade of type 2 cyclo-oxygenase and prostaglandin synthesis. In summary, our present data document the ontogeny, sensitivity and intracellular signals for the stimulatory action of kisspeptin on the GnRH/LH axis in the rat. Although LH responses to low doses of kisspeptin appeared to be enhanced at puberty, kisspeptin was able to readily activate the GnRH system at early stages of postnatal maturation. These observations further stress the essential role of kisspeptin in normal, and eventually pathological, timing of puberty.

The effect of progesterone on oxytocin-stimulated intracellular mobilization of Ca 2+ and prostaglandin E 2 and F 2α secretion from porcine myometrial cells

Our past studies have shown that porcine myometrium produce prostaglandins (PG) during luteolysis and early pregnancy and that oxytocin (OT) and its receptor (OTr) support myometrial secretion of prostaglandins E 2 and F 2α (PGE 2 and PGF 2α) during luteolysis. This study investigates the role of intracellular Ca 2+ [Ca 2+] i as a mediator of OT effects on PG secretion from isolated myometrial cells in the presence or absence of progesterone (P 4). Basal [Ca 2+] i was similar in myometrial cells from cyclic and pregnant pigs (days 14–16). OT (10 −7 M) increased [Ca 2+] i in myometrial cells of cyclic and pregnant pigs, although this effect was delayed in myometrium from pregnant females. After pre-incubation of the myocytes with P 4 (10 −5 M) the influence of OT on [Ca 2+] i was delayed during luteolysis and inhibited during pregnancy. Myometrial cells in culture produce more PGE 2 than PGF 2α regardless of reproductive state of the female. OT (10 −7 M) increased PGE 2 secretion after 6 and 12 h incubation for the tissue harvested during luteolysis and after 12 h incubation when myometrium from gravid females was used. In the presence of P 4 (10 −5 M), the stimulatory effect of OT on PG secretion was diminished. In conclusion: (1) porcine myometrial cells in culture secrete PG preferentially during early pregnancy and produce more PGE 2 than PGF 2α, (2) OT controls myometrial PGF 2α secretion during luteolysis, (3) release of [Ca 2+] i is associated with the influence of OT on PG secretion, and (4) the effects of OT on PG secretion and Ca 2+ accumulation are delayed by P 4 during luteolysis and completely inhibited by P 4 during pregnancy.

Regulation of acetylcholinesterase expression by calcium signaling during calcium ionophore A23187- and thapsigargin-induced apoptosis

We have recently reported that acetylcholinesterase expression was induced during apoptosis in various cell types. In the current study we provide evidence to suggest that the induction of acetylcholinesterase expression during apoptosis is regulated by the mobilization of intracellular Ca 2+. During apoptosis, treatment of HeLa and MDA-MB-435s cells with the calcium ionophore A23187 resulted in a significant increase in acetylcholinesterase mRNA and protein levels. Chelation of intracellular Ca 2+ by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid–acetoxymethyl ester), an intracellular Ca 2+ chelator, inhibited acetylcholinesterase expression. A23187 also enhanced the stability of acetylcholinesterase mRNA and increased the activity of acetylcholinesterase promoter, effects that were blocked by BAPTA-AM. Perturbations of cellular Ca 2+ homeostasis by thapsigargin resulted in the increase of acetylcholinesterase expression as well as acetylcholinesterase promoter activity during thapsigargin induced apoptosis in HeLa and MDA-MB-435s cells, effects that were also inhibited by BAPTA-AM. We further demonstrated that the transactivation of the human acetylcholinesterase promoter by A23187 and thapsigargin was partially mediated by a CCAAT motif within the −1270 to −1248 fragment of the human acetylcholinesterase promoter. This motif was able to bind to CCAAT binding factor (CBF/NF-Y). These results strongly suggest that cytosolic Ca 2+ plays a key role in acetylcholinesterase regulation during apoptosis induced by A23187 and thapsigargin.

Basiliolides, a Class of Tetracyclic C19 Dilactones from Thapsia garganica, Release Ca2+ from the Endoplasmic Reticulum and Regulate the Activity of the Transcription Factors Nuclear Factor of Activated T Cells, Nuclear Factor-κB, and Activator Protein 1 in T Lymphocytes

Calcium concentration within the endoplasmic reticulum (ER) plays an essential role in cell physiology. We have investigated the effects of basiliolides, a novel class of C19 dilactones isolated from Thapsia garganica, on Ca(2+) mobilization in T cells. Basiliolide A1 induced a rapid mobilization of intracellular Ca(2+) in the leukemia T-cell line Jurkat. First, a rapid calcium peak was observed and inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. This initial calcium mobilization was followed by a sustained elevation, mediated by the entry of extracellular calcium through store-operated calcium release-activated Ca(2+) (CRAC) channels and sensitive to inhibition by EGTA, and by the CRAC channel inhibitor N-{4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2). Basiliolide A1 mobilized Ca(2+) from ER stores, but in contrast to thapsigargin, it did not induce apoptosis. Basiliolide A1 induced nuclear factor of activated T cells 1 dephosphorylation and activation that was inhibited by BTP-2 and cyclosporine A. In addition, we found that basiliolide A1 alone did not mediate IkappaBalpha degradation or RelA phosphorylation (ser536), but it synergized with phorbol 12-myristate 13-acetate to induce a complete degradation of the nuclear factor-kappaB inhibitory protein and to activate the c-Jun NH(2)-terminal kinase. Moreover, basiliolide A1 regulated both interleukin-2 and tumor necrosis factor-alpha gene expression at the transcriptional level. In basiliolide B, oxidation of one of the two geminal methyls to a carboxymethyl group retained most of the activity of basiliolide A1. In contrast, basiliolide C, where the 15-carbon is oxidized to an acetoxymethine, was much less active. These findings qualify these compounds as new probes to investigate intracellular calcium homeostasis.

Impaired Ca++ mobilization in rituximab-resistant cells (RRCL) is associated with changes in the structure of CD20 antigen, down-regulation of Bax/Bak pro-apoptotic proteins and up-regulation of the endoplasmic reticulum (ER) Ca++ pump protein SERCA-3

2516 Ca++ mobilization leading to apoptosis has been observed following rituximab biding to CD20 in lymphoma cells. Extending rituximab (R) exposure (e.g. as via R maintenance regimens) could potentially accelerate the emergence of resistance to rituximab. To define the molecular basis for rituximab resistance we developed several RRCL and demonstrated changes in CD20 expression/structure and membrane reorganization following rituximab therapy. Specifically, changes in the N-terminal and the C-terminal region of the internal domain of CD20 were observed in RRCL as determined by Western blotting using various antibodies recognizing epitopes located in the internal (GST77 and 1439) and external domain (B1) of CD20. In our current work we evaluated the effects that CD20 structure/expression changes have upon Ca++ mobilization. Extracellular and intracellular Ca++ mobilization was measured by flow cytometric analysis using FLUO-3 AM (acetoxymethyl ester). Optimization of the Ca++ indicator and calibration curves were performed for each cell line. Raji and RRCL were labeled under optimal conditions with FLUO-3 AM/Pluronic Acid F-127. Subsequently, cells were then re-suspended in HANKS media with or without Ca++ and exposed to rituximab or isotype (10μg/ml) ± human serum (25%). Surface CD20 expression was similar between Raji cells and RRCL. Expression of pro- or anti-apoptotic proteins and Ca++ regulatory proteins SERCA3, SERCA2 and Calreticulin was also determined by western blotting. In vitro exposure of Raji cells to rituximab + HS resulted in Ca++ mobilization even in Ca++ depleted media. Notably, Ca++ mobilization was impaired in RRCL when compared to Raji parental cells. In addition, down-regulation of Bax/Bak and up-regulation of SERCA3 was demonstrated in RRCL. Our data suggest that the acquirement of rituximab resistance is associated with changes in the intracellular domain of CD20 and in Ca++ regulator/pro-apoptotic proteins (SERCA3 and Bax/Bak) resulting in a decrease in the intracellular mobilization of Ca++. No significant financial relationships to disclose.

Involvement of ryanodine-operated channels intert-butylhydroperoxide-evoked Ca2+ mobilisation in pancreatic acinar cells

Reactive oxygen species and related oxidative damage have been implicated in the initiation of acute pancreatitis, a disease characterised in its earliest stages by disruption of intracellular Ca2+ homeostasis. The present study was carried out in order to establish the effect of the organic pro-oxidant, tert-butylhydroperoxide (tBHP), on the mobilisation of intracellular Ca2+ stores in isolated rat pancreatic acinar cells and the mechanisms underlying this effect. Cytosolic free Ca2+ concentrations ([Ca2+]c) were monitored using a digital microspectrofluorimetric system in fura-2 loaded cells. In the presence of normal extracellular Ca2+ concentrations ([Ca2+]o), perfusion of pancreatic acinar cells with 1 mmol l-1 tBHP caused a slow sustained increase in [Ca2+]c. This increase was also observed in a nominally Ca2+-free medium, indicating a release of Ca2+ from intracellular stores. Pretreatment of cells with tBHP abolished the typical Ca2+ response of both the physiological agonist CCK-8 (1 nmol l-1) and thapsigargin (TPS, 1 micromol l-1), an inhibitor of the SERCA pump, in the absence of extracellular Ca2+. Similar results were observed with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 0.5 micromol l-1), a mitochondrial uncoupler. In addition, depletion of either agonist-sensitive Ca2+ pools by CCK-8 or TPS or mitochondrial Ca2+ pools by FCCP were unable to prevent the tBHP-induced Ca2+ release. By contrast, simultaneous administration of TPS and FCCP clearly abolished the tBHP-induced Ca2+ release. These results show that tBHP releases Ca2+ from agonist-sensitive intracellular stores and from mitochondria. On the other hand, simultaneous application of FCCP and of 2-aminoethoxydiphenylborane (2-APB), a blocker of IP3-mediated Ca2+ release, was unable to suppress the increase in [Ca2+]c induced by tBHP, while the application of 50 micromol l-1 of ryanodine (which is able to block the ryanodine channels) inhibits tBHP-evoked Ca2+ mobilisation. These findings indicate that tBHP releases Ca2+ from non-mitochondrial Ca2+ pools through ryanodine channels.

Platelet-derived growth factor receptor independent proliferation of human glioblastoma cells: selective tyrosine kinase inhibitors lack antiproliferative activity

The aim of this study was to investigate the role of platelet-derived growth factor (PDGF) and PDGF receptors (PDGFRs) in the proliferation of human glioblastoma cells as a prerequisite for a new therapeutic approach to the treatment of malignant brain tumors with selective tyrosine kinase inhibitors such as imatinib. In the human glioblastoma cell lines U-87 MG, U-118 MG and U-373 MG different PDGF and PDGFR mRNAs were detected by RT-PCR, and the expression of the receptor proteins was demonstrated by immunostaining and flow cytometry. Moreover, functional activity of PDGFRs was demonstrated in PDGFRbeta expressing glioblastoma cell variants by measuring the mobilization of intracellular Ca(2+) upon PDGF-BB stimulation. However, addition of PDGF-BB to the serum-free culture medium had no stimulatory effect on cell proliferation. Furthermore, cell growth in serum-supplemented and serum-free medium was not affected by imatinib, leflunomide and AG-1296 at therapeutically relevant concentrations. Our results suggest that clinical antitumor effects of imatinib on glioblastoma, if any, are not mediated by the PDGFR.

Effects of dexamethasone andl-canavanine on the intracellular calcium-contraction relation of the rat tail artery during septic shock

The intracellular mechanism by which sepsis lowers vascular reactivity and the subsequent reversal by dexamethasone or nitric oxide synthase (NOS) inhibitors remain unclear. We measured the sensitivity of contraction of the rat tail artery to intracellular Ca2+ in a model of polymicrobial septic shock. At 22 h after cecal ligation and puncture (CLP), rats were treated with an anti-inflammatory glucocorticoid (dexamethasone, 1 mg/kg ip), an inducible NOS inhibitor (L-canavanine, 100 mg/kg ip), or saline. At 24 h after CLP, endothelium-denuded, perfused segments of tail artery were loaded with the intracellular Ca2+-sensitive dye fura 2 in vitro. Intracellular Ca2+ concentration and perfusion pressure were measured simultaneously. The rightward shift of the perfusion pressure-intracellular Ca2+ mobilization curve after norepinephrine stimulation subsequent to CLP indicates decreased intracellular Ca2+ sensitivity of contraction. The relation was restored by dexamethasone (which also restored in vivo blood pressure and flow), but not by L-canavanine (which restored perfusion pressure by further mobilization of intracellular Ca2+). We conclude that CLP lowers vasomotion by lowering intracellular Ca2+ sensitivity, which can be restored with glucocorticoid treatment. The involvement of inducible NOS does not solely account for the sepsis-induced reduction in Ca2+ sensitivity of contraction.

Effect of ammonia on astrocytic glutamate uptake/release mechanisms

Hyperammonemic disorders such as acute liver failure (ALF) or urea cycle enzymopathies are associated with hyperexcitability, seizures, brain edema and increased extracellular brain glutamate. Mechanisms responsible for increased glutamate content in the extracellular space of the brain include decreased uptake by perineuronal astrocytes and/or increased release from neurons and/or astrocytes. Exposure of astrocytes to millimolar concentrations of ammonia results in cell swelling, loss of expression of the glutamate transporters excitatory amino acid transporter (EAAT-1) and EAAT-2 and increased release of glutamate. Three distinct mechanisms are theoretically possible to explain ammonia-induced glutamate release from astrocytes namely: release due to swelling; reversal of glutamate transporters and due to Ca2+-dependent vesicular release. Recent identification of vesicular docking and fusion proteins in astrocytes together with glutamate-release (due to intracellular alkanization and mobilization of intracellular Ca2+-stores) studies implies that vesicular release is a predominant mechanism responsible for ammonia-induced release of glutamate from astrocytes.

Pharmacological Evidence for a Functional Serotonin-2B Receptor in a Human Uterine Smooth Muscle Cell Line

The present study investigated the serotonin-induced increase in phosphoinositide hydrolysis and mobilization of intracellular Ca2+ ([Ca2+]i) in human uterine smooth muscle cells (HUSMCs) to identify the serotonergic receptor positively coupled to phospholipase C in these cells. In phosphoinositide (PI) assays, serotonin (5-HT) and alpha-methyl-5-HT were potent, full agonists (EC50 = 20 and 4.1 nM, respectively), whereas the phenylethylamine, R-(-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride, was less active (EC50 = 63 nM). Proposed 5-HT2B-selective agonists, BW-723C86 [alpha-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine hydrochloride] and (+)-norfenfluramine, exhibited strong agonist potency and efficacy comparable with 5-HT (EC50 = 18 and 33 nM, respectively) and approximately 15-fold more potency than (-)-norfenfluramine (EC50 = 500 nM). 5-HT2C receptor agonists m-chlorophenylpiperazine and MK-212 [6-chloro-2-(1-piperaxinyl)pyrazine] were weak agonists in these cells, with potencies of 110 and 880 nM, respectively. A similar rank order of potency was observed in [Ca2+]i mobilization assays (r = 0.9, p < 0.005) in the HUSMC and with contraction of rat stomach fundus strips that contain a 5-HT2B receptor (r = 0.9, p < 0.001). Antagonist studies revealed that a 5-HT2B-selective antagonist, RS-127445 [2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine] (Ki = 0.13 nM), was significantly more effective at inhibiting 5-HT-induced activity than a 5-HT2A antagonist, M-100907 (R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol]) (Ki= 914 nM) and the 5-HT2C antagonists RS-102221 (8-[5-(2,4-dimethoxy-5-(4-trifluoromethylsulfo-amido)phenyl-5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione hydrochloride) (Ki = 2.5 microM) and SB-242084 (6-chloro-5-methyl-1-[6-92-methylpyridin-3-yloxy) pyridine-3-ylcarbamoyl] indoline) (Ki = 42.4 nM) in the HUSMC PI turnover assays. Taken together, these studies strongly suggest the presence of a functionally active 5-HT2B receptor subtype in HUSMCs. The physiological role of this receptor in these cells remains to be defined.

Induction of intracellular signalling in human endothelial cells by the hyaluronan-binding protease involves two distinct pathways

Recently a novel plasma serine protease with high affinity to hyaluronic acid and glycosaminoglycans, such as heparin and heparan sulfate, has been described and termed hyaluronan-binding protease (HABP). HABP cleaves kininogen in vitro, releasing the vasoactive peptide bradykinin, and activates plasminogen activators, suggesting a vascular cell-directed physiological function of this novel plasma protease. Here we show that HABP stimulates human umbilical vein endothelial cells (HUVECs) by activating two distinct cell-surface receptors. On the one hand, HABP releases bradykinin from cell surface-bound or soluble kininogen and triggers a bradykinin B2-receptor-dependent mobilisation of intracellular Ca2+. On the other hand, HABP activates the p44/42-dependent MAPK (ERK1/2) signalling cascade independent of the B2-receptor, but involving the fibroblast growth factor receptor-1 and basic fibroblast growth factor. This signalling pathway leads to phosphorylation of the kinases Raf, MEK1/2 and ERK1/2. The extracellular activity of HABP also affects the gene expression level through phosphorylation of two transcription factors, the cAMP-responsive element binding protein CREB and the proto-oncogene c-Myc. Our results indicate a proangiogenic potential of HABP, which, in combination with a profibrinolytic activity, directs the physiological function of this plasma protease to processes in which clot lysis, cell motility and neovascularisation are pivotal processes, e.g., in wound healing, tissue repair and tumour progression.

3-O-(2,3-Dimethylbutanoyl)-13-O-decanoylingenol from Euphorbia kansui Suppresses IgE-Mediated Mast Cell Activation

Aggregation of the high affinity receptor for IgE (FcepsilonRI) on mast cells by antigen and IgE complex induces release of chemical mediators, leading to acute allergic inflammation. We recently found that 3-O-(2,3-dimethylbutanoyl)-13-O-decanoylingenol (DBDI), purified from the Euphorbia kansui L., inhibits degranulation in rat basophilic leukemia 2H3 cells upon aggregation of the FcepsilonRI. In the present study, we demonstrated that the DBDI significantly inhibits release of beta-hexosaminidase, synthesis of eicosanoids, and mobilization of intracellular Ca(2+) in the bone marrow-derived mouse mast cells stimulated with IgE and antigen. Furthermore, we revealed that phosphorylation of Syk, phospholipase C-gamma(2), and extracellular signal-related kinase 1/2 is significantly suppressed in the DBDI-treated mast cells. These findings suggest that the DBDI may have a therapeutic potential for allergic diseases by inhibiting intracellular signaling pathways for activation and chemical mediator release in mast cells.

CD16b associates with high-density, detergent-resistant membranes in human neutrophils

CD16b is unique in that it is the only Fc receptor linked to the plasma membrane by a GPI (glycosylphosphatidylinositol) anchor. GPI-anchored proteins often preferentially localize to DRMs (detergent-resistant membranes) that are rich in sphingolipids and cholesterol and play an important role in signal transduction. Even though the responses to CD16b engagement have been intensively investigated, the importance of DRM integrity for CD16b signalling has not been characterized in human neutrophils. We provide direct evidence that CD16b constitutively partitions with both low- and high-density DRMs. Moreover, upon CD16b engagement, a significant increase in the amount of the receptor is observed in high-density DRMs. Similarly to CD16b, CD11b also resides in low- and high-density DRMs. In contrast with CD16b, the partitioning of CD11b in DRMs does not change in response to CD16b engagement. We also provide evidence for the implication of Syk in CD16b signalling and its partitioning to DRMs in resting and activated PMNs (polymorphonuclear neutrophils). Additionally, DRM-disrupting agents, such as nystatin and methyl-beta-cyclodextrin, alter cellular responses to CD16b receptor ligation. Notably, a significant increase in the mobilization of intracellular Ca2+ and in tyrosine phosphorylation of intracellular substrates after CD16b engagement is observed. Altogether, the results of this study provide evidence that high-density DRMs play a role in CD16b signalling in human neutrophils.