CTRP3/cartducin promotes proliferation and migration of endothelial cells

Nicotinic acid (niacin) has been used clinically to manage dyslipidemia for many years. The molecular target of nicotinic acid was unknown until the recent revelation of human G-coupled receptor HM74a as the high affinity receptor for nicotinic acid. In searching for a cell line expressing endogenous human HM74a receptor, we have identified that the A431 cell line, a human epidermoid cell line, expresses a high level of HM74a receptor. An HM74a-specific real time PCR probe set was designed and the mRNA levels of HM74a in A431 and 32 other cultured cell lines were measured quantitatively. When the mRNA expression of HM74a in A431 cells was compared to that in human primary preadipocytes, adipocytes and adipose tissue, we found that the level in A431 was about 10- fold higher than that in adipocytes and adipose tissue. The ratio of HM74a:HM74 mRNA was measured quantitatively and it was determined to be 3:2 in A431 cells. The function of the HM74a receptor in A431 cells was evaluated for its ability to inhibit forskolin-induced cAMP production. Pertussis toxin treatment abolished the inhibition. Our data suggest that the A431 cell line may serve as a cellular model for further investigation of niacin/HM74a-mediated signal transduction in modulating metabolism. A431 cell line may also provide a valuable cell model to study prostaglandin production upon HM74a activation to improve our understanding of niacin/HM74a-mediated skin flushing. The first two author contributed equally.     

Niacin mediates lipolysis in adipose tissue through its G-protein coupled receptor HM74A

A G-protein coupled receptor to niacin (nicotinic acid) was identified recently but the physiological/pharmacological role of the receptor remains poorly defined. We present our studies to demonstrate that HM74A, but not HM74, binds niacin at high affinities and effectively mediates Gi signaling events in human embryonic kidney HEK293 cells as well as in 3T3L1 adipocytes expressing HM74A. Furthermore, HM74A, but not HM74, expressed in differentiated 3T3L1 adipocytes effectively mediated inhibition of lipolysis by niacin. Our results provided direct evidence indicating that HM74A, but not HM74, was sufficient to mediate anti-lipolytic effect of niacin in adipose tissue.     

Triglyceride modulation by acifran analogs: activity towards the niacin high and low affinity G protein-coupled receptors HM74A and HM74

Niacin is known to exert profound beneficial effects on cholesterol levels in humans, although its use is somewhat hampered by the gram quantities necessary to exert effects and the prevalence of compliance-limiting skin flushing side effects that occur. Recently, two G protein-coupled receptors (GPCRs) for niacin were identified and characterized as high (HM74A; GPR109A) and low (HM74; GPR109B) affinity receptors based on the binding affinities of niacin. These receptors also bind acifran (AY-25,712), which is known to modulate lipid levels like niacin, with similar affinities. Twelve analogs of acifran were chemically synthesized. One analogue demonstrated a dose-dependent decrease in serum triglycerides in rats within 3h of oral administration. Next, the acifran analogs were assessed for their activity towards the high and low affinity niacin receptors expressed in CHO-K1 cells. Constructs expressing HM74A or HM74 were stably transfected into CHO-K1 cells and shown to elicit phosphorylation of p42 and p44 mitogen-activated protein kinase (ERK1/ERK2) phosphorylation upon addition of niacin or acifran. The presence of functionally coupled GPCRs was further confirmed using Pertussis toxin, which completely inhibited the ability of either niacin or acifran to elicit phospho-ERK1/ERK2. The EC(50) of p-ERK1/ERK2 for niacin for the high and low affinity receptors was 47nM and indeterminate (i.e., >100microM), respectively, while the EC(50) for acifran was 160 and 316nM, respectively. Two chemical analogs of acifran demonstrated robust phosphorylation of ERK1/ERK2. Collectively, these data suggest that the synthesis of acifran analogs may be a suitable path for developing improved HM74A agonists.     

Endothelins regulate arachidonic acid release and mitogen-activated protein kinase activity in Schwann cells

Immortalized rat Schwann cells (iSC) express endothelin (ET) receptors coupled to inhibition of adenylyl cyclase and stimulation of phospholipase C (PLC). These effects precede phenotypic changes and increased DNA synthesis. We have investigated the role of ETs in the regulation of arachidonic acid (AA) release and mitogen-activated protein kinases (MAPKs). Both ET-1 and ET-3 increased AA release in iSC. This effect was sensitive to the phospholipase A(2) (PLA(2)) inhibitors E:-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H:-pyran-2-one and arachidonyl-trifluoromethyl ketone but was insensitive to inhibitors of PLC or phospholipase D-dependent diacylglycerol generation. ET-1-dependent AA release was also unaffected by removal of extracellular Ca(2+) and blocking the concomitant elevation in [Ca(2+)](i), consistent with participation of a Ca(2+)-independent PLA(2). Treatment of iSC with ETs also resulted in activation of extracellular signal-regulated kinase, c-Jun-NH(2)-terminal kinase (JNK), and p38 MAPK. A cause-effect relationship between agonist-dependent AA release and stimulation of MAPKs, but not the opposite, was suggested by activation of JNK by exogenous AA and by the observation that inhibition of MAPK kinase or p38 MAPK was inconsequential to ET-1-induced AA release. Similar effects of ETs on AA release and MAPK activity were observed in cultures expanded from primary SC and in iSC. Regulation of these effectors may mediate the control of proliferation and differentiation of SC by ETs during peripheral nerve development and regeneration.     

Lysophosphatidic acid is a mediator of Trp-Lys-Tyr-Met-Val-d-Met-induced calcium influx

Intracellular calcium (Ca(2+)) homeostasis is very strictly regulated, and the activation of G-protein-coupled receptor (GPCR) can cause two different calcium changes, intracellular calcium release, and calcium influx. In this study, we investigated the possible role of lysophosphatidic acid (LPA) on GPCR-induced Ca(2+) signaling. The addition of exogenous LPA induced dramatic Ca(2+) influx but not intracellular Ca(2+) release in U937 cells. LPA-induced Ca(2+) influx was not affected by pertussis toxin and phospholipase C inhibitor (U73122), ruling out the involvement of pertussis toxin-sensitive G-proteins, and phospholipase C. Stimulation of U937 cells with Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), which binds to formyl peptide receptor like 1, enhanced phospholipase A(2) and phospholipase D activation, indicating LPA formation. The inhibition of LPA synthesis by phospholipase A(2)-specific inhibitor (MAFP) or n-butanol significantly inhibited WKYMVm-induced Ca(2+) influx, suggesting a crucial role for LPA in the process. Taken together, we suggest that LPA mediates WKYMVm-induced Ca(2+) influx.     

Distinct Kinetic and Spatial Patterns of Protein Kinase C (PKC)- and Epidermal Growth Factor Receptor (EGFR)-dependent Activation of Extracellular Signal-regulated Kinases 1 and 2 by Human Nicotinic Acid Receptor GPR109A

Nicotinic acid (niacin) has been widely used as a lipid-lowering drug for several decades, and recently, orphan G protein-coupled receptor GPR109A has been identified as a receptor for niacin. Mechanistic investigations have shown that, upon niacin activation, GPR109A couples to a G_i protein and inhibits adenylate cyclase activity, leading to inhibition of liberation of free fatty acid. However, the underlying molecular mechanisms for GPR109A signaling remain largely unknown. Using CHO-K1 cells stably expressing GPR109A and A431 cells, which are a human epidermoid cell line with high levels of endogenous expression of functional GPR109A receptors, we found that activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by niacin was rapid, peaking at 5 min, and was significantly blocked by pertussis toxin. Furthermore, time course experiments with different kinase inhibitors demonstrated that GPR109A induced ERK1/2 activation via the matrix metalloproteinase/epidermal growth factor receptor transactivation pathway at both early and later time points (2–5 min); this pathway was distinct from the PKC pathway-mediated ERK1/2 phosphorylation that occurs at early time points (≤2 min) in response to niacin. Overexpression of Gβγ subunit scavengers βARK1-CT and the Gα subunit of transducin led to a significant reduction of ERK1/2 phosphorylation, suggesting a critical role for βγ subunits in GPR109A-activated ERK1/2 phosphorylation. Using arrestin-2/3-specific siRNA and an internalization-deficient GPR109A mutant, we found that arrestin-2 and arrestin-3 were not involved in GPR109A-mediated ERK1/2 activation. In conclusion, our findings demonstrate that upon binding to niacin GPR109A receptors initially activate G_i, leading to dissociation of the Gβγ subunit from activated G_i, and subsequently induce ERK1/2 activation via two distinct pathways, one PKC-dependent pathway occurring at a peak time of ≤2 min and the other matrix metalloproteinase-dependent growth factor receptor transactivation occurring at both early and later time points (2–5 min).     

TLR-4 mediated group IVA phospholipase A2 activation is phosphatidic acid phosphohydrolase 1 and protein kinase C dependent

Group IVA phospholipase A₂ (GIVA PLA₂) catalyzes the release of arachidonic acid (AA) from the sn-2 position of glycerophospholipids. AA is then further metabolized into terminal signaling molecules including numerous prostaglandins. We have now demonstrated the involvement of phosphatidic acid phosphohydrolase 1 (PAP-1) and protein kinase C (PKC) in the Toll-like receptor-4 (TLR-4) activation of GIVA PLA₂. We also studied the effect of PAP-1 and PKC on Ca^+ 2 induced and synergy enhanced GIVA PLA₂ activation. We observed that the AA release induced by exposure of RAW 264.7 macrophages to the TLR-4 specific agonist Kdo₂-Lipid A is blocked by the PAP-1 inhibitors bromoenol lactone (BEL) and propranolol as well as the PKC inhibitor Ro 31-8220; however these inhibitors did not reduce AA release stimulated by Ca^+ 2 influx induced by the P2X7 purinergic receptor agonist ATP. Additionally, stimulation of cells with diacylglycerol (DAG), the product of PAP-1 mediated hydrolysis, initiated AA release from unstimulated cells as well as restored normal AA release from cells treated with PAP-1 inhibitors. Finally, neither PAP-1 nor PKC inhibition reduced GIVA PLA₂ synergistic activation by stimulation with Kdo₂-Lipid A and ATP.     

Brevetoxin activation of voltage-gated sodium channels regulates Ca dynamics and ERK1/2 phosphorylation in murine neocortical neurons

Voltage-gated sodium channels (VGSC) are involved in the generation of action potentials in neurons. Brevetoxins (PbTx) are potent allosteric enhancers of VGSC function and are associated with the periodic 'red tide' blooms. Using PbTx-2 as a probe, we have characterized the effects of activation of VGSC on Ca(2+) dynamics and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling in neocortical neurons. Neocortical neurons exhibit synchronized spontaneous Ca(2+) oscillations, which are mediated by glutamatergic signaling. PbTx-2 (100 nm) increased the amplitude and reduced the frequency of basal Ca(2+) oscillations. This modulatory effect on Ca(2+) oscillations produced a sustained rise in ERK1/2 activation. At 300 nm, PbTx-2 disrupted oscillatory activity leading to a sustained increase in intracellular Ca(2+) ([Ca(2+)](i)) and induced a biphasic, activation followed by dephosphorylation, regulation of ERK1/2. PbTx-2-induced ERK1/2 activation was Ca(2+) dependent and was mediated by Ca(2+) entry through manifold routes. PbTx-2 treatment also increased cAMP responsive element binding protein (CREB) phosphorylation and increased gene expression of brain-derived neurotrophic factor (BDNF). These findings indicate that brevetoxins, by influencing the activation of key signaling proteins, can alter physiologic events involved in survival in neocortical neurons, as well as forms of synaptic plasticity associated with development and learning.     

Endoplasmic reticulum Ca2+ release modulates endothelial nitric-oxide synthase via extracellular signal-regulated kinase (ERK) 1/2-mediated serine 635 phosphorylation

Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca(2+) and protein phosphorylation, but the interrelationship between intracellular Ca(2+) mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca(2+) release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca(2+) release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca(2+) release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca(2+) and selective to Ser-635 because the phosphorylation status of other eNOS sites, including Ser-1179 or Thr-497, was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca(2+) release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca(2+)/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK1/2 directly phosphorylated the eNOS Ser-635 residue in vitro. Further studies demonstrated that ER Ca(2+) release-induced ERK1/2 activation mediated the enhancing action of purine or bradykinin receptor stimulation on eNOS Ser-635/633 phosphorylation in bovine/human endothelial cells. Mutating the Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. Taken together, these studies reveal that ER Ca(2+) release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca(2+) is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca(2+)-ERK1/2-eNOS Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function.     

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis?2-aminophenoxy?thane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.     

SHP-2 modulates interleukin-1-induced Ca2+ flux and ERK activation via phosphorylation of phospholipase Cgamma1

Interleukin-1 (IL-1) signaling is dependent on focal adhesions, structures that are enriched with tyrosine kinases and phosphatases. Because the non-receptor tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is enriched in focal adhesions and IL-1-induced ERK activation requires increased Ca(2+), we determined whether SHP-2 modulates IL-1-induced Ca(2+) signaling. In SHP-2-deficient fibroblasts, IL-1-induced Ca(2+) signaling and ERK activation were markedly diminished compared with cells expressing SHP-2. IL-1-induced Ca(2+) release from the endoplasmic reticulum occurred in the vicinity of focal adhesions and was strongly inhibited by the blockage of phospholipase C (PLC) catalytic activity. Immunoprecipitation and immunostaining showed that SHP-2, the endoplasmic reticulum-specific protein calnexin, and PLCgamma1 were associated with focal adhesions; however, these associations and IL-1-induced ERK activation dissipated after cells were plated on non-integrin substrates. IL-1 promoted phosphorylation of SHP-2 and PLCgamma1. IL-1-induced phosphorylation of PLCgamma1 was diminished in SHP-2-deficient cells but was restored by stable transfection with SHP-2. BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)) blocked IL-1-induced phosphorylation of SHP-2 and PLCgamma1, indicating mutually dependent interactive roles for Ca(2+), SHP-2, and PLCgamma1 in IL-1 signaling. We conclude that SHP-2 is critical for IL-1-induced phosphorylation of PLCgamma1 and thereby enhances IL-1-induced Ca(2+) release and ERK activation. Focal adhesions co-localizing with the endoplasmic reticulum may provide molecular staging sites required for ERK activation.     

Ca2+-Independent, Ca2+-Dependent, and Carbachol- Mediated Arachidonic Acid Release from Rat Brain Cortex Membrane

Synaptoneurosomes obtained from the cortex of rat brain prelabeled with [14C]arachidonic acid [( 14C]AA) were used as a source of substrate and enzyme in studies on the regulation of AA release. A significant amount of AA is liberated in the presence of 2 mM EGTA, independently of Ca2+, primarily from phosphatidic acid and polyphosphoinositides (poly-PI). Quinacrine, an inhibitor of phospholipase A2 (PLA2), suppressed AA release by about 60% and neomycin, a putative inhibitor of phospholipase C (PLC), reduced AA release by about 30%. An additive effect was exhibited when both inhibitors were given together. Ca2+ activated AA release. The level of Ca2+ present in the synaptoneurosomal preparation (endogenous level) and 5 microM CaCl2 enhance AA liberation by approximately 25%, whereas 2 mM CaCl2 resulted in a 50% increase in AA release relative to EGTA. The source for Ca(2+)-dependent AA release is predominantly phosphatidylinositol (PI); however, a small pool may also be liberated from neutral lipids. Carbachol, an agonist of the cholinergic receptor, stimulated Ca(2+)-dependent AA release by about 17%. Bradykinin enhanced the effect of carbachol by about 10-15%. This agonist-mediated AA release occurs specifically from phosphoinositides (PI + poly-PI). Quinacrine almost completely suppresses calcium-and carbachol-mediated AA release. Neomycin inhibits this process by about 30% and totally suppresses the effect of bradykinin. Our results indicate that both phospholipases PLA2 and PLC with subsequent action of DAG lipase are responsible for Ca(2+)-independent AA release. Ca(2+)-dependent and carbachol-mediated AA liberation occurs mainly as the result of PLA2 action. A small pool of AA is probably also released by PLC, which seems to be exclusively responsible for the effect of bradykinin.     

A complex signaling cascade links the serotonin2A receptor to phospholipase A2 activation: the involvement of MAP kinases

Previous studies in our laboratory have shown that in NIH3T3-5HT2A cells, 5-HT-induced AA release is PLA2-coupled and independent of 5-HT2A receptor-mediated PLC activation. Although 5-HT2A receptor-mediated PLC activation is known to be Galphaq-coupled, much less is understood about 5-HT2A receptor-mediated PLA2 activation. Therefore, the studies presented here were aimed at elucidating the signal transduction pathway linking stimulation of the 5-HT2A receptor to PLA2 activation. By employing various selective inhibitors, toxins, and antagonistic peptide constructs, we propose that the 5-HT2A receptor can couple to PLA2 activation through two parallel signaling cascades. Initial experiments were designed to examine the role of pertussis toxin-sensitive G proteins, namely Galphai/o, as well as pertussis toxin-insensitive G proteins, namely Galpha12/13, in 5-HT-induced AA release. Furthermore, inactivation of both Gbetagamma heterodimers and Rho proteins resulted in decreased agonist-induced AA release, without having any effect on PLC-IP accumulation. We also demonstrated 5-HT2A receptor-mediated phosphorylation of ERK1,2 and p38. Moreover, pretreatment with selective ERK1,2 and p38 inhibitors resulted in decreased 5-HT-induced AA release. Taken together, these results suggest that the 5-HT2A receptor expressed in NIH3T3 cells can couple to PLA2 activation though a complex signaling mechanism involving both Galphai/o-associated Gbetagamma-mediated ERK1,2 activation and Galpha12/13-coupled, Rho-mediated p38 activation.     

Differential participation of phospholipase A(2) isoforms during iron-induced retinal toxicity. Implications for age-related macular degeneration

Both elevated iron concentrations and the resulting oxidative stress condition are common signs in retinas of patients with age-related macular degeneration (AMD). The role of phospholipase A(2) (PLA(2)) during iron-induced retinal toxicity was investigated. To this end, isolated retinas were exposed to increasing Fe(2+) concentrations (25, 200 or 800μM) or to the vehicle, and lipid peroxidation levels, mitochondrial function, and the activities of cytosolic PLA(2) (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)) were studied. Incubation with Fe(2+) led to a time- and concentration-dependent increase in retinal lipid peroxidation levels whereas retinal cell viability was only affected after 60min of oxidative injury. A differential release of arachidonic acid (AA) and palmitic acid (PAL) catalyzed by cPLA(2) and iPLA(2) activities, respectively, was also observed in microsomal and cytosolic fractions obtained from retinas incubated with iron. AA release diminished as the association of cyclooxigenase-2 increased in microsomes from retinas exposed to iron. Retinal lipid peroxidation and cell viability were also analyzed in the presence of cPLA(2) inhibitor, arachidonoyl trifluoromethyl ketone (ATK), and in the presence of iPLA(2) inhibitor, bromoenol lactone (BEL). ATK decreased lipid peroxidation levels and also ERK1/2 activation without affecting cell viability. BEL showed the opposite effect on lipid peroxidation. Our results demonstrate that iPLA(2) and cPLA(2) are differentially regulated and that they selectively participate in retinal signaling in an experimental model resembling AMD.     

Inhibition of phospholipase A2-mediated arachidonic acid release by cyclic AMP defines a negative feedback loop for P2Y receptor activation in Madin-Darby canine kidney D1 cells

In Madin-Darby canine kidney D1 cells extracellular nucleotides activate P2Y receptors that couple to several signal transduction pathways, including stimulation of multiple phospholipases and adenylyl cyclase. For one class of P2Y receptors, P2Y2 receptors, this stimulation of adenylyl cyclase and increase in cAMP occurs via the conversion of phospholipase A2 (PLA2)-generated arachidonic acid (AA) to prostaglandins (e.g. PGE2). These prostaglandins then stimulate adenylyl cyclase activity, presumably via activation of prostanoid receptors. In the current study we show that agents that increase cellular cAMP levels (including PGE2, forskolin, and the beta-adrenergic agonist isoproterenol) can inhibit P2Y receptor-promoted AA release. The protein kinase A (PKA) inhibitor H89 blocks this effect, suggesting that this feedback inhibition occurs via activation of PKA. Studies with PGE2 indicate that inhibition of AA release is attributable to inhibition of mitogen-activated protein kinase activity and in turn of P2Y receptor stimulated PLA2 activity. Although cAMP/PKA-mediated inhibition occurs for P2Y receptor-promoted AA release, we did not find such inhibition for epinephrine (alpha1-adrenergic) or bradykinin-mediated AA release. Taken together, these results indicate that negative feedback regulation via cAMP/PKA-mediated inhibition of mitogen-activated protein kinase occurs for some, but not all, classes of receptors that promote PLA2 activation and AA release. We speculate that receptor-selective feedback inhibition occurs because PLA2 activation by different receptors in Madin-Darby canine kidney D1 cells involves the utilization of different signaling components that are differentially sensitive to increases in cAMP or, alternatively, because of compartmentation of signaling components.     

A new mode of Ca2+ signaling by G protein-coupled receptors: gating of IP3 receptor Ca2+ release channels by Gbetagamma

The most common form of Ca(2+) signaling by Gq-coupled receptors entails activation of PLCbeta2 by Galphaq to generate IP(3) and evoke Ca(2+) release from the ER. Another form of Ca(2+) signaling by G protein-coupled receptors involves activation of Gi to release Gbetagamma, which activates PLCbeta1. Whether Gbetagamma has additional roles in Ca(2+) signaling is unknown. Introduction of Gbetagamma into cells activated Ca(2+) release from the IP(3) Ca(2+) pool and Ca(2) oscillations. This can be due to activation of PLCbeta1 or direct activation of the IP(3)R by Gbetagamma. We report here that Gbetagamma potently activates the IP(3) receptor. Thus, Gbetagamma-triggered [Ca(2+)](i) oscillations are not affected by inhibition of PLCbeta. Coimmunoprecipitation and competition experiments with Gbetagamma scavengers suggest binding of Gbetagamma to IP(3) receptors. Furthermore, Gbetagamma inhibited IP(3) binding to IP(3) receptors. Notably, Gbetagamma activated single IP(3)R channels in native ER as effectively as IP(3). The physiological significance of this form of signaling is demonstrated by the reciprocal sensitivity of Ca(2+) signals evoked by Gi- and Gq-coupled receptors to Gbetagamma scavenging and PLCbeta inhibition. We propose that gating of IP(3)R by Gbetagamma is a new mode of Ca(2+) signaling with particular significance for Gi-coupled receptors.     

Role of mitogen-activated protein kinase-mediated cytosolic phospholipase A2 activation in arachidonic acid metabolism in human eosinophils.

The objective of this investigation was to determine the role of secretory and cytosolic isoforms of phospholipase A(2) (PLA(2)) in the induction of arachidonic acid (AA) and leukotriene synthesis in human eosinophils and the mechanism of PLA(2) activation by mitogen-activated protein kinase (MAPK) isoforms in this process. Pharmacological activation of eosinophils with fMLP caused increased AA release in a concentration (EC(50) = 8.5 nM)- and time-dependent (t(1/2) = 3.5 min) manner. Both fMLP-induced AA release and leukotriene C(4) (LTC(4)) secretion were inhibited concentration dependently by arachidonic trifluoromethyl ketone, a cytosolic PLA(2) (cPLA(2)) inhibitor; however, inhibition of neither the 14-kDa secretory phospholipase A(2) by 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid nor cytosolic Ca(2+)-independent phospholipase A(2) inhibition by bromoenol lactone blocked hydrolysis of AA or subsequent leukotriene synthesis. Pretreatment of eosinophils with a mitogen-activated protein/extracellular signal-regulated protein kinase (ERK) kinase inhibitor, U0126, or a p38 MAPK inhibitor, SB203580, suppressed both AA production and LTC(4) release. fMLP induced phosphorylation of MAPK isoforms, ERK1/2 and p38, which were evident after 30 s, maximal at 1-5 min, and declined thereafter. fMLP stimulation also increased cPLA(2) activity in eosinophils, which was inhibited completely by 30 microM arachidonic trifluoromethyl ketone. Preincubation of eosinophils with U0126 or SB203580 blocked fMLP-enhanced cPLA(2) activity. Furthermore, inhibition of Ras, an upstream GTP-binding protein of ERK, also suppressed fMLP-stimulated AA release. These findings demonstrate that cPLA(2) activation causes AA hydrolysis and LTC(4) secretion. We also find that cPLA(2) activation caused by fMLP occurs subsequent to and is dependent upon ERK1/2 and p38 MAPK activation. Other PLA(2) isoforms native to human eosinophils possess no significant activity in the stimulated production of AA or LTC(4).     

ADP-ribose stimulates the calcium release channel RyR1 in skeletal muscle of rat

The Ca(2+) mobilizing metabolite cyclic ADP-ribose has been shown to release Ca(2+) from intracellular ryanodine sensitive stores in many cells. However, the activation of the ryanodine receptor of skeletal muscle by cADP-ribose (cADPr) and its precursor and metabolite (beta-NAD(+) and ADPr) remains to be discussed. We studied the effect of ADPr on the Ca(2+) release channel of skeletal muscle RyR1 after incorporation of microsomes isolated from fast muscles of rat in planar lipid bilayers. We observed an increase in the electrophysiological activity of the channel after addition of ADPr (10 microM) at micromolar Ca(2+) concentrations, characterized by a time-lag. The increase in P(o) is mainly due to an increase in the open frequency. The long time course observed for the development of the ADPr effect may indicate that this activation induces a change in the conformation of the RyR1 channel, which increases its sensitivity to calcium.