Acid Release In Response Research Articles

Abstract 016: Sex-Specific Effects of Neuropeptide Y2 Receptor (y2) Deletion in Adipose Tissue From Mice Exposed to Early Life Stress

Neuropeptide Y (NPY) plays a critical role in the regulation of energy homeostasis acting in the central nervous system as an orexigenic agent. Peripherally, NPY is co-released by catecholaminergic neurons and stimulates adipocyte precursor proliferation, differentiation, via NPY receptor 2 (Y2), and inhibit lipolysis, via the cAMP/PKA pathway. We have shown that female mice exposed to a model of early life stress, maternal separation and early weaning (MSEW), display greater increases in fat mass in response to high fat diet (HF). Thus, we hypothesized that Y2 deletion in adipose tissue could prevent fat expansion in female MSEW mice. MSEW was performed by separating pups from the dam during postnatal days 2 to 16, and weaned early on day 17. Control (C) mice remained undisturbed and were weaned on day 21. Genotyping was completed to determine WT (Y2 fl/fl) and Y2 KO (Y2 fl/fl) littermates. All groups were placed on a HF (60% kcal from fat) for 18 weeks. Following, mice were subjected to 6-weeks of chronic restraint stress (1 hr/day) combined with high fat/high sucrose diet (RS+HFS). No differences in fat mass were found between male WT MSEW and C mice (37.5±2.0 vs. 41.9±2.3 % body fat, respectively), although Y2 KO reduced fat mass in both groups (23.2±2.3 vs. 38.1±2.9 % body fat, p MSEW=0.084, p genotype=0.011, p interaction=0.401). Conversely, female WT MSEW mice displayed increased fat mass compared to C (26.4±2.6 vs. 19.1±2.6 % body fat), and Y2 KO further increased fat deposition in MSEW female mice compared to C (36.1±4.3 vs. 15.8±1.2 % body fat, p MSEW=0.0001, p genotype=0.047, p interaction=0.007). In addition, female WT MSEW mice displayed reduced free fatty acid release in response to an in vivo lipolysis test in compared to C (101±21 vs. 167±12 uM/ml, p=0.066). After RS+HFS, MSEW increased fat mass in male WT mice more than in female WT mice, while Y2 KO attenuated this effect in both sexes. These data suggests that Y2 in adipose tissue may contribute to reduced lipolysis in HF-fed female mice exposed to MSEW. However, Y2-mediated effects of chronic stress on fat expansion in adult life are greater in male than in female MSEW mice. Overall, our data shows that there is possibly a sex-specific imbalance between adipogenesis and lipolysis in response to different stressors.

Suppression of the C/EBP family of transcription factors in adipose tissue causes lipodystrophy.

Adipose-specific inactivation of both AP-1 and CCAAT-enhancer-binding protein (C/EBP) families of B-ZIP transcription factors in transgenic mice causes severe lipoatrophy. To evaluate whether inactivation of only C/EBP members was critical for lipoatrophy, A-C/EBP, a dominant-negative protein that specifically inhibits the DNA binding of the C/EBP members, was expressed in adipose tissue. For the first 2 weeks after birth, aP2-A-C/EBP mice had no white adipose tissue (WAT), drastically reduced brown adipose tissue (BAT), and exhibited marked hepatic steatosis, hyperinsulinemia, and hyperlipidemia. However, WAT appeared during the third week, coinciding with significantly improved metabolic functioning. In adults, BAT remained reduced, causing cold intolerance. At 30 weeks, the aP2-A-C/EBP mice had only 35% reduced WAT, with clear morphological signs of lipodystrophy in subcutaneous fat. Circulating leptin and adiponectin levels were less than the wild-type levels, and these mice exhibited impaired triglyceride clearance. Insulin resistance, glucose intolerance, and reduced free fatty acid release in response to β3-adrenergic agonist suggest improper functioning of the residual WAT. Gene expression analysis of inguinal WAT identified reduced mRNA levels of several enzymes involved in fatty acid synthesis and glucose metabolism that are known C/EBPα transcriptional targets. There were increased levels for genes involved in inflammation and muscle differentiation. However, when dermal fibroblasts from aP2-A-C/EBP mice were differentiated into adipocytes in tissue culture, muscle markers were elevated more than the inflammatory markers. These results demonstrate that the C/EBP family is essential for adipose tissue development during the early postnatal period, the regulation of glucose and lipid homeostasis in adults, and the suppression of the muscle lineage.

Alginate film prepared on polyethylene nonwoven sheet and its function for ellagic acid release in response to sodium ions

Calcium alginate film containing ellagic acid as a functional material was formed on polyethylene nonwoven sheet by the reaction of sodium alginate with calcium chloride in the presence of ellagic acid. The film had the intelligent function of release of ellagic acid triggered by sodium ion. This response resulted from conversion of the water-insoluble calcium alginate to water-soluble sodium alginate. The optimal conditions for the intelligent calcium alginate film prepared on the polyethylene surface were 0.5% CaCl2 solution and 0.05 or 0.1% sodium alginate solution.

Activation of Cytosolic Phospholipase A2α in Resident Peritoneal Macrophages by Listeria monocytogenes Involves Listeriolysin O and TLR2

Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2alpha). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (DeltahlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and DeltahlyLM. The attenuated release of arachidonic acid that is observed in TLR2-/- and MyD88-/- macrophages infected with WTLM and DeltahlyLM correlates with diminished MAPK activation. WTLM but not DeltahlyLM increases intracellular calcium, which is implicated in regulation of cPLA2alpha. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2alpha+/+ but not cPLA2alpha-/- macrophages in response to WTLM and DeltahlyLM. Tumor necrosis factor (TNF)-alpha production is significantly lower in cPLA2alpha+/+ than in cPLA2alpha-/- macrophages infected with WTLM and DeltahlyLM. Treatment of infected cPLA2alpha+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFalpha production to the level produced by cPLA2alpha-/- macrophages implicating prostaglandins in TNFalpha down-regulation. Therefore activation of cPLA2alpha in macrophages may impact immune responses to L. monocytogenes.

The immunosuppressant drug FTY720 inhibits cytosolic phospholipase A2 independently of sphingosine-1-phosphate receptors

AbstractFTY720 is a potent immunomodulator drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. FTY720 is phosphorylated in vivo by sphingosine kinase 2 to FTY720-phosphate, which acts as a potent sphingosine-1-phosphate (S1P) receptor agonist. However, in contrast to S1P, FTY720 has no effect on mast-cell degranulation, yet significantly reduces antigen-induced secretion of PGD2 and cysteinyl-leukotriene. Unexpectedly, this effect of FTY720 was independent of its phosphorylation and S1P receptor functions. The rate-limiting step in the biosynthesis of all eicosanoids is the phospholipase A2 (PLA2)–mediated release of arachidonic acid from glycerol phospholipids. Although FTY720 also reduced arachidonic acid release in response to antigen, it had no effect on translocation of cPLA2 or ERK1/2 activation, suggesting that it does not interfere with FcϵRI-mediated events leading to cPLA2 activation. Remarkably, however, FTY720 drastically inhibited recombinant cPLA2α activity, whereas FTY720-phosphate, sphingosine, or S1P had no effect. This study has uncovered a unique action of FTY720 as an inhibitor of cPLA2α and hence on production of all eicosanoids. Our results have important implications for the potential therapeutic mechanism of action of FTY720 in eicosanoid-driven inflammatory disorders such as asthma and multiple sclerosis.

Homeostatic Regulation of Glutamate Release in Response to Depolarization

Proper nervous system function requires a balance between excitation and inhibition. Systems of homeostasis may have evolved in neurons to help maintain or restore balance between excitation and inhibition, presumably because excessive excitation can cause dysfunction and cell death. This article reviews evidence for homeostatic mechanisms within the hippocampus that lead to differential regulation of glutamate and gamma-aminobutyric acid release in response to conditions of excess depolarization. We recently found differential effects on glutamate release at the level of action potential coupling to transmitter release, vesicular release probability, and vesicle availability. Such differential regulation may help to prevent excitotoxicity and runaway excitation.

Expression of Cytosolic Phospholipase A2α in Murine C12 Cells, a Variant of L929 Cells, Induces Arachidonic Acid Release in Response to Phorbol Myristate Acetate and Ca2+ Ionophores, but Not to Tumor Necrosis Factor-α

Tumor necrosis factor-α (TNFα)-induced cell death is regulated through the release of arachidonic acid (AA) by group IVA cytosolic phospholipase A2 (cPLA2α) in the murine fibroblast cell line L929. However, the signaling pathway by which TNFα activates cPLA2α remained to be solved. We examined AA release in L929 cells, in a variant of L929 (C12 cells) lacking cPLA2α, and in C12 cells transfected with cPLA2α expression vectors. In transient and stable clones of C12 cells expressing cPLA2α, Ca2+ ionophore A23187 and phorbol myristate acetate (PMA) stimulated AA release within 90 min, although no response to TNFα was observed within 6 h. These results suggest that C12 cells may lack the components necessary for TNFα-induced AA release, in addition to cPLA2α. PMA is known to stimulate AA release via phosphorylation of Ser505 in cPLA2α by activating extracellular signal-regulated kinases (ERK1/2). However, PMA-induced AA release from C12 cells expressing mutant cPLA2αS505A (mutation of Ser505 to Ala), which is not phosphorylated by ERK1/2, was similar to that from L929 cells and C12 cells expressing wild-type cPLA2α. The role of Ser505 phosphorylation in AA release induced by PMA is also discussed.

Regulation of kinin receptors in airway epithelial cells by inflammatory cytokines and dexamethasone

The two kinin receptors, B 1 and B 2, are upregulated in inflammation and may play a role in diseases such as asthma. In pulmonary A549 cells, TNF-α or interleukin-1β dramatically increased bradykinin B 1 and B 2 receptor mRNA expression and this response was prevented by dexamethasone. In primary human bronchial epithelial cells, bradykinin B 1 receptor mRNA expression showed a similar trend, whereas bradykinin B 2 receptor showed almost constitutive expression. Radioligand-binding studies revealed significant increases in bradykinin B 2 receptor protein expression following both interleukin-1β and TNF-α treatment of A549 cells; however, no evidence was found for bradykinin B 1 receptor. Functionally, the bradykinin B 2 receptor ligand, bradykinin, but not the B 1 ligand, des-Arg 10-kallidin, produced a marked increase in prostaglandin E 2 release when administered following interleukin-1β treatment. Arachidonic acid release in response to bradykinin was markedly enhanced by prior incubation with interleukin-1β and this was prevented by the prior addition of dexamethasone.

Evidence for a Role for Phospholipase C, but not Phospholipase A2, in Platelet Activation in Response to Low Concentrations of Collagen

The release of arachidonic acid is a key component in platelet activation in response to low concentrations (1-20 microg/ml) of collagen. The precise mechanism remains elusive although a variety of pathways have been implicated. In the present study the effects of inhibitors of several potentially key enzymes in these pathways have been examined. Collagen 1-10 microg/ml) caused maximal platelet aggregation which was accompanied by the release of arachidonic acid, the synthesis of thromboxane A2, and p38MAPK phosphorylation. Preincubation with the dual cyclooxygenase/lipoxygenase inhibitor BW755C inhibited aggregation and thromboxane production, and reduced p38MAPK phosphorylation. A phospholipase C inhibitor, U73122, blocked collagen-induced aggregation and reduced arachidonic acid release, thromboxane synthesis and p38MAPK phosphorylation. Pretreatment with a cytosolic phospholipase A2 inhibitor, AACOCF3, blocked collagen-induced aggregation, reduced the levels of thromboxane formation and p38MAPK phosphorylation but had no significant effect on arachidonic acid release. In contrast inhibition of PKC by Ro31-8220 inhibited collagen-induced aggregation. did not affect p38MAPK phosphorylation but significantly potentiated arachidonic acid release and thromboxane formation. Collagen caused the tyrosine phosphorylation of phospholipase Cgamma2 which was inhibited by pretreatment with U73122, unaffected by AACOCF3 and enhanced by Ro31-8220. These results suggest that cytosolic phospholipase A2 plays no role in the arachidonic acid release in response to collagen. In contrast, the data are consistent with phospholipase Cgamma2 playing a role in an intricately controlled pathway, or multiple pathways, mediating the release of arachidonic acid in collagen-stimulated platelets.

N-terminal and C-terminal plasma membrane anchoring modulate differently agonist-induced activation of cytosolic phospholipase A2.

The 85 kDa cytosolic phospholipase A2 (cPLA2) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA2 undergoes calcium-dependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA2 activation, we constructed chimeras of cPLA2 constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA2) or the C-terminal targeting signal of K-Ras4B (cPLA2-Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the alpha2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [3H]arachidonic acid, indicating that constitutive association of cPLA2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA2 increased [3H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA2-Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts of recombinant wild-type cPLA2. The lack of stimulation of cPLA2-Ras was not due to a decreased enzymatic activity as measured using an exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.

Modulatory actions of progesterone on gonadotropin-releasing hormone-induced arachidonic acid liberation from perifused rat pituitary cells.

The stimulatory action of gonadotropin-releasing hormone (GnRH) on gonadotropin secretion from cultured rat pituitary cells is modulated by estradiol and progesterone. Recent studies provided evidence that both steroids exert effects on different pathways of GnRH signal transduction, which might be responsible for their actions on luteinizing hormone (LH) release. Here we investigated whether the steroids are able to modulate GnRH-induced liberation of arachidonic acid, which is thought to be involved in GnRH signal transduction. Pituitary cells obtained from female rats were treated for 48 h with vehicle, 1 nmol/l estradiol or 1 nmol/l estradiol + 100 nmol/l progesterone, 48 h with 1 nmol/l estradiol and 2 h with 100 nmol/l progesterone. In addition, these cells were prelabeled with [3H]arachidonic acid. Then the cells were transferred to a perifusion system and challenged with a 6-min pulse of 100 nmol/l GnRH. Estradiol treatment enhanced the LH secretory response while GnRH-induced [3H]arachidonic acid liberation remained unaffected. However, progesterone modulated both LH secretion and [3H]arachidonic acid release in response to the GnRH stimulus. The short-term progesterone treatment paradigm enhanced the LH and arachidonic acid responses by up to 160 +/- 13 and 204 +/- 18%, respectively, while long-term treatment was inhibitory (59 +/- 9 and 63 +/- 4% vs control). Because arachidonic acid has been shown to be involved in GnRH signal transduction, it seems reasonable to speculate that the actions of progesterone described in the present study are related to its modulatory effect on GnRH-induced LH secretion.

Basic fibroblast growth factor-stimulated arachidonic acid release in rat pancreatic acini: Sequential action of tyrosine kinase, phospholipase C, protein kinase C and diacylglycerol lipase

This study was performed to evaluate the effect of human recombinant basic fibroblast growth factor on arachidonic acid release from rat pancreatic acini and to determine the cellular mechanism involved. From enzymatic assays, basic fibroblast growth factor did not significantly stimulate phospholipase A 2 activity, whereas it significantly increased diacylglycerol lipase activity. Validity of phospholipase A 2 or diacylglycerol lipase inhibitors was confirmed by their ability to inhibit phospholipase A2 or diacylglycerol lipase activities. Basic fibroblast growth factor increased intracellular accumulation and extracellular release of arachidonic acid from metabolically labelled acinar cells in a concentration- and time-dependent manner. This effect was maximal with 50 pM basic fibroblast growth factor and became significant after a 5-min incubation period. The protein tyrosine kinase inhibitor, 0.5 mM genistein, inhibited arachidonic acid release in basic fibroblast growth factor-stimulated acini, whereas 100 μM vanadate, a protein tyrosine phosphatase inhibitor, enhanced arachidonic acid release. Two phospholipase A 2 inhibitors, mepacrine and aristolochic acid, failed to attenuate basic fibroblast growth factor-stimulated arachidonic acid release. A diacylglycerol lipase inhibitor RHC 80267 at 150 μM and 50 μM completely inhibited 50 pM basic fibroblast growth factor-induced intracellular accumulation and extracellular release of arachidonic acid, respectively. Furthermore, basic fibroblast growth factor stimulated arachidonic acid release was also inhibited by 10 μM U73122 and by 100 nM staurosporine, phospholipase C and protein kinase C respective inhibitors. Wortmannin, an inhibitor of basic fibroblast growth factor-stimulated phospholipase D, did not affect arachidonic acid release. 100 nM 4β-phorbol 12-myristate 13-acetate also increased arachidonic acid release, an effect also inhibited by staurosporine. Taken together, these data demonstrate activation of diacylglycerol lipase and arachodonic acid release in pancreatic acini upon stimulation by basic fibroblast growth factor, and strongly indicate that arachidonic acid release in response to basic fibroblast growth factor depends upon the sequential action of tyrosine kinase, phospholipase C, protein kinase C and diacylglycerol lipase but not from phospholipase A 2 nor phospholipase D activation.

Ethanol inhibits zymosan-stimulated eicosanoid production in mouse peritoneal macrophages

Resident peritoneal macrophages synthesized and released eicosanoids when challenged by zymosan, a phagocytosable particle. Incubation of these cells with ethanol resulted in dose-dependent inhibition of arachidonic acid release and eicosanoid generation in response to zymosan. Ethanol affected the extent but not the ratio of eicosanoids released. When assayed in a cell-free system, endogenous phospholipase A 2 activity was neither affected by the presence of ethanol in the incubation medium nor by preincubation of the cells with ethanol. Ethanol also inhibited arachidonic acid release in response to phorbol myristate acetate, a compound that, like zymosan, triggered a pertussis-toxin-sensitive response. When cells that had been previously treated with pertussis toxin were used, no further inhibitory effect of ethanol was seen in response to both zymosan and phorbol myristate acetate. On the other hand, ethanol had no effect on arachidonic acid release stimulated by ionophore A23187 or lipopolysaccharide, two compounds that triggered a pertussis-toxin-insensitive response. Moreover, ethanol was able to nearly abolish arachidonic acid release in response to fluoroaluminate, a direct activator of G-proteins. Altogether, the results of this study suggest that ethanol inhibits zymosan-stimulated eicosanoid production by interacting with a G-protein — or a G-protein-mediated process — that is critically involved in arachidonic acid mobilization.

Regulation of phospholipase A2 activation by phosphorylation in mouse peritoneal macrophages.

The regulation of phospholipase A2 (PLA2) activation by phosphorylation, and phosphorylation of an 85-kDa, arachidonoyl-hydrolyzing PLA2 was investigated in mouse peritoneal macrophages. Phorbol 12-myristate 13-acetate (PMA) and okadaic acid, an inhibitor of serine/threonine phosphatases, stimulated arachidonic acid release, supporting a role for phosphorylation events in regulating PLA2 activation. In response to zymosan, PMA, or A23187, arachidonic acid was released at a linear rate up to 30-45 min after stimulation, whereas there was a 30-min lag preceding arachidonic acid release in response to okadaic acid. The 85-kDa PLA2 was phosphorylated on serine in the macrophages, and the level of phosphorylation increased in response to zymosan, PMA, okadaic acid, and, to a lesser extent, A23187. Two-dimensional phosphopeptide mapping revealed multiple phosphopeptides, several of which showed increased phosphorylation in response to zymosan, okadaic acid, and PMA. Zymosan, PMA, A23187, or okadaic acid stimulated time-dependent increases in PLA2 activity in the cytosolic fraction. PLA2 activation was most rapid in response to PMA, whereas activation in response to okadaic acid was delayed similar to the time course of arachidonic acid release. The cytosolic PLA2 had characteristics of the 85-kDa enzyme, including kinetic properties and substrate preference. Phosphatase treatment of the cytosols dephosphorylated the 85-kDa PLA2 and reversed the increase in activity. The results provide evidence that phosphorylation of the 85-kDa PLA2, induced by stimuli that induce arachidonic acid release, is an important mechanism for activation of the enzyme in macrophages.

Release of amino acids into regional superfusates of the spinal cord by mechano-stimulation of the reproductive tract

Based on pharmacological evidence that inhibitory amino acids mediate vaginocervical mechano-stimulation produced analgesia (VSPA), we hypothesized that inhibitory amino acids would be released endogenously in the spinal cord in response to vaginocervical mechano-stimulation (VS). This hypothesis was tested by HPLC analysis of the amino acid content of 5-min superfusates of the spinal cord before, during and after VS (400 g force applied against the cervix) in urethane-anesthetized rats. Utilizing an in vivo push-pull superfusion method, artificial cerebrospinal fluid was continuously superfused over the spinal cord through the intrathecal space surrounding the sacral-lower thoracic region. In addition, concentrations of amino acids in the superfusate were measured in response to KCl stimulation (increasing the superfusion medium from 3.4 to 40.0 mM KCl to produce non-specific depolarization), and to noxious hind paw mechano-stimulation (pinching the hind paw to produce a sustained flexor response in ipsilateral hind leg). There was a significant increase in the concentration of Gly, Tau, Asp, Glu and Lys in the superfusate in response to VS ( n = 8) and to KCl ( n = 8), but not to hind paw stimulation ( n = 5). Also, GABA concentrations increased in response to KCl, and the concentration of Ala, Ser, Gln, Thr, Arg and Phe increased in response to VS, however, GABA levels were sometimes below the limits of detection. In contrast, there was no significant change in any amino acid concentration in response to hind paw pinch stimulation, and VS did not significantly affect the concentrations of Tyr, His, Ile, Leu, Met, Trp or Val. The present findings support our hypothesis that VS releases inhibitory amino acids in the spinal cord. Moreover, other amino acids, including ‘excitatory’ amino acids, are released into the superfusate. The profile of amino acid release in response to VS differs from that in response to paw pinch or KCl administration.

Interleukin-1 selectively potentiates bradykinin-stimulated arachidonic acid release from human synovial fibroblasts

Exposure of human synovial fibroblasts prelabelled with [ 3H]arachidonic acid to bradykinin causes a rapid and sustained increase in arachidonic acid release, a transient increase in cytosolic calcium and an increase in radiolabelled diacylglycerol. Activation of arachidonic acid release by bradykinin was potentiated by interleukin-1 added either simultaneously with bradykinin or to cultures 24 h before addition of bradykinin. In contrast, interleukin-1 did not modify bradykinin-induced increases in cytosolic calcium or diacylglycerol. The stimulation of arachidonic acid release in response to bradykinin, in the absence or presence of interleukin-1, was not affected by RHC-80267, an inhibitor of diacylglycerol kinase, suggesting that deacylation of diacylglycerol was not an important pathway of arachidonic acid production in cultures exposed to bradykinin. This conclusion is supported by the observation that increased release of arachidonic acid was not accompanied by increased release of [ 14C]stearic acid in cultures labelled with both isotopes. Bradykinin-stimulated release of arachidonic acid was prevented by down-regulating protein kinase C by pretreatment with phorbol 12-myristate 13-acetate and was unaffected by inhibitors of protein synthesis actinomycin D or cycloheximide. On the other hand, interleukin-1 amplification of bradykinin-stimulated release of arachidonic acid was blocked by actinomycin D and cycloheximide. The results from this study point to activation of phospholipase A 2 as the source of arachidonic acid in response to bradykinin. Our data further indicate that interleukin-1 selectively potentiates bradykinin activation of a phospholipase A 2 by a mechanism requiring protein synthesis, but has no effect on bradykinin activation of phospholipase C.

Coupling of two endothelin receptor subtypes to differing signal transduction in transfected Chinese hamster ovary cells.

We examined the intracellular signal transduction of two endothelin receptor subtypes (ETA and ETB) by transfection and stable expression of individual receptor cDNAs in Chinese hamster ovary cells. Both receptors showed a rapid and marked stimulation of phosphatidylinositol hydrolysis and arachidonic acid release in response to agonist interaction. The two receptors, however, exhibited different responses in the cyclic AMP transduction cascades. ETA mediated the accumulation of cyclic AMP formation, whereas ETB displayed an inhibitory action on the forskolin-stimulated cyclic AMP accumulation. In both receptors, the responses of phosphatidylinositol hydrolysis, arachidonic acid release, and cyclic AMP formation were induced in complete agreement with the endothelin-binding selectivity of each receptor subtype. Endothelin, added together with GTP, activated the adenylate cyclase activity in membrane preparations of ETA-expressing cells, indicating the direct linkage of ETA to the adenylate cyclase system. Pertussis toxin treatment of ETA-expressing cells resulted in partial inhibition of the endothelin-induced cyclic AMP accumulation, whereas the same treatment of ETB-expressing cells completely abolished the endothelin-induced inhibition of cyclic AMP formation. Thus, the two endothelin receptor subtypes are coupled to multiple but distinct signal transduction cascades through different G proteins.

Human decidua is a target tissue for bradykinin and kallikrein: phosphoinositide hydrolysis accompanies arachidonic acid release in uterine decidua cells in vitro.

The endocrine and intracellular mechanisms regulating prostaglandin precursor release in the uterine decidua during labor are unknown. This in vitro study investigates a potential role for a kallikrein-kinin system in the activation of phospholipid hydrolysis and arachidonic acid release in human decidua cells. Primary cultures of human decidua cells were prelabeled with [3H]inositol or [14C]arachidonic acid to monitor phosphoinositide hydrolysis and prostaglandin precursor release, respectively. Bradykinin (100 nmol/L) stimulated a rapid release of arachidonic acid (within 2 min) associated with an increase in inositol trisphosphate which was detectable after 20 s. Protein kinase C activation by phorbol ester enhanced arachidonic acid release in response to both bradykinin and the Ca++ ionophore A23187 but inhibited bradykinin-stimulated phosphoinositide hydrolysis. Epidermal growth factor also enhanced arachidonate release in response to both bradykinin and A23187. Kallikrein stimulated both phosphoinositide hydrolysis and arachidonic acid release in decidua cells. Kallikrein action was inhibited by the kallikrein protease inhibitor aprotinin and D-Arg[Hyp3Thi5,8,D-Phe7] bradykinin, a B2 receptor antagonist. Bradykinin also stimulated prostaglandin F2 alpha production in both primary decidua cell cultures and fibroblasts in the presence of interleukin-1 beta. These findings are consistent with a mediatory role for bradykinin in the action of kallikrein on decidua cells and suggest that inositol phospholipid hydrolysis is instrumental for arachidonic acid release in response to bradykinin in these cells. This study supports a novel role for a kallikrein-kinin system in the human uterine decidua.

Receptor-mediated activation of arachidonic acid release in mouse peritoneal macrophages is linked to extracellular calcium influx

The role of external calcium in platelet-activating factor- and zymosan-stimulated arachidonic acid release from mouse macrophages was investigated. Deprivation of external Ca2+ led to strong inhibition of receptor-mediated arachidonic acid release, which was completely restored when Ca2+ was added to the incubation medium. When arachidonic acid release was examined in Ca(2+)-depleted cells, the response took place only in presence of external Ca2+. Verapamil, a voltage-dependent Ca2+ channel blocker, nearly abolished arachidonic acid release in response to both platelet-activating factor and zymosan. These results suggest that extracellular Ca2+ influx is functionally linked to arachidonic acid release and hence to phospholipase A2 activation in mouse peritoneal macrophages.

Regulation of bradykinin receptor level by cholera toxin, pertussis toxin and forskolin in cultured human fibroblasts.

1. The effect of bacterial toxins on bradykinin-triggered release of arachidonic acid was studied in serum-deprived human foreskin (HSWP) fibroblasts prelabelled with [3H]-arachidonic acid. An 18-h exposure of HSWP cells to cholera toxin, pertussis toxin, or forskolin enhanced the bradykinin-stimulated release of arachidonic acid and metabolites. 2. Prolonged treatment of HSWP cells with these agents also caused a 3 to 4 fold rise in cell surface [3H]-bradykinin binding. The rise was inhibited by concurrent incubation with cycloheximide or actinomycin D. In addition, cholera toxin and foreskolin increased [3H]-bradykinin binding in wildtype PC12 cells, but not in mutant PC12 cells with reduced cyclic AMP-dependent protein kinase type II activity. 3. In conclusion, cholera toxin, pertussis toxin and forskolin enhanced arachidonic acid release in response to bradykinin, and increased the number of bradykinin receptors in HSWP fibroblasts. A cyclic AMP-dependent mechanism appears to mediate the actions of the toxins and forskolin.