scholarly journals Modulation of Cell-Substrate Adhesion by Arachidonic Acid: Lipoxygenase Regulates Cell Spreading and ERK1/2-inducible Cyclooxygenase Regulates Cell Migration in NIH-3T3 Fibroblasts

2001 ◽  
Vol 12 (7) ◽  
pp. 1937-1956 ◽  
Author(s):  
Rebecca A. Stockton ◽  
Bruce S. Jacobson
Keyword(s):  
Arachidonic Acid ◽  
Cell Migration ◽  
Cell Attachment ◽  
Cell Spreading ◽  
Cyclooxygenase 2 ◽  
Arachidonic Acid Release ◽  
Cell Substrate ◽  
Acid Release ◽  
Nih 3T3 ◽  
Arachidonate Release

Adhesion of cells to an extracellular matrix is characterized by several discrete morphological and functional stages beginning with cell-substrate attachment, followed by cell spreading, migration, and immobilization. We find that although arachidonic acid release is rate-limiting in the overall process of adhesion, its oxidation by lipoxygenase and cyclooxygenases regulates, respectively, the cell spreading and cell migration stages. During the adhesion of NIH-3T3 cells to fibronectin, two functionally and kinetically distinct phases of arachidonic acid release take place. An initial transient arachidonate release occurs during cell attachment to fibronectin, and is sufficient to signal the cell spreading stage after its oxidation by 5-lipoxygenase to leukotrienes. A later sustained arachidonate release occurs during and after spreading, and signals the subsequent migration stage through its oxidation to prostaglandins by newly synthesized cyclooxygenase-2. In signaling migration, constitutively expressed cyclooxygenase-1 appears to contribute ∼25% of prostaglandins synthesized compared with the inducible cyclooxygenase-2. Both the second sustained arachidonate release, and cyclooxygenase-2 protein induction and synthesis, appear to be regulated by the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2. The initial cell attachment-induced transient arachidonic acid release that signals spreading through lipoxygenase oxidation is not sensitive to ERK1/2 inhibition by PD98059, whereas PD98059 produces both a reduction in the larger second arachidonate release and a blockade of induced cyclooxygenase-2 protein expression with concomitant reduction of prostaglandin synthesis. The second arachidonate release, and cyclooxygenase-2 expression and activity, both appear to be required for cell migration but not for the preceding stages of attachment and spreading. These data suggest a bifurcation in the arachidonic acid adhesion-signaling pathway, wherein lipoxygenase oxidation generates leukotriene metabolites regulating the spreading stage of cell adhesion, whereas ERK 1/2-induced cyclooxygenase synthesis results in oxidation of a later release, generating prostaglandin metabolites regulating the later migration stage.

Norepinephrine stimulates arachidonic acid release from vascular smooth muscle via activation of cPLA2

1998 ◽  
Vol 274 (4) ◽  
pp. C1129-C1137 ◽  
Author(s):  
Edward F. LaBelle ◽  
Erzsebet Polyak
Keyword(s):  
Arachidonic Acid ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Adrenergic Receptors ◽  
Phospholipase A ◽  
Maximal Effect ◽  
Arachidonic Acid Release ◽  
Tail Artery ◽  
Acid Release ◽  
Arachidonate Release

The mechanism of agonist-activated arachidonate release was studied in segments of rat tail artery. Tail artery segments were prelabeled with [3H]arachidonate and then stimulated with norepinephrine (NE), and the radioactivity of the extracellular medium was determined. NE stimulated arachidonate release from the tissue without increasing arachidonic acid levels within cellular cytosol or crude membranes. About 90% of the extracellular radioactivity was shown to be unmetabolized arachidonate by TLC. Arachidonic acid release was not inhibited by the removal of the endothelium from the artery. NE exerted a half-maximal effect at a concentration of 0.2 μM. NE-stimulated arachidonate release was not inhibited by blockers of phospholipase C (U-73122), diacylglycerol lipase (RHC-80267), secretory phospholipase A2 (manoalide), calcium-insensitive phospholipase A2 (HELSS), or β-adrenergic receptors (propranolol). NE-stimulated arachidonic acid release was inhibited by blockers of cytosolic phospholipase A2(cPLA2) (AACOCF3), α1-adrenergic receptors (prazosin), and specific G proteins (pertussis toxin). This indicated that NE stimulated arachidonate release from vascular smooth muscle via activation of α-adrenergic receptors, either Gi or Go, and cPLA2. NE-activated arachidonic acid release from vascular smooth muscle may play a role in force generation by the tissue. Perhaps arachidonic acid extends the effect of NE on one specific smooth muscle cell to its nearby neighbor cells.

scholarly journals Arachidonic acid mobilization is suppressed during mitosis: role of cytosolic phospholipase A2 activation

1995 ◽  
Vol 309 (1) ◽  
pp. 91-97 ◽  
Author(s):  
R D Berlin ◽  
S F Preston
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Hela Cells ◽  
Arachidonic Acid Release ◽  
Cytosolic Phospholipase ◽  
Interphase Cells ◽  
Acid Release ◽  
Arachidonate Release ◽  
Stimulation Of ◽  
Mitotic Cells

In interphase HeLa cells, incubation with histamine or thapsigargin led to the rapid release of arachidonic acid. The release was absolutely dependent on Ca2+, consistent with the activation of an 85 kDa cytosolic phospholipase A2 (cPLA2). In metaphase-arrested HeLa cells, by contrast, the stimulation of arachidonate release by these agents was inhibited by more than 90%. The lack of arachidonic acid release by mitotic cells was at least partly expected, since histamine- or thapsigargin-induced Ca2+ influx and elevations of cytosolic free Ca2+ are known to be strongly inhibited during mitosis [Preston, Sha'afi and Berlin (1991) Cell Regul. 2, 915-925]. Indeed, incubation of interphase cells with the Ca2+ ionophore A23187 alone induced a high level of arachidonate release. However, even A23187 failed to elicit release from mitotic cells. Since the Ca(2+)-dependent release of arachidonate by many cell types is promoted by preincubation with ligands that activate receptors of the tyrosine kinase class, and tumour promoters that lead to the phosphorylation of cPLA2, we determined if the responses of mitotic HeLa cells could be modified by this ‘priming’ process. We first established that epidermal growth factor and phorbol 12-myristate 13-acetate were effective priming agents in interphase cells: cells preincubated with the hormone or tumour promoter showed a 2-fold stimulation of thapsigargin- or A23187-induced arachidonic acid release. However, none of the priming agents reversed the lack of mitotic cell response. This refractoriness was not caused by destruction of cPLA2 during mitosis: by Western blotting, cPLA2 of interphase and mitotic cells was shown to be present in comparable amounts. Moreover, cPLA2 activities measured in extracts of interphase and mitotic cells were also comparable. Surprisingly, mitotic cPLA2 appeared to be constitutively phosphorylated in non-hormone-treated (control) cells. The results indicate a novel mechanism of regulation by cPLA2 activity in mitotic cells.

scholarly journals Regulation of arachidonic acid release in vascular endothelium. Ca2+-dependent and -independent pathways

1991 ◽  
Vol 280 (2) ◽  
pp. 281-287 ◽  
Author(s):  
B J Buckley ◽  
A Barchowsky ◽  
R J Dolor ◽  
A R Whorton
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Combined Treatment ◽  
Arachidonic Acid Release ◽  
Aortic Endothelial Cells ◽  
Protein Activator ◽  
Kinase C ◽  
Acid Release ◽  
Arachidonate Release

Ca2+ metabolism and its relationship to arachidonic acid release were studied in cultured pig aortic endothelial cells. When cells were treated with bradykinin, a rapid rise in intracellular Ca2+ concentration ([Ca2+]i) occurred. Arachidonic acid release from cells prelabelled with [3H]arachidonic acid and subjected to flow-through conditions closely followed the changes in [Ca2+]i. Attenuation of the Ca2+ response by chelating extracellular and intracellular Ca2+ or by desensitization of receptors led to comparable attenuation of arachidonate release. Activation of protein kinase C inhibited Ca2+ mobilization in response to bradykinin and stimulated arachidonic acid release. Inhibition of protein kinase C had no effect on bradykinin-stimulated arachidonic acid release, suggesting that protein kinase C does not mediate the bradykinin response. The role of GTP-binding regulatory proteins (G-proteins) in mediating the bradykinin response was also investigated. Bradykinin-stimulated arachidonic acid release was not diminished by preincubation with pertussis toxin. Treatment with the G-protein activator AlF4- resulted in the release of a large pool of arachidonic acid and the formation of lysophospholipids. Combined treatment with AlF4- and bradykinin resulted in a greater than additive effect on arachidonic acid release. In contrast with bradykinin, AlF(4-)-stimulated arachidonic acid release was not dependent on the presence of extracellular Ca2+ or the mobilization of intracellular Ca2+. These results demonstrate Ca(2+)-dependent (bradykinin) and Ca(2+)-independent (AlF4-) pathways of phospholipase A2 activation.

scholarly journals Effect of temperature on bradykinin-induced arachidonate release and calcium mobilization in vascular endothelial cells

1993 ◽  
Vol 291 (3) ◽  
pp. 803-809 ◽  
Author(s):  
O L Wang ◽  
Y T Xuan ◽  
Z Mirza ◽  
A R Whorton
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Arachidonic Acid Release ◽  
Acid Release ◽  
Lower Temperature ◽  
Arachidonate Release

The effect of decreased temperature on Ca(2+)-dependent arachidonic acid release was studied in vascular endothelial cells by investigating bradykinin (BK)-stimulated Ca2+ mobilization, inositol phosphate formation and arachidonic acid release. At both 37 degrees C and 22 degrees C, BK efficiently increased cytosolic Ca2+ concn. ([Ca2+]i). At 22 degrees C, peak [Ca2+]i was higher and returned to basal levels more slowly. Although this response was preceded by rapid formation of Ins(1,4,5)P3, the activity of phospholipase C was significantly impaired at 22 degrees C. To determine if Ins(1,4,5)P3 effectively mobilized intracellular Ca2+, we used saponin-permeabilized cells. Ins(1,4,5)P3, mobilized sequestered Ca2+ to a similar degree at 37 degrees C and 22 degrees C, although Ca2+ release was prolonged at 22 degrees C. In intact cells, BK mobilized intracellular Ca2+ stores and activated Ca2+ entry. The rate of 45Ca2+ entry was approx. 2-fold slower at 22 degrees C, even though the peak and duration of the rise in [Ca2+]i were higher and sustained at the lower temperature. TG mobilized intracellular Ca2+, activated Ca2+ entry and elevated [Ca2+]i at both temperatures. As with BK, the peak [Ca2+]i reached after thapsigargin treatment was higher at 22 degrees C. This effect of lower temperature on [Ca2+]i was most probably due to decreased Ca2+ efflux after a decrease in activity of the Ca(2+)-ATPase on the plasma membrane. Both A23187 and BK were shown to stimulate phospholipase A2 and arachidonic acid release at 22 degrees C. In each case, the rate and extent of release were decreased compared with that at 37 degrees C. Among several effects, lowering the temperature decreases the activity of phospholipase C, Ca(2+)-ATPase(s), Ca(2+)-entry mechanisms and phospholipase A2. Together, these effects lead to a higher and more prolonged elevation of [Ca2+]i, but a decrease in arachidonate release in response to BK.

PGE2 release by bradykinin in human airway smooth muscle cells: involvement of cyclooxygenase-2 induction

1997 ◽  
Vol 273 (6) ◽  
pp. L1132-L1140 ◽  
Author(s):  
Linhua Pang ◽  
Alan J. Knox
Keyword(s):  
Arachidonic Acid ◽  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Arachidonic Acid Release ◽  
Human Airway Smooth Muscle ◽  
Short Term ◽  
Human Airway ◽  
Acid Release ◽  
Cox 2

Prostanoids may be involved in bradykinin (BK)-induced bronchoconstriction in asthma. We investigated whether cyclooxygenase (COX)-2 induction was involved in prostaglandin (PG) E2 release by BK in cultured human airway smooth muscle (ASM) cells and analyzed the BK receptor subtypes responsible. BK stimulated PGE2release, COX activity, and COX-2 induction in a concentration- and time-dependent manner. It also time dependently enhanced arachidonic acid release. In short-term (15-min) experiments, BK stimulated PGE2 generation but did not increase COX activity or induce COX-2. In long-term (4-h) experiments, BK enhanced PGE2 release and COX activity and induced COX-2. The long-term responses were inhibited by the protein synthesis inhibitors cycloheximide and actinomycin D and the steroid dexamethasone. The effects of BK were mimicked by the B2-receptor agonist [Tyr(Me)8]BK, whereas the B1 agonist des-Arg9-BK was weakly effective at high concentrations. The B2antagonist HOE-140 potently inhibited all the effects, but the B1 antagonist des-Arg9,(Leu8)-BK was inactive. This study is the first to demonstrate that BK can induce COX-2. Conversion of increased arachidonic acid release to PGE2 by COX-1 is mainly involved in the short-term effect, whereas B2 receptor-related COX-2 induction is important in the long-term PGE2 release.

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