Elevated extracellular K+ enhances arachidonic acid release in MDCK-D1 cells

1990 ◽  
Vol 259 (2) ◽  
pp. C224-C231 ◽  
Author(s):  
M. J. Howard ◽  
P. A. Insel
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Mdck Cells ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Membrane Receptors ◽  
Phosphoinositide Hydrolysis ◽  
Ionophore A23187 ◽  
K Concentration ◽  
The Impact

Depolarization can alter the expression of membrane receptors and change certain receptor-mediated events, but previous studies have not assessed the impact of depolarization on generation of arachidonic acid and its metabolites (AA) in nonexcitable tissues. We assessed AA generation in Madin-Darby canine kidney (MDCK) cells grown for 3 days in increased extracellular [K+], which is known to acutely depolarize these cells. Growth under these conditions resulted in decreases in the number of alpha 1-adrenergic receptors (alpha 1 AR), a small decrease in receptor-mediated phosphoinositide hydrolysis, but increases in alpha 1 AR-mediated prostaglandin E2 formation and AA release. Calcium ionophore (A23187)-, melittin-, and bradykinin-stimulated AA release were also enhanced. The reduction in alpha 1 AR number and increased AA release were substantially reduced or eliminated when K(+)-treated cells were grown in the absence of extracellular calcium. The results provide evidence that hormone-stimulated AA release and prostaglandin production can be enhanced by chronic exposure to elevated extracellular K+ concentration, perhaps as a consequence of a depolarization-induced enhancement in phospholipase A2 activity. The results provide evidence for the parallel and independent regulation of the pathways for receptor-mediated phosphoinositide hydrolysis (phospholipase C activation) and AA release (phospholipase A2 activation) in MDCK cells.

scholarly journals Demonstration of calcium-dependent phospholipase A2 activity in membrane preparation of rabbit neutrophils. Absence of activation by fMet-Leu-Phe, phorbol 12-myristate 13-acetate and A-kinase

1988 ◽  
Vol 250 (2) ◽  
pp. 343-348 ◽  
Author(s):  
T Matsumoto ◽  
W Tao ◽  
R I Sha'afi
Keyword(s):  
Arachidonic Acid ◽  
Cyclic Amp ◽  
Phospholipase A2 ◽  
Kinase Inhibitor ◽  
Calcium Ionophore ◽  
Membrane Preparation ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Pla2 Activity ◽  
Intact Cells

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.

Reduced prostacyclin formation after reoxygenation of anoxic endothelium

1990 ◽  
Vol 259 (5) ◽  
pp. C738-C745 ◽  
Author(s):  
S. L. Hempel ◽  
D. L. Haycraft ◽  
J. C. Hoak ◽  
A. A. Spector
Keyword(s):  
Arachidonic Acid ◽  
Umbilical Vein ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
High Rate ◽  
Ionophore A23187 ◽  
Arachidonic Acid Release ◽  
Cyclooxygenase Activity ◽  
Platelet Adherence ◽  
Acid Release

Human umbilical vein endothelial cells subjected to 24 h of anoxia followed by reoxygenation released less prostacyclin (PGI2) in response to thrombin, calcium ionophore A23187, or arachidonic acid. This was associated with a substantial increase in stimulated platelet adherence. Increased lactate dehydrogenase and 51Cr release occurred after 1 h of reoxygenation, but the high rate of release did not persist during the subsequent 23 h of reoxygenation. The changes in platelet adherence and PGI2 release partially resolved over 24 h. PGI2 formation from prostaglandin H2 was not reduced, suggesting that cyclooxygenase activity, but not prostacyclin synthase, is affected by reoxygenation. A decrease in arachidonic acid release from cellular lipids also occurred. The reduction in cyclooxygenase activity, but not arachidonic acid release, was prevented by the presence of ibuprofen during reoxygenation. Addition of catalase or superoxide dismutase during reoxygenation increased PGI2 release but did not completely overcome the reduction relative to control cultures. These findings suggest that the increase in platelet adherence during reoxygenation may be mediated in part by a change in cyclooxygenase activity. This is only partly overcome by extracellular oxygen species scavengers but is prevented by the presence of a reversible cyclooxygenase inhibitor during reoxygenation.

Transepithelial prostacyclin gradient in isolated canine trachea

1989 ◽  
Vol 67 (1) ◽  
pp. 276-281 ◽  
Author(s):  
D. H. Eidelman ◽  
W. S. Powell ◽  
S. Bellofiore ◽  
J. G. Martin
Keyword(s):  
Arachidonic Acid ◽  
Airway Epithelium ◽  
High Performance ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Ussing Chambers ◽  
Alpha Production ◽  
Acid Potential ◽  
Mucosal Side

Cyclooxygenase products of arachidonic acid, potential modulators of airway smooth muscle, have recently been described in bronchoalveolar lavage from canine lungs. To evaluate the possibility that airway epithelium represents a barrier to movement of prostacyclin (PGI2), an important bronchodilator synthesized by isolated airway, we measured the concentrations of 6-oxoprostaglandin F1 alpha (6-oxo-PGF1 alpha), the stable degradation product of PGI2, on the mucosal and serosal sides of isolated canine tracheal segments (CTS) mounted in Ussing chambers. 6-oxo-PGF1 alpha was measured by radioimmunoassay after purification by high-performance liquid chromatography. The concentration of 6-oxo-PGF1 alpha was significantly higher on the serosal than the mucosal side of CTS (1,262 +/- 252 vs. 390 +/- 168 pg.min-1.g-1, n = 8, P less than 0.05). A significant correlation was present between 6-oxo-PGF1 alpha measured on both sides of each CTS (r = 0.778, n = 26, P less than 0.01). 6-oxo-PGF1 alpha production from CTS stripped of mucosa was significantly greater than from isolated mucosa. Radiochromatograms obtained after incubation with [3H]arachidonic acid and calcium ionophore A23187 confirmed PGI2 as the predominant cyclooxygenase product of the submucosa, whereas the mucosa produced only small amounts of PGI2 in proportion to other cyclooxygenase products. PGI2 (10(-8) to 10(-6) M) applied to the mucosal surface of closed tracheal segments precontracted with histamine resulted in no significant relaxation, whereas serosal application showed a concentration-dependent effect. Radiolabeled 6-oxo-PGF1 alpha did not cross the isolated epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Leukotriene generation by eosinophils.

1982 ◽  
Vol 155 (2) ◽  
pp. 390-402 ◽  
Author(s):  
A Jörg ◽  
W R Henderson ◽  
R C Murphy ◽  
S J Klebanoff
Keyword(s):  
Arachidonic Acid ◽  
Calcium Ionophore ◽  
Leukotriene B4 ◽  
Arachidonic Acid Metabolism ◽  
Calcium Ionophore A23187 ◽  
Eicosatetraenoic Acid ◽  
Gas Chromatography Mass Spectrometry ◽  
Ionophore A23187 ◽  
Lipoxygenase Pathway ◽  
Leukotriene D4

Horse eosinophils purified to greater than 98% generated slow reacting substance (SRS) when incubated with the calcium ionophore A23187. On a per cell basis, eosinophils generated four to five times the SRS produced by similarly treated horse neutrophils. Eosinophil SRS production was inhibited by 5,8,11,14-eicosatetraynoic acid and augmented by indomethacin and arachidonic acid, suggesting that it was a product(s) of the lipoxygenase pathway of arachidonic acid metabolism. Compounds with SRS activity were purified by high-pressure liquid chromatography (HPLC) and identified by ultraviolet spectra, spectral shift on treatment with lipoxygenase, incorporation of [14C]arachidonic acid, gas chromatography-mass spectrometry, and comparison of retention times on HPLC to authentic standards. The eosinophil products characterized were 5-(S), 12-(R)-dihydroxy-6-cis-8, 10-trans-14-cis-eicosatetraenoic acid (leukotriene B4) and its 5-(S), 12-(R)-6-trans and 5-(S), 12-(S)-6-trans isomers, 5-(S)-hydroxy-6-(R)-S-glutathionyl-7,9-trans-11, 14-cis-eicosatetraenoic acid (leukotriene C4) and its 11-trans isomer, and 5-(S)-hydroxy-6-(R)-S-cysteinylglycine-7,9-trans-11,14-cis-eicosatetraenoic acid (leukotriene D4).

Effects of prostaglandin F2α and other potential secretagogues on oxytocin secretion and second messenger metabolism in the ovine corpus luteum in vitro

1990 ◽  
Vol 126 (1) ◽  
pp. 89-98 ◽  
Author(s):  
T. J. McCann ◽  
A. P. F. Flint
Keyword(s):  
Arachidonic Acid ◽  
Lactate Dehydrogenase ◽  
Calcium Ionophore ◽  
Prostaglandin F2α ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Lactate Dehydrogenase Release ◽  
Intracellular Ca

ABSTRACT Release of oxytocin by sliced or minced sheep luteal tissue in vitro was stimulated up to 1·6- and 2·3-fold by arachidonic acid and the calcium ionophore A23187 respectively. Prostaglandin (PG) F2α and the PGF2α analogue cloprostenol, and other potential agonists known to be active in vivo, including noradrenaline and acetylcholine, were ineffective, as was the phorbol ester tetradecanoylphorbol acetate (TPA). The ineffectiveness of PGF2α was not due to a general unresponsiveness of the tissue in vitro, as PGF2α reduced LH stimulation of tissue concentrations of cyclic AMP and activated inositol lipid hydrolysis. The effect of arachidonic acid was accompanied by release from the tissue of the cytosolic enzyme lactate dehydrogenase (at arachidonic acid concentrations below those required to release oxytocin) and its effect on oxytocin and lactate dehydrogenase release was mimicked by oleic and linolenic acids; arachidonic acid was concluded to act by a non-physiological physicochemical effect without conversion to an eicosanoid. As PGF2α in vitro is known to raise intracellular Ca2+ concentrations in the large luteal cells that secrete oxytocin, and as A23187 stimulates oxytocin release in vitro in the presence and absence of TPA, it is concluded that in-vitro incubation results in an artifactual blockade of the oxytocin-releasing action of PGF2α at an unidentified point distal to the effect on intracellular Ca2+. Journal of Endocrinology (1990) 126, 89–98

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