scholarly journals Human pulmonary macrophage-derived mucus secretagogue.

1984 ◽  
Vol 159 (3) ◽  
pp. 844-860 ◽  
Author(s):  
Z Marom ◽  
J H Shelhamer ◽  
M Kaliner
Keyword(s):  
Arachidonic Acid ◽  
Gel Filtration ◽  
Receptor Activation ◽  
Surface Activation ◽  
Sequential Fractionation ◽  
Human Lung Tissue ◽  
Lipoxygenase Inhibitors ◽  
Dose Dependent Fashion ◽  
Pulmonary Macrophages ◽  
Human Airways

Human pulmonary macrophages (PM) obtained from surgically removed human lung tissue released a factor after exposure to activated zymosan that caused cultured human airways to release increased amounts of radiolabeled mucous glycoproteins. The factor was released maximally after 4-8 h of zymosan exposure and caused a dose-related increase in glycoprotein release; it was termed macrophage-derived mucus secretagogue (MMS). MMS release was produced in a dose-dependent fashion by activated but not by nonactivated zymosan. The activation of zymosan was C3 dependent, and C3b-coated Sepharose was also an effective stimulant. The data suggested that cell surface activation of the PM was a sufficient stimulus to cause MMS release and that both C3-dependent activation as well as Fc receptor activation were effective. The synthesis of MMS was sensitive to cycloheximide, and no active MMS was detectable intracellularly. To determine if MMS might be one of the oxidative derivatives of arachidonic acid, PM were incubated with cyclooxygenase and lipoxygenase inhibitors before activation. These maneuvers did not influence MMS generation. MMS-rich supernatants were then extracted into organic solvents or exposed to lipophilic resin; in both cases, MMS remained in the aqueous phase. Thus, MMS is not a derivative of arachidonic acid. Sequential fractionation of MMS on ultramembrane and gel filtration followed by isoelectric focusing and gel filtration indicated that MMS is a small (approximately 2000 daltons), acidic (pI, 5.15) molecule. Therefore, surface activation of human PM results in the synthesis and release of a small acidic molecule that causes airway mucous glands to secrete increased quantities of mucous glycoproteins.

Decreased Platelet Vitamin C in Diabetes Mellitus; Possible Role in Peraggregatoon

1979 ◽  
Author(s):  
K.E. Sarji ◽  
J. Gonzalez ◽  
H. Hempling ◽  
J.A. Colwell
Keyword(s):  
Ascorbic Acid ◽  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Vitamin C ◽  
Platelet Rich Plasma ◽  
Solution Ph ◽  
Dose Dependent Fashion ◽  
Insulin Dependent Diabetic ◽  
Induced Aggregation

To determine whether Vitamin C might relate to the increased platelet sensitivity in the diabetic, we have measured levels of platelet Vitamin C and studied the effects of Vitamin C on platelet aggregation. Ascorbic acid levels in washed platelets from diabetics were significantly lower than from normals (4s.2±3 μg/1010 platelets vs. 2s.s±2 μg/1010 platelets, p<.001). The effects of ascorbic acid on platelet aggregation in vitro were studied by adding ascorbic acid in buffered solution (pH 7.35) prior to-aggregating agents. Ascorbic acid in platelet-rich plasma consistently inhibited platelet aggregation with threshold concentrations of ADP, epinephrine, and collagen. With washed platelets, ascorbic acid inhibited arachidonic, acid-induced aggregation. When platelets were incubated at 37°C for 10 minutes with varying concentrations of ascorbic acid, rewashed, and aggregation with arachidonic acid tested, aggregation was inhibited in a linear dose-dependent fashion. Oral ingestion of ascorbic acid (2 gm/day) for seven days by normal non-smoking males produced a marked inhibition of aggregation. In a similar study, platelets from an insulin-dependent diabetic showed no change in aggregation. These results suggest that platelet levels of ascorbic acid may relate to the hyperaggregat ion of platelets from diabetics.

Arachidonic acid enhances contraction and intracellular Ca2+ transients in individual rat ventricular myocytes

1997 ◽  
Vol 272 (1) ◽  
pp. H350-H359 ◽  
Author(s):  
D. S. Damron ◽  
B. A. Summers
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cardiac Muscle ◽  
Ventricular Myocytes ◽  
Contractile Function ◽  
Receptor Activation ◽  
K Channels ◽  
Rat Ventricular Myocytes ◽  
Kinase C

Modulation of intracellular free Ca2+ concentration ([Ca2+]i) by inotropic stimuli alters contractility in cardiac muscle. Arachidonic acid (AA), a precursor for eicosanoid formation, is released in response to receptor activation and myocardial ischemia and has been demonstrated to alter K+ and Ca2+ channel activity. We investigated the effects of AA on contractility by simultaneously measuring [Ca2+]i and shortening in single field-stimulated rat ventricular myocytes. [Ca2+]i transients were measured using fura 2, and myocyte shortening was assessed using video edge detection. AA stimulated a doubling in the amplitude of the [Ca2+]i transient and a twofold increase in myocyte shortening. In addition, AA stimulated a 30% increase in the time to 50% diastolic [Ca2+]i and a 35% increase in the time to 50% relengthening. These effects of AA were mediated by AA itself (56 +/- 5%) and by cyclooxygenase metabolites. Pretreatment with the protein kinase C inhibitors staurosporine and chelerythrine nearly abolished (> 90% inhibition) these AA-induced effects. Inhibition of voltagegated K+ channels with 4-aminopyridine mimicked the effects of AA. Addition of AA to the 4-aminopyridine-treated myocyte had no additional effect on parameters of contractile function. These data indicate that AA alters the amplitude and duration of Ca2- transients and myocyte shortening via protein kinase C-dependent inhibition of voltage-gated K+ channels. Release of AA by phospholipases in response to receptor activation by endogenous mediators or pathological stimuli may be involved in mediating inotropic responses in cardiac muscle.

Aspirin Preferentially Inhibits Arachidonic Acid Metabolism In Collagen Vs. Thrombin-Stimulated Platelets

1981 ◽  
Author(s):  
D Deykin ◽  
R Vaillancourt
Keyword(s):  
High Performance Liquid Chromatography ◽  
Arachidonic Acid ◽  
Liquid Chromatography ◽  
High Performance ◽  
Acid Metabolism ◽  
Gel Filtration ◽  
Human Platelets ◽  
Arachidonic Acid Metabolism ◽  
Selective Effect ◽  
Oxygenase Activity

The purpose of this study was to compare the effect of aspirin on the release of metabolites of arachidonic acid from thrombin and collagen stimulated platelets. Human platelets were incubated with tritium-labeled arachidonic acid and then isolated by gel filtration. The labeled platelets were stimulated with varied doses of either thrombin or collagen for 15 minutes. The platelets were then pelleted and the released metabolites of arachidonic acid were separated by high-performance liquid chromatography. In experiments with aspirin, the aspirin was added 5 minutes before either thrombin or collagen. The total release of radioactivity was comparable at 15 μg/ml of collagen and 1.0 units/ml of thrombin (approximately 10% of the total) and at 100 μg/ml of collagen and 5 units/ml of thrombin (approximately 30%). Aspirin (25 μg/ml) preferentially inhibited collagen-stimulated release of radioactivity (62% inhibition of release with 15 μg/ml of collagen vs. 25% inhibition of release with 1.0 units/ml of thrombin; 54% inhibition of release with 100 μg/ml of collagen vs. 8% inhibition of release with 5.0 units/ml of thrombin). At all concentrations of collagen or thrombin, cyclo-oxygenase activity was markedly reduced by aspirin. The selective effect of aspirin on collagen reflects primarily preferential suppression of HETE formation. We conclude that aspirin inhibits the formation of both lipoxygenase and cyclooxygenase-derived products in collagen-stimulated platelets.

Inhibition Of Platelet Aggregation, Malonyldialdehyde And Thromboxane Formation By Hydroperoxides Of Arachidonic Acid

1981 ◽  
Author(s):  
D Aharonv ◽  
J B Smith ◽  
M J Silver
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Lipoxygenase Pathway ◽  
Dose Dependent Fashion ◽  
Induced Aggregation ◽  
Layer Chromatography ◽  
Dose Dependent ◽  
Thromboxane Formation

The arachidonate hydroperoxides 12-HPETE and 15-HPETE were biosynthesized from arachidonic acid using partially purified human platelet lipoxygenase or soybean lipoxidase respectively, and isolated by thin layer chromatography. Both compounds inhibited the arachidonic acid- induced aggregation of washed human platelets, suspended in calcium-free Krebs Henseleit solution, in a dose dependent fashion at concentrations between 1 and 50 uM. No inhibition was seen with up to 100 uM of these hydroperoxides when platelet -rich plasma was used. 12-HPETE (in micromolar concentrations) inhibited the formation of both thromboxane B2 (radioimmunoassay) and malonyldialdehyde (spectrophotometrie assay) when washed platelets were incubated with arachidonic acid. The 12-hydroxide, 12-HETE also inhibited platelet aggregation and thromboxane formation, but was less potent than 12-HPETE. We suggest that arachidonate hydroperoxide generated in platelets via the lipoxygenase pathway modulates platelet aggregation induced by arachidonic acid by inhibiting thromboxane formation.

scholarly journals A New Pathway for Arachidonic Acid Metabolism in Human Platelets

1977 ◽  
Author(s):  
S. Rittenhouse-Simmons ◽  
F. A. Russell ◽  
D. Deykin
Keyword(s):  
Arachidonic Acid ◽  
Acid Metabolism ◽  
De Novo ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Kinetic Studies ◽  
Arachidonic Acid Metabolism ◽  
Biologically Active ◽  
Resting Cells ◽  
Active Metabolites

We are reporting a novel pathway of arachidonic acid metabolism in the phosphatides of thrombin-activated platelets. For kinetic studies of arachidonic acid turnover, platelet phosphatides were labeled by incubation of platelet rich plasma with (3H)-arachidonic acid for 15 min. Unincorporated isotope was removed during subsequent gel-filtration. Platelet phosphatides were resolved and quantitated following two-dimensional silica paper chromatography of chloroform/methanol extracts of incubated platelets. Plasmalogen phosphatidylethanolamine (PPE) was examined on paper chromatograms after its breakdown to lysoPPE with HgCl2. In other experiments, gel-filtered platelets were incubated with (14C)-glycerol to monitor de novo phosphatide synthesis. (3H)-Arachidonic acid was released from phosphatidylcholine and phosphatidylinositol of pre-labeled platelets exposed to thrombin and appeared increasingly in PPE in acyl linkage at glycerol-C-2. (3H)-Arachidonic acid was not found in PPE of resting cells. Maximum transfer occurred with 5 U/ml of thrombin and 15 min, of incubation, with t½ of 2½ min., and was Ca+2 dependent. The presence of aspirin, indomethacin, or eicosatetraynoic acid did not prevent the thrombin-activated transfer of (3H)-arachidonic acid to PPE. The stimulated incorporation of (3H)-arachidonic acid into PPE was not accompanied by a stimulation of (14C)-glycerol uptake into this phosphatide. We suggest that perturbation of the platelet may activate a phospholipase A2 leading to turnover of arachidonic acid in PPE, which is rich in this fatty acid. Such turnover may provide substrate for conversion by cyclo-oxygenase and lipoxydase to biologically active metabolites, and therefore, may offer a locus for regulation of prostaglandin synthesis in the human platelet.

scholarly journals Orientation of palmitoylated CaVβ2a relative to CaV2.2 is critical for slow pathway modulation of N-type Ca2+ current by tachykinin receptor activation

2009 ◽  
Vol 134 (5) ◽  
pp. 385-396 ◽  
Author(s):  
Tora Mitra-Ganguli ◽  
Iuliia Vitko ◽  
Edward Perez-Reyes ◽  
Ann R. Rittenhouse
Keyword(s):  
Amino Acids ◽  
Arachidonic Acid ◽  
Palmitic Acid ◽  
Receptor Activation ◽  
Slow Pathway ◽  
Channel Activity ◽  
Tachykinin Receptor ◽  
Previous Hypothesis ◽  
Inhibitory Site ◽  
Neurokinin 1

The Gq-coupled tachykinin receptor (neurokinin-1 receptor [NK-1R]) modulates N-type Ca2+ channel (CaV2.2 or N channel) activity at two distinct sites by a pathway involving a lipid metabolite, most likely arachidonic acid (AA). In another study published in this issue (Heneghan et al. 2009. J. Gen Physiol. doi:10.1085/jgp.200910203), we found that the form of modulation observed depends on which CaVβ is coexpressed with CaV2.2. When palmitoylated CaVβ2a is coexpressed, activation of NK-1Rs by substance P (SP) enhances N current. In contrast, when CaVβ3 is coexpressed, SP inhibits N current. However, exogenously applied palmitic acid minimizes this inhibition. These findings suggested that the palmitoyl groups of CaVβ2a may occupy an inhibitory site on CaV2.2 or prevent AA from interacting with that site, thereby minimizing inhibition. If so, changing the orientation of CaVβ2a relative to CaV2.2 may displace the palmitoyl groups and prevent them from antagonizing AA's actions, thereby allowing inhibition even in the presence of CaVβ2a. In this study, we tested this hypothesis by deleting one (Bdel1) or two (Bdel2) amino acids proximal to the α interacting domain (AID) of CaV2.2's I–II linker. CaVβs bind tightly to the AID, whereas the rigid region proximal to the AID is thought to couple CaVβ's movements to CaV2.2 gating. Although Bdel1/β2a currents exhibited more variable enhancement by SP, Bdel2/β2a current enhancement was lost at all voltages. Instead, inhibition was observed that matched the profile of N-current inhibition from CaV2.2 coexpressed with CaVβ3. Moreover, adding back exogenous palmitic acid minimized inhibition of Bdel2/β2a currents, suggesting that when palmitoylated CaVβ2a is sufficiently displaced, endogenously released AA can bind to the inhibitory site. These findings support our previous hypothesis that CaVβ2a's palmitoyl groups directly interact with an inhibitory site on CaV2.2 to block N-current inhibition by SP.

scholarly journals Effects of calmodulin and lipoxygenase inhibitors on LH (lutropin)- and LHRH (luliberin)-agonist-stimulated steroidogenesis in rat Leydig cells

1985 ◽  
Vol 232 (1) ◽  
pp. 55-59 ◽  
Author(s):  
M H Sullivan ◽  
B A Cooke
Keyword(s):  
Arachidonic Acid ◽  
Leydig Cells ◽  
Acid Metabolism ◽  
Arachidonic Acid Metabolism ◽  
Ionophore A23187 ◽  
Lhrh Agonist ◽  
Lipoxygenase Pathway ◽  
Lipoxygenase Inhibitors ◽  
Testosterone Production

The results of this study, carried out with purified rat Leydig cells, indicate that there are no major differences in the stimulating effects of lutropin (LH) and luliberin (LHRH) agonists on steroidogenesis via mechanisms that are dependent on Ca2+. This was demonstrated by using inhibitors of calmodulin and the lipoxygenase pathways of arachidonic acid metabolism. All three calmodulin inhibitors used (calmidazolium, trifluoperazine and chlorpromazine) were shown to block LH- and LHRH-agonist-stimulated steroidogenesis. This probably occurred at the step of cholesterol transport to the mitochondria. Similarly, three lipoxygenase inhibitors (nordihydroguaiaretic acid, BW755c and benoxaprofen), inhibited both LH- and LHRH-agonist-stimulated steroidogenesis. The amounts of the inhibitors required were similar for LH- and LHRH-agonist-stimulated steroidogenesis. Steroidogenesis stimulated by the Ca2+ ionophore A23187 was also inhibited, but higher concentrations of the inhibitors were required. Indomethacin (a cyclo-oxygenase inhibitor) increased LHRH-agonist-stimulated steroidogenesis;this is consistent with the role of the products of arachidonic acid metabolism via the alternative, lipoxygenase, pathway. The potentiation of LH-stimulated testosterone production by LHRH agonist was unaffected by indomethacin or by lipoxygenase inhibitors at concentrations that inhibited LH-stimulated testosterone production by 75-100%. It was not possible to eliminate a role of calmodulin in modulating the potentiation, although higher concentrations of the inhibitors were generally required to negate the potentiation than to inhibit LH- or LHRH-agonist-stimulated testosterone production.

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