Role of arachidonic acid in the regulation of adrenocorticotropin release from rat anterior pituitary cell cultures

In addition to cAMP-dependent mechanisms, stimulation of pituitary ACTH secretion by various stimuli, including CRF, may involve phospholipid and arachidonic acid turnover. To determine the role of phospholipase A2 activation in corticotroph function, we studied the effect of exogenous arachidonic acid, phospholipase A2, and the phospholipase A2 activator melittin on ACTH release in cultured rat anterior pituitary cells. Incubation with 1-100 micron arachidonic acid, 0.01-1 micron melittin, 0.1-10 U/ml phospholipase A2, and 0.01-10 nM CRF caused dose-dependent increases in ACTH release to 8.1 +/- 1.1- (+/- SE), 16.2 +/- 0.9-, 13.6 +/- 1.2-, and 2.9 +/- 0.3-fold; respectively. The participation of the major pathways of arachidonic acid metabolism in the control of ACTH release was analyzed in cells treated with nordihydroguaiaretic acid, a lipoxygenase inhibitor; indomethacin, a cycloxygenase inhibitor; and 5,8,11,14-eicosatetraynoic acid, an inhibitor of both pathways. The effects of arachidonic acid, melittin, and CRF were partially blocked by 10 micron nordihydroguaiaretic acid and 5,8,11,14-eicosatetraynoic acid, but were significantly enhanced by 10 micron indomethacin. These results suggest that arachidonic acid is mainly metabolized through the lipoxygenase pathway to a stimulatory metabolite and, to a lesser extent, through the cycloxygenase pathway to an inhibitory metabolite. Arachidonic acid release from anterior pituitary cells labeled with [3H]arachidonic was analyzed during cell column perifusion and stimulation by CRF and other secretagogues. Two-minute pulses of CRF (10 nM), vasopressin (10 nM) and phorbol 12-myristate 13-acetate (100 nM) caused immediate 1.5- to 2-fold increases in [3H]arachidonic acid release, and melittin (100 nM) caused a 5-fold increase in [3H]arachidonic acid release. The ability of both exogenously added and endogenously generated arachidonic acid to stimulate ACTH secretion, together with the stimulation of arachidonic acid release by ACTH secretagogues and the attenuation of stimulated ACTH release by lipoxygenase blockers, indicate that lipoxygenase products of arachidonic acid metabolism participate in the control of ACTH secretion.     

Norepinephrine and thyrotropin stimulation of iodide efflux in FRTL-5 thyroid cells involves metabolites of arachidonic acid and is associated with the iodination of thyroglobulin

Ca2+-dependent and TSH-, norepinephrine (NE)-, and A23187-induced iodide (I-) efflux from FRTL-5 rat thyroid cells is inhibited by quinacrine and trifluoroperazine, agents that inhibit phospholipase A2 activity. Furthermore, I- efflux can be stimulated by an activator of phospholipase A2 activity, melittin. Phospholipase A2 action releases arachidonic acid from phospholipids; arachidonic acid enhances I- efflux in FRTL-5 cells. Inhibitors of arachidonic acid metabolism via the lipoxygenase pathway, 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid, and via the cytochrome P450-linked epoxygenase pathway, piperonyl butoxide and 2-diethylaminoethyl-2,2-diphenyl valerate, but not an inhibitor of the cyclooxygenase pathway, indomethacin, can inhibit TSH-, NE-, and A23187-induced I- efflux. TSH, NE, and arachidonic acid stimulation of I- efflux in FRTL-5 cells is associated with increased iodination of thyroglobulin, which is blocked by 10 microM 5,8,11,14-eicosatetraynoic acid and 50 microM piperonyl butoxide. The data thus suggest that TSH- and NE-induced I- efflux from FRTL-5 thyroid cells involves lipoxygenase and/or epoxygenase metabolites of arachidonic acid, released from phospholipids upon Ca2+-dependent activation of phospholipase A2. Since this process is associated with the iodination of thyroglobulin, TSH- and NE-induced I- efflux in FRTL-5 cells may represent the transport of I- from the cell into the follicular lumen in vivo.     

Neutrophil chemotactic activity generation by alveolar macrophages after bleomycin injury

Saline lavage was performed on rat lungs after the intratracheal injection of saline or bleomycin. An increase (p less than 0.025) in total cells recovered, an increase (p less than 0.001) in neutrophils, and an increase (p less than 0.001) in albumin concentration were noted in lavage fluid recovered from rats subsequent to bleomycin injury. At 5, 10, 15, and 20 days after injury, macrophages recovered from bleomycin-treated rats generated increased (p less than 0.05) amounts of neutrophil chemotactic activity in vitro compared with macrophages recovered from saline-treated rats. The chemotactic activity was attributable to a factor or factors of low molecular weight and hydrophobic in nature, characteristics similar to previously described alveolar macrophage-derived neutrophil chemotactic factors. The generation of neutrophil chemotactic activity was suppressed (p less than 0.025) by hydrocortisone and 5,8,11,14-eicosatetraynoic acid (ETYA), suggesting that the neutrophil chemotactic activity generation is dependent upon the lipoxygenase pathway of arachidonic acid metabolism.     

Somatostatin potentiates the alpha 1-adrenergic activation of phospholipase C in striatal astrocytes through a mechanism involving arachidonic acid and glutamate.

As previously shown with adenosine, somatostatin, which is ineffective alone, enhanced the alpha 1-adrenergic-agonist-stimulated production of inositol phosphates in cultured striatal astrocytes. This effect was suppressed in cells pretreated with pertussis toxin. It required external calcium and was selectively antagonized by both mepacrine, an inhibitor of phospholipase A2, and 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable analog of arachidonic acid. In addition, a long-lasting elevation of cytosolic calcium and a release of arachidonic acid were observed only under the combined stimulation of somatostatin and alpha 1-adrenergic receptors. Arachidonic acid could in turn inhibit glutamate uptake into astrocytes, and the resulting external accumulation of glutamate could account for the somatostatin-evoked amplification of the alpha 1-adrenergic-agonist-stimulated hydrolysis of inositol-phospholipids. The effect of somatostatin was indeed reproduced by glutamate or glutamate uptake inhibitors and suppressed by enzymatic removal of external glutamate. Thus, astrocytes may contribute to long-term plasticity events in glutamatergic synapses through regulation of external glutamate levels.     

Arachidonic acid metabolism in polymorphonuclear leukocytes: Effects of ionophore A23187*

Addition of arachidonic acid and the divalent cation ionophore A23187 to a suspension of human peripheral blood polymorphonuclear leukocytes led to the formation of (5S)-hydroxy-6,8,11,14-icosatetraenoic acid, (15S)-hydroxy-5,8,11,13-icosatetraenoic acid, and (5S,12R)-dihydroxy-6,8,10,14-icosatetraenoic acid. A method based on high-pressure liquid chromatography has been developed for assay of these metabolites. The addition of arachidonic acid to human polymorphonuclear leukocytes always resulted in formation of the isomeric monohydroxy acids. However, cells prepared from blood of different subjects were found to vary with respect to formation of the 5,12-dihydroxy acid. Addition of the ionophore alone strongly stimulated the formation of the 5-monohydroxy acid and more specifically the 5,12-dihydroxy acid from endogenous arachidonic acid. In all experiments performed the formation of the 5-hydroxy acid and the 5,12-dihydroxy acid was maximally stimulated when both arachidonic acid and the ionophore were added to the incubation mixture. Under these conditions, stimulation of 40-fold or more of the formation of both compounds was observed. The data demonstrate that, in addition to causing release of endogenous substrate, the ionophore also activated the enzymatic system involved in the further transformations of arachidonic acid. This finding raises the possibility that this pathway of arachidonic acid metabolism is involved in the biological response (e.g., release of lysosomal enzymes, the slow reacting substance of anaphylaxis, and chemotactic factors) of leukocytes to A23187 and other stimuli.     

Chemoattractant receptor functions in human polymorphonuclear leukocytes are divergently altered by membrane fluidizers.

The chemotactic factor receptor on leukocytes initiates several cellular responses including chemotaxis, lysosomal enzyme secretion, and O2- production. The latter two responses require approximately 10-100 times more chemoattractant than is required for chemotaxis. We determined the effects of membrane fluidizers on the binding characteristics and the functional activities of the oligopeptide fMet-Leu-Phe chemotactic factor receptor on polymorphonuclear leukocytes. Fluidization was induced by aliphatic alcohols and monitored by diphenylhexatriene fluorescence polarization. Low doses of n-butanol (0.25%) and n-pentanol (0.1%) were nontoxic to the leukocytes yet reduced their diphenylhexatriene-induced polarization, indicating increased membrane fluidity. At these doses of alcohols, the affinity of the fMet-Leu-Phe receptor was enhanced from Kd = 25.5 +/- 7.6 nM to Kd = 5.2 +/- 0.9 nM and Kd = 6.0 +/- 0.9 nM, respectively. Chemotaxis was also increased, as indicated by the decrease, by a factor of approximately 1/3 in the ED50 for fMet-Leu-Phe, as well as by a 1.5-fold increase in the maximal distance of migration in the presence of 0.25% butanol or 0.1% pentanol. In contrast to chemotaxis, the alcohols depressed fMet-Leu-Phe stimulation of O2- production by 90% although they had no effect on phorbol 12-myristate 13-acetate-induced O2- production. Secretion of lysozyme was also inhibited. Thus, the affinity of the fMet-Leu-Phe receptor can be modulated by membrane fluidizers. The higher affinity state of the receptor induced by the alcohols is more efficient in transducing chemotactic signals but is deficient in mediating O2- production or secretion. Thus, the transduction mechanisms for the various biological activities of the chemotactic factor receptor are heterogeneous and can be differentially manipulated by membrane fluidizers.     

Stimulation of leukotriene biosynthesis in human blood leukocytes by platelet-derived 12-hydroperoxy-icosatetraenoic acid.

Addition of arachidonic acid to suspensions of human blood leukocytes induces the synthesis of small amounts only of the C-5 lipoxygenase products as demonstrated by HPLC. However, the coincubation of blood platelets with the leukocytes always resulted in an activation of the C-5 lipoxygenase and formation of (5S)-5-hydroxy-6,8,11,14-icosatetraenoic acid, (5S,12S)-5,12-dihydroxy-6,8,10,14-icosatetraenoic acid, and leukotriene B4 from exogenous arachidonic acid. It was found that the activation of arachidonic acid metabolism in leukocytes was caused by a labile compound because the synthesis of the C-5 lipoxygenase products did not occur when platelets were preincubated for 1 min or more with the substrate prior to the addition of the leukocytes. The use of cyclooxygenase inhibitors did not suppress the activation of the leukocytes by the platelets. However, the addition of 5,8,11,14-icosatetraynoic acid, an inhibitor of cyclooxygenase and C-12 and C-15 lipoxygenases, completely suppressed the formation of leukotrienes, although this substance is not an inhibitor of the C-5 lipoxygenase in human leukocytes. This indicated that a product of the C-12 lipoxygenase was likely the mediator of the stimulatory effect of platelets on leukocyte arachidonic acid metabolism. The finding that the direct addition of (12S)-12-hydroperoxy-5,8,10,14-icosatetraenoic acid, but not of the corresponding hydroxy derivative, could activate the leukocyte's C-5 lipoxygenase confirmed this hypothesis. These data demonstrate that an interaction between C-12 and C-5 lipoxygenases can promote the formation of leukotrienes and support the possibility of a cooperation between platelets and leukocytes in inflammation and hypersensitivity reactions. Furthermore, the finding provides a new interest for the platelet C-12 lipoxygenase.     

Substrate autoregulation of glucose transport: hexose 6-phosphate mediates the cellular distribution of glucose transporters

Summary Exposure of rat skeletal muscle and skeletal muscle cell lines to high glucose levels results in a time- and dose-dependent reduction of the rate of hexose uptake, paralleled by a reduction in the plasma membrane density of glucose transporters. The mechanism of this process was investigated in cultured L8 myocytes. Low concentrations (0.5–2.0 mmol/l) of deoxyglucose mimicked the downregulatory action of 20 mmol/l glucose both regarding the time-course and magnitude of the effect, but in an irreversible manner. A dose-dependent relationship between intracellular accumulation of deoxyglucose 6-phosphate and the magnitude of the downregulatory response was observed. Depletion of intracellular deoxyglucose 6-phosphate restored the rate of hexose transport to the control level. The reduction of hexose transport activity by deoxyglucose occurred independently of ATP depletion which by itself produced the opposite effect. The effects of deoxyglucose and high glucose on hexose transport were associated with reduced transport maximal velocity and GLUT1 transporter abundance in the plasma membranes of myocytes, as assessed by cell surface biotinylation. The reduction of myocyte GLUT1 mRNA content, observed after exposure to high glucose, did not accompany the transport downregulatory action of deoxyglucose. We suggest that hexose 6-phosphate is the mediator of the downregulatory signal for subcellular redistribution of GLUT1 in L8 myocytes. The signal responsible for reducing the GLUT1 mRNA level may be related to glucose metabolites downstream of the hexokinase reaction. [Diabetologia (1997) 40: 30–39] Received: 1 August 1996 and in revised form: 17 October 1996     

Stimulation of cyclic AMP accumulation by arachidonic and 8, 11, 14-eicosatrienoic acids in rat anterior pituitary gland.

The effects of fatty acids on adenosine 3',5'-monophosphate (cAMP) accumulation in rat anterior pituitary gland were studied in vitro. Of all the fatty acids tested, namely oleic, lifiolenic, γ-linolenic, cis-5-eicosaenoic, cis-8, 11, 14-eicosatrienoic, cis-11, 14, 17-eicosatrienoic, arachidonic, and cis-5, 8, 11, 14, 17-pentaenoic, only the direct precursors of prostaglandin e₁ (cis-8, 11, 14-eicosatrienoic acid) and prostaglandin E₂ (arachidonic acid) increased intracellular cAMP levels. Stimulation was apparent within l min of incubation and reached a maximum at 15 min. At longer time intervals, the concentration of cAMP decreased to basal level. Calcium is not essential for the action of fatty acids while theophylline does not result in any qualitative change. It is concluded that fatty acids stimulate adenylate cyclase activity in rat anterior pituitary tissue and that prostaglandin synthesis is probably involved as mediator of their action.     

Arachidonate metabolism by human polymorphonuclear leukocytes stimulated by N-formyl-Met-Leu-Phe or complement component C5a is independent of phospholipase activation.

Release of arachidonic acid by the membrane phospholipase and metabolism by the 5-lipoxygenase pathway was examined in human polymorphonuclear leukocytes (PMNs). The 5-lipoxygenase pathway is activated when PMNs are given arachidonic acid in ethanol and there is extensive metabolism to 5-hydroxyicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4). This activation event was shown to be altered by the ethanol because resting PMNs given arachidonic acid with bovine serum albumin fail to metabolize arachidonic acid. However, cells activated by the inflammatory agents N-formyl-Met-Leu-Phe (fMLF) or complement component C5a recruit the 5-lipoxygenase to metabolize exogenous arachidonic acid to 5-HETE and LTB4. When PMNs were incubated with arachidonic acid-bovine serum albumin and challenged with fMLF or C5a (des-Arg-C5a) they produced 49-75 pmol of LTB4 and 310-440 pmol of 5-HETE per 10(7) cells. PMNs stimulated by fMLF or C5a (des-Arg-C5a) do not induce membrane phospholipases to mobilize endogenous arachidonic acid and neither 5-HETE nor LTB4 is formed. In contrast, PMN stimulation by the ionophore A23187 activates both the membrane phospholipase and the 5-lipoxygenase to produce 5-HETE and LTB4 from endogenous arachidonic acid. Our results indicate that the lipoxygenase pathway is inoperative in resting PMNs but can be recruited by chemotactic factors to act on arachidonate from extracellular sources. It was previously believed that formation of 5-HETE and LTB4 by the PMN depends solely on phospholipase to mobilize endogenous arachidonic acid. The results reported here refute this concept and indicate that the role of phospholipase activation in PMN may be overestimated. Therefore, subsequent involvement of lipoxygenase products in mediating stimulation of PMN by inflammatory factors (e.g., as in aggregation and chemotaxis) remains in question unless an exogenous source of arachidonate can be identified.     

Arachidonic acid metabolism and the adhesion of human polymorphonuclear leukocytes to cultured vascular endothelial cells

Polymorphonuclear leukocytes (PMN) adhere to the vascular endothelial lining in vivo and to the surfaces of cultured endothelial cells in vitro, but the mechanisms of these cellular interactions remain unclear. Arachidonic acid metabolites, both cyclooxygenase- and lipoxygenase-derived, have been shown to influence PMN locomotion, secretion, and adhesion to artificial surfaces. To determine whether such mediators also are involved in regulating PMN-endothelial cell interactions, we have examined the effects of prostacyclin and various inhibitors of arachidonic acid metabolism on the adherence of radiolabeled PMN to cultured bovine aortic endothelial cells. Confluent endothelial monolayers were incubated with washed suspensions of radiolabeled human PMN (which contained less than 1% platelet contamination) at 37 degrees C for 30 min, then subjected to a standardized wash procedure and the number of adherent leukocytes determined radiometrically. Under basal conditions, i.e., in the absence of exogenous activating stimuli, 4,163 +/- 545 PMN adhered per square millimeter of endothelial surface (mean +/- SEM, n = 12). This basal adhesion (which corresponds to approximately 4-5 leukocytes per endothelial cell) was unaffected when the leukocytes and endothelial monolayers were pretreated with cyclooxygenase inhibitors (100 microM aspirin or 1-5 microM indomethacin) or PGI2 (10(-9)-10(6) M). Thus, basal PMN-endothelial adhesion in this in vitro model system does not appear to be dependent on endogenous cyclooxygenase derivatives of arachidonate or to be sensitive to inhibition by exogenous prostacyclin. In contrast, leukocyte adhesion was significantly reduced by pretreatment with 5,8,11,14- or 4,7,10,13-eicosatetraynoic acid, 0.5- 5 mM sodium salicylate, or 10-1,000 microM indomethacin, antiinflammatory agents that can interfere with the metabolism of arachidonic acid via non-cyclooxygenase-dependent mechanisms. These observations may be relevant to the interactions of circulating PMN with vascular endothelium under both physiologic and pathophysiologic conditions in vivo.     

Stimulation of sugar uptake and glycolysis in chicken embryo fibroblasts by the major glycoprotein from avian myeloblastosis virus.

Addition of purified major glycoprotein from avian myeloblastosis virus to growing or quiescent chicken embryo fibroblasts rapidly stimulates the rate of hexose transport and increases the lactic acid production. These stimulatory effects are dependent on the time of exposure and the dose of viral glycoprotein. In contrast, the glycoprotein only marginally affects hexose transport in chicken cells transformed by Rous sarcoma virus. Some effects of the glycoprotein on serum-starved quiescent cells were similar to those observed upon re-addition of serum; however, the viral glycoprotein did not stimulate DNA synthesis. Quiescent cells stimulated by saturating levels of serum showed little further stimulation of hexose uptake upon exposure to viral glycoprotein for 3 hr. This behavior suggests that the glycoprotein may be acting on a system that is also a target for serum action.     

Specific receptor sites for chemotactic peptides on human polymorphonuclear leukocytes.

Synthetic N-formylmethionyl peptides are chemotactic attractants for human polymorphonuclear leukocytes. The well-defined structure-activity relationship of these peptides in eliciting a chemotactic response suggests that the interaction of the peptides with a specific cellular binding site may initiate chemotaxis. By using tritiated N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-[3H]Phe), a potent chemotactic peptide with high specific radioactivity, we have directly identified binding sites on human polymorphonuclear leukocytes. Binding of fMet-Leu-[3H]Phe to polymorphonuclear leukocytes is rapid (t1/2 less than 2 min) and reversible. The equilibrium dissociation constant (KD) for the interaction of fMet-Leu-[3H-A1Phe with the binding site is 12-14 nM at 37 degrees. The number of binding sites is approximately 2000 per cell. The specificity of the binding sites for a series of N-formylmethionyl peptides exactly reflects the specificity of the chemotactic response to the peptides in that they compete for the binding sites and initiate chemotaxis with the same order of potency (fMet-Leu-Phe greater than fMet-Met-Met greater than fMet-Phe greater than fMet-Leu greater than fMet),fPhe-Met is a competitive antagonist of the chemotactic activity of N-formylmethionyl peptides and has a calculated KD of 6x10-5 M. FPhe-Met also half-maximally inhibits binding of fMet-Leu[3H]Phe binding was the highest in polymorphonuclear leukocytes. No binding of fMet-Leu-[3H]Phe to human erythrocytes could be detected. These data indicate that fMet-Leu-[3H]Phe can be used to identify binding sites for chemotactic peptides on human polymorphonuclear leukocytes. It is likely that these binding sites initiate the specific response of motile cells to N-formylmethionyl peptides.     

Leukocyte-connective tissue cell interaction. II. The specificity,duration, and mechanism of interaction effects

Human fibroblasts grown in vitro as monolayer cultures were “activated” by syngeneic lymphocytes, polymorphonuclear leukocytes, and fibroblasts as well as by allogeneic leukocytes, human embryonic kidney cells, fibroblasts, and thrombocytes. “Activation” was characterized by increased medium acidity, increased glucose uptake, increased lactate formation, and marked stimulation of hyaluronic acid formation. The “activation” process was blocked by preincubation with actinomycin D, but not by incubation with 2,4 dinitrophenol. “Activated” fibroblasts continued to exhibit increased mucopolysaccharide synthesis for 5 to 28 days following a single exposure to stimulatory materials.     

Thiourea causes endothelial cells in tissue culture to produce neutrophil chemoattractant activity

We describe neutrophil chemoattractant activity that is produced by cultured bovine aortic and pulmonary arterial endothelial cells when incubated with thiourea, a substance that causes increased permeability pulmonary edema in animals. The chemoattractant activity was present in culture supernates and cell lysates of endothelial cells incubated with thiourea but was not present in untreated cells. Production of chemoattractant activity was not associated with cell death; viable cell counts and cell homogenate angiotensin converting enzyme levels were not affected, and Cr release was only slightly elevated after incubation with thiourea. At least 1.5 h of incubation with 0.5 mM thiourea was necessary for generation of neutrophil chemoattractant activity. Culture supernates from pulmonary vascular smooth muscle cells and lung fibroblasts did not show increased neutrophil chemoattractant activity after incubation with thiourea. The chemoattractant had both chemokinetic and chemotactic properties, was heat stable, and was extractable into organic solvents. Meclofenamate, a cyclooxygenase inhibitor, minimally inhibited chemoattractant production, whereas 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of both cyclooxygenase and lipoxygenase, completely abolished generation of chemoattractant activity, suggesting that the activity could be a product of arachidonic acid metabolism. These results demonstrate that endothelial cells can produce a substance(s) with neutrophil chemotactic activity. Production of neutrophil chemoattractant activity by endothelial cells could be important in polymorphonuclear leukocyte accumulation at injured vascular sites.     

Possible role of phospholipase A2 action and arachidonic acid metabolism in angiotensin II-mediated aldosterone secretion

When [3H]arachidonic acid-labeled calf adrenal glomerulosa cells are stimulated by angiotensin II (AII), free [3H]arachidonic acid is released. AII treatment significantly decreases radioactivity in phosphatidylinositol but not in other phospholipids. Inhibitors of phospholipase A2 (PL-A2) activity, quinacrine and p-bromophenacyl bromide, inhibit AII-stimulated aldosterone secretion from glomerulosa cells in a dose-dependent manner. The effect of these inhibitors is irreversible when used at high concentration, but not when employed at lower concentration. Exogenous PL-A2 as well as arachidonic acid stimulates both radiocalcium efflux and aldosterone secretion. Unlike AII, stimulation of aldosterone secretion by PL-A2 is only transient. Radiocalcium efflux induced by PL-A2 is greater than that induced by AII and is not inhibited by either nitrendipine or dantrolene. Pretreatment with PL-A2 abolishes the radiocalcium efflux response to subsequent AII, whereas AII pretreatment does not abolish the subsequent PL-A2-mediated radiocalcium efflux response. The aldosterone secretory response to AII is not affected by 0.3 microM indomethacin but is inhibited by either of three compounds which inhibit lipoxygenase activity; 5,8,11,14-eicosatetraynoic acid, BW755c, or caffeic acid. In a static incubation system, AII-stimulated aldosterone secretion is inhibited 40-50% by any of these lipoxygenase inhibitors. In a perifusion system, BW755c partially inhibits only the sustained phase of AII-stimulated aldosterone secretion. However, BW755c has no effect on the secretion of aldosterone in response to combined A23187 plus 12-O-tetradecanoyl-phorbol-13-acetate. These results suggest that PL-A2 action is not obligatory in AII-induced aldosterone secretion and that lipoxygenase, but not cyclooxygenase, products of arachidonic acid metabolism may play a role in AII action as positive feed forward mediators.     

Effect of phorbol myristate acetate on the oxidative metabolism of human polymorphonuclear leukocytes.

The addition of 0.1 mug/ml of phorbol myristate acetate (PMA) to a suspension of resting human neutrophils causes a marked stimulation of all aspects of cellular oxidative metabolism normally associated with phagocytosis. PMA induces a greatly increased rate of glucose oxidation via the hexose monophosphate shunt, increased production of superoxide anion and of hydrogen peroxide, increased cellular chemiluminescence, and increased iodination of protein material. The time course of hexose monophosphate shunt activation and of chemiluminescence are similar to those observed following phagocytosis of opsonized zymosan; the levels of activation achieved in all cases approximate those seen following phagocytosis. These phenomena are not simply reflections of altered cellular permeability, since PMA actually inhibits the uptake of radioactive 2-deoxyglucose and of uniformly labeled amino acids. The addition of PMA similarly inhibits the uptake of 14C-labeled bacteria, suggesting a competition between the effect of the chemical and the process of phagocytosis. These results suggest that PMA activates the cell in the same manner as does phagocytosis. This compound should provide a useful tool for elucidating the metabolic events underlying the phenomena of phagocytosis and bacterial killing by polymorphonuclear leukocytes.     

Effects of quercetin on magnesium-dependent adenosine triphosphatase and the metabolism of human polymorphonuclear leukocytes

The bioflavinoid quercetin was found to exert at least three separate effects on human polymorphonuclear leukocytes. (1) Concentrations of approximately 100 microM inhibited the membrane-associated magnesium adenosine triphosphatase by 60%-80% in either broken cell preparations or intact cells. Lineweaver-Burk plots showed the inhibition to be uncompetitive in nature. (2) Similar concentrations of quercetin inhibited respiratory burst activity of the cells as measured by oxygen consumption, glucose oxidation, or iodination of protein. All inhibitions were dose-dependent and were observed with either opsonized zymosan or phorbol myristate acetate as stimulus. (3) Quercetin likewise inhibited the transport of the nonmetabolizable hexose, 3H-2- deoxyglucose. These observations are most consistent with the hypothesis that quercetin exerts a generalized effect at the level of the cell membrane of the neutrophil.     

Age-related decline in lysosomal enzyme release from polymorphonuclear leukocytes after N-formyl-methionyl-leucyl-phenylalanine stimulation

Aging is assumed to decrease lysosomal enzyme release from polymorphonuclear leukocytes (PMN). A synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) was utilized to stimulate enzyme release of PMN from 45 human subjects, 21 males and 24 females, ranging in age from 22-83 yr old. Results of the studies showed no sex differences in the stimulation of enzyme release for either age group. However, stimulation was found to significantly decline in both males and females over 50 yr old compared to subjects under 50 yr old. The linear formulae for beta-glucuronidase, beta-galactosidase and lysozyme in male subjects were Y = 6.5X + 617.2, Y = -1.9X + 311.5 and Y = -1.9X + 327.3 with correlation coefficient of -0.685, -0.352 and -0.401, respectively. The linear formulae in females were Y = -5.2X + 536.6, Y = -3.0X + 340.6 and Y = -1.7X + 333.6 with correlation coefficient of -0.582, -0.303 and -0.462, respectively. These findings suggest that there was an age-related decline of response to the stimulant, fMet-Leu-Phe.     

Effect of cyanide on NADPH oxidation by granules from human polymorphonuclear leukocytes.

Cyanide has been shown to stimulate both oxygen uptake and hexose monophosphate shunt activity in phagocytizing human polymorphonuclear leukocytes. It also stimulates the oxidation of NADPH by a particulate fraction derived from phagocytizing cells. This stimulation of NADPH oxidase is not observed in the presence of exogenous Mn2+. Studies with purified enzymes have shown that CN- also stimulates NADPH oxidation by horseradish peroxidase or lactoperoxidase, suggesting that the respiratory burst might be initiated by activation of a peroxidase-like enzyme in the human polymorphonuclear leukocyte. Based on studies of others, however, it does not appear as though the enzyme is identical to myeloperoxidase. The mechanism of the CN- stimulation appears to involve an oxidatic chain reaction, since it stimulates markedly NADPH oxidation in the presence of an artificial superoxide-generating system.