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Biomedical subjects

D J Hanahan

Publications and source records attributed to D J Hanahan.

At least 55 records · Page 3Linked to original sources
PubMed1987

Binding and internalization of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in washed rabbit platelets.

The binding profile of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC, platelet-activating factor) to washed rabbit platelets was investigated through the use of structural analogs of AGEPC, e.g. U66985, which specifically suppressed AGEPC biological activities on rabbit platelets. This interaction of AGEPC with platelets could be divided into three different components termed A, B, and C. Component A was considered as one of high affinity (Kd = 0.5 X 10(-9) M) and with a low capacity (about 400 sites/platelet). The binding of AGEPC to component A was reversible and was blocked by the inhibitory analogs of AGEPC. This was considered to be the AGEPC receptor site(s). Component B was irreversible in nature and was presumed to be associated with internalization of AGEPC. The latter process was sensitive to the structural inhibitors. Component C was not affected by the inhibitors and probably represented a nonspecific binding to the lipid layer of the membrane. The binding profile of 1-O-alkyl-2-(lyso)-sn-glycero-3-phosphocholine, a biologically inactive and noninhibitory analog of AGEPC, was observed to consist of a single component and was (also) unaffected by the inhibitors. Internalization of AGEPC into rabbit platelets was further examined by the bovine serum albumin extraction method, which was originally developed by Mohandas et al. (Mohandas, N., Wyatt, J., Mel, S. F., Rossi, M. E., and Shohet, S. B. (1982) J. Biol. Chem. 257, 6537-6543). AGEPC was instantly taken up by the cell and internalization into its membrane, where it remained and was not released into cytosol. The internalization of AGEPC was suppressed by pretreating the cells with AGEPC analogs. In platelets desensitized to AGEPC, no down-regulation of the receptor site(s) was observed. The internalization of AGEPC in the desensitized cells was clearly enhanced and this was obvious even in the presence of the AGEPC inhibitor(s). Even in the presence of the inhibitors, effective internalization of AGEPC was also evident in thrombin-treated cells. These results suggested that the internalization of AGEPC was irreversibly enhanced in the platelets which were activated by AGEPC itself as well as by thrombin.

Animals↗
PubMed1987

Platelet-activating factor stimulation of rabbit platelets is blocked by serine protease inhibitor (chymotryptic protease inhibitor).

The serine protease inhibitors diethyl p-nitrophenyl phosphate and phenylmethylsulfonyl fluoride (chemical modifiers of serine residue) and N-acetyl-l-tryptophan ethyl ester (competitive inhibitor of chymotryptic protease) inhibited 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC; platelet-activating factor)-induced platelet aggregation and secretion. The inhibition was dependent on the preincubation time with the serine protease inhibitor and on the concentration of AGEPC and inhibitor. The IC50 value of diethyl-p-nitrophenyl phosphate, phenylmethylsulfonyl fluoride, and N-acetyl-l-tryptophan ethyl ester towards 5 X 10(-10) M AGEPC in serotonin release was 2.2 X 10(-4), 8.0 X 10(-4), and 5.0 X 10(-4) M, respectively. In experiments where platelets were incubated with these inhibitors and then washed with buffer, the inhibition of AGEPC stimulation was not observed. Prostaglandin H2 analog U46619 (10(-6) to 10(-5) M)- and thrombin (0.1 unit/ml)-induced platelet activation were also blocked by 1 mM diethyl p-nitrophenyl phosphate and 1 mM N-acetyl-l-tryptophan ethyl ester. The binding of AGEPC (1.5 X 10(-11) to 9.4 X 10(-10) M) to platelets and the platelet cyclic AMP level were not affected by diethyl p-nitrophenyl phosphate, phenylmethylsulfonyl fluoride, and N-acetyl-l-tryptophan ethyl ester. However, 1 mM diethyl p-nitrophenyl phosphate and 1 mM N-acetyl-l-tryptophan ethyl ester suppressed 10(-9) M AGEPC-induced breakdown of phosphatidylinositol 4,5-bisphosphate and formation of phosphatidic acid to 10-12 and 39-42%, 40-kDa protein phosphorylation to 4 and 30%, and arachidonic acid release to 17 and 28% of controls, respectively. On the other hand, 5 mM diethyl p-nitrophenyl phosphate did not inhibit diacylglycerol production and arachidonic acid release initiated by 2.5 mM deoxycholate treatment, suggesting that receptor-mediated phospholipase C and phospholipase A2 activation were inhibited by the serine protease inhibitor, but the deoxycholate (physicochemical stimulant)-initiated activation was not. AGEPC-induced 20-kDa protein phosphorylation and the inhibitory action of AGEPC on cyclic AMP accumulation were abolished in the presence of diethyl p-nitrophenyl phosphate and phenylmethylsulfonyl fluoride. However, a tryptic protease inhibitor, 1 mM p-aminobenzamidine and 1 mM benzoyl-l-arginine methyl ester, did not prevent the AGEPC-induced platelet secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Glycogenolytic and haemodynamic responses to heat-aggregated immunoglobulin G and prostaglandin E2 in the perfused rat liver.

Infusion of heat-aggregated immunoglobulin G (HAG) into perfused livers from fed rats caused transient increases in hepatic glycogenolysis and portal-vein pressure, accompanied by a transient increase in hepatic glycogen phosphorylase alpha content. The hepatic responses to HAG were inhibited by indomethacin (2 microM). In contrast, HAG was without effect on phosphorylase alpha content and glucose output in isolated hepatocytes. HAG infusion caused a transient decrease in hepatic cyclic AMP. Lowering the extracellular Ca2+ concentration to 6 or 50 microM attenuated markedly the glycogenolytic and haemodynamic responses to HAG; efflux of Ca2+ from the liver was not observed in response to HAG. Co-infusion of the specific platelet-activating-factor antagonist U-66985 (1-O-octadecyl-2-O-acetyl-sn-glycero-3-phosphoric acid 6'-trimethylammoniumhexyl ester) did not attenuate the glycogenolytic response to HAG. Infusion of prostaglandin E2 caused increases in glucose output, portal-vein pressure and the reduction state of the cytosolic NAD(H) redox couple similar to those seen with HAG. The present study suggests that the glycogenolytic activation after HAG infusion may be an indirect consequence of the haemodynamic response of the hepatic vasculature to stimulation of the reticuloendothelial cells of the liver.

Animals↗
PubMed1987

Mobilization of hepatic calcium pools by platelet activating factor.

In the perfused rat liver, platelet activating factor, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), infusion produces an extensive but transient glycogenolytic response which at low AGEPC concentrations (i.e., 10(-11) M) is markedly dependent upon the perfusate calcium levels. The role of calcium in the glycogenolytic response of the liver to AGEPC was investigated by assessing the effect of AGEPC on various calcium pools in the intact liver. Livers from fed rats were equilibrated with 45Ca2+, and the kinetics of 45Ca2+ efflux were determined in control, AGEPC-stimulated, and phenylephrine-stimulated livers during steady-state washout of 45Ca2+. AGEPC treatment had only a slight if any effect on the pattern of steady-state calcium efflux from the liver, as opposed to major perturbations in the pattern of calcium efflux effected by the alpha-adrenergic agonist phenylephrine. Infusion of short pulses of AGEPC during the washout of 45Ca2+ from labeled livers caused a transient release of 45Ca2+ which was not abolished at low calcium concentrations in the perfusate. Moreover, there occurred no appreciable increase in the total calcium content in the liver perfusate at either high or low concentrations of calcium in the perfusion fluid. Infusion of latex beads, which are removed by the reticuloendothelial cells, caused the release of hepatic 45Ca2+ in a fashion similar to the case with AGEPC. Our findings indicate that AGEPC does not perturb a major pool of calcium within the liver as occurs upon alpha-adrenergic stimulation; it is likely that AGEPC mobilizes calcium from a smaller yet very important pool, very possibly from nonparenchymal cells in the liver.

Animals↗
PubMed1987

Occurrence of an endogenous inhibitor of platelet-activating factor in rat liver.

Hepatocytes obtained from livers derived from fed rats perfused with a collagenase-containing mixture were found to contain significant levels of platelet-activating factor activity as isolated by Silica Gel G thin layer chromatography. However, when soybean trypsin inhibitor was included in the collagenase-containing perfusion medium for hepatocyte preparation, platelet-activating factor activity could not be detected on Silica Gel G chromatograms. Examination of the lipids extracted from freeze clamped perfused rat livers revealed low, but detectable, levels of platelet-activating factor. Further investigation of these observations indicated that a lipid-like inhibitor was present in freeze-clamped perfused livers as well as in hepatocytes isolated in the presence of soybean trypsin inhibitor. In each instance platelet-activating factor and this newly discovered inhibitor, which comigrated at the same RF value on Silica Gel G thin layer chromatography plates, could be separated by further chromatography on high performance thin layer plates. The present study shows that platelet-activating factor is present in unstimulated liver and that its detection is masked by an endogenous lipid-like inhibitor.

Animals↗
PubMed1987

Metabolism of platelet-activating factor (alkylacetylphosphocholine) by type-II epithelial cells and fibroblasts from rat lungs.

The uptake and metabolism of 3H-labeled platelet-activating factor by interstitial and epithelial cells from rat lungs was investigated. The uptake of 1-O-[3H]octadecyl-2-acetyl-sn-glycero-3-phosphocholine (3H-AGEPC) by alveolar type-II cells was linear with time from 5 to 60 min, with an average rate of 660 and 450 fmol/10(6) cells for cells in primary culture for 48 to 72 h, respectively. AGEPC was rapidly metabolized and by 10 min 60% of AGEPC was converted into long-chain acylphosphatidylcholine (PC) (50%) and 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-GEPC) (10%). By 60 min radioactivity in AGEPC was less than 10% of the total intracellular activity. Lyso-GEPC remained at about 10% throughout the incubation period. The uptake of 3H-AGEPC by fibroblasts was very similar to type II cells, but the rate of metabolism was slower. AGEPC in fibroblasts constituted 85% of the cellular counts after 10 min of incubation, and 50% by 60 min. After 60 min only 30% of the AGEPC was converted to alkylacyl-PC. Characterization of the fatty acids in the alkylacyl-PC of both the type-II cells and lung fibroblasts indicated that arachidonic acid was preferentially (more than 90%) inserted at the 2-position.

Animals↗
PubMed1987

Some unique features of the metabolic conversion of platelet-activating factor (AGEPC) to alkyl acyl PC by washed rabbit platelets.

Recently several synthetic analogs of 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC; platelet-activating factor) were characterized as selective inhibitors of this agonist's effects on rabbit platelets (Tokumura, A., Homma, H., and Hanahan, D. J. (1985) J. Biol. Chem. 260, 12710-12714). In this current investigation, these studies have been extended to include a further inquiry into the biochemical nature of the metabolic inactivation of AGEPC in rabbit platelets, and the effect of these analogs on this process. Two of the latter components (U66985 and CV3988), which blocked AGEPC biological activity on rabbit platelets, also blocked the metabolism of this agonist. The metabolic conversion of AGEPC to alkyl acyl PC was inhibited nearly sevenfold by the most potent analog, U66985. Those analogs with low (U68043) or no biological inhibitory activity (lysoGEPC) had marginal effects on the metabolism of AGEPC. The effects of these compounds on the metabolism of AGEPC was not simply due to competitive inhibition. In platelets which had been pretreated with AGEPC in absence of extracellular Ca2+ (desensitized) and washed, the metabolic conversion of AGEPC to alkyl acyl PC was actually enhanced. This enhanced metabolic inactivation of AGEPC was also observed upon the treatment of the cells with thrombin, collagen, or ionophore A23187, indicating that the metabolism of AGEPC in platelets was enhanced not only by AGEPC itself but by other agonists as well. Nearly 85% of the fatty acyl residues was arachidonate in the alkyl acyl PC derived from AGEPC. This specific acylation with arachidonate was observed in the presence and absence of the inhibitor and in desensitized cells, indicating that selectivity for arachidonate is not dependent on the enhancement of the metabolism of AGEPC. The alkyl acyl PC found in the cells treated with thrombin, collagen, or A23187 was also predominantly alkyl arachidonoyl PC. Thus it has been shown that the inactivation of AGEPC by its conversion to alkyl acyl PC by rabbit platelets is enhanced by this agonist itself and that excess amounts of AGEPC could be further inactivated by the enhanced capacity of the metabolism process.

Animals↗
PubMed1986

Alkylacetylglycerophosphocholine stimulates Na+-Ca2+ exchange, protein phosphorylation and polyphosphoinositide turnover in rat ileal plasmalemmal vesicles.

The novel ether phospholipid, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), isometrically contracted helically cut rat ileal smooth muscle strips in a dose- and time-dependent manner. Utilizing an enriched plasma membrane vesicular preparation from rat ileal longitudinal smooth muscle, AGEPC specifically stimulated Na+-Ca2+ exchange in a dose- and time-dependent manner. Concomitant with the AGEPC stimulation of Na+-dependent Ca2+ influx in plasma membrane vesicles is an enhanced turnover of the polyphosphoinositides, an elevated concentration of phosphatidic acid and also an enhanced phosphorylation of an Mr 40,000 plasmalemmal protein. The mechanisms by which AGEPC may regulate ileal plasmalemmal Ca2+ flux and contractility are considered.

Adenosine Triphosphate↗
PubMed1986

Effects of beta-adrenergic stimulation on 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine-mediated vasoconstriction and glycogenolysis in the perfused rat liver.

The beta-adrenergic agonist isoproterenol inhibited the glycogenolytic response of platelet-activating factor (AGEPC, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) in perfused livers derived from fed rats. AGEPC-stimulated hepatic vasoconstriction, measured by increases in portal vein pressure, also was inhibited by prior isoproterenol infusion. Isoproterenol-mediated inhibition of these hepatic responses to AGEPC was not apparent when isoproterenol (10 microM) was coinfused with the beta-receptor antagonist propranolol (75 microM) or when isoproterenol was replaced with the alpha-adrenergic agonist phenylephrine (10 microM). alpha-Agonist-induced glycogenolysis and vasoconstriction in the perfused liver was unaffected by isoproterenol infusion. Glucagon (2.3 nM) had no effect on the glycogenolytic or vasoconstrictive responses of the liver to AGEPC despite the fact that glucagon increased hepatic cAMP levels to a far greater extent than isoproterenol. Additionally, inhibition of the hepatic responses to AGEPC by isoproterenol occurred in perfused livers from mature rats (i.e. greater than 300 g) in which liver parenchymal cells lack functional beta-adrenergic receptors. The data presented in this study illustrate a specific inhibition of AGEPC-induced hepatic glycogenolysis and vasoconstriction by beta-adrenergic stimulation of the perfused liver. This inhibition appears to be mediated by interaction of isoproterenol with nonparenchymal cells within the liver. These findings are consistent with the concept that AGEPC stimulates hepatic glycogenolysis by an indirect mechanism involving hepatic vasoconstriction.

Animals↗
PubMed1986

Effect of acetylglyceryl ether phosphocholine (platelet-activating factor) and thrombin on acylation of exogenous lysophosphoglycerides in rabbit platelets.

Exogenous lysophosphoglycerides such as 1-O-acyl-2-(lyso)-sn-glycero-3-phosphocholine (lysoPC), 1-O-acyl-2-(lyso)-sn-glycero-3-phosphoethanolamine (lysoPE) and 1-O-alkyl-2-(lyso)-sn-glycero-3-phosphocholine (lysoGEPC) radiolabelled on the 1-O-aryl moiety were found to be acylated to the corresponding phosphoglycerides by rabbit platelets at different initial rates and to different extents. LysoPC and lysoGEPC were found to be the best precursors for specifically labelling the corresponding phosphoglyceride pool. Interestingly, synthetic 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and thrombin were able to increase the initial rate of acylation of all these lysophosphoglycerides up to 4-6-fold in a concentration-dependent manner. The finding that stimulation of the acylation rate of the various lysophosphoglycerides was relatively independent of the stimuli used supports the hypothesis that this activation could be a new phenomenon associated with the reversible shape change observed in platelets after stimulation with AGEPC or thrombin.

Acylation↗
PubMed1986

Characterization of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC)-induced protein phosphorylation in rabbit platelets: inhibitory effects of AGEPC analogs.

1-O-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) induced phosphorylation of two proteins having molecular masses of approximately 20- and 40-kDa in washed rabbit platelets in a concentration- and time-dependent manner. Sequential stimulation with AGEPC did not induce additional protein phosphorylation, supporting the concept of desensitization of the AGEPC receptors responsible for biological activity. AGEPC analogs 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphoric acid-6'-trimethylammonium hexyl ester and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphoric acid-10'-trimethylammonium decyl ester (U66985 and U66982), containing polar head groups with methylene chain lengths of C6 and C10, did not cause protein phosphorylation, but they did inhibit the AGEPC-induced events. Thus protein phosphorylation is closely associated with the receptor-mediated stimulation of platelets and is a useful indicator of the signaling process initiated through the receptors. Other synthetic analogs of AGEPC such as rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate and 1-(N-n-pentadecylcarbamoyloxy)-2-methoxy-rac-glycero-3-phosphochol ine (CV3988 and U68043) were also shown to be inhibitors of the AGEPC-induced protein phosphorylation. Inhibition by these analogs was specific for AGEPC since there was no observed effect of thrombin, ADP, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and arachidonic acid-induced changes. The extent of inhibition was dependent on the concentration of AGEPC and its analogs and did not change with time after the addition of AGEPC. In platelets incubated with AGEPC analogs before and simultaneously with the addition of AGEPC, protein phosphorylation was prevented; however, addition of AGEPC to platelets shortly before the addition of these analogs showed a high response. In experiments where platelets were previously incubated with AGEPC analogs and washed with buffer containing 0.5% bovine serum albumin, AGEPC-induced protein phosphorylation was recovered to a level of 80%. These observations support the conclusion that AGEPC stimulates platelets through its specific receptor, and that the AGEPC analogs bind to the AGEPC receptor and block that pathway sensitive to AGEPC stimulation but not because of the desensitization of its receptor. On the other hand, in platelets where phosphorylation of the 40-kDa protein was induced by a 2-min preincubation with 3 X 10(-10) M TPA, 5 X 10(-10) M AGEPC-induced serotonin release decreased by 51% compared to a control value.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Specific antagonists of platelet activating factor-mediated vasoconstriction and glycogenolysis in the perfused rat liver.

Stimulation of hepatic glycogenolysis and vasoconstriction of the hepatic vasculature in response to acetyl glyceryl ether phosphocholine (AGEPC; platelet activating factor) was inhibited by two structural analogues of AGEPC, U66985 (1-O-octadecyl-2-O-acetyl-sn-glycero-3-phosphoric acid-6'-trimethyl ammonium hexyl ester) and CV3988 [rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxy-propyl-2-thiazolioethyl+ ++ phosphate]. Infusion of CV3988, 10(-7) M, increased the AGEPC dose needed for half-maximal hemodynamic response by approximately 5-fold, while U66985 at 10(-7) M increased by twenty times the dose of AGEPC required to give the half-maximal response. Glucose output responses were similarly inhibited. U66985, 10(-6) M, completely abolished both hemodynamic and glycogenolytic responses to AGEPC, 2 X 10(-10) M, while in the presence of CV3988, 10(-6) M, approximately 15% of the uninhibited responses remained. Perfusion of livers for 20 min after termination of inhibitor infusion, in the absence or presence of bovine serum albumin, resulted in only a slightly smaller extent of inhibition than simultaneous infusion of agonist and antagonist. Specificity of the inhibitors was demonstrated by only a minimal inhibition of glycogenolytic response to the alpha-adrenergic agonist phenylephrine at a sub-maximal dose.

Animals↗
PubMed1986

Platelet-activating factor-mediated vasoconstriction and glycogenolysis in the perfused rat liver.

Infusion of platelet-activating factor (alkyl acetylglycerophosphocholine (AGEPC] into isolated perfused rat livers caused a dose-dependent, transient increase in portal vein pressure, indicative of constriction of the hepatic vasculature. A close correlation was observed between the changes in portal pressure and concomitant transient increases in hepatic glucose output. The two processes displayed similar dose dependence and were attenuated to a similar extent by reducing the perfusate calcium concentration. Reducing the perfusate free calcium concentration to 1 nM by co-infusion of EGTA did not abolish completely the hepatic responses to AGEPC. Verapamil inhibited both the hemodynamic and glycogenolytic responses to AGEPC in a dose-dependent fashion; the IC50 was approximately 10 microM at an AGEPC concentration of 6.6 X 10(-11) M. Also, both responses displayed similar degrees of tachyphylaxis in response to repeated short infusions of AGEPC. Measurement of glycogen phosphorylase a in extracts from freeze-clamped livers demonstrated a rapid increase in phosphorylase a in response to infusion of AGEPC. A small but significant increase in whole tissue ADP was found in response to AGEPC (2 X 10(-8) M); cAMP levels were not changed by AGEPC infusion. It is concluded that glycogenolysis in the perfused liver in response to AGEPC may be a result of the hemodynamic effects of AGEPC, rather than a direct effect of the phospholipid mediator on the hepatocyte.

Adenosine Diphosphate↗
PubMed1986

Alkylacetylglycerophosphocholine effects on the metabolism of phospholipids in rabbit platelets: effects of extracellular Ca2+ and prostacyclin.

Alkylacetylglycerophosphocholine (AGEPC) stimulation of 32P-labeled lysophosphatidic acid formation in washed rabbit platelets was dependent on extracellular Ca2+. Its accumulation was slower and required a higher concentration of AGEPC in comparison to the degradation of inositol phospholipids and production of phosphatidic acid induced by the same agonist. These results suggest that the formation of lysophosphatidic acid is not directly related to the primary activation of rabbit platelets by AGEPC. AGEPC elicited a preferential degradation of inositol phospholipids in the following order: phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. The degradation of inositol phospholipids and subsequent production of phosphatidic acid were affected by pretreatment of platelets with prostacyclin or ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Synergistic inhibitions of these metabolic changes were observed in the platelets pretreated with both prostacyclin and EGTA. These results were compared with effects of prostacyclin and EGTA on serotonin release induced by AGEPC, and the possible roles of metabolic changes in phospholipids induced by AGEPC are discussed with respect to the mechanism of platelet activation.

Animals↗
PubMed1985

Structural analogs of alkylacetylglycerophosphocholine inhibitory behavior on platelet activation.

Several analogs of alkylacetylglycerophosphocholine (AGEPC; platelet-activating factor) were investigated as potential selective inhibitors of AGEPC-induced activation of washed rabbit platelets. Two particular compounds, CV-3988 (rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl-2-thiazolioethyl++ + phosphate) and U66985 (1-O-octadecyl-2-acetyl-sn-glycero-3-phosphoric acid-6'-trimethylammonium-hexyl ester) emerged as particularly active and effective inhibitors. Aggregation and secretion profiles, as well as the degradation of inositol phospholipids and production of phosphatidic acid, were used as monitors of their inhibitory capabilities. U66985 was the most effective inhibitor, giving an IC50 value of 4.1 +/- 1.5 X 10(-8) M against a challenge of 1 X 10(-10) M AGEPC in the secretion assay. Phospholipid turnover was blocked completely at this inhibitor concentration. On the other hand, while CV-3988 was an effective inhibitor, a higher concentration was required and a more restricted range of activity was noted with an IC50 value of 5.9 +/- 1.3 X 10(-7) M against a challenge of 1 X 10(-10) M AGEPC in the secretion assay. While CV-3988 did indeed completely block the turnover of inositol phospholipids and phosphatidic acid formation, these effects were noted at a higher concentration than with U66985. On the basis of data obtained in desensitization experiments with AGEPC and U66985, it appears that each inhibitor occupies the same receptor site as the agonist, AGEPC. These results illustrate the usefulness of these AGEPC analogs in exploring the biochemical characteristics of the interaction of AGEPC with a cell.

Animals↗
PubMed1985

Occurrence of glyceryl ethers in the phosphatidylcholine fraction of surfactant from dog lungs.

The molecular species of ether-linked lipids in the phosphatidylcholine (PC) fraction of the pulmonary surfactant obtained from the lavage fluid of dog were characterized. A combination of base-catalyzed methanolysis, phospholipase C treatment, gas-liquid chromatography, and mass spectrometry procedures were applied. The phospholipid composition of the surfactant, obtained by phosphorus assay of lipids separated by silica gel G thin-layer chromatography (TLC), was: PC (75%), phosphatidylglycerol (10%), phosphatidylethanolamine (7%), plus small amounts of sphingomyelin, phosphatidylinositol, and phosphatidylserine. The major components of the PC were 1,2-diacylPC (95%), and 1-O-alkyl-2-acylPC (5%). No detectable amounts of 1-O-alkyl-1'-enyl-2-acylPC or di-alkyl-1-enylPC were observed. The acyl groups present in the diacylPC were 14:0 (5%), 16:0 (68%), 16:1 (12%), 18:0 (6%), 18:1 (7%) and 18:2 (2%). The predominant alkyl ether chains located at the carbon 1 position of the 1-O-alkyl-2-acylPC were 16:0 (84%), 18:0 (5%) and 18:1 (14%). At the carbon 2 position only a 16:0 fatty acyl residue was detected. In three out of seven animals platelet-activating factor-like activity, as determined by a platelet aggregation assay, was isolated by TLC. This aggregating activity was lost upon base-catalyzed methanolysis, but was restored by functional levels after acetylation.

Animals↗