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Biomedical subjects

D J Hanahan

Publications and source records attributed to D J Hanahan.

At least 91 records · Page 5Linked to original sources
PubMed1983

Acetylglyceryl ether phosphorylcholine. A potent activator of hepatic phosphoinositide metabolism and glycogenolysis.

Acetylglyceryl ether phosphorylcholine (AGEPC), or 1-O-Alkyl-2-acetyl-sn-glyceryl 3-phosphorylcholine, has been shown to have a dramatic influence on phosphoinositide metabolism in isolated rat hepatocytes and upon glycogenolysis in the intact perfused rat liver. Addition of 5 X 10(-10) M AGEPC to 32Pi-labeled rat hepatocytes resulted in up to a 30 to 40% decrease in the [32Pi]phosphatidylinositol 4,5-bisphosphate within 10 s. The 32P content of phosphatidylinositol 4-phosphate decreased approximately 25% within 60 s, while a 5 to 8% decrease in [32P]phosphatidylinositol was observed only after 2 to 5 min of incubation of hepatocytes with AGEPC. Infusion of AGEPC (2 X 10(-10) M) into perfused livers resulted in a 3-fold increase in the glucose output in the effluent perfusate within 2 min. Interestingly, when a 500-fold higher concentration, i.e. 1 X 10(-7) M, of 1-O-alkyl-sn-glyceryl 3-phosphorylcholine or the stereoisomer 3-O-alkyl-2-acetyl-sn-glyceryl 1-phosphorylcholine was infused, no increase in the hepatic glucose output was seen. These observations lead to the conclusion that AGEPC exerts a potent influence on the polyphosphoinositide metabolism and glycogenolysis in rat liver and establishes the liver as an ideal system in which to conduct a detailed inquiry into the biochemical mechanism(s) responsible for the biological action of this unusual phospholipid.

Animals↗
PubMed1983

Metabolic behavior of acetyl glyceryl ether phosphorylcholine on interaction with rabbit platelets.

The metabolic fate of 1-O-[3H]alkyl-2-acetyl-sn-glycero-3-phosphorylcholine ([3H]-AGEPC) upon interaction with rabbit platelets was investigated. [3H]AGEPC was converted to a product identified as the long-chain fatty acyl analog. The reaction was unaffected by extracellular calcium. After a lag time of 30 to 60 s the kinetics of the conversion was linear. The rate of the reaction was found to be a function of platelet and AGEPC concentrations. Of the [3H]AGEPC (10(-9) M) 85 +/- 5% was processed into the-long chain fatty acyl analog within 1 h when incubated at 37 degrees C with a 1.25 X 10(9) platelets per milliliter suspension. A maximal number of 1200 to 3600 [3H]AGEPC molecules were converted to the long-chain fatty acyl derivative per minute per platelet in the presence of 2 mM EDTA. Under similar conditions the 1-O-[3H]alkyl-2-(lyso)-sn-glycero-3-phosphorylcholine ([3H]lysoGEPC) also was transformed to a comparable long-chain fatty acyl derivative at a much slower rate and to a lower extent. No significant increase in lysoGEPC was noted in incubation mixtures containing [3H]AGEPC. The possible direct transacylation of AGEPC upon interaction with platelets is discussed as well as the possible involvement of this reaction in directly triggering the platelet response to AGEPC stimuli.

Animals↗
PubMed1983

An early transient decrease in phosphatidylinositol 4,5-bisphosphate upon stimulation of rabbit platelets with acetylglycerylether phosphorylcholine (platelet activating factor).

Washed rabbit platelets labeled with [3H]inositol were stimulated with AGEPC (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) (5 X 10(-10) M) for various time periods. Within 5 s of the mixing of these platelets with AGEPC, an approximately 25% decrease in the [3H]TPI (phosphatidylinositol 4,5-bisphosphate) was evident; immediately thereafter the radioactivity in TPI increased. These labeled platelets treated with various concentrations of AGEPC for only 5 s indicated a characteristic dose-related decrease in [3H]TPI. Radioactivity in phosphatidylinositol 4-phosphate also appeared to increase after AGEPC-induced stimulation of platelets. Interestingly, within 15 s a 15 to 20% decrease in [3H]PI (phosphatidylinositol) and an increase in [3H]lysoPI was observed. However, [3H]lysoPI could be related only to one-third of the decrease in [3H]PI. LysoGEPC (lyso-1-O-alkyl-sn-glyceryl-3-phosphorylcholine), which is ineffective in the activation of platelets, was unable to cause any changes in the phosphoinositides. The fact that the status of TPI was influenced in a time- and dose-dependent manner and the rapidity with which these changes take place suggest that this inositol phospholipid may be associated closely with the early processes which accompany the interaction of AGEPC with platelets.

Animals↗
PubMed1983

Thromboxane B2 (TxB2) release following acetyl glyceryl ether phosphorylcholine (AGEPC) infusion in the rabbit.

Intravenous infusion of acetyl glyceryl ether phosphorylcholine (AGEPC) into rabbits resulted in an AGEPC dose-dependent elevation of plasma thromboxane B2 (TxB2) levels within 30 seconds. In contrast to AGEPC, the deacetylated derivative, lyso-GEPC (36.8 micrograms), did not increase intravascular TxB2 levels when similarly infused into rabbits. Intravascular TxB2 levels were maximal at 60 seconds after the infusion of 0.61 micrograms AGEPC whereas at higher doses of AGEPC (1.21-2.4 micrograms), plasma TxB2 levels were maximally elevated within 30 seconds after initiation of AGEPC infusion. These acute increases in the intravascular levels of TxB2 were accompanied by the development of thrombocytopenia, neutropenia, and basopenia which occurred concomitantly with AGEPC dose-dependent elevations in the plasma levels of platelet factor 4. Elevations in the plasma TxB2 levels returned to pre-infusion levels within 10-20 minutes after the initiation of AGEPC infusion. Thus, in vivo, one potent phospholipid, AGEPC, stimulates the production of another class of potent lipid mediators, the thromboxanes.

Animals↗
PubMed1983

Differential susceptibility of mono- and di-O-alkyl ether phosphoglycerides to acetolysis.

The effectiveness of acetolysis as a tool in structural characterization of mono- and di-O-alkyl phosphoglycerides was investigated. Surprisingly, it was found that the di-O-alkyl phosphoglycerides were resistant to attack during acetolysis, whereas the mono-ether types, with a free hydroxyl function or an ester on carbon-2, were easily attacked at the glycerol-phosphate bond. On the other hand, Vitride reduction occurred readily with the mono-ether or di-ether phosphoglycerides. The implications of these findings as they relate to identification of ether phospholipids in tissues are discussed.

Acetates↗
PubMed1982

Identification of domains of phosphatidylcholine in human erythrocyte plasma membranes. Differential action of acidic and basic phospholipases A2 from Agkistrodon halys blomhoffii.

Highly purified acidic (pI 4.9) and basic (pI 8.7) phospholipases A2 from snake (Agkistrodon halys blomhoffii) venom hydrolyzed approximately 20% and 60%, respectively, of the phosphatidylcholine (PC) of intact human erythrocytes prior to hemolysis. Sequential use of the acidic enzyme followed by the basic phospholipase A2 or vice versa manifested a characteristic PC hydrolysis pattern. For example, when acidic enzyme had hydrolyzed nearly 20% of this substrate, a subsequent treatment with the basic enzyme hydrolyzed only an additional 40% of the PC before hemolysis. On the other hand, in experiments where hydrolysis of about 20% of PC of erythrocytes was achieved by a short term incubation with the basic enzyme, then a further treatment of the same cells with the acidic enzyme caused only 10% additional PC hydrolysis before hemolysis. This demonstrated that the acidic enzyme hydrolyzed one domain of PC in the intact erythrocytes, whereas the basic enzyme hydrolyzed not only the same but also another domain of PC in membranes. Analysis of fatty acids released by the action of these two phospholipases A2 on erythrocytes indicated further characteristic differences. In particular, the ratio of released saturated to unsaturated fatty acids was significantly higher with the acidic enzyme as compared with the basic phospholipase A2. These results provide firm support to the conclusion that there are different domains of PC in human erythrocyte membranes and that the acidic and basic phospholipase A2 of A. halys blomhoffii can be used to identify them.

Erythrocyte Membrane↗
PubMed1982

Vasoactive properties of acetyl glyceryl ether phosphorylcholine and analogues.

Intradermal injection of 0.1 pmole (52 pg.) of acetyl glyceryl ether phosphorylcholine (AGEPC) in guinea pigs induced increased vascular permeability as assessed by a skin-blueing model. Rabbits and rats showed increased vascular permeability with 1.0 pmole (520 pg.) of AGEPC. On a molar basis, from 1,000 to 10,000 times more histamine was required to induce skin blueing in the same animals. In rabbits and guinea pigs, the increased vascular permeability induced by AGEPC occurred as an early, transient phase and as a delayed, prolonged phase. The early, transient phase was not inhibited by chlorpheniramine doses which markedly reduced or abrogated histamine-induced blueing. AGEPC was 100 to 1000 times more potent than histamine on a molar basis for the induction of vasoconstriction in a blued skin assay in guinea pigs. These studies demonstrate that AGEPC has potent vasoactive properties and provide additional information that implicates this acetylated alkyl phosphoglyceride as a mediator of the acute inflammatory process.

Animals↗
PubMed1982

Induction of leukocytic infiltrates in rabbit skin by acetyl glyceryl ether phosphorylcholine.

Intracutaneous injection of 5 to 50 pmoles of acetyl glyceryl ether phosphorylcholine (AGEPC) induced neutrophil (PMN) infiltrates in rabbit skin. PMN margination and plugging were present in dermal venules within 15 minutes after AGEPC injection, and extravascular PMN were most abundant after 3 hours. There was a sharp decline in PMN numbers when sites were examined 6 hours after AGEPC injection. PMN emigration appeared to occur predominantly from venules in the deep portion of the cutaneous venous plexus, and PMN accumulation was greatest in the deep dermis. Few if any PMN were present in lyso-glyceryl ether phosphorylcholine and human serum albumin sites at all time points studied. This demonstration that extravascularly introduced AGEPC induces PMN infiltrates in vivo provides evidence that this acetylated alkyl phosphoglyceride may be a significant mediator of acute allergic and inflammatory processes with a broader role than its previously described platelet-activating properties.

Animals↗
PubMed1981

Modification of the polar head group of acetyl glyceryl ether phosphorylcholine and subsequent effects upon platelet activation.

A series of phosphoglycerides containing the same structural unit, 1-O-alkyl-2-acetyl-sn-glycerol, was prepared with the following polar head groups: -3-phosphoric acid (AGEPA), -3-phosphorylethanol (AGEPEt), -3-phosphorylethanolamine (AGEPE), -3-phosphoryl-N-monomethylethanolamine (AGEPMME), -3-phosphoryl-N,N-dimethylethanolamine (AGEPDME), and -3-phosphorylcholine (AGEPC). These compounds were synthesized primarily by phospholipase D modification of AGEPC. The characterization of these derivatives was achieved through thin layer chromatography, infrared spectrometry, gas-liquid chromatography, and combined gas-liquid chromatograph-mass spectrometry. Their ability to induce irreversible aggregation and 50% secretion of serotonin from washed rabbit platelets was investigated. The latter secretory activity, expressed on a molar basis (60 s following platelet stimulation) was shown to be particularly sensitive to the nature of the polar head group as follows: AGEPA, 8.3 X 10(-7) M; AGEPE, 6.8 X 10(-7) M; AGEPEt, 4.0 x 10(-7) M; AGEPMME, 3.7 X 10(-9) M; AGEPDME, 4.5 X 10(-10) M; and AGEPC, 1.8 X 10(-10) M. It was concluded that the polar head group of the various acetyl glyceryl ether phosphoglycerides had an important role in expressing the biological activity of this unique type of phosphoglyceride towards platelets. It appears likely that the polar head group of the various acetyl glyceryl ether phosphoglycerides had an important role in expressing the biological activity of this unique type of phosphoglyceride towards platelets. It appears likely that the recognition site (receptor site) on platelets is more reactive to the dimethylethanolamine or teh quaternary ammonium base, choline, than to any of the other polar head groups under consideration.

Animals↗
PubMed1981

Activation of rabbit platelet phospholipase and thromboxane synthesis by 1-O-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (platelet activating factor).

1-O-Hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), structurally identical with platelet activating factor, is a potent stimulus for rabbit platelet aggregation and serotonin secretion. AGEPC at concentrations between 10(-10) and 10(-8) M induced stimulation of rabbit platelet synthesis of thromboxane B2. The dose vs. response curve for platelet thromboxane B2 synthesis was displaced slightly towards higher stimulus concentrations compared to [3H]serotonin secretion, with half-maximal concentrations of 2.5 . 10(-9) and 8 . 10(-10) M, respectively. Rates of thromboxane B2 synthesis and secretion were similar with a t 1/2 max of 4.0-4.5 s for both processes. AGEPC induced a decrease in platelet [14C]arachidonic acid in both phosphatidylinositol and phosphatidylcholine, although [14C]arachidonic acid turnover in phosphatidylcholine was not observed below 1 . 10(-8) M AGEPC. Concomitantly, this decrease in phospholipid [14C]arachidonic acid was associated with a marked increase of radiolabel in platelet diacylglycerol and phosphatidic acid 15 s after AGEPC addition, suggesting the possibility of a phospholipase C-diacylglycerol lipase mechanism of fatty acid cleavage. As observed previously with secretion and aggregation, removal of the 2-acetyl group from AGEPC abrogated all capacity of this molecule to stimulate platelet phospholipase. This study indicates that AGEPC (or platelet activating factor) activation of rabbit platelet phospholipase occurs in a time-course and concentration range similar to that required for [3H]serotonin secretion.

Animals↗
PubMed1981

Contraction of guinea pig ileal smooth muscle by acetyl glyceryl ether phosphorylcholine.

Acetyl glyceryl ether phosphorylcholine (AGEPC) is a chemical that has the biological activity of what was formerly termed platelet-activating factor. We report here that synthetic AGEPC induces the contraction of guinea pig ileal smooth muscle. Antagonists of histamine, acetylcholine, and slow-reacting substances (SRS) do not block AGEPC-induced contraction. These responses were long lasting, resistant to washing, and displayed complete agonist specific desensitization. Histamine- and SRS-induced contractions were unaffected by AGEPC. These studies show that AGEPC has the potential to produce a component of anaphylactically induced smooth muscle contraction.

Animals↗