The stimulation of arachidonic acid metabolism in human platelets by hydrodynamic stresses

Even though shear-induced platelet activation and aggregation have been studied for about 20 years, there remains some controversy concerning the arachidonic acid metabolites formed during stress activation and the role of thromboxane A2 in shear-induced platelet aggregation. In this study, platelets were labelled with [1-14C]arachidonic acid to follow the metabolism of arachidonic acid in stimulated platelets using HPLC and scintillation counting. Platelets activated by thrombin formed principally thromboxane A2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). In contrast, for platelets activated by shear--though arachidonic acid metabolism was stimulated--only 12-HETE was formed and essentially no cyclooxygenase metabolites were detected. This indicates that physical forces may initiate a different pathway for eicosanoid metabolism than most commonly used chemical stimuli and perhaps also implies that regulation of the cyclooxygenase activity may be a secondary level of regulation in eicosanoid metabolism.     

Effects of estradiol on the metabolism of arachidonic acid by aortas and platelets in rats

We have previously reported that estradiol treatment stimulates prostacyclin production by cultured rat aortic smooth muscle cells, through the stimulation of fatty acid cyclooxygenase and prostacyclin synthetase activities. In order to see whether estradiol stimulates the fatty acid cyclooxygenase activity in platelets, intact rats were treated with estradiol, and thromboxane biosynthesis in platelets and prostacyclin production by aortas were investigated. Estradiol significantly stimulates prostacyclin production by aortas. However, no significant effect on thromboxane biosynthesis in platelets is observed. Our present results support the idea that estradiol would be a protective hormone in atherosclerotic heart disease.     

Cytokine- and calcium ionophore A23187-mediated arachidonic acid metabolism in neutrophils

Arachidonic acid (AA) metabolism is implicated as an intracellular and/or intercellular second messenger system for the transmission of cytokine-initiated signals that affect neutrophils and mediate systemic toxicity. The purpose of the present study is to ascertain if cytokines that are known to affect neutrophil function in vivo and in vitro directly stimulate neutrophil AA metabolism in vitro. The recombinant human cytokines multi-colony stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 1, tumor necrosis factor (TNF), and interleukin 6 and the calcium ionophore A23187 were incubated with purified 14C-AA radiolabeled human peripheral blood neutrophils and the effects were assayed by one- and two-dimensional thin layer lipid chromatography. None of the cytokines appeared to induce the release of cell-incorporated AA or to increase the level of radiolabeled phosphatidic acid. TNF induces severe systemic toxicity that is inhibited by cyclooxygenase inhibitors, which suggests a role for AA metabolites in the pathophysiologic effects of TNF; we have confirmed that TNF and endotoxin act synergistically to induce indomethacin-inhibitable fatal shock in rats. However, when in 3H-AA radiolabeled human neutrophils were incubated with TNF in kinetic, cold-chase, and TNF preincubation experiments, TNF was not found to increase AA metabolism, although changes in the intracellular neutral lipid content were noted. GM-CSF, which has been reported by previous investigators to directly induce the release of AA, did not release neutrophil-associated 3H-AA. In conclusion, the direct release of AA from membrane-associated phospholipids does not appear to be a major second messenger pathway for cytokine-initiated activation of neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of dimethyl sulfoxide on human platelet aggregation and arachidonic acid metabolism

DMSO inhibited human platelet aggregation induced by ADP, AA, PAF, or collagen in a concentration-related manner, in vitro. DMSO was a more effective inhibitor for aggregation induced by ADP and collagen than PAF or AA. However, in vivo experiments on rabbits showed that DMSO did not protect rabbits against death from pulmonary platelet thrombosis induced by AA. On the other hand, DMSO (1-30% v/v) had no effect on thromboxane production by platelets incubated with [14C]AA. Moreover, DMSO stimulated PGE2 production by bovine seminal vesicle PG synthase. DMSO also stimulated the production of 12-HETE but inhibited the production of tri-HETE produced via lipoxygenase pathway. Since lipoxygenase products play an important role in inflammation, our data suggest that the anti-inflammatory effects of DMSO are probably not mediated via its action on AA metabolism.     

In vitro effect of trichosanic acid, a major component of Trichosanthes japonica on platelet aggregation and arachidonic acid metabolism in human platelets

The in vitro effect of trichosanic acid (TCA; C18:3, omega-5), a major component of Trichosanthes japonica, on platelet aggregation and arachidonic acid (AA) metabolism in human platelets was studied. TCA dose-dependently suppressed platelet aggregation of platelet rich plasma and washed platelets. TCA decreased collagen (50 micrograms/ml)-stimulated production of thromboxane B2 (TXB2) and 12-hydroxyhepta-decatrienoic acid (HHT) in a dose-dependent manner, while that of 12-hydroxyeicosatetraenoic acid (12-HETE) was rather enhanced. The conversion of exogenously added [14C]AA to [14C]TXB2 and [14C]HHT in washed platelets was dose-dependently reduced by the addition of TCA, while that to [14C]12-HETE was increased. Similar observations were obtained when linolenic acid (LNA; C18:3, omega-3) was used. These results suggest that TCA may decrease TXA2 formation in platelets, probably due to the inhibition of cyclooxygenase pathway, and thereby reduce platelet aggregation.     

Differential effects of 15-HPETE on arachidonic acid metabolism in collagen-stimulated human platelets

The 15-hydroperoxyeicosatetraenoic acid (15-HPETE) has been shown to affect platelet aggregation induced by collagen, arachidonic acid (AA), and PGH2-analogue. Furthermore, it also inhibits the platelet cyclooxygenase and lipoxygenase enzymes, and prostacyclin synthase. The present study was designed to test the effect of 15-HPETE on the mobilization of endogenous AA in collagen-stimulated human platelets. For this purpose, human platelets pretreated with BW755C (a dual inhibitor of cyclooxygenase and lipoxygenase) were stimulated with collagen in the presence of varied concentrations of 15-HPETE. We observed a significant inhibition of oxygenases at all concentrations of 15-HPETE. In contrast, our results indicate that 15-HPETE at lower concentrations (10 microM and 30 microM) significantly stimulated the collagen-induced release of AA from phospholipid sources. Although higher concentrations of 15-HPETE (50 microM and 100 microM) caused some inhibition of AA accumulation in the free fatty acid fraction (25% and 60%), the degree of inhibition was significantly lower than the inhibition observed for the oxygenases (65% and 88% for cyclooxygenase and 77% and 94% for lipoxygenase respectively). These results provide support that hydroperoxides also regulate phospholipases presumably by a different mechanism, which may be important in the detoxification of phospholipid peroxides.     

In vitro effects of synthetic antioxidants and vitamin E on arachidonic acid metabolism and thromboxane formation in human platelets and on platelet aggregation

The “in vitro” effects of α-tocopherol, butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) were studied on aggregation of human platelets induced by collagen and arachidonic acid (AA), on the metabolic conversion of ¹⁴C AA through the cyclooxygenase and lipoxygenase pathways and on the formation of thromboxane B₂ (TXB₂) in washed platelets after stimulation with collagen.Vitamin E completely inhibited AA induced platelet aggregation only at high concentration (mM) and after 10 minutes of preincubation, with limited effects on AA metabolism in platelets and no effect on TXB₂ formation from endogenous substrate. BHA completely inhibited platelet aggregation in the 10⁻⁶M range, gave 50% inhibition of AA metabolism in the 10⁻⁵M range and almost complete inhibition of thromboxane formation in the 10⁻⁴M range. BHT was about 100 times less active on platelet aggregation and AA metabolism. The lipoxygenase and cyclooxygenase pathways were differentially affected at low concentrations of BHA and only at concentrations greater than 5×10⁻⁵M were both pathways depressed.     

The metabolism of arachidonic and eicosapentaenoic acids in human neutrophils stimulated by A23187 and FMLP

A23187 stimulates the metabolism of endogenous as well as exogenous arachidonic acid (AA) and eicosapentaenolc acid (EPA) to their corresponding leukotrienes in human neutrophils. In contrast, conflicting results have been obtained concerning the effect of FMLP on the metabolism of these fatty acids. In the present study we compared the effect of A23187 and FMLP on the release and metabolism of these fatty acids in neutrophils. Stimulation of neutrophils with A23187, but not with FMLP, resulted in detectable levels of AA in the presence or absence of BW755C (a dual inhibitor of cyclooxygenase and lipoxygenase). The absolute amount of nonesterified AA in the extracts of neutrophils exposed to the agonist A23187 in the presence of BW755C was 20% higher than that obtained in the absence of BW755C, indicating that only a small fraction of the released AA was converted to lipoxygenase products. Furthermore, significant quantities of AA and EPA metabolites were detected only after treatment of neutrophils with A23187, but not with FMLP. Both A23187 and FMLP stimulated the conversion of exogenous EPA to 5-lipoxygenase products, with A23187 being somewhat more effective. In addition, significant differences were noted on the effect of EPA and DHA on the conversion of AA to its metabolites in A23187-stimulated neutrophils. Our results provide strong evidence that the amounts of eicosanoid precursors mobilized in response to FMLP are extremely small, if any, and this appears to be the likely explanation for the lack of eicosanoid detection by HPLC in FMLP-stimulated neutrophils.     

Mobilization of arachidonic acid in collagen-stimulated human platelets.

Stimulation of platelets with collagen results in the mobilization of arachidonic acid (AA) from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). In this study the effect of aspirin, indomethacin, BW755C and prostaglandin H2 (PGH2) on labelled AA release in response to varied concentrations of collagen was investigated. Our results indicate that aspirin (0.56 mM) and indomethacin (5.6 microM) not only inhibited the collagen-mediated formation of cyclo-oxygenase metabolites, but also caused a significant reduction in the accumulation of free labelled AA and 12-hydroxyeicosatetraenoic acid (12-HETE) (21-64%). Aspirin and indomethacin also inhibited the release of [3H]AA from PC (37-75%) and PI (33-63%). The inhibition of AA release caused by aspirin was reversed partially by PGH2 (1 microM). In contrast, a smaller/no inhibition of collagen-stimulated labelled AA and 12-HETE accumulation (0-11%) and of collagen-stimulated AA loss from PC and PI was observed in the presence of BW755C. The results obtained in the presence of aspirin, indomethacin and BW755C at lower concentrations of collagen further demonstrate that AA release from PI (45-61% inhibition at 10 micrograms of collagen), but not from PC, was affected by the inhibition of cyclo-oxygenase. The results obtained on the effect of PGH2 further support that deacylation of phospholipids occurs independently of cyclo-oxygenase metabolites, particularly at higher concentrations of collagen. These results also demonstrate that aspirin and indomethacin, but not BW755C, cause a direct inhibition of collagen-induced [3H]AA liberation from PC as well as from PI. We also conclude that the diacylglycerol lipase pathway is a minor, but important, route for AA release from PI in collagen-stimulated human platelets. The mechanisms underlying the regulation of AA release by collagen in the absence of cyclo-oxygenase metabolites are not clear.     

Thiopental enhances human platelet aggregation by increasing arachidonic acid release.

Conflicting results have been reported regarding the effect of thiopental on aggregation and cytosolic calcium levels in platelets. The present study attempted to clarify these phenomena. Using platelet-rich plasma or washed suspensions, platelet aggregation, thromboxane (TX) B2 formation, arachidonic acid (AA) release, and cytosolic free calcium concentrations ([Ca2+]i) were measured in the presence or absence of thiopental (30-300 microM). Platelet activation was induced by adenosine diphosphate (ADP, 0.5-15 microM), epinephrine (0.1-20 microM) arachidonic acid (0.5-1.5 mM), or (+)-9,11-epithia-11,12-methano-TXA2 (STA2, 30-500 nM). Measurements of primary aggregation were performed in the presence of indomethacin (10 microM). Low concentrations of ADP and epinephrine, which did not induce secondary aggregation in a control study, induced strong secondary aggregation in the presence of thiopental (> or = 100 microM). Thiopental (> or = 100 microM) also increased the TXB2 formation induced by ADP and epinephrine. Thiopental (300 microM) increased ADP- and epinephrine-induced 3H-AA release. Thiopental (300 microM) also augmented the ADP- and epinephrine-induced increases in [Ca2+]i in the presence of indomethacin. Thiopental appears to enhance ADP- and epinephrine-induced secondary platelet aggregation by increasing AA release during primary aggregation, possibly by the activation of phospholipase A2.     

Influence of short term dietary supplementation of different lipids on aggregation and arachidonic acid metabolism in rabbit platelets

Semisynthetic diets containing 8% by weight of either corn oil or butter were fed to male New Zealand rabbits for three weeks. The plasma cholesterol values were determined, the threshold concentrations for aggregation of platelet rich plasmas were measured for collagen and Na arachidonate, and the conversion of ¹⁴C arachidonic acid to thromboxane B₂ and hydroxy fatty acids (HETE and HHT) at 10, 20 and 40 μM substrate concentrations were studied. The thresholds for arachidonate induced aggregation were lower and the amplitudes of collagen induced aggregations were greater in the butter fed than in the corn oil fed rabbits. Conversions of arachidonic acid to thromboxane B₂ but not to hydroxy fatty acids were greater in the butter fed rabbits at 10 and 20 μM substrate. The observed changes were accompanied by only slight modifications of plasma cholesterol levels.     

Metabolism of [14C]arachidonic acid by human platelets.

A time dependent incorporation of [1-14C] arachidonic acid into platelet phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine was observed in platelet-rich plasma. When platelets, so labelled, were washed and treated with thrombin, there was a major decrease in the radioactivity of phosphatidylcholine and phosphatidylinositol. This decrease was accounted for by the appearance of several previously identified (Hamberg and Samuelsson (1974) Proc. Natl. Acad. Sci. U.S. 71, 3400) 14C-labelled oxygenated products of arachidonic acid.     

Rantes potentiates human macrophage aggregation and activation responses to calcium ionophore (A23187) and activates arachidonic acid pathways

Regulated on activation, normal T-cell expressed and presumably secreted (RANTES), which generally mediates monocyte-macrophage (MO) activation and recruitment, is a protein of 8-10 kD that chemoattracts eosinophils, monocytes and certain T leukocyte subsets. RANTES is coded for by a gene cluster located on human chromosome 17 and is a human T-cell specific molecule. RANTES is a member of a beta intercrine subfamily reported to be a selective chemoattractant for human monocytes rather than neutrophils, and is also a chemoattractant for memory T lymphocytes, CD4+ cells. RANTES is a modulator of many important macrophage functions in addition to aggregation, such as chemotaxis and phagocytosis. Our investigations focussed on the ability to modulate the aggregation of macrophages induced by calcium ionophore A23187. The ionophore A23187 directly induced potent aggregation of MO which was markedly enhanced when the cells were pretreated with RANTES. However, the addition of RANTES in the absence of other co-stimuli did not directly induce aggregation. Additional cytokines examined for possible induction of macrophage aggregation were interleukin-1 (IL-1), tumor necrosis alpha (TNF-alpha), and IL-6; all proved to be incapable of inducing aggregation directly, nor did they enhance the effects of A23187 on macrophage aggregation. Additionally, we found that RANTES can directly stimulate MO to activate specific pathways of arachidonic acid cascade, inducing a synthesis and release of thromboxane (TxA2) and leukotriene B4 (LTB4). RANTES did not augment the potent ability of A23187 to induce increased production of LTB4 or TxA2 by human MO. These data suggest that RANTES can contribute directly to monocyte-leukocyte-activation during inflammatory responses, resulting in greater cell aggregation, activation, and specific pro-inflammatory arachidonic acid products release, such as TxA2 and LTB4.     

Role of arachidonic acid in platelet aggregation induced by S. typhimurium endotoxin

The platelet aggregation reaction was used to assess the influence of arachidonic acid (AA), endotoxin (E) S. typhimurium and ADP on platelet aggregation properties. All the three substances induced platelet aggregation. A higher degree of aggregation was attained by the application of E combined with AA and ADP as compared with the effects produced by E and ADP alone. Prolonged incubation of platelet-rich plasma (PRP) samples with E led to an essential decrease of the aggregation degree on ADP addition. Incubation of PRP samples with E and ADP did not evoke any analogous decrease in the platelet aggregation degree. The data obtained indicate that AA stimulates platelet aggregation induced by E and ADP.     

Effects of retinoic acid treatment on release of arachidonic acid by chondrogenic cells in response to ionophore A23187

Chondrogenic differentiation in mouse limb bud mesenchymal cells cultured at high density was suppressed by supplementation of the medium with retinoic acid in a dose-dependent fashion. Cells prelabeled with (3H) arachidonic acid were treated with 0.3 microgram/ml retinoic acid. Treatment with retinoic acid increased the (3H) fatty acid in the triglyceride fraction. Furthermore, treatment with retinoic acid enhanced the release of (3H) fatty acid upon stimulation of these cells with the divalent ionophore A23187. These data permit the suggestion that there may be a correlation between altered lipid metabolism and retinoic acid's ability to disrupt chondrogenic differentiation.     

Synthesis of thromboxane A2 by non-aggregating dog platelets challenged with arachidonic acid or with prostaglandin H2.

Dog platelets challenged with arachidonic acid fail to aggregate but synthesize a substance which aggregates rabbit and human platelets, this aggregation being suppressed by dibutyryl cyclic AMP. The aggregating substance contracts strips of rabbit aorta and of coeliac and mesenteric arteries, is soluble in diethyl ether, has a half-life of about 40 seconds at 37 degrees C and of 100 seconds at 22 degrees C. Its generation is blocked by various inhibitors of prostaglandin biosynthesis. The thromboxane A2 synthetase inhibitor imidazole and its analogue benzimidazolamine also suppress generation of vessel contracting activity in incubates of dog platelets and prostaglandin H2. Since dog platelets also transform prostaglandin H2 into thromboxane A2 their failure to aggregate, when stimulated by arachidonic acid or by prostaglandin H2, is not due to lack of thromboxane synthesizing ability.     

Mobilization of arachidonic acid in thrombin-stimulated human platelets

In the present work we investigated the effect of serine esterase inhibitors such as 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC) and phenylmethylsulfonyl fluoride (PMSF), as well as the effect of mepacrine on thrombin-induced mobilization of arachidonic acid (AA) in human platelets. The inhibitor NCDC (0.6 mM) completely abolished the thrombin-induced activation of phospholipase C, phospholipase A2, and transacylase enzymes, whereas the pretreatment of platelets with PMSF (2 mM) resulted in a highly selective inhibition of phospholipase A2 and transacylase activities, with no marked effect on thrombin-induced activation of phospholipase C. The thrombin-induced release of [3H]AA from phosphatidylcholine and phosphatidylinositol was reduced by 90 and 56%, respectively, in the presence of PMSF. This inhibitor also caused a parallel inhibition in the accumulation of [3H]AA (85%) with little effect on thrombin-induced formation of [3H]phosphatidic acid (5%), whereas mepacrine (0.4 mM) caused a selective inhibition of phospholipase A2 and transacylase activities with concomitant stimulation of [3H]phosphatidic acid formation in intact human platelets. These results demonstrate that NCDC and PMSF (serine esterase inhibitors) do not affect agonist-induced activation of phospholipases that mobilize arachidonic acid through a common site. Our results further demonstrate that the inhibition of [3H]AA release observed in the presence of NCDC, PMSF, and mepacrine is primarily due to their direct effects on enzyme activities, rather than due to their indirect effects through formation of complexes between inhibitors and membrane phospholipids. Based upon these results, we also conclude that the combined hydrolysis of phosphatidylcholine and phosphatidylinositol by phospholipase A2 serves as a major source for eicosanoid biosynthesis in thrombin-stimulated human platelets.     

The inhibition of arachidonic acid metabolism in human platelets by RHC 80267, a diacylglycerol lipase inhibitor

The diacylglycerol lipase inhibitor, RHC 80267, 1,6-di(O-(carbamoyl)cyclohexanone oxime)hexane, was tested for its ability to block the release of arachidonic acid from human platelets. At a concentration (10 microM) reported to completely inhibit diacylglycerol lipase in fractions of broken platelets, RHC 80267 had no effect on diacylglycerol lipase activity or the release of arachidonic acid from washed human platelets stimulated with collagen. At a high concentration (250 microM), the compound inhibited the formation of arachidonyl-monoacylglycerol by 70% and the release of arachidonate by 60%. However, at this concentration RHC 80267 was found to inhibit cyclooxygenase activity, phospholipase C activity and the hydrolysis of phosphatidylcholine (PC) (presumably by inhibiting phospholipase A2). The phospholipase C inhibition was attributed to the inhibition of prostaglandin H2 formation, as it was alleviated by the addition of the endoperoxide analog, U-46619. PC hydrolysis was only partially restored with U-46619, suggesting that RHC 80267 directly alters phospholipase A2 activity. The inhibition of arachidonate release observed was accounted for by the inhibition of PC hydrolysis. We conclude that RHC 80267, because of its lack of specificity at concentrations needed to inhibit diacylglycerol lipase, is an unsuitable inhibitor for studying the release of arachidonic acid in intact human platelets.     

Metabolism of icosa-5,11,14-trienoic acid in human platelets and the inhibition of arachidonic acid metabolism in human platelets by icosa-5,8,14-triynoic and icosa-5,11,14-triynoic acids

Two fatty acids differing from arachidonic acid in lacking one of the internal double bonds (20:35,8,14 and 20:35,11,14) and their 1-C¹⁴ and acetylenic analogues were synthesized. 20:35,8,14 was not metabolized by human platelets but 20:35,11,14 yielded a small amount (1.5% conversion) of two hydroxy fatty acids in a three (11-hydroxy-5,12,14-icosatrienoic acid) to one (15-hydroxy-5,11,13-icosatrienoic acid) proportion. Indomethacin inhibited formation of both hydroxy fatty acids indicating that they are produced via cyclooxygenase. Both ethylenic acids were weak inhibitors of cyclooxygenase (substrate 20 μM arachidonic acid) (ID₅₀: 8.8 μM 20:3^{5,8,14; 11.2 μM} 20:3^5,11,14) but were inactive against lipoxygenase (RD₅₀ > 100 μM). Similarly, both acetylenic analogues were poor inhibitors of lipoxygenase (ID₅₀: 23.4 μM 20:3^5,8,14; 47.8 μM 20:3^5,11,14) but although 20:3^5,8,14 was inactive against cyclooxygenase (ID₅₀ > 100 μM) the 20:3^5,11,14 was a potent inhibitor (ID₅₀: 0.35 μM). The results are interpreted on the basis that hydrogen removal by the lipoxygenase is from C10 and by the cyclooxygenase from C13 but only in 20:3^5,11,14 are these hydrogens (C13) located at the center of a 1,4 pentadiene system (ethylenic) or a 1,4 pentadiyne system (acetylenic).     

Metabolism of icosa-5,11,14-trienoic acid in human platelets and the inhibition of arachidonic acid metabolism in human platelets by icosa-5,8,14-triynoic and icosa-5,11,14-triynoic acids

Two fatty acids differing from arachidonic acid in lacking one of the internal double bonds (20:35,8,14 and 20:35,11,14) and their 1-C14 and acetylenic analogues were synthesized. 20:35,8,14 was not metabolized by human platelets but 20:35,11,14 yielded a small amount (1.5% conversion) of two hydroxy fatty acids in a three (11-hydroxy-5,12,14-icosatrienoic acid) to one (15-hydroxy-5,11,13-icosatrienoic acid) proportion. Indomethacin inhibited formation of both hydroxy fatty acids indicating that they are produced via cyclooxygenase. Both ethylenic acids were weak inhibitors of cyclooxygenase (substrate 20 microM arachidonic acid) (ID50: 8.8 microM 20:35,8,14; 11.2 microM 20:35,11,14) but were inactive against lipoxygenase (ID50 greater than 100 microM). Similarly, both acetylenic analogues were poor inhibitors of lipoxygenase (ID50: 23.4 microM 20:35,8,14; 47.8 microM 20:35,11,14) but although 20:35,8,14 was inactive against cyclooxygenase (ID50 greater than 100 microM) the 20:35,11,14 was a potent inhibitor (ID50: 0.35 microM). The results are interpreted on the basis that hydrogen removal by the lipoxygenase is from C10 and by the cyclooxygenase from C13 but only in 20:35,11,14 are these hydrogens (C13) located at the center of a 1,4 cis cis pentadiene system (ethylenic) or a 1,4 pentadiyne system (acetylenic).