The role of thrombocyte prostanoids and products secreted by dense granules in the platelet attachment, spreading and aggregation on collagen substrates

The role of platelet prostanoids and substances released from dense bodies (ADP and serotonin) in the initial attachment, spreading and aggregation of platelets on surfaces coated with I, III, IV and V genetic types of collagen was investigated. A positive linear correlation was found to exist between thrombi-like aggregate formation on collagen substrates and platelet prostanoid synthesis. No correlation was established between platelet aggregate formation and 14C-serotonin release. The cyclooxygenase inhibitor indomethacin and the antagonists of PG endoperoxides and TXA2 (13-APA and BM 13.177) completely block thrombi-like aggregate formation. Neither 13-APA nor BM 13.177 affect platelet spreading, while indomethacin inhibits this process by 25%. The ADP-scavenger CP/CPK inhibits platelet aggregation and spreading by 25-30%. The inhibitors of cyclooxygenase and CP/CPK do not influence the initial attachment of platelets. The data obtained suggest that thrombi-like aggregate formation on collagen substrates is mediated by the synthesis of PG endoperoxides and TXA2; however, in platelet spreading this synthesis plays a limited role. Spreading and aggregation of platelets on collagen substrates is only partly mediated by ADP and serotonin. Initial attachment of platelets does not depend on ADP and serotonin release and PG endoperoxide/TXA2 synthesis.     

Up-regulation of endothelial cyclooxygenase-2 and prostanoid synthesis by platelets. Role of thromboxane A2

Platelet-vascular endothelial cell interactions are central to the maintenance of vascular homeostasis. Thromboxane A2 (TXA2) and prostacyclin (prostaglandin (PG)I2) are the major products of cyclooxygenase (COX) metabolism by platelets and the vascular endothelium, respectively. Here we report the effects of platelet-endothelial interactions on human umbilical vein endothelial cells (HUVECs) COX-2 expression and prostanoid synthesis. Co-incubation of platelets with HUVECs resulted in a dose-dependent induction in COX-2 expression. This was accompanied by a relatively small increase in thromboxane B2 synthesis (2 ng) by comparison to the production of 6-keto-PGF1alpha and PGE2, which increased by approximately 14 and 12 ng, respectively. Abrogation of platelet-HUVEC interactions excluded direct cell-cell contact as a required event. Preincubation of HUVECs with SQ29548, a TXA2 receptor antagonist, dose-dependently inhibited platelet-induced COX-2 expression and prostanoid synthesis. Similarly, if platelet TXA2 synthesis was inhibited no induction of COX-2 was observed. Furthermore, a TXA2 analog, carbocyclic TXA2, induced HUVEC COX-2 expression and the synthesis of 6-keto-PGF1alpha and PGE2. This was also associated with an increase in the expression and activity of PGI synthase and PGE synthase but not TX synthase. Platelet co-incubation (or TXA2) also selectively activated the p44/42 mitogen-activated protein kinase pathway to regulate HUVEC COX-2 expression. Thus it seems that platelet-derived TXA2 can act in a paracrine manner to up-regulate endothelial COX-2 expression and PGI2 synthesis. These observations are of particular importance given the recent observations regarding selective COX-2 inhibitors and the suppression of PGI2 synthesis.     

Release of tumor necrosis factor-alpha and prostanoids in whole blood cultures after in vivo exposure to low-dose aspirin

BACKGROUND: The preventive effect of low-dose aspirin in cardiovascular disease is generally attributed to its antiplatelet action caused by differential inhibition of platelet cyclooxygenase-1. However, there is evidence that aspirin also affects release of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha). It is not known whether this is caused by direct action on the cytokine pathway or indirectly through cyclooxygenase inhibition and altered prostanoid synthesis, or both. METHODS: We assessed the capacity of lipopolysaccharide-activated leukocytes in whole blood cultures of eight healthy subjects following a single oral dose of 80 mg aspirin to release TNF-alpha, prostanoid E2 (PGE2) and prostanoid I2 (PGI2), and thromboxane A2 (TXA2). TNF-alpha and prostanoids were determined by enzyme-linked immunoassays. RESULTS: In seven subjects, TNF-alpha release in blood cultures decreased 24h after intake of aspirin. The effect of aspirin on prostanoid release was assessed in three individuals: PGE2 increased in all subjects, PGI2 increased in two and remained unchanged in one, and TXA2 was reduced in two and unchanged in one individual The presence of DFU, a specific inhibitor of cyclooxygenase 2, did not affect the reduction of TNF-alpha release by aspirin, but abolished prostanoid production in all three individuals. Conclusion: The capacity of activated leukocytes to release TNF-alpha is reduced by ingestion of low-dose aspirin, independent of changes in prostanoid biosynthesis.     

NSAIDs induce both necrosis and apoptosis in guinea pig gastric mucosal cells in primary culture

A major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin is gastropathy. In this study, we examined, using guinea pig gastric mucosal cells in primary culture, how NSAIDs damage gastric mucosal cells. The short-term treatment of cells with high concentrations of indomethacin decreased cell viability in the absence of apoptotic DNA fragmentation, chromatin condensation, or caspase activation. Cells lost membrane integrity with this short-term indomethacin treatment, suggesting that indomethacin induced necrosis under these conditions. In contrast, the long-term treatment of cells with low concentrations of indomethacin decreased cell viability and was accompanied by apoptotic DNA fragmentation, chromatin condensation, and caspase activation. Pretreatment of cells with inhibitors of caspases or protein synthesis suppressed cell death caused by long-term indomethacin treatment, suggesting that apoptosis was induced when the inhibitors were not present. These results imply that NSAIDs cause gastric mucosal damage through both necrosis and apoptosis of gastric mucosal cells.     

Cyclooxygenase-2 selective and nitric oxide-releasing nonsteroidal anti-inflammatory drugs and gastric mucosal responses.

Occurrence of gastrointestinal damage and delayed healing of pre-existing ulcer are commonly observed in association with clinical use of nonsteroidal antiinflammatory drugs (NSAIDs). We examined the effects of NS-398, the cyclooxygenase (COX)-2 selective inhibitor, and nitric oxide (NO)- releasing aspirin (NCX-4016) on gastric mucosal ulcerogenic and healing responses in experimental animals, in comparison with those of nonselective COX inhibitors such as indomethacin and aspirin. Indomethacin and aspirin given orally were ulcerogenic by themselves in rat stomachs, while either NS-398 or NCX-4016 was not ulcerogenic at the doses which exert the equipotent antiinflammatory action with indomethacin or aspirin. Among these NSAIDs, only NCX-4016 showed a dose-dependent protection against gastric lesions induced by HCl/ethanol in rats. On the other hand, the healing of gastric ulcers induced in mice by thermal-cauterization was significantly delayed by repeated administration of these NSAIDs for more than 7 days, except NCX-4016. Gastric mucosal prostaglandin contents were reduced by indomethacin, aspirin and NCX-4016 in both normal and ulcerated mucosa, while NS-398 significantly decreased prostaglandin generation only in the ulcerated mucosa. Oral administration of NCX-4016 in pylorus-ligated rats and mice increased the levels of NO metabolites in the gastric contents. In addition, both NS-398 and NCX-4016 showed an equipotent anti-inflammatory effect against carrageenan-induced paw edema in rats as compared with indomethacin and aspirin. These results suggest that both indomethacin and aspirin are ulcerogenic by themselves and impair the healing of pre-existing gastric ulcers as well. The former action is due to inhibition of COX-1, while the latter effect may be accounted for by inhibition of COX-2 and mimicked by NS-398, the COX-2 selective NSAID. NCX-4016, despite inhibiting both COX-1 and COX-2, protects the stomach against damage and preserves the healing response of gastric ulcers, probably because of the beneficial action of NO.     

Regulation of microvascular prostacyclin and thromboxane with inhibitors of arachidonic acid metabolism

The levels of the stable degradation products of prostacyclin (PGI2) and thromboxane A2 (TXA2): 6-oxo-prostaglandin E1 alpha (6-oxo-PGE1 alpha) and thromboxane B2 (TXB2) respectively were determined in the effluent of the rabbit epigastric skin flap after infusion of exogenous arachidonic acid. The blood to the flap passes through the microcirculation and thus the changes in eicosanoid biosynthesis in this part of the vasculature were recorded. The aim was to use inhibitors of arachidonic acid metabolism to increase the PGI2/TXA2 ratio. This may be potentially beneficial to ischaemic skin flaps by reducing platelet aggregation associated with damaged microvascular endothelium, overcoming vasospasm and increasing microvascular blood flow. Increased PGI2/TXA2 ratios (up to 5-fold) were best achieved using TXA2 synthetase inhibitors such as dazoxiben hydrochloride. These were significantly more potent than the phosphodiesterase inhibitor dipyridamole, and the lipoxygenase inhibitor Bay g6575. No increase in blood flow was achieved. The cyclooxygenase inhibitor indomethacin did slow the blood flow at high concentrations (above 10(-5) M), and inhibited both PGI2 and TXA2 synthesis. Approximately 2-fold higher concentrations of dazoxiben hydrochloride and dipyridamole were required to produce the same TXA2 synthetase inhibition in the flap microvasculature in vivo compared with platelets in vitro.     

Effect of indomethacin, tiaprofenic acid and dicrofenac on rat gastric mucosal damage and content of prostacyclin and prostaglandin E2

Gastric ulcerogenicity and depletion of endogenous prostaglandins (PGs) content induced by tiaprofenic acid, dicrofenac and indomethacin were examined using the same antiinflammatory effective doses. Male Wistar rats were given each of these drugs intragastrically 24, 18, and 3 hrs before sacrifice in the following doses (mg/kg): indomethacin (0.8, 4 and 20); tiaprofenic acid (1.2, 6 and 30); dicrofenac (0.8, 4 and 20). Endogenous prostacyclin (PGI2) and PGE2 in fundic mucosa were determined by radioimmunoassay. The three compounds produced fundic mucosal lesions in a dose-dependent manner. However, tiaprofenic acid and dicrofenac were both less potent than indomethacin in producing gastric mucosal lesions at similar antiinflammatory doses. Mucosal PGE2 content was abolished by the three compounds in the following doses (mg/kg): indomethacin (4 and 20); tiaprofenic acid (6 and 30); dicrofenac (20). Mucosal PGI2 was maintained around 50% of the control value in rats given tiaprofenic acid in a dose of 6 mg/kg or dicrofenac in a dose of 4 mg/kg, while indomethacin in a dose of 4 mg/kg markedly reduced mucosal PGI2 to 17% of the control value. In larger doses, tiaprofenic acid and dicrofenac were also significantly less potent in reducing mucosal PGI2 than indomethacin. These results suggest that the difference in ulcerogenicity between indomethacin and the other two compounds was closely related to their potency in decreasing PGI2 in the gastric (fundic) mucosa.     

Gastric mucosal cell model for estimating relative gastrointestinal toxicity of non-steroidal anti-inflammatory drugs

The study objective was to characterize the AGS human gastric mucosal cell line as a model for estimating gastrointestinal toxicity of COX-inhibiting compounds. Rofecoxib, celecoxib, nimesulide, ibuprofen, indomethacin, aspirin, salicylic acid, naproxen and acetaminophen were tested for inhibition of COX-2-mediated prostaglandin E2 synthesis in A549 and AGS cells. The IC50 ratio AGS/A549 was calculated as an estimate of the therapeutic index (TI) for gastrointestinal toxicity. Calculated IC50 values of non-steroidal anti-inflammatory drugs (NSAIDs) in A549 cells were in excellent agreement with published values (r = 0.996; P < 0.005). Calcium ionophore induction of arachidonic acid release in AGS cells provided TI similar to those using platelets and A549 cells (r = 0.918; P < 0.01). The AGS/A549 model exhibited lower TI than the platelet/A549 model. Spearman ranking correlated clinical NSAID gastropathy with lower AGS TI values. The AGS cell line has excellent potential to serve as a model for assessing the gastrointestinal effects of COX-inhibiting compounds.     

Salicylic acid blocks indomethacin-induced cyclooxygenase inhibition and lesion formation in rat gastric mucosa

Salicylic acid has been shown to decrease gastric mucosal lesions induced by indomethacin in the rat. In vitro, it has also been shown to counteract the inhibitory effect of indomethacin and aspirin on the cyclooxygenase enzyme system in seminal vesicle microsomes and in platelets and vascular tissue. The hypothesis that the mechanism of salicylic acid "protection" against indomethacin-induced gastric lesions involves interference with indomethacin-induced mucosal cyclooxygenase inhibition was tested. Male, fasted rats were treated with intragastric salicylic acid in doses of 50, 100, 200, 300, or 400 mg/kg concomitantly with a sc injection of 20 mg/kg of indomethacin. Gastric mucosal lesions and mucosal cyclooxygenase activity (as measured by ex vivo prostaglandin F2 alpha synthesis) were examined 3 hr later. Intragastric salicylic acid, 200-400 mg/kg, significantly reduced indomethacin-induced lesion formation, while counteracting significantly indomethacin inhibition of prostaglandin synthesis. Salicylic acid alone did not significantly change cyclooxygenase activity. It is concluded that topical salicylic acid can decrease indomethacin-induced gastric mucosal lesion in the rat, in part, by counteracting the inhibitory effect of indomethacin at the cyclooxygenase level.     

Adaptive cytoprotection against alcohol injury in the rat stomach is not due to increased prostanoid synthesis

This study evaluated the effects of 25% ethanol, a mild irritant, on endogenous prostanoid synthesis in the rat stomach before and after exposure to oral 100% ethanol. Rats received water or 25% ethanol orally. After 15 min, a portion of each group was sacrificed and the remaining animals treated with 100% ethanol prior to sacrifice one minute later. Microsomal membrane fractions were prepared from the glandular gastric mucosa in all groups and incubated with 14C arachidonic acid in the presence of cofactors. Endogenous mucosal prostanoid synthesis was analyzed by radiochromatography and results correlated with the presence or absence of gastric injury macroscopically. Prostanoids measured included PGI2, PGF2 alpha, PGE2, PGD2, PGA2, and thromboxane A2. Additional experiments were performed in like manner to those just described with the exception that indomethacin (5 mg/kg intraperitoneally) pretreatment was rendered. Stomachs exposed to water or 25% ethanol alone demonstrated a modest and equivalent level of synthesis of all prostanoids measured. Exposure to 100% ethanol (with and without mild irritant pretreatment) significantly increased prostanoid synthesis (especially PGI2, PGF2 alpha, and PGE2) compared with stomachs exposed to water or 25% ethanol alone; only mild irritant treated mucosa was protected from injury by 100% ethanol. Indomethacin pretreatment reversed the increased prostanoid synthesis in mucosa exposed to 100% ethanol, with or without mild irritant pretreatment, and partially reversed the protective effect of 25% ethanol. Other experiments using tissue slices in which perturbations in mucosal levels of prostanoids were measured by radioimmunoassay under identical experimental conditions exhibited similar results. These data dispute the notion that adaptive cytoprotection is mediated by increased endogenous prostanoid synthesis. The partial reversal of this process by indomethacin was most likely secondary to some other action of this agent, such as a reduction in gastric blood flow, rather than direct effects on prostanoid synthesis.     

Prostaglandin production in cultured gastric mucosal cells: role of cAMP on its modulation

The effect of cAMP on prostaglandin production may depend on cell types. To clarify the relationship between PG and cAMP, we examined arachidonate's effects on PG synthesis and intracellular cAMP accumulation in monolayers of rat gastric mucosal cells. These cells produced PGE2, PGI2 and thromboxaneA2 (TXA2) in amounts of 316 +/- 18, 100 +/- 7 and 30 +/- 5 pg per 10(5) cells in 10 min, respectively, in response to 10 microM arachidonic acid (AA). The production of these PG, however, leveled off subsequently. Cells initially exposed to AA responded poorly to a subsequent stimulation by AA. AA simultaneously stimulated intracellular cAMP accumulation; this stimulatory effect on cAMP production was abolished by the pretreatment with indomethacin. Nevertheless, the pretreatments with dibutyryl cAMP (0.1-5 mM) did not alter the amount of subsequent AA-induced PGE2 production. Furthermore, the preincubation with 1mM isobutyl methyl xanthine also failed to affect PGE2 synthesis, while it increased intracellular cAMP accumulation. Our studies suggest AA stimulates intracellular cAMP formation in cultured gastric mucosal cells, linked with conversion of AA to cyclooxygenase metabolites, AA-induced PG production is limited in these cells, and it seems, however, unlikely that intracellular cAMP modulates AA metabolism to PG.     

Prostaglandin and thromboxane synthesis by tissue slices from human aortic aneurysms

Sliced portions of the walls of human aortic aneurysms were incubated with extracts of human plasma and serum to determine the profile of prostanoid production. 6-Oxo-prostaglandin (PG) F1 alpha, PGE2, PGF2 alpha, and thromboxane (TX) B2 were measured by gas chromatography/electron capture mass spectrometry. 6-Oxo-PGF1 alpha, the stable hydrolysis product of PGI2, was the major cyclooxygenase product but substantial amounts of TXB2 (the hydrolysis product of TXA2), with smaller amounts of PGE2 and PGF2 alpha were also synthesised. These prostanoids could contribute to the response of the vascular wall to injury, thereby influencing the disease process. Serum extracts stimulated PGI2 and TXA2 synthesis, probably as a result of their Ca2+ content.     

Roles of cyclooxygenase (COX)-1 and COX-2 in prostanoid production by human endothelial cells: selective up-regulation of prostacyclin synthesis by COX-2

The two cyclooxygenase (COX) isoforms, COX-1 and COX-2, both metabolize arachidonic acid to PGH(2), the common substrate for thromboxane A(2) (TXA(2)), prostacyclin (PGI(2)), and PGE(2) synthesis. We characterized the synthesis of these prostanoids in HUVECs in relation to COX-1 and COX-2 activity. Untreated HUVEC expressed only COX-1, whereas addition of IL-1beta caused induction of COX-2. TXA(2) was the predominant COX-1-derived product, and TXA(2) synthesis changed little with up-regulation of COX-2 by IL-1beta (2-fold increase). By contrast, COX-2 up-regulation was associated with large increases in the synthesis of PGI(2) and PGE(2) (54- and 84-fold increases, respectively). Addition of the selective COX-2 inhibitor, NS-398, almost completely abolished PGI(2) and PGE(2) synthesis, but had little effect on TXA(2) synthesis. The up-regulation of COX-2 by IL-1beta was accompanied by specific up-regulation of PGI synthase and PGE synthase, but not TX synthase. An examination of the substrate concentration dependencies showed that the pathway of TXA(2) synthesis was saturated at a 20-fold lower arachidonic acid concentration than that for PGI(2) and PGE(2) synthesis. In conclusion, endothelial prostanoid synthesis appears to be differentially regulated by the induction of COX-2. The apparent PGI(2) and PGE(2) linkage with COX-2 activity may be explained by a temporal increase in total COX activity, together with selective up-regulation of PGI synthase and PGE synthase, and different kinetic characteristics of the terminal synthases. These findings have particular importance with regard to the potential for cardiovascular consequences of COX-2 inhibition.     

Engineering of a protein with cyclooxygenase and prostacyclin synthase activities that converts arachidonic acid to prostacyclin

Prostacyclin (PGI2), a vascular protector with vasodilation and antithrombotic properties, is synthesized by coupling reactions of cyclooxygenase (COX, the first enzyme) with PGI2 synthase (PGIS, the second enzyme) using arachidonic acid (AA) as an initial substrate. The first COX product, prostaglandin H2 (PGH2) is also a command substrate for other prostanoid enzymes that produce distinct eicosanoids, such as thromboxane A2 (TXA2). The actions of TXA2 to cause vasoconstriction and platelet aggregation oppose the vasodilatory and anti-aggregatory effects of PGI2. Specifically upregulating PGI2 biosynthesis is an ideal model for the prevention and treatment of the TXA2-mediated thrombosis involved in strokes and myocardial infarctions. Here, we report that a single protein was constructed by linking COX-2 and PGIS together to form a new fusion enzyme through a transmembrane domain with 10 or 22 residues. The engineered protein expressed in HEK293 and COS-7 cells was able to continually convert AA to prostaglandin (PG) G2 (catalytic step 1), PGH2 (catalytic step 2), and PGI2 (catalytic step 3). The studies first demonstrate that a single protein with three catalytic functions could directly synthesize PGI2 from AA with a Km of approximately 3.2 microM. Specific upregulation of PGI2 biosynthesis through expression of the engineered single protein in the cells has shown strong activity in inhibiting platelet aggregation induced by AA in vitro, which creates a great potential for the fusion enzyme to be used as one of the new therapeutic interventions for strokes and heart attacks. The studies have also provided a model linking COX with its downstream enzymes to specifically regulate biosynthesis of eicosanoids which have potent biological functions.     

Effect of taurine on arterial, uterine and cardiac PGI2 and TXA2 synthesis in the rat

The influence of taurine (in drinking water for 6 weeks) on PGI2 and TXA2 synthesis by some female rat organs was investigated using radioimmunoassay and platelet antiaggregatory bioassay. Taurine 100 and 200 mg/kg/day increased aortic PGI2 release from 0.59 +/- 0.04 (control) to 0.85 +/- 0.05 and 1.01 +/- 0.06 ng/mg, respectively and that by the myometrium from 0.24 +/- 0.02 (control) to 0.38 +/- 0.01 and 0.50 +/- 0.04 ng/mg wet tissue, respectively (P less than 0.05, n = 6). It did not affect PGI2 and TXA2 production in the heart or TXA2 in the aorta. Taurine 200 mg/kg depressed uterine TXA2 synthesis from 148.6 +/- 9.8 (control) to 85.4 +/- 6.8 pg/mg (P less than 0.05, n = 6). Furthermore taurine 0.4 and 0.8 mM in vitro stimulated PGI2 release by the myometrial and aortic tissues from pregnant rats. The stimulant effect of taurine on PGI2 may be related to its antioxidant effect whereas its inhibitory effect on uterine TXA2 may result from direction of synthesis towards PGI2. It is concluded that endogenous taurine may participate in regulation of PGs synthesis and that prostanoids may contribute to its known actions. On broad basis, taurine-induced release of PGI2 may prove of potential value in those ailments characterised by deficiency in PGI2 release.     

Gastrotoxic activity and inhibitory effects on gastric mucosal PGE2 production with different non-steroidal anti-inflammatory drugs: Modifications induced by pretreatment with zinc acexamate

Gastrotoxic activities of different non-steroidal anti-inflammatory drugs (NSAIDs) (diclofenac, indomethacin, ketoprofen, naproxen and piroxicam) administered per os were compared with their ability to inhibit gastric prostaglandin E₂ (PGE₂) synthesis in the rat. In a parallel study, effects of pretreatment with zinc acexamate (ZAC) were also assessed. NSAIDs invariably caused gastric mucosal damage and a decrease of PGE₂ levels. A good correlation between the decrease of PGE₂ levels and the index of gastric lesion (r = 0.41; p < 0.021) was observed when results obtained with the different NSAIDs were pooled. ZAC pretreatment significantly decreased the overall severity of lesions induced by NSAIDs. However, no correlation between gastric lesion index and depletion of PGE₂ gastric levels was observed after treatment with ZAC (r = 0.012; p < 0.948). These data corroborate the hypothesis that preservation of the capability to synthesize endogenous PGs is of critical importance in the maintenance of gastric mucosal integrity. The gastroprotective action observed with ZAC involves alternative mechanisms other than modification of PGE₂ levels.     

Plasma prostaglandin levels and circulating fuel levels in rats with diabetic ketoacidosis: effects of cyclooxygenase inhibitors and of alpha and beta adrenergic blockade

We studied the effects of two structurally unrelated inhibitors of the fatty acid cyclooxygenase and of alpha and beta adrenergic blockade on the elevated plasma levels of 13,14-dihydro-15-keto-prostaglandin (PG)E2, 6-keto-PGF1 alpha and thromboxane (TX)B2, the stable derivatives of PGE2, PGI2 (prostacyclin) and TXA2, respectively, in rats with streptozotocin-induced diabetic ketoacidosis (DKA). Meclofenamic acid and indomethacin each produced a significant decrease in the elevated plasma levels of 13,14-dihydro-15-keto-PGE2, 6-keto-PGF1 alpha and TXB2. Phentolamine significantly reduced the plasma level of TXB2 but had no effect on the elevated circulating levels of glucose, free fatty acids, total ketones, 13,14-dihydro-15-keto-PGE2 or 6-keto-PGF1 alpha. Propranolol significantly reduced the elevated circulating levels of glucose, free fatty acids and total ketones but had no effect on the levels of the three prostaglandin derivatives. The ability of meclofenamic acid and indomethacin to reduce the plasma levels of 13,14-dihydro-15-keto-PGE2, 6-keto-PGF1 alpha and TXB2 confirms that the plasma levels of these three derivatives are elevated in rats with DKA. Since abnormalities in the production of PGI2 and perhaps other cyclooxygenase derivatives may contribute to the pathogenesis of certain important hemodynamic and gastrointestinal features of DKA, cyclooxygenase inhibitors may play a role in the management of selected patients with this disorder. Alpha adrenergic activity is essential for the maintenance of the elevated plasma TXB2 level in rats with DKA. The fall in the plasma TXB2 level during alpha adrenergic blockade appears to reflect inhibition of platelet aggregation and platelet TXA2 production, but other sources of the elevated plasma TXB2 level in DKA are not excluded. Beta adrenergic activity contributes to the maintenance of elevated circulating levels of glucose, free fatty acids and total ketones in experimental DKA but not to the elevated plasma levels of the prostaglandin derivatives.     

A copper-complex reduced gastric damage caused by acetylsalicylic acid and ethanol

We investigated the effect of oral administration of CuNSN, a bis(2-benzimidazolyl)thioether (see structure 1) on gastric lesions induced in rats by acetylsalicylic acid (ASA) or ethanol. The involvement of endogenous eicosanoids and nitric oxide in protection by CuNSN was evaluated with indomethacin and N^G-nitro-L-arginine (L-NNA), inhibitors of prostaglandin and NO synthesis respectively. L-arginine and its enantiomer D-arginine were also used. Pretreatment with graded doses of CuNSN inhibited ASA- and ethanol-induced mucosal injury. CuNSN increased PGE₂ output in rat ex vivo gastric mucosal pieces after administration of 100 mg/kg of ASA. Pretreatment with indomethacin only partially counteracted the protective activity of CuNSN against ethanol-induced damage. L-NNA did not attenuate the protection by CuNSN, which was reduced but not prevented by indomethacin, suggesting that prostanoids contribute to the CuNSN protective effect, together with some mechanism(s) other than NO synthesis.     

Differential effects of megavitamin E on prostacyclin and thromboxane synthesis in streptozotocin-induced diabetic rats

Diabetic subjects tend to develop microvascular complications believed to be due to platelet hyperaggregability. This increased platelet sensitivity is though to be the result of an imbalance of PGI2 and TXA2 production in diabetes. This study sought to determine whether megavitamin E supplementation could restore PGI2/TXA2 balance in streptozotocin-diabetic rats. Endogenous release of PGI2 by isolated aorta, determined via radioimmunoassay of its stable metabolite, 6-keto-PGF1 alpha, was significantly greater (P less than 0.05) in rats receiving 100x the normal vitamin E requirement than in untreated diabetic rats. PGI2 synthesis was negatively correlated with plasma glucose levels (r = -0.87, P less than 0.05) in non-fasted rats at sacrifice. Vitamin E supplementation, at both the 10x and the 100x level, significantly depressed (P less than 0.05) thrombin-stimulated synthesis of TXA2 in washed platelet. PGI2 and TXA2 production were expressed as a ratio. Megavitamin E therapy appears to increase this ratio over that seen in the diabetic animal. The data suggest that vitamin E, at high levels, exerts an ameliorating influence of the PGI2/TXA2 imbalance of diabetes.     

Characterization of 11-HETE and 15-HETE, together with prostacyclin, as major products of the cyclooxygenase pathway in cultured rat aorta smooth muscle cells

Arachidonic acid is the precursor of several potent derivatives that regulate physiological functions in the cardiovascular system. Thromboxane (TXA2) and prostacyclin (PGI2) are synthesized by the cyclooxygenase enzyme. The proaggregatory and vasoconstrictive TXA2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of PGI2 synthesized by blood vessels. Arachidonic acid is also converted via a 5-lipoxygenase to leukotrienes, the vasoconstrictive components of SRSA. We have shown that this latter pathway is regulated by 15-HETE, a product of the 15-lipoxygenase present in lymphocytes. Confluent cultures of rat aorta smooth muscle cells (RSM) were superfused briefly with [14C]arachidonic acid. The products were isolated and analyzed by thin-layer chromatography-radioautography, high performance liquid chromatography, and gas-liquid chromatography-mass spectrometry. Prostacyclin (PGI2) was identified as the major product both by its biological properties in a platelet aggregation assay and by the mass spectrum of its tetra-trimethylsilylether-methyl ester derivative. Minor quantities of PGE2, PGD2, and PGF2 alpha were also synthesized. Three other compounds with chromatographic properties of mono-hydroxy eicosanoic acids were also formed in major amounts. These were shown to be cyclooxygenase products since their synthesis, together with that of prostacyclin, was blocked by the cyclooxygenase inhibitors aspirin (0.2 mM) and indomethacin (10 microM). Quantities of the hydroxy-eicosanoids were isolated from large scale incubations by silicic acid chromatography. Following methylation and reduction with platinum oxide/H2, the compounds were converted to their trimethylsilylether derivatives and analyzed by gas-liquid chromatography-mass spectrometry. The compounds were identified as 11-hydroxy-5,8,12,14-eicosatetraenoic acid (11-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), and hydroxy-5,8,10-heptadeca-trienoic acid (HHT) by simultaneous ion monitoring of characteristic ions at M/e ratios of 287, 258, 229 for 11-HETE and 343, 314, 173 for 15-HETE, and by comparison with the mass spectra of authentic samples. Rat smooth muscle cells, prelabeled by 24-hour incubation with [14C]arachidonic acid, released large amounts of prostacyclin together with enhanced amounts of 11- and 15-HETE in response to physiological levels of thrombin (0.5-5 units/ml). These experiments demonstrate that, in addition to the thromboxane antagonist prostacyclin, vascular smooth muscle cells produce significant quantities of the leukotriene inhibitor 15-HETE via the cyclooxygenase pathway in response to physiological stimuli such as thrombin. The release of both prostacyclin and 15-HETE by vascular smooth muscle cells may thus play an important role in vascular homeostasis.