Availability Of Arachidonic Acid Research Articles

Prevention of Stress‐Induced Erosive Gastritis by Parenteral Administration of Arachidonic Acid

Stress-induced mucosal ulcerations are associated with a decreased synthesis of mucosal prostaglandin (PG) E2. This phenomenon is poorly understood. To investigate whether it is due to a decreased availability of the necessary substrates to the mucosa, four groups of 10 Holtzman rats were studied: group 1 received normal saline by intraperitoneal (ip) injection; group 2 also received ip normal saline, then were submitted to stress, by the cold restraint method; group 3 received a solution of arachidonic acid (AA) ip; and group 4 also received ip AA, then were submitted to stress. After sacrifice, the number of gastric ulcerations were counted and specimens of nonulcerated mucosa were assayed for PGE2 by high-performance liquid chromatography; the mean numbers of ulcers were 0, 5.8, 0.8, and 3 and the mean levels of PGE2 were 55, 41, 125, and 62 pg/mg of wet tissue for groups 1, 2, 3, and 4, respectively. It is concluded that parenteral administration of AA reduces but does not completely eliminate stress-induced gastric ulcerations and that the stressed animals synthesized half as much PGE2 as the nonstressed ones after ip administration of equal amounts of AA, suggesting that stress reduces the availability of AA to the gastric mucosa, possibly by vascular spasm.

Effects of moderate dietary supplementations with n — 3 fatty acids on macrophage and lymphocyte phospholipids and macrophage eicosanoid synthesis in the rat

The effects of a moderate dietary intake of n − 3 polyunsarurated fatty acids were determined on the eicosanoid synthesis by rat peritoneal macrophages: the availability of arachidonic acid (20:4 ( n − 6)) and eicosapentaenoic acid (20:5 ( n −3)) in the membrane phospholipids of peritoneal macrophages and splenic lymphocytes and the synthesis of the main eicosanoids were evaluated in parallel. The n− 6 n−3 ratio was decreased from 13.5 in the control diet rich in 18:2( n −6) to about 2 by an addition of either linseed oil providing 18:3( n −3) (linseed oil diet) or fish oil providing 20:5( n −3) and 22:6( n −3) (fish oil diet). The dietary linoleic acid content was at least 3.5% of energy in each diet. In peritoneal macrophage and splenic lymphocyte membrane phospholipids, arachidonic acid content was significantly decreased with the linseed oil and fish oil diets. 20:5( n −3) content was significantly raised up in the linseed oil group (3-fold in macrophage phospholipids) and more strikely in the fish oil group (7-fold). In response to a stimulation by the calcium ionophore A23187, peritoneal macrophages released 6-ketoPGFlα, TXB2, PGE2 and LTB4 and/or 5, but no or few peptidoleukotrienes. The linseed oil did not affect significantly the synthesis of these eicosanoids, except for LTB5, which was increased 2-fold. In contrast, the fish oil diet led to significant decreases in the productions of 6-ketoPGFlα and PGE2 (−50%) and LTB4 (−40%) and to a 10-fold increase in the release of LTB5. TXB2 was also decreased (−35%), but not significantly. These results and the parallel evolutions of the two ratios 20:4(n−6) 20:5(n−3) and LTB4/LTB5 showed that eicosanoid synthesis is greatly dependent on the relative availability of the two substrates in competition.

Arachidonate metabolism increases as rat alveolar type II cells differentiate in vitro

Rat type II alveolar epithelial cells are known to undergo morphological and functional changes when maintained in culture for several days. Having previously demonstrated that these cells can deacylate free arachidonic acid (AA) and metabolize it to products of the cyclooxygenase pathway, the present study was undertaken to determine whether in vitro differentiation was accompanied by alterations in the availability and metabolism of AA. We assessed the constitutive and ionophore A23187-induced deacylation and metabolism of endogenous AA, as well as the metabolism of exogenously supplied AA, in primary cultures of rat type II cells at days 2, 4, and 7 after isolation. Levels of free endogenous AA were increased at day 4, whereas eicosanoid synthesis, predominantly prostaglandin E2 and prostacyclin, increased markedly only at day 7. A similar time course of augmentation of prostanoid release was seen in response to exogenous AA. Type II cells cultured on fibronectin, intended to hasten cell flattening and spreading, demonstrated accelerated increases in available free AA in response to A23187; cells cultured on basement membrane derived from Engelbreth-Holm-Swarm mouse sarcoma, known to maintain the type II phenotype, exhibited diminished levels of available free AA. From these findings, we conclude that alterations in arachidonate metabolism are linked to alterations in cellular phenotype. The potentiation of eicosanoid synthesis accompanying in vitro differentiation suggests a possible role for the alveolar epithelium in the modulation of inflammation and fibrosis in the distal lung.

Sex related differences in platelet TXA 2 generation

Platelet lipid composition, 1–14C arachidonic acid (AA) metabolism by platelets (stimulated with thrombin), serum thromboxane (Tx)B 2 production and plasma lipid composition were investigated in 53 healthy females (18–45 years) and 65 males (19–45 years) with similar dietary habits. In males, serum TxB 2 production and cholesterol platelet membrane levels were found significantly higher (p<0.001 and p<0.05) than in females. No differences were observed between the two groups in the AA conversion through cyclo-oxygenase and lipoxygenase pathways or in the platelet phospholipid fatty acid composition. These findings indicate that in males the platelet proaggregatory capacity is greater than in females and the higher platelet TxB 2 production does not depend on a larger AA availability or on enzyme activation for its conversion. The increased TxB 2 production may be, at least in part, induced by functional differences such as a different membrane cholesterol content inducing, in its turn, an increased microviscosity and/or higher number of platelet receptors for thrombin.

Inhibition of prostacyclin formation by cyclosporin is not due to reduced availability of arachidonic acid in membrane phospholipids of cultured human endothelial cells

Our studies have shown that CS inhibits PGI2 production in HUVEC, that this inhibition is not overcome when exogenous AA is supplied, that the inhibitory action of CS is proximal to PGI2 synthetase and finally that there is abundant free AA available in membrane phospholipids of CS treated HUVEC [4,5]. In conclusion, CS does not appear to inhibit PGI2 synthesis by reducing the availability of free AA in the endothelial cell membrane. Although CS appears to inhibit cyclo-oxygenase, we can not exclude an additional effect on acyltransferase.

Production of prostaglandin E 2 by human amnion in vitro in response to addition of media conditioned by microorganisms associated with chorioamnionitis and preterm labor

To examine the potential role of bacterial infection in the cause of spontaneous preterm labor, human amnion cells in tissue culture were exposed to medium conditioned by culturing each of 21 microorganisms previously found in association with chorioamnionitis and preterm labor. At a final concentration of 0.1 % bacterial conditioned medium, a significant stimulation of prostaglandin E2 production from amnion cells was observed for this range of microorganisms. Conditioned medium obtained from culturing Bacteroides fragilis caused a dose-related increase in prostaglandin production, final concentrations of 0.02% to 0.1% being stimulatory but greater concentrations (0.1% to 10%) causing a progressive inhibition of prostaglandin synthesis. A similar concentration-related response in which stimulation was followed by inhibition occurred on addition of increasing concentrations of phosphilipase A2 to amnion cells. These data suggest that bacterial phospholipase may release arachidonic acid from amnion leading to prostaglandin E2 synthesis, but excessive addition of phospholipase and consequent increased arachidonic acid availability may give rise to substrate inhibition of cyclooxygenase enzyme and inhibit prostaglandin E2 synthesis. Overall it appears that a wide variety of microorganisms associated with preterm labor may secrete phospholipase, which liberates amnion arachidonic acid for conversion to the oxytocic agent prostaglandin E2.

Altered arachidonic acid metabolism during differentiation of the human monoblastoid cell line U937

The human cell line U937 was used as a model for differentiation along the mononuclear phagocyte lineage. Following treatment with the phorbol ester TPA, PGE 2 and TxB 2 secretion was induced 50–100-fold, and both PGF 2α and PGI 2 levels became detectable in the supernatant of TPA-differentiated U937 cells. The content of the prostaglandin precursor, arachidonic acid, remained unchanged in the cellular phospholipids of undifferentiated and TPA-differentiated U937 cells. Of the enzymes involved in the availability and metabolism of arachidonic acid, phospholipase A 2 activity was increased 2-fold in the membranes of TPA-differentiated U937 cells, whereas lysophosphatide acyltransferase activity remained unaltered. Cyclooxygenase activity, however, was enhanced 5–10-fold, which was due to enhanced expression of the enzyme as demonstrated by dot-blot analysis. The data suggest that the capacity to secrete prostaglandins is acquired during differentiation with TPA and results mainly from an increased Cyclooxygenase activity. Despite the capacity of TPA-differentiated U937 cells to synthesize prostaglandins, none of the known monocytic stimuli further stimulated prostaglandin secretion in TPA-differentiated U937 cells. Generation of leukotrienes appears to represent a later state in the differentiation along the monocyte-macrophage lineage, since neither LTB 4 nor cysteinyl-leukotrienes were detectable in the supernatant of either undifferentiated or TPA-differentiated U937 cells.

Enhanced Secretion of Prostaglandin E&lt;sub&gt;2&lt;/sub&gt; by Tissue-Fixed Macrophages in Colonic Carcinoma

Prostaglandin E2 (PGE2) secretion by peripheral blood and tissue-fixed macrophages from patients with colorectal carcinoma was assessed. There was no significant difference between PGE2 production by peripheral blood mononuclear cells between patients with colorectal carcinoma and normal controls. However, secretion of PGE2 by tissue-fixed macrophages from within the colorectal carcinomata in response to opsonised zymosan was significantly higher than in the uninvolved colonic tissue. PGE2 production by tissue-fixed macrophages from within colonic polyps was found to be normal. These results could not be explained on the basis of increased availability of substrate arachidonic acid since addition of excess arachidonic acid resulted in similar findings. The enhanced production of PGE2 correlated with Dukes staging but not the level of differentiation. The production of PGE2 from epithelial cells in response to ionophore A23187 was not significantly enhanced. Leukotriene B4 secretion by intestinal macrophages in response to opsonised zymosan was not significantly elevated in the colonic tumour tissue. Modulation of levels of prostaglandin production within colonic tumours may play a role in the rate of growth and vascularity of these tumours and in the regulation of the local immune response to malignancy.

Essential fatty acid deficiency delays the onset of puberty in the female rat.

This study assessed the effect of a dietary deficiency in the essential fatty acids (EFA) linoleic and linolenic acids on the onset of female puberty. EFA deficiency was produced in female rats by means of a semipurified diet and was biochemically documented by analyzing serum and erythrocyte fatty acid levels of more than 30 fatty acids, including all members of the n-6 and n-3 series. Levels of linoleic acid (18:2 n-6) and all n-6 derivatives, particularly arachidonic acid, were strikingly reduced. A less pronounced but clear-cut decrease in n-3 fatty acids, including docosahexaenoic acid (22:6 n-3) was also found. The times of puberty and first ovulation, as assessed by the ages at vaginal opening and first diestrus, were significantly delayed in EFA-deficient rats. The mechanisms underlying this delay appear to reside at both hypothalamic and ovarian sites. Simulation of preovulatory plasma estradiol (E2) levels via implantation of E2-containing Silastic capsules evoked a LH surge 30 h later in control juvenile rats, but not in EFA-deficient animals, indicating a delay in the development of the hypothalamic component of E2-positive feedback in the latter group. This delay appears to be due at least in part to reduced prostaglandin E2 (PGE2) synthesis, as the ability of the neurotransmitter norepinephrine to induce PGE2 release from median eminence nerve terminals was markedly reduced in EFA-deficient rats compared with that in controls. The decrease in hypothalamic PGE2 release was related to the EFA deficiency and not to reduced PG synthase activity, as determined by HPLC analysis of PG synthase products derived from exogenous [14C]arachidonic acid. Basal and hCG-stimulated PGE2 synthesis was also compromised in ovaries from EFA-deficient rats. Depressed gonadal function resulting from the EFA deficiency was further evidenced by a reduced gonadotropin receptor content, a blunted E2 response to hCG in vitro, and an increase in mean serum FSH levels. These results suggest that the delay in puberty resulting from EFA deficiency is due to a reduced availability of arachidonic acid for synthesis of bioactive metabolites. This results in delayed development of both the hypothalamic and ovarian components of the reproductive axis.

The influence of mono- and divalent cations on the cardiac metabolism of arachidonic acid

Our previous study indicated that, in the isolated rabbit heart, perfusion with Ca 2+ free Krebs Henseleit buffer (KHB) results in increased conversion of exogenous arachidonic acid to PGE 2 and 6-keto-PGF 1α, probably as the result of increased availability of substrate to cuclooxygenase. Since perfusion with Ca 2+ free buffer is known to cause alterations in the cardiac content of various mono- and divalent cations, the present study was performed to determine: a) The relationship between the conversion of exogenous arachidonic acid to prostaglandins and cardiac content of Na +, K +, Ca 2+ and Mg 2+; and b) Whether enhanced arachidonic acid conversion to prostaglandins during Caa 2+ free perfusion is due to reduced incorporation of this fatty acid into tissue lipids. Perfusion of the rabbit heart with Ca 2+ free buffer produced a significant reduction in the tissue content of Na +, K +, Ca 2+ and Mg 2+. However, the production of 6-keto-PGF 1α from exogenous arachidonic acid was linearly correlated with tissue Mg 2+. These observations, together with our finding that perfusion with Ca 2+ free KHB reduced the incorporation of [ 3H] arachidonic acid into tissue lipids, suggests that Ca 2+ free perfusion may, by reducing the activity of arachidonyl CoA synthetase (a Mg 2+ dependent enzyme), decrease the acylation of arachidonic acid into lipids, thus increasing the availability of arachidonic acid to cyclooxygenase.

Phospholipases and acyltransferases in macrophages

In contrast to many other cells, macrophages contain a phospholipase A2, which preferentially liberates arachidonic acid from the main phospholipids. In unstimulated macrophages this acylchain-specific phospholipase A2 is localized in the lipid-free cytosol and thus without function. After activation of protein kinase C with diacylglycerols, the cytosolic phospholipase A2 is translocated to cellular membranes. The same activator of protein kinase C causes an inhibition of the acyl-CoA: lysophosphatide acyltransferase. This enzyme regulates the availability of free arachidonic acid for eicosanoid synthesis by reacylation into phospholipids. Thus protein kinase C seems to regulate the level of free arachidonic acid by opposite effects on the two major enzymes, which are responsible for the control of free arachidonic acid.

Synergy of interleukin 1 (IL-1) with arachidonic acid and A23187 in stimulating PGE synthesis in human fibroblasts: IL-1 stimulates fibroblast cyclooxygenase

The stimulation of prostaglandin E (PGE) synthesis by interleukin 1 (IL-1) has important physiologic effects in many target tissues. The mechanisms(s) whereby IL-1 stimulates PGE synthesis is not well understood. Two alternative mechanisms have been postulated: hydrolysis of membrane phospholipid with release of arachidonic acid substrate and induction of cyclooxygenase activity. We examined these mechanisms in IL-1 stimulation of human dermal fibroblast PGE synthesis. IL-1 failed to induce release of previously incorporated radiolabeled arachidonic acid from fibroblasts under conditions where there was a 30-fold or greater stimulation of PGE synthesis. The calcium ionophore, A23187, gave much less stimulation of PGE synthesis while inducing 30–100% release of incorporated [ 14C]arachidonic acid. There was marked synergy between IL-1 and agents which increased availability of arachidonic acid. For example, the combination of IL-1 and A23187, used at concentrations where neither agent alone stimulated PGE synthesis, increased PGE synthesis from 3.1 to 22.5 ng/ml; similar synergy was seen between IL-1 and exogenous arachidonic acid. To examine a possible effect of IL-1 on cyclooxygenase synthesis, fibroblast cyclooxygenase was measured by radioimmunoassay. Following treatment with IL-1, fibroblasts demonstrated a two- to threefold increase in content of immunoreactive cyclooxygenase. These results suggest that IL-1 increases fibroblast PGE synthesis by a mechanism(s) other than making available increased membrane arachidonic acid, and that at least part of its effect may be mediated by induction of cyclooxygenase. Furthermore, the effect of IL-1 on fibroblast PGE synthesis is markedly potentiated by agents which increase available substrate.

Prostaglandin E2 inhibits and indomethacin and aspirin enhance, A23187‐stimulated leukotriene B4 synthesis by rat peritoneal macrophages

1. The calcium ionophore, A23187, stimulated leukotriene B4 (LTB4), thromboxane B2 (TxB2) and prostaglandin E2 (PGE2) synthesis by 4 day carrageenin-elicited rat peritoneal macrophages. 2. At concentrations of 2 x 10(-7)-2 x 10(-5) M indomethacin and aspirin enhanced A23187-stimulated LTB4 synthesis and inhibited PGE2 and TXB2 formation. 3. PGE2 inhibited A23187-stimulated LTB4 and TXB2 formation as well as the augmentation of LTB4 release caused by aspirin and indomethacin. However, PGE2 was ineffective when the cells were challenged with arachidonic acid (AA). 4. Dibutyryl adenosine 3':5'-cyclic monophosphate (db-cyclic AMP) partially inhibited A23187-stimulated LTB4 production. 5. Our results suggest that PGE2 inhibits macrophage LTB4 synthesis by limiting the availability of AA. Indomethacin and aspirin, possibly by removing the regulatory effect of PGE2, promote the synthesis of the pro-inflammatory LTB4.

Requirement and role of arachidonic acid in the differentiation of pre-adipose cells

The terminal adipose differentiation of Ob1771 cells, characterized by glycerol-3-phosphate dehydrogenase activity and triacylglycerol accumulation, was studied in serum-free hormone-supplemented medium containing growth hormone, tri-iodothyronine, insulin, transferrin and fetuin. Arachidonic acid was able to substitute for a crude adipogenic fraction isolated from fetal bovine serum but not for growth hormone or tri-iodothyronine. Arachidonic acid was also able to increase in a rapid and dramatic manner cyclic AMP production; moreover it was able to amplify the adipose conversion promoted by other agents elevating cyclic AMP concentrations and to induce inositol phospholipid breakdown. Both phorbol 12-myristate 13-acetate, a protein kinase C activator and ionomycin, a Ca2+-mobilizing agent, showed potent synergy with agents elevating cyclic AMP concentrations for the promotion of adipose conversion, whereas 8-bromo cyclic GMP and 4 alpha-phorbol 12,13-dibutyrate were ineffective. The triggering of both the cyclic AMP and inositol phospholipid pathways was accompanied by a single round of cell division, and within a few days all the cells became differentiated. Similar results were obtained, after exposure to arachidonic acid, with preadipose 3T3-F442A cells and with rat adipose precursor cells in primary culture. The availability of arachidonic acid from intracellular stores and/or of exogenous origin should play a major role for the onset of critical mitoses leading to terminal differentiation in pre-adipose cells.

Azathioprine's inhibitory effect on prostaglandin E 2 production is not via cyclooxygenase inhibition

Azathioprine, a widely used antimetabolite, is also known for its anti-inflammatory action in rheumatic disorders and in uveitis, an inflammation of the eye, both of which are associated with increased production of prostaglandin E2. Recently we demonstrated that prostaglandin E2 production by rabbit retina/choroid was inhibited by azathioprine and suggested that this inhibitory effect may underlie the drug's antiinflammatory action. In the present study we showed that azathioprine's inhibition of prostaglandin E2 synthesis by the rat retina/choroid was reversed by addition of arachidonic acid, indicating that inhibition occurred through lack of availability of arachidonic acid, similar to the mechanism underlying the inhibitory effect of the steroidal anti-inflammatory drugs on prostaglandin E2. This study rules out the possibility that azathioprine's suppressive activity is via inhibition of the cyclooxygenase pathway.

Lipid and glucose metabolism in human placental culture

Placental culture models have been used to increase the understanding of endocrinology and pathophysiology in pregnancy. This article describes glucose and lipid metabolism in several of these models. Of special interest is the availability of arachidonic acid for the production of prostanoids. Ten placentas were collected at the time of cesarean section in term pregnancies without labor. Minced villous tissue was incubated for 48 hours in media with a glucose concentration of 100, 200, or 500 mg/dl. Tissue was dispersed in the media or was left as a single clump during the incubation. Glucose levels in the culture media were measured at 8, 20, 32, and 48 hours. Tissue lipid levels were measured before and after incubation in seven placentas. At 8 hours, glucose utilization ranged from 2.38 +/- 0.40 to 9.44 +/- 1.22 mumol/gm tissue/hr (mean +/- SEM). By 48 hours the cumulative glucose utilization ranged from 1.56 +/- 0.09 to 6.87 +/- 0.38 mumol/gm tissue/hr. Tissue lipid analysis showed most of the fatty acids to be in the phospholipids initially (4477 +/- 179 micrograms/gm tissue). Subsequent to incubation for 48 hours, phospholipid levels fell to a range of 2686 +/- 90 to 3466 +/- 157 micrograms/gm tissue in various culture conditions (p less than 0.005 compared with initial values). Whereas phospholipid levels decreased during incubation, levels of triglycerides and nonesterified fatty acids increased significantly in placental tissue. Arachidonic acid, the precursor of prostaglandins, thromboxane, and prostacyclin, makes up about one quarter of the fatty acid in the initial placental phospholipid. Arachidonic acid follows the pattern of total fatty acids during incubation; it is released from phospholipid and is converted to nonesterified fatty acid and triglyceride. We may conclude from this study that each placenta has a unique glucose utilization rate and a unique capacity to produce triglyceride. In tissue culture, arachidonic acid is released to its nonesterified state much more quickly than it can be converted to prostanoids by cyclooxygenase. The choice of initial glucose concentration, tissue preparation (dispersed in media or left as single clump), and time of incubation all may determine the rate of glucose metabolism, the rate of phospholipid breakdown, the rate of triglyceride production, and the quantity of nonesterified arachidonic acid in placental tissue culture.

Enhancement of eicosanoid synthesis in mouse peritoneal macrophages by the organic mercury compound thimerosal.

Availability of the common precursor arachidonic acid represents the fundamental prerequisite of the cellular eicosanoid synthesis. The amount of free arachidonic acid is regulated not only by phospholipases, which liberate this polyunsaturated fatty acid from lipid pools, but also by the reacylating enzyme acylCoA:lysophosphatide acyltransferase. We have previously shown (Goppelt-Strübe, G., C.-F. Körner, G. Hausmann, D. Gemsa, and K. Resch. Control of Prostanoid Synthesis: Role of Reincorporation of Released Precursor Fatty Acids. Prostaglandins 32:373. 1986.) that the organic mercury compound thimerosal in murine peritoneal macrophages inhibits arachidonic acid reincorporation into cellular lipids, thereby leading to an enhanced prostanoid synthesis. In this report we show that the production of leukotriene C4 was also increased after the addition of thimerosal to mouse peritoneal macrophages in a time and dose dependent manner. Concomitantly, thimerosal led to a significant rise of the intracellular calcium concentration as measured by fura-2 fluorescence. Simultaneous addition of thimerosal and indomethacin or exogeneous arachidonic acid to the cells resulted in a synergistic enhancement of leukotriene C4 synthesis. On the other hand, another sulfhydryl group blocking agent, ethacrynic acid, was found to be ineffective in increasing leukotriene C4 levels even in combination with exogeneous arachidonic acid. Thimerosal therefore provides a helpful tool in studying the basic regulatory mechanisms of the cellular leukotriene synthesis.

Gingival tissue-produced inhibition of platelet aggregation and the loss of inhibition in streptozotocin-induced diabetic rats.

Addition of medium incubated with normal rat gingival tissue to platelet‐rich plasma inhibited ADP‐induced platelet aggregation. The ability of rat gingiva to produce activity inhibiting platelet aggregation was enhanced by the addition of arachidonic acid. Diabetic rat gingiva failed to inhibit platelet aggregation but did produce the anti‐platelet aggregating activity in the presence of arachidonic acid. Indomethacin blocked the production of anti‐platelet aggregating activity. There was no difference in conversion of [1‐14C]arachidonic acid to prostaglandins by normal and diabetic rat gingiva. These results suggest that an arachidonic acid metabolite released from gingiva during incubation inhibits platelet aggregation, and the synthesis of the metabolite is impaired in diabetic rat gingiva. A decrease in availability of arachidonic acid may be a causal factor of the defect in diabetic rat gingiva.

Platelet synthesis of cyclooxygenase and lipoxygenase products in type I and type II diabetes

In 24 type I and 22 type II diabetic patients without vascular complications and in 25 controls platelet thromboxane A2 (TxA2) and prostaglandin E2 (PGE 2) production (by radioimmunoassay-RIA) and 1–14C arachidonic acid (AA) metabolism (by high pressure liquid chromatography-HPLC) after thrombin stimulation were studied. Platelets both from type I and type II diabetics generated larger amounts of TxB2 (p < 0.001) and PGE 2 (p<0.005) than controls, independently of the presence of retinopathy. No significant differences in platelet AA uptake or metabolism via the cyclooxygenase (CO) route, after thrombin stimulation (5 NIH U/ml), were observed in diabetic patients: lipoxygenase metabolites were found to be slightly, but significantly decreased. A positive linear relationship (r=0.64, p < 0.001) was found between HbA-1c and TxB2 production, but not with fasting plasma glucose. These results indicate that metabolic alterations can affect platelet function independently of vascular complications. The absence of alterations in intraplatelet 1–14C AA metabolism via CO, in the presence of increased TxB2 and PGE 2 production from endogenous AA, suggests that the activation of CO is not the only possible mechanism of platelet activation and that probably an increased availability of platelet AA plays an important role in the enhanced platelet aggregation commonly found in diabetics.

Characteristics of bradykinin and TPA increases in the PGE2 levels of human urothelial cells.

Prostaglandins play a potential key role in the pathogenesis of urinary bladder cancer. Bradykinin and TPA increases in prostaglandin (PG)E2 levels were compared in primary cultures of human urothelial cells. Increased PGE2 levels were dependent upon the dose of TPA and were not apparent until 30-60 min after addition of TPA, with larger increases occurring between 60 and 120 min. Stimulation was inhibited by cycloheximide. Addition of arachidonic acid to TPA-stimulated cells increased PGE2 to a level similar to that seen in arachidonic acid-stimulated controls, and this level was not altered by cycloheximide. In contrast to TPA, the bradykinin-increased PGE2 levels were maximal at 5 min (the earliest time-point assessed) and were not inhibited by cycloheximide. Increases in PGE2 levels by both TPA and bradykinin required calcium. Excessive stimulation by TPA resulted in a desensitization to subsequent stimulation by TPA, but not bradykinin. Combination of TPA with bradykinin produced at least an additive effect on PGE2 levels. Both agonists increased the release of [3H]arachidonic acid over a time-course similar to their PGE2 response. Bradykinin and TPA appear to increase PGE2 levels by enhancing arachidonic acid availability through separate phospholipase pathways. Thus, human urothelial cells exhibit similar, but yet distinct profiles for prostaglandin stimulation by TPA and bradykinin.