Plasmenylethanolamine is the major storage depot for arachidonic acid in rabbit vascular smooth muscle and is rapidly hydrolyzed after angiotensin II stimulation

The present study demonstrates that rabbit aortic intimal smooth muscle cells contain the majority of their endogenous arachidonic acid mass in plasmenylethanolamine molecular species. To demonstrate the potential significance of these plasmenylethanolamines as substrates for the smooth muscle cell phospholipases that are activated during agonist stimulation, aortic rings were prelabeled with [3H]arachidonic acid and stimulated with angiotensin II. Although the specific activities of the choline and inositol glycerophospholipid pools were similar after the labeling interval, ethanolamine glycerophospholipids had a specific activity of only 20% of the specific activity of choline and inositol glycerophospholipids. Despite the marked disparity in the specific activities of these three phospholipid classes, angiotensin II stimulation resulted in similar fractional losses (35-41%) of [3H]arachidonic acid from vascular smooth muscle choline, ethanolamine, and inositol glycerophospholipid classes. Reverse-phase HPLC demonstrated that greater than 60% of the [3H]arachidonic acid released from ethanolamine glycerophospholipids after angiotensin II stimulation originated from plasmenylethanolamine molecular species. Taken together, the results demonstrate that the major phospholipid storage depot for arachidonic acid in vascular smooth muscle cells are plasmenylethanolamine molecular species which are important substrates for the phospholipase(s) that are activated during agonist stimulation.     

Phospholipase A2 and phospholipase C are activated by distinct GTP-binding proteins in response to alpha 1-adrenergic stimulation in FRTL5 thyroid cells.

In FRTL5 rat thyroid cells, norepinephrine, by interacting with alpha 1-adrenergic receptors, stimulates inositol phosphate formation, through activation of phospholipase C, and arachidonic acid release. Recent studies have shown that GTP-binding proteins couple several types of receptors to phospholipase C activation. The present study was undertaken to determine whether GTP-binding proteins couple alpha 1-adrenergic receptors to stimulation of phospholipase C activity and arachidonic acid release. When introduced into permeabilized FRTL5 cells, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]), which activates many GTP-binding proteins, stimulated inositol phosphate formation and arachidonic acid release. Neomycin inhibited GTP[gamma-S]-stimulated inositol phosphate formation but was without effect on GTP[gamma-S]-stimulated arachidonic acid release, suggesting that separate GTP-binding proteins mediate each process. In addition, pertussis toxin inhibited norepinephrine-stimulated arachidonic acid release but not norepinephrine-stimulated inositol phosphate formation. Norepinephrine-stimulated arachidonic acid release but not inositol phosphate formation was also inhibited by decreased extracellular calcium and by TMB-8, suggesting a role for a phospholipase A2. To confirm that arachidonic acid was released by a phospholipase A2, FRTL5 membranes were incubated with 1-acyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine. GTP[gamma-S] slightly stimulated arachidonic acid release, whereas norepinephrine acted synergistically with GTP[gamma-S] to stimulate arachidonic acid release. The results show that phospholipase C and phospholipase A2 are activated by alpha 1-adrenergic agonists. Both phospholipases are coupled to the receptor by GTP-binding proteins. That coupled to phospholipase A2 is pertussis toxin-sensitive, whereas that coupled to phospholipase C is pertussis toxin-insensitive.     

Evidence supporting a correlation between arachidonic acid release and prolactin secretion from GH3 cells

In this study, pharmacological agents that alter phospholipase A2 activity were examined for their effects on PRL release and arachidonic acid mobilization in GH3 cells, a pituitary tumor cell line. Stimulators of phospholipase A2 activity, melittin and mastoparan, increased PRL release during short term incubation. This stimulation was reduced by carbachol, a cholinergic receptor ligand that inhibits PRL release from GH3 cells. Melittin also caused release of [3H]arachidonic acid that had previously been incorporated into phospholipids. Increased levels of free [3H]arachidonic acid in the medium were associated with a loss of radiolabel from the phospholipid fraction of the cells. The [3H]arachidonic acid in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol was reduced during melittin exposure. In contrast, two inhibitors of phospholipase A2, dibromoacetophenone (BAP) and U10029A, inhibited spontaneous PRL release. BAP also decreased basal release of [3H]arachidonic acid, blocked melitin-induced PRL secretion, and inhibited melittin-induced [3H] arachidonic acid release. Exogenous arachidonic acid at doses from 10 nM to 1 microM stimulated PRL secretion. The phospholipase A2 inhibitor BAP blocked TRH- and vasoactive intestinal peptide-induced PRL release, whereas U10029A blocked cAMP-induced and blunted TRH- and vasoactive intestinal peptide-induced PRL release. The hydrolysis of membrane phospholipids generating free arachidonic acid and lysophospholipid under our experimental conditions correlated with PRL secretion in GH3 cells. Addition of arachidonic acid to the culture medium stimulated PRL secretion. These data suggest that release of arachidonic acid and its subsequent actions may participate in the intracellular regulation of PRL secretion.     

Multiple signaling pathways of V1-vascular vasopressin receptors of A7r5 cells.

We explored the nature and time course of the multiple signal transduction pathways for V1-vascular vasopressin (AVP) receptors of A7r5 aortic smooth muscle cells in culture by using radioligand binding techniques, intracellular calcium monitoring, and polyphosphoinositide and phospholipid analyses. V1-vascular AVP receptors of A7r5 cells were characterized by the agonist radioligand [3H]AVP and the antagonist radioligand [3H]d(CH2)5Tyr(Me)AVP. Affinity and capacity of agonist but not antagonist binding were modulated by MgCl2 and aluminum fluoride, suggesting that the receptors are coupled to a guanine nucleotide regulatory protein. In fura-2-loaded A7r5 cells, AVP induced within seconds a dose-dependent increase of free intracellular Ca++ ([Ca++]i) consisting of a rapid transient spike and a sustained increase lasting for 3-5 min. The baseline [Ca++]i was 136 +/- 18 nM, the maximum [Ca++]i response to AVP was 1,582 +/- 297 nM, and AVP ED50 was 1.87 +/- 0.15 nM. Diverse experiments performed with EGTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethylester, Mn++, ionomycin, terbutylbenzo hydroquinone, and nicardipine suggested that the initial spike resulted from both intracellular Ca++ release from the endoplasmic reticulum and extracellular Ca++ influx, whereas the sustained phase depended on dihydropyridine-insensitive extracellular Ca++ influx. Experiments done with indomethacin and arachidonic acid indicated that AVP-induced extracellular Ca++ influx was in part dependent on phospholipase A2 activation. In [3H]myoinositol and [3H]arachidonate-labeled A7r5 cells, AVP stimulated inositol 1,4,5 trisphosphate and 1,2 diacylglycerol production via activation of phospholipase C. Also, AVP stimulated a transphosphatidylation reaction through activation of phospholipase D in A7r5 cells labeled with [3H]1-O-alkyl lysoglycerophosphocholine. Thus, the stimulation of V1-vascular AVP receptors of A7r5 cells triggers several signaling pathways. The immediate and transient [Ca++]i rise due to mobilization of intracellular and extracellular Ca++ is associated with the activation of phospholipases A2 and C, and the sustained activation of phospholipase D.     

Effect of various plasminogen activators on prostacyclin synthesis in cultured vascular cells

In this study, we examined the effects of various plasminogen activators on arachidonic acid release and prostacyclin biosynthesis in cultured rat aortic smooth muscle cells and bovine pulmonary artery endothelial cells. Prostacyclin was the major product formed from arachidonic acid in aortic smooth muscle cells and endothelial cells. When intact cells were incubated with streptokinase, one of the plasminogen activators, a significant stimulatory effect on prostacyclin biosynthetic activity in cells was evident without any cellular damage at all concentrations used (1-5,000 units/ml). Streptokinase also caused a marked release of arachidonic acid. However, when it was incubated with cell-free homogenates and [3H]arachidonic acid, it did not show any effects on prostacyclin biosynthesis. The addition of urokinase and tissue-type plasminogen activator had no effect on prostacyclin biosynthesis. Urokinase stimulated the release of arachidonic acid from cells, but it did not show any effect on prostacyclin release at any concentration of urokinase (1-5,000 units/ml). The release of arachidonic acid and the increased prostacyclin synthesis were not observed when tissue-type plasminogen activator was added. These results indicate that, among various plasminogen activators investigated, only streptokinase causes the release of arachidonic acid and prostacyclin. This could be a beneficial effect in thrombolytic therapy.     

Hydrolysis of inositol phospholipids precedes cellular proliferation in asbestos-stimulated tracheobronchial epithelial cells.

Metabolism of inositol phospholipids and phosphatidylcholine was investigated in tracheobronchial epithelial cells exposed to mitogenic concentrations of crocidolite asbestos. Alterations in levels of diacylglycerol, the endogenous activator of protein kinase C, and inositol polyphosphates, presumed mobilizers of intracellular calcium, were examined. Cultures labeled with [3H]glycerol and exposed to proliferative concentrations of crocidolite asbestos demonstrated significant elevations in [3H]diacylglycerol. In contrast, crocidolite-exposed cells labeled with [3H]myristic acid or [3H]choline did not display elevated production of [3H]diacylglycerol or release of [3H]choline metabolites--i.e., evidence of phosphatidylcholine hydrolysis. The soluble tumor promoter phorbol 12-myristate 13-acetate catalyzed both of these changes. myo-[3H]Inositol-labeled cells exposed as briefly as 10 min to mitogenic concentrations of crocidolite demonstrated elevations in [3H]inositol mono-, tris-, and terakisphosphates, phenomena indicating turnover of inositol phospholipids. The detection of diacylglycerol and inositol phosphates in crocidolite asbestos-exposed cells suggests that this fibrous tumor promoter activates phospholipase C as it stimulates cellular proliferation.     

Gonadotropin-releasing hormone (GnRH) rapidly stimulates the formation of inositol phosphates and diacyglycerol in rat granulosa cells: further evidence for the involvement of Ca2+ and protein kinase C in the action of GnRH

GnRH provokes a phospholipid response in rat granulosa cells that has been characterized by increased incorporation of radioactive precursors into phosphatidic acid and phosphatidylinositol, and by depletion of 32P-prelabeled polyphosphoinositides. In this report, rat granulosa cells from mature Graafian follicles were incubated with GnRH under various conditions to follow the hydrolysis of phosphoinositides and the generation of the metabolic byproducts of phospholipase C action. Granulosa cells were prelabeled for 3 h with myo[2-3H]inositol. GnRH provoked rapid (10 sec) and sustained (up to 60 min) increases in the levels of inositol monophosphates, inositol bisphosphates, and inositol trisphosphates (IP3). Time-course studies revealed that IP3 was formed more rapidly than inositol bisphosphate and inositol monophosphate after GnRH treatment. The response to GnRH was concentration dependent (maximal at 10 ng/ml) and was prevented by a specific GnRH antagonist. Lithium chloride (1-10 mM) greatly enhanced the GnRH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. No changes were observed in the levels of free [3H] inositol and [3H]phosphatidylinositol in GnRH-treated cells. However, treatment with both lithium and GnRH for 30 min significantly reduced the levels of free [3H]inositol and [3H] phosphatidylinositol. In the presence of lithium, the rate of hormone-stimulated inositol phosphate formation was not altered by 30 min of prior treatment with GnRH, indicating that phospholipase C activity is not readily desensitized. GnRH also increased the formation of diacylglycerol (DAG), another product of phospholipase C action. In cells prelabeled with [3H] arachidonic acid, GnRH significantly increased levels of DAG in incubations lasting 2-5 min. Concomitant increases in [3H] phosphatidic acid were also observed in GnRH-treated cells. In conjunction with these studies, intracellular free Ca2+ levels were measured by Quin 2 fluorescence. GnRH and its agonistic analog rapidly increased (5 sec) cytosolic free Ca2+ levels (approximately double). The results demonstrate that an early event in the action of GnRH is the hydrolysis of phosphoinositides by a phospholipase C-dependent mechanism. The products resulting from this action of GnRH, i.e. IP3 and DAG, may serve as intracellular mediators for the mobilization of intracellular calcium, or the activation of protein kinase C and arachidonic acid release.     

Group I secreted PLA2 in the maintenance of human lower esophageal sphincter tone

BACKGROUND & AIMS: In cat spontaneous lower esophageal sphincter (LES), tone is maintained by the activity of group I secreted phospholipase A2 (sPLA2-I) that produces arachidonic acid. Arachidonic acid metabolites activate G proteins linked to phospholipases, producing second messengers and activation of a protein kinase C-dependent pathway to maintain tone. We examined the role of sPLA2-I in the maintenance of tone in human LES samples obtained from organ donors. METHODS: In vitro LES tone and sPLA2-I-induced contraction of enzymatically isolated LES smooth muscle cells were measured in the absence or presence of inhibitors. Cell permeabilization by saponin allowed use of G-protein antibodies. RESULTS: In vitro LES tone was reduced by inhibitors of sPLA2-I, by indomethacin, by the phosphatidylcholine-specific phospholipase C inhibitor D609, and by the protein kinase C inhibitor chelerythrine. sPLA2-I-induced contraction of isolated LES smooth muscle cells was reduced by indomethacin, pertussis toxin, Gi3 antibodies, D609, and by chelerythrine. CONCLUSIONS: Human LES tone is maintained by the activity of sPLA2-I that produces arachidonic acid and metabolites and activation of Gi3-linked receptors and of phosphatidylcholine-specific phospholipase C, resulting in production of diacylglycerol, activation of PKC, and maintenance of tone through a protein kinase C-dependent contractile pathway.     

Effect of elastase on aortic smooth muscle cell proliferation

We studied the effects of elastase on [3H]thymidine incorporation into aortic smooth muscle cells. When elastase was added to cultured aortic smooth muscle cells, [3H]thymidine incorporation was inhibited in a dose-dependent manner in the presence of fetal bovine serum. Elastase also inhibited this incorporation in cells treated with epidermal growth factor. Epidermal growth factor (50 ng/ml) stimulated thymidine incorporation, without elastase, but with the addition of 20 units/ml of elastase the incorporation was inhibited 70%. The incorporation of thymidine into cells treated with 50 ng/ml epidermal growth factor was also inhibited 50% by a low concentration of elastase (5 units/ml). These inhibitory effects on thymidine incorporation were also observed in cells stimulated with platelet-derived growth factor. Platelet-derived growth factor (20 units/ml) markedly stimulated thymidine incorporation into cells, and elastase inhibited its activity in a dose-dependent manner. These results suggest that elastase has the potential to prevent the development of atherosclerosis by inhibiting smooth muscle proliferation.     

Enhanced phosphodiesteratic breakdown and turnover of phosphoinositides during reperfusion of ischemic rat heart

In this study, we examined phosphoinositide metabolism during ischemia and reperfusion using an isolated and perfused rat heart. When myocardial phosphoinositides were prelabeled with [3H]inositol, reperfusion after 30 minutes of normothermic global ischemia resulted in significant accumulations of radiolabeled inositol phosphate, inositol bisphosphate, and inositol trisphosphate. Isotopic incorporation of [3H]inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly in the heart reperfused with [3H]inositol after 30 minutes of ischemia compared with that perfused with [3H]inositol after 30 minutes of nonischemic perfusion. However, isotopic incorporation of [3H]glycerol into diacylglycerol, phosphatidic acid, and all of the three phosphoinositides was diminished in the reperfused hearts. Reperfusion of the ischemic heart prelabeled with [14C]arachidonic acid resulted in significant increases in [14C]diacylglycerol and [14C]phosphatidic acid. The enhanced accumulations of [3H]inositol phosphates during reperfusion were not affected by treatment with prazosin plus atropine or indomethacin, but were inhibited by hypoxic reperfusion, reperfusion with Ca2+-free buffer, or by mepacrine. These results suggest that myocardial reperfusion stimulates phosphodiesteratic breakdown and turnover of phosphoinositides, and increased Ca2+ influx caused by reperfusion may be involved in the mechanism of stimulation of phosphatidylinositol-specific phospholipase C activity in the rat heart.     

Activation of phospholipase D by glyceraldehyde in isolated islet cells follows protein kinase C activation.

Our recent studies have demonstrated the presence in neonatal islet cells and intact adult islets of a phosphatidylcholine-directed phospholipase D (PLD) which is activated after phorbol ester stimulation. The present study describes PLD activation in the presence of a carbohydrate insulin secretagogue. At the highest concentration tested (20 mM) the triose, glyceraldehyde, induced formation of phosphatidic acid in cells prelabeled with [14C]arachidonic acid or [3H]myristic acid (164 +/- 7 and 210 +/- 9% of basal phosphatidic acid values, respectively). Experimental confirmation of a concentration-dependent specific activation of PLD was provided by the formation of a transphosphatidylation product, phosphatidylethanol, after stimulation with glyceraldehyde in the presence of added ethanol (1.5%). Additionally, there was an early (within 5 min) rise in [14C]arachidonate-labeled diacylglycerol (139 +/- 7% of basal) accompanied by an increase in intracellular diacylglycerol mass (51 +/- 2 pmol/mg protein) and an increase in membrane-associated protein kinase C activity (183 +/- 5% of basal) which preceded the activation of PLD, as indicated by the time course of glyceraldehyde-stimulated phosphatidylethanol formation in the presence of ethanol. Pretreatment of islet cells with 2 microM 12-O-tetradecanoylphorbol-13-acetate for 18 h, to down-regulate protein kinase C, was without effect on diacylglycerol and phosphatidic acid production after 5 min but inhibited completely the production of phosphatidylethanol at 30 min. The phosphohydrolase inhibitor propranolol (100 microM) potentiated the accumulation of phosphatidic acid and phosphatidylethanol incubation following incubation with glyceraldehyde. These findings demonstrate for the first time that a physiological nutrient activates a phospholipase directed against endogenous phosphatidylcholine in intact islet cells; furthermore, they indicate a role for PLD in a delayed formation of phosphatidic acid in the islet cell. The finding of an early rise in glyceraldehyde-stimulated diacylglycerol (which may be formed de novo or by the action of phospholipase C), suggests that PLD is recruited by the activation of protein kinase C by this nutrient.     

Receptor-mediated release of endothelium-derived relaxing factor and prostacyclin from bovine aortic endothelial cells is coupled

Bovine aortic endothelial cells were grown on microcarrier beads and were perfused with Krebs-Ringer solution. Endothelium-derived relaxing factor (EDRF) was bioassayed on a cascade of four strips of rabbit aorta, and prostacyclin was analyzed by RIA of 6-oxo-prostaglandin F1 alpha. The endothelial cells released EDRF and prostacyclin when stimulated with bradykinin and its analogues, or with ADP, ATP, arachidonic acid, and phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3). The detection of EDRF was potentiated by superoxide dismutase, and the relaxation of rabbit aortic strips induced by EDRF was antagonized by methylene blue. The release of EDRF and prostacyclin was inhibited by phorbol myristate acetate, R59022 (a diacylglycerol kinase inhibitor), and gentamycin. We suggest that the release of EDRF and prostacyclin is coupled and the initial common step is activation of a phospholipase C.     

Thyrotropin (TSH)-releasing hormone decreases phosphatidylinositol and increases unesterified arachidonic acid in thyrotropic cells: possible early events in stimulation of TSH secretion

TRH stimulated the metabolism of lipids of the phosphatidylinositol (PI)-phosphatidic acid (PA) cycle and caused an increase in the level of free or unesterified arachidonic acid in mouse pituitary thyrotropic tumor (TtT) cells. In cells labeled with [32P]orthophosphate for 45 min, TRH caused a rapid specific increase in [32P]PA to 190 +/- 8% (+/- SE) of the control value at 15 sec (P less than 0.005) and in [32P]PI to 158 +/- 8% at 2 min (P less than 0.005). In cells labeled to isotopic steady state with [3H]inositol, TRH caused a decrease in [3H]PI to 92 +/- 1.8% of the control value at 1 min (P less than 0.01) and increased the level of [3H]inositolmonophosphate. In cells labeled to isotopic steady state with [14C]stearic acid, TRH caused a transient rise in [14C]diacylglycerol and a more prolonged increase in [14C]PA. In cells labeled to isotopic steady state with [3H]arachidonic acid, TRH stimulated a rise in free [3H]arachidonic acid to 210 +/- 8% of the control value at 15 sec (P less than 0.001), with a return to a level of 125 +/- 2% of the control value by 5 min. Arachidonic acid added exogenously caused efflux of 45Ca2+ from prelabeled cells and stimulated TSH secretion. Hence, in TtT cells, TRH 1) rapidly stimulated a decrease in the level of PI and increased inositolmonophosphate, diacylglycerol, and PA; and 2) caused a rapid increase in the level of free arachidonic acid. These effects may be important in stimulation of TSH secretion by TRH. Because arachidonic acid, when added exogenously, mobilized cellular Ca2+ and stimulated TSH secretion, arachidonic acid may mediate, at least in part, TRH-stimulated TSH secretion. The action of TRH on lipid metabolism in TtT cells is different from that in mammotropic pituitary cells, since TRH does not cause an increase in the level of free arachidonic acid in GH3 cells.     

Activation of protein kinase C by elevation of glucose concentration: proposal for a mechanism in the development of diabetic vascular complications.

Hyperglycemia is believed to be the major cause of diabetic vascular complications involving both microvessels and arteries as in the retina, renal glomeruli, and aorta. It is unclear by which mechanism hyperglycemia is altering the metabolism and functions of vascular cells, although changes in nonenzymatic protein glycosylation and increases in cellular sorbitol levels have been postulated to be involved. Previously, we have reported that the elevation of extracellular glucose levels with cultured bovine retinal capillary endothelial cells causes an increase in protein kinase C (PKC) activity of the membranous pool with a parallel decrease in the cytosol without alteration of its total activity. Now we demonstrate that the mechanism for the activation of PKC is due to an enhanced de novo synthesis of diacylglycerol as indicated by a 2-fold increase of [14C]diacylglycerol labeling from [14C]glucose. The elevated diacylglycerol de novo synthesis is secondarily due to increased formation of precursors derived from glucose metabolism; this formation is enhanced by hyperglycemia as substantiated by elevated [3H]glucose conversion into water. This effect of hyperglycemia on PKC is also observed in cultured aortic smooth muscle and endothelial cells and the retina and kidney of diabetic rats, but not in the brain. Since PKC in vascular cells has been shown to modulate hormone receptor turnover, neovascularization in vitro, and cell growth, we propose that this mechanism of enhancing the membranous PKC activities by hyperglycemia plays an important role in the development of diabetic vascular complications.     

Serotonin stimulates phospholipase A2 and the release of arachidonic acid in hippocampal neurons by a type 2 serotonin receptor that is independent of inositolphospholipid hydrolysis.

Serotonin (5-HT) stimulated the release of arachidonic acid in hippocampal neurons cocultured with glial cells but not in glial cultures alone. Similar results were observed for the 5-HT-stimulated release of inositol phosphates. These results suggest a neural but not glial origin of both responses. Pharmacological studies suggested that release of arachidonic acid and inositol phosphates was mediated by a type 2 5-HT (5-HT2) receptor. 5-HT-stimulated release of arachidonic acid was also detected in cortical neurons, which contain high levels of 5-HT2 receptors, but not striatum, spinal cord, or cerebellar granule cells, which have very low levels or are devoid of 5-HT2 receptors. The phorbol ester phorbol 12-myristate 13-acetate augmented the 5-HT-stimulated release of arachidonic acid but inhibited the 5-HT-stimulated release of inositol phosphates. 5-HT-stimulated release of arachidonic acid, but not inositol phosphates, was dependent on extracellular calcium. 5-HT stimulated the release of [3H]lysophosphatidylcholine from [3H]choline-labeled cells with no increase in the release of [3H]choline or phospho[3H]choline. These data suggest that 5-HT stimulated the release of arachidonic acid in hippocampal neurons through the activation of phospholipase A2, independent of the activation of phospholipase C.     

Dual pathways for carbamylcholine-stimulated arachidonic acid release in rat pancreatic acini

Recent studies suggested the involvement of arachidonic acid in the mediation of pancreatic amylase release. However, an effect of carbamylcholine on arachidonic acid release has not yet been reported in the exocrine pancreas. This study was performed to evaluate the effect of carbamylcholine on arachidonic acid release and determine the underlying intracellular mechanisms. From enzymatic assays, phospholipase A₂ and diacylglycerol lipase were activated by carbamylcholine and these activations were inhibited by the phospholipase A₂ inhibitors, mepacrine and aristolochic acid, and by the diacylglycerol lipase inhibitor RHC 80267. Carbamylcholine also increased arachidonic acid release in a concentration-dependent manner. Both phospholipase A₂ and diacylglycerol inhibitors partially inhibited carbamylcholine-stimulated arachidonic acid release. The phospholipase C inhibitor U73122 and the protein kinase C inhibitor staurosporine also caused partial inhibition. Arachidonic acid release by carbamylcholine was suppressed by the simultaneous addition of RHC 80267 with either phospholipase A₂ inhibitors. Our data demonstrate that phospholipase A₂ and diacylglycerol lipase are activated and arachidonic acid is released in pancreatic acini by carbamylcholine. Dual pathways are responsible for carbamylcholine-induced arachidonic acid release. One such pathway involves the sequential action of phospholipase C, protein kinase C and diacylglycerol lipase, whereas the other involves phospholipase A₂ activation.     

Vascular injuries induced by materials released from platelet-rich thrombus in vivo

Polyethylene tubing was inserted into the ascending aorta of rabbits via the right common carotid artery and placed for one or four weeks continuously to induce vessel wall injury and thrombotic events, and then the direct non-injured segments from the descending thoracic and abdominal aorta were examined morphologically and for [3H]thymidine incorporation into endothelial cells and smooth muscle cells. The descending aortas of experimental rabbits showed endothelial damage and increased mitosis of endothelial cells. [3H]Thymidine incorporation into the intima and media was significantly increased in the experimental group. This experiment indicates that materials released from activated platelets and/or thrombi into the circulation can cause endothelial damage and smooth muscle cell proliferation at downstream and remote aortic segments.     

Arachidonic acid elicits endothelium-dependent release from the rabbit aorta of a constrictor prostanoid resembling prostaglandin endoperoxides

This study was designed to investigate the mediator(s) of endothelium-dependent arterial constrictor responses evoked by arachidonic acid in vitro. A segment of descending rabbit thoracic aorta was isolated and perfused (1-2 ml/min) with oxygenated Krebs' bicarbonate buffer. Changes in the vascular smooth muscle-contracting activity of the aortic effluent were detected by superfusion bioassay using either strips of rabbit aorta or rings of dog saphenous vein, both denuded of endothelium and exposed to indomethacin (10 microM). Arachidonic acid (5-50 micrograms) injected into the inflow of the perfused aorta caused a dose-related increase in the vascular smooth muscle-contracting activity of the aortic effluent, whereas arachidonic acid added directly into the aortic effluent did not. The arachidonic acid-induced elevation of vascular smooth muscle-contracting activity in the aortic effluent was not apparent when indomethacin (10 microM) was added to the aortic inflow to inhibit cyclooxygenase, when the endothelium of the perfused aorta was removed by rubbing, or when the thromboxane A2/prostaglandin H2 receptors of the vascular tissues used for bioassay were blocked with an antagonist (1 microM SQ29548), and was unaffected when an inhibitor of thromboxane synthase (10 microM CGS 13080) was added to the aortic inflow. This effect of arachidonic acid was accompanied by release of prostaglandin H2 (measured as prostaglandin F2 alpha after reduction with SnCl2) in amounts sufficient to elicit contraction of the vascular tissues used for bioassay and was attenuated when a reducing agent (2 mM FeCl2) that converts prostaglandin H2 to 12-heptadecatrienoic acid was added to the aortic effluent. Collectively, these observations suggest that arachidonic acid stimulates endothelium-dependent release from the perfused aorta of a prostanoid that contracts vascular smooth muscle via interaction with thromboxane A2/prostaglandin H2 receptors. The study also suggests that the prostanoid responsible for the vascular smooth muscle-contracting activity of the aortic effluent is a prostaglandin endoperoxide(s) rather than thromboxane A2.     

Effect of diacylglycerol lipase inhibitor RHC 80267 on pancreatic mouse islet metabolism and insulin secretion

Summary The effect of interference with diacylglycerol metabolism was investigated in pancreatic mouse islets. In the presence of the diacylglycerol lipase inhibitor RHC 80267, glucose-induced insulin secretion was reduced 50–60%; whereas carbacholin-induced insulin secretion was unaffected. Addition of the diacylglycerol kinase inhibitor R 59022 did not change glucose-stimulated insulin secretion but abolished the inhibition seen in the presence of RHC 80267. RHC 80267 increased islet glucose utilisation, measured as formation of tritiated water from 5-[³H]-glucose, 3-fold but did not affect glucose oxidation to CO₂, lactate production or islet ATP levels. Glucose utilisation in leucocytes and hepatocytes was not increased by addition of RHC 80267. Islet lipid production from glucose was augmented 4-fold in the presence of RHC 80267 but only accounted for about 5% of the increase in glucose utilisation. The activity of adenylate cyclase and phosphoinositide-specific phospholipase C was unaffected by RHC 80267. Concentrations of RHC 80267 below 35 mol/l did not alter the activity of phospholipase A₂; whereas higher concentrations of the drug inhibited phospholipase A₂ activity approx 25%. The data support the hypothesis that production of arachidonic acid from diacylglycerol may be involved in regulation of insulin secretion.Abbreviations RHC 80267 (1,6-di(O-(carbamoyl)cyclohexanone oxime)hexane) - R 59022 6-[2-[4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl]ethyl]-7-methyl-5H-thiazolo[3,2-]pyrimidin-5-one