The effects of fatty acids on phosphoinositide synthesis and myo-inositol accumulation in exocrine pancreas

The effects of arachidonic acid (20:4) on phosphoinositide turnover were examined in rat pancreatic acinar cells prelabeled with myo-[3H]inositol. Arachidonic acid (50 microM) increased the accumulation of myo-[3H]inositol, but not that of [3H]inositol monophosphate, [3H]inositol bisphosphate, or [3H]inositol trisphosphate. By contrast, 10 microM carbamoylcholine increased the accumulation of all four compounds. A combination of arachidonic acid plus carbamoylcholine caused a selective and marked accumulation of myo-[3H]inositol, which was abolished by 10 mM LiCl. Arachidonic acid (10-100 microM) produced a concentration-dependent inhibition of myo-[3H]inositol incorporation into phosphoinositides and markedly depressed carbamoylcholine-induced increases in myo-[3H]inositol incorporation into inositol phospholipids. Several other unsaturated and saturated fatty acids failed to elicit a synergistic response with carbamoylcholine in stimulating myo-[3H]inositol accumulation and did not retard the incorporation of myo-[3H]inositol into phosphoinositides. The fact that eicosapentaenoic acid (20:5), but not arachidic acid (20:0), mimicked the depressant effect of arachidonate on phosphoinositide labeling suggests that the degree of unsaturation of the fatty acid, rather than chain length, is important for inhibition of phosphoinositide synthesis. The arachidonate-induced decrease in myo-[3H]inositol incorporation was accompanied by a reduction in the steady state level of [32P]phosphatidylinositol 4,5-bisphosphate. The mass of arachidonic acid liberated in response to carbamoylcholine was measured by gas chromatography-mass spectrometry, and the time course of stimulated arachidonate accumulation paralleled that of inositol phosphate accumulation and amylase release. These observations suggest that in exocrine pancreas, endogenous arachidonic acid serves as a negative feedback regulator of phosphoinositide turnover.     

The incorporation of myo-inositol into phosphatidylinositol derivatives is stimulated during hormone-induced meiotic maturation of amphibian oocytes

The incorporation of myo-[3H]inositol into phosphatidylinositol and its phosphorylated derivatives was studied by microinjection of the radioactive precursor into Xenopus laevis oocytes. Induction of meiotic maturation of the oocytes by treatment with either progesterone one or insulin resulted in a significant increase in the incorporation of myo-[3H]inositol into the phospholipid fraction. This increase occurred 3-6 h after hormonal treatment, a time coincident with the start of the breakdown of the nuclear envelope, and requires protein synthesis. The effect of progesterone and insulin contrasts with the effect of acetylcholine, which acts through a muscarinic receptor causing the activation of phospholipase C, since the latter effector causes an increase in myo-[3H]inositol incorporation, which is more rapid and does not require protein synthesis. These results suggest that the meiotic maturation process is connected with changes in inositol metabolism in the amphibian oocyte.     

Differential presynaptic effects of hexachlorocyclohexane isomers on noradrenaline release in cerebral cortex.

To investigate presynaptic effects of hexachlorocyclohexane (HCH) isomers, the release of noradrenaline (NA) in brain tissue was analyzed using rat cerebral cortical slices preloaded with [3H]-NA. gamma-HCH (lindane) 50 microM significantly enhanced the [3H]-NA release evoked by 15-25 mM K+. alpha- and beta-HCH (50 microM) did not produce any significant effect on K(+)-evoked [3H]-NA release. delta-HCH (50 microM) induced a significant decrease of the 25 mM K(+)-evoked release of [3H]-NA. The effect of the gamma- and delta-HCH isomers on the presynaptic action of the alpha 2-agonist clonidine and the alpha 2-antagonist yohimbine was also studied. The presynaptic inhibitory effect of clonidine and the stimulatory effect of yohimbine on [3H]-NA release was attenuated by lindane and delta-HCH, respectively. These results are consistent with a presynaptic action of the HCH isomers on noradrenergic release processes.     

The effect of phentolamine on synaptosomal phosphatidylinositol in experimental galactose toxicity

Abnormal myo-[2-³H]inositol incorporation into phosphatidylinositol has been found in phentolamine-treated synaptosomes that were isolated from the cerebral hemispheres of galactose toxic rats and incubated with [³³P]P_i and myo-[2-³H] inositol. In galactose toxic rats phentolamine-stimulated myo-[2-³H]inositol labeling of phosphatidylinositol was 70% greater than in normal animals. This enhanced labeling of synaptosomal phosphatidylinositol in galactose toxic rats during stimulation with phentolamine is in marked contrast to the depressed myo-inositol labeling of phosphatidylinositol reported with acetylcholine stimulation.     

Stimulation of phosphoinositide hydrolysis by oxytocin and the mechanism by which oxytocin controls prostaglandin synthesis in the ovine endometrium.

Slices of caruncular endometrium from steroid-treated ovariectomized sheep were incubated with myo-[2-3H]inositol to label tissue phosphatidylinositol. Effects of oxytocin were determined on the rate of incorporation of radioactivity into phosphatidylinositol and on the hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol. Incorporation of radioactivity into phosphatidylinositol was linear during 2 h incubations; 10(-7) M (100 nM)-oxytocin caused a 2.8-fold increase in the rate of incorporation. In the presence of Li+, addition of 10(-7) M-oxytocin to slices in which phosphatidylinositol was pre-labelled caused mean increase of 40-fold in the incorporation of radioactivity into inositol mono-, bis- and tris-phosphates. Inositol 1,3,4-trisphosphate was quantitatively the major trisphosphate formed. The action of oxytocin on phosphoinositide hydrolysis was dose- and time-dependent, occurring at concentrations within the range observed in plasma during episodes of secretion in vivo, and with a time course comparable with that of the action of oxytocin on uterine prostaglandin production. The effect of oxytocin on incorporation of radioactivity into inositol phosphates was not affected by inhibitors of prostaglandin synthesis. Diacylglycerol 1- and 2-lipases in caruncular endometrium converted up to 72% of added 2-[3H]arachidonyldiacylglycerol into [3H]arachidonic acid during 30 min incubations at pH 7.0. Caruncular endometrium contained 1.49 mumol of phosphatidylinositol/g, representing approx. 0.2 mumol/g of phosphatidylinositol arachidonic acid. It is proposed that the stimulation of endometrial prostaglandin synthesis by oxytocin is accounted for by increased hydrolysis of phosphoinositides to diacylglycerol and inositol phosphates with subsequent release of arachidonic acid from diacylglycerol.     

Relationship between hormonal activation of phosphatidylinositol hydrolysis, fluid secretion and calcium flux in the blowfly salivary gland.

The addition of 5-hydroxytryptamine to the isolated blowfly salivary gland stimulates fluid secretion, transepithelial calcium transport and the breakdown of 32P- or 3H-labelled phosphatidylinositol The breakdown of [32P]phosphatidylcholine and [32P]-phosphatidylethanolamine was not stimulated by 5-hydroxytryptamine. In salivary glands incubated with myo-[2-3H]inositol for 1--3 h, more than 95% of the label retained by the tissue was in the form of phosphatidylinositol. The addition of 5-hydroxytryptamine resulted in an increase in the accumulation of label in intracellular inositol 1:2-cyclic phosphate, inositol 1-phosphate and free inositol along with an increase in the release of [3H]inositol to the medium and saliva. The release of [3H]inositol to the medium served as a sensitive indicator of phosphatidylinositol breakdown. The release of [3H]inositol was not increased by cyclic AMP or the bivalent-cation ionophore A23187 under conditions in which salivary secretion was accelerated. The stimulation of fluid secretion by low concentrations of 5-hydroxytryptamine was potentiated by 3-isobutyl-1-methylxanthine, which had no effect on inositol release. The stimulation of fluid secretion by 5-hydroxytryptamine was greatly reduced in calcium-free buffer, but the breakdown of phosphatidylinositol continued at the same rate in the absence of calcium. These results support the hypothesis that breakdown of phosphatidylinositol by 5-hydroxytryptamine is involved in the gating of calcium.     

L-myo-inositol 1,4,5,6-tetrakisphosphate is present in both mammalian and avian cells.

When myo-[3H]inositol-prelabelled primary-cultured murine bone-marrow-derived macrophages were challenged with platelet-activating factor (PAF; 200 ng/ml), there was a rapid (2.5-fold at 10 s) rise in the intracellular concentration of D-myo-[3H]inositol 1,4,5-trisphosphate, followed by a rise in myo-[3H]inositol tetrakisphosphate. myo-[3H]Inositol tetrakisphosphate fractions were isolated by high-performance anion-exchange chromatography from myo-[3H]inositol-prelabelled chick erythrocytes and primary-cultured macrophages. In both cases [3H]iditol and [3H]inositol were the only significant products (greater than 90% of recovered radioactivity) after oxidation to completion with periodic acid, reduction with NaBH4 and dephosphorylation with alkaline phosphatase. The presence of [3H]inositol after this procedure is consistent with the occurrence of [3H]inositol 1,3,4,5-tetrakisphosphate in the cell extracts, whereas [3H]iditol could only be derived from D- or L-inositol 1,4,5,6-tetrakisphosphate. When [3H]inositol tetrakisphosphate fractions obtained from (A) unstimulated macrophages, (B) macrophages that had been stimulated with PAF for 40s or (C) chick erythrocytes were subjected to the above procedure, radioactivity was recovered in these polyols in the following proportions: A, 60-90% in iditol, with 10-40% in inositol; B, total radioactivity increased by a factor of 9.8, 94% being recovered in inositol and 8% in iditol; C, 70-80% in iditol and 20-30% in inositol. [3H]Iditol derived from myo-[3H]inositol tetrakisphosphate fractions from macrophages and chick erythrocytes was oxidized to sorbose by L-iditol dehydrogenase (L-iditol:NAD+2-oxidoreductase, 1.1.1.14) at the same rate as authentic L-iditol. D-[14C]Iditol, derived from D-myo-inositol 1,4,5-trisphosphate, was not oxidized by L-iditol dehydrogenase. This result indicates that the [3H]iditol was derived from L-myo-inositol inositol 1,4,5,6-tetrakisphosphate. The data are consistent with rapid PAF-sensitive synthesis of D-myo-[3H]inositol 1,3,4,5-tetrakisphosphate in macrophages, and demonstrate that L-myo-inositol 1,4,5,6-tetrakisphosphate is synthesized in both mammalian and avian cells. The levels of L-myo-[3H]inositol 1,4,5,6-tetrakisphosphate in primary-cultured macrophages are not acutely sensitive to PAF.     

Effect of lindane on phosphatidylinositol synthesis by cerebral cortex after acute and subchronic treatment

• 1.1. The incorporation of myo-[2-³H]inositol into phosphatidylinositols was unmodified in brain cortex miniprisms from convulsant rats.
• 2.2. However, the incorporation had increased by 300–400% in non convulsant rats which had received the same amount of lindane at a lower concentration.
• 3.3. This result suggests that phosphatidylinositols are implicated in the convulsion syndrome.
• 4.4. Experiments with lindane added in vitro were performed with both subchronically lindane intoxicated and untreated rats.
• 5.5. The results show an interesting lack of parallelism.
• 6.6. This might indicate the development of some resistance to the effects of lindane, possibly as the result of complex compensatory changes in inositol lipid biosynthesis.

     

An analysis of myo-[3H]inositol trisphosphates found in myo-[3H]inositol prelabelled avian erythrocytes.

Evidence is presented to show that acid extracts of avian erythrocytes prelabelled for 24-48 h with myo-[3H]inositol contain the following myo-[3H]inositol trisphosphates (expressed as a percentage of total myo-[3H]inositol trisphosphates extracted): 36% myo-[3H]inositol 1,4,5-trisphosphate; 33.7% myo-[3H]inositol 1,3,4-trisphosphate; 13% myo-[3H]inositol 3,4,5-trisphosphate; 9.7% myo-[3H]inositol 3,4,6-trisphosphate; 4.4% myo-[3H]inositol 1,4,6-trisphosphate and 3.3% myo-[3H]inositol 1,3,6-trisphosphate. The only phosphatidyl-myo-[3H]inositol bisphosphate that could be detected in [3H]Ins-prelabelled avian erythrocytes was phosphatidyl-myo-[3H]inositol 4,5-bisphosphate. Cellular myo-[3H]inositol 3,4,5-trisphosphate may be synthesized by dephosphorylation of myo-[3H]inositol 3,4,5,6-tetrakisphosphate. D- and L-myo-[3H]inositol 1,4,6-trisphosphate and D- and L-myo-[3H]inositol 1,3,6-trisphosphate may be dephosphorylation products of myo-[3H]inositol 1,3,4,6-tetrakisphosphate.     

Etomoxir mediates differential metabolic channeling of fatty acid and glycerol precursors into cardiolipin in H9c2 cells

We examined the effect of etomoxir treatment on de novo cardiolipin (CL) biosynthesis in H9c2 cardiac myoblast cells. Etomoxir treatment did not affect the activities of the CL biosynthetic and remodeling enzymes but caused a reduction in [1-14C]palmitic acid or [1-14C]oleic acid incorporation into CL. The mechanism was a decrease in fatty acid flux through the de novo pathway of CL biosynthesis via a redirection of lipid synthesis toward 1,2-diacyl-sn-glycerol utilizing reactions mediated by a 35% increase (P < 0.05) in membrane phosphatidate phosphohydrolase activity. In contrast, etomoxir treatment increased [1,3-3H]glycerol incorporation into CL. The mechanism was a 33% increase (P < 0.05) in glycerol kinase activity, which produced an increased glycerol flux through the de novo pathway of CL biosynthesis. Etomoxir treatment inhibited 1,2-diacyl-sn-glycerol acyltransferase activity by 81% (P < 0.05), thereby channeling both glycerol and fatty acid away from 1,2,3-triacyl-sn-glycerol utilization toward phosphatidylcholine and phosphatidylethanolamine biosynthesis. In contrast, etomoxir inhibited myo-[3H]inositol incorporation into phosphatidylinositol and the mechanism was an inhibition in inositol uptake. Etomoxir did not affect [3H]serine uptake but resulted in an increased formation of phosphatidylethanolamine derived from phosphatidylserine. The results indicate that etomoxir treatment has diverse effects on de novo glycerolipid biosynthesis from various metabolic precursors. In addition, etomoxir mediates a distinct and differential metabolic channeling of glycerol and fatty acid precursors into CL.     

Prolactin induces the formation of inositol bisphosphate and inositol trisphosphate in cultured mouse mammary gland explants

The effect of prolactin on [3H]inositol metabolism in cultured mouse mammary gland explants derived from 12-14-day pregnant mice was determined. In mammary gland explants that were prelabeled by culturing the tissues with 3 microCi/ml myo-[3H]inositol for 48 h, the levels of 3H in inositol derivatives were determined. The temporal effect of prolactin on the quantity of 3H present in phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP), phosphatidylinositol bisphosphate (PIP2) and various inositol phosphate containing fractions were examined. Prolactin significantly stimulated the accumulation of 3H label in inositol monophosphate (IP1), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) 1-3 h after addition of prolactin. An effect of prolactin on the accumulation of inositol derivatives was not apparent at prolactin-exposure periods of less than 60 min; nor was an effect of prolactin apparent when exposure periods of 4 h or longer were employed. Prolactin did not significantly decrease the 3H label in PI, PIP or PIP2 except at 1 and 2 h. These data when considered with other apropos studies are compatible with the conclusion that the turnover of inositol lipid derivatives may be involved in the mechanism by which prolactin regulates metabolic processes in the mammary gland. The primary action of prolactin on mammary cells, however, would not appear to involve its action on the metabolism of the inositol derivatives in view of the extended time required (1 h) before effects of prolactin on perturbations of inositide metabolism are manifested.     

Polyphosphoinositides are present in plant tissue culture cells

Polyphosphoinositides have been isolated from wild carrot cells grown in suspension culture. This is the first report of polyphosphoinositides in plant cells. The phospholipids were identified by comigration with known standards on thin-layer plates. After overnight labeling of the cells with myo-[2-3H] inositol, the phosphoinositides as percent recovered inositol were 93% phosphatidylinositol., 3.7% lysophosphatidylinositol, 1.7% phosphatidylinositol monophosphate, 0.8% phosphatidylinositol bisphosphate.     

Manganese Stimulates the Incorporation of [3H]Inositol into a Pool of Phosphatidylinositol in Brain That Is Not Coupled to Agonist-Induced Hydrolysis

Mn2+ greatly increases the incorporation of myo-[3H]inositol into phosphatidylinositol (PI) of brain and other tissues by stimulating the activity of a PI-myo-inositol exchange enzyme. This study examined the ability of norepinephrine (NE) and carbachol to stimulate the hydrolysis of [3H]PI formed in the absence and presence of Mn2+-stimulated [3H]inositol exchange. Rat cerebral cortical slices were incubated with myo-[3H]inositol for 60 min in an N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES) buffer without or with MnCl2 (1 mM). The tissue was washed and further incubated with unlabeled myo-inositol and LiCl (10 mM). Prelabeled slices were then incubated with NE (0.1 mM) or carbachol (1 mM) to induce agonist-stimulated [3H]PI hydrolysis. Mn2+ treatment resulted in eight- and sixfold increases in control levels of [3H]PI and [3H]inositol monophosphate [( 3H]IP), respectively. Both NE and carbachol stimulated [3H]IP formation in tissue prelabeled without or with manganese. However, the degree of stimulation (percentage of control values) was greatly attenuated in the presence of Mn2+. In the absence of Mn2+ treatment, NE decreased [3H]PI radioactivity in the tissue to 80% of control values. However, NE did not decrease [3H]PI radioactivity in the Mn2+-treated tissue. These data demonstrate that Mn2+ stimulates incorporation of myo-[3H]inositol into a pool of PI in brain that has a rapid turnover but is not coupled to agonist-induced hydrolysis.     

Analysis of the regulation of phosphatidylinositol-4,5-bisphosphate synthesis by arachidonic acid in exocrine pancreas

In pancreatic acinar cells prelabeled with either 32Pi or myo-[3H]inositol, arachidonic acid (10-50 microM) rapidly decreased the steady-state levels of [32P]phosphatidylinositol 4',5'-bisphosphate [( 32P]PtdIns4,5P2) and inhibited carbachol-stimulated accumulation of [3H]inositol trisphosphate [( 3H]InsP3). Both actions of arachidonic acid were rapidly reversed by bovine serum albumin (BSA). Indomethacin and nordihydoguaiaretic acid failed to block the inhibitory effects of arachidonic acid on [32P]PtdIns4,5P2 levels. Arachidonic acid (10-50 microM) also caused a prompt depletion of cellular ATP which was rapidly reversed by BSA. The ATP-depleting action of arachidonate paralleled in terms of concentration dependence and time course its inhibitory effects on [32P]PtdIns4,5P2 and [3H]InsP3 levels. Exposure of acinar cells to 50 microM arachidonic acid produced an increase in oxygen consumption which exceeded that elicited by either carbachol or ionomycin. Arachidonic acid (10-50 microM) also caused a concentration-dependent rise in cytosolic Ca2+, which was partially obtunded by Ca2+ deprivation. A proposed mechanism involving arachidonic acid as a negative feedback regulator of polyphosphoinositide turnover in exocrine pancreas is discussed.     

Interleukin 2 rapidly stimulates synthesis and breakdown of polyphosphoinositides in interleukin 2-dependent, murine T-cell lines

Several T-cell functions are controlled by the regulatory peptide interleukin 2 (IL-2). Binding of IL-2 with specific receptors has been well documented, but the molecular mechanism by which IL-2/IL-2 receptor interaction is transduced is not known. We have found that treatment of IL-2-dependent T-cell lines with IL-2 is followed by a rapid stimulation of inositol phospholipid metabolism, as determined by isotopic methodology employing myo-[1,2-3H]inositol. Increased incorporation of the metabolic precursor into phosphatidylinositol and phosphatidylinositol 4-monophosphate, together with the appearance of radiolabeled phosphatidylinositol 4,5-bisphosphate, occurred within minutes of treatment with IL-2 of factor-dependent CT6 cells. Analysis of labeled water-soluble compounds from prelabeled cells indicated a rapid (within 1 min) stimulation of inositol phospholipid hydrolysis following IL-2 treatment. Increased recovery of [3H] inositol phosphates and appearance of [3H]inositol trisphosphate were observed after treatment with IL-2 of CT6 cells, as well as of a second IL-2-dependent cell line, CTB6. These findings suggests that inositol phospholipid-derived metabolites (i.e. diacylglycerol and inositol trisphosphate) may be part of the mechanism by which certain IL-2 signals are transduced.     

Influence of potassium and calcium ions on the phosphatidylinositol and phosphatidylcholine metabolism in rat fibroblasts after growth stimulation by calf serum.

In secondary cultures of embryonic rat fibroblasts which were arrested in G1 (G0) by serum depletion and subsequently triggered into the cell cycle by readdition of growth factors isolated from fetal calf serum the influence of the potassium and calcium concentrations in the medium on phosphatidylinositol and phosphatidylcholine metabolism was investigated. The incorporation of inorganic [32P]phosphate into phosphatidylinositol is dependent on the potassium content of the culture medium. The specific activity of 32P in phosphatidylinositol is increased at K+ concentrations between 0.1 and 1 mM. Also calcium (between 0.01 and 2 mM) slightly stimulates phosphatidylinositol metabolism. Also the incorporation of myo-[3H]inositol is increased at potassium concentrations between 0.2 and 1 mM, whereas calcium is slightly inhibitory. The labelling of phosphatidylcholine with either [32P]phosphate or [3H]choline is not dependent on the potassium and calcium concentrations of the culture medium. Moreover, the phospholipid metabolism of permanently growing epithelioid and fibroblastoid cells lines, which were investigated, is considerably less dependent on the K+ and Ca2+ ions.     

Phospholipid metabolism and lysosomal enzyme secretion by leukocytes. Effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP.

The effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP on isotope incorporation into phospholipids and the release of beta-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3':5'-monophosphate (0.1--1.0 mM) reduced beta-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited 32Pi incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-3H]inositol into the phosphoinositides. Added ATP (0.1--1.0 MM), but not other nucleotides, was found to potentiate beta-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-3H]inositol. The mechanism of the inhibition the isotope incarparation into these acidic phospholipids by the two mucleotides has not been defined. Dibutyryl cyclic adenosine 3':5'-monophosphate at 2--4 mM concentration was not found to appreciably alter the incorporation of [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.     

Lithium inhibits muscarinic-receptor-stimulated inositol tetrakisphosphate accumulation in rat cerebral cortex.

The effects of Li+ on carbachol-stimulated phosphoinositide metabolism were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. The muscarinic agonist carbachol evoked an enhanced steady-state accumulation of [3H]inositol monophosphate ([3H]InsP1), [3H]inositol bisphosphate ([3H]InsP2), [3H]inositol 1,3,4-trisphosphate ([3H]Ins(1,3,4)P3), [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) and [3H]inositol tetrakisphosphate ([3H]InsP4). Li+ (5 mM), after a 10 min lag, severely attenuated carbachol-stimulated [3H]InsP4 accumulation while simultaneously potentiating accumulation of both [3H]InsP1 and [3H]InsP2 and, at least initially, of [3H]Ins(1,3,4)P3. These data are consistent with inhibition of inositol mono-, bis- and 1,3,4-tris-phosphate phosphatases to different degrees by Li+ in brain, but are not considered to be completely accounted for in this way. Potential direct and indirect mechanisms of the inhibitory action of Li+ on [3H]InsP4 accumulation are considered. The present results stress the complex action of Li+ on cerebral inositol metabolism and indicate that more complex mechanisms than are yet evident may regulate this process.     

Activation of phospholipase A2 by endothelin in cultured vascular smooth muscle cells

The ability of endothelin to promote phospholipid hydrolysis has been studied in myo-[2-3H]-inositol-, [3H]-arachidonic acid- or methyl-[3H]choline chloride-prelabelled cultured vascular smooth muscle cells (VSMC) from rat and bovine thoracic aortae and human omental vessels. The biochemical responses to endothelin were comparable between the different VSMC isolates. Endothelin promoted the accumulation of glycerolphospho[3H]inositol and concomitant loss of [3H]-inositol label from phosphatidylinositol. Exposure of [3H]choline-labelled VSMC to endothelin resulted in a loss of radioactivity from phosphatidylcholine that was inversely parallelled by an increase in water-soluble [3H]-choline metabolites. In [3H]-arachidonic acid ([3H]-AA)-labelled VSMC, endothelin induced extracellular release of [3H]-AA which derived from both phosphatidylcholine and phosphatidylinositol. Half-maximally effective concentrations of endothelin for all these responses were approximately 2-7 nM and did not vary between VSMC types. Endothelin-induced release of [3H]-AA into VSMC medium-overlay was inhibited by quinacrine and nordihydroguaiaretic acid but not by neomycin or indomethacin. The data herein implicate activation of phospholipase A2 by endothelin with subsequent metabolism of arachidonic acid via the lipoxygenase pathway.     

Subcellular localization of inositol lipids in blood platelets as deduced from the use of labelled precursors.

1. By rapid fractionation of blood platelet lysates on Percoll density gradients at alkaline pH (9.6), a very pure plasma-membrane fraction was obtained, as well as discrimination between endoplasmic reticulum and lysosomes. 2. Labelling of intact platelets with [32P]Pi followed by subcellular fractionation showed an exclusive localization of all inositol lipids in the plasma membrane. 3. Preincubation of whole platelets with myo-[3H]inositol in a buffer containing 1 mM-MnCl2 allowed incorporation of the label into PtdIns (phosphatidylinositol) of both plasma and endoplasmic-reticulum membrane, whereas [3H]PtdIns4P (phosphatidylinositol 4-phosphate) and [3H]PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) were exclusively found on the plasma membrane. 4. It is concluded that PtdIns4P and PtdIns(4,5)P2 are exclusively localized in the plasma membrane, whereas PtdIns is present in both plasma and endoplasmic-reticulum membranes. This could provide an explanation for previously reported data on hormone-sensitive and -insensitive inositol lipid pools.