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.     

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.     

Inositol metabolism in WRK-1 cells. Relationship of hormone-sensitive to -insensitive pools of phosphoinositides

Previous studies have indicated the existence of two separate pools of phosphoinositides in WRK-1 cells; one is labile and hormone-sensitive with respect to turnover, while the other is stable. Hormonal stimulation results in a rapid increase in 32Pi incorporation into the sensitive pool, while in the absence of hormone, incorporation of 32Pi into this pool is slow. Results are quite different when [3H]inositol is the precursor utilized. Incorporation of [3H]inositol into hormone-sensitive phosphoinositides is not stimulated in the presence of hormone, suggesting entry of this exogenous precursor into the cycle by a route other than the resynthetic phase of the cycle. Furthermore, failure of hormone to induce loss of [3H]phosphoinositide in pulse-chase experiments in the absence of lithium suggests reutilization of the [3H]inositol moiety generated by phosphodiesteratic cleavage of hormone-sensitive phosphoinositide. Time course studies indicate that the relative rates of incorporation of [3H]inositol into sensitive and insensitive phosphoinositide remain constant from 2 to 24 h. Several factors are capable of increasing [3H]inositol incorporation into hormone-insensitive phosphoinositide including vasopressin, calcium ionophores, and manganese. On the other hand, vasopressin treatment appears to decrease incorporation of [3H]inositol into the hormone-sensitive pool, probably by shifting the equilibrium between phosphoinositides and inositol phosphates, since the decrease in radioactivity observed in the phosphoinositides is equaled by the increase observed in that in the inositol phosphates.     

Enhanced Coupling of Neonatal Muscarinic Receptors in Rat Brain to Phosphoinositide Turnover

The relationship between the density of the muscarinic receptor in developing rat cerebral cortex and its coupling to phosphoinositide turnover is examined. Tissue slices from rats of various ages were incubated with myo-[2-3H]inositol, and the effect of carbamoylcholine on the release of total inositol phosphates was determined. Binding of [3H]quinuclidinyl benzilate was determined in the same tissue. Although muscarinic receptor density in day-18 embryonic cortex was only 5% of that in the adult, the maximal response of stimulated phosphoinositide turnover to carbamoylcholine (1-10 mM) was at the adult level (i.e., three-fold increase). Comparison of the dependence of the turnover on carbamoylcholine concentration revealed that in neonates, the dose-response curve was shifted to the left, giving a half-maximal effect at concentrations approximately tenfold lower than that in the adult. In addition, the partial muscarinic agonists oxotremorine-2 and bethanechol were both more efficacious in young rats than in adults. The differences could not be accounted for either by alterations in agonist affinity for the receptor or by the presence of "spare" muscarinic receptors. These results indicate that muscarinic receptors in fetal and newborn rat cerebral cortex are more efficiently coupled to stimulation of phosphoinositide turnover than in the adult.     

In Vivo Electrical Stimulation of the Sympathetic Nerve of the Eye Increases Inositol Phosphate Production and Prostaglandin Release in the Rabbit Iris Muscle

The effects of in vivo electrical stimulation of the sympathetic nerve of the eye on phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis in rabbit iris and release of arachidonate and prostaglandin (PG) E2 into aqueous humor were investigated. myo-[3H]Inositol or [1-14C]arachidonate was injected intracamerally into each eye 3 h before electrical stimulation of one of the sympathetic trunks. Tissue phosphoinositides were determined by TLC, and 3H-labeled inositol phosphates were analyzed by either ion-exchange chromatography or HPLC. The aqueous humor was analyzed for 14C-labeled arachidonate and PGE2 by radiochromatography and for unlabeled PGE2 by radioimmunoassay. The results obtained from this study can be summarized as follows: (a) The rates of in vivo incorporation of myo-[3H]inositol into phosphoinositides and accumulation of 3H-labeled inositol phosphates in the iris muscle increased with time and then leveled off between 3 and 5 h. (b) Distribution of 3H radioactivity in inositol phosphates, as determined by HPLC, showed that of the total radioactivity in inositol phosphates, 53.6% was recovered in myo-inositol 1-phosphate, 36% in myo-inositol bisphosphate, 0.95% in myo-inositol 1,3,4-trisphosphate (1,3,4-IP3), and 2.6% in 1,4,5-IP3. (c) Electrical stimulation of the sympathetic nerve resulted in a significant loss of 3H radioactivity from PIP2 and a concomitant increase of that in IP3, an observation indicating that PIP2 is the physiological substrate for alpha 1-adrenergic receptors in this tissue. (d) Release of IP3 and liberation of arachidonate for PGE2 synthesis are dependent on the duration of stimulation and the intensity (voltage) of stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphoinositides and inositol phosphates in Discopyge tschudii electrocyte membranes

• 1.1. The metabolism of inositol (Ins)-containing phospholipids and inositol phosphates has been studied by following the incorporation and distribution of myo-[³ H]Ins in metabolically active electrocytes from the electric ray Discopyge tschudii.
• 2.2. The apparent initial rate of myo-[³H]Ins incorporation into total phosphoinositides was ca 8.2 fmol/mg protein/hr. Phosphatidylinositol (Ptdlns) displayed the highest levels of labelling. Lithium inhibited this incorporation probably by limiting the recycling of myo-[³H]Ins from [³H]Ins-monophosphate.
• 3.3. The formation of water-soluble products of phosphoinositides between 7 and 24 hr was 4.1 ± 0.2, 0.4 ± 0.2 and 3.0 ± 1.0 fmol/μmmol total lipid phosphorus for myo-[³H]InsP, -InsP₂ and Ins-P₃ respectively.
• 4.4. Lithium ions are shown to modulate phosphoinositide synthesis and Ins-phosphate accumulation. Ins-mono-, bis- and tris-phosphate production was enhanced 5-, 3- and 2-fold by Li +.
• 5.5. The above results suggest the participation of a C-type phospholipase and of Li-sensitive phosphatases in the modulation of phosphoinositide metabolism in the electrocyte.

     

Arachidonic Acid Inhibits Choline Uptake and Depletes Acetylcholine Content in Rat Cerebral Cortical Synaptosomes

The effects of arachidonic acid on [3H]choline uptake, on [3H]acetylcholine accumulation, and on endogenous acetylcholine content and release in rat cerebral cortical synaptosomes were investigated. Arachidonic acid (10-150 microM) produced a dose-dependent inhibition of high-affinity [3H]choline uptake. Low-affinity [3H]choline uptake was also inhibited by arachidonic acid. Fatty acids inhibited high-affinity [3H]choline uptake with the following order of potency: arachidonic greater than palmitoleic greater than oleic greater than lauric; stearic acid (up to 150 microM) had no effect. Inhibition of [3H]choline uptake by arachidonic acid was reversed by bovine serum albumin. In the presence of arachidonic acid, there was an increased accumulation of choline in the medium, but this did not account for the inhibition of [3H]choline uptake produced by the fatty acid. Arachidonic acid inhibited the synthesis of [3H]acetylcholine from [3H]choline, and this inhibition was equal in magnitude to the inhibition of high-affinity [3H]choline uptake produced by the fatty acid. A K+-stimulated increase in [3H]acetylcholine synthesis was inhibited completely by arachidonic acid. Arachidonic acid also depleted endogenous acetylcholine stores. Concentrations of arachidonic acid and hemicholinium-3 that produced equivalent inhibition of [3H]choline uptake also produced equivalent depletion of acetylcholine content. In the presence of eserine, arachidonic acid had no effect on acetylcholine release. The results suggest that arachidonic acid may deplete acetylcholine content by inhibiting high-affinity choline uptake and subsequent acetylcholine synthesis. This raises the possibility that arachidonic acid may play a role in the impairment of cholinergic transmission seen in cerebral ischemia and other conditions in which large amounts of the free fatty acid are released in brain.     

Local anesthetics: stimulation of incorporation of inositol into phosphoinositides in guinea pig cerebral cortical synaptoneurosomes

Various local anesthetics enhanced the incorporation of [3H]inositol into phosphoinositides in guinea pig cerebral cortical synaptoneurosomes. Dibucaine, QX-572 and dimethisoquin showed maximum stimulation at 100 microM, tetracaine and diphenhydramine at 300 microM, and QX-314 at 1 mM, while quinacrine, lidocaine and cocaine showed no or only slight stimulation. There was no correlation between local anesthetic activity, estimated by inhibition of the 22Na+ flux elicited by the sodium channel activator batrachotoxin, and the potency for stimulation of inositol incorporation. A quaternary, relatively weak, local anesthetic, QX-572, was the most potent agent in stimulation of inositol incorporation, while the next most potent agent was dibucaine, a tertiary, very potent, local anesthetic. Dibucaine did not affect the uptake of [3H]inositol by synaptoneurosomes. The incorporation of [3H]inositol into phosphoinositides was increased in calcium-free buffer. The presence of dibucaine resulted in further stimulation of [3H]inositol incorporation in calcium-free buffer. Although dibucaine and QX-572 markedly stimulated incorporation of [3H]inositol into phosphoinositides, only QX-572 significantly enhanced the incorporation of 32PO4(3-) into phosphoinositides. The results suggest that certain local anesthetics enhance a pathway involving an exchange reaction between inositol and the phosphoinositol ester bond of phosphatidylinositol, but do not markedly affect the de novo pathway of phosphoinositide synthesis.     

Acetylcholine stimulates selective liberation and re-esterification of arachidonate and accumulation of inositol phosphates and glycerophosphoinositol in C62B glioma cells

Glioma C62B cells, incubated for 18 h with either an unsaturated (arachidonate or oleate) or saturated (palmitate or stearate) radioactive fatty acid, incorporated label into most species of cellular glycerolipids. Treatment of prelabeled C62B cells with 1 mM acetylcholine (ACh) resulted in an accumulation of radioactive phosphatidate irrespective of which fatty acid was used as a label. However, only in cells prelabeled with unsaturated fatty acids were increases in radioactive fatty acids observed. When exogenous radioactive arachidonate was added to C62B cells in the presence of 1 mM ACh, there was a rapid, selective, and transiently enhanced incorporation of label (several times the control) into phosphatidylinositol (PI). The ACh-enhanced incorporation into PI was not preceded by enhanced incorporation of label into sn-1,2-diacylglycerol or phosphatidate but was followed by an increased labeling of polyphosphoinositides. Similarly, incorporation of oleate into PI was enhanced by ACh. In contrast, ACh did not enhance the incorporation of label into any glycerolipids when saturated fatty acids were used. C62B cells, incubated with [2-3H]inositol for 18 h selectively incorporated label into phosphoinositides. Stimulation of [2-3H]inositol-labeled cells with 1 mM ACh in the presence of 25 mM LiCl resulted in a rapid accumulation of radioactive inositol phosphates (mono-, bis-, and trisphosphates) and glycerophosphoinositol. The accumulation of inositol trisphosphates preceded that of inositol monophosphate and glycerophosphoinositol, while the accumulation of glycerophosphoinositol paralleled the time required for the ACh-stimulated esterification of arachidonate. These results suggest that ACh stimulates activation of a phospholipase C in C62B cells and release of 1,4,5-inositol trisphosphate. There is subsequent activation of phospholipase A2, which in turn liberates arachidonate from PI. The resulting lyso PI is either rapidly reesterified with unsaturated fatty acid to resynthesize PI, or further deacylated to yield glycerophosphoinositol.     

Bradykinin Stimulates Phosphoinositide Hydrolysis and Mobilization of Arachidonic Acid in Dorsal Root Ganglion Neurons

Cultures of fetal rat dorsal root ganglion neurons (7 days in culture) were prelabeled with myo-[3H]inositol or [3H]arachidonic acid for 24 h and stimulated with 10 microM bradykinin for time intervals of 5-300 s. The incubation was terminated by addition of 5% perchloric acid to extract inositol phosphates or organic solvent to extract lipids. Inositol phosphates were resolved by anion-exchange HPLC; lipids were resolved by TLC. Bradykinin stimulation resulted in a 10-fold increased accumulation of inositol 1,4,5-trisphosphate (IP3) and inositol bisphosphate (IP2) (fivefold) by 5 s. The increase in IP3 was transient (half maximal by 1 min), whereas stimulated IP2 levels were sustained for several minutes. Even longer term increases were observed in inositol monophosphate. Stimulation also resulted in a threefold increase in arachidonic acid which was preceded by transient increases in diacylglycerol (twofold) and arachidonoyl-monoacylglycerol (threefold). The temporal lag in the accumulation of arachidonic acid with respect to diglyceride and monoglyceride suggested the involvement of di- and monoglyceride lipases in arachidonic acid mobilization. A role for phospholipase A2 is also possible, because pretreatment of cultures with quinacrine partially blocked arachidonic acid release. Bradykinin-stimulated arachidonic acid release was decreased in the presence of calcium channel blockers nifedipine or verapamil (50 microM), or EDTA (2.5 mM). The role of calcium was verified further in that accumulation of phosphatidic acid, diacylglycerol, and arachidonic acid was maximally stimulated by treatment with the calcium ionophore A23187 (20 microM).     

Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).     

Muscarinic Cholinergic Receptor-Mediated Phosphoinositide Metabolism in Peripheral Nerve

Few receptor-mediated phenomena have been detected in peripheral nerve. In this study, the ability of the muscarinic cholinergic receptor agonist carbamylcholine to enhance phosphoinositide (PPI) breakdown in sciatic nerve was investigated by measuring the accumulation of inositol phosphates. Rat sciatic nerve segments were prelabeled with myo-[3H]inositol and then incubated either with or without carbamylcholine in the presence of Li+. [3H]Inositol monophosphate ([3H]IP) accumulation contained most of the radioactivity in inositol phosphates, with [3H]inositol bisphosphate ([3H]IP2) and [3H]inositol trisphosphate ([3H]IP3) accounting for 7-8% and 1-2% of the total, respectively. In the presence of 100 microM carbamylcholine, [3H]IP accumulation increased by up to 150% after 60 min. The 50% effective concentration for the response was determined to be 20 microM carbamylcholine and stimulated IP generation was abolished by 1 microM atropine. Enhanced accumulation of IP2 and IP3 was also observed. Determination of the pA2 values for the muscarinic receptor antagonists atropine (8.9), pirenzepine (6.5), AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) (5.7), and 4-diphenylacetoxy-N-methylpiperidinemethiodide (4-DAMP) (8.6) strongly suggested that the M3 muscarinic receptor subtype was predominantly involved in mediating enhanced PPI degradation. Following treatment of nerve homogenates and myelin-rich fractions with pertussis toxin and [32P]NAD+, the presence of an ADP-ribosylated approximately 40-kDa protein could be demonstrated. The results indicate that peripheral nerve contains key elements of the molecular machinery needed for muscarinic receptor-mediated signal transduction via the phosphoinositide cycle.     

[3H]Arachidonic acid metabolism in rat brain minces: Effects of nucleotide triphosphates,CDPcholine and CMP

Rat brain minces were used to investigate the effects of nucleotides on the metabolism of arachidonic acid in nerve tissue. Brain free fatty acids, neutral lipids and phospholipids, were radiolabeled in vivo following intracerebral injection of [³H]arachidonic acid. Minces were prepared from the radiolabeled cerebra and were incubated in a modified Krebs-Ringer buffer with and without various nucleotides. The incubation-induced accumulation of unesterified [³H]arachidonate was reduced in the presence of CDPcholine, ATP, CTP, GTP, and UTP. These nucleotides inhibited choline and inositol glycerophospholipid hydrolysis. They also reduced the amount of labeled diglycerides. However, CDPethanolamine had no effect on arachidonic acid metabolism in the mince preparation and CMP appeared to stimulate further hydrolysis of choline glycerophospholipids, resulting in increased accumulation of [³H]arachidonic acid and labeled diglycerides. We suggest that the production of unesterified [³H]arachidonate and labeled diglycerides is due to the involvement of more than one catabolic reaction, since the high energy nucleotides had similar effects on fatty acid accumulation, but different effects on phospholipid labeling.     

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.     

Calcitonin gene-related peptide and cyclic AMP stimulate phosphoinositide turnover in skeletal muscle cells. Interaction between two second messenger systems

Calcitonin gene-related peptide (CGRP) has previously been shown to coexist with acetylcholine in spinal cord motoneurons and to stimulate adenylate cyclase in skeletal muscle cells. We now demonstrate that in cultured chick myotubes whose phosphoinositides have been labeled with [3H]inositol, CGRP enhanced the accumulation of [3H]inositol mono-, bis-, and trisphosphates. Rat CGRP-I (rCGRP) (0.1 microM) elicited a transient increase in [3H]inositol 1,4,5-trisphosphate, as well as a more sustained elevation of [3H]inositol 1,3,4-trisphosphate levels. In the presence of Li+, rCGRP evoked an approximately 3-fold increase of [3H]inositol monophosphate levels, which persisted for up to 1 h. This effect of rCGRP was concentration-dependent, the half-maximal response being obtained at 1 nM. Since rCGRP also accelerated the rate of synthesis of [3H]inositol-containing lipids, it appears that the peptide acts by stimulating phosphoinositide turnover in chick myotubes. Agents that either mimic or elevate intracellular cyclic AMP also enhanced the synthesis of [3H]inositol-containing lipids, and the accumulation of inositol phosphates, suggesting that the effects of rCGRP are mediated, at least in part, via the activation of adenylate cyclase. This hypothesis was strengthened by the non-additivity of the inositol phosphate responses elicited by rCGRP and other cAMP-mobilizing agents, and by the sensitivity of these responses to various pharmacological treatments. The present results provide an example of positive interaction between cAMP and the phosphoinositide signaling system. They further suggest that a coexisting neuropeptide may exert pleiotropic actions upon its target cell by stimulating multiple signal transduction pathways.     

Enhanced arterial contractility to noradrenaline in diabetic rats is associated with increased phosphoinositide metabolism.

The purpose of this study was to investigate whether the increased contractile responsiveness of aortae from male rats with 12-14 week streptozotocin-induced diabetes to noradrenaline is associated with alterations in phosphoinositide metabolism. The contractile response to noradrenaline (10 microM) in both the presence and absence of extracellular calcium was significantly enhanced in aortae from diabetic rats. No significant differences were found between control and diabetic arteries in the basal incorporation of 32P and [3H]myo-inositol into phosphoinositides, or in the basal accumulation of [32P]phosphatidic acid and [3H]inositol phosphates. However, noradrenaline (10 microM) caused significantly greater breakdown of [32P]phosphatidylinositol 4,5-bisphosphate and formation of [32P]phosphatidic acid and [3H]inositol phosphates in diabetic aortae than in control preparations. The production of [3H]inositol phosphates induced by noradrenaline was selectively reduced by the alpha 1-adrenoceptor antagonist, prazosin, in both control and diabetic tissues. These results indicate that phosphoinositide metabolism in response to noradrenaline via stimulation of alpha 1-adrenoceptors is enhanced in aortae from chronic streptozotocin-diabetic rats. The increase in inositol 1,4,5-trisphosphate and 1,2-diacylglycerol production that presumably results could be responsible, at least in part, for the enhanced contractile response of aortae from diabetic rats to noradrenaline.     

Long-term atropine treatment lowers the efficacy of carbachol to stimulate phosphatidylinositol breakdown in the cerebral cortex and hippocampus of rats.

The effect of long-term treatment with atropine, a muscarinic antagonist, known to cause up-regulation of receptor numbers, was examined on the muscarinic-receptor-mediated stimulation of phosphoinositide breakdown in the rat cerebral cortex and hippocampus. Although the numbers of both M1 muscarinic receptors, as measured by [3H]pirenzepine binding, and M1 and M2 receptors increased in both brain regions, the maximal breakdown of myo-[3H]inositol-labelled phosphoinositides was unaltered in the presence of carbachol at a saturating concentration (10(-2) M). In fact the efficacy of carbachol was decreased in slices from atropine-treated cerebral cortex [EC50 (concentration producing half-maximal effect) = 93 microM] as compared with the saline-treated control (EC50 = 23 microM)(P less than 0.005). Similarly the EC50 value (23 microM) in hippocampal slices from saline-treated rats increased in atropine-treated rats to 126 microM (P less than 0.005). This lowered efficacy of muscarinic stimulation could not be explained in terms of residual atropine in the tissue from treated rats. The noradrenaline- or serotonin (5-hydroxytryptamine)-stimulated breakdown or the K+ potentiation of the muscarinic-receptor-stimulated breakdown of [3H]phosphoinositides was not affected by the atropine treatment. Chromatography of the released [3H]inositol phosphates shows that atropine treatment did not cause any qualitative change in the pattern of [3H]inositol phosphates released by carbachol stimulation.     

The alpha 1-adrenergic transduction system in hamster brown adipocytes. Release of arachidonic acid accompanies activation of phospholipase C.

Previous studies of brown adipocytes identified an increased breakdown of phosphoinositides after selective alpha 1-adrenergic-receptor activation. The present paper reports that this response, elicited with phenylephrine in the presence of propranolol and measured as the accumulation of [3H]inositol phosphates, is accompanied by increased release of [3H]arachidonic acid from cells prelabelled with [3H]arachidonic acid. Differences between stimulated arachidonic acid release and formation of inositol phosphates included a requirement for extracellular Ca2+ for stimulated release of arachidonic acid but not for the formation of inositol phosphates and the preferential inhibition of inositol phosphate formation by phorbol 12-myristate 13-acetate. The release of arachidonic acid in response to phenylephrine was associated with an accumulation of [3H]arachidonic acid-labelled diacylglycerol, and this response was not dependent on extracellular Ca2+ but was partially prevented by treatment with the phorbol ester. The release of arachidonic acid was also stimulated by melittin, which increases the activity of phospholipase A2, by ionophore A23187, by lipolytic stimulation with forskolin and by exogenous phospholipase C. The arachidonic acid response to phospholipase C was completely blocked by RHC 80267, an inhibitor of diacylglycerol lipase, but this inhibitor had no effect on release stimulated with melittin or A23187 and inhibited phenylephrine-stimulated release by only 40%. The arachidonate response to forskolin was additive with the responses to either phenylephrine or exogenous phospholipase C. These data indicate that brown adipocytes are capable of releasing arachidonic acid from neutral lipids via triacylglycerol lipolysis, and from phospholipids via phospholipase A2 or by the sequential activities of phospholipase C and diacylglycerol lipase. Our findings also suggest that the action of phenylephrine to promote the liberation of arachidonic acid utilizes both of these reactions.     

G-protein involvement in central-nervous-system muscarinic-receptor-coupled polyphosphoinositide hydrolysis.

Potentiation of muscarinic-agonist-stimulated polyphosphoinositide (PPI) hydrolysis was demonstrated in a rat cerebral-cortical membrane preparation prelabelled with myo-[3H]inositol. Accumulation of myo-[3H]inositol 1,4-bisphosphate ([3H]IP2) was used to assess brain [3H]phosphatidylinositol 4,5-bisphosphate hydrolysis as its immediate metabolite, myo-[3H]inositol 1,4,5-trisphosphate, was rapidly hydrolysed to [3H]IP2. Inclusion of ATP (100 microM) and Mg2+ (5 mM) in the assay medium was necessary to demonstrate the effect of GTP analogues on carbachol-stimulated brain [3H]PPI turnover. Carbachol (100 microM) induced only a small increment in [3H]IP2 accumulation (142% of control) in 1 min. However, its effect was markedly enhanced, to 800% and 300% of control, by 100 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) respectively. GTP[S] and p[NH]ppG also stimulated [3H]IP2 accumulation by over 500% and 200% of control, respectively. The GTP-analogue-potentiated carbachol effect was antagonized by 10 microM-atropine, whereas the GTP-analogue stimulation was unaffected. This report confirms the involvement of a G (GTP-binding) protein(s) in brain PPI metabolism and provides new evidence for the role of G protein(s) in the coupling of stimulated muscarinic receptors to PPI hydrolysis in the central nervous system.     

PGJ2 and delta 12PGJ2 inhibit cell growth accompanied with inhibition of phosphoinositide turnover in human astrocytoma cells

PGJ2 and delta 12PGJ2 (1 microM to 30 microM) inhibited the growth of human astrocytoma cells (1321N1) in a time-dependent manner within 48 hrs, determined by [3H]thymidine incorporation into acid-insoluble fraction or amounts of protein. The EC50 values for PGJ2 and delta 12PGJ2 were approximately 8 microM and 6 microM, respectively. [3H]Thymidine incorporation to acid insoluble fraction was inhibited by these PGs within 1 hr, indicating that these PGs rapidly affect cell functions. Although it has been reported that an increase in cyclic AMP inhibits cell growth, PGJ2 and delta 12PGJ2, but not PGE1, reduced isoproterenol (10 microM)-induced accumulation of cyclic AMP, suggesting that PGJ2 and delta 12PGJ2 may disturb adenylate cyclase system, which might be independent on cell growth. On the other hand, these PGs inhibited the incorporation of [3H]inositol into phospholipid fraction within 6 hrs. Furthermore, PGJ2 and delta 12PGJ2 inhibited carbachol- and/or histamine-induced accumulation of inositol phosphates with a similar dose-dependency to their inhibitions of cell growth. In membrane preparations, however, PGJ2 and delta 12PGJ2 failed to inhibit GTP gamma S (10 microM)- nor Ca2+ (1 mM)-induced accumulation of inositol phosphates. The site of PGJ2 or delta 12PGJ2 in inhibition of inositol phosphate accumulation would not be phospholipase C nor a putative GTP binding protein involved in activation of phospholipase C. The present results indicate that PGJ2 and delta 12PGJ2 inhibit cell growth in human astrocytoma cells and the inhibition of phosphoinositide turnover by these PGs might be involved in the inhibition of cell growth.