Labeling Of Phosphatidylinositol Research Articles

Rapid and selective uptake, metabolism, and cellular distribution of docosahexaenoic acid among rod and cone photoreceptor cells in the frog retina

The uptake, metabolism, and cellular distribution of 3H-docosahexaenoic acid (3H-22:6) in the frog retina during in vitro incubation were studied. An initial diffuse labeling throughout the retina was detected by autoradiography and was followed by an active steady increase in labeled photoreceptor cells. After 6 hr of incubation, 92% of the label was concentrated in photoreceptor cells. Among these cells, 435-rods (green rods) labeled heavily and showed two to three times higher uptake than the 502-rods (red rods). Cone uptake labeling was the lowest, showing negligible labeling throughout the cytoplasm. However, oil droplets of the 575-cones actively concentrated 22:6. The high uptake of 3H-22:6 by photoreceptor cells was followed by its rapid esterification into phospholipids. After 6 hr of labeling, only 5% of the radioactivity in the retina was free 22:6, whereas 88% was esterified into phospholipids. The remaining 22:6 was distributed equally in triacylglycerols (TAGs) and diacylglycerols. When 3H-22:6 (0.11 microM) of high specific activity was used, early incubation times showed phosphatidylinositol (PI) labeling to be of the same order of magnitude or greater than that of phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Although the amount of endogenous 22:6 esterified into PI accounted for less than 2% of the 22:6 in retinal phospholipids, 27% of 3H-22:6 labeling was recovered in this phospholipid. When 14C-22:6 at a final concentration of 70 microM was used, a different profile of lipid labeling was observed. Forty percent of the labeling remained in the free fatty acid pool, followed by TAG (24%), PC (14%), and PE (12%). PI showed the smallest increase in picomoles of 14C-22:6 incorporated, when compared with 3H-22:6. In conclusion, a selective and differential uptake of 3H-22:6 by photoreceptor cells is coupled to its active utilization for phospholipid biosynthesis, mainly that of PC, PE, and PI. The differential uptake of 3H-22:6 among photoreceptor cells may reflect involvement of this fatty acid in cell-specific functions.

Swiss 3T3 cells preferentially incorporate sn-2-arachidonoyl monoacylglycerol into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol

The sn-1-stearoyl-2-arachidonoyl phospholipids of animal cells appear to be formed by special mechanisms. To determine whether monoacylglycerol (MG) incorporation pathways are involved we incubated quiescent Swiss 3T3 cells with [3H]glycerol-labeled sn-2-arachidonoyl MG, then analyzed the radioactive cell lipids that accumulated. We also examined cell homogenates to identify enzyme activities that might promote the incorporation of sn-2-arachidonoyl MG into other cell lipids. The cell incubation experiments demonstrated rapid labeling of several lipids, including diacylglycerol, lysophosphatidic acid, phosphatidic acid, and phosphatidylinositol. They also demonstrated selective labeling of sn-1-stearoyl-2-arachidonoyl species of phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. The cell homogenate experiments identified an sn-2-acyl MG acyltransferase activity, an MG kinase activity that phosphorylates sn-2-arachidonoyl MG in preference to sn-2-oleoyl MG, and a stearoyl-specific acyl transferase activity that converts sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidic acid. The results also showed that this stearoyl transferase could act with other enzymes to convert sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol. The combined results indicate that Swiss 3T3 cells incorporate sn-2-arachidonoyl MG into phospholipids by at least two different pathways, including one that specifically forms sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.

Developmental assembly of calcium-mobilizing systems for excitatory amino acids in rat cerebellum.

The postnatal development of calcium-mobilizing systems was studied by both microfluorometric imaging analysis of Ca2+ on living rat cerebellar slices and immunohistochemical labeling of phosphatidylinositol 4,5-bisphosphate (PIP2) and inositol 1,4,5-trisphosphate binding protein (IP3BP) in fixed rat cerebellum. Stimulation with quisqualate (QA) or N-methyl-D-aspartate (NMDA) enhanced the Ca2+ level only diffusely on postnatal day (PND) 3, but more discretely on PNDs 7 and 15. On PND 21, QA-induced responses were localized in the molecular layer especially, but not in the granular layer. By contrast, NMDA mobilized Ca2+ prominently in the granular layer, but only weakly in the molecular layer. Localized expression of PIP2 in the molecular layer paralleled QA-induced Ca2+ mobilization, but IP3BP was expressed more diffusely. The present study offers the first direct evidence that PIP2, but not IP3BP, is essential for QA-induced Ca2+ mobilization in the cerebellar cortex.

Phosphatidylinositol synthase and phosphatidylinositol/inositol exchange reactions in turkey erythrocyte membranes

Unlike human erythrocytes, those from avian species, such as turkeys and chicks, rapidly incorporate myo-[3H]inositol into membrane phospholipids. The mechanisms regulating [3H]Ins labelling of phosphatidylinositol have been investigated using turkey erythrocyte membranes. In the absence of added nucleotides, [3H]inositol incorporation appears to proceed via phosphatidylinositol/inositol exchange, with a Km for inositol of 0.01 mM. The reaction was dependent upon divalent cations, either Mg2+ or Mn2+, with the latter metal ion being the more effective. [3H]Inositol incorporation was accelerated by CMP, especially when the concentration of Ins was greater than the Km for the exchange reaction. CMP-dependent labelling of PtdIns had a Km for inositol of 0.3 mM and for CMP of 0.015 mM. Divalent cations were also required for this reaction: activity peaked at 0.5 mM-Mn2+ and declined at higher concentrations. At relatively high concentrations, Mg2+ was more effective than Mn2+, with peak activity being achieved above 10 mM. CMP-dependent incorporation of [3H]inositol appears to reflect an exchange reaction catalysed by PtdIns synthase. Definitive evidence for the occurrence of PtdIns synthase in turkey erythrocyte membranes was obtained by demonstrating the formation of [14C]CMP-phosphatidate from [14C]CMP. The radioactivity could be efficiently chased from [14C]CMP-phosphatidate in the presence of unlabelled inositol. The detection of PtdIns synthase activity in morphologically simple turkey erythrocytes should help to clarify the subcellular distribution of this important component of the phosphatidylinositol cycle.

Role of lipid metabolism in cell killing by calcium plus ionophore A23187

Cultured fibroblasts treated with divalent cation ionophore A23187 in the presence of extracellular calcium provide a useful model system for studying mechanisms of cell death associated with elevated intracellular calcium concentrations. Cell death induced by A23187 plus calcium can be conveniently monitored as membrane permeabilization to Trypan blue dye. Because lipids are a major component of cell membranes and play an important role in determining membrane permeability, the present study was initiated to identify changes in cell lipid composition that occur during membrane permeabilization induced by calcium plus A23187. The percent label in each of the major structural lipids in biosynthetically labeled NIH3T3 fibroblasts changed less than 10% during the time course of membrane permeabilization. During the course of membrane permeabilization there was significantly increased label in lysophosphatidylinositol and lysophosphatidylcholine and reduced label in phosphatidylinositol 4,5-bisphosphate. The time course of these changes corresponded to that of the arachidonic acid release response stimulated by calcium plus A23187, not to the time course of membrane permeabilization, which occurs later. These observations are consistent with lipid metabolism induced by A23187 plus calcium playing only a possible regulatory or intermediatory role in membrane permeabilization, rather than causing direct permeabilization of the lipid phase of the membrane.

α1-Adrenoceptor subtypes in aorta (α1A) and liver (α1B)

The effect of several alpha 1 adrenoceptor antagonists on the alpha 1-adrenoceptor-mediated stimulation of phosphatidylinositol labeling was studied comparatively in rat hepatocytes and rabbit aorta. It was observed that 5-methyl urapidil and WB 4101 were much more potent in rabbit aorta than in hepatocytes. The orders of potency were prazosin much greater than 5-methyl urapidil greater than or equal to WB 4101 in liver cells and WB 4101 greater than or equal to 5 methyl urapidil = prazosin in aorta. Treatment with chlorethylclonidine inhibited 70-80% of the stimulation of labeling induced by epinephrine in rat liver, but only 30-40% of that in aorta. Our data suggest the existence of two pharmacologically distinct receptors in these tissues i.e.m alpha 1A-adrenoceptors in aorta and alpha 1B in liver cells.

Influence of lithium on second messenger accumulation in NG 108-15 cells

Treatment of NG108-15 cells with bradykinin for 10 min resulted in a reduction of phosphatidylinositol levels with no change in the other inositol lipids. Accompanying this was a small increase in diacylglycerol but no modification in the level of inositol phosphates. Addition of bradykinin to cells incubated for 30 min in buffer containing lithium did not cause a further decrease in the labeling of phosphatidylinositol, but was accompanied by an increase in mass of the three inositol phosphates. The increase was about 150% for inositol monophosphate, 40% for inositol bisphosphate and 80% for inositol trisphosphate. Under these conditions there was a marked increase in the ability of bradykinin to elevate the content of diacylglycerol. Moreover lithium by itself induced an increase in diacylglycerol six times as great as the increase in total inositol phosphates. The excess diacylglycerol may have its origin in phospholipids other than phosphoinositides.

Pelanserin inhibition of serotonin‐induced phosphatidylinositol turnover and contraction in rabbit aorta

AbstractStimulation of rabbit aortic rings with serotonin or quipazine increased the incorporation of [32P]Pi into phosphatidylinositol (Pl) and induced a dose‐related contraction. The effects of serotonin and quipazine were blocked by 5‐HT2 antagonists with the following order of potency: pelanserin = ketanserin ≫ methysergide. Indorenate, a 5‐HT1 agonist, failed to modify phosphatidylinositol labeling in rabbit aorta but elicited a very weak contraction at high concentrations. Pelanserin blocked epinephrine‐stimulated phosphatidylinositol labeling, being two orders of magnitude less potent than prazosin. The results demonstrate that pelanserin is a potent antagonist of the stimulation of Pl labeling and vasoconstrictor effects of 5‐hydroxytryptamine and suggest that 5‐HT2 blockade is involved as its major pharmacologic action.

Preferential uptake and metabolism of docosahexaenoic acid in membrane phospholipids from rod and cone photoreceptor cells of human and monkey retinas

The uptake, metabolism, and cellular distribution of [3H]docosahexaenoic acid (DHA) in human and monkey retinas were studied with biochemical and autoradiographic techniques. In specimens from two human retina biopsies, incubated for 4 hr or 6 hr with [3H]docosahexaenoic acid (110 nM), 80% of the esterified [3H]fatty acid was recovered in phospholipids and the remainder in triacylglycerols and diacylglycerols. The distribution of [3H]DHA in individual phospholipids (PL) was similar in both retinas, with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) accounting for most of the label. A similar labeling profile was observed in glycerolipids from monkey retina, and after 1 hr of incubation, high labeling of phosphatidic acid (PA, 11%) and phosphatidylinositol (PI, 20%) was observed. In both human and monkey retinas, a preferential uptake of [3H]DHA by photoreceptor cells was revealed by autoradiography. Cone photoreceptors showed a slightly higher density of silver grains in their inner segments than did rod photoreceptors. Photoreceptors accounted for 59% and 79% of the total [3H]DHA taken up by the human and monkey retinas, respectively, the remainder being distributed throughout the neural retina. In conclusion, this study shows for the first time that in human and monkey retinas, DHA is taken up with a high degree of selectivity by photoreceptor cells, and then becomes esterified mainly into phospholipids that will be subsequently utilized for the synthesis of new disc membranes.

Angiotensin II stimulates phosphoinositide turnover and phosphorylase through AII-1 receptors in isolated rat hepatocytes

Angiotensin II stimulated the activity of phosphorylase a (EC50 ≈ 3 nM). The effect of two receptor subtype-selective nonpeptide antagonists, DuP 753 (AII-1 selective) and PD123177 (AII-2 selective), was studied. It was observed that DuP 753 inhibited the effect of angiotensin II (IC 50 100 nM) but in contrast, PD123177 was without effect on this action of the peptide hormone. Angiotensin II stimulated the labeling of phosphatidylinositol (resynthesis) and the release of inositol phosphates (breakdown). These effects of angiotensin II were blocked by DuP 753 but not by PD123177. The antagonists were without effect by themselves on these parameters. The results clearly indicate that angiotensin II receptors of the AII-1 subtype are coupled to phosphoinositide turnover and mediate phosphorylase activation in isolated rat hepatocytes.

Effects of 5-hydroxytryptamine (serotonin) on the incorporation of32P-inorganic phosphate into phospholipids inHymenolepis diminuta(Cestoda)

5-Hydroxytryptamine (5-HT) (1,2 and 5 mM) significantly stimulated the incorporation of radioactive inorganic phosphate (32Pi) into phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid and also total phospholipid fraction of Hymenolepis diminuta after one hour of incubation. Such effect was both time and concentration dependent. In the presence of 5-HT early labelling of phosphatidylinositol was observed. Also, the percentage stimulation by 5-HT was the highest in this fraction under all experimental conditions. The inorganic, organic, total and phosphatidylcholine-bound phosphate of H. diminuta incubated with 5-HT were not significantly different from those of the control under all incubation conditions. Results reported herein suggest that messenger molecules that are derived from phosphoinositides may be involved in the stimulatory mechanism of 5-HT in H. diminuta.

Contrasting effects of phorbol dibutyrate and phorbol myristate acetate in rabbit aorta

In rat hepatocytes, active phorbol esters inhibited the alpha 1-adrenergic stimulation of phosphatidylinositol labeling with the expected potency order: phorbol myristate acetate (PMA) > phorbol dibutyrate (PDB). In contrast, in rabbit aorta the alpha 1-adrenergic action was inhibited dose-dependently by PDB but not by PMA. Similarly PDB (but not PMA) induced a strong contraction in rabbit aorta. The phorbol ester-induced contraction developed slowly, was dose-dependent and independent of extracellular calcium. These effects of PDB in rabbit aorta were neither inhibited by the protein kinase inhibitor H-7 nor mimicked by the synthetic diacylglycerol, OAG. Our results raise some doubts on the mechanism(s) through which the actions of PDB take place in rabbit aorta.

Selective incorporation of (15S)-hydroxyeicosatetraenoic acid in phosphatidylinositol of human neutrophils: agonist-induced deacylation and transformation of stored hydroxyeicosanoids.

The uptake and mobilization of (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE), a major product of arachidonic acid metabolism, was examined with human neutrophils (polymorphonuclear leukocytes; PMNs). Upon exposure to labeled 15-HETE, PMNs rapidly (15 sec to 20 min) incorporated approximately 20% of the label into phosphatidylinositol, while less than 4% was associated with other phospholipid classes and neutral lipids. This pattern was distinct from that of either labeled arachidonate or labeled(5S)-hydroxy-8,11,14-cis-6-trans-eicosatetraenoic acid (5-HETE), which within 20 min were predominantly associated with triglycerides and phosphatidylcholine. After reversed-phase HPLC, greater than 98% of the label in phosphatidylinositol, isolated from PMNs, was released with phospholipase A2. Upon exposure to either chemotactic peptide (FMLP), phorbol 12-myristate 13-acetate, or an ionophore (A23187), 15-HETE-labeled PMNs released 15-HETE from phosphatidylinositol and displayed an impaired ability to generate leukotriene B4 (LTB4), 20-OH-LTB4, and 20-COOH-LTB4. Deacylated [3H]15-HETE was converted to (5S,15S)-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid (5,15-DHETE), lipoxin A4, and lipoxin B4, each carrying 3H label. PMNs labeled with 5-HETE also released and transformed this HETE when stimulated. However, the profile of labeled products differed between PMNs with either esterified 15-HETE or 5-HETE. When activated, 5-HETE-labeled PMNs generated both 5,20-DHETE and 5,15-DHETE but not labeled lipoxins. Threshold aggregation induced by FMLP with 15-HETE-labeled PMNs was inhibited (approximately 2 orders of magnitude), while the threshold response was relatively unimpaired with either A23187 or phorbol 12-myristate 13-acetate-induced aggregation. Results indicate that 15-HETE is rapidly esterified into phosphatidylinositol of PMNs, which can be mobilized and transformed upon exposure of the cells to a second signal. Moreover, they suggest that eicosanoid intermediates other than arachidonic acid can be stored by cells, released via signal transduction, and oxygenated to generate alternative profiles of eicosanoids.

Ca 2+-dependence of arachidonic acid redistribution among phospholipids of cultured mouse keratinocytes

Mouse keratinocytes cultured in a medium containing less than 0.1 mM Ca 2+ (low Ca 2+) incorporated [1− l4C]arachidonic acid (AA) into phospholipids by kinetics including; (i) a rapid labelling of phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer) and both acid-stable and alkenylacyl forms of phosphatidylcholine (PtdCho); and (ii) a slow but long-lasting radiolabel incorporation into both acid-stable and alkenylacyl forms of phosphatidylethanolamine (PtdEtn), partly associated with a net radioactivity loss from acid stable-PtdCho. Under low Ca 2+ conditions no radioactivity transfer apparently occurred between PtdIns and other phospholipid classes. When cells were prelabelled for 24 h with [1− 14C]AA and reincubated in label-free medium containing 1.2 mM Ca 2+ (normal Ca 2+), an early and extensive loss of radioactivity from PtdIns was observed, reasonably in connection with Ca 2+ stimulation of phosphoinositide turnover [1]. Cell shift to normal Ca 2+ did not result in an increased synthesis of labelled eicosanoids, but was consistent with an increase of radioactivity incorporation into diacylglycerol (DAG) and with a complex pattern of [1− 14C]AA redistribution, eventually leading to a marked radioactivity incorporation into acid stable-PtdEtn (but not into alkenylacyl-PtdEtn) and to a labelling decrease of acid stable-PtdCho. The possible mechanisms driving AA recycling after cell shift to normal Ca 2+ are discussed.

Self suppression of phosphoinositide turnover and contraction by stimulating release of endogenous endothelium derived relaxing factor in vascular action of histamine

The aim of the study was to examine the relationship between phosphoinositide turnover and vascular contraction in response to histamine, and the role of endothelium derived relaxing factor (EDRF) in these activities. Thoracic aortic strips were studied under isometric tension in an organ bath. Phosphoinositide turnover was studied using [32P]Pi labelling. Cumulative concentration-response curves were obtained for histamine, and for histamine plus H1 and H2 antagonists. The effect of endothelial denudation was examined, as was that of removing Ca2+ from buffer medium, and of adding methylene blue, an inhibitor of EDRF. EXPERIMENTAL PREPARATIONS: Thoracic aortic rings were obtained from 81 male Japanese white rabbits, weight 2.0-2.4 kg. Histamine (0.1 mumol-0.1 mmol.litre-1) caused concentration dependent contractions and increases of [32P]Pi incorporation into phosphatidylinositol (ED50 = 4.6 mumol.litre-1 and 4.0 mumol.litre-1 respectively). Diphenhydramine inhibited contractile response and phosphatidylinositol labelling by histamine. Cimetidine potentiated contractile response but had no effect on phosphatidylinositol labelling. Endothelial denudation potentiated maximum contraction by 15% and augmented phosphatidylinositol labelling at 0.1 mmol.litre-1 histamine by 30%. In intact aortic rings, methylene blue (10 mumol.litre-1), an inhibitor of EDRF, potentiated histamine induced phosphatidylinositol labelling, but had no influence on denuded rings. A-23187 (0.01 mumol.litre-1), which caused release of EDRF from intact endothelium, suppressed histamine induced phosphatidylinositol labelling in intact aortic rings. The results suggest that stimulation of the release of EDRF in response to histamine causes self suppression of phosphoinositide turnover and contraction.

The phosphatidylinositol synthase of proximal tubule cells

Phosphatidylinositol (PI) is a precursor for an important class of phospholipids, the phosphatidylinositol polyphosphates. Because renal myo-inositol levels may vary under both physiological (e.g., antidiuretic) and pathophysiological (e.g., diabetic) conditions, the formation of PI from CDP-diacylglyceroI (CDP-DG) and myo-inositol via phosphatidylinositol synthase and the regulation of this enzyme have important implications for the cellular biology of renal epithelia. We sought to understand the role of PI synthase by determining its subcellular localization, kinetic properties and regulation in rabbit proximal tubule cells. Proximal tubule cells were isolated from New Zealand White rabbits. The subcellular synthesis of PI was assessed by [ 32P]orthophosphate labelling with subsequent subcellular fractionation. Labelling of PI was time-dependent and consistent with the rapid incorporation of 32PO 4 into basolateral, brush-border, microsomal and nuclear fractions. Pulse-chase labelling of proximal tubule cells was consistent with the formation of PI in microsomal fraction of the proximal tubule cells in addition to both brush-border and basolateral membranes. Conversely, phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol displayed radiolabelling patterns consistent with microsomal synthesis alone. The in situ formation of phosphatidylinositol was substantiated by the direct measurement of phosphatidylinositol synthase activity in basolateral, brush-border and microsomal fractions. The apparent K m values for myo-inositol were 0.32 ± 0.19, 0.39 ± 0.21 and 0.23 ± 0.05 mM, and for CDP-DG were 0.12 ± 0.02, 0.14 ± 0.05 and 0.12 ± 0.02 mM in basolateral, brush-border and microsomal fractions, respectively. V max values for phosphatidylinositol formation were slightly, but not significantly greater, in microsomal than for plasma membrane fractions. Moreover, based on enzymatic enrichment data, plasma membrane PI synthase activity could not be explained by microsomal cross-contamination alone. PI synthase activity was inhibited by co-incubation with PI without differences among the cellular fractions. Intracellular myo-inositol concentration in the proximal tubule cells as measured by gas-liquid chromatography was 20.5 mM, significantly greater than the apparent K m values for myo-inositol. In conclusion, the in situ synthesis of phosphatidylinositol occurs in several membrane fractions; the kinetic properties of phosphatidylinositol synthase appear to be similar in each fraction; and phosphatidylinositol synthase in proximal tubule cells is inhibited by its own formation product. These data suggest that myo-inositol concentration alone is unlikely to be an important regulator of the chemical mass of phosphatidylinositol at the levels of this polyol observed in rabbit kidney.

Signalling mechanism in the lysophosphatidylserine-induced activation of mouse mast cells

Lysophosphatidylserine (0.1–1 μM) elicits histamine release in isolated mouse peritoneal mast cells. The effect becomes manifest after a lag of 30 s and reaches completion in 5 min. Maximal activity is observed when serine is in l-configuration. As shown by the activity of a lysophosphatidylserine analogue lacking the OH group in C 2 position of glycerol, conversion into phosphatidylserine is not required. When 32PO 4-labeled mast cells are challenged 2–5 min with lysophosphatidylserine, the labeling of phosphatidate, phosphatidylinositol and phosphatidylcholine is increased. When [ 3H]arachidonate-labeled mast cells are used, lysophosphatidylserine increases the appearance of isotopic diacylglycerol and phosphatidate. Like the secretory response, these effects are independent of the presence of extracellular Ca 2+. Incubations in the presence of [ 3H]glycerol show that lysophosphatidylserine does not activate the de novo synthesis of phospholipids. In agreement with a participation of phosphoinositidase C in the action of lysophosphatidylserine, we observe accumulation of inositol phosphates in [ 3H]inositol labeled mast cells incubated in the presence of Li +. The results suggest that lysophosphatidylserine delivers its stimulus to mast cells, by the activation of phosphoinositide-dependent signalling mechanism.

Decapitation-induced changes in inositol phosphates in rat brain

Decapitation resulted in a time-dependent production of inositol phosphates in rat brain. This production was analyzed by measuring both the radioactivity and the concentrations of inositol phosphates generated from [3H]inositol-labeled phospholipids. Both measurements produced the same time-dependent changes, including a rapid decrease in inositol 1,4,5-trisphosphate within 1.5 min, a 6-fold increase in inositol 1,4-bisphosphate to a maximum at 1.5 min, a 5-fold rise in inositol 4-monophosphate to a maximum at 2.5 min, and little change in inositol 1-monophosphate. The temporal changes in the mass and radioactivity of these compounds, together with the decrease in labeling of phosphatidylinositol 4,5-bisphosphates, support the idea that the inositol phosphates originate from the hydrolysis of phosphatidylinositol 4,5-bisphosphates and not from either the direct hydrolysis of phosphatidylinositol 4-phosphates or phosphatidylinositols.

Regulation of histamine H 1 receptor-mediated phosphoinositide hydrolysis by histamine and phorbol esters in DDT 1 MF-2 cells

The regulation of histamine-stimulated phosphoinositide turnover by histamine and phorbol esters was examined in intact DDT 1 MF-2 cells grown in suspension culture. Histamine increased the incorporation of 32P into phosphatidylinositol (PI) in these cells, and this stimulation was inhibited by the H 1 antagonist diphenhydramine but not by the H 2 antagonist cimetidine. Pretreatment of cells with histamine or with phorbol 12-myristate 13-acetate (PMA) or other activators of protein kinase C induced a marked decrease in the subsequent stimulation by histamine. PMA, but not histamine, also decreased the ability of epinephrine to stimulate PI labelling through α 1-adrenoceptors. Thus, histamine appears to induce homologous desensitization of histamine H 1 receptor-mediated PI turnover, whereas direct activation of protein kinase C in the absence of receptor occupancy by agonist induces nonspecific heterologous desensitization of both histamine H 1- and α 1-adrenoceptor-mediated responses.

Epidermal growth factor increases the labeling of phosphatidylinositol 3,4-bisphosphate in MA-10 Leydig tumor cells.

Previous studies from this laboratory have shown that mouse epidermal growth factor (mEGF) modulates the hormonal responsiveness of MA-10 Leydig tumor cells without affecting cell multiplication. In an attempt to characterize the intracellular signaling systems activated by mEGF in this cell type, we examined its effects on the labeling of phosphatidylinositols in cells that had been preincubated with different radioactive precursors. Here we report that exposure of MA-10 cells to mEGF, but not other ligands that affect their differentiated function, results in an increase in the labeling of an unusual phosphatidylinositol that does not appear to be present in unstimulated cells. This phosphatidylinositol has been identified as phosphatidylinositol 3,4-bisphosphate.