The high turnover rate of phosphatidylinositol in chicken heart ventricular cells during the earlier stages of development

The number of the chicken ventricular cells develops exponentially up to Day 12 in the developmental stages of embryos, after which the number gradualy decreases. The phosphatidylinositol metabolism at various stages in development (Days 5 to 21) has been studied. Ventricular cells were incubated in a physiological solution containing ³²P_i, or [1,3-³H]glycerol. Radioactivities incorporated into the phosphatidylinositol were estimated. The specific activity of [1,3-³H]glycerol, taken into phosphatidylinositol, at Day 12, was shown to be approximately equal to that of the other classes of phospholipids. However, the rate of labeling of ³²P_i into the phosphatidylinositol was extremely high in comparison with the other classes of phospholipids in the same ventricles. These results show that there is a rapid turnover of the phosphorylinositol moiety in the phosphatidylinositol in the earlier stages of development. This high turnover rate of the phosphatidylinositol was observed up to Day 12, after which it began to decrease. This turning point of the phosphatidylinositol metabolism coincided well with the decrease of the rate of cell proliferation. Therefore, this rapid turnover of phosphatidylinositol could have a specific functional role related to cell division. This rapid turnover of the phosphorylinositol moiety of the phosphatidylinositol associated with ventricular cell proliferation at different embryonic stages is reported for the first time.     

Phospholipid metabolism in Ehrlich ascites tumor cells. I. Turnover rate of phosphatidylinositol.

1. Radioactive precursors, 32 PI, [1-14C]glycerol, and [1-14C]acetate, were individually injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor, and the rates of incorporation into phospholipid fraction of Ehrlich ascites tumor cells were estimated. Although no distinct difference in specific activities was observed between phosphatidylinositol and other phospholipid classes as regards the incorporation of [1-14C]acetate of [1-14C]glycerol, a higher rate of incorporation of 32Pi into phosphatidylinositol was observed. The specific activity of phosphatidylinositol reached more than ten times that of phosphatidylcholine in the first hour. 2. The radioactivities incorporated into the phospholipids of Ehrlich ascites tumor cells and liver were estimated after simultaneous injection 32Pi and [2-3H]inositol. The incorporation of 32Pi into phosphatidylinositol of liver was similar in specific activity to those of other phospholipids. The ratio (3H/32Pi) of phosphatidylinositol only slightly in the ascites tumor cells, while an appreciable decrease of the ratio was observed in the liver during the first 3 hr. 3. These results suggest that phosphatidylinositol synthesis through pathways other than de novo synthesis is rapid in ascites tumor cells.     

Enhanced turnover of arachidonic acid-containing species of phosphatidylinositol and phosphatidic acid of concanavalin A-stimulated lymphocytes

The incorporation of [32P]orthophosphate into phosphatidylinositol (PI) of pig lymphocytes was markedly increased by stimulation with concanavalin A. The labeling of PI with [3H]glycerol was also enhanced significantly, indicating that both de novo synthesis and recircular system (PI response) of PI were accelerated. This rapid labeling of PI might be related to the rapid breakdown of phosphatidylinositol 4,5-bisphosphate which was observed in various stimulated tissues. Concanavalin A also accelerated the labeling of phosphatidic acid with 32P and [3H]glycerol. To determine the dependence of this phenomenon on the fatty acid composition of both phospholipids, we separated PI and phosphatidic acid into individual molecular species. The predominant molecular species in PI was tetraene (81.6%) and those in phosphatidic acid were monoene (53.0%), diene (15.8%) and tetraene (19.2%), respectively. Interestingly, the incorporation of 32P into arachidonic acid-containing species (tetraene) was most rapidly elevated. On the other hand, the increment of 32P into saturated + monoene, diene and triene was relatively smaller and resembled that of [3H]glycerol. Similarly, the incorporation of 32P into tetraene of phosphatidic acid was preferentially accelerated. This is the first report concerning the metabolism of molecular species of phosphatidic acid in stimulated cells. These results indicate that the PI recirculating system is virtually dependent on tetraenoic species and that the participation of other molecular species is small. The increased de novo synthesis mainly depends upon molecular species other than tetraene. Arachidonic acid-containing species which turn over rapidly via the PI cycle may have an important role in the mitogenic triggering.     

Turnover rates of the molecular species of alkenyl ether phospholipids of Ehrlich ascites tumor cells.

[1-14C]Glycerol was injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor cells and the lipids were extracted from the cells after selected periods. 1-O-Alkenyl-2-acyl-3-acetylglycerols were prepared from the ethanolamine phosphoglycerides and fractionated by AgNO3-impregnated thin-layer chromatography into five molecular species. Bands 1--5 obtained in this way can be designated as saturated, monoene, diene, tetraene and hexaene species, respectively. Band 5, composed mainly of hexaene species was predominant among those derived from alkenyl ether phospholipids. The specific radioactivities of the fractionated species were determined. Hexaene molecular species turned over most rapidly and disaturated and tetraene species turned over at a high rate, while monoene and diene molecular species turned over very slowly.     

Effect of calmodulin antagonists on lysosomal enzyme secretion and phospholipid metabolism in guinea-pig macrophages

The effects of calmodulin antagonists on the secretion of lysosomal enzyme and lipid metabolism in guinea-pig peritoneal macrophages were studied. Calmodulin antagonists, such as trifluoperazine, dibucaine and quinacrine, inhibited the secretion of N-acetyl-β-d-glucosaminidase from cytochalasin B-treated macrophages when the macrophages were stimulated by the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (f Met-Leu-Phe) or the Ca²⁺ ionophore A23187. The effect of calmodulin antagonists on the incorporation of [³²P]P_i or [³H]glycerol into glycerolipids as well as on the redistribution of [¹⁴C]glycerol or [³H]arachidonic acid in [¹⁴C]glycerol- or [³H]arachidonic acid-prelabelled lipids were examined. Trifluoperazine, dibucaine or quinacrine stimulated [³²P]P_i incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) without significant effect on the labelling of phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), lysophosphatidylcholine (lyso-PtdCho) and lysophosphatidylethanolamine (lyso-PtdEtn). The incorporation of [³²P]P_i into phosphatidylcholine (PtdCho) was, on the contrary, inhibited. When calmodulin antagonists were added to macrophages stimulated by fMet-Leu-Phe, [³²P]P_i incorporation into PtdIns and PtdA was synergistically increased compared with that induced only by calmodulin antagonists. Trifluoperazine inhibited the incorporation of [³H]glycerol into PtdCho, triacylglycerol and PtdEtn. Also in this case, the incorporation of [³H]glycerol into PtdA and PtdIns was greatly enhanced. But [³H]glycerol incorporation into PtdSer, lyso-PtdEtn and lyso-PtdCho was not affected by the drug. On the other hand, diacylglycerol labelling with [³H]glycerol was maximally activated by 10μm-trifluoperazine and levelled off with the increasing concentration. When the effect of calmodulin antagonists on the redistribution of [¹⁴C]glycerol among lipids was examined in pulse-chase experiments, no significant effect on [¹⁴C]glycerol redistribution in PtdEtn, PtdCho, PtdIns, PtdSer, PtdA and tri- and di-acylglycerol could be detected. When macrophages prelabelled with [³H]arachidonic acid were treated with trifluoperazine, dibucaine or quinacrine, the [³H]arachidonic acid moiety in PtdEtn and PtdCho was decreased and that in PtdA was increased. The formation of [arachidonate-³H]diacylglycerol and non-esterified [³H]-arachidonic acid was also enhanced, but the increase in [³H]arachidonic acid was only observed at concentrations between 1 and 50μm. [Arachidonate-³H]PtdIns was not significantly affected. The activated formation of [arachidonate-³H]PtdA, diacylglycerol and non-esterified arachidonic acid by these drugs was synergistically enhanced in the presence of fMet-Leu-Phe.     

Phosphate transport and its relationship to cation movements in Ehrlich Lettré ascites tumor cells.

In view of the importance of P_i in the control of cell metabolism, it was of interest to study the mechanism and regulation of P_i uptake by ascites tumor cells. For this purpose, the incorporation of ³²P_i into Ehrlich Lettré cells was compared when competitive anions and inhibitors which alter cation movements were present. Anions such as sulfanilate (35 mm) and succinate (30 mm) decrease ³²P_i uptake by ca. 35%, suggesting that transport is mediated by a protein similar to the 100,000 M_r anion carrier isolated from erythrocyte membranes. Furosemide, a diuretic which bears a structural analogy to sulfanilate inhibitors of anion transport, also decreases ³²P_i incorporation at concentrations as low as 2 × 10^?5m. This inhibitor blocks cation exchange in ascites tumor cells, and from the present data, it is suggested that a possible function of the furosemidesensitive cation exchange protein is to facilitate anion transport. Ouabain, known to inhibit (Na⁺ + K⁺)-ATPase and its dephosphorylation, stimulates the rate of incorporation of ³²P_i into cells and also raises the net inorganic phosphate level. The stimulation of ³²P_i incorporation is decreased by sulfanilate or succinate. In contrast to the effects of ouabain, addition of 10 mm K⁺, which is known to stimulate (Na⁺ + K⁺)-ATPase and its dephosphorylation, decreases ³²P_i incorporation. These observations suggest that anion transport and energy-dependent Na⁺ and K⁺ movements may be closely coupled to the intact cell.     

Stimulation of the incorporation of 32Pi and myo-(2-3H)inositol into the phosphoinositides in polymorphonuclear leukocytes during phagocytosis

The effect of phagocytosis on the incorporation of ³²P_i and myo-[2-³H]inositol into the phosphoinositides (phosphatidylinositol, diphosphoinositide, and triphosphoinositide) by polymorphonuclear leukocytes from guinea pig peritoneal exudates has been studied. The results show that phagocytosis enhanced the incorporation of ³²P_i and myo-[2-³H]inositol into all three inositides in polymorphonuclear leukocytes. Pulse-chase experiments revealed that phagocytosis did not stimulate the loss of the label from the inositides. The findings indicate that the increased radioactivity of the phosphoinositides in polymorphonuclear leukocytes during phagocytosis is due to a greater rate of synthesis of these phospholipids at the time of labeling, rather than due to an increase in the rate of their turnover.     

THE METABOLIC TURNOVER OF MOLECULAR SPECIES OF PHOSPHATIDYLINOSITOL and ITS PRECURSOR PHOSPHATIDIC ACID IN GUINEA-PIG CEREBRAL HEMISPHERES

Abstract– The molecular species composition of phosphatidylinositol from guinea-pig cerebral hemispheres was studied and found similar to that of phosphatidylinositol from ox cerebral hemispheres. In both cases the tetraenoic species was predominant. Phosphatidic acid from guinea-pig cerebral hemispheres contained two major molecular species; the monoenoic and hexaenoic (33.4 and 24 mol/100 mol respectively). In order to study the metabolism of molecular species of phosphatidic acid and phosphatidylinositol in the cerebral hemispheres, guinea-pigs were injected intracisternally with ³²P_i and [U-¹⁴C]glucose. After 5 min of isotopic exchange, the specific radioactivity of ³²P in phosphatidylinositol was nearly equal to that in phosphatidic acid, whereas specific radioactivity of ¹⁴C in the glycerol was 1.4 times and in the fatty acids nearly 0.5 times that in the phosphatidic acid respectively, indicating metabolic heterogeneity of both phospholipids. The glycerol specific radioactivity was different in all the molecular species of phosphatidic acid being greatest in the monoenoic and least in the tetranenoic species. When the molecular species were arranged in this way, the order was representative of their relative rates of synthesis by acylation of glycerol-3-phosphate. An almost opposite order was obtained when the molecular species were arranged according to their phosphate/glycerol radioactivity ratios, indicating the relative contribution of the diacylglycerol kinase pathway to their formation. When the specific radioactivity values and ratios of phosphatidylinositol were similarly considered, the orders of the molecular species were, on the whole, similar to that of phosphatidic acid. This indicated that synthesis de novo (Paulus & Kennedy , 1960) was operative in the formation of most of its molecular species, but due to other considerations it was concluded that part of the tetraenoic, and probably the whole of saturated phosphatidylinositol may be formed by transacylation reactions. The results are discussed in terms of the experimental limitations of previous and present techniques for the analysis of phospholipid molecular species.     

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.     

Ca2+ translocation in Ehrlich ascites tumor cells

Summary Ca²⁺ uptake into Ehrlich ascites tumor cells was studied at 0°C in the presence of mitochondrial inhibitors, conditions that minimized complications caused by sequestration of Ca²⁺ into organelles or by excretion. Under these conditions Ruthenium Red inhibited Ca²⁺ uptake, but other previously implicated ions, such as P_i or Mg²⁺, had no effect. Valinomycin either inhibited or slightly stimulated Ca²⁺ uptake depending on the presence of excess K⁺ on the outside or inside of the cell, respectively. Nigericin inhibited Ca²⁺ transport. Based on these data we propose an electrogenic uptake of Ca²⁺, possibly via a Ca²⁺/H⁺ antiport mechanism.The observation that glucose inhibited Ca²⁺ uptake suggested that in Ehrlich ascites tumor cells an energy-driven Ca²⁺ expulsion mechanism is operative, similar to that in erythrocytes. Plasma membrane preparations of ascites tumor cells were found to contain a Ca²⁺-dependent ATPase. These preparations, when incorporated into liposomes in an inside-out orientation, catalyzed an ATP-dependent uptake of Ca²⁺.     

The distribution of phosphatidylinositol in microsomal membranes from rat liver after biosynthesis de novo. Evidence for the existence of different pools of microsomal phosphatidylinositol by the use of phosphatidylinositol-exchange protein

1. The phosphatidylinositol-exchange protein from bovine brain was used to determine to what extent phosphatidylinositol in rat liver microsomal membranes is available for transfer. 2. The microsomal membranes used in the transfer reaction contained either phosphatidyl[2-³H]inositol or ³²P-labelled phospholipid. The ³²P-labelled microsomal membranes were isolated from rat liver after an intraperitoneal injection of [³²P]P_i. The ³H-labelled microsomal membranes and rough- and smooth-endoplasmic-reticulum membranes were prepared in vitro by the incorporation of myo-[2-³H]inositol into phosphatidylinositol by either exchange in the presence of Mn²⁺ or biosynthesis de novo in the presence of CTP and Mg²⁺. 3. Tryptic or chymotryptic treatment of the microsomes impaired the biosynthesis de novo of phosphatidylinositol. It was therefore concluded that the biosynthesis of phosphatidylinositol and/or its immediate precursor CDP-diacylglycerol takes place on the cytoplasmic surface of the microsomal membrane. 4. Under the conditions of incubation 42% of the microsomal phosphatidyl[2-³H]inositol was transferred with an estimated half-life of 5min; 38% was transferred with an estimated half-life of about 1h; the remaining 20% was not transferable. Identical results were obtained irrespective of the method of myo-[2-³H]inositol incorporation. 5. Both measurement of phosphatidylinositol phosphorus in the microsomes after transfer and the transfer of microsomal [³²P]phosphatidylinositol indicate that phosphatidyl[2-³H]-inositol formed by exchange or biosynthesis de novo was homogeneously distributed throughout the microsomal phosphatidylinositol. 6. We present evidence that the slowly transferable pool of phosphatidylinositol does not represent the luminal side of the microsomal membrane; hence we suggest that this phosphatidylinositol is bound to membrane proteins.     

Distribution of acetylated forms of nucleosomal histones in fractionated chromatin

Hyperacetylated chromatin was isolated from Ehrlich ascites tumor cells grown in n-butyrate containing medium and fractionated by mild digestion with deoxyribonuclease II and precipitation with MgCl₂. The most highly acetylated forms of histones H4 and H3 as well as the acetylated subspecies of H2B were found almost entirely associated with the putatively active Mg²⁺-soluble chromatin fraction. The Mg²⁺-insoluble fraction contained mainly mono- and diacetylated molecules of H4 and H3.     

Increased labelling of polyphosphoinositide in chemically transformed cell line C3H10T1/2 CL8

The effect of malignant transformation of cells on phosphatidylinositol metabolism was investigated using C3H10T1/2 cells and its chemically transformed cell line, MCA CL-16 cells. We found that incorporation of [³²P]P_i into polyphosphoinositide was greatly increased in the transformed cells. A similar tendency was observed when myo-[2-³H]inositol was used as a labelling reagent. It is also observed that influx of labelled inorganic phosphate is enhanced 2-fold by the cell transformation. Therefore, promotion of polyphosphoinositide labelling in the transformed cell might be caused not only by the enhanced metabolism of phosphatidylinositol but also by the increased membrane permeability for radioactive labelling reagents.     

EVIDENCE FOR INVOLVEMENT OF Ca2+ IN THE INCORPORATION OF 32Pi INTO THE PHOSPHATIDYLINOSITOL OF RAT DIAPHRAGM

Abstract— Ethyleneglycol-bis (β-aminoethyl ether)-N-N'-tetraacetic acid (EGTA) inhibited the incorporation of ³²P_i into phosphatidylinositol (PI) in rat diaphragm incubated in Ca²⁺-free Krebs-Ringer medium. Only the labelling of the PI was altered, and no effects on the pool size of PI or on the incorporation of ³²P_i into other phospholipids were observed. The effect of EGTA was concentration-dependent and appeared to be related to its Ca^a+-chelating properties; the inhibition of the incorporation of ³²P_i could be completely reversed by the addition of excess Ca²⁺ but not Mg²⁺. The inhibitory effect of the EGTA was progressively enhanced by lengthening the preincubation of the tissue with EGTA, an observation suggesting that chelation of intracellular or membrane-bound Ca²⁺, rather than extracellular Ca²⁺, was involved in the effect. In contrast to its inhibition of the incorporation of ³²P_i EGTA enhanced the incorporation of [³H]inositol into PI, but this effect was accompanied by an appreciable increase in total uptake of [³Hlinositol by the tissue. Our results suggest that the level of intracellular Ca²⁺ plays a role in the regulation of the incorporation of ³²P_i into PI. Addition of unlabelled α-glycerophosphate to the incubation medium of tissues which had been preincubated with 2-deoxy-d -glucose failed to cause a significant diminution in the inhibition by EGTA of the incorporation of ³²P_i into PI. This experiment suggests, but does not prove, that the effect of EGTA was not at the level of incorporation of ³²P_i into α-glycerophosphate.     

A simple modification to an enzymic method for the preparation of[γ-32P]ATP free of salt and buffer

An existing enzymic method for preparing [γ-³²P]ATP from 32P_i has been modified toyield [γ-³²P]ATP free of salt and buffer. ³²P is incorporated into the γ-position of ATP by isotopic exchange in the presence of glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase. Unreacted ³²P_i is separated from [γ-³²P]ATP by column chromatography on Dowex 1 bicarbonate. [γ-³²P]ATP is eluted with 2 m triethylammonium bicarbonate, which is then completely removed by freeze-drying.     

The rat retina is a useful in vivo model to study membrane lipid synthesis: Rates of biosynthesis of neutral glycerides and phospholipids

The phospholipid composition was studied in the whole rat retina, as well as in its subcellular fractions. A relative enrichment of phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine was observed in rod outer segments (ROS) in comparison with entire retina: nuclear-photoreceptor inner segmentssynaptic bodies (P₁) and synaptosomal-mitochondrial (P₂) fractions. Phosphatidylcholine was the predominant phospholipid class found in all subcellular fractions analyzed. The microsomal fraction was relatively enriched in phosphatidic acid and in phosphatidylinositol. In addition, the rat eye has been used as an in vivo system to study membrane lipid synthesis. After intravitreal injections of [2-³H]glycerol a rapid labeling of retinal glycerolipids took place. Up to 120 min after injection only the glycerol backbone of lipids was labeled. Phosphatidic acid and diacylglycerol displayed rapid rates of synthesis and breakdown. Fastest rates of labeling were attained by phosphatidylcholine followed by phosphatidylinositol. Differences were found when in vitro labeling by [2-³H]glycerol was compared with intravitreal injections. Labeling of phospholipids of subcellular fractions by intravitreally injected [2-³H]glycerol showed that most of the label accumulated in microsomal phosphatidylcholine and phosphatidylinositol. Diacylglycerols and phosphatidylethanolamine also took up 10 and 20% respectively of the precursor. It is concluded that the rat eye is a useful experimental model to study synthesis and metabolism of membrane lipids in the retina.     

PHOSPHATIDYLINOSITOL AND OTHER LIPIDS IN A MAMMALIAN SYMPATHETIC GANGLION: EFFECTS OF NEURONAL ACTIVITY ON INCORPORATION OF LABELLED INOSITOL,PHOSPHATE, GLYCEROL AND ACETATE1

Abstract— Superior cervical ganglia from adult rats were incubated for 1–6 h in a physiological salt solution containing ³²P_i [2-³H]inositol, [U-¹⁴C]glycerol, or [U-¹⁴C]acetate. Control ganglia were at rest throughout incubation, while the preganglionic nerves of the experimental ganglia were stimulated at 5/s, starting after 1 h of incubation. Responses were monitored by recording the action potentials in a postganglionic nerve. Radioactivity of phospholipids was counted after separation of the lipids by paper chromatography. Specific activity of free inositol and the gamma-phosphate of ATP were measured, the latter by using the hexokinase reaction with [¹⁴C]glucose, isolating the product, and counting its content of ³²P and ¹⁴C. At rest, labelling of phosphatidylinositol (PI), phosphatidylcholine and phosphatidylethanolamine proceeded at constant rates for at least 8 h with all precursors which entered them, except that labelling with glycerol slowed after 2–4 h. During stimulation the rate of incorporation of ³²P into PI approximately doubled, as previously reported. The increased rate remained constant for 3 h and then reverted to approximately the resting rate, although the electrical response continued unabated for 16 h. This decrease in rate of ³²P-labelling of PI in the ganglion could not be accounted for by transport into the postganglionic nerves. In stimulated preparations, after 4 h of incubation the labelling of PI was increased above the resting level by 53 ± 5% (mean ±s.e.m. ) with [³H]inositol, 97 ± 6% with ³²P_i, 24 ± 6% with [¹⁴C]glycerol and ?3 ± 10% with [¹⁴C]acetate. The increase with glycerol was thus statistically significant, in contrast with the findings of others on brain, where an increase of this size has neither been demonstrated nor excluded. There were no accompanying effects of stimulation on the specific activities of the gamma-P of ATP or of the free inositol within the ganglion that were sufficient to explain the difference between the labelling of PI with P and that with inositol.     

Phospholipid turnover in isolated rat pancreatic acini. Consideration of the relative roles of phospholipase A2 and phospholipase C

The purpose of the present study was to explore the interaction of phosphatidylinositol breakdown and the turnover of arachidonic acid in isolated rat pancreatic acini by using receptor agonists and the calcium ionophore ionomycin. Acini prelabelled with myo-[³H]inositol in vivo responded to carbachol with a rapid breakdown of phosphatidylinositol. In the presence of [³²P]P_i, carbachol increased labelling of phosphatidic acid and phosphatidylinositol within 1 and 5 min respectively. Carbachol also rapidly stimulated the incorporation of [¹⁴C]arachidonic acid into phosphatidylinositol within 2 min, and the peptidergic secretagogue caerulein caused the loss of radioactivity from phospholipids prelabelled with arachidonic acid. Ca²⁺ deprivation partially impaired the stimulatory action of carbachol on arachidonic acid turnover. In contrast with its stimulatory effects on [³²P]P_i and [¹⁴C]arachidonate incorporation, carbachol inhibited the incorporation of the saturated fatty acid stearic acid into phosphatidylinositol. Whereas ionomycin stimulation of phosphatidylinositol breakdown and [³²P]P_i labelling of phospholipids was slower in onset and less effective than carbachol stimulation, the ionophore effectively promoted (arachidonyl) phosphatidylinositol turnover within 2 min. These results implicate two separate pathways for stimulated phosphatidylinositol degradation in the exocrine pancreas, involving phospholipases A₂ and C. Whereas mobilization of cellular Ca²⁺ appears sufficient to cause activation of phospholipase A₂ and amylase secretion, additional events triggered by receptor activation may be required to act in concert with Ca²⁺ to optimally stimulate phospholipase C. The nature of the interaction between phospholipases A₂ and C and their specific physiological roles in pancreatic secretion remain to be elucidated.     

Conversion of dCTP to CTP by isolated nuclei of Ehrlich ascites tumor cells

Isolated nuclei of Ehrlich ascites tumor cells synthesize DNA in vitro using endogenous template and enzymes. Deoxycytidine nucleoside triphosphate (dCTP) is incorporated into acid-insoluble material to a much greater extent than the other substrates, even in the absence of the other triphosphates. Much of the [³H] dCTP is converted to [³H]CTP, some of which is incorporated into RNA, as evidenced by alkali-lability and density on cesium sulfate gradients.     

Continuous measurement of metabolically produced 14CO2 within the range of minutes

A new device is presented for the continuous evolution, expulsion, and collection of metabolically produced ¹⁴CO₂. It permits the investigation of short-term changes of ¹⁴CO₂ production within minutes. In combination with liquid seintillation counting of the chemically trapped and fractionated ¹⁴CO₂ the method is extremely sensitive. The incubation vessel is specifically designed for susceptible eucaryotic cells. The capacity of this method is demonstrated by the measurement of the complex regulation of the pyruvate dehydrogenase activity by means of ¹⁴CO₂ evolution from [1-¹⁴C]pyruvate within intact Ehrlich ascites tumor cells.