Labeling Of Phosphatidylinositol Research Articles

Tetradecanoyl phorbol-13-acetate counteracts the responsiveness of cultured thyroid cells to thyrotropin.

We have studied the effects of TPA on the metabolism of porcine thyroid cells cultured for 1-4 days in the absence (control cells) and in the presence of 0.1 mU/ml TSH (TSH cells). The phospholipid turnover, evaluated after a 2 hr incorporation of 32P-phosphate into phospholipids, is markedly modified by the presence of TPA (1.5 microM, 2 hr) in the incubation medium of control and TSH treated cells. The total incorporation is 3-4 times higher than untreated cells, the labelling of phosphatidylinositol (PI) is slightly decreased or unchanged whereas that of phosphatidylcholine (PC) is strongly increased. The increased labelling of PI, promoted by an acute TSH treatment is counteracted by TPA. This TPA effect is not observed when prelabelled cells are challenged for 5 min with the drug. A similar effect is observed when 10 nM TPA is added in the culture medium for 20 hr. The addition of TPA does not affect significantly the protein iodine content in 3 or 4 days control cells incubated for 45 min or 2 hr with 125I-iodine, but dramatically decreases the very high iodination rate of TSH cells. We have tested the TPA effect on the cyclic AMP accumulation for the last 5 min of a 2 hr incubation. TPA inhibits by about 50-80% the stimulation evoked by TSH and only by 10% that evoked by forskolin (0.1 mM). These results suggest a possible link between the PC turnover and the adenylate cyclase responsiveness to TSH and the iodination rate.

Turnover of phospholipids isolated from fractions of lung lavage fluid.

To clarify the steps following surfactant secretion, we injected adult rabbits with radioactive palmitate, lavaged the airways, removed the cells, separated by ultracentrifugation lavage components into two fractions (B and C), and followed the labeling of phospholipids of these fractions. The results were compatible with the view that total and saturated phosphatidylcholine are transferred from B to C. Furthermore, the fluxes of total and saturated phosphatidylcholine through fraction C (0.45 and 0.30 mumol . h-1 . g lung-1, respectively) were compatible with the actual estimates of surfactant recycling. The labeling of phosphatidylglycerol ruled out a simple precursor-product relationship between B and C but was compatible with a nonideal first-order relationship. The labeling of phosphatidylinositol, cardiolipin, and phosphatidylethanolamine was incompatible with the existence of a direct precursor-product relationship between B and C. The labeling of total and saturated phosphatidylcholine suggests that fraction B may be made by active surfactant, whereas fraction C may contain surfactant modified for reuptake or for reuptake and catabolism.

The effect of angiotensin II on arachidonate metabolism in adrenal glomerulosa cells

The effect of angiotensin II on arachidonate metabolism was examined in rat adrenal glomerulosa cells. Incorporation of both [ 3H]arachidonate and [ 32P]phosphate into phosphatidylinositol (PI) were significantly stimulated by angiotensin II. These effects were abolished by lithium, a cation, which was found suitable to prevent increased synthesis of PI in our previous study (T. Balla et al., FEBS Letters 171, 179, 1984). On the other hand, the phospholipase A 2 inhibitor mepacrine failed to inhibit the increased labelling of PI. These observations suggest that the increased 3H labelling of PI occurs via CDP-diacylglycerol, and not via enhanced deacylation-reacylation cycle. The validity of this assumption was further supported, since angiotensin II failed to stimulate the formation of lyso-PI, as examined by both [ 32P]phosphate incorporation and pulse-chase techniques. Angiotensin II decreased the incorporation of [ 3H]arachidonate into phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Considering that we did not find arachidonate release either from phospholipids or from other possible arachidonate sources this decrease may not be due to dilution of the tracer. Thus we assume that angiotensin II may induce a shift in phospholipid synthesis from PC and PE to phosphoinositides. These observations indicate that the enhanced hydrolysis and synthesis of PI in response to angiotensin II is not associated with increased phospholipase A 2 activity in adrenal glomerulosa cells.

Calcium-independent phosphatidylinositol response in gonadotropin-releasing-hormone-stimulated pituitary cells.

This paper describes the effect of gonadotropin-releasing hormone (GnRH, gonadoliberin) on phospholipid metabolism in cultured rat pituitary cells. The cells were incubated with [32P]Pi to label endogenous phospholipids (10-60 min) and then stimulated with GnRH for up to 60 min. Cellular phospholipids were separated by two-dimensional t.l.c. and the radioactivity was determined. Phosphatidylinositol (PI), a minor constituent of cellular phospholipids (7.7%), was the major labelled phospholipid, accounting for 45% of the total radioactivity, at early periods after pulse labelling. On the other hand, phosphatidylcholine, the major cellular phospholipid (37%), was labelled only to 32% of the total radioactivity. The remaining label was distributed among phosphatidylethanolamine (4.2%), cardiolipin (3.4%), phosphatidic acid (PA, 2.5%), and phosphatidylserine (1.8%). GnRH doubled 32P labelling of PA and PI significantly at 1 and 5 min of incubation respectively in the presence or absence of extracellular Ca2+. Labelling of other phospholipids was not affected by GnRH treatment. The half-maximal stimulating dose (ED50) for PI labelling and lutropin release was 0.75 nM and 0.5 nM respectively, and the stimulatory effect was blocked by the potent GnRH antagonist [D-Glp1,pClPhe2,D-Trp3,6]GnRH. GnRH-stimulated PA and PI labelling could not be demonstrated after 1 and 45 min of incubation respectively, or when the prelabelling was conducted for 60 min rather than 10 min. These results suggest heterogeneous compartmentalization of gonadotroph PA and PI pools and that increased PI turnover might be a transducing signal for Ca2+ gating that follows gonadotroph GnRH-receptor activation.

Insulin-stimulated phosphoinositide metabolism in isolated fat cells.

Treatment of isolated fat cells with insulin produced increases of up to 4.8-fold in the incorporation of [3H]inositol into phosphatidylinositol. This effect of insulin was both time- and dose-dependent with half-maximal stimulation at 30 microunits/ml of insulin. Insulin increased the labeling of phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate but not phosphatidylinositol 4-monophosphate in cells which had been preincubated with [3H]inositol for 90 min. Incubation of the cells in a Ca2+-free buffer increased the basal level of phosphatidylinositol labeling and enhanced the effect of insulin. Glucagon and isoprenaline, both of which stimulate lipolysis, had no effect on phosphatidylinositol labeling but did potentiate insulin-stimulated incorporation of [3H]inositol into phosphatidylinositol. Phosphoinositide breakdown was measured by the accumulation of inositol phosphates. Insulin did not increase the level of the inositol phosphates at all concentrations of the hormone tested. By comparison, phenylephrine and vasopressin were able to stimulate phosphoinositide breakdown. Pretreatment of the cells with insulin enhanced the effect of phenylephrine on inositol phosphates' accumulation, suggesting that insulin may potentiate phenylephrine-mediated phosphoinositide turnover. From these data we conclude that insulin stimulates the de novo synthesis of phosphatidylinositol and phosphatidylinositol 4,5-biphosphate, but has no effect on phosphoinositide breakdown.

Effects of platelet-activating factor on the release of arachidonic acid and prostaglandins by rabbit iris smooth muscle. Inhibition by calcium channel antagonists.

Addition of physiological concentrations (10(-12)-10(-8)M) of platelet-activating factor (PAF) to rabbit iris muscle induced a rapid release (in 15s) of prostaglandin (PG)E2 and 6-oxo-PGF1 alpha, measured by radioimmunoassay and rapid release of 14C-labelled arachidonate and PGE2 in muscle prelabelled with [14C]arachidonic acid, measured by radiochromatography. These PAF actions are concentration- and time-dependent. The effect of PAF on PG release is not mediated through the cyclo-oxygenase pathway. The studies on the properties and mechanism of arachidonate release from phosphatidylinositol and other phospholipids in prelabelled irides by PAF suggest the involvement of a phospholipase A2. This conclusion is supported by the findings: (a) that both the removal of arachidonate and formation of lysophosphatidylinositol, from phosphatidylinositol, by PAF occur concomitantly in a time-dependent manner, (b) that Ca2+ is required for the agonist-induced release of arachidonate and PGE2, and (c) that in contrast to the rapid release of [3H]myo-inositol phosphates by carbachol and other Ca2+-mobilizing agonists previously reported in the iris muscle [Akhtar & Abdel-Latif (1984) Biochem. J. 224, 291-300], PAF (10(-12)-10(-8)M) did not appreciably enhance the release of [14C]myo-inositol phosphates and 32P labelling of phosphatidate and phosphatidylinositol in this tissue. Ca2+-channel antagonists, such as nifedipine, verapamil, diltiazem and manganese inhibited PAF-induced arachidonate and PGE2 release in a dose-dependent manner. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not increase the release of arachidonate and PGE2. The ability of Ca2+ antagonists to inhibit arachidonate release by PAF in this tissue probably reflects interference with PAF binding to its receptor. The PAF-induced release of arachidonate and PGE2 occur independently of the cyclo-oxygenase and lipoxygenase pathways. Whether the PAF-induced release of arachidonate and PG in the iris muscle is involved in the pathogenesis of inflammatory and/or physiological reactions in the eye, and how much the inhibitory effects of Ca2+-entry blockers on the PAF actions contribute to the therapeutic use of these drugs, remain to be established.

Differential effects of adrenergic agonists and phorbol esters on the α1-adrenoceptors of hepatocytes and aorta

Epinephrine, norepinephrine and phenylephrine stimulate phosphatidylinositol labeling with [ 32P]Pi in both rat hepatocytes and rabbit aorta. Methoxamine was a full agonist for this effect in rabbit aorta whereas cirazoline and oxymetazoline were partial agonists. In contrast, these three agents (methoxamine, cirazoline and oxymetazoline) were unable to stimulate phosphatidylinositol labeling in rat hepatocytes. Furthermore, cirazoline and oxymetazoline were able to displace the dose-response curve to epinephrine in rat hepatocytes, i.e., they behaved as antagonists. Binding competition curves of these agents with labeled adrenergic ligands indicate that the affinity of α 1-adrenergic receptors in these two tissues (aorta and liver) for the different agents tested was very similar. In addition it was observed that phorbol myristate-acetate inhibited in a dose-dependent fashion the epinephrine-mediated stimulation of phosphatidylinositol labeling in hepatocytes but was without effect on the action of the amine in aorta. Our data suggest that stereochemical differences for α 1-adrenergic activation in liver and aorta may exist and indicate that the ability of phorbol esters to inhibit α 1-adrenergic effects is not universal.

Increased phosphatidylinositol metabolism is an important but not an obligatory early event in B lymphocyte activation.

The phosphatidylinositol (PI) response has been implicated in membrane signaling and cell activation. The role of phospholipid metabolism among the early events in B cell activation has not been clear. We have treated murine B cells with anti-Ig antibody and lipopolysaccharide (LPS) and have found that, although anti-IgM induces the PI response, LPS does not. The increase in metabolic labeling of PI is specific to PI, and not the phosphatidylinositols. Anti-IgM unresponsive B cells from CBA/N mice, which may correspond to a specific functional subpopulation of normal B cells, do not increase PI metabolism in response to anti-IgM, nor do they undergo blastogenesis or DNA synthesis. Moreover, when these deficient B cells are given a stimulus sufficient to drive them into S (LPS + anti-IgM), there is still no corresponding activation of PI metabolism. These results are consistent with a two-signal model of xid B cell activation by anti-IgM. One very early signal primes the cells but does not induce the PI response. A second early signal is supplied by LPS. This signal sustains cells in the activated state, allowing them to receive yet other signals to proceed through G1 and progress further along the cell cycle. A similar sequence of events may occur in the normal B cell, with the first signal provided by priming with anti-IgM, and the second signal, the PI response, supported by a sufficiently high dose of anti-IgM to induce PI turnover and maintain the cell in G1.

A tumor promoter enhances the phosphorylation of polyphosphoinositides while decreasing phosphatidylinositol labelling in lymphocytes

12-O-Tetradecanoyl-phorbol-13-acetate, a comitogen for lymphocytes, suppresses the concanavalin A-induced accumulation of 32P-phosphatidylinositol, in mouse spleen lymphocytes incubated with 32P-orthophosphate. The comitogenic tumor promoter does not affect the rate of de , novo synthesis of phosphatidylinositol, as measured by [ 3H]-glycerol incorporation. Mitogen stimulates the incorporation of [ 32P]-phosphate into phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate, but the tumor promoter suppresses only the increased labelling of the phosphatidylinositol, while it enhances phosphorylation of the polyphosphoinositides.

Effects of prostaglandins and luteinizing hormone-releasing hormone on phosphatidic acid-phosphatidylinositol labeling in rat granulosa cells.

In cultures of rat granulosa cells, luteinizing hormone-releasing hormone (LHRH) increases 32P incorporation into both phosphatidylinositol (PI) and phosphatidic acid (PA). After 20 min, the level of radioactivity was three- to four-fold (p less than 0.01) above control in the PI and PA fractions, respectively. The stimulatory effect of LHRH on 32P incorporation was limited to PI and PA. Similar to the effects of LHRH, a rapid and marked increase of 32P incorporation into both PI and PA is observed upon addition of prostaglandin F2 alpha (PGF2 alpha) (10(-5)M) to rat granulosa cells. Incorporation of radioactivity into PA was already increased (p less than 0.05) by 2 min following PGF2 alpha addition, while the increase in 32P-labeled PI became significant (p less than 0.01) by 5 min. In contrast to PGF2 alpha, the labeling of PI and PA following the addition of PGE2 (10(-5)M) was not significantly different from control levels during the entire 10 min of incubation. The sensitivity of the increased PA-PI labeling induced by LHRH and PGF2 alpha is compared in another experiment. After 20 min incubation 10(-6)M LHRH increased PI and PA labeling by six- and four-fold, respectively. Although the effect of PGF2 alpha is less than that of LHRH, 10(-5)M PGF2 alpha significantly (p less than 0.01) increased PI and PA labeling by three- and two-fold, respectively. By contrast, 10(-6)M PGE2 failed to affect 32P incorporation into the various phospholipid fractions, but a small enhancement (p less than 0.05) of PI and PA labeling was observed only at 10(-5)M PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin rapidly stimulates phosphatidic acid-phosphatidylinositol turnover in rat anterior pituitary cells

In cultured rat anterior pituitary cells, the agonist [Asu 1,6, Arg 8]vasopressin (AVP-A) increased by 1.5-fold 32P i incorporation into phosphatidic acid (PA), as early as 15 s after its addition. Increased phosphatidylinositol (PI) labeling became significant 4 min after AVP-A addition. Dose-response measurements with AVP-A showed ED 50 values of 76 and 62 nM for PA and PI labeling, respectively. Peptide corticotropin-releasing factor (CRF) (0.1μM) did not affect the stimulatory effect of AVP-A on PA and PI labeling. These data suggest that stimulation of PI metabolism in corticotrophs may be one of the early events involved in the stimulation of ACTH release induced by vasopressin. Vasopressin Corticotropin releasing factor Adrenocorticotropin Corticotroph Pituitary Phospholipid

Interactions of vasopressin, cAMP, and prostaglandins in toad urinary bladder.

Prostaglandin (PG) inhibits the hydroosmotic effect of vasopressin. We therefore reexamined the interaction of vasopressin (VP), cAMP, and prostaglandins in toad bladder epithelial cells. Vasopressin slightly, but reproducibly, stimulated PGE2 and thromboxane B2 (TXB2) synthesis in cells prepared by the use of collagenase. When cells were prepared in the presence of a readily reversible cyclooxygenase inhibitor, ibuprofen, subsequent PGE2 synthesis was enhanced sevenfold but that of TXB2 was not. Increasing cAMP by either phosphodiesterase inhibition or 8-bromo-cAMP significantly inhibited both basal and VP-stimulated PGE2 synthesis. This inhibition was overcome by addition of arachidonic acid. Future studies employing these agents will have to consider these effects. VP enhanced 32P labeling of phosphatidylinositol (PI) and phosphatidic acid. This effect was prevented by the phosphodiesterase inhibitor, which also decreased phosphatidylcholine labeling. The results indicate that the phosphodiesterase inhibitor for cAMP may decrease PG formation by interfering with phospholipase activation. Furthermore, VP, similar to its effect in the liver, also increases PI turnover in toad bladder. This may initiate PG synthesis and provide a link among VP, cAMP, and calcium. A double-reciprocal feedback is proposed, whereby VP stimulates PG synthesis in a cAMP-independent manner and also inhibits PG synthesis in a cAMP-dependent manner.

Mechanisms of gonadotropin-releasing hormone and prostaglandin action on luteal cells.

Both gonadotropin-releasing hormone (GnRH) and prostaglandin F2 alpha (PGF2 alpha) can inhibit cAMP and progesterone production in the corpus luteum; however, their mechanism of action is not known. GnRH or PGF2 alpha causes a rapid and marked increase of labelling of phosphatidylinositol (PI) and phosphatidic acid (PA) in rat luteal cells in culture. The incorporation of radioactivity is increased as early as 2 and 5 min into PA and PI, respectively. The labelling of the other phospholipids is not affected. GnRH and PGF2 alpha exert their stimulatory effects on PA-PI turnover at a mean effective dose value of ca. 15 and 100 nM, respectively. Their effects appeared to be additive when both agents were present in the same incubations. Interestingly, addition of the calcium ionophore A23187 also causes a dramatic increase of PA-PI turnover in luteal cells. By contrast, human chorionic gonadotropin and isoproterenol, agents that stimulate cAMP and progesterone production in luteal cells, as well as PGE2 (1 microM), all fail to alter phospholipid labelling; dibutyryl or 8-bromo-cAMP (2-5 mM) actually attentuates the GnRH or PGF2 alpha effect on PI and PA. A very similar PA-PI response to GnRH and PGF2 alpha has also been observed using rat granulosa cells in culture. It seems that following their binding to membrane receptors, GnRH and PGF2 alpha may share a common mechanism in the ovarian cell, possibly involving the stimulation of PA-PI metabolism.

Tetraenoic species are conserved in muscarinically enhanced inositide turnover.

Carbamylcholine enhances the labeling of phosphatidate and phosphatidylinositol from 32Pi in nerve endings. Approximately 74% of labeled phosphatidate and 85% of labeled phosphatidylinositol produced on muscarinic stimulation are accounted for by tetraenoic species, as detected by argentation TLC. Incubation of membranes derived from nerve endings with [gamma-32P]ATP under conditions of phosphodiesteratic degradation of endogenous polyphosphoinositides resulted in increased labeling of phosphatidate. Approximately 78% of the newly formed phosphatidate was in a tetraenoic fraction. It is concluded that in muscarinically stimulated nerve endings, the diacylglycerol moiety is conserved following diacylglycerol release from polyphosphoinositides through its resynthesis to inositol lipid via phosphatidate.

Phosphorus Labeling of Proteins and Phospholipids in Intact Platelets in Response to pH 5.3

Previous studies had shown that when gel-filtered or washed human platelets were incubated at pH 5.3, the cells secreted their granule-stored materials suggesting that low pH can act as a platelet activator. We determined here whether the effects of low pH on platelet protein phosphorylation and on platelet lipid metabolism were consistent with this view. When washed human platelets were incubated for 20 min at pH 5.3 and electrophoresed on SDS-PAGE, there was a great increase in 32P-label in the 20,000 and 47,000 dalton protein bands. There was also an increase in the labeling of phosphatidic acid and a small decrease in phosphatidyl inositol. When the platelets were returned to pH 7.6, the 32P labeling of the 20,000 and 47,000 dalton bands was greatly reduced, and that of phosphatidic acid reduced to the control value, while the labeling of phosphatidyl inositol was increased above control. Incubation at pH 5.3 for 60 min gave the same pattern, but return to pH 7.6 resulted in only partial reversal of labeling of the two protein bands and little decrease in the label associated with phosphatidic acid, but the radioactivity in phosphatidyl inositol was greatly increased. The changes in the 32P-labeling of phospholipids and proteins after incubation of platelets at pH 5.3 may reflect an increase in cytoplasmic Ca++ resulting from leakage of Ca++ from intracellular storage sites, a process which becomes irreversible after longer time exposure to the low pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of luteinizing hormone on phosphoinositide metabolism in rat granulosa cells.

Rat granulosa cells isolated from mature Graafian follicles were incubated with luteinizing hormone under various conditions in order to follow the synthesis and degradation of phospholipids. During acute incubations, luteinizing hormone provoked rapid and concentration-dependent increases in the incorporation of 32PO4 into phosphatidic acid, phosphatidylinositol, and the polyphosphoinositides. Similarly, luteinizing hormone provoked increases in labeling of phosphatidylinositol and the polyphosphoinositides when granulosa cells were incubated with myo-[2-3H]inositol. When granulosa cells were prelabeled with 32PO4 in order to label phosphatidylinositol to constant specific radioactivity (4 h), luteinizing hormone treatment significantly increased 32P-phosphatidylinositol levels (23%). Comparable increases (27%) in the cellular concentrations of phosphatidylinositol were observed in response to luteinizing hormone. In pulse-chase experiments employing 32PO4 - or [3H]inositol-prelabeled cells, luteinizing hormone did not alter phospholipid degradation. In addition, luteinizing hormone did not stimulate degradation of polyphosphoinositides. These results demonstrate that: (a) luteinizing hormone has selective effects on phospholipid metabolism in rat granulosa cells which involve phosphatidic acid, phosphatidylinositol, and the polyphosphoinositides, (b) luteinizing hormone increases net levels of phosphatidylinositol and presumably phosphatidic acid and the polyphosphoinositides, and (c) luteinizing hormone does not increase phospholipid degradation. Our findings suggest that luteinizing hormone provokes increases in de novo synthesis of phosphatidylinositol in rat granulosa cells. These changes in phospholipid metabolism may be important for steroidogenesis and other enzymatic processes during treatment with luteinizing hormone.

Enhanced turnover of arachidonic acid-containing species of phosphatidylinositol and phosphatidic acid of concanavalin astimulated 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 phosphatidie 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.

Phosphatidylinositol 4,5-bisphosphate: Light-mediated breakdown in the vertebrate retina

Isolated frog ( Rana Pipiens ) retinas were labeled in the dark with either [ 32P]PO 4-orthophosphate or myo -[2- 3H]inositol for 2.5–4 hrs. After washing the retinas with cold buffer, they were exposed to brief flashes of light (5 secs or 15 secs) and their rod outer segments isolated. Upon separation of labeled phospholipids, a specific decrease in label in phosphatidylinositol 4,5-bisphosphate was observed, whereas there was no significant effect on the labeling of phosphatidylinositol 4-phosphate, phosphatidylinositol, or phosphatidic acid. These results are indicative of a light-activated phosphatidylinositol 4,5-bisphosphate-specific phospholipase C in frog rod outer segments.

Interactions between thyreostimulin and PAF-acether on thyroid cell metabolism

1-O-Alkyl, 2-acetyl sn-glycerylphosphorylcholine (platelet activating factor (PAF)-acether) originally described as a platelet activating factor, is effective on various parameters in different cells. It seemed interesting to us to test it on porcine thyroid cells cultured for 1 to 5 days in the absence (control cells) or in the presence of 0.1 mU/ml thyreostimulin (TSH cells). At concentrations ranging from 0.5 to 2.5 μM, PAF-acether inhibited significantly the accumulation of cyclic AMP resulting from a 5 min incubation of the cells with TSH (40 mU/ml) or forskolin (0.1 mM). PAF-acether alone did not affect basal cyclic AMP accumulation. The maximal inhibition was obtained on a 3 day culture and amounted to 40–50%. The inhibition was transient and vanished after a 30 min incubation. The effects of PAF-acether (0.5 μM) on phospholipid metabolism depended closely upon the physiological state of the cells and upon the age of the culture. When PAF-acether was incubated for 2 h with [ 32P]phosphate, it mimicked the effects of TSH, i.e. it increased phosphatidylinositol (PI) labelling on 1 day control cells (expected effect) and decreased it with 1 day TSH cells (reverse effects). The PAF-acether effect was rapid in onset. After cell prelabelling for 2 h in the presence of TSH, PAF-acether added for 15 min completely counteracted the hormone effects on PI and phosphatidylcholine (PC) but increased the phosphatidic acid (PA) labelling. The effect of PAF-acether on PI labelling was partially antagonized by forskolin. Protein iodination, which is very dependent on the culture conditions, reached a very high rate in 3 or 4 day TSH-treated cells. This rate was not markedly enhanced by acute TSH stimulation and was decreased by 37% in 45 min by 2.5 μM PAF. This negative effect was completely abolished in the presence of TSH. On the contrary PAF, like TSH but more efficiently, increased the protein iodination of control cells by 60% in a 45 min period. The positive effect of PAF was not additive to that of TSH. The partial sharing of TSH properties by forskolin on the one hand and PAF on the other suggests that the cyclic AMP-dependent effects of the hormone are controlled by yet uncharacterized agents linked to phospholipid turnover and vice-versa.

Stimulation of phosphatidylinositol turnover by acetylcholine, angiotensin II and ACTH in bovine adrenal fasciculata cells

The effect of acetylcholine, angiotensin II and adrenocorticotropin (ACTH) on phosphatidylinositol (PI) metabolism was examined using bovine adrenocortical fasciculata cell suspensions. The three agents, which acutely stimulate glucocorticoid production by these cells, were all able to increase [ 32P]P i incorporation into cellular PI. However, whereas the relative steroidogenic potency (at maximally active concentrations) was ACTH ⩾ angiotensin II > acetylcholine, the effect on PI labeling was in the order angiotensin II > acetylcholine > ACTH. The dose-response curves for steroidogenesis and that for PI labeling were superimposable in the case of angiotensin II (ED 50=1·10 −8 M) and of acetylcholine (ED 50=5·10 −7 M), while the two responses were dissociated under graded ACTH challenge. Both steroidogenic response and increased PI labeling elicited by angiotensin II and acetylcholine were respectively inhibited by (Sar 1-Ala 8)-angiotensin II and muscarinic antagonists. Time-course study showed that in the case of angiotensin II and acetylcholine, the sequence of events was: increased phosphatidic acid labeling, increased PI labeling, activated steroidogenesis. By sharp contrast, under ACTH stimulation, increased steroidogenesis was detected well before activation of PI metabolism. These data suggest that in bovine adrenocortical fasciculata cell, steroidogenesis may be activated by two different pathways. The first one would act mainly through cyclic AMP-dependent intracellular events and is usually accepted in the mechanism of action of ACTH. The other, cyclic AMP-independent pathway, as in the case of angiotensin II and acetylcholine actions, may involve phospholipid-mediated intracellular processes.