Stimulation by prostaglandin F2 alpha of phosphatidic acid-phosphatidylinositol turnover in rat luteal cells

Prostaglandin F2 alpha (PGF 2 alpha) causes a rapid and marked increase of [32P]-orthophosphate incorporation into 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 after PGF 2 alpha addition into PA and PI, respectively, and by 10 min has reached a 2-fold stimulation over control in both lipid moieties. The labeling of other phospholipids is not affected. PGF 2 alpha exerts its stimulatory effect at an ED50 value of approximately 200 and 60 nM on PI and PA labeling, respectively. By contrast, human chorionic gonadotropin has no effect alone and does not interfere with the PGF 2 alpha-induced stimulation of PA-PI labeling. The striking similarity between the effects of PGF 2 alpha and LHRH on PA-PI labeling suggests that the two agents may exert their direct action on the corpus luteum via a common intracellular mechanism involving acidic phospholipid metabolism.     

Insulin increases the synthesis of phospholipid and diacylglycerol and protein kinase C activity in rat hepatocytes

The effects of insulin on phospholipid metabolism and generation of diacylglycerol (DAG) and on activation of protein kinase C in rat hepatocytes were compared to those of vasopressin and angiotension II. Insulin provoked increases in [3H]glycerol labeling of phosphatidic acid (PA), diacylglycerol (DAG), and other glycerolipids within 30 s of stimulation. Similar increases were also noted for vasopressin and angiotensin II. Corresponding rapid increases in DAG mass also occurred with all three hormones. As increases in [3H]DAG (and DAG mass) occurred within 30-60 s of the simultaneous addition of [3H]glycerol and hormone, it appeared that DAG was increased, at least partly, through the de novo synthesis of PA. That de novo synthesis of PA was increased is supported by the fact that [3H]glycerol labeling of total glycerolipids was increased by all three agents. Increases in [3H]glycerol labeling of lipids by insulin were not due to increased labeling of glycerol 3-phosphate, and were therefore probably due to activation of glycerol-3-phosphate acyltransferase. Unlike vasopressin, insulin did not increase the hydrolysis of inositol phospholipids. Insulin- and vasopressin-induced increases in DAG were accompanied by increases in cytosolic and membrane-associated protein kinase C activity. These findings suggest that insulin-induced increases in DAG may lead to increases in protein kinase C activity, and may explain some of the insulin-like effects of phorbol esters and vasopressin on hepatocyte metabolism.     

Microwave induced stimulation of32Pi incorporation into phosphoinositides of rat brain synaptosomes

Summary Exposure of synaptosomes to microwave radiation at a power density of 10 mW/sq cm or more produced stimulation of the³²Pi-incorporation into phosphoinositides. The extent of³²Pi incorporation was found to be much more pronounced in phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP₂) as compared to phosphatidylinositol (PI) and phosphatidic acid (PA). Other lipids were also found to incorporate³²Pi but no significant changes in their labeling were seen after exposure to microwave radiation. Inclusion of 10 mM lithium in the medium reduced the basal labeling of PIP₂, PIP and PI and increased PA labeling. Li⁺ also inhibited the microwave stimulated PIP₂, PIP and PI labeling but had no effect on PA labeling. Calcium ionophore, A₂₃₁₈₇, inhibited the basal and microwave stimulated³²Pi labeling of PIP and PIP₂, stimulated basal labeling of PA and PI and had no effect on microwave stimulated PA and PI labeling. Calcium chelator, EGTA, on the other hand, had no effect on basal labeling of PA and PI, stimulated basal PIP and PIP₂ labeling but did not alter microwave stimulated labeling of these lipids. Exposure of synaptosomes to microwave radiation did not alter the chemical concentration of phosphoinositides indicating that the turnover of these lipids was altered. These results suggest that low frequency microwave radiation alter the metabolism of inositol phospholipids by enhancing their turnover and thus may affect the transmembrane signalling in the nerve endings.     

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)     

Molecular mechanisms involved in T cell activation. II. The phosphatidylinositol signal-transducing mechanism mediates antigen-induced lymphokine production but not interleukin 2-induced proliferation in cloned cytotoxic T lymphocytes

The phospholipid metabolism of cloned murine cytotoxic T lymphocytes (CTL) was examined under conditions in which the induction of proliferation by interleukin 2 (IL 2) and the stimulated production of lymphokine (macrophage-activating factor (MAF] by concanavalin A (Con A) and specific antigen occurred independently of each other. Activation of the CTL by either of the latter two stimuli resulted in changes in the metabolism of phosphatidylinositol (PI) that were early (less than 2.5 min), specific, and prolonged (6 to 8 hr). These changes were primarily characterized by an increase in phosphatidic acid (PA) and PI, with a decrease in phosphatidylinositol-4,5-bisphosphate. The duration of these phospholipid responses, particularly PA and PI, approximated the minimum time of CTL-stimulus interaction required to produce maximal titers of MAF. No changes were observed in other major classes of phospholipids during 8 hr of continuous stimulation. Stimulation with an irrelevant antigen had no effect on CTL phospholipid metabolism. In contrast to specific antigen or Con A, the T cell growth factor IL 2 failed to elicit specific and early biosynthetic responses from PA and PI. Instead, there were nonspecific biosynthetic responses from all major phospholipid classes (including phosphatidylcholine and phosphatidylethanolamine, as well as PA and PI) which occurred between 1 and 6 hr after IL 2 stimulation. Both 1,2-diacylglycerol (DAG) and inositol phosphates (IP), the hydrolytic products of PI turnover, were produced in response to MAF-inducing stimuli, but neither were detected in response to the proliferative stimulus IL 2. Together, these results indicate that the hydrolysis of PI and the concomitant production of the putative second messengers DAG and IP are involved in signaling the production of lymphokines (MAF) by CTL. On the other hand, the failure of IL 2 to elicit a full-spectrum PI response suggests that signals mediating CTL proliferation may utilize an alternate and still undefined pathway.     

Effects of Na+, Ca2+, and Acetylcholine on Phosphoinositide- and ATP-Phosphate Turnover in 32P-Labeled Rabbit Iris Smooth Muscle

Parallel studies were carried out in the rabbit iris on (a) the effects of Na+ and/or Ca2+ on the acetylcholine-stimulated 32P labeling of phosphatidic acid (PA) and phosphatidylinositol (PI) and the breakdown of polyphosphoinositides (poly PI), and (b) the effects of these cations on the specific radioactivity of [gamma-32P]ATP. Incorporation of 32P1 into ATP and phosphoinositides is time-dependent, and it is remarkably dependent upon Na+ concentration in the incubation medium. The Na+ effect is reversible. Calcium ion, in the absence of Na+, had no effect on the specific radioactivity of ATP in 32P-labeled iris muscle; however, it moderately stimulated the 32P labeling of PA and PI and the breakdown of poly PI. In contrast, the addition of Na+, in the presence or absence of Ca2+, significantly reduced the specific radioactivity of ATP and 32P labeling of phospholipids in the 32P-labeled iris muscle. Acetylcholine had no measurable effect on the specific radioactivity of ATP. Furthermore, the neurotransmitter stimulated the 32P labeling of PA and PI and the breakdown of poly PI in the 32P-labeled muscle only in the presence of both Na+ and Ca2+. These data provide additional support for the concept that in the rabbit iris receptor-activated Ca2+ fluxes mediate or precede the effects of alpha-adrenergic and cholinergic muscarinic agents on phosphoinositide breakdown into 1,2-diacylglycerol and inositol phosphates and that restoration of the polar head groups to the 1,2-diacylglycerol (i.e., the recovery stage) is probably associated with Na+ outflux, via the Na+ -pump mechanism.     

Endogenous phospholipid metabolism in stimulated neutrophils differential activation by FMLP and PMA

Endogenous phospholipid metabolism was examined during the initial 0–120 seconds of neutrophil (PMN) stimulation. When PMN were exposed to the chemotactic peptide FMLP (10^?7 M) or the tumor promotor, phorbol myristate acetate (PMA, 1 μg/ml) extensive changes in specific phospholipid (PL) classes were evident within 15 seconds. The profile and kinetics of stimulus-induced PL changes were stimulus-dependent. Five seconds after the addition of FMLP, PMN content of PC, PS and PA increased, while the level of PI decreased. Kinetic studies revealed that only PA levels remained elevated (0–120 s) while other PL decreased. In contrast, when cells were exposed to PMA (1 μg/ml), the levels of PC and PS rapidly increased (< 15 s). With PMA as stimulus, changes in PI and PA were not observed until > 60 s. Results indicate that exposure to PMN to stimuli leads to rapid changes in specific PL. In addition, they support the concept that neutrophils rapidly “remodel” endogenous PL upon stimulation.     

Anti-mu antibody stimulates the phosphatidylinositol cycle and immunoglobulin secretion in a human lymphoblastoid B-cell line, LA350

Within 5 min of the binding of anti-mu antibody (anti-mu) to surface IgM on LA350, a human lymphoblastoid B-cell line, there was a significantly enhanced incorporation of 32P into the phosphatidic acid (PA) and phosphatidylinositol (PI) fractions of cellular phospholipids and the magnitude of the early increase in PA was twice as great as that in PI. This anti-mu-enhanced incorporation of 32P into PA and PI required the binding of a divalent form of antibody (IgG or F(ab')2), was blocked by coincubation with micromolar concentrations of soluble IgM, was decreased by incubation of cells at temperatures lower than 37 degrees C, and was inhibited by coincubation with millimolar concentrations of dibutyryl cyclic AMP and theophylline. Similar incorporation studies with [3H]inositol demonstrated a selective and significant increase in labeling of PI. In LA350 labeled with [3H]inositol for 30 hr (equilibrium) and acutely stimulated by anti-mu, specific hydrolysis of phosphorylated PI (PI 4,5-bisphosphate) was measured by the significantly increased release at 15 min of radioactive inositol 1,4,5-trisphosphate, inositol 1,4 bisphosphate, and inositol 1-phosphate. The release of these inositol phosphates was significantly augmented by coincubation with 0.01 M LiCl which prevented their simultaneous enzymatic degradation. All of these findings are consistent with an activation of a linked series of metabolic events known as the PI cycle. In similar cell cultures anti-mu significantly stimulated the secretion of IgM by LA350 as measured at 48 hr in a reverse hemolytic plaque assay. Two other IgM-bearing human lymphoblastoid B-cell lines which gave no evidence of turnover of 32P in PA and PI in response to binding by anti-mu likewise failed to enhance their secretion of IgM. We conclude that the binding of surface IgM on LA350 by anti-mu results in the generation of a transmembrane signal which causes a rapid activation of the PI cycle which itself may play a role in the subsequent increase in IgM secretion.     

Cardiolipin is essential for higher proton translocation activity of reconstituted Fo

The Fo membrane domain of FoF1-ATPase complex had been purifiedfrom porcine heart mitochondria. SDS-PAGE with silver staining indicated that the purity of Fo was about 85% and the sample contained no subunits of F1-ATPase. The purified Fo was reconstituted into liposomes with different phospholipid composition, and the effect of CL (cardiolipin), PA (phosphatidic acid), PI (phosphatidylinositol) and PS (phosphatidylserine) on the H+ translocation activity of Fo was investigated. The results demonstrated that CL, PA and PI could promote the proton translocation of Fo with the order of CL＞PA＞＞PI, while PS inhibited it. Meanwhile ADM (adriamycin) severely impaired the proton translocation activity of Fo vesicles containing CL, which suggested that CL's stimulation of the activity of reconstituted Fo might correlate with its non-bilayer propensity. After Fo was incorporated into the liposomes containing PE (phosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) as well as DEPE (dielaidoylphosphatidylethanolamine), it was found that the proton translocation activity of Fo vesicles increased with the increasing content of PE or DOPE, which has high propensity of forming non-bilayer structure, but was independent of DEPE. The dynamic quenching of the intrinsic fluorescence of tryptophan by HB (hypocrellin B) as well as fluorescent spectrum of acrylodan labeling Fo at cysteine indicated that CL could induce Fo to a suitable conformation resulting in higher proton translocation activity.     

Phosphatidic Acid and Phosphoinositide Turnover in Myelin and Its Stimulation by Acetylcholine

Brain slices obtained from the forebrains of adult female rats were incubated with [32P]phosphate and [3H]glycerol for 60 min, and lipids extracted and analyzed by TLC. The 32P in brain slice lipids was primarily in polyphosphoinositides, phosphatidylinositol (PI), and phosphatidate (PA). Distribution of the 32P-labeled lipids in isolated myelin was biased toward PA, 38%, relative to 16% in whole tissue slice lipids. About 33% of the total labeled PA in brain slices was accounted for by that in myelin. On a per milligram protein basis, PA labeling in myelin is about 2.5-fold greater than that of whole brain slice. Since incorporation of [3H]glycerol (indicative of synthesis by the de novo synthetic pathway) was at very low levels, we conclude that [32P]phosphate entered into myelin PA primarily through a pathway involving phospholipase C activity. Much of the production of PA relates to hydrolysis of phosphoinositides, yielding diacylglycerol which is then phosphorylated within myelin. The distribution of label among the inositol-containing lipids suggests that only a fraction of the myelin polyphosphoinositides serve as substrate for rapid diglyceride production. In the presence of 10 mM acetylcholine (ACh) there was a 20-60% stimulation of [32P]phosphate incorporation into PA and PI of brain slice lipids and purified myelin. Stimulation by ACh was blocked by atropine. The observed increase in the 32P/3H ratio, relative to controls, indicated that for both total lipids and myelin lipids there was selective stimulation of a phospholipase C-dependent cycle relative to de novo biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Ca2+-dependent and Ca2+-independent mechanisms for phosphatidylinositol hydrolysis and 32P-labeling during cholinergic stimulation of the rat submaxillary gland in vitro

Ca²⁺ was required for carbachol-induced decreases in phosphatidylinositol (PI) and increases in phosphatidic acid (PA) concentrations during incubation of rat submaxillary gland fragments, but was not required for increases in [³²P]P_i incorporation into these phospholipids. Like carbachol, A23187 provoked a Ca²⁺-dependent decrease in PI mass. These results suggest concomitant operation of two separate mechanisms for stimulating PI hydrolysis and ³²P labeling of PA and PI during carbachol action: one mechanism is not dependent on external Ca²⁺ and is manifested by rapid labeling in a relatively small PA-PI pool; the other mechanism is dependent on Ca²⁺ and involves a large PA-PI pool which appears to have a relatively slow renewal (labeling) rate.     

Mechanism of action of TRH: involvement of the phosphatidylinositol (PI) response in the action of TRH in rat anterior pituitary cells

In order to further investigate the molecular basis for the action of TRH, experiments were performed to examine the ability of rat anterior pituitary cells to incorporate [32P]orthophosphate into phospholipids in response to the neurohormone. Addition of 0.1 microM TRH rapidly stimulates the phosphatidylinositol (PI) response. The incorporation of radioactivity into PA and PI is increased as early as in 2 and 5 min after TRH addition, respectively, and reaches 40 and 140% above control, respectively, at 20 min. By contrast, 0.1 microM somatostatin is inactive. TRH exerts its stimulatory effect at an ED50 value of ca. 10 nM. The present results suggest that the PI response is an early event associated with the action of TRH in the anterior pituitary gland.     

The relative degradation of [14C]eicosapentaenoyl and [3H]arachidonoyl species of phosphatidylinositol and phosphatidylcholine in thrombin-stimulated human platelets

The platelet-rich plasma from volunteers who had consumed a supplement containing eicosapentaenoate (EPA) was incubated with [3H]arachidonic acid (AA) and [14C]EPA so as to provide for the labelling of these fatty acids in the individual platelet phospholipids. Washed dual-labelled platelet suspensions were prepared and incubated with and without thrombin in the presence of BW755C and in the presence and absence of trifluoperazine (TFP) or indomethacin. The platelet lipids were extracted and the individual phospholipids, as well as diacylglycerol and phosphatidic acid, were separated by thin-layer chromatography and the radioactivity in each fraction was determined. The [3H]AA/[14C]EPA dpm ratio for the loss of radioactivity from phosphatidylcholine (PC) upon thrombin stimulation, as well as that in the residual PC remaining after stimulation, was similar to that in PC in the resting platelets. This suggests no marked selectivity in the degradation of EPA-versus AA-containing species of PC during platelet activation. The [3H]/[14C] ratios for the increased radioactivity appearing in diacylglycerol (DG) and phosphatidic acid (PA) upon thrombin stimulation were not significantly different from the corresponding ratio in phosphatidylinositol (PI) from resting platelets, suggesting little or no preference for 1-acyl-2-eicosapentaenoyl PI over 1-acyl-2-arachidonoyl PI in the pathway from PI to DG to PA. These results suggest that the relative formation of the 2- and 3-series prostaglandins, including thromboxane (Tx) A2 and A3, in stimulated platelets is not regulated by a preferential loss of one of the corresponding eicosanoid precursors over the other from membrane PC and PI.     

Concerted stimulation of PI-turnover, Ca2+-influx and histamine release in antigen-activated rat mast cells

Phospholipid metabolism in rat mast cells activated by antigen was examined with reference to phosphatidylinositol (PI) turnover. Upon antigen stimulation, histamine release from passively sensitized mast cells with IgE was potentiated by adding phosphatidylserine (PS). The addition of antigen to [3H]glycerol-prelabeled and sensitized mast cells induced a marked loss of radioactivity of PI and a concurrent accumulation of 1,2-diacylglycerol (DG) and phosphatidic acid (PA) within 5 to 60 sec. Furthermore, this antigen-induced PI breakdown was enhanced in the presence of Mg2+. Histamine release occurred in parallel with PI breakdown. On the other hand, the transient Ca2+ influx into mast cells, as measured by uptake of 45Ca2+, was found to occur quickly after cells were activated by antigen, which was concerted with PI breakdown. These results suggest that enhanced PI turnover may be an important step in the biochemical sequence of events leading to release of histamine, and that not only Ca2+ but also Mg2+ appears to take a part in stimulus-response coupling in rat mast cells.     

Cardiolipin is essential for higher proton translocation activity of reconstituted F_o

The Fo membrane domain of FoF1-ATPase complex had been purified from porcine heart mitochondria. SDS-PAGE with silver staining indicated that the purity of Fo was about 85% and the sample contained no subunits of F1-ATPase. The purified Fo was reconstituted into liposomes with different phospholipid composition, and the effect of CL (cardiolipin), PA (phosphatidic acid), PI (phosphatidylinositol) and PS (phosphatidylserine) on the H+ translocation activity of Fo was investigated. The results demonstrated that CL, PA and PI could promote the proton translocation of Fo with the order of CL>PA>>PI, while PS inhibited it. Meanwhile ADM (adriamycin) severely impaired the proton translocation activity of Fo vesicles containing CL, which suggested that CL's stimulation of the activity of reconstituted Fo might correlate with its non-bilayer propensity. After Fo was incorporated into the liposomes containing PE (phosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) as well as DEPE (dielaidoylphospha     

PLCgamma participates in insulin stimulation of glucose uptake through activation of PKCzeta in brown adipocytes

Based on recent studies showing that PLCgamma associates to insulin receptor, we investigated its role in insulin stimulation of glucose transport in brown adipocytes. Insulin stimulation induced rapid PLCgamma association to phosphorylated insulin receptor, and activation of PLCgamma, as assessed by the mobilization of Ca(2+) from intracellular stores and by the production of the second messenger DAG. Both events are dependent on activation of PI3-kinase. Inhibition of PLCgamma activity either with the chemical compound U73122 or with an inhibitor peptide precluded insulin stimulation of glucose uptake, GLUT4 translocation, and actin reorganization, as wortmannin did. In contrast, the inactive analog U73343 did not have an inhibitory effect. Furthermore, translocation of GLUT4-GFP in response to insulin was completely abolished by cotransfection with a PLCgamma-inactive mutant in HeLa cells, a cell model sensitive to insulin that express PLCgamma. U73122 did not affect PI3-kinase nor Akt activation, but impaired PKCzeta activation by insulin, as wortmannin did. PLC activity renders two products, IP(3) and DAG, and DAG can be metabolized to PA by the action of DAG-kinase. Using the compound R54494, a DAG-kinase inhibitor, insulin-induced PKCzeta activation was also suppressed, this activity being restored by addition of PA. In summary, these data indicate that PLCgamma, activated at least partially by PI3-kinase, is a link between insulin receptor and PKCzeta through the production of PA and could mediate insulin-induced glucose uptake and GLUT4 translocation.     

The role of phosphatidylinositol turnover in initiation of prostaglandin biosynthesis in concanavalin A-induced activation of rat peritoneal macrophages

When macrophages were stimulated by concanavalin A (50 micrograms/ml) in the presence of 32Pi and 3H-arachidonic acid (AA), rapid incorporation of these radiolabeled substances into phosphatidylinositol (PI) and phosphatidic acid (PA) were induced within 10 min. The pool of PI and PA incorporating 3H-AA was the same as that of PI and PA which incorporated 32P. However, significant increase in incorporation of 32P and 3H-AA did not occur in phosphatidylcholine, phosphatidylethanolamine or phosphatidylserine within this time interval as compared with control levels. These results indicate that PI turnover might play a role in the initiation of prostaglandin biosynthesis in the early stages of macrophage activation induced by concanavalin A.     

A new model system for studying the phosphatidylinositol cycle

An early manifestation of the response of WRK-1 rat mammary tumor cells to vasopressin is an increase in incorporation of (³²P)P_i into phospholipids. Incorporation into all classes of phospholipids is stimulated; however, incorporation into phosphatidylinositol (PI) is increased to the greatest degree (3- to 10-fold as compared with 1.3- to 2-fold for the other phosholipids). Furthermore, increased incorporation into PI is accompanied by an increased rate of PI turnover; turnover rates of the other phospholipids are unaffected by vasopressin.     

The effects of ACTH, serotonin, K+ and angiotensin analogues on 32P incorporation into phospholipids of the rat adrenal cortex: basis for an assay method using zona glomerulosa cells

The effects of various concentrations of serotonin, ACTH, K+, angiotensin II (AII), angiotensin III (AIII) and [Sar1]angiotensin II (SAII) on steroidogenesis and the incorporation of 32P (after preincubation to near equilibrium with the ATP pool) into phosphatidylinositol (PI), phosphatidic acid (PA) and phosphatidylcholine (PC) in a preparation of capsular cells from rat adrenals, consisting of 95% zona glomerulosa (z.g.) and 5% zona fasciculata plus reticularis (z.f.r.) cells, were investigated. Serotonin and ACTH stimulated steroidogenesis in the usual manner but had little or no effect on 32P incorporation into any of the three phospholipids. However, AII, AIII and SAII stimulated steroidogenesis and also 32P incorporation into PA and PI (maximally to about 280% of control values) but not into PC. These results taken together with other data on effects on the cAMP output and Ca2+ fluxes of z.g. cells suggest that stimulation by ACTH and serotonin is mediated by cAMP as second messenger. However, the angiotensins probably act through Ca2+, with associated changes in phospholipid metabolism. The 32P incorporation into PA as a function of lg concentration of AII was linear and showed a reasonable index of precision (0.36 +/- 0.03, eight experiments, 0.23 +/- 0.02 for a further eight experiments) and correlation with steroidogenesis. The corresponding incorporation into PI showed a maximum effect and a much poorer index of precision (1.02 +/- 0.30 (4.69 +/- 3.7] over the same full range of AII concentration used. The effects of AIII and SAII showed similar characteristics for 32P incorporation into both PA and PI, but, as for stimulation of steroidogenesis, at higher concentrations for AIII than for AII. The effects of different doses of AII, AIII and ACTH on the corticosterone output and 32P incorporation into PA, PI and PC of a preparation of cells, consisting of more than 98% z.f.r. cells, from rat decapsulated adrenals were also studied. ACTH, at low doses, which nevertheless markedly stimulated corticosterone output, had a small (maximally to about 125% of control values) but significant effect on 32P incorporation into PA, PI and PC. The maximum effect was usually at about 10(-10) M ACTH and was not significant at 10(-8) M.