Polyphosphoinositides are present in plasma membranes isolated from fusogenic carrot cells

Fusogenic carrot cells grown in suspension culture were labeled 12 hours with myo-[2-³H]inositol. Plasma membranes were isolated from the prelabeled fusogenic carrot cells by both aqueous polymer two-phase partitioning and Renografin density gradients. With both methods, the plasma membrane-enriched fractions, as identified by marker enzymes, were enriched in [³H]inositol-labeled phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP₂). An additional [³H]inositol-labeled lipid, lysophosphatidylinositol monophosphate, which migrated between PIP and PIP₂ on thin layer plates, was found primarily in the plasma membrane-rich fraction of the fusogenic cells. This was in contrast to lysophosphatidylinositol which is found primarily in the lower phase, microsomal/mitochrondrial-rich fraction.     

Phosphorylation of lysophosphatidylinositol by carrot membranes.

sn-1 Palmitoyl lysophosphatidylinositol is found in carrot suspension culture cells and can be phosphorylated to [32P]lysophosphatidylinositol monophosphate (LPIP) when [gamma 32P]ATP is added to isolated membranes. Based on in vivo labeling studies, [3H]inositol sn-1 palmitoyl LPIP was found predominantly in the plasma membrane-rich fraction or upper phase isolated by aqueous two-phase partitioning and LPI was found in the intracellular membrane-rich fraction or lower phase (Wheeler and Boss, Plant Physiol. 85, 389-392, 1987). While both membrane fractions phosphorylated LPI in vitro, the apparent Km for LPI in the intracellular membrane fraction was 180 microM and for the plasma membrane was 580 microM. When cells were treated with the ionophore, monensin, the percentage of [3H]inositol LPIP increased in the whole cell lipid extract. However, the monensin treatment decreased the amount of [3H]inositol LPIP and PIP recovered in the plasma membrane fraction relative to the sum of the individual lipid, [3H]inositol LPIP or PIP, respectively, recovered in both membrane fractions.     

Phosphatidylinositol 4,5-Bisphosphate Phospholipase C and Phosphomonoesterase in Dunaliella salina Membranes

In comparison with other cell organelles, the Dunaliella salina plasma membrane was found to be highly enriched in phospholipase C activity toward exogenous [³H]phosphatidylinositol 4,5-bisphosphate (PIP₂). Based on release of [³H]inositol phosphates, the plasma membrane exhibited a PIP₂-phospholipase C activity nearly tenfold higher than the nonplasmalemmal, nonchloroplast `bottom phase' (BP) membrane fraction and 47 times higher than the chloroplast membrane fraction. The majority of phospholipase activity was clearly of a phospholipase C nature since over 80% of [³H]inositol phosphates released were recovered as [³H]inositol trisphosphate (IP₃). These results suggest a plausible mechanism for the rapid breakdown of PIP₂ and phosphatidylinositol 4-phosphate (PIP) following hypoosmotic shock. Quantitative analysis of major [³H]inositol phospholipids during these assays revealed that some of the [³H]-PIP₂ was converted to [³H]phosphatidylinositol 4-monophosphate (PIP) and to [³H]phosphatidyl-inositol (PI) in the BP fraction of membrane remaining after removal of plasmalemma and chloroplasts. This latter fraction is enriched more than fivefold in PIP₂/PIP phosphomonoesterase activity when compared to the plasmalemma or chloroplast membrane fractions. We have also examined some of the in vitro characteristics of the plasma membrane phospholipase C activity and have found it to be calcium sensitive, reaching maximal activity at 10 micromolar free [Ca²⁺]. We also report here that 100 micromolar GTPγS stimulates phosphospholipase C activity over a range of free [Ca²⁺]. Together, these results provide evidence that the plasma membrane PIP₂-phospholipase C of D. salina may be subject to Ca²⁺ and G-protein regulation.     

Neomycin inhibits the phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate stimulation of plasma membrane ATPase activity

The inositol phospholipids, phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP₂), have been shown to increase the vanadate-sensitive ATPase activity of plant plasma membranes (AR Memon, Q Chen, WF Boss [1989] Biochem Biophys Res Commun 162: 1295-1301). In this paper, we show the effect of various concentrations of phosphatidyinositol, PIP, and PIP₂ on the plasma membrane vanadate-sensitive ATPase activity. PIP and PIP₂ at concentrations of 10 nanomoles per 30 microgram membrane protein per milliliter of reaction mixture caused a twofold and 1.8-fold increase in the ATPase activity, respectively. The effect of these negatively charged phospholipids on the ATPase activity was inhibited by adding the positively charged aminoglycoside, neomycin. Neomycin did not affect the endogenous plasma membrane ATPase activity in the absence of exogenous lipids.     

Polyphosphoinositides are present in plant tissue culture cells

Polyphosphoinositides have been isolated from wild carrot cells grown in suspension culture. This is the first report of polyphosphoinositides in plant cells. The phospholipids were identified by comigration with known standards on thin-layer plates. After overnight labeling of the cells with myo-[2-3H] inositol, the phosphoinositides as percent recovered inositol were 93% phosphatidylinositol., 3.7% lysophosphatidylinositol, 1.7% phosphatidylinositol monophosphate, 0.8% phosphatidylinositol bisphosphate.     

Plasma membrane lipid metabolism of petunia petals during senescence

The specific activities of 6 enzymes, which are involved in the synthesis and catabolism of membrane lipids, were monitored in plasma membranes isolated from petunia petals during senescence. These included phosphatidylinositol (PI) kinase (EC 2.7.1.67), phosphatidylinositol monophosphate (PIP) kinase (EC 2.7.1.68). diacylglycerol (DAG) kinase (EC 2.7.1.107), phospholipase A (EC 3.1.1.4) and PIP- and PIP₂-phospholipase C˙(EC 3.1.4.3). Using endogenous substrate, the [³²P]PA and [³²P]PIP₂ formation increased to 140 and 200%, respectively, of the day 1 value by 4 days after harvest. There was no significant change in [³²P]PIP formation during the same time period. On the fifth day the petals wilted and the [³²P]PA and [³²P]PIP formation declined significantly. In contrast, the [³²P]PIP₂ formation remained high in the day 5 petals. When the lipid kinase activities were assayed in the membranes in the presence of exogenous substrate the specific activity of all of the enzymes increased. and the changes in [³²P]PA production over the 5-day period were similar to those observed with endogenous substrate. When exogenous PI and PIP were added, however, there was no longer an increase in [³²P]PIP₂ formation by plasma membranes of day 4 petals and [³²P]PIP formation significantly decreased. The relative decrease in PIP and PIP₂ formation by day 4 membranes when exogenous substrate was added may have resulted from differences in the lipase activities in the day 1 and day 4 membranes. The plasma membrane A-type phospholipase activity increased throughout the 5 day period, and phospholipase C activity increased two-fold between day 1 and day 4. Such changes in the metabolism of the plasma membrane lipids during flower senescence would affect the ability of the petals to use inositol phospholipid-based signal transduction pathways.     

Uptake and phosphorylation of phosphatidylinositol by rat liver nuclei. Role of phosphatidylinositol transfer protein

The incorporation of phosphatidyl[2-3H]inositol ([3H]PI) from vesicles or microsomal membranes into rat liver nuclei is greatly stimulated by phosphatidylinositol transfer protein (PI-TP). The nuclei are able to phosphorylate [3H]PI, with the production of phosphatidylinositol 4-phosphate (PIP). Recovery of tritiated inositol trisphosphate, inositol phosphate, glycerophosphoinositol and inositol, suggests that in isolated nuclei a large set of enzymes of the PI cycle is present, similar to the enzymes involved in the plasma membrane PI cycle. Incubation with [gamma-32P]ATP shows that isolated nuclei are able to phosphorylate endogenous PI to PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). In the presence of exogenous PI and detergent the synthesis of PIP is increased, indicating that in nuclei the PI pool is suboptimal for the PI-kinase activity. The present study suggests that PI-TP may be involved in providing substrates for PI metabolism at the nuclear level.     

Kinetic analysis of guanosine 5'-O-(3-thiotriphosphate) effects on phosphatidylinositol turnover in NRK cell homogenates

Addition of the guanine nucleotide analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to [3H]inositol-labeled NRK cell homogenates resulted in rapid breakdown of cellular polyphosphoinositides. GTP gamma S stimulated phospholipase C, resulting in a more than 4-fold increase in the hydrolysis rates of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bis(phosphate) (PIP2). No significant effect of GTP gamma S on direct phosphatidylinositol (PI) hydrolysis was detected. There was an increase in water-soluble inositols, with inositol tris(phosphate) (IP3) levels increasing at least 10 times over the decrease seen in PIP2, indicating that PIP kinase activity was also accelerated following GTP gamma S addition. Inositol 1,4,5-tris(phosphate) peaked rapidly after GTP gamma S addition (less than 2 min) while inositol 1,3,4-tris-(phosphate) was produced more slowly and leveled off after approximately 10 min. The differential equations describing conversion between intermediates in the PI turnover pathway were solved and fitted to data obtained from both [3H]inositol and [32P]phosphate fluxes by nonlinear least-squares analysis. GTP gamma S effects on the pseudo-first-order rate constants for the lipase, kinase, and phosphatase steps were determined from the analysis. From these measurements it can be estimated that, in the presence of GTP gamma S and calcium buffered to 130 nM, hydrolysis of PIP2 accounts for at least 10 times as much diacylglycerol as direct PI breakdown despite the 100-fold excess of PI over PIP2. From the kinetic model it is predicted that small changes in the activities of PI and PIP kinases can have large but different effects on the level of IP3 and diacylglycerol following GTP gamma S addition. These results argue that regulation of PI and PIP kinases may be important for determining both cellular IP3 and diacylglycerol levels.     

Platelet phosphoinositide turnover in streptozotocin-induced diabetes

Increased platelet aggregation and secretion in response to various agonists has been described in both diabetic humans and animals. Alterations in the platelet membrane fatty acid composition of phospholipids and changes in the prostacyclin and thromboxane formation could only partly explain the altered platelet function in diabetes. In the present study, we have examined the role of phosphoinositide turnover in the diabetic platelet function. We report alterations in 2-[³H] myo-inositol uptake, phosphoinositide turnover, inositol phosphate and diacylglycerol (DAG) formation, phosphoinositide mass, and phospholipase C activity in platelets obtained from streptozotocin (STZ)-induced diabetic rats. There was a significant increase in the 2-[³H) myo-inositol uptake in washed platelets from diabetic rats. Basal incorporation of 2-[³H] myo-inositol into phosphatidylinositol 4,5-bisphosphate (PIP₂), phosphatidylinositol 4-phosphate (PIP) or phosphatidylinositol (PI) in platelets obtained from diabetic rats was, however, not affected. Thrombin stimulation of platelets from diabetic rats induced an increase in the hydrolysis of [³²P]PIP₂ but indicated no change in the hydrolysis of [³²P]PIP and [³²P]PI as compared to their basal levels. Thrombin-induced formation of [³H]inositol phosphates was significantly increased in both diabetic as well as in control platelets as compared to their basal levels. This formation of [³H]inositol phosphates in diabetic platelets was greater than controls at all time intervals studied. Similarly, there was an increase in the release of DAG after thrombin stimulation in the diabetic platelets. Based on these results, we conclude that there is an increase in the transport of myoinositol across the diabetic platelet membrane and this feature, along with alterations in the hydrolysis of PIP₂, inositol phosphates and DAG in the diabetic platelets, may play a role in increased phosphoinositide turnover which could explain the altered platelet function in STZ-induced diabetes.     

Deoxycholate induces the preferential hydrolysis of polyphosphoinositides by human platelet and rat corneal phospholipase C

Deoxycholate promotes phospholipase C degradation of endogenous phosphatidyl[3H]inositol (Pl), phosphatidyl[3H]inositol monophosphate (PIP) and phosphatidyl[3H]inositol bisphosphate (PIP2) in rat cornea and human platelets. Hydrolysis of phosphatidyl[3H]inositol significantly lags polyphospho[3H]inositide degradation. Concomitantly, formation of [3H]inositol monophosphate (IP1) lags behind [3H]inositol bisphosphate (IP2) and [3H]inositol trisphosphate (IP3) production. These results demonstrate that rat cornea and human platelet phospholipase C cause a preferential hydrolysis of the endogenous polyphosphoinositides rather than phosphatidylinositol.     

Plasma membrane associated phospholipase C from human platelets: synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5'-O-(3-thiotriphosphate)

The effects of thrombin and GTP gamma S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous [3H]inositol-labeled membranes or with lipid vesicles containing either [3H]phosphatidylinositol or [3H]phosphatidylinositol 4,5-bisphosphate. GTP gamma S (1 microM) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP3), inositol bisphosphate (IP2), or inositol phosphate (IP) from [3H]inositol-labeled membranes. IP2 and IP3, but not IP, from [3H]inositol-labeled membranes were, however, stimulated 3-fold by GTP gamma S (1 microM) plus thrombin (1 unit/mL). A higher concentration of GTP gamma S (100 microM) alone also stimulated IP2 and IP3, but not IP, release. In the presence of 1 mM calcium, release of IP2 and IP3 was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) by platelet membrane associated PLC was also markedly enhanced by GTP gamma S (100 microM) or GTP gamma S (1 microM) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP2 was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP gamma S (100 microM) or calcium (1 mM) dependent PIP2 breakdown, while TPA inhibited GTP gamma S-dependent but not calcium-dependent phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence of two isomers of phosphatidylinositol in plant tissue

The structure of phosphatidylinositol in barley (Hordeum vulgare) aleurone layers was investigated by chemical degradation. In vivo myo-[2-³H]inositol-labeled phosphatidylinositol was first converted to glycerophosphoinositol and, subsequently, after removal of the glycerol moiety, to inositol monophosphate. Here, we present data that show that, in addition to the commonly occurring 1,2-diacylglycero-3-(d-myo-inositol-1-phosphate), barley aleurone cells contain a novel second isomer of phosphatidylinositol that differs in structure of the head group.     

Metabolism of lysopolyphosphoinositides by rat brain and liver microsomes

Lysophosphatidylinositol 4,5-bisphosphate has been reported to form ion-conducting channels in artificial membranes. If formed in vivo, mechanisms for its removal from cellular membranes would be required. Thus, possible pathways were explored in rat brain and liver microsomes. Since neither lysophosphatidylinositol 4-phosphate nor lysophosphatidylinositol 4,5-bisphosphate were acylated in experiments with [3H]arachidonic acid or [14C]oleoyl CoA, polyphosphoinositides do not participate directly in a deacylation-reacylation cycle as proposed for the postsynthesis enrichment of phosphatidylinositol with arachidonic acid. Similar enrichment in polyphosphoinositides can occur only via the rapid phosphorylation-dephosphorylation cycle linking all three phosphoinositides. Lysophosphatidyl[2-3H]inositol 4,5-bisphosphate and lysophosphatidyl[2-3H]inositol 4-phosphate were rapidly dephosphorylated to 1-acyl-sn-glycero(3)phospho(1)-D-myo-inositol by microsomes from both tissues. Appearance of only trace quantities of radioactive lysophosphatidylinositol monophosphate during the catabolism of lysophosphatidyl[2-3H]inositol 4,5-bisphosphate indicated that the second dephosphorylation step, which was cation independent, was at least as fast as the first step which required Mg2+. In the presence of ATP, CoA, and arachidonic acid, the lysophosphatidylinositol was converted to phosphatidylinositol. This acylation reaction was rate limiting in brain microsomes. Dephosphorylation of lysophosphatidylinositol 4,5-bisphosphate was rate limiting in liver microsomes. Neither the lysopolyphosphoinositides nor the lysophosphatidylinositol produced from them in the reactions were degraded by acyl hydrolases or phosphodiesterases in microsomes from either tissue. Therefore, any lysopolyphosphoinositide formed in vivo would probably be removed by dephosphorylation and recycled to phosphatidylinositol.     

ACTH stimulates turnover of the phosphatidylinositol-glycan

Primary cultures of calf adrenal glomerulosa cells were prelabeled for 3 days with [3H]inositol or [3H]glucosamine and stimulated with 10 nM ACTH. Labeled phosphatidylinositol (PI), polyphosphoinositides (PIP and PIP2) and a novel phosphatidylinositol-glycan (PI-glycan) were measured after separation by TLC. [3H]-Inositol labeling of PI, PIP and PIP2 increased rapidly, whereas labeling of the PI-glycan showed an initial decrease at 1 minute followed by a subsequent increase. Similar results were obtained when cells were prelabeled with [3H]glucosamine, viz. the PI-glycan label decreased at 1 min and subsequently increased. These results suggest that ACTH provokes (a) coordinated increases in the synthesis of PI, PIP, PIP2 and the PI-glycan, and (b) the increase in PI-glycan synthesis is preceded by initial decrease, presumably reflecting hydrolysis of this lipid.     

Platelet-activating factor stimulates metabolism of phosphoinositides via phospholipase A2 in primary cultured rat hepatocytes

Addition of platelet-activating factor (PAF) to cells doubly labeled with [14C]glycerol plus [3H]arachidonic acid resulted in a transient decrease of [14C]glycerol-labeled phosphatidylinositol (PI) and a transient increase of [14C]glycerol-labeled lysophosphatidylinositol (LPI). [3H]Arachidonate-labeled PI, on the other hand, decreased in a time-dependent manner. The radioactivity in phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and phosphatidylserine did not change significantly. The 3H/14C ratio decreased in PI in a time-dependent manner, suggesting the involvement of a phospholipase A2 activity. Although PAF also induced a gradual increase of diacylglycerol (DG), the increase of [14C]glycerol-labeled DG paralleled the loss of triacyl [14C]glycerol and the 3H/14C ratio of DG was 16 times smaller than that of PI. Thus, DG seemed not to be derived from PI. In myo- [3H]inositol-prelabeled cells, PAF induced a transient decrease of [3H]phosphatidylinositol-4,5-bis-phosphate (TPI) and [3H]phosphatidylinositol-4-phosphate (DPI) at 1 min. PAF stimulation of cultured hepatocytes prelabeled with 32Pi induced a transient decrease of [32P]polyphosphoinositides at 20 sec to 1 min. [32P]LPI appeared within 10 sec after stimulation and paralleled the loss of [32P]PI. [3H]Inositol triphosphate, [3H]inositol diphosphate, and [3H]inositol phosphate, which increased in a time-dependent manner upon stimulation with adrenaline, did not accumulate with the stimulation due to PAF. These observations indicate that PAF causes degradation of inositol phospholipids via phospholipase A2 and induces a subsequent resynthesis of these phospholipids.     

Stimulation of phosphoinositide degradation and phosphatidylinositol-4-phosphate phosphorylation by GTP exclusively in plasma membrane of rat brain

The effect of GTP on the hydrolysis of [³H]phosphatidyinositol (PI), [³H]phosphatidylinositol-4-phosphate (PIP) and [³H]phosphatidylinositol-4,5-bisphosphate (PIP₂) by phospholipase C of rat brain plasma membrane, microsomes and cytosol was determined. Moreover the regulation of PI and PIP phosphorylation by GTP in brain plasma membrane was investigated.In the presence of EGTA PIP₂ was actively degradted, opposite to PI and PIP which require Ca²⁺ for their hydrolysis. Addition of calcium ions in each case caused stimulation of inositide phosphodiesterase(s). GTP independently of calcium ions activates by about 3 times phospholipase C acting on PIP and PIP₂ exclusively in the plasma membrane. PI degradation was unaffected by GTP. In the presence of Ca²⁺ guanine nucleotides have synergistic stimulatory effect on plasma membrane bound phospholipase C acting on PIP₂. PIP kinase of brain plasma membrane was stimulated by GTP by about 20–100% in the presence of exogenous and endogenous substrate respectively. PI kinase was negligible activated by about 20% exclusively in the presence of endogenous substrate. These results indicated that guanine nucleotide modulates the level of second messengers as diacylglycerol and IP₃ through the activation of phospholipase C acting on PIP₂ exclusively in brain plasma membrane. The stimulation of phospholipase C by GTP may occur directly or through the enhancement of substrate level PIP₂ due to stimulation of PIP kinase.     

Carbachol induces a rapid and sustained hydrolysis of polyphosphoinositide in bovine tracheal smooth muscle measurements of the mass of polyphosphoinositides, 1,2-diacylglycerol, and phosphatidic acid

The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of [3H]inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2-diacylglycerol and phosphatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60-min period of carbachol stimulation. The addition of atropine reverses these responses completely. Carbachol stimulates a rapid loss in [3H]inositol radioactivity from phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate associated with production of [3H]inositol trisphosphate. The carbachol-induced change in the mass of phosphoinositides and phosphatidic acid is not affected by removal of extracellular Ca2+ and does not appear to be secondary to an increase in intracellular Ca2+. These results indicate that carbachol causes phospholipase C-mediated polyphosphoinositide breakdown, resulting in the production of inositol trisphosphate and a sustained increase in the actual content of 1,2-diacylglycerol. These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphosphoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,2-diacylglycerol.     

Guanine nucleotides stimulate hydrolysis of phosphatidylinositol and polyphosphoinositides in permeabilized Swiss 3T3 cells

Hydrolysis-resistant analogues of GTP specifically stimulate the formation of [3H]inositol mono-, bis- and trisphosphates by saponin-permeabilized Swiss 3T3 cells prelabelled with [3H]inositol. Each inositol phosphate is formed largely by hydrolysis of its parent lipid and not by dephosphorylation of inositol 1,4,5-trisphosphate [(1,4,5)IP3]. Although hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is most sensitive to guanine nucleotides, hydrolysis of phosphatidyl-inositol (PI) and phosphatidylinositol 4-phosphate (PIP) is quantitatively more important. These results suggest that a guanine nucleotide-dependent regulatory protein(s) (G-protein) is involved in regulating the hydrolysis of PI and PIP, as well as PIP2, and so may allow formation of diacylglycerol (DG) without simultaneous production of (1,4,5)IP3 and mobilization of intracellular Ca2+.     

Prolactin induces the formation of inositol bisphosphate and inositol trisphosphate in cultured mouse mammary gland explants

The effect of prolactin on [3H]inositol metabolism in cultured mouse mammary gland explants derived from 12-14-day pregnant mice was determined. In mammary gland explants that were prelabeled by culturing the tissues with 3 microCi/ml myo-[3H]inositol for 48 h, the levels of 3H in inositol derivatives were determined. The temporal effect of prolactin on the quantity of 3H present in phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP), phosphatidylinositol bisphosphate (PIP2) and various inositol phosphate containing fractions were examined. Prolactin significantly stimulated the accumulation of 3H label in inositol monophosphate (IP1), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) 1-3 h after addition of prolactin. An effect of prolactin on the accumulation of inositol derivatives was not apparent at prolactin-exposure periods of less than 60 min; nor was an effect of prolactin apparent when exposure periods of 4 h or longer were employed. Prolactin did not significantly decrease the 3H label in PI, PIP or PIP2 except at 1 and 2 h. These data when considered with other apropos studies are compatible with the conclusion that the turnover of inositol lipid derivatives may be involved in the mechanism by which prolactin regulates metabolic processes in the mammary gland. The primary action of prolactin on mammary cells, however, would not appear to involve its action on the metabolism of the inositol derivatives in view of the extended time required (1 h) before effects of prolactin on perturbations of inositide metabolism are manifested.     

Changes in phosphatidylinositol metabolism in response to hyperosmotic stress in Daucus carota L. cells grown in suspension culture.

Carrot (Daucus carota L.) cells plasmolyzed within 30 s after adding sorbitol to increase the osmotic strength of the medium from 0.2 to 0.4 or 0.6 osmolal. However, there was no significant change in the polyphosphorylated inositol phospholipids or inositol phosphates or in inositol phospholipid metabolism within 30 s of imposing the hyperosmotic stress. Maximum changes in phosphatidylinositol 4-monophosphate (PIP) metabolism were detected at 5 min, at which time the cells appeared to adjust to the change in osmoticum. There was a 30% decrease in [3H]inositol-labeled PIP. The specific activity of enzymes involved in the metabolism of the inositol phospholipids also changed. The plasma membrane phosphatidylinositol (PI) kinase decreased 50% and PIP-phospholipase C (PIP-PLC) increased 60% compared with the control values after 5 min of hyperosmotic stress. The PIP-PLC activity recovered to control levels by 10 min; however, the PI kinase activity remained below the control value, suggesting that the cells had reached a new steady state with regard to PIP biosynthesis. If cells were pretreated with okadaic acid, the protein phosphatase 1 and 2A inhibitor, the differences in enzyme activity resulting from the hyperosmotic stress were no longer evident, suggesting that an okadaic acid-sensitive phosphatase was activated in response to hyperosmotic stress. Our work suggests that, in this system, PIP is not involved in the initial response to hyperosmotic stress but may be involved in the recovery phase.