Perturbation of glycerolipid content and biosynthesis in hepatocytes of rats continuously infused with Escherichia coli endotoxin

The effect of chronic, nonlethal endotoxemia on the endogenous content and de novo biosynthesis of glycerolipids was investigated in rat hepatocytes. Continuous E. coli endotoxin (ET) infusion for 30 hours through a subcutaneously implanted mini-pump greatly altered the composition of membrane phospholipids. Sphingomyelin (SPH) and phosphatidylserine (PS) content increased by 56% and 29%, respectively, while the content of phosphatidylcholine (PC) decreased slightly (6%) as compared with saline-infused rats. These effects contrasted with those observed in pair-fed rats (whose food intake was matched to that voluntarily consumed by ET-infused animals). Food restriction induced a great depletion of phospholipid content, mainly phosphatidylethanolamine (PE), PC, phosphatidylinositol (PI), and PS, with no changes at the level of SPH as compared with control (fed ad libitum) rats. Triacylglycerol (TG) content was greatly decreased (66%) in ET-infused rats and the magnitude of the change and the fatty acid composition followed a pattern similar to that observed in pair-fed rats. The kinetics of [2-3H]-glycerol incorporation reflected efficient utilization of the precursor for de novo biosynthesis of glycerolipids. Labeling of the intermediate metabolite phosphatidic acid (PA) peaked at an earlier time (1 min) in ET-infused, and in pair-fed rats, as compared with saline-infused and control rats (3 min) respectively, and was followed by a later peak in diacylglycerol (DG) labeling. The metabolic flux thereafter in endotoxemia reflected a redirection toward the synthesis of TG and PI, while in pair-fed animals the label went mainly to PC, concomitantly with a great reduction in the uptake of label into PI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impairments in vasopressin-stimulated inositol lipid metabolism in hepatocytes of septic rats

Vasopressin (VP)-stimulated 32P-inositol lipid metabolism was studied in hepatocytes obtained from rats rendered septic by cecal ligation and puncture. Basal 32P-phosphatidylinositol (PI) labeling, as well as its hormone-stimulated turnover, were greatly reduced in septic rats compared with sham-operated rats. The earliest VP-induced degradation of 32P-polyphosphoinositides (poly-PI) was greatly attenuated in septic rats. Moreover, while 32P-poly-PI labeling reached its lowest value by 60 sec of VP stimulation in cells from sham-operated rats, maximal changes in 32P-phosphatidylinositol 4,5-bisphosphate (32PIP2) occurred within 30 sec in septic rats. In contrast, the recovery of 32PIP2 labeling was more active in cells from septic rats, overcoming the impairment in its resynthesis triggered by surgical trauma in cells from sham-operated rats. The lower uptake of 32P into phosphatidic acid (PA) at the different time points analyzed was a sensitive indicator of the lower production of diacylglycerols from the VP-induced degradation of inositol phospholipids in septic rats. These observations support the idea that sepsis is associated with perturbations in the earliest events of the hepatocyte signal transmission pathway, namely, at the level of a receptor coupled to inositol lipid metabolism. Such perturbations are likely to be involved in the previously reported defective cell physiologic response to external hormone stimulation.     

Reduction of norepinephrine-induced tonic contraction and phosphoinositide turnover in arteries of spontaneously hypertensive rats. A possible role for protein kinase C

Experiments were conducted to determine whether a difference in receptor-induced phosphatidylinositol hydrolysis occurred in aorta from spontaneously hypertensive rats (SHR) v Wistar-Kyoto (WKY) rats, and whether such a difference was correlated with contractile response. Basal incorporation of 32P into phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidylinositol diphosphate (PIP2) and phosphatidic-acid (PA) was not different between SHR and WKY groups. However, after five minutes of norepinephrine (NE; 10 mumol) exposure, increases in 32P labeling were markedly lower in SHR arteries. The percentage decrease amounted to 45% for PI, 68% for PIP, 100% for PIP2 and 58% for PA. Basal incorporation of 3H-myo-inositol into inositol monophosphate (IP) was similar for SHR and WKY groups. However, after 30 minutes of NE (10 mumol), SHR arteries failed to show an increase in 3H-IP levels, whereas labeling was increased 219% in WKY arteries. The contractile response of SHR arteries to 10 mumol NE showed a marked reduction in the rate of development of the tonic phase that has previously been shown to be supported by activity of protein kinase C. Higher Ca2+ levels failed to augment the SHR response, whereas WKY responses were significantly increased. Contractions in the presence of the phorbol ester tetradecanoylphorbolacetate exhibited a similar reduction in NE-induced tonic phase tension. These results indicate an impairment in SHR arteries at the level of receptor-induced formation of inositol cycle second messengers, possibly due to elevated basal levels of protein kinase C. These differences may be important in explaining altered vascular responses in primary hypertension.     

Effects of Ethanol on Phosphorylation of Lipids in Rat Synaptic Plasma Membranes

Synaptic plasma membranes (SPM) isolated from rat cerebral cortex contain lipid kinases for conversion of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol (DG) to PIP, phosphatidylinositol 4,5-bisphosphate (PIP₂), and phosphatidic acid (PA), respectively. These anionic phospholipids are important in signal transduction mechanisms and are required for synaptic function. The effect of ethanol and other aliphatic alcohols on phosphorylation of these lipids in SPM has not been established. incubation of SPM with [Y³²P]ATP resulted in labeling of PIP, lyso-PIP, PIP₂, and PA. Ethanol (50–200 mM) added to the incubation system showed a dose-dependent decrease in labeling of PIP₂ but not PIP or PA. To a lesser extent, labeling of PIP₂ was also inhibited by 1-propanol, but neither isopropanol nor 1-butanol could alter the PIP₂ labeling pattern. Under similar incubation conditions, labeling of PIP and PA in SPM was not altered by ethanol, 1-propanol, isopropanol, but l-butanol stimulated PIP labeling with a peak at 25 mM. Addition of exogenous PIP to the incubation mixture led to an increase in labeling of PIP₂, suggesting that the endogenous PIP pool in SPM is limiting for the synthesis of PIP₂ in SPM. Interestingly, when SPM were incubated with exogenous PIP, addition of ethanol (50–100 mM) to this incubation mixture resulted in an increase in PIP₂ labeling. Taken together, these results suggest a specific effect of ethanol on PIP kinase in SPM, and this effect seems to be dependent on the location and/or amount of PIP in the membrane.     

Identification of minor metabolites of phospholipid signal molecules in [(32)P]P(i)-labelled platelets

Incubation of blood platelets with (32)P-labelled inorganic phosphate for 60 min leads to incorporation of radioisotope mainly into phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP) and phosphatidyl-4,5-bisphosphate (PIP(2)) in resting platelets and into phosphatidic acid (PA) in activated platelets. Small amounts of other important phosphoinositide isomers also become labelled following platelet activation, among them the 3-phosphorylated derivatives. In addition, several other faintly labelled spots are visible on TLC separations. Three of these lipids have now been identified as lysophosphatidylinositol (lysoPI), lysophosphatidic acid (lysoPA) and CDP-diacylglcerol (CDP-DAG).[(32)P]LysoPI was present in resting and activated platelets, whereas [(32)P]lysoPA and [(32)P]CDP-DAG were observed only upon platelet activation. The phosphoinositide cycle turns over without accumulation of [(32)P]PA and [(32)P]CDP-DAG in resting platelets. A large increase (as much as 40-fold) in the steady-state level of [(32)P]PA is seen in thrombin-activated platelets. A slight increase in the steady-state levels of [(32)P]CDP-DAG is accompanied by a similar increase in [(32)P]PI and larger increases in [(32)P]PIP and [(32)P]PIP(2) (about 50%), which is indicative of a general increase in flux in the PPI cycle. Elevation of CDP-DAG levels is probably only a reflection of increased flux, whereas lysoPA and lysoPI have been reported to have diverse signalling functions in various cells.     

Identification of minor metabolites of phospholipid signal molecules in [ 32 P]P i -labelled platelets

Incubation of blood platelets with 32 P-labelled inorganic phosphate for 60 min leads to incorporation of radioisotope mainly into phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP) and phosphatidyl-4,5-bisphosphate (PIP 2 ) in resting platelets and into phosphatidic acid (PA) in activated platelets. Small amounts of other important phosphoinositide isomers also become labelled following platelet activation, among them the 3-phosphorylated derivatives. In addition, several other faintly labelled spots are visible on TLC separations. Three of these lipids have now been identified as lysophosphatidylinositol (lysoPI), lysophosphatidic acid (lysoPA) and CDP-diacylglcerol (CDP-DAG).[ 32 P]LysoPI was present in resting and activated platelets, whereas [ 32 P]lysoPA and [ 32 P]CDP-DAG were observed only upon platelet activation. The phosphoinositide cycle turns over without accumulation of [ 32 P]PA and [ 32 P]CDP-DAG in resting platelets. A large increase (as much as 40-fold) in the steady-state level of [ 32 P]PA is seen in thrombin-activated platelets. A slight increase in the steady-state levels of [ 32 P]CDP-DAG is accompanied by a similar increase in [ 32 P]PI and larger increases in [ 32 P]PIP and [ 32 P]PIP 2 (about 50%), which is indicative of a general increase in flux in the PPI cycle. Elevation of CDP-DAG levels is probably only a reflection of increased flux, whereas lysoPA and lysoPI have been reported to have diverse signalling functions in various cells.     

Effect of growth hormone-releasing factor on phosphoinositide hydrolysis in somatotrophs

We studied the role of the phosphatidylinositol system in the action of growth hormone-releasing factor (GRF). We asked whether GRF stimulates the activity of phospholipase C by determining GRF-induced changes in 32P labeling of the individual phosphoinositides and inositol phosphates in purified rat somatotrophs. The somatotrophs were challenged with GRF (10(-7)M) for 0.33, 1, 3, 10, 30, and 90 min. GRF did not significantly or consistently alter 32P incorporation into phosphatidylinositol bisphosphate (PIP2), phosphatidylinositol monophosphate (PIP), or phosphatidylinositol (PI), except for a small reduction in PIP labeling at 90 min. In general the level of 32P incorporation into the inositol phosphates did not increase but instead decreased with GRF. There was a small but significant reduction of labeling of inositol trisphosphate (IP3) at 90 min of GRF incubation. There were also small but significant decreases in 32P incorporation into inositol bisphosphate (IP2) at 0.33, 3, and 30 min. GRF did not significantly alter 32P labeling of inositol monophosphate (IP). These results indicate that GRF does not stimulate phospholipase C activity in somatotrophs. We conclude that the phosphatidylinositol second messenger system does not play an essential role in the action of GRF.     

The inhibition of phosphatidylinositol turnover: a possible postreceptor mechanism for the prolactin secretion-inhibiting effect of dopamine

We studied the association between the inhibition of phosphatidylinositol (PI) turnover and the inhibition of PRL secretion in the presence of dopamine. The incorporation of radiolabeled phosphate into anterior pituitary gland PI as well as serum PRL levels were significantly (P less than 0.01) greater in female than in male rats. No significant sex-related difference was found in the incorporation by pituitary tissue of 32P into phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Dopamine decreased the incorporation of 32P into PI, but not into PC or PE, by female rat pituitary glands; this effect was reversed by two dopamine receptor-blocking agents, haloperidol and pimozide. After dopamine was removed from the incubation medium, basal 32P incorporation into PI was restored within 10 min. The administration of bromocriptine (500 micrograms/kg, ip, 4 h earlier) significantly reduced pituitary PI turnover. Conversely, in vivo injection of alpha-methyl-p-tyrosine (alpha MpT; 200 mg, ip, 2.5 h before death), an inhibitor of catecholamine biosynthesis, dramatically increased serum PRL levels. In vitro incorporation of 32P into PI, but not into PC or PE, increased (+130%) when these glands were incubated for 30 min with radiolabeled phosphate. The in vitro addition of 0.5 microM dopamine to glands from alpha MpT-treated rats counteracted the stimulation of 32P incorporation into PI produced by alph MpT treatment. In rats bearing the transplantable PRL-secreting tumor MtTW15, the hyperprolactinemia produced by the tumor stimulates hypothalamic turnover of dopamine, with a consequent inhibition of pituitary gland PRL secretion. 32P incorporation into PI, but not into PC or PE, was significantly (P less than 0.01) inhibited (-41%) in pituitary glands from these rats. The injection of alpha MpT (200 mg/kg, ip) or haloperidol (2 mg/kg, ip) 12 and 3 h before death into MtTW15 tumor-bearing rats abolished the inhibition of 32P incorporation into pituitary PI. Dopamine also decreased PI turnover in the 7315a PRL-secreting pituitary tumor. Our data indicate that the PI cycle may be an intracellular mechanism controlling PRL release in the rat and that the changes in its cleavage and turnover may be an early postreceptor event responsible for the inhibition of PRL secretion produced by factors such as dopamine.     

Age-dependent changes in the mechanisms controlling prolactin secretion and phosphatidylinositol turnover in male rats: effect of phosphatidylserine

The present study was undertaken in order to better characterize the functional state of anterior pituitary gland in young and old rats by using prolactin secretion and incorporation of radioactive phosphate into phosphatidylinositol (PI) as markers. The in vitro incorporation of radiolabeled phosphate into anterior pituitary PI was significantly (p less than 0.01) greater in young (3-5 months) than in aged (24-25 months) male Sprague-Dawley rats. No significant difference was found in the incorporation by pituitary tissue of 32P into phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Also, the extent of prolactin secretion from isolated pituitary was significantly greater in young than in aged rats, while the prolactin pituitary content was significantly higher in aged animals. In vitro dopamine (DA) decreased the incorporation of 32P into PI, both in young and old pituitary glands, and inhibited prolactin secretion into the incubation medium. Brain cortex-phosphatidylserine (BC-PS), a pharmacologically active purified phospholipid, capable of stimulating the dopaminergic system in the hypothalamus and of decreasing prolactin secretion both in humans and rats in vitro and in vivo, inhibited the incorporation of labeled phosphate into PI of pituitary glands from either young or old rats, but did not alter the prolactin secretion from the glands incubated in vitro. Baseline prolactin plasma levels did not differ significantly between young and old rats either when blood was collected from the trunk after decapitation or underwent sampling from chronically cannulated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombin-induced platelet aggregation,phosphoinositide metabolism and protein phosphorylation in NIDDM patients treated by diet,sulphonylurea or insulin

Summary We studied thrombin-induced metabolism of phosphoinositide, protein phosphorylation and platelet aggregation in platelets from 32 NIDDM patients and 12 control subjects. To clarify the effect of diet, sulphonylureas, or insulin treatment, the subjects were divided into three groups based on the type of treatment. Thrombin-induced platelet aggregation was measured with an aggregometer. Low-dose thrombin (0.25 U/ml)-stimulated platelet aggregation in diabetic patients was significantly increased compared with the control subjects. Platelet aggregation in the sulphonylurea and insulin groups was significantly lower than in the diet group. On the other hand, in platelets incubated with [³²P]orthophosphate, thrombin-induced incorporation of ³²P radioactivity into phosphatidic acid (PA) was significantly lower in the sulphonylurea and insulin groups than in the diet group. Thrombin-induced incorporation of [³²P] radioactivity into phosphatidylinositol (PIP) for 10 s was significantly higher in the sulphonylurea group than in the diet group. There were no differences in thrombin-induced 47 kDa protein phosphorylation between platelets from the diet, sulphonylurea, or insulin groups. These results suggest that sulphonylureas and insulin induce suppression of thrombin-induced activation of phospholipase C, which mediates hydrolysis of PIP and PIP₂ and production of PA, which leads to inhibition of platelet aggregation.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - IDDM insulin-dependent diabetes mellitus - PA phosphatidic acid - PI phosphatidylinositol - PIP phosphatidylinositol 4-monophosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - TPA 12-O-tetradecanoylphorbol-13-actate - IP₃ inositol 1,4,5-trisphophate - E/I expiration/inspiration - HPLTC high performance thin layer chromatography     

Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity

Among various second messengers, phosphatidylinositol 3,4,5-triphosphate (PIP3) and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] regulate a variety of cellular processes, such as cell survival, polarization, and proliferation. Many of these functions are achieved via activation of serine/threonine kinase Akt. To investigate the spatiotemporal regulation of these lipids, we constructed a genetically targetable phosphoinositide (PI) indicator by sandwiching pleckstrin homology (PH) domain of Akt and a "pseudoligand" containing acidic amino acid residues, between cyan and yellow mutants of GFP. In living cells, elevations in PIP3 and PI(3,4)P2 by growth factor-induced activation of phosphatidylinositol 3-kinase (PI3K) resulted in a change in fluorescence resonance energy transfer (FRET) between the fluorescent proteins, increasing yellow to cyan emission ratios by 10-30%. This response can be reversed by inhibiting PI3K and abolished by mutating the critical residues responsible for PI binding. Differential dynamics of PIs were observed at plasma membrane of NIH 3T3 cells, stimulated by various growth factors. On the other hand, the nuclear targeted indicator showed no response within an hour after platelet-derived growth factor stimulation, suggesting that no appreciable amounts of accessible PIP3 and PI(3,4)P2 were produced in the nucleus. Furthermore, simultaneous imaging of a plasma membrane-targeted PI indicator and a nuclear-targeted Akt activity reporter revealed a gradual and sustained accumulation of Akt activity in the nucleus after rapid and transient production of PIP3 and PI(3,4)P2 at plasma membrane in the same cell. Thus, signal propagation from the lipid messengers at plasma membrane to the effectors in the nucleus is precisely controlled by kinases as well as lipid and protein phosphatases.     

Rapid glucose-dependent increases in phosphatidic acid and phosphoinositides in rat pancreatic islets

Glucose effects on islet phospholipids were examined during direct incubation or after 3 days of 32P prelabeling in primary culture. In both cases, glucose increased the 32P content of phosphatidic acid (PA), phosphatidylinositol (PI), and polyphosphoinositides (PPI). Glucose-induced increases in PA, PI, and PPI in the culture-prelabeling experiments were evident within 1 min, dose related, and reflective of increases in phospholipid mass, which was confirmed in direct incubations by measurement of PI phosphorus. Thus, in addition to increasing PI-PPI hydrolysis, glucose increases de novo phospholipid synthesis in pancreatic islets. The latter may result from enhanced glycolysis and substrate availability for PA-PI-PPI synthesis, since glyceraldehyde and pyruvic acid also increased PI levels. Our findings raise the possibility that increases in PA, PI, and PPI synthesis could serve as a mechanism to enhance the generation of intracellular mediators, which are purported to regulate insulin secretion.     

LHRH rapidly stimulates phosphatidylinositol metabolism in enriched gonadotrophs

The effects of luteinizing hormone-releasing hormone (LHRH) and human pancreatic growth hormonereleasing factor (hpGRF(l-40)-NH₂) on phospholipid metabolism were studied in rat anterior pituitary cells in primary culture. In a 4-fold enriched population of gonadotrophs, 30 nM LHRH increased ³²P; incorporation into phosphatidic acid (PA) as early as l min after its addition. Phosphatidylinositol (PI) labeling was increased l min later. The stimulatory action of LHRH was observed in both phospholipids up to 100 min, the last time interval studied. The decapeptide did not affect ³²P_i labeling of phosphatidylcholine (PC), lysoPC, phosphatidylethanolamine or phosphatidylserine. Dose-response studies performed after 25 min of incubation showed an ED₅₀ value of LHRH action at approximately 1 nM for PI labeling. In contrast, the addition of 0.1 μM GRF to anterior pituitary cells enhanced ³²P_i incorporation only into PC after a 60 min incubation period. The present data suggest that stimulation of acidic phospholipid metabolism, particularly an increase in PA-PI turnover, may represent an early event in the mechanism of action of LHRH but not GRF in the anterior pituitary gland.     

Stimulation of phosphatidic acid and phosphatidylinositol labeling in luteal cells by luteinizing hormone releasing hormone

Luteinizing hormone-releasing hormone (LHRH) causes a rapid and marked increase of [32P]orthophosphate incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA) in rat luteal cells in culture. The neurohormone exerts its stimulatory effect at an ED50 value of approximately 15 nM. Human chorionic gonadotropin (hCG) has no effect alone and does not interfere with the LHRH-induced PA-PI labeling. The rapidity and the specificity of the effect of LHRH suggest that the stimulation of the PA-PI cycle may well serve as a potent transducing mechanism responsible for the direct action of LHRH and its agonists at the ovarian level.     

The Role of Phosphatidylinositol Transfer Proteins (PITPs) in Intracellular Signalling.

Phosphatidylinositol transfer protein (PITP) has been identified as a key player in numerous signalling pathways relying on phosphatidylinositol (PI) metabolites. Although its cellular function is most likely linked to its PI/phosphatidylcholine (PC) transfer activity-an in vitro activity shared by all known PITPs-this feature cannot explain all findings from studies with PITP. Here, we review evidence suggesting that one of the main functions of PITP in cellular signalling is to present PI to lipid kinases for localized production of phosphatidylinositol (4,5)-bisphosphate (PIP(2)), either to be used as a signalling molecule (for example, in exocytosis) or as a substrate (for example, by phospholipases).     

Thyrotropin (TSH)-releasing hormone decreases phosphatidylinositol and increases unesterified arachidonic acid in thyrotropic cells: possible early events in stimulation of TSH secretion

TRH stimulated the metabolism of lipids of the phosphatidylinositol (PI)-phosphatidic acid (PA) cycle and caused an increase in the level of free or unesterified arachidonic acid in mouse pituitary thyrotropic tumor (TtT) cells. In cells labeled with [32P]orthophosphate for 45 min, TRH caused a rapid specific increase in [32P]PA to 190 +/- 8% (+/- SE) of the control value at 15 sec (P less than 0.005) and in [32P]PI to 158 +/- 8% at 2 min (P less than 0.005). In cells labeled to isotopic steady state with [3H]inositol, TRH caused a decrease in [3H]PI to 92 +/- 1.8% of the control value at 1 min (P less than 0.01) and increased the level of [3H]inositolmonophosphate. In cells labeled to isotopic steady state with [14C]stearic acid, TRH caused a transient rise in [14C]diacylglycerol and a more prolonged increase in [14C]PA. In cells labeled to isotopic steady state with [3H]arachidonic acid, TRH stimulated a rise in free [3H]arachidonic acid to 210 +/- 8% of the control value at 15 sec (P less than 0.001), with a return to a level of 125 +/- 2% of the control value by 5 min. Arachidonic acid added exogenously caused efflux of 45Ca2+ from prelabeled cells and stimulated TSH secretion. Hence, in TtT cells, TRH 1) rapidly stimulated a decrease in the level of PI and increased inositolmonophosphate, diacylglycerol, and PA; and 2) caused a rapid increase in the level of free arachidonic acid. These effects may be important in stimulation of TSH secretion by TRH. Because arachidonic acid, when added exogenously, mobilized cellular Ca2+ and stimulated TSH secretion, arachidonic acid may mediate, at least in part, TRH-stimulated TSH secretion. The action of TRH on lipid metabolism in TtT cells is different from that in mammotropic pituitary cells, since TRH does not cause an increase in the level of free arachidonic acid in GH3 cells.     

Human cardiac phospholipase D activity is tightly controlled by phosphatidylinositol 4,5-bisphosphate

Phospholipase D (PLD) plays a central role in receptor-mediated breakdown of choline phospholipids and formation of phosphatidic acid (PA), an important regulator of cardiac function. However, specific mechanisms that regulate myocardial PLD activity remain largely unknown, particularly in the human heart. We hypothesized that phosphatidylinositol 4,5-bisphosphate (PIP2), best known as substrate for phospholipase C (PLC) isozymes, plays a critical role in regulating myocardial PLD activity. We examined the effect of PIP2 on human myocardial PLD activity in vitro by utilizing a fluorescence HPLC assay. PIP2 increased 10-fold the maximal activity of a partially solubilized PLD from human atrial myocardium. PIP2-stimulated PLD activity was accompanied by a consecutive increase in diacylglycerol, indicating dephosphorylation of PA by PA phosphohydrolase. Likewise, phosphatidylinositol 3,4,5-trisphosphate, which is produced from PIP2 by phosphatidylinositol 3-kinase, increased PLD activity with about the same potency but with somewhat lower efficacy. In contrast, other phospholipids were ineffective, indicating that the action of PIP2 on PLD is highly specific. Neomycin, a high-affinity ligand of PIP2, inhibited PLD activity in human atrial myocardium, but had no effect on the activity of partially solubilized enzyme. The addition of PIP2 restored the sensitivity of solubilized PLD to neomycin inhibition, indicating that neomycin inhibits PLD activity by binding to endogenous PIP2. Our results demonstrate a critical role for PIP2 in human cardiac PLD activity and suggest that PIP2 synthesis (by phosphatidylinositol 4-phosphate 5-kinase) and hydrolysis (by PIP2-specific PLC) could be important determinants in regulating PLD signal transduction in the human heart.     

Inositol affects the intracellular turnover of pulmonary surfactant phospholipids in the rat

Rats, given a diet supplemented with 20% inositol, had threefold increased plasma inositol concentrations. The pool size of their alveolar surfactant fraction and their lamellar body fraction were the same as in the control rats and differences in phospholipid composition of the surfactant fractions were mainly restricted to changes in the percentages phosphatidylinositol (PI) and phosphatidylglycerol (PG). The change in phospholipid composition did not affect the pressure-volume relationship of the lungs. The labeling of phosphatidylcholine (PC), saturated phosphatidylcholine (SPC) or PI in the alveolar lavage fraction was the same for both groups, whereas labeling of alveolar PG was delayed in the inositol-fed rats. The specific activity-time relationships of the lamellar body phospholipids differed significantly between the control and inositol-fed rats and the differences in disappearance rate of the label from these fractions suggest that approximately 25-30% of the lamellar body material in inositol-fed rats is directed to a third, intracellular pool. We conclude that an increase in PI and a concomitant decrease in PG content of surfactant do not affect the clearance of alveolar surfactant, but enlarge the turnover of the lamellar body fraction because of intracellular degradation.     

Phosphoinositide turnover in erythrocyte membranes in human and experimental hypertension

The metabolism of phosphoinositides, a class of membrane lipids involved in Ca2+ -transport and/or mobilization systems was investigated in patients with moderate essential hypertension and in Sabra rats. Experiments were performed in vitro on isolated erythrocyte membranes by measuring the 32P-labelling of phosphatidylinositol 4,5-bisphosphate (PI-P2) and of phosphatidylinositol 4-phosphate (PI-P) following the incubation of membranes with [gamma-32P] ATP. In untreated essential hypertensives (n = 31) or in hypertensive patients whose blood pressure was controlled by beta-blocker therapy (n = 20), 32P-PI-P2 was significantly higher than in normotensive controls (n = 30); no significant difference was observed between the two groups of hypertensive patients. In Sabra rats fed on a low Na diet, 32P-PI-P2 levels were significantly higher in hypertensive-prone animals (SBH) than in hypertensive-resistant animals (SBN). When the animals were fed a high Na diet or were DOCA/salt treated, 32P-PI-P2 did not change in either substrain, although such conditions differentially affected the blood pressure of SBH and SBN. Our data indicate that the modification of phosphoinositide metabolism is not a consequence of the blood pressure elevation, but can be considered as an intrinsic membrane defect which may be associated with functional alterations of Ca2+ fluxes which in hypertensives result in an enhanced intracellular Ca2+ level.     

Effects of prostaglandin F2 alpha and a gonadotropin-releasing hormone agonist on inositol phospholipid metabolism in isolated rat corpora lutea of various ages

The sensitivity of rat corpora lutea to luteolytic agents increases with luteal age. We examined the effect of prostaglandin F2 alpha (PGF2 alpha) and [D-Ala6,Des-Gly10]GnRH ethylamide (GnRHa) on inositol phospholipid metabolism in day 2 and day 7 corpora lutea from PMSG-treated rats. Isolated corpora lutea were incubated with 32PO4 or [3H]inositol and were treated with LH, PGF2 alpha, or GnRHa. Phospholipids were purified by TLC, and the water-soluble products of phospholipase-C activity (inositol phosphates) were isolated by ion exchange chromatography. In day 2 corpora lutea, PGF2 alpha, (10 microM) and GnRHa (100 ng/ml) significantly increased 32PO4 incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI), but not into other fractions. LH provoked slight increases in PA. Results were similar with 30 min of prelabeling or simultaneous addition of 32PO4 and stimulants. In other experiments, PGF2 alpha and GnRHa provoked rapid increases (1-5 min) in the accumulation of inositol mono-, bis-, and trisphosphates. LH did not significantly increase inositol phosphate accumulation, but stimulated cAMP accumulation in 2-day-old corpora lutea. Inositol phospholipid metabolism was increased in day 7 corpora lutea compared to that in day 2 corpora lutea. This increase was associated with increased incorporation of 32PO4 into PA and PI and increased accumulation of [3H]inositol phosphates. In day 7 corpora lutea, which are very sensitive to the luteolytic effect of PGF2 alpha, the PG-induced increase in PA labeling was small and inconsistent, whereas PI labeling was unaffected in 30-min incubations. GnRHa was without effect in such corpora lutea. LH, PGF2 alpha, or GnRHa did not increase inositol phosphate accumulation in 7-day-old corpora lutea. These studies demonstrate that the transformation of young (day 2) to mature (day 7) corpora lutea is associated with an increase in luteal inositol phospholipid metabolism. The results also show that PGF2 alpha and GnRHa stimulate phospholipase-C activity in young corpora lutea, but are ineffective in mature corpora lutea, and suggest that an increase in inositol phospholipid metabolism by itself is not sufficient to explain the acute luteolytic action of PGF2 alpha and GnRH in vitro. However, phospholipase-C-derived second messengers may be involved in the action of hormones that control luteal function.