The effects of lithium on platelet phosphoinositide metabolism.

The effects on phosphoinositide metabolism of preincubation of platelets for 90 min with 10 mM-Li+ were studied. Measurements were made of [32P]phosphate-labelled phosphoinositides and of [3H]inositol-labelled inositol mono-, bis- and tris-phosphate (InsP, InsP2 and InsP3). Li+ had no effect on the basal radioactivity in the phosphoinositides or in InsP2 or InsP3, but it caused a 1.8-fold increase in the basal radioactivity in InsP. Li+ caused a 4-, 3- and 2-fold enhanced thrombin-induced accumulation of label in InsP, InsP2 and InsP3 respectively. Although the elevated labelling of InsP2 and InsP3 returned to near-basal values within 30-60 min, the high labelling of InsP did not decline over a period of 60 min after addition of thrombin to Li+-treated platelets, consistent with inhibition of InsP phosphatase by Li+. The effect of Li+ was not due to a shift in the thrombin dose-response relationship; increasing concentrations of thrombin enhanced the initial rate of production of radiolabelled inositol phosphates, whereas Li+ affected either a secondary production or the rate of their removal. The only observed effect of Li+ on phosphoinositide metabolism was a thrombin-induced decrease (P less than 0.05) in labelled phosphatidylinositol 4-phosphate in Li+-treated platelets; this suggests an effect on phospholipase C. Li+ enhanced (P less than 0.05) the thrombin-induced increase in labelled lysophosphatidylinositol, suggesting an effect on phospholipase A2. It is concluded that Li+ inhibits InsP phosphatase and has other effects on phosphoinositide metabolism in activated platelets. The observed effects occur too slowly to be the mechanism by which Li+ potentiates agonist-induced platelet activation.     

Effects of diabetes and insulin on phosphoinositide metabolism in R3230AC mammary tumors.

The effects of diabetes and insulin administration on certain aspects of phosphoinositide metabolism in R3230AC mammary tumors were studied in vivo. Three weeks after diabetes was induced by streptozotocin, [3H]myoinositol incorporation into PI, PIP and PIP2 was increased in R3230AC tumors, whereas the formation of [3H]IP, [3H]IP2 and [3H]IP3 was decreased. Administration of protamine zinc insulin (3IU, twice daily, for 3 days) to diabetic rats decreased [3H]myoinositol incorporation into phosphoinositides and inositol phosphates in these mammary tumors. The R3230AC tumor from insulin-treated diabetic hosts had lower levels of unmetabolized [3H]-myoinositol compared to tumors from diabetic animals. Enzymatically-dissociated tumor cells from insulin-treated animals displayed decreased myoinositol transport in vitro. These findings suggest that the insulin-induced decrease in the turnover of inositol lipids in vivo in R3230AC mammary tumors could have resulted from the decreased level of [3H]myoinositol in these cells.     

Insulin and oxytocin effects on phosphoinositide metabolism in adipocytes

The effects of hormones on phosphoinositide metabolism were examined in rat adipocytes prelabeled with 32Pi or [3H]inositol. Oxytocin and vasopressin produced large decreases in labeled polyphosphoinositides and increases in phosphatidic acid and inositol phosphates, whereas insulin was without effect, although it stimulated lipogenesis from glucose. Likewise, insulin did not elevate 1,2-diacylglycerol measured chemically by high pressure liquid or thin-layer chromatography in fat cells or pads. It also did not increase the radioactivity in 1,2-diacylglycerol in ghosts prepared from fat cells previously labeled with [3H]arachidonic acid, although oxytocin and vasopressin increased this. It is therefore concluded that insulin does not stimulate the breakdown of polyphosphoinositides to yield 1,2-diacylglycerol and inositol phosphates in adipocytes and that the insulin-like actions of oxytocin must be due to other changes. Insulin induced small, but significant and equal increases (40% at 30 min) in the incorporation of [3H] inositol into phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in adipocytes. The effects were not dependent upon glucose and were not evident before 15 min. Oxytocin also produced large increases in the labeling of the three phosphoinositides. Insulin stimulated the incorporation of [3H]glycerol into the three phosphoinositides and also phosphatidic acid, phosphatidylserine, and phosphatidylethanolamine by 50-100% in cells incubated without glucose. No changes in the labeling of glycerol 3-phosphate, lysophosphatidic acid, phosphatidylcholine, and triacylglycerol were detected, and there was a small increase (30%) in 1,2-diacylglycerol labeling. It is concluded that insulin increases the synthesis of phosphatidylinositol, phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, phosphatidylethanolamine, and phosphatidylserine in fat cells partly by stimulating a reaction(s) located between glycerol 3-phosphate and phosphatidic acid in the biosynthetic pathway.     

S-adenosyl-l-methionine inhibits phosphoinositide metabolism in the rat brain synaptosomal suspensions

S-adenosyl-l-methionine (AdoMet) has been reported to affect events linked to noradrenergic neurotransmission. In the present work, we studied the effect of AdoMet on norepinephrine (NE)-stimulated inositol phosphate production in³H-inositol-labelled crude synaptosomal suspensions of rat brain. AdoMet (50–1000 M) decreased both the synthesis of labelled polyphosphoinositide (30–50%) and the release of inositol mono- and bisphosphate (40–50%). The AdoMet effect was not dependent on NE concentration (10–1000 M), suggesting that the inhibition of inositol phosphate release was not the result of a modification of the norepinephrine binding to its receptor sites. S-adenosyl-L-homocysteine (AdoHcy) (1 mM) an inhibitor of methyltransferase activities, partially inhibited (70%) the AdoMet (0.1 mM) effect, indicating that the methylation processes cannot explain all the effects observed. We conclude that, in addition to previously reported effects of AdoMet on NE transport, AdoMet may reduce NE-linked intracellular signalling.     

Sympathetic nervous system effects on rat brain metabolism.

Brain tissue lactate and pyruvate were measured in rats under normoxic and normocapnic conditions. A significant increase in brain lactate was observed following (i) 15 minutes of unilateral carotid ligation, (ii) 30 minutes of norepinephrine infusion and (iii) 30 minutes of electrical stimulation of the superior cervical ganglion. Lactate values were not significantly altered after (i) a 30 minute epinephrine infusion, and (ii) a partial chemical sympathectomy obtained through an injection of 6-Hydroxydopamine. No alterations in brain tissue pyruvate concentration were obtained. These findings suggest that sympathetic stimulation causing the release of norepinephrine in the cerebral vessels results in increased anaerobic metabolism.     

Glucose metabolism in murine fetal cortical brain cells: lack of insulin effects

Glucose uptake and oxidation were markedly higher in cultured than in freshly isolated neural cells, prepared from murine fetal brain cortices. The hexose transport process--measured as 3-O-methyl-D-glucose uptake--appeared comparable in both conditions, and proceeded proportionally to the extracellular sugar concentration up to 6 mM. In contrast, glucose oxidation occurred independently of the prevailing glucose concentration from 1.4 mM on. Acute or chronic exposure to insulin exerted no effect upon cellular glucose uptake or oxidation. These results suggest that glucose handling by maturing fetal cortical cells is mainly determined by the rate of cellular glucose breakdown rather than by the rate of glucose transport into the cell; the marked rise in cellular glucose metabolism during culture might result from the synthesis and/or activation of a key enzyme in glucose catabolism. Our observations also indicate that the previously described neurotrophic effects of insulin are not mediated via enhanced glucose handling.     

Differential effects of chlorpromazine on secretion, protein phosphorylation and phosphoinositide metabolism in stimulated platelets.

Increasing concentrations of chlorpromazine (30-500 microM) caused a progressive lysis of gel-filtered platelets, as monitored by the extracellular appearance of cytoplasmic ([14C]adenine-labelled) adenine nucleotides. The chlorpromazine-induced lysis was markedly enhanced by thrombin and phorbol ester, and complete cytolysis was found at chlorpromazine concentrations of 100 microM and above in the presence of thrombin. At non-lytic concentrations, chlorpromazine caused a dramatic increase in the thrombin- or phorbol ester-mediated incorporation of 32P into phosphatidylinositol 4-phosphate and, to a lesser extent, into phosphatidylinositol 4,5-bisphosphate in platelets pulse-labelled with [32P]Pi. Chlorpromazine alone also caused an incorporation of 32P into the phosphoinositides. Non-lytic concentrations of chlorpromazine had no effect on the phosphorylation of the 47 kDa protein (regarded as the substrate for protein kinase C), but markedly inhibited the accompanying secretion of ATP + ADP and beta-hexosaminidase when platelets were incubated with 0.17 microM-phorbol ester or 0.1-0.2 unit of thrombin/ml. At lower concentrations of thrombin, chlorpromazine did not inhibit, but slightly enhanced, secretion. A protein of 82 kDa was phosphorylated during the interaction of platelets with thrombin and phorbol ester, and this phosphorylation was enhanced by chlorpromazine (non-lytic). These results suggest that the previously reported inhibition of protein kinase C by chlorpromazine is probably non-specific and due to cytolysis. However, since non-lytic concentrations of chlorpromazine inhibit secretion, but not protein kinase C, in platelets, activation of protein kinase C is not involved in the stimulation-secretion coupling, or chlorpromazine acts at a step after kinase activation. Possible mechanisms of this inhibition by chlorpromazine are discussed in the light of its effect on phosphoinositide metabolism and protein phosphorylation.     

The effects of cycloheximide and chloroquine on insulin receptor metabolism. Differential effects on receptor recycling and inactivation and insulin degradation

The effects of protein synthesis inhibitors and the lysosomotropic agent chloroquine on the metabolism of the insulin receptor were examined. Through the use of the heavy-isotope density shift technique, cycloheximide was found to inhibit both the synthesis of new insulin receptor and the inactivation of old cellular insulin receptor. Upon investigation of the locus of this effect of protein synthesis inhibition, it was found that cycloheximide did not inhibit 1) the translocation of receptor from the cell surface to an intracellular site, 2) the recycling of receptor from the internal site back to the plasma membrane, nor 3) the degradation of insulin. Cycloheximide did, however, rapidly and completely inhibit the inactivation of the insulin receptor. In the presence of extracellular insulin, this effect of cycloheximide resulted in the long-term (6 h) accumulation of receptor in a trypsin-resistant intracellular compartment. Puromycin and pactamycin, protein synthesis inhibitors with mechanisms of action which differ from cycloheximide, produced the same effects on insulin receptor metabolism as cycloheximide, indicating that this effect on receptor metabolism is due to the inhibition of protein synthesis and not a secondary effect of cycloheximide. Actinomycin D also inhibited the inactivation of receptor. Chloroquine inhibited the receptor-mediated degradation of insulin, but had no effect on either the internalization or inactivation of the insulin receptor. The insulin-induced recycling of the internalized receptor was inhibited by chloroquine, possibly through the inhibition of the discharge of insulin from the insulin-receptor complex. From these observations, we suggest that 1) a protein factor is required to inactivate the insulin receptor, 2) this protein and the messenger RNA coding for the protein have short cellular half-lives, and 3) insulin degradation and insulin receptor inactivation are distinct, separable processes which not only occur at different rates, but possibly occur in distinct subcellular locations.     

Microwave effects on energy metabolism of rat brain

Rat brain was exposed to 591-MHz, continuous-wave (CW) microwaves at 13.8 or 5.0 mW/cm² to determine the effect on nicotinamide adenine dinucleotide, reduced (NADH), adenosine triphosphate (ATP) and creatine phosphate (CP) levels. On initiation of the in vivo microwave exposures, fluorimetrically determined NADH rapidly increased to a maximum of 4.0%–12.5% above pre-exposure control levels at one-half minute, then decreased slowly to 2% above control at three minutes, finally increasing slowly to 5% above control level at five minutes. ATP and CP assays were performed on sham- and microwave-exposed brain at each exposure time. At 13.8 mW/cm², brain CP level was decreased an average of 39.4%, 41.1%, 18.2%, 13.1%, and 36.4% of control at exposure points one-half, one, two three, and five minutes, respectively, and brain ATP concentration was decreased an average of 25.2%, 15.2%, 17.8%, 7.4%, and 11.2% of control at the corresponding exposure periods. ATP and CP levels of rat brain exposed to 591-MHz cw microwaves at 5 mW/cm² for one-half and one minute were decreased significantly below control levels at these exposure times, but were not significantly different from the 13.8 mW/cm² exposures. For all exposures, rectal temperature remained constant. Heat loss through the skull aperture caused brain temperature to decrease during the five-minute exposures. This decrease was the same in magnitude for experimental and control subjects. Changes in NADH, ATP, and CP levels during microwave exposure cannot be attributed to general tissue hyperthermia. The data support the hypothesis that microwave exposure inhibits mitochondrial electron transport chain function, which results in decreased ATP and CP levels in brain.     

Effects of lithium on phosphoinositide metabolism in vivo

All of the known pathways for metabolizing the phospholipase C (EC 3.1.4.10) products of phosphoinositide metabolism eventually lead to myo-inositol monophosphates and products that are hydrolyzed by myo-inositol 1-phosphatase (EC 3.1.3.25). That enzyme is inhibited by lithium (Ki about 1 mM). In animals treated with LiCl, elevations of myo-inositol 1-phosphate (1-IP) occur in brain that appear to result from endogenous neural activity for they are diminished by the anesthetics halothane and pentobarbital. Lithium is thus a useful tool for assessing endogenous in vivo cerebral phosphoinositide metabolism. The 1-IP elevation is also useful for revealing in vivo central nervous system (CNS) receptor activity that is stimulated by endogenous or exogenous processes such as the effects of centrally acting drugs and of seizures. Stimulation of the CNS in the presence of lithium causes myo-inositol to be sequestered in 1-IP in proportion to the amount of stimulation. Thus if the inositol level falls sufficiently resynthesis of the phosphoinositides may be compromised and receptor response to stimuli may be reduced. Evidence for such an occurrence would support the theory that this is one mechanism by which lithium acts in the therapy of manic illness. We extended our efforts to identify such a lowering of phosphoinositide levels to mice where cerebral metabolism can be halted more rapidly than in rats. However, the only change detected was a small elevation in phosphatidylinositol 4-phosphate. We were successful, however, in causing all of the phosphoinositides to be reduced in rat cerebral cortex by pilocarpine stimulation after lithium treatment, a procedure that causes seizures. The same procedure causes the largest reduction in cortical myo-inositol levels that we have observed, and thus may represent the point where the inositol decrement is sufficient to interfere with resynthesis of the lipids.     

Dual effect of fluoride on phosphoinositide metabolism in rat brain cortex. Stimulation of phospholipase C and inhibition of polyphosphoinositide synthesis.

We have studied the effects of fluoride, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and carbachol on phospholipase C and polyphosphoinositide synthesis. The experimental system consisted of membranes from rat brain cortex, with exogenous [3H]phosphatidylinositol ([3H]PtdIns) as substrate. In such systems, we have not found evidence to support carbachol and/or GTP[S] stimulation of PtdIns phosphorylation. Fluoride inhibited synthesis of PtdIns4P and PtdIns(4,5)P2 from PtdIns. Consequently, under conditions where breakdown of polyphosphoinositides by phospholipase C was dependent on PtdIns kinase activity, fluoride inhibited activation by GTP[S] plus carbachol of phospholipase C. When conditions allowed direct breakdown of PtdIns and precluded PtdIns kinase activity, the stimulatory effects of fluoride and GTP[S] plus carbachol on phospholipase C activity were additive.     

Glucagon effects on brain carbohydrate and ketone body metabolism of rainbow trout.

The levels of glycogen in brain, lactate and acetoacetate in brain and plasma, glucose in plasma and the activities of brain key enzymes of glycogen metabolism (glycogen phosphorylase, GPase, glycogen synthetase, GSase), gluconeogenesis (fructose 1,6-bisphosphatase, FBPase), and glycolysis (6-phosphofructo 1-kinase, PFK) were evaluated in rainbow trout, Oncorhynchus mykiss, from 0.5 to 3 hr after intraperitoneal injection of 1 ml/kg(-1) body weight of saline alone (controls) or containing bovine glucagon at three different doses: 10, 50, and 100 ng/g(-1) body weight. The results obtained demonstrate, for the first time in a teleost fish, the existence of changes in brain carbohydrate and ketone body metabolism following peripheral glucagon treatment. A clear stimulation of brain glycogenolytic potential was observed after glucagon treatment, as judged by the time- and dose-dependent changes observed in brain glycogen levels (up to 88% decrease), and GPase (up to 30% increase) and GSase (up to 42% decrease) activities. In addition, clear time- and dose-dependent increased and decreased levels were observed in brain of glucagon-treated rainbow trout for lactate (up to 60% increase) and acetoacetate (up to 67% decrease), respectively. In contrast, no significant changes were observed after glucagon treatment in those parameters related to glycolytic/gluconeogenic capacity of rainbow trout brain. Altogether, these in vivo results suggest that glucagon may play a role (direct or indirect) in the regulation of carbohydrate and ketone body metabolism in brain of rainbow trout.     

Essential role of phosphoinositide metabolism in synaptic vesicle recycling.

Growing evidence suggests that phosphoinositides play an important role in membrane traffic. A polyphosphoinositide phosphatase, synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that synaptojanin 1-deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings. In cell-free assays, reduced phosphoinositide phosphatase activity correlated with increased association of clathrin coats with liposomes. Intracellular recording in hippocampal slices revealed enhanced synaptic depression during prolonged high-frequency stimulation followed by delayed recovery. These results provide genetic evidence for a crucial role of phosphoinositide metabolism in synaptic vesicle recycling.     

Role of calcium ions in rapid effects of L-thyroxine on phosphoinositide metabolism in rat liver cells

The role of calcium ions in the L-thyroxine-induced initiation of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) and also the course of releasing individual fractions of inositol phosphates and diacylglycerides (DAG) were studied in liver cells during early stages of the hormone effect. L-Thyroxine stimulated a rapid hydrolysis in hepatocytes of PtdInsP₂ labeled with [¹⁴C]linoleic acid and [³H]inositol mediated by phosphoinositide-specific phospholipase C. This was associated with accumulation of [¹⁴C]DAG, total inositol phosphates, [³H]inositol 1,4,5-trisphosphate (Ins1,4,5P₃) and [³H]inositol 1,4-bisphosphate (Ins1,4P₂). Elimination of calcium ions from the incubation medium of hepatocytes did not abolish the effect of thyroxine on the accumulation of [¹⁴C]DAG and total [³H]inositol phosphates. Preincubation of liver cells with TMB-8 increased the stimulatory effect of L-thyroxine on the accumulation of [¹⁴C]DAG. During the incubation of hepatocytes in the presence of the hormone the content of ¹⁴C-labeled fatty acids did not change. The L-thyroxineinduced accumulation of [³H]Ins1,4,5P₃ and [³H]Ins1,4P₂ did not depend on the presence of calcium ions in the incubation medium of the cells.     

Inositol cycling and phosphoinositide metabolism in rat pancreatic islets.

The effects of extracellular inositol and LiCl on intra-islet inositol cycling were investigated in isolated rat islets. Islets were cultured for 7 days in inositol-free RPMI 1640 containing 11.1 mM glucose and labeled with 3.7 MBq myo-[2-3H] inositol for the final 3 days. The labeled islets were then perifused under various conditions. There was a persistent increase in [3H] efflux from labeled islets stimulated with 16.7 mM glucose for 60 min. Addition of 5 mM inositol resulted in marked release of [3H] from islets and a decrease in radioactive inositol-lipid. When islets were perifused with 5 mM LiCl, the glucose-induced efflux of [3H] was greatly inhibited. The inhibitory effect of LiCl on [3H] efflux was partially corrected by the addition of 5 mM inositol. A prominent effect of LiCl was an increase in inositol monophosphate, indicating increased phospholipase C activity. This was detected within 5 min after glucose stimulation. The present data suggest that there is always very active intra-islet inositol cycling and that glucose can augument inositol-lipid metabolism.