Increased turnover of platelet phosphatidylinositol in schizophrenia.

The potential role of receptor-stimulated phosphatidylinositol (PI) hydrolysis in a signal transduction mechanism has been increasingly recognized. Earlier studies have suggested a defect in alpha-adrenergic receptor function in the platelets of schizophrenic patients. Little is known, however, about the mechanisms for PI synthesis, breakdown, and regulation in schizophrenia. The present study was undertaken to investigate the metabolic turnover of inositol phospholipids and inositol phosphates by incorporation of [3H]myoinositol or [32P]orthophosphate into resting and activated platelets of normal controls and schizophrenic patients with and without neuroleptic treatment. After 5 h incubation at 37 degrees C, the majority of [3H]myoinositol was incorporated into platelet PI. Following thrombin-induced platelet activation, there was rapid formation of 3H-labeled inositol phosphates (IPs) with inositol monophosphate (IP1) being the most abundant product. The thrombin-induced formation of platelet IPs was found significantly higher in both haloperidol-stabilized and drug-free schizophrenics than in normal control subjects. When platelets were prelabeled with [32P]orthophosphates, thrombin-induced formation of phosphatidic acid (PA) was also significantly higher in haloperidol-stabilized schizophrenics than in normal controls. It is thought that thrombin-induced platelet activation is mediated through hydrolysis of polyphosphoinositides (poly-PI). The present data thus may reflect an increased signal transduction in schizophrenia, which is mediated through neuroleptic-regulated inositol phospholipid hydrolysis.     

Adenosine kinase and 5'-nucleotidase activity after prolonged wakefulness in the cortex and the basal forebrain of rat

The effect of prolonged wakefulness on adenosine kinase (AK), ecto-5'-nucleotidase and endo-5'-nucleotidase activity was assessed in the present study. Rats were sleep deprived for 3 or 6h, and one group was allowed to sleep 2h of recovery sleep after the 6h deprivation. The cortex and the basal forebrain were dissected, and frozen rapidly on dry ice. The enzyme activity of adenosine kinase was measured by monitoring the conversion of [2-3H]-adenosine into [3H]-adenosine monophosphate (AMP) and the ecto-5'-nucleotidase and endo-5'-nucleotidase activities by monitoring the conversion of [2-3H]-AMP into [3H]-adenosine. The enzyme activities did not change during deprivation or recovery sleep in either cortex or basal forebrain when compared to unhandled controls. Significant diurnal variation in enzyme activities was noted in both brain areas. In the basal forebrain adenosine kinase and both nucleotidases showed their lowest activity in the middle of the rest phase, 6h after lights on, suggesting a low level of adenosine metabolism, both production and degradation at this time point. In the cortex adenosine kinase had a diurnal activity pattern similar to the basal forebrain and the ecto-5'-nucleotidase activity was low already early in the rest phase, 3h after lights on, and remained low until the end part of the rest phase, 8h after lights on. Endo-5'-nucleotidase lacked diurnal variation. These activity patterns may be associated with the lower level of energy metabolism during sleep compared to wakefulness.     

Relationship between stroke volume and sympathetic nerve activity: new insights about autonomic mechanisms of syncope

Head-up tilt table experiments conducted in astronauts prior to and immediately after the NASA Neurolab Space Mission (STS-90) revealed that a reduction in stroke volume induced by moving from the supine to upright posture was associated with increased muscle sympathetic nerve activity (MSNA). Although this finding was not unexpected, lower average stroke volume and greater average MSNA measured after space flight in both supine and upright postures were positioned in a linear fashion on the same stroke volume-MSNA stimulus-response relationship as the average pre-flight stroke volume and MSNA responses. Since all astronauts who participated in the Neurolab orthostatic experiments completed the 10-min tilt table tests, these observations supported the notion that sympathetic reflex responses were not altered but functioned adequately after space flight in non-presyncopal subjects. In contrast to the Neurolab results, development of orthostatic hypotension and presyncopal events reported in astronauts during standing after space flight have been accompanied by attenuated peripheral vasoconstriction and less elevation in plasma concentrations of norepinephrine. The association between circulating norepinephrine (NE) and peripheral vascular resistance in presyncopal astronauts after space flight led to the conclusion that postflight presyncope can be attributed to a combination of inherently low-resistance responses, a strong dependence on volume status, and relative hypoadrenergic function. In the present investigation, we used graded levels of lower body negative pressure (LBNP) to produce linear reductions in stroke volume and performed direct measurements of MSNA to test the hypotheses that (1) elevations in MSNA during central hypovolemia are proportional (i.e., linear) with reductions in stroke volume and; (2) that the slope of the stroke volume-MSNA relationship will be reduced in presyncopal subjects.     

Chronic Lithium Treatment Impairs Phosphatidylinositol Hydrolysis in Membranes from Rat Brain Regions

Membranes prepared from rat brain regions were used to measure the receptor-coupled and/or guanine nucleotide-binding protein (G protein)-mediated hydrolysis of exogenous [3H]phosphatidylinositol ([3H]PI). Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and NaF (in the presence of AlCl3) caused concentration-dependent stimulations of [3H]PI hydrolysis, supporting the conclusion that G proteins mediating [3H]PI hydrolysis can be activated in this preparation. Neither of these responses was altered by in vitro incubation with 8 mM LiCl, but both were reduced in hippocampal, striatal, and cortical membranes from rats that had been treated with lithium for 4 weeks compared with controls. Two cholinergic agonists, carbachol and pilocarpine, induced no hydrolysis of [3H]PI unless GTP gamma S was also present, in which case each equally stimulated [3H]PI hydrolysis above that obtained with GTP gamma S alone. In the presence of GTP gamma S several excitatory amino acid agonists stimulated [3H]PI hydrolysis to an extent similar to that of carbachol. After chronic lithium treatment, [3H]PI hydrolysis stimulated by carbachol was significantly attenuated, but the response to quisqualate was unaffected. Therefore, lithium added in vitro does not have an effect on cholinergic receptor- or G protein-mediated [3H]PI hydrolysis, but each of these is reduced by chronic lithium treatment. Because exogenous [3H]PI was provided as the substrate, it is evident that the inhibitory effect of chronic lithium treatment cannot be due to substrate depletion. Impaired function of G proteins appears to be the most likely mechanism accounting for attenuated [3H]PI hydrolysis after chronic administration of lithium.     

The effect of intruded events on peak time: the role of reinforcement history during the intruded event

Pigeons were studied in an extension of a study by Aum et al. [Aum, S., Brown, B.L., Hemmes, N.S. 2004. The effects of concurrent task and gap events on peak time in the peak procedure. Behav. Process. 65, 43-56] on timing behavior under a discrete-trial fixed-interval (FI) procedure during which 6-s intruded events were superimposed on peak-interval (PI) test trials. In Aum et al., one event consisted in termination of the timing cue (gap trial); the other was a stimulus in the presence of which subjects had been trained to respond under an independent random-interval (RI) schedule of reinforcement (concurrent task trial). Aum et al. found a disruption of timing on concurrent task trials that was greater than that on gap trials. The present study investigated history of reinforcement associated with intruded events as a possible explanation of this earlier finding. After training to peck a side key on a 30-s PI procedure, discrimination training was conducted on the center key in separate sessions; red or green 6-s stimuli were associated with RI 24s or EXT (extinction) schedules. During testing under the PI procedure, three types of intruded events were presented during probe trials--the stimulus associated with the RI (S+) or EXT (S-) schedule during discrimination training, or a gap (termination of the side-keylight). Intruded events occurred 3, 9, or 15s after PI trial onset. Effects of reinforcement history were revealed as substantial disruption of timing during the S+ event and relatively little disruption during the S- event. Intermediate effects were found for the gap event. Results indicate that postcue effects are at least partially responsible for the disruptive effects of the S+ event.     

The Phosphoinositide Signal Transduction System Is Impaired in Bipolar Affective Disorder Brain

Abstract: The function of the phosphoinositide second messenger system was assessed in occipital, temporal, and frontal cortex obtained postmortem from subjects with bipolar affective disorder and matched controls by measuring the hydrolysis of [³H]phosphatidylinositol ([³H]PI) incubated with membrane preparations and several different stimulatory agents. Phospholipase C activity, measured in the presence of 0.1 mM Ca²⁺ to stimulate the enzyme, was not different in bipolar and control samples. G proteins coupled to phospholipase C were concentration-dependently activated by guanosine 5′-O-(3-thiotriphosphate) (GTPγS) and by NaF. GTPγS-stimulated [³H]PI hydrolysis was markedly lower (50%) at all tested concentrations (0.3–10 µM GTPγS) in occipital cortical membranes from bipolar compared with control subjects. Responses to GTPγS in temporal and frontal cortical membranes were similar in bipolars and controls, as were responses to NaF in all three regions. Brain lithium concentrations correlated directly with GTPγS-stimulated [³H]PI hydrolysis in bipolar occipital, but not temporal or frontal, cortex. Carbachol, histamine, trans-1-aminocyclopentyl-1,3-dicarboxylic acid, serotonin, and ATP each activated [³H]PI hydrolysis above that obtained with GTPγS alone, and these responses were similar in bipolars and controls except for deficits in the responses to carbachol and serotonin in the occipital cortex, which were equivalent to the deficit detected with GTPγS alone. Thus, among the three cortical regions examined there was a selective impairment in G protein-stimulated [³H]PI hydrolysis in occipital cortical membranes from bipolar compared with control subjects. These results directly demonstrate decreased activity of the phosphoinositide signal transduction system in specific brain regions in bipolar affective disorder.     

Phosphate turnover of phosphatidylinositol in resting and thrombin-stimulated platelets

Human platelets were pulse-labelled with [32P]Pi and extracts were analyzed for masses and radioactivities of ATP and phosphoinositides. Immediately after pulse-labelling, the specific 32P radioactivity of phosphatidylinositol (PI) was only 3.4% of that of the gamma-phosphoryl of ATP. Upon incubation of the platelets at 37 degrees C, the specific 32P radioactivity of ATP (beta- and gamma-phosphoryls) remained constant. However, specific 32P radioactivity in PI increased continuously to 17% of specific [gamma-32P]ATP at 90 min of incubation. Stimulation with 0.5 U/ml of thrombin induced a 35% decrease in mass of PI which was unaffected by the time after the pulse-labelling. In contrast, the thrombin-induced changes in [32P]PI differed markedly at the various times after the [32P]Pi-pulse. Immediately after pulse-labelling, [32P]PI initially decreased but increased thereafter to 260% of control values after 180 s. With increasing specific 32P-radioactivity in PI before stimulation, the thrombin-induced increase in [32P]PI gradually disappeared. After 90 min of incubation, thrombin induced a continuous decrease in [32P]PI that almost parallelled mass. The data are explained by an initial breakdown of PI to diacylglycerol through the PI cycle or the polyphosphoinositide cycle, followed by resynthesis of PI through phosphatidic acid. In contrast to pre-existing PI, the resynthesized PI is in full isotopic equilibrium with ATP. This allowed us to estimate that 14% of the PI that is consumed between 30 and 180 s of stimulation, is recycles. From our data we calculate that the rate of PI resynthesis increased from 2.4 to 20 nmol/min per 10(11) cells upon thrombin stimulation of platelets.     

Secretogogue-stimulated phosphatidylinositol breakdown in the exocrine pancreas liberates arachidonic acid, stearic acid, and glycerol by sequential actions of phospholipase C and diglyceride lipase

When mouse pancreatic "minilobules" prelabeled with either [14C]arachidonic acid (AA), [14C]stearic acid (SA), or [3H]glycerol were stimulated with the secretogogue, caerulein, there was a 60-70% loss in radioactivity in phosphatidylinositol (PI) at 30 min. This loss was accompanied by the formation of [14C] phosphatidic acid (PA), [14C]diacylglycerol (DG), [14C] triacylglycerol (TG), and free [14C]AA, [14C]SA, and [3H]glycerol. The loss in radioactive PI was the same as the loss in chemically measured PI-phosphorus. Thirty to fifty per cent of the caerulein-induced loss of prelabeled PI could be accounted for as free [14C]AA, [14C]SA, or [3H]glycerol. Increased incorporation of fatty acid or glycerol residues into DG, PA, and TG accounted for the balance of the loss in PI. The specific DG-lipase inhibitor, RHC 80267, markedly inhibited the caerulein-stimulated release of [14C]AA, [14C]SA, and [3H]glycerol and roughly doubled the caerulein-induced increment in [14C]AA-, [14C]SA-, or [3H]glycerol-labeled DG, showing that the source of the caerulein-induced increment in fatty acids and glycerol was DG. When the PI was prelabeled with either [32P] orthophosphate, [3H]myoinositol, or [3H]glycerol, only 1% or less of the radioactivity in PI was in lysophosphatidylinositol (LPI), and there was no increase in radioactivity in LPI on stimulation with caerulein. These observations, taken together, argue strongly for a phospholipase C-catalyzed breakdown of PI followed by DG-lipase and argue against any significant involvement of phospholipase A2 in PI degradation in mouse pancreas. The formation of substantial amounts of free [14C]AA on stimulation supports the view that, among other things, the phosphoinositide effect in the exocrine pancreas serves to generate arachidonate (and its metabolites). The release of appreciable amounts of free fatty acids and glycerol shows that a significant portion of the DG formed as a result of caerulein-stimulated PI breakdown is not conserved in the phosphoinositide cycle.     

Platelet alpha-2 adrenergic receptor-mediated phosphoinositide responses in endogenous depression.

We have previously indicated that epinephrine stimulates phosphoinositide (PI) hydrolysis by activating alpha-2 adrenergic receptors in human platelets [H. Mori et. al. Life Sci., 741-747 44 (1989)]. This method involves the measurement of the accumulation of [3H]-inositol-1-phosphate (IP-1) as an index of PI hydrolysis; lithium is added to inhibit the metabolism of IP-1, thus giving an enhanced signal. In the present study, we assessed the platelet alpha-2 adrenergic receptor-mediated PI responses in samples from 15 unmedicated patients with endogenous depression and 15 age- and sex-matched control subjects. The responses to epinephrine (10 microM and 100 microM) in the depressed patients were significantly higher than those of the controls, whereas the basal values did not differ significantly. These results support the hypothesis that platelet alpha-2 adrenergic receptors may be supersensitive in patients with endogenous depression.     

Co-mitogenic tumor promoters suppress the phosphatidylinositol response in lymphocytes during early mitogenesis

The tumor-promoting agents 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phorbol-12,13-dibenzoate inhibited the increased accumulation of [32P]phosphatidylinositol (PI) induced in mouse spleen lymphocytes by mitogenic lectins in the presence of [32P]orthophosphate. Similar inhibition of [32P]PI levels by TPA was seen in human tonsil T-lymphocytes stimulated with phytohemagglutinin. Only co-mitogenic phorbol esters prevented the [32P]PI accumulation during early mitogenesis. No increased 32P-labelling due to mitogen or decreases due to TPA was observed when cells were equilibrated with [32P]orthophosphate for 24 h prior to stimulation with mitogen, from which it is concluded that the total concentrations of phosphatidylcholine (PC) and PI are unaffected by mitogen or co-mitogen. The [32P]PI elevation but not the [32P]PC elevation was proportional to T-cell mitogenic potency for the lectins concanavalin A, divalent succinyl concanavalin A and phytohemagglutinin, and was prevented in each case by 5 X 10(-8) M TPA. Escherichia coli lipopolysaccharide did not give increased 32P incorporation into PI or PC, and TPA had no effect on 32P labelled phospholipid levels in the presence of this B-cell mitogen. The results indicate that the phosphatidylinositol response is not an invariable correlate of T-cell mitogenesis by polyclonal mitogens.     

Plasma hypoxanthine and ammonia in humans during prolonged exercise

In this study we examined the time course of changes in the plasma concentration of oxypurines [hypoxanthine (Hx), xanthine and urate] during prolonged cycling to fatigue. Ten subjects with an estimated maximum oxygen uptake (VO2(max)) of 54 (range 47-67) ml x kg(-1) x min(-1) cycled at [mean (SEM)] 74 (2)% of VO2(max) until fatigue [79 (8) min]. Plasma levels of oxypurines increased during exercise, but the magnitude and the time course varied considerably between subjects. The plasma concentration of Hx ([Hx]) was 1.3 (0.3) micromol/l at rest and increased eight fold at fatigue. After 60 min of exercise plasma [Hx] was >10 micromol/l in four subjects, whereas in the remaining five subjects it was <5 micromol/l. The muscle contents of total adenine nucleotides (TAN = ATP+ADP+AMP) and inosine monophosphate (IMP) were measured before and after exercise in five subjects. Subjects with a high plasma [Hx] at fatigue also demonstrated a pronounced decrease in muscle TAN and increase in IMP. Plasma [Hx] after 60 min of exercise correlated significantly with plasma concentration of ammonia ([NH(3)], r = 0.90) and blood lactate (r = 0.66). Endurance, measured as time to fatigue, was inversely correlated to plasma [Hx] at 60 min (r = -0.68, P < 0.05) but not to either plasma [NH(3)] or blood lactate. It is concluded that during moderate-intensity exercise, plasma [Hx] increases, but to a variable extent between subjects. The present data suggest that plasma [Hx] is a marker of adenine nucleotide degradation and energetic stress during exercise. The potential use of plasma [Hx] to assess training status and to identify overtraining deserves further attention.     

STS-95 Space Experiments (plants and cell biology).

We report the outline of Space Experiments conducted on Space Shuttle (STS-95) launched in autumn of 1998. In this STS-95 mission, Japanese astronaut Dr. Chiaki Mukai achieved her 2nd space flight and conducted a part of 82 space experiments including Japanese experiments. US astronaut Senator John Glenn also achieved his second space flight, 36 years after his first space flight. Senator Glenn was a leader of the original (the first) 7 US astronauts and very famous in US because he succeeded US first orbital space flight around the earth. NASDA had started the project of space experiment using STS-95 at the summer of 1997, therefore we had only one year for the all preparation Yamashita, et al. Biological Sciences in Space, Vol.12 No.3(1998). Scientific results will be reported by investigators, therefore we report here how we had been developing the space experiment plan, on board operation procedure and ground operations including ground control experiments about four plant experiments and one cell biology experiment.     

U73122 inhibits Ca2+ oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells.

Stimulation of rat pancreatic acinar cells with low concentrations of phosphatidylinositol (PI)-linked secretagogues induces [Ca2+]i oscillations, without measurable changes in the formation of inositol 1,4,5-trisphosphate. Therefore, we tested U73122 a new phospholipase C inhibitor to determine if PI turnover is necessary for the generation of [Ca2+]i oscillations. In acini prelabeled with [3H]inositol, PI hydrolysis on stimulation with either cholecystokinin or carbachol was inhibited dose-dependently by U73122, with a maximal effect seen at 10 microM; the formation of inositol 1,4,5-trisphosphate, measured using a radioreceptor assay, was also similarly inhibited. By contrast secretin- or vasoactive intestinal peptide-stimulated production of cAMP was unaffected by 10 microM U73122. These studies indicate that U73122 is a relatively specific inhibitor of G-protein-mediated phospholipase C activation in pancreatic acini. In fura-2-loaded acini, U73122 inhibited the increases in [Ca2+]i stimulated by these high concentrations of secretagogues which can be demonstrated to elicit PI turnover. The [Ca2+]i signal generated by directly stimulating G-proteins with sodium fluoride was also inhibited by U73122; however, the [Ca2+]i rise induced by thapsigargin was unaffected. These data indicate that the mechanism of inhibition was distal to the occupation of cell surface receptors but did not involve an interference of Ca2+ metabolism in general. When [Ca2+]i oscillations were elicited by low concentrations of cholecystokinin or carbachol, U73122 rapidly inhibited the oscillating [Ca2+]i signal. In contrast, oscillations induced by an analogue of cholecystokinin, JMV-180, which does not stimulate changes in PI metabolism at any concentration, were unaffected. This indicates that cholecystokinin- and carbachol-induced oscillations are probably initiated by small, localized changes in PI metabolism, which are not readily detectable. However, the inability of U73122 to inhibit JMV-180-induced oscillations indicates that PI metabolism may not necessarily be a prerequisite for the generation of [Ca2+]i oscillations.     

C5a reduces formyl peptide-induced actin polymerization and phosphatidylinositol(3,4,5)trisphosphate formation, but not phosphatidylinositol (4,5) bisphosphate hydrolysis and superoxide production, in human neutrophils.

We investigated phospholipid signal transduction, calcium flux, O2- anion production and actin polymerization after stimulation with the C fragment and chemoattractant, C5a, and then determined how C5a pretreatment affected subsequent responses to formyl peptide in human neutrophils. We have previously demonstrated that the novel lipids, phosphatidylinositol trisphosphate (PIP3) and phosphatidylinositol(3,4)P2 (PI(3,4)P2), rise transiently in neutrophils after activation with formyl peptide. Furthermore, the rise in PIP3 parallels actin polymerization. In this study, neutrophils activated with C5a exhibited two distinct G protein-dependent signal pathways involving different phosphoinositides: 1) [32P]PI(4,5)P2 hydrolysis and [32P]PA production, and 2) the transient formation of D-3-phosphorylated phosphoinositides, [32P]PIP3 and [32P]PI(3,4)P2. When neutrophils were preincubated with C5a for 5 min before stimulation with formyl peptide, [32P]PI(4,5)P2 hydrolysis was unchanged, and [32P]PA production and O2- formation were slightly enhanced compared with controls stimulated with formyl peptide in the absence of C5a. In contrast, [32P]PIP3 production, right angle light scatter, and actin polymerization were all reduced 35 to 40%. Therefore, these data support the hypothesis that PIP3 plays a role in chemotaxis but not superoxide formation.     

Influence of osmolytes on in vivo glucose monitoring using optical coherence tomography

Diabetes mellitus and its complications are the third leading cause of death in the world, exceeded only by cardiovascular disease and cancer. Tighter monitoring and control of blood glucose could minimize complications associated with diabetes. Recently, optical coherence tomography (OCT) for noninvasive glucose monitoring was proposed and tested in vivo. The aim of this work was to investigate the influence of changes in blood glucose concentration ([glu]) and sodium concentration ([Na+]) on the OCT signal. We also investigated the influence of other important analytes on the sensitivity of glucose monitoring with OCT. The experiments were carried out in anesthetized female pigs. The OCT images were acquired continuously from skin, while [glu] and [Na+] were experimentally varied within their physiological ranges. Correlations of the OCT signal slope with [glu] and [Na+] were studied at different tissue depths. The tissue area probed with OCT was marked and cut for histological examination. The correlation of blood [glu] and [Na+] with the OCT signal slope was observed in separate tissue layers. On average, equimolar changes in [glu] produced 2.26 +/- 1.15 greater alterations of the OCT signal slope than changes in [Na+]. Variation of concentrations of other analytes did not influence the OCT signal slope. The influence of [Na+] on relative changes in the OCT signal slope was generally less than [glu]-induced changes. OCT is a promising method for noninvasive glucose monitoring because of its ability to track the influence of changing [glu] on individual tissue layers.     

Agonist-Induced, GTP-Dependent Phosphoinositide Hydrolysis in Postmortem Human Brain Membranes

Abstract: Membranes prepared from postmortem human brain were used to measure the activities of three components of the phosphoinositide second messenger system. [³H] Phosphatidylinositol ([³H] PI) hydrolysis was stimulated by directly activating phospholipase C with calcium, by activating guanine nucleotide-binding proteins (G proteins) with guanosine-5′-O-(3-thiotriphosphate) (GTPγS) or with AIF₄, and by receptors activated with several agonists (in the presence of GTPγS), including (in order of increasing magnitudes of responses) carbachol, pilocarpine, histamine, trans-1-aminocyclopentyl-1, 3-dicarboxylic acid (a selective excitatory amino acid metabotropic receptor agonist), serotonin, and ATP. G_q/11 was identified as the G protein most likely to mediate [³H] PI hydrolysis in human brain membranes based on the findings that this process was not impaired by pretreatment with pertussis toxin and it was inhibited by antibodies specific for the α-subunit of G_q/11 but not by antibodies for G_o or G₁₁. The effects of postmortem delay on [³H] PI hydrolysis were examined by studying tissues obtained 6–21 h postmortem. A slight increase in basal [³H] PI hydrolysis was associated with increased postmortem time, suggesting a slow loss of the normal inhibitory control of phospholipase C. GTPγS- stimulated [³H] PI hydrolysis was unaffected by postmortem times within this range, but carbachol-induced [³H] PI hydrolysis tended to decrease with increasing postmortem times. These results demonstrate that the entire phosphoinositide complex remains functional and experimentally detectable in postmortem human brain membranes. This method provides a means to study the function, regulation, effects of diseases, and responses to drugs of the phosphoinositide system in human brain.     

Phospholipase A2 in macrophage plasma membrane releases arachidonic acid from phosphatidylinositol

A high level of arachidonic acid release from [2-14C]arachidonylphosphatidylinositol (PI) was observed at neutral pH (6.0-7.0) in the presence of purified plasma membranes of guinea pig peritoneal macrophages. This activity was at least 10-fold higher than that with arachidonylphosphatidylcholine (PC) or phosphatidylethanolamine (PE) as substrate. The accumulation of [14C]diacylglycerol and [14C]phosphatidic acid was not detected at any time, and arachidonic acid release from [14C]arachidonyldiacylglycerol was not detectable either. The data suggest that arachidonic acid release from PI may not occur via the phospholipase C pathway. In this paper, we demonstrate the possibility that arachidonic acid release from PI at neutral pH in the macrophage plasma membrane is dependent on the action of phospholipase A2 (EC 3.1.1.4) -like activity. The maximum arachidonic acid release was dependent upon both pH and substrate. Particularly, the activity of arachidonic acid release from PI at neutral pH was very high compared with that from PC or PE. We suggest that phosphatidylinositol phospholipase A2 (EC 3.1.1.52) may play an important role in providing arachidonic acid for subsequent metabolic activity in the macrophages.     

Muscarinic Cholinergic Stimulation of Exogenous Phosphatidylinositol Hydrolysis Is Regulated by Guanine Nucleotides in Rabbit Brain Cortical Membranes

Rabbit brain cortical membranes, which have been extracted with 2 M KCl, hydrolyze exogenously added [3H]phosphatidylinositol [( 3H]PI) in a guanine nucleotide- and carbachol-dependent manner. Both oxotremorine-M and carbachol are full agonists with EC50 values of 8 and 73 microM, respectively. Pirenzepine and atropine inhibit carbachol-stimulated [3H]PI hydrolysis. The hydrolysis-resistant guanine nucleotide analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) is the most potent in supporting carbachol-stimulated hydrolysis of PI. There is no effect of carbachol in the absence of guanine nucleotides or in the presence of 100 microM adenosine 5'-O-(3-thiotriphosphate), adenosine-5'-(beta, gamma-imido)triphosphate, or sodium pyrophosphate. Guanylyl-5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] in the presence of carbachol also stimulates PI hydrolysis although much less than that seen with GTP gamma S. GDP and Gpp(NH)p are potent antagonists of the GTP gamma S-dependent carbachol response. Optimal stimulation by carbachol and GTP gamma S was observed at 0.3-1 microM free Ca2+ and 6 mM MgCl2. Limited trypsinization resulted in loss of receptor-regulated PI breakdown and a slight decrease in basal activity. These results demonstrate that phospholipase C hydrolysis of exogenous PI by rabbit cortical membranes may be stimulated by carbachol in a guanine nucleotide-dependent manner.     

Identification of 5-hydroxytryptamine binding substances from rat brain stem

Abstract— Godwin & Sneddon (1975) reported the binding of 5-hydroxy-[³H]tryptamine (5-HT) on a Sephadex LH-20 column to‘proteolipid material’extracted with n-butanol from rat brain stem. An examination of this‘proteolipid material’with TLC showed the main constituents to be cerebroside sulfate (CS), monophosphoinositide (PI), and diphosphoinositide. The elution profiles of [³H]5-HT incubated with purified CS or with a mixture of CS and PI were similar to that of the brain extract on the same column. Because the elution profile of the mixture of CS and PI was more similar to that of the brain extract, it was concluded that what was suggested to be a possible proteolipid‘5-HT receptor’was mainly two acidic lipids. The elution profile of [³H]5-HT incubated with purified PI, however, was similar to [³H]5-HT eluted alone. This suggested that either PI did not bind to 5-HT or that the PI-5-HT complex possesses different Chromatographie behavior than PI. To test this latter possibility, [¹⁴C]5-HT and [³H]PI were incubated then eluted on a Sephadex LH-20 column with a continuous gradient of increasing polarity. The gradient first eluted PI, then an apparent PI-5-HT complex, and finally 5-HT. This demonstrated that PI will bind to 5-HT on a Sephadex LH-20 column and that the PI-5-HT complex is probably more polar than PI.     

MIP-1alpha induces activation of phosphatidylinositol-3 kinase that associates with Pyk-2 and is necessary for B-cell migration.

The chemokine macrophage inflammatory protein-1 alpha [MIP-1alpha] causes migration of B cells and also induces changes in antibody secretion. However, the signal transduction pathways leading to these phenotypic changes remain undefined. We have identified a signal transduction pathway initiated by MIP-1alpha in B cells. Here we report that stimulation of tonsil B cells with MIP-1alpha induces phosphatidylinositol 3-kinase [PI3-K] activation. Kinase activity was transient with peak induction occurring within 2.5 to 5 min after stimulation and was dose-dependent. In addition stimulation with MIP-1alpha induces tyrosine phosphorylation of the proline-rich tyrosine kinase Pyk-2. Immunoprecipitation analysis showed a constitutive association between Pyk-2 and PI3-K and pretreatment of MIP-1alpha-stimulated B cells with wortmannin, a specific inhibitor of PI3-K, resulted in a loss of PI3-K activity. The PI3-K inhibitor wortmannin prevented B cells from migrating in response to MIP-1alpha. Hence, PI3-K and Pyk-2 seem to be components of a signal transduction pathway induced by stimulation of B cells with MIP-1alpha, and this pathway may play a role in B-cell migration.