Type I phosphatidylinositol-4,5-bisphosphate 4-phosphatase regulates stress-induced apoptosis

A recently discovered phosphatidylinositol monophosphate, phosphatidylinositol 5-phosphate (PtdIns-5-P), plays an important role in nuclear signaling by influencing p53-dependent apoptosis. It interacts with a plant homeodomain finger of inhibitor of growth protein-2, causing an increase in the acetylation and stability of p53. Here we show that type I phosphatidylinositol-4,5-bisphosphate 4-phosphatase (type I 4-phosphatase), an enzyme that dephosphorylates phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P(2)), forming PtdIns-5-P in vitro, can increase the cellular levels of PtdIns-5-P. When HeLa cells were treated with the DNA-damaging agents etoposide or doxorubicin, type I 4-phosphatase translocated to the nucleus and nuclear levels of PtdIns-5-P increased. This action resulted in increased p53 acetylation, which stabilized p53, leading to increased apoptosis. Overexpression of type I 4-phosphatase increased apoptosis, whereas RNAi of the enzyme diminished it. The half-life of p53 was shortened from 7 h to 1.8 h upon RNAi of type I 4-phosphatase. This enzyme therefore controls nuclear levels of PtdIns-5-P and thereby p53-dependent apoptosis.     

The phosphatidylinositol (PI)-5-phosphate 4-kinase type II enzyme controls insulin signaling by regulating PI-3,4,5-trisphosphate degradation

Phosphatidylinositol-5-phosphate (PI-5-P) is a newly identified phosphoinositide with characteristics of a signaling lipid but no known cellular function. PI-5-P levels are controlled by the type II PI-5-P 4-kinases (PIP4K IIs), a family of kinases that converts PI-5-P into phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). The PI-5-P pathway is an alternative route for PI-4,5-P2 synthesis as the bulk of this lipid is generated by the canonical pathway in which phosphatidylinositol-4-phosphate (PI-4-P) is the intermediate. Here we examined the effect of activation of the PI-5-P pathway on phosphoinositide 3-kinase (PI3K) signaling by expressing PIP4K II beta in cells that lack this enzyme. Although PIP4K II generates PI-4,5-P2, a substrate for PI3K, expression of this enzyme reduced rather than increased phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) levels in cells stimulated with insulin or cells expressing activated PI3K. This reduction in PI-3,4,5-P3 levels resulted in decreased activation of the downstream protein kinase, Akt/PKB. Consistent with these results, expression of IpgD, a bacterial phosphatase that converts PI-4,5-P2 to PI-5-P, resulted in Akt activation, and this effect was partially reversed by PIP4K II beta. PIP4K II beta expression did not impair insulin-dependent association of PI3K with insulin receptor substrate 1 (IRS1) but abbreviated Akt activation, indicating that PIP4K II regulates PI-3,4,5-P3 degradation rather than synthesis. These data support a model in which the PI-5-P pathway controls insulin signaling that leads to Akt activation by regulating a PI-3,4,5-P3 phosphatase.     

The protein deficient in Lowe syndrome is a phosphatidylinositol-4,5-bisphosphate 5-phosphatase.

Lowe syndrome, also known as oculocerebrorenal syndrome, is caused by mutations in the X chromosome-encoded OCRL gene. The OCRL protein is 51% identical to inositol polyphosphate 5-phosphatase II (5-phosphatase II) from human platelets over a span of 744 aa, suggesting that OCRL may be a similar enzyme. We engineered a construct of the OCRL cDNA that encodes amino acids homologous to the platelet 5-phosphatase for expression in baculovirus-infected Sf9 insect cells. This cDNA encodes aa 264-968 of the OCRL protein. The recombinant protein was found to catalyze the reactions also carried out by platelet 5-phosphatase II. Thus OCRL converts inositol 1,4,5-trisphosphate to inositol 1,4-bisphosphate, and it converts inositol 1,3,4,5-tetrakisphosphate to inositol 1,3,4-trisphosphate. Most important, the enzyme converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The relative ability of OCRL to catalyze the three reactions is different from that of 5-phosphatase II and from that of another 5-phosphatase isoenzyme from platelets, 5-phosphatase I. The recombinant OCRL protein hydrolyzes the phospholipid substrate 10- to 30-fold better than 5-phosphatase II, and 5-phosphatase I does not cleave the lipid at all. We also show that OCRL functions as a phosphatidylinositol 4,5-bisphosphate 5-phosphatase in OCRL-expressing Sf9 cells. These results suggest that OCRL is mainly a lipid phosphatase that may control cellular levels of a critical metabolite, phosphatidylinositol 4,5-bisphosphate. Deficiency of this enzyme apparently causes the protean manifestations of Lowe syndrome.     

Neurotensin stimulates polyphosphoinositide breakdown and prolactin release in anterior pituitary cells in culture

Biochemical events underlying neurotensin action at the pituitary were investigated in primary culture of anterior pituitary cells prelabeled with [³H]inositol. Incubation with the tridecapeptide produced a dose-dependent increase in the content of total [³H]inositol phosphates. The time-course showed that the effect was rapid and significant within l min. Fractionation of [³H]inositol phosphates revealed that inositol triphosphate (IP₃) and inositol diphosphate (IP₂) increased earlier than inositol monophosphate (IP₁) Structure/activity correlation studies demonstrated the specificity of neurotensin effect, showing that acetylneurotensin(8–13) displayed an action similar to the natural peptide, while neurotensin(1–6) hexapeptide did not exhibit any effect. The neurotensin analog [d-Trp¹¹]-neurotensin antagonized in a concentration-dependent manner the effect of neurotensin both on prolactin release and on [³H]inositol phosphate production. The loss of prelabeled phosphoinositides was also investigated. Phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P₂) and phosphatidylinositol-4-phosphate (PtdIns-4-P) decreased significantly within 15 s, while a slight decline in phosphatidylinositol (PtdIns) level appeared only l min after neurotensin addition.These results suggest that neurotensin action at the pituitary is mediated by the early hydrolysis of polyphosphoinositides, leading to the production of 1,2-diacylglycerol and inositol phosphates which may initiate intracellular processes responsible for hormonal release.     

Angiotensin-stimulated production of inositol trisphosphate isomers and rapid metabolism through inositol 4-monophosphate in adrenal glomerulosa cells.

The production and metabolism of inositol phosphates in rat adrenal glomerulosa cells prelabeled with [3H]inositol and stimulated with angiotensin II were analyzed by high-performance anion-exchange chromatography. Exposure to angiotensin II was accompanied by a rapid and substantial decrease in the phospholipid precursor, phosphatidylinositol (PtdIns) 4,5-bisphosphate with only a slight and transient increase in the level of the biologically active product, inositol 1,4,5-trisphosphate (Ins-1,4,5-P3), to a peak at about 5 sec. Inositol 1,3,4-trisphosphate (Ins-1,3,4-P3), the putative metabolite of Ins-1,4,5-P3, was also formed rapidly and maintained an elevated steady-state level during stimulation by angiotensin II. Inositol 1,4-bisphosphate (Ins-1,4-P2) exhibited a simultaneous and prominent increase that could not be accounted for solely by direct breakdown of PtdIns 4-phosphate, indicating that large amounts of Ins-1,4,5-P3 must also have been produced and metabolized. The rapid formation of a substantial amount of inositol 4-monophosphate (Ins-4-P), with no significant change in the level of inositol 1-monophosphate (Ins-1-P) during the first minute of stimulation, was a notable feature of the glomerulosa cell response to angiotensin II. These observations indicate that PtdIns-4,5-P2 catabolism in the angiotensin-stimulated glomerulosa cell initially proceeds via Ins-1,4,5-P3 through Ins-1,3,4-P3 and Ins-1,4-P2 to form Ins-4-P rather than Ins-1-P and that direct hydrolysis of PtdIns by phospholipase C does not occur during the initial phase of angiotensin action. In glomerulosa cells stimulated by angiotensin II in the presence of Li+, the progressive accumulation of both Ins-4-P, and after a short lag period, Ins-1-P indicated that dephosphorylation of both isomers was inhibited by Li+. The increase of Ins-P isomers in the presence of Li+ was associated with increased and progressive accumulation of Ins-1,4-P2 and Ins-1,3,4-P3 but not of Ins-1,4,5-P3. These data demonstrate that sustained and massive breakdown of PtdIns phosphates begins within seconds during cell activation by angiotensin II. The Ca2+-mobilizing metabolite, Ins-1,4,5-P3, is rapidly converted to Ins-1,3,4-P3 and degraded through Ins-1,4-P2 and Ins-4-P, in contrast to the previous view that conversion to Ins-1-P is the major route of PtdIns 4,5-bisphosphate metabolism.     

Mass determination of polyphosphoinositides and inositol triphosphate in rat adrenal glomerulosa cells with a microspectrophotometric method

A method is described for the assay of subnanogram amounts of phosphorus in phospholipids and organic phosphates. The formation of a complex with a high molar absorption coefficient at 600 nm when malachite green is added to phosphomolybdate at low pH and the adaptation of a microspectrophotometer to quantify the color in 10 microliters solution have made it possible for a dose-response curve from 0.1-1.2 ng phosphorus to be developed. The method has been applied to the assay of phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns 4-P), phosphatidylinositol-4,5-diphosphate (PtdIns 4,5-P2), and inositol-1,4,5-triphosphate (Ins 1,4,5-P3) in rat adrenal glomerulosa cells after stimulation with angiotensin II (AII), K+, and ACTH for 0, 2, 4, 6, 8, 10, 12, 15, and 60 sec. A control (nonstimulated) sample was incubated concomitantly for every time period. Nonstimulated cell levels (mean +/- SEM; n = 216) were: PtdIns, 577 +/- 6.4; PtdIns 4-P, 183 +/- 3.1; PtdIns 4,5-P2, 59 +/- 1.8; and Ins 1,4,5-P3, 94 +/- 1.3 pmol/incubate. Maximum increase in levels of PtdIns, PtdIns 4-P, PtdIns 4,5-P2, and Ins 1,4,5-P3 above control values was obtained after 8 sec with AII (10(-8) M) and after 6 sec with K+ (8.7 mM) stimulation. The values (picomoles per 2 X 10(5) cell incubate; n = 4) were: PtdIns, 808 +/- 28; PtdIns 4-P, 263 +/- 20; PtdIns 4,5-P2, 112 +/- 10; and Ins 1,4,5-P3, 136 +/- 4 for AII stimulation, and PtdIns, 925 +/- 76, PtdIns 4-P, 308 +/- 11; PtdIns 4,5-P2 146 +/- 28; and Ins 1,4,5-P3, 149 +/- 5 for K+ stimulation. No increase above control levels could be found at any incubation time after ACTH stimulation. Thus, both AII and K+ stimulate a short-lived increase in the mass of several elements of the phosphatidylinositol pathway. The discrepancy between these mass determinations and isotope study suggests that only some, but not all, pools are labeled by currently available techniques.     

Inhibition of Phosphatidylinositol 4-phosphate 5-kinase by Cyclic AMP in Human Platelets

The effects of cyclic AMP (cAMP) on phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) synthesis were examined in human platelets. In (32)P-prelabeled intact platelets, although the level of [(32)P]phosphatidylinositol 4-phosphate (PI 4-P) was increased by forskolin and prostaglandin-I(2) (PGI(2)), the formation of [(32)P]PI 4,5-P(2) time-and concentration-dependently decreased, suggesting inhibition of phosphatidylinositol 4-phosphate 5-kinase (PI 4-P 5-kinase). In saponin-permeabilized platelets, formation of PI 4-P and PI 4,5-P(2) can be measured by utilizing [γ-(32)P] ATP. In this system, PGI(2) and cAMP inhibited the generation of [(32)P)PI 4,5-P(2). The PI 4-P 5-kinase activity was mostly located in the platelet membrane fraction and was inhibited by cAMP; H-8 and H-89, inhibitors of cAMP-dependent protein kinase (PKA), abolished this inhibitory effect, suggesting that cAMP exerted its action on PI 4-P 5-kinase via PKA. Adenosine, which is reported to directly inhibit phosphatidylinositol 4-kinase (PI 4-kinase) in some types of cells, had no effect on platelet membrane PI 4-P 5-kinase activity. In dbc AMP-pretreated membranes, PI 4-P 5-kinase activity was lower than that of control membranes. The involvement of PKA with the inhibitory action of cAMP in PI 4-P 5-kinase activity was further confirmed using the catalytic subunit of PKA. The synthesis of [(32)P] PI 4,5-P(2) in permeabilized platelets and the specific activity of partially purified PI 4-P 5-kinase were decreased by incubation with the PKA catalytic subunit. The present results indicate that the cAMP-PKA system inhibits PI 4-P 5-kinase activity, leading to decreased formation of PI 4,5-P(2) in human platelets.     

Phosphatidylinositol phosphate kinase type I gamma regulates dynamics of large dense-core vesicle fusion

Phosphatidylinositol-4,5-bisphosphate was proposed to be an important regulator of large dense-core vesicle exocytosis from neuroendocrine tissues. Here, we have examined the kinetics of secretion in chromaffin cells from mice lacking phosphatidylinositol phosphate kinase type I gamma, the major neuronal phosphatidylinositol-4-phosphate 5-kinase. Absence of this enzyme caused a reduction of the readily releasable vesicle pool and its refilling rate, with a small increase in morphologically docked vesicles, indicating a defect in vesicle priming. Furthermore, amperometry revealed a delay in fusion pore expansion. These results provide direct genetic evidence for a key role of phosphatidylinositol-4,5-bisphosphate synthesis in the regulation of large dense-core vesicle fusion dynamics.     

Inhibition of Phosphatidylinositol 4-phosphate 5-kinase by Cyclic AMP in Human Platelets

The effects of cyclic AMP (cAMP) on phosphatidylinositol 4,5-bisphosphate (PI 4,5-P₂) synthesis were examined in human platelets. In ³²P-prelabeled intact platelets, although the level of [³²P]phosphatidylinositol 4-phosphate (PI 4-P) was increased by forskolin and prostaglandin-I₂ (PGI₂), the formation of [³²P]PI 4,5-P₂ time-and concentration-dependently decreased, suggesting inhibition of phosphatidylinositol 4-phosphate 5-kinase (PI 4-P 5-kinase). In saponin-permeabilized platelets, formation of PI 4-P and PI 4,5-P₂ can be measured by utilizing [γ-³²P] ATP. In this system, PGI₂ and cAMP inhibited the generation of [³²P)PI 4,5-P₂. The PI 4-P 5-kinase activity was mostly located in the platelet membrane fraction and was inhibited by cAMP; H-8 and H-89, inhibitors of cAMP-dependent protein kinase (PKA), abolished this inhibitory effect, suggesting that cAMP exerted its action on PI 4-P 5-kinase via PKA. Adenosine, which is reported to directly inhibit phosphatidylinositol 4-kinase (PI 4-kinase) in some types of cells, had no effect on platelet membrane PI 4-P 5-kinase activity. In dbc AMP-pretreated membranes, PI 4-P 5-kinase activity was lower than that of control membranes. The involvement of PKA with the inhibitory action of cAMP in PI 4-P 5-kinase activity was further confirmed using the catalytic subunit of PKA. The synthesis of [³²P] PI 4,5-P₂ in permeabilized platelets and the specific activity of partially purified PI 4-P 5-kinase were decreased by incubation with the PKA catalytic subunit. The present results indicate that the cAMP-PKA system inhibits PI 4-P 5-kinase activity, leading to decreased formation of PI 4,5-P₂ in human platelets.     

Gonadotropin-releasing hormone-stimulated phosphoinositide hydrolysis in the anterior pituitary. Modulation by protein kinase C but not by cyclic nucleotides

Gonadotropin-releasing hormone (GnRH) produces a rapid and concentration-dependent hydrolysis of polyphosphoinositides in rat anterior pituitary cells in culture. Evaluation of the action of the decapeptide by measurement of [3H]-inositol phosphates and of prelabeled phosphoinositides demonstrated an effect on phosphatidylinositol-4,5-bis-phosphate and phosphatidylinositol-4-phosphate earlier than on phosphatidylinositol. The receptor antagonist [D-pGlu1,D-Phe2,D-Trp3,6]-luteinizing hormone-releasing hormone blocked the effect of GnRH on [3H]-inositol phosphate production. Protein kinase C activators attenuated GnRH-induced phosphoinositide hydrolysis, while neither cyclic AMP analogs nor cyclic GMP analogs were effective. These results indicate that phosphoinositide hydrolysis represents an important postreceptor transducing mechanism for GnRH action at the gonadotroph and that protein kinase C (but not cyclic nucleotides) may exert a negative feedback control on GnRH receptor-coupling mechanisms.     

Src homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1) negatively regulates TLR4-mediated LPS response primarily through a phosphatase activity- and PI-3K-independent mechanism

Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1) plays important roles in negatively regulating the activation of immune cells primarily via the phosphoinositide 3-kinase (PI-3K) pathway by catalyzing the PI-3K product PtdIns-3,4,5P3 (phosphatidylinositol-3,4,5-triphosphate) into PtdIns-3,4P2. However, the role of SHIP1 in Toll-like receptor 4 (TLR4)-mediated lipopolysaccharide (LPS) response remains unclear. Here we demonstrate that SHIP1 negatively regulates LPS-induced inflammatory response via both phosphatase activity-dependent and -independent mechanisms in macrophages. SHIP1 becomes tyrosine phosphorylated and up-regulated upon LPS stimulation in RAW264.7 macrophages. SHIP1-specific RNA-interfering and SHIP1 overexpression experiments demonstrate that SHIP1 inhibits LPS-induced tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) production by negatively regulating the LPS-induced combination between TLR4 and myeloid differentiation factor 88 (MyD88); activation of Ras (p21(ras) protein), PI-3K, extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun NH2-terminal kinase (JNK); and degradation of IkappaB-alpha. SHIP1 also significantly inhibits LPS-induced mitogen-activated protein kinase (MAPK) activation in TLR4-reconstitited COS7 cells. Although SHIP1-mediated inhibition of PI-3K is dependent on its phosphatase activity, phosphatase activity-disrupted mutant SHIP1 remains inhibitory to LPS-induced TNF-alpha production. Neither disrupting phosphatase activity nor using the PI-3K pathway inhibitor LY294002 or wortmannin could significantly block SHIP1-mediated inhibition of LPS-induced ERK1/2, p38, and JNK activation and TNF-alpha production, demonstrating that SHIP1 inhibits LPS-induced activation of MAPKs and cytokine production primarily by a phosphatase activity- and PI-3K-independent mechanism.     

Attenuation of anterior pituitary phosphoinositide phosphorylase activity by the D2 dopamine receptor

We have examined the influences of dopamine and the D2 receptor agonist bromocriptine on phosphoinositide metabolism in primary cultures of rat anterior pituitary cells, monitoring changes in the levels of phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate [PtdIns(4)P], and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Basal incorporation of [3H]inositol ([3H]Ins) into phosphoinositides was progressive, and radioisotopic equilibrium was attained in all three species within 48 h. The inclusion of dopamine or bromocriptine in the incubation medium promoted concentration-dependent reductions in the rate, but not the magnitude, of phosphoinositide radiolabeling. The onset of this effect was rapid; inhibition of [3H]Ins incorporation by dopamine (500 nM) and bromocriptine (100 nM) could be detected within 2 h. This treatment also produced a comparable reduction in the incorporation of [32P]orthophosphate into PtdIns(4,5)P2. In extended time-course studies, bromocriptine dramatically retarded the radiolabeling of PtdIns(4)P and PtdIns(4,5)P2, and apparent equilibria in these species were attained only after 96 h. We also assessed the ability of dopamine to modify the concentration-response characteristics of [3H]Ins-labeled inositol phosphate ([3H]InsPx) production by TRH, angiotensin II (AII), neurotensin (NTS), bombesin (BBS), and vasoactive intestinal polypeptide (VIP). Neither dopamine nor bromocriptine altered the rate or magnitude of TRH-, AII-, NTS-, or BBS-related InsPx generation. VIP was completely ineffective in stimulating InsPx generation. PRL release was significantly reduced in all dopamine-treated groups. That the InsPx concentration-response relationships for each of these peptides remained unimpaired by exposure to dopamine or bromocriptine extends our previous observation that the phosphoinositide-specific phospholipase-C is insensitive to dopaminergic tone. Consistent with our earlier findings, these data indicate that activation of the D2 dopamine receptor attenuates the activity of mechanisms associated with the serial phosphorylations of PtdIns and PtdIns(4)P, reactions that give rise to PtdIns(4)P and PtdIns(4,5)P2, respectively. It is our conclusion that dopamine, in addition to its other actions, attenuates the phosphorylation, rather than the hydrolysis, of anterior pituitary phosphoinositide. This attenuation appears to be mediated by an inhibitory coupling of the D2 receptor with the phosphoryltransferase activities that catalyze PtdIns(4)P and PtdIns(4,5)P2 formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of sodium intake on phosphoinositides and inositol trisphosphate response to angiotensin II, K+ and ACTH in rat glomerulosa cells

To assess the impact of sodium intake on the adrenal phosphoinositide system, rats were maintained on a low or normal salt diet for 5 days, and glomerulosa cell preparations (2 x 10(5) cells) were stimulated by angiotensin II (AII; 10 nmol/l), potassium (K+; 8.7 mmol/l) or ACTH (0.1 nmol/l) for 0, 2, 4, 6, 12, 15 and 60 s. Levels of phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns 4-P), phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) and inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) + inositol 1,3,4-trisphosphate (Ins 1,3,4-P3) were assayed by a microspectrophotometric procedure. Non-stimulated levels of PtdIns, PtdIns 4-P, PtdIns 4,5-P2 and Ins 1,4,5-P3 (+ Ins 1,3,4-P3) (means +/- S.E.M.; n = 36) in cells from rats on the low Na+ intake were 580 +/- 6.5, 187 +/- 2.6, 82 +/- 3 and 95 +/- 1.2 pmol per incubate respectively, indistinguishable from those observed in rats on a normal Na+ intake, except for the significantly (P less than 0.025) greater PtdIns 4,5-P2 level. In response to AII stimulation, all four compounds showed an earlier and greater peak response when cells were obtained from animals on a low rather than a high sodium intake. All values has returned to control levels by 12-15 s, regardless of the level of sodium intake. In contrast, with K+ stimulation there were no differences in the peak response of cells from rats on the two dietary intakes, but there was a shift of the peak to a longer time-interval (6 versus 8 s) in animals maintained on a low sodium intake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin F2 alpha stimulates inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate formation in bovine luteal cells.

The present studies were conducted to further evaluate inositol phosphate formation and metabolism in prostaglandin F2 alpha (PGF2 alpha)-stimulated bovine luteal cells. Corpora lutea were dispersed with collagenase, and luteal cells were prelabeled for 3 h with [3H]inositol. Inositol phosphates produced in response to PGF2 alpha were analyzed by ion exchange column chromatography and HPLC. Time-course experiments revealed that significant increases in inositol trisphosphate (InsP3) were apparent within 5 sec of incubation with PGF2 alpha. Increases in inositol bisphosphate (InsP2) were also apparent within 5 sec. InsP1 and InsP4 were observed after a short (5-sec) lag period. HPLC revealed that PGF2 alpha provoked rapid (5 sec) increases in inositol 1,4,5-trisphosphate (Ins 1,4,5-P3), which was rapidly converted to inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4) and inositol 1,3,4-trisphosphate (Ins 1,3,4-P3). The primary inositol bisphosphate isomer present in PGF2 alpha-stimulated bovine luteal cells was inositol 1,4-bisphosphate (Ins 1,4-P2), with lesser amounts of Ins 1,3-P2. Inositol monophosphates were also increased. These findings were confirmed in studies in which the metabolism of purified [3H]Ins 1,4,5-P3 was followed temporally in saponin-permeabilized bovine luteal cells. Additional studies demonstrated the presence of an enzyme, InsP3-3-kinase, in the cytosolic fraction of bovine corpora lutea. InsP3-3-kinase phosphorylated Ins 1,4,5-P3 to form Ins 1,3,4,5-P4. The activity of InsP3-3-kinase was calcium dependent and was enhanced by calmodulin at low calcium concentrations. Calmidazolium, a calmodulin inhibitor, reduced InsP3-3-kinase activity in a concentration-dependent manner. These results demonstrate the presence of multiple polyphosphorylated inositol phosphates in PGF2 alpha-stimulated bovine luteal cells. The isomers were formed via the action of a specific calcium/calmodulin-regulated kinase (InsP3-3-kinase), which phosphorylated Ins 1,4,5-P3 during agonist-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. These data suggest that the inositol tris/tetrakisphosphate pathway is an important sequelae to PGF2 alpha-stimulated inositol phospholipid hydrolysis, and that the pathway may be activated during agonist-mediated calcium mobilization.     

Role for a novel signaling intermediate, phosphatidylinositol 5-phosphate, in insulin-regulated F-actin stress fiber breakdown and GLUT4 translocation

The cellular functions and regulation of phosphatidylinositol (PtdIns) 5-phosphate (5-P), the newest addition to the family of phosphoinositides (PIs), are still elusive. Here we have examined a plausible role of PtdIns 5-P as a signaling intermediate in acute insulin action. A wortmannin-insensitive transient increase of PtdIns 5-P mass levels that peaked at 10 min, and declined 20-30 min after insulin stimulation, was observed in both Chinese hamster ovary (CHO)-T cells stably expressing the insulin receptor and 3T3-L1 adipocytes. Similarly to insulin, found to induce a rapid disassembly of Texas-Red phalloidin-labeled actin stress fibers in CHO-T cells, microinjected PtdIns 5-P, but not other PIs, decreased the number and length of F-actin stress fibers in this cell type to a magnitude seen in response to insulin. Likewise, increases of PtdIns 5-P by ectopic expression of the PtdIns 5-P-producing enzyme PIKfyve yielded a similar effect. As with insulin, the PtdIns 5-P-induced loss of actin stress fibers was independent of PI 3-kinase activation. Furthermore, sequestration of functional PtdIns 5-P, either by ectopic expression of 3xPHD domains that bind selectively PtdIns 5-P or by microinjecting the GST-3xPHD fusion peptide, abrogated insulin-induced F-actin stress fiber disassembly in CHO-T cells. In 3T3-L1 adipocytes, microinjected PtdIns 5-P, but not other PIs, partially mimicked insulin's effect of translocating enhanced green fluorescent protein-GLUT4 to the cell surface. Conversely, insulin-induced myc-GLUT4 vesicle dynamics was arrested in the presence of coexpressed enhanced green fluorescent protein-3xPHD. Involvement of PIKfyve membrane recruitment, but not activation, and/or a decrease in PtdIns 4,5-bisphosphate levels are likely to be among the mechanisms underlying the insulin-induced PtdIns 5-P increase. Together, these results identify PtdIns 5-P as a novel key intermediate for insulin signaling in F-actin remodeling and GLUT4 translocation.     

Independent signal-transduction pathways for vanadate and for insulin in the activation of glycogen synthase and glycogenesis in rat adipocytes

The activating effect of vanadate on glycogenesis and on glycogen synthase (uridine diphosphate-glucose-glycogen glucosyl transferase) activity was studied in rat adipocytes and compared with that of insulin. Using several approaches and specific blockers, we found that vanadate and insulin resemble each other, in the activation of glycogen synthase, in several aspects: both require nonarrested protein phosphatase 1 activity; they are equally suppressed by conditions that elevate cAMP-levels; and both depend on the activation of phosphatidylinositol-3 kinase. The basic differences between them are as follows: 1) vanadate promotes glycogenesis through the activation of a cytosolic protein tyrosine kinase, in an insulin-receptor-independent manner; 2) vanadate elevates glucose-6-phosphate (G-6-P) to a higher level than insulin; 3) vanadate-activated glycogenesis is accompanied by an increase in the cellular content of immunoreactive glycogen synthase, an effect less noticeable with insulin; 4) adipose glucose-6-phosphatase is inhibited by vanadate (dose for 50% inhibition, IC50 = 7 +/- 0.7 microM) but not by insulin. We have concluded that insulin and vanadate activate glycogenesis through a phosphatidylinositol-3 kinase and dephosphorylation-dependent mechanism. Vanadate, however, uses a receptor-independent pathway and is superior to insulin in elevating the level of G-6-P, a key metabolite for activating glycogen synthase. This is attributed to the combined effect of vanadate in enhancing glucose entry and in inhibiting dephosphorylation of endogenously formed G-6-P. The latter effect is not exerted by insulin.     

Hexose metabolism in pancreatic islets. Absence of glucose-6-phosphatase in rat islet cells

Homogenates of either rat or mouse pancreatic islets, pure rat B cells or insulin-producing cells of the RINm5F line catalyzed the hydrolysis of D-glucose-6-phosphate. Relative to protein content, the enzymic activity, which was mainly associated with particulate rather than soluble subcellular material, was much lower in endocrine pancreatic cells than in liver. The rat islet enzyme differed from liver glucose-6-phosphate by its lower affinity for D-glucose-6-phosphate, its lower pH optimum, its greater relative efficiency towards L-glycerol-3-phosphate as distinct from D-glucose-6-phosphate, its restricted lability during exposure to pH 5.0, its inability to act as a glucose-6-phosphate:glucose phosphotransferase, and its insensitivity to inhibition by D-glucose. It is concluded that rat islet cells are virtually devoid of true glucose-6-phosphatase activity.     

Regulation of Phosphatidylinositol 4-phosphate 5-kinase by Protein Kinase C in Human Platelet Membranes

Phorbol myristate acetate (PM A) increased the formation of |(32)P | PI 4,5-P, in (32)P-prelabeled human platelet. In saponin-permeabilized platelets, in which (32)P from exogenous |γ-(32)P| ATP was incorporated into PI 4-P and PI 4,5-P(2), addition of 10 nM PMA resulted in increased formation of |(32)P|PI 4,5-P(2) and |(32)P|PI 4-P. In order to distinguish whether increased [(32)P]PI 4,5-P(2) formation by PMA reflected merely an increase of [(32)P]PI 4-P, the substrate for PI 4-P 5-kinase, or activation of PI 4-P Skinase, we examined the membrane fraction in which most of the kinase activity was located. Although PMA itself did not affect the PI 4-P 5-kinase activity in the control membranes, the kinase activity was increased nearly 2-fold in membranes pretreated with 10 nM PMA but not 4α-phorbol didecanoate which does not activate protein kinase C (PKC). These results suggested that membrane PI 4-P 5-kinase activity was stimulated by the activation of PKC. However, 100 nM PMA did not stimulate [(32)P]PI 4,s-P(2) formation in saponin-permeabilized platelets, and the PI 4-P 5-kinase activity in membranes from platelets pretreated with 100 nM PMA was almost the same as that in control membranes. This can be explained by product inhibition, since PI 4,5-P(2) inhibited concentration-dependently the membrane PI 4-P 5-kinase activity. The Ca(2+) -dependent PKC fraction partially purified from the platelet cytosol stimulated the membrane PI 4-P 5-kinase activity, whereas the Ca(2)'-independent PKC fraction inhibited the kinase activity. Taken together, the present results suggest that the platelet membrane PI 4-P 5-kinase activity is stimulated by Ca(2+) -dependent PKC (cPKC) and is negatively regulated by PI 4,s-p(2) and Ca(2+) -independent PKC(nPKC).     

Low level of sarcolemmal phosphatidylinositol 4,5-bisphosphate in cardiomyopathic hamster (UM-X7.1) heart

OBJECTIVE: Phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P(2)) is not only a precursor to inositol 1,4,5-trisphosphate (Ins 1,4, 5-P(3)) and sn-1,2 diacylglycerol, but also essential for the function of several membrane proteins. The aim of this study was to evaluate the changes in the level of this phospholipid in the cell plasma membrane (sarcolemma, SL) of cardiomyopathic hamster (CMPH) heart. METHODS: We examined the cardiac SL PtdIns 4,5-P(2) mass and the activities of the enzymes responsible for its synthesis and hydrolysis in 250-day-old UM-X7.1 CMPH at a severe stage of congestive heart failure (CHF) and in age-matched controls (Syrian Golden hamsters). RESULTS: The SL PtdIns 4,5-P(2) mass in CMPH was reduced by 72% of the control value. The activities of PtdIns 4 kinase and PtdIns 4-P 5 kinase were depressed by 69 and 50% of control values, respectively. Although, the total phospholipase C (PLC) activity was moderately, although significantly, decreased (by 18% of control), PLCdelta(1) isoenzyme activity in the SL membrane was elevated, with a concomitant increase in its protein content, whereas PLCbeta(1) and gamma(1) isoenzyme activities were depressed despite the increase in their protein levels. A 2-fold increase in the Ins 1,4,5-P(3) concentration in the cytosol of the failing heart of CMPH was also observed. CONCLUSIONS: Reduced SL level of PtdIns 4, 5-P(2) may severely jeopardize cardiac cell function in this hamster model of CHF. In addition, the profound changes in the profile of heart SL PLC isoenzyme could alter the complex second messenger responses of these isoenzymes, and elevated Ins 1,4,5-P(3) levels may contribute to intracellular Ca(2+) overload in the failing cardiomyocyte.     

Phosphatidylinositol 4,5-bisphosphate turnover is transient while phosphatidylinositol turnover is persistent in thyrotropin-releasing hormone-stimulated rat pituitary cells.

Stimulated inositolphospholipid turnover has been proposed to be initiated and sustained by hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], which may be replenished by an enhanced flux of phosphatidylinositol (PtdIns) to PtdIns 4-phosphate (PtdIns4P) to PtdIns(4,5)P2. To determine whether there is continued hydrolysis and resynthesis of PtdIns(4,5)P2 in rat pituitary cells (GH3 cells) during stimulation by thyrotropin-releasing hormone (TRH), we investigated the turnover kinetics of the inositolphospholipids and of phosphatidic acid (PtdOH). In cells incubated with 32Pi for 1 min, TRH rapidly and persistently (for at least 30 min) enhanced the rate of 32P-labeling of PtdOH. After a lag time of 1 min, TRH markedly and persistently increased 32P-labeling of PtdIns also. In contrast, TRH caused only a transient increase in 32P-labeling of PtdIns(4,5)P2 that lasted less than 2 min. There was no rapid (before 10 min) effect of TRH on 32P-labeling of PtdIns4P. By 2 min of TRH stimulation, specific 32P radioactivity in PtdOH increased from 3.6% (control) of that in the gamma-phosphate of ATP to 15%; in PtdIns, from 0.07% to 1.3%; and in PtdIns(4,5)P2, from 3.8% to 5.4% (specific 32P radioactivity in PtdIns4P was 1.7% of that in ATP in control and TRH-stimulated cells). In cells exposed to TRH for 4 min and then to 32Pi, 32P-labeling of PtdOH and PtdIns increased, but that of PtdIns(4,5)P2 was not affected. Last, persistent turnover of PtdOH and PtdIns was not caused by initial hydrolysis of PtdIns(4,5)P2 because the turnover of PtdOH and PtdIns could be terminated by displacement of TRH from its receptor by chlordiazepoxide and restarted by reoccupying the receptors with TRH. These data demonstrate that turnover of PtdIns(4,5)P2 is stimulated only transiently, whereas turnover of PtdIns and PtdOH is stimulated persistently by TRH in GH3 cells. Hence, inositolphospholipid turnover in GH3 cells does not occur via continued hydrolysis of PtdIns(4,5)P2 accompanied by enhanced flux of PtdIns to PtdIns4P to PtdIns(4,5)P2, but there is direct and persistent hydrolysis of PtdIns. The dissociation of these actions suggests that there are separate mechanisms involved in coupling TRH-receptor complexes to stimulation of PtdIns(4,5)P2 and PtdIns hydrolysis in GH3 cells.