Inhibition of phosphatidylinositol kinase in vascular smooth muscle membranes by adenosine and related compounds

Adenosine and 5'-chloro-5'-deoxyadenosine inhibited the phosphorylation of phosphatidylinositol in membranes prepared from aortic smooth muscle. The nucleosides did not affect the breakdown of phosphatidylinositol-4-phosphate. Under certain conditions, the membrane-bound phosphatidylinositol kinase phosphorylated exogenous phosphatidylinositol. The nucleosides inhibited the enzyme competitively with respect to magnesium-ATP and non-competitively with respect to phosphatidylinositol. Adenosine analogs modified in the ribose moiety were inhibitors with potencies comparable to that of adenosine, whereas adenine nucleotides and purine-modified adenosine analogs were much weaker inhibitors. Density gradient fractionation studies showed that phosphatidylinositol kinase is primarily associated with the sarcoplasmic reticulum. Vascular smooth muscle contraction is associated with increased phosphatidylinositol turnover. Inhibition of phosphatidylinositol kinase by intracellular adenosine may, therefore, be a factor involved in regulating vasodilation.     

Effects of heparin and other chemicals on phosphorylation of phosphatidylinositol in pig erythrocyte membrane

In the presence of Mg2+, the pig erythrocyte membranes were incubated with [gamma-32P]ATP at 30 degrees C for 3 min to study the effects of some chemicals on the phosphorylation of phosphatidylinositol. The incubations were stopped by the addition of chloroform/methanol (2:1, vol/vol) and the phospholipids were extracted with acid chloroform/methanol and separated on silica gel TLC plates. [gamma-32P] phosphatidyl-inositol-4-phosphate was quantitated by scintillation counting and autoradiography. The results indicated that heparin and neomycin inhibited the phosphorylation of phosphatidylinositol in a concentration-dependent manner, while dimethylsulfoxide stimulated the phosphorylation at low concentration (less than 10%), but inhibited at high concentration (greater than 10%). Hexachlorocyclohexane stimulated the phosphorylation within a certain limit of concentration up to 6.4 micrograms/ml. Li2SO4 (10 mmol/L), EGTA (100 mumol/L) and theophylline (100 mumol/L) had no significant effects.     

The development of phosphatidylinositol ether lipid analogues as inhibitors of the serine/threonine kinase, Akt

The serine/threonine kinase Akt is a component of the phosphatidylinositol 3'-kinase/Akt signal transduction pathway that is activated by receptor tyrosine kinases, activated Ras and integrins. As Akt regulates many processes crucial to carcinogenesis, and Akt activation has been observed in human cancers, intense efforts are underway to develop Akt inhibitors as cancer therapeutics. Towards this aim, phosphatidylinositol ether lipid analogues (PIAs), which are structurally similar to the products of phosphatidylinositol 3'-kinase, have been synthesised. PIAs inhibit Akt translocation, phosphorylation and kinase activity. Furthermore, they selectively induce apoptosis in cancer cell lines that depend on Akt for survival. This review will trace the development of PIAs, cover the biological activities of PIAs and discuss future steps and challenges in their development.     

The development of phosphatidylinositol ether lipid analogues as inhibitors of the serine/threonine kinase,Akt

The serine/threonine kinase Akt is a component of the phosphatidylinositol 3′-kinase/Akt signal transduction pathway that is activated by receptor tyrosine kinases, activated Ras and integrins. As Akt regulates many processes crucial to carcinogenesis, and Akt activation has been observed in human cancers, intense efforts are underway to develop Akt inhibitors as cancer therapeutics. Towards this aim, phosphatidylinositol ether lipid analogues (PIAs), which are structurally similar to the products of phosphatidylinositol 3′-kinase, have been synthesised. PIAs inhibit Akt translocation, phosphorylation and kinase activity. Furthermore, they selectively induce apoptosis in cancer cell lines that depend on Akt for survival. This review will trace the development of PIAs, cover the biological activities of PIAs and discuss future steps and challenges in their development.     

Activation of phospholipase D in porcine tracheal smooth muscle: role of phosphatidylinositol 3-kinase and RhoA activation.

Muscarinic receptor agonists transiently activate phospholipase D in tracheal smooth muscle. Muscarinic activation of phospholipase D in this tissue is dependent on activation of protein kinase C and an unidentified pathway that is not protein kinase C dependent. Cholinergic agents have also been shown to activate phospholipase D by pathways linked to the small G protein, RhoA. This study explores the relationship between muscarinic activation of phophatidylinositol 3-kinase and activation of RhoA, and examines whether phospholipase D activation is dependent on either pathway in tracheal smooth muscle. Wortmannin or 2-(4-morphonyl)-8-phenyl-4H-1-benzopyran-4-one (LY-294002), putative specific inhibitors of phophatidylinositol 3-kinase, significantly inhibit acetylcholine-induced formation of phosphatidylethanol and also block acetylcholine-induced translocation of RhoA to the membrane. In previous experiments calphostin C, a protein kinase C inhibitor, partially inhibited both acetylcholine-induced and phorbol-12-myristate-13-acetate (PMA)-induced phosphatidylethanol formation. In the present study calphostin C did not block acetylcholine-induced RhoA translocation to the membrane. However, the Rho kinase inhibitor, N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632), significantly inhibited acetylcholine-induced phosphatidylethanol formation, but had no effect on activation of phospholipase D by PMA. Acetylcholine treatment also stimulated the phosphorylation of the 110-kDa subunit of phosphatidylinositol 3-kinase. Phosphorylation of phosphatidylinositol 3-kinase 110-kDa subunit could be blocked by wortmannin in a concentration-dependent manner, and acetylcholine-induced phosphatidylinositol 3-kinase activity was significantly inhibited by wortmannin. LY-294002 also inhibited acetylcholine-induced phosphorylation of 110-kDa subunit and activation of phosphatidylinositol 3-kinase. These results suggest that acetylcholine stimulation translocates RhoA to the membrane by a phosphatidylinositol 3-kinase-dependent mechanism and acetylcholine-induced phospholipase D stimulation is at least partly mediated via phosphatidylinositol 3-kinase, however, protein kinase C appears to activate phospholipase D independent of phosphatidylinositol 3-kinase or RhoA activation in porcine tracheal smooth muscle.     

Inhibition of phosphatidylinositol kinase by toyocamycin

We have screened toyocamycin as an inhibitor of phosphatidylinositol kinase. It inhibited the enzyme of A431 cell membrane with an IC50 of 3.3 micrograms/ml. Adenosine and formycin A also inhibited the enzyme, but other 6 related nucleosides did not. Although orobol and 2,3-dihydroxybenzaldehyde that inhibit phosphatidylinositol kinase inhibited in situ phosphatidylinositol turnover, toyocamycin did not.     

Effects of isoproterenol pretreatment on phosphatidylinositol turnover in rat parotid gland

The effects of isoproterenol pretreatment on phosphatidylinositol turnover in rat parotid slices were studied to elucidate the relationship between beta- and alpha 1-adrenoceptors. 32P-Labeling of phosphatidylinositol in parotid slices was increased by an alpha 1- and alpha 2-agonist (epinephrine and norepinephrine) and alpha 1-agonists (methoxamine and phenylephrine), but not by an alpha 2-agonist (clonidine) and a beta-agonist (isoproterenol). Prazosin inhibited the increase in phosphatidylinositol turnover elicited by epinephrine, but propranolol did not. These results indicate that the stimulation of phosphatidylinositol turnover elicited by adrenergic agonists is mediated by activation of alpha 1-adrenoceptors in the parotid glands. Isoproterenol pretreatment of the parotid slices caused a significant increase in 32P-labeling of phosphatidylinositol and a decrease in that of phosphatidic acid. The epinephrine- or phenylephrine-induced increase in 32P-labeling of phosphatidylinositol were further enhanced by the isoproterenol pretreatment of the slices. In the isoproterenol-treated membranes of the parotid glands, [3H]prazosin binding to alpha 1-receptors increased, but [3H]dihydroalprenolol binding to beta-receptors did not. These findings indicate that the acceleration of phosphatidylinositol turnover induced by the isoproterenol pretreatment may be associated with an increase in alpha 1-adrenoceptor binding sites which might have appeared as a result of the isoproterenol pretreatment of the parotid slices.     

磷脂酰肌醇-3-激酶在胰岛素与表皮生长因子激活MAPK信号通路中的不同作用

目的　研究磷脂酰肌醇 3 激酶 (phosphatidylinositol3 kinase, PI3K)在胰岛素和表皮生长因子 (epidermalgrowthfactor, EGF)刺激丝裂原激活的蛋白激酶 (mitogen activatedproteinkinase,MAPK)信号通路中的作用。方法　主要应用免疫印迹方法分析MAPK的磷酸化水平,利用PI3K特异性抑制剂wortmannin了解PI3K在其中所起的作用。结果　胰岛素和EGF均能有效、快速地刺激MAPK的磷酸化;wort mannin抑制胰岛素刺激的MAPK磷酸化,但对EGF刺激的MAPK磷酸化却没有明显影响。与此相反,胰岛素和EGF刺激的蛋白激酶B的磷酸化,均可被wortmannin同样有效地抑制。另外,wortmannin抑制胰岛素刺激的MAPK磷酸化具浓度依赖性;其不能抑制EGF刺激的MAPK磷酸化的性质并不随EGF刺激浓度的改变而改变。结论　PI3K在胰岛素和EGF刺激的MAPK磷酸化信号通路中起不同的作用。     

Effect of progesterone on the stimulation of phosphatidylinositol turnover by epinephrine in guinea pig ductus deferens

Progesterone enhanced the contractile effect of epinephrine on the ductus deferens of the guinea pig in vitro. In relation to this mechanical phenomenon, we examined the phosphatidylinositol metabolism. In the 3H-myoinositol labeled ductus deferens, radioactivity in phosphatidylinositol bisphosphate was about 2.6 times as high as that in phosphatidylinositol. Phosphatidylinositol and phosphatidylinositol phosphate were not changed by epinephrine (100 microM), but phosphatidylinositol bisphosphate was increased at 10 sec and 1 min after the administration of epinephrine (100 microM). Progesterone (100 microM) added 5 min before the administration of epinephrine increased the stimulatory effect of epinephrine on the phosphatidylinositol bisphosphate metabolism, but had no effect on the phosphatidylinositol and phosphatidylinositol phosphate metabolism. These studies suggest that progesterone expresses its activity not through the cytoplasmic progesterone receptor but through the epinephrine mediated smooth-muscle contractile mechanism.     

Clonidine attenuates the carbachol-induced contractile and phosphatidylinositol responses of rat trachea

Although clonidine is known to affect vascular smooth muscle, its effects on airway smooth muscle are not fully understood. This study was designed to examine the effects of clonidine on carbachol-induced contractile and phosphatidylinositol responses of rat trachea. Clonidine, at a dose of 100 microM or greater, attenuated carbachol-induced contraction and the accumulation of carbachol-induced inositol monophosphate (IP1). Clonidine also attenuated the accumulation of aluminium fluoride-induced IP1. The concentration-effect relationship of IP1 accumulation was similar to that of carbachol-induced contraction; r = 0.797, P < 0.001. These results suggest that clonidine attenuates contractile responses, at least in part, through the inhibition of phospholipase C (coupled with G-proteins) in phosphatidylinositol responses.     

dl-3n-Butylphthalide promotes angiogenesis via the extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase/Akt-endothelial nitric oxide synthase signaling pathways

We have previously demonstrated that dl-3n-butylphthalide (NBP) has a potential angiogenic activity. In this study, we investigated the angiogenic effect of NBP and the molecular mechanisms underlying NBP-mediated angiogenesis. Zebrafish embryos and human umbilical vein endothelial cells were treated with various doses of NBP and several signaling pathway inhibitors. NBP induced ectopic subintestinal vessel production in zebrafish embryos and induced invasion, migration, and endothelial cell tube formation of human umbilical vein endothelial cells in a dose-dependent manner. These NBP-induced angiogenic effects were partially suppressed by SU5402, a fibroblast growth factor receptor 1 inhibitor; U0126, an extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor; LY294002, a phosphatidylinositol 3-kinase inhibitor; 1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate, an Akt inhibitor; cavtratin, an endothelial nitric oxide synthase (eNOS) inhibitor and completely inhibited by a combination of U0126 and LY294002. NBP enhanced phosphorylation of ERK1/2 and fibroblast growth factor receptor 2 expression, which were inhibited by U0126. NBP increased the phosphorylation of Akt and eNOS at serine 1177, which was blocked by LY294002. NBP-stimulated nitric oxide production, which was reduced by LY294002. Our data demonstrated that (1) NBP promoted angiogenesis and (2) the angiogenic effects of NBP were mediated by the ERK1/2 and phosphatidylinositol 3-kinase/Akt-eNOS signaling pathways. Our findings suggest that NBP could be a novel agent for therapeutic angiogenesis in ischemic diseases.     

Separation of phosphatidylinositol 4-phosphate from a mixture with phosphatidylserine and phosphatidylinositol by Sep-Pak C18 cartridge

A simple and efficient method for the separation of phosphatidylinositol 4-phosphate (PI 4-P) from phosphatidylinositol (PI) and phosphatidylserine (PS) is described. A mixture of PI, PI 4-P and PS was injected onto a Sep-Pak C18 cartridge. PI and PS were flushed through the cartridge with solvent 1 [methanol-chloroform (3: 1)] while PI 4-P remained in it. Then the cartridge was inverted, and PI 4-P was eluted backward with solvent 2 [chloroform-methanol-0.5 M aqueous ammonium hydroxide (9:7:2)].     

Alpha 1-adrenergic stimulation of phosphatidylinositol turnover and respiration of brown fat cells

The alpha-adrenergic agonist phenylephrine (in the presence of the beta-adrenergic antagonist alprenolol) stimulated respiration and incorporation of [3H]glycerol and [32P] Pi into phosphatidylinositol of hamster brown fat cells in a concentration-dependent manner. Both responses were preferentially inhibited by prazosin as compared with yohimbine, indicating alpha 1 specificity. Uniquely, prazosin inhibition of phenylephrine-stimulated phosphatidylinositol metabolism had two components, since 30% of the response was inhibited by less than 1 nM prazosin, 10 nM gave no further inhibition, and 100 nM prazosin completely inhibited the response. The phosphatidylinositol response was still present in Ca2+-free buffer, although reduced in magnitude. The concentration relationships of the effects of agonists and antagonists were compared with those of previous results of [3H]prazosin binding and with phenylephrine potency to compete for binding. On the basis of these comparisons, it is suggested that the highly prazosin-sensitive part of the phosphatidylinositol response may be closely associated with receptor occupation.     

Pelanserin inhibition of serotonin-induced phosphatidylinositol turnover and contraction in rabbit aorta

Stimulation of rabbit aortic rings with serotonin or quipazine increased the incorporation of [³²P]Pi into phosphatidylinositol (Pl) and induced a dose-related contraction. The effects of serotonin and quipazine were blocked by 5-HT₂ antagonists with the following order of potency: pelanserin = ketanserin ? methysergide. Indorenate, a 5-HT₁ agonist, failed to modify phosphatidylinositol labeling in rabbit aorta but elicited a very weak contraction at high concentrations. Pelanserin blocked epinephrine-stimulated phosphatidylinositol labeling, being two orders of magnitude less potent than prazosin. The results demonstrate that pelanserin is a potent antagonist of the stimulation of Pl labeling and vasoconstrictor effects of 5-hydroxytryptamine and suggest that 5-HT₂ blockade is involved as its major pharmacologic action.     

Class I phosphatidylinositol 3-kinase inhibitors for cancer therapy

The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers. Class I PI3Ks are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate (PIP2) at the 3-OH of the inositol ring to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3), which in turn activates Akt and the downstream effectors like mammalian target of rapamycin (mTOR) to play key roles in carcinogenesis. Therefore, PI3K has become an important anticancer drug target, and currently there is very high interest in the pharmaceutical development of PI3K inhibitors. Idelalisib has been approved in USA and Europe as the first-in-class PI3K inhibitor for cancer therapy. Dozens of other PI3K inhibitors including BKM120 and ZSTK474 are being evaluated in clinical trials. Multifaceted studies on these PI3K inhibitors are being performed, such as single and combinational efficacy, resistance, biomarkers, etc. This review provides an introduction to PI3K and summarizes key advances in the development of PI3K inhibitors.     

Regulation of human cone cyclic nucleotide-gated channels by endogenous phospholipids and exogenously applied phosphatidylinositol 3,4,5-trisphosphate

Cyclic nucleotide-gated (CNG) channels are critical components of the vertebrate visual transduction cascade involved in converting light-induced changes in intracellular cGMP concentrations into electrical signals that can be interpreted by the brain as visual information. To characterize regulatory mechanisms capable of altering the apparent ligand affinity of cone channels, we have expressed heteromeric (CNGA3 + CNGB3) human cone CNG channels in Xenopus laevis oocytes and characterized the alterations in channel activity that occur after patch excision using patch-clamp recording in the inside-out configuration. We found that cone channels exhibit spontaneous changes in current at subsaturating cGMP concentrations; these changes are enhanced by application of ATP and seem to reflect alterations in channel gating. Similar to rod CNG channels, lavendustin A prevented this regulation, suggesting the involvement of a tyrosine phosphorylation event. However, the tyrosine residue in CNGB3 (Tyr545) that is equivalent to the critical tyrosine residues in rod and olfactory CNG channel subunits does not participate in cone channel regulation. Furthermore, the changes in ligand sensitivity of CNGA3 + CNGB3 channels were prevented by inhibition of phosphatidylinositol 3-kinase (PI3-kinase) using wortmannin or 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), which suggests that phospholipid metabolism can regulate the channels. Direct application of phosphatidylinositol 3,4,5-trisphosphate (PIP3) to the intracellular face of excised patches also resulted in down-regulation of channel activity. Thus, phospholipid metabolism and exogenously applied PIP3 can modulate heterologously expressed cone CNG channels.     

The phosphatidylinositol 3-kinase/Akt pathway negatively regulates Nod2-mediated NF-kappaB pathway

Nucleotide-binding oligomerization domain containing proteins (Nods) are intracellular pattern recognition receptors (PRRs) that recognize conserved moieties of bacterial peptidoglycan and activate downstream signaling pathways, including NF-kappaB pathway. Here, we show that Nod2 agonist muramyldipeptide (MDP) induces Akt phosphorylation in time and dose-dependent manner. The pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) (wortmannin) and dominant-negative forms of p85 (the regulatory subunit of PI3K) or Akt enhance, while constitutive active forms of p110 (the catalytic subunit of PI3K) or Akt inhibit, NF-kappaB activation and the target gene interleukin (IL)-8 induced by MDP. In addition, the pharmacological inhibitors of PI3K (wortmannin and LY294002) enhance phosphorylation of NF-kappaB p65 on Ser529 and Ser536 residues, which result in enhanced p65 transactivation activity. Furthermore, we show that the inhibition of PI3K by the pharmacological inhibitors prevent the inactivation of glycogen synthase kinase (GSK)-3beta, suggesting that the negative regulation of PI3K/Akt on MDP-induced NF-kappaB activation is at least in part mediated through inactivation of GSK-3beta. Taken together, our results demonstrate that PI3K/Akt pathway is activated by Nod2 agonist MDP and negatively regulates NF-kappaB pathway downstream of Nod2 activation. Our results suggest that PI3K/Akt pathway may involve in the resolution of inflammatory responses induced by Nod2 activation.     

Thyrotropin-releasing hormone stimulates rapid breakdown of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate in GH3 pituitary tumor cells

Thyrotropin-releasing hormone (TRH) induced a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5P2) and phosphatidylinositol 4-phosphate (PtdIns 4P) in GH3 cells labeled to isotopic equilibrium with [3H]inositol. Within 10 sec of the addition of TRH (1 microM), there was a maximal 60% decrease in PtdIns 4,5P2 and 40% decrease in PtdIns 4P. Breakdown of phosphatidylinositol (PtdIns) occurred only after a lag of 30 sec. While the reduced levels of the polyphosphoinositides had almost returned to control values by 5 min, the GH3 cell PtdIns content remained at around 85% of controls for at least 2 hr. Both phosphatidic acid (PA) and 1,2-diacylglycerol levels increased in response to TRH in [32P]PO4- and [3H]glycerol-labeled GH3 cells. 1,2-Diacylglycerol accumulated in a biphasic manner with an early peak 10 sec after addition of the peptide. This early rise in 1,2-diacylglycerol levels coincided in time and was equivalent in lipid mass with the decrease in the polyphosphoinositide content, suggesting the involvement of a phospholipase C-type enzyme. 1,2-Diacylglycerol levels subsequently fell toward control values and, after 3 min of treatment with TRH, rose again to levels 50% above normal. PA levels reached a peak value approximately 2-fold above normal 1 min after the addition of TRH. At all times after TRH addition, the bulk of the inositol phospholipid lost was recovered as 1,2-diacylglycerol. These results suggest that TRH stimulates a cycle of events in which the breakdown of the polyphosphoinositides, PtdIns 4,5P2 and, perhaps, PtdIns 4P by a phospholipase C enzyme could be the initiating event.     

A calcium requirement for the phosphatidylinositol response following activation of presynaptic muscarinic receptors.

The labelling in vitro by [³²P]phosphate of phosphatidic acid and phosphatidylinositol in synaptosomes from guinea-pig brain was studied. Acetylcholine increased the labelling and evidence is provided that pre-synaptic muscarinic receptors are involved. The increase was not seen in the presence of EGTA. Experiments with various calcium buffers indicated that concentrations of greater than 10^?7 M free Ca²⁺ are required in the incubation medium for this phospholipid effect. A similar muscarinic effect in parotid gland is unaffected by EGTA. It is suggested that, in the parotid, postsynaptic receptors mediate increased phosphatidylinositol labelling and increased availability of Ca²⁺ for stimulus-secretion coupling. Presynaptic receptors similarly mediate increased labelling, which differs in being sensitive to EGTA and associated with decreased availability of Ca²⁺.