Nuclear phosphoinositide kinases and inositol phospholipids

Stimulated hydrolysis of the inositol phospholipids phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was investigated by studying the phosphoinositides produced in a suspended preparation of plasma membranes by transference of 32P from [gamma-32P]ATP. At basal Ca2+ concentration (calculated free Ca2+, 150 nM) phospholipid hydrolysis was stimulated either by the muscarinic agonists carbamoylcholine and bethanecol or by the addition of the non-hydrolysable analogue of GTP, guanosine 5′-[beta gamma-imido]triphosphate [p(NH)ppG]. GTP was without effect on basal hyrolysis. Both GTP and p(NH)ppG enhanced the rapid (within 10 s) hydrolysis of PtdIns4P and PtdIns(4,5)P2 induced by carbamoylcholine in a dose-dependent manner. A rightward shift in the competition curve of carbamoylcholine for bound L-[3H]quinuclidinyl benzilate was seen on addition of GTP or p(NH)ppG (100 microM) under phosphorylating conditions. Pretreatment of intact islet cells with Bordetella pertussis toxin, islet-activating protein (IAP) or treatment of membranes with IAP under conditions which elicited ADP-ribosylation of a protein of Mr 41,000 was without effect on muscarinic binding, phosphoinositide phosphorylation or subsequent hydrolysis by carbamoylcholine. The findings indicate the involvement of a GTP-binding protein in the coupling of the muscarinic receptor to phosphoinositide hydrolysis in the islet cell and suggest that this is distinct from the GTP-binding regulatory component of adenylate cyclase which is covalently modified by IAP.

Download Full-text

[13] Inositol phospholipids and phosphates for investigation of intact cell phospholipase C substrates and products

Methods in Enzymology - Phospholipases ◽

10.1016/0076-6879(91)97141-k ◽

1991 ◽

pp. 149-158 ◽

Cited By ~ 5

Author(s):

Michael R. Hanley ◽

David R. Poyner ◽

Phillip T. Hawkins

Keyword(s):

Phospholipase C ◽

Intact Cell ◽

Inositol Phospholipids

Download Full-text

Biosynthesis of inositol phospholipids

Inositol Phospholipid Metabolism and Phosphatidyl Inositol Kinases - Laboratory Techniques in Biochemistry and Molecular Biology ◽

10.1016/s0075-7535(03)30023-3 ◽

2003 ◽

pp. 335-402

Keyword(s):

Inositol Phospholipids

Download Full-text

Changes In Triphosphatidylinositol Metabolism During PGE1- Induced Shape Change Of Washed Rabbit Platelets

10.1055/s-0038-1652880 ◽

1981 ◽

Author(s):

J D Vickers ◽

R L Kinlough-Rathbone ◽

J F Mustard

Keyword(s):

Phosphatidic Acid ◽

Shape Change ◽

Specific Radioactivity ◽

Inositol Phospholipids ◽

Rabbit Platelets ◽

Turn Over ◽

Camp Levels ◽

Camp Formation ◽

Platelet Shape ◽

Stimulation Of

Since the inositol phospholipids are present in small amounts in platelets and turn over rapidly during platelet shape change, aggregation and release, they are thought to have a functional rather than structural role in platelets. We have previously reported that within 10 sec of stimulation of prelabeled, washed rabbit platelets with ADP, the amount of triphosphatidylinositol (TPI) is significantly reduced while the specific radioactivity of its [32p]phosphate is increased. One explanation of this result is that ADP- stimulation may divert ATP required for phosphorylation of diphosphatidylinositol (DPI) to TPI, leading to a decrease in the amount of TPI. PGE1 (10 μM) causes conversion of ATP to cAMP and induces a transient platelet shape change. The shape change may be due to the reduction in ATP with a concomitant fall in TPI. We have therefore studied whether PGE1-stimulation of washed rabbit platelets prelabeled with [32P] causes a change in TPI. Within 10 sec the amount of TPI in PGE1-treated platelets was reduced from 2.22 nmoles/ 109 platelets to 1.98 nmoles/109 platelets (p<0.05) although neither the [32P] labeling (51.1 × 103 dpm/109 platelets) nor specific radioactivity (24.1 × 103 dpm/nmole) were significantly changed. These results are compatible with the theory that diversion of ATP by PGE1-stimulation of cAMP formation from ATP, may reduce the amount of TPI. A similar effect was observed previously with ADP-stimulation. PGE1 caused no change in the [32p] labeling of phosphatidic acid (PA) (ADP caused a 290% increase) and caused only a small increase in its specific radioactivity (16% compared to 270% with ADP). If the rates of turnover of TPI and PA which are reflected in their specific radioactivities are Ca2+- dependent, Ca2+ sequestration due to increased cAMP levels induced by PGE1 would, after the initial effects, terminate these changes. The results further support the suggestion that reduction in the amount of TPI may be involved in platelet shape change and initiation of aggregation.

Download Full-text