Phosphatidylinositol resynthesis

IP3 is rapidly degraded to inactive IP2 and then on to inositol. Meanwhile, diacylglycerol is phosphorylated and then converted to CDP-diacylglycerol, which combines with inositol to form phosphatidylinositol. The latter is subsequently phosphorylated in two steps to PIP2. The degradation and resynthesis of PIP2 completes the so-called phosphatidylinositol cycle. 

Scheme 1. The hydrolysis and resynthesis of phosphoinositides. Hydrolysis of phosphat-idylinositol (PI), phosphatidylinositol 4-phosphate (PIP), or phosphatidylinositol 4,5-bisphosphate (PIP2) is catalyzed by PLC in the plasma membrane. The resynthesis of phosphoinositides proceeds in the endoplasmic reticulum...

Breakdown and resynthesis of phosphatidylinositol and phosphatidic acid are closely associated with the events following depolarization of isolated synaptosomes. The most important of these events is transmitter release and the weight of the present evidence favours the view that exocytosis is the mechanism of release. This is supported by our finding that major phospholipid effects are seen in the synaptic vesicle fraction. Work with pre-labelled synaptosomes showed that stimulation led to loss of phosphatidylinositol from this fraction. The enzyme converting it to diacylglyce rol is most likely to be involved. Entry of calcium... 

Resynthesis of (18 0>20 4)phosphatidylinositol from (18 0>20 4)phos-phatidic acid during reversion to the unstimulated state... 

To summarize these changes which I have discussed, stimulation of salt gland and pancreas with acetylcholine leads to a breakdown of phosphatidylinositol. Under some conditions, the (18 0,20 4)-diglyceride moiety of this phosphatidylinositol is converted almost quantitatively to (18 0,20 4)phosphatidic acid. During the stimulated state, this novel species of phosphatidic acid undergoes continuous turnover of its phosphate group, presumably with (18 0, 20 4)diglyceride as an intermediate. It is specifically used for the resynthesis of phosphatidylinositol when stimulated tissue reverts to the unstimulated state (Fig. 1). 

In pancreas tissue incubated in the presence ofPh glycerol, there is some incorporation of Ph glycerol into phosphatidic acid. In acetylcholine-stimulated tissue to which atropine is added, some of this II glycerol-labeled phosphatidic acid is used for the resynthesis of phosphatidylinositol when the tissue reverts to the unstimulated state. The specific activity of phosphatidylinositol rises dramatically, to become only slightly less than that of phosphatidic acid. The rise in phosphatidylinositol specific activity does not occur in response to atropine if the tissue has not previously been exposed to acetylcholine. This confirms that the rise is not a direct response to atropine, but is due to resynthesis of phosphatidylinositol after acetylcholine-induced breakdown. In pancreas tissue which has been labeled in this manner by an acetyl-choline-atropine sequence, addition of pancreozymin causes a significant fall in the specific activity of the h]glycerol-... 

The NaK-ATPase activity which is initiated in response to acetylcholine can be monitored by measurement of the 3-fold increase in the rate of respiration which it evokes. This increase in the respiratory rate is blocked by ouabain, which specifically blocks NaK-ATPase activity, and by atropine, which specifically blocks acetylcholine receptors. In the salt gland, an almost maximal increase in secretory NaK-ATPase activity occurs in response to O.lyM acetylcholine and in this tissue, in contrast to the pancreas, the breakdown of phosphatidylinositol, as measured by the amount resynthesized after addition of atropine to stimulated tissue, also occurs almost maximally at this same low concentration of acetylcholine (Table 5). These observations lead me to propose that in the salt gland cell, the function of stimulated phosphatidylinositol breakdown, and of its resynthesis during the reversion to the unstimulated state, is to exert on-off control of the activity of the secretory NaK-ATPase molecules. These are "turned on" when this tissue is stimulated, and are "turned off when the tissue reverts to the non-secreting state. A simple mechanism for this might be that when the responsive lipid molecules are in the phosphatidylinositol form, an active site of the secretory NaK-ATPase is buried in a hydrophobic area of the membrane, and that when the phosphatidylinositol is broken down, this results in a membrane change... 

Figure 2. The so-called canonical phosphoinositide pathway . The continuous phosphorylation/dephosphorylation reactions allow a steady-state level of Ptdins, PtdIns(4)P and PtdIns(4,5)P2 in the plasma membrane (PM). Cleavage of PtdIns(4,5)P2 by phospholipase C (PLC) generates the two well-known second messengers, inositol 1,4,5-trisphosphate (Ins(l,4,5)P3) and diacylglycerol (DAG). Besides its role as a protein kinase C (PKC) activator, DAG can be phosphorylated to phosphatidic acid (PA). The resynthesis of Ptdins from inositol and PA occurs mainly in the endoplasmic reticulum (ER). PPi, inorganic phosphate. PA-Pase, phosphatidic acid phosphatase. PA-TP, phosphatidic acid transport protein. PtdIns-TP, phosphatidylinositol transport protein. CDP-DAG, cytidine diphosphate-diacylglycerol. CMP, CDP and CTP, cytidine mono-, di- and triphosphate, respectively.

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