Phosphatidylinositol stimulated breakdown

When acetylcholine-stimulated breakdown of phosphatidylinositol was first observed in the avian salt gland, it seemed logical to assume that this would be by cleavage of phosphatidylinositol to form diglyceride and inositol phosphate (Hokin, M.R. Hokin, L.E., 1964). A Ca -dependent enzyme with phospholipase C type activity which catalyzed this reaction had been demonstrated by Kemp et al. (1961). More recently, Dawson et al. (1971) showed that the products are inositol 1,2-cyclic phosphate, inositol 1-phosphate and diglyceride. There is an enzyme with these properties in mouse pancreas. The enzyme is not activated by acetylcholine in soluble extracts, and we have found no evidence that it is concerned in acetylcholine-stimulated breakdown of phosphatidylinositol in the intact cell, nor that the function of phosphatidylinositol breakdown is to form inositol 1,2-cyclic phosphate, as has been suggested by Michell Lapetina (1972). 

Inositol lipids play an important role in controlling cellular metabolism. They do this through the inositol cycle in which the key event is a phospholipase C-stimulated breakdown of phosphatidylinositol-4,5-bisphosphate. Agonist binding to receptors causes the stimulation of this... 

Hormonal factors and other stimuli by activating phospholipase C-(3 or -y isoforms stimulate the breakdown of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol, a reaction called PI response. 

In Uver, adrenaline binds to the a-receptor, and the hormone-receptor complex activates a membrane-bound phospholipase enzyme which hydrolyses the phospholipid phosphatidylinositol 4,5-bisphosphate. This produces two messengers, inositol trisphosphate (IP3) and diacylglycerol (DAG) (Figure 12.5). The increase in IP3 stimulates release of Ca ions from the endoplasmic reticulum into the cytosol, the effect of which is glycogen breakdown and release into the blood (see Figure 12.5 and Chapter 6). 

I nositol is a simple substance present normally in the diet at about 1 g/day and is an isomer of glucose. The phosphatidylinositol (PI) cycle is an important second messenger system for several brain neurotransmitters (Figure 9-1). Receptor (R) stimulation by an activator (A) leads to breakdown of membrane phosphatidylinositol 4,5-biphosphate (PIP2) to... 

Two of the more studied effector proteins of G-proteins are adenylate cyclase (AC) and phospholipase C (PLC). AC converts adenosine triphosphate (ATP, 5.4) into 3, 5 -cyclic adenosine monophosphate (cAMP, 5.5) (Scheme 5.4). cAMP is a secondary messenger that can activate certain kinases (phosphorylation enzymes) and stimulate the breakdown of fats and glycogen. PLC hydrolyzes phosphatidylinositol 3,4-bisphosphate (PIP2, 5.6) to form two secondary messengers, diacylglycerol (DAG, 5.7) and inositol... 

Activation of phospholipase C leads to cleavage of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate into inositol trisphosphate (IP3) and diacylglycerol (DAG). 1P3 promotes release of Ca2+ from storage organelles, whereby contraction of smooth muscle cells, breakdown of glycogen, or exocytosis may be initiated. DAG stimulates protein kinase C, which phosphorylates certain serine- or threonine-containing enzymes. 

Initial studies in rat brain showed that intracistemal injection of histamine stimulated the incorporation of [33P]P into inositol phospholipids [235, 236]. Studies with selective H,- and H2-agonists and antagonists further suggested that this response was mediated by H,-receptors [236]. Increased turnover of inositol phospholipids is nevertheless a rather indirect measure of the initial receptor-mediated event, namely breakdown of phosphatidylinositol 4,5-bisphosphate. A more direct and sensitive method of monitoring inositol phospholipid breakdown is now available, however, following the discovery that Li+ ions can cause an accumulation of inositol 1-phosphate in slices of rat cerebral cortex and parotid gland as a result of inhibition of inositol 1-phosphatase [237],... 

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... 

Although there is an increased turnover of phosphatidylinositol in stimulated tissue, the primary action of acetylcholine is to cause a net breakdown of this phosphatide. The breakdown was first observed as a loss of radioactivity from prelabeled phosphatidylinositol in response to acetylcholine in the avian salt gland (Hokin, M.R. ... 

Hokin, L.E., 1964 Hokin, M.R., 1965, 1967) and later in the mouse pancreas (Hokin, M.R., 1974). The extent of breakdown of prelabeled phosphatidylinositol suggested that there was probably a net change in the level of phosphatidylinositol in the stimulated tissue. Phosphatidylinositol is a relatively minor component of the total phospholipids of animal tissues. Its separation and quantitative estimation by early methods was difficult and somewhat unreliable. 

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). 

When we examined the water-soluble products of stimulated phosphatidylinositol breakdown in pancreas we did not find any increase... 

The lipid-soluble product of stimulated phosphatidylinositol breakdown can appear as (18 0,20 4)phosphatidic acid or (18 0,20 4)... 

The hormone pancreozymin (cholecystokinin-pancreoz3miin) stimulates the protein secretory cycle in the exocrine pancreas (Harper Raper, 1943) it also elicits phosphatidylinositol breakdown, formation of phosphatidic acid, and increased turnover of the polar headgroups of these phosphatides. These effects are essentially the same as those observed in response to acetylcholine (Hokin, M.R.,... 

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-... 

If the major site of acetylcholine-stimulated phosphatidylinositol breakdown in the pancreas is the rough endoplasmic reticular membranes rather than at the cell surface, then it is necessary to postulate that some second messenger system is needed to carry the information from the receptor at the cell surface to the site of the response. The nature of this is not known. The dibutyryl derivatives of cyclic AMP and cyclic GMP do not give rise to phosphatidylinositol breakdown in the pancreas nor does the influx of Ca ion... 

Acetylcholine and pancreozymin both stimulate zymogen extrusion in the pancreas. However, many lines of evidence have indicated that the phosphatidylinositol response is not closely related to the process of exocytosis in the pancreas or in other glands. One point of difference between stimulation of exocytosis and stimulation of phosphatidylinositol breakdown in mouse pancreas is that maximum extrusion of enzjnne occurs in vitro in response to O.lyM acetylcholine stimulation of H phosphatidylinositol breakdown increases linearly between concentrations of acetylcholine of O.lpM to lOOyM (Hokin, M.R. 1974). 

Table 4. Comparison of the stimulation by acetylcholine of protein synthesis and of phosphatidylinositol breakdown in mouse pancreas...

Table 5. Comparison of acetylcholine stimulation of ouabain sensi-tive respiration and of phosphatidylinositol breakdown in goose...

This "microsomal" fraction from salt gland consists largely of smooth membrane fragments which appear to be derived from the extensive plasma membrane of cell (Slautterback, Hokin, L.E. Hokin, M.R., unpublished). The major site of acetylcholine-stimulated phosphatidylinositol breakdown in the salt gland appears therefore to be this extensive plasma membrane, which contains the NaK-ATPase and carries out the secretory function of the gland. 

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... 

A variety of physiological stimuli which cause the opening of cell-surface Ca2+ gates also stimulate phosphatidylinositol breakdown. Studies of various receptors and tissues have shown that the breakdown of phosphatidylinositol is not caused by an increase in the intracellular Ca + concentration. It seems most likely that the function of stimulated PI breakdown lies in the coupling between activated cell-surface receptors and the opening of membrane Ca " " gates, and some possible mechanisms for this coupling have been briefly discussed. 

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