Volume-dependent regulation of ion transport and membrane phosphorylation in human and rat erythrocytes

Summary Osmotic swelling of human and rat erythrocytes does not induce regulatory volume decrease. Regulatory volume increase was observed in shrunken erythrocytes of rats only. This reaction was blocked by the inhibitors of Na⁺/H⁺ exchange. Cytoplasmic acidification in erythrocytes of both species increases the amiloride-inhibited component of²²Na influx by five- to eight-fold. Both the osmotic and isosmotic shrinkage of rat erythrocytes results in the 10- to 30-fold increase of amiloride-inhibited²²Na influx and a two-fold increase of furosemide-inhibited⁸⁶Rb influx. We failed to indicate any significant changes of these ion transport systems in shrunken human erythrocytes. The shrinking of quin 2-loaded human and rat erythrocytes results in the two- to threefold increase of the rate of⁴⁵Ca influx, which is completely blocked by amiloride. The dependence of volume-induced²²Na influx in rat erythrocytes and⁴⁵Ca influx in human erythrocytes on amiloride concentration does not differ. The rate of⁴⁵Ca influx in resealed ghosts was reduced by one order of magnitude when intravesicular potassium and sodium were replaced by choline. It is assumed that the erythrocyte shrinkage increases the rate of a nonselective Ca _o ²⁺ (Na _i ⁺ , K _i ⁺ ) exchange. Erythrocyte shrinking does not induce significant phosphorylation of membrane protein but increases the³²P incorporation in diphosphoinositides. The effect of shrinkage on the³²P labeling of phosphoinositides is diminished after addition of amiloride. It is assumed that volume-induced phosphoinositide response plays an essential role in the mechanism of the activation of transmembrane ion movements.     

Dissecting the gelsolin-polyphosphoinositide interaction and engineering of a polyphosphoinositide-sensitive gelsolin C-terminal half protein

Gelsolin and other proteins in the villin/gelsolin family are regulated by polyphosphoinositides (PPIs), and manipulation of cellular PIP(2) levels alters the structure of the actin cytoskeleton coincident with the dissociation of gelsolin-actin complexes. This work explores the structure-function relationship of the gelsolin-PPI interaction. Circular dichroism experiments show that upon binding to PPIs, the PPI-sensitive N-terminal half of gelsolin undergoes significant secondary and tertiary structural changes that do not occur in the structurally homologous but PPI-insensitive C-terminal half. Secondary structure modeling algorithms predict an alpha-helical conformation for one of the gelsolin PPI-binding sites, P2, which differs from the conformation of P2 in the structure of gelsolin determined by X-ray crystallography, whereas structure prediction of the C-terminal homolog of P2 agrees well with the X-ray crystallography structure. Simulation of a change to helical conformation for P2 using molecular modeling indicates that such a structural transition will destabilize the F-actin-binding sites in domain 2. A hypothesis is proposed that PPIs initiate conformational changes at the PPI-binding site(s) that destabilize the protein structure, and subsequently disrupt the actin-binding sites. To further evaluate the role of P2 in the gelsolin-PPI interaction, a Ct mutant P2Ct is constructed by inserting P2 in place of its C-terminal homologous site. P2Ct interacts with actin in the same way as the wild-type protein. In contrast to Ct, however, P2Ct interacts strongly with PPIs, and its monomeric actin-binding activity becomes regulated by PPIs. It is concluded that the P2 site is sufficient for PPI-sensitivity in gelsolin. Furthermore, the P2 site in P2Ct and the actin-binding sites of Ct do not overlap, suggesting that PPIs regulate actin binding of P2Ct through induction of structural changes, rather than through direct competition.     

Glucose inhibits insulin release when not promoting the entry of calcium into the beta-cells

The effect of antigen on the metabolism of polyphosphoinositides was investigated in sensitized rat peritoneal mast cells. Addition of antigen to rat peritoneal mast cells prelabelled with [3H]arachidonic acid resulted in a very rapid decrease in the level of phosphatidylinositol 4-phosphate (DPI) within 5 sec, which appeared to precede the breakdown of phosphatidylinositol (PI), while there was no significant decline of PI 4,5-bisphosphate (TPI). The reduced levels of these phosphoinositides returned almost to control or even slightly higher values by 300 sec in parallel with the antigen-stimulated [32P]phosphate incorporation into these lipids. This early and transient disappearance in DPI prior to that in PI was also observed in [3H]glycerol-prelabelled cells. These data suggest that DPI degradation upon stimulation by antigen in mast cells may be an initial step in the histamine release process.     

Effects of β-phenylethylamine on polyphosphoinositide turnover in rat cerebral cortex

The effects of the trace amine, -phenylethylamine, on the hydrolysis of inositol phospholipids in rat cerebral cortical slices was studied using a direct assay involving prelabeling with [³H]inositol and then examining the production of [³H]inositol phosphates in the presence of lithium. Phenylethylamine exhibited two different effects. Millimolar concentrations of phenylethylamine stimulated the production of [³H]inositol phosphates to about 200% of control, while much smaller concentrations (micromolar) inhibited noradrenaline(NE)-stimulated [³H]inositol phosphate formation dose-dependently. The ₁-antagonist, prazosin, inhibited the increases in [³H]polyphosphoinositide turnover stimulated by phenylethylamine and by NE, though it inhibited phenylethylamine to a lesser extent than NE. It appears, therefore, that phenylethylamine affects [³H]inositol phosphate formation by acting as a partial ₁-agonist.     

Stimulation by d-glucose of the synthesis of polyphosphoinositides in pancreatic islets

d-glucose (16.7 mM) stimulates the synthesis of polyphosphoinositides in in intact pacreatic islets prelabelled with tritiated myo-inositol and incubated in the absence of extracellular Ca²⁺. ATP (1.0 mM) exerts a comparable effect in sonicates of prelabelled normal or tumoral islet cells. In the acellular system, ATP fails to affect the generation of tritiated inositol phosphates in the absence of Ca²⁺, but augments the Ca²⁺-stimulated production of inositol mono-, bis- and triphosphates. The latter effect is not reproduced by α, ß-methylene ATP, suggesting that it is not attributable to a purinergic mechanism. Whether in the absence or presence of ATP, the Ca²⁺-induced increment in inositol phosphates production coincides with a comparable decrease in tritiated polyphosphoinositides. It is proposed, therefore, that the increased production of inositol phosphates in intact islets stimulated by nutrient secretagogues is attributable, in part at least, to an accelerated generation of polyphosphoinositides, possibly resulting from a rise in cytosolic ATP concentration.     

The Inositol Lipids of Paramecium tetraurelia and Preliminary Characterizations of Phosphoinositide Kinase Activity in the Ciliary Membrane

Inositol glycerolipids make up less than 10% of total phospholipids of Paramecium tetraurelia cells. Unlike inositol lipids found in mammalian and other cell types, these lipids from Paramecium lack arachidonic acid. It was demonstrated that kinase and possibly phosphatase enzymes that interconvert phosphatidylinositol (PI), phosphatidylinositol phosphate (PI-P) and phosphati-dylinositol-bis-phosphate (PI-P₂) exist in ciliary membranes of this ciliate. When exogenous soybean PI and [γ-³²P]ATP were provided as substrates, isolated cilia preparations exhibited PI and PI-P kinase activities as demonstrated by the incorporation of radiolabel into PI-P and PI-P₂. Kinase activity was activated by millimolar [Mg²⁺] and inhibited by millimolar [Ca²⁺]. Significant inhibition of kinase activity in the presence of unlabeled excess ATP suggested that ATP is the preferred phosphate donor for this reaction. Of 4 suborganellar fractions of isolated cilia, the membrane fraction had the greatest kinase activity indicating that the enzyme(s) is membrane-associated