Sodium ion and the neutrotransmitter-stimulated 32P labelling of phosphoinositides and other phospholipids in the iris muscle
Abstract
The effects of Na+, other cations and the neurotransmitters, acetylcholine and norepinephrine on 32Pi incorporation into phospholipids of the rabbit iris smooth muscle were investigated [1]. The basal 32P-labelling of phospholipids, including phosphatidic acid, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine and the polyphosphoinositides increased with Na+ concentration [2]. The neurotransmitter-stimulated 32P-labelling of phosphotidic acid, phosphatidylinositol and phosphatidylcholine is dependent on the presence of extracellular Na+ [3]. The monovalent cation requirement for Na+ is specific. Of the monovalent cations Li+, NH4+, K+, choline+ and Tris, only Li+ partially substituted for Na+ [4]. A significant decrease in 32P labelling of phospholipids in response to acetylcholine was observed when Ca2+ and/or K+ were added to an isoosmotic medium deficient of Na+ [5]. Ouabain, which blocks the Na+-pump, inhibited the basal 32Pi incorporation into phosphatidylcholine and the acetylcholine-stimulated 32P labelling of phosphatidic acid, phosphatidylinositol and phosphatidylcholine [6]. It was suggested that phosphoinositide breakdown is associated with Ca2+ influx as we have previously reported (Akhtar, R.A. and Abdel-Latif, A.A. (1978) J. Pharmacol. Exp. Ther. 204, 655–668) and that the enhanced 32P-labelling of phosphoinositides could be associated with Na+ outflux, via the Na+-pump mechanism.
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Choline<sup>+</sup> is a low-affinity ligand for α<inf>1</inf>-adrenoceptors
1994, Biochemical PharmacologyThe effect of choline+, a commonly used Na+, on ligand binding to α1-adrenoceptors was investigated. It was found that replacement of 25% of the Na+ in a Krebs-Ringer bicarbonate buffer with choline+ led to a 3-fold decrease in the apparent affinity of [3H]prazosin for its binding site (i.e. the α1-receptor) in a membrane preparation from brown adipose tissue, while no decrease in the total number of binding sites was observed. Similar effects were seen in membrane preparations from liver and brain. In competition experiments, it was found that choline+ could inhibit [3H]prazosin binding; from the inhibition curve, an affinity (Ki) of 31 mM choline+ for the [3H]prazosin-binding site could be calculated. In fully choline+-substituted buffers, where the level of [3H]prazosin binding was substantially reduced, both phentolamine and norepinephrine could still compete with [3H]prazosin for its binding site, with virtually unaltered affinity; thus choline+ did not substantially affect the characteristics of those receptors to which it did not bind. Choline+ did not affect the binding characteristics of the β1/β2 radioligand [3H]CGP-12177; thus, the effect on α1-receptors was not due to general, unspecific effects on the membrane preparations. It is concluded that choline + possesses characteristics similar to those of a competitive ligand for the α1adrenoceptor; it has a low affinity but the competitive type of interaction of choline may nonetheless under experimental conditions interfere with agonist interaction with the α1-receptor.
Compartmentation of phosphorylated precursors of phospholipid biosynthesis in cultured neuroblastoma cells
1987, BBA - BiomembranesThe continuous turnover of membrane phospholipids requires a steady supply of biosynthetic precursors. We evaluated the effects of decreasing extracellular Na+ concentration on phospholipid metabolism in cultured neuroblastoma (N1E 115) cells. Incubating cultures with 145 to 0 mM NaCl caused a concentration-dependent inhibition of [32P]phosphate uptake into the water-soluble intracellular pool and incorporation into phospholipid. Phospholipid classes were differentially affected; [32P]phosphate incorporated into phosphatidylethanolamine (PE) and phosphatidylcholine (PC) was consistently less than into phosphatidylinositol (PI) and phosphatidylserine (PS). This could not be attributed to decreased phospholipid synthesis since under identical conditions, there was no effect on arachidonic acid or ethanolamine incorporation, and choline utilization for PC synthesis was increased. The effect of Na+ was highly specific since reducing phosphate uptake to a similar extent by incubating cultures in a phosphate-deficient medium containing Na+ did not alter the relative distribution of [32P]phosphate in phospholipid. Of several cations tested only Li+ could partially (50%) replace Na+. Incubation in the presence of ouabain or amiloride had no effect on [32P]phosphate incorporation into phospholipid. The differential effects of low Na+ on [32P]phosphate incorporation into PI relative to PC and PE suggests preferential compartmentation of [32P]phosphate into ATP in pools used for phosphatidic acid synthesis and relatively less in ATP pools used for synthesis of phosphocholine and phosphoethanolamine, precursors of PC and PE, respectively. This suggestion of heterogeneous and distinct pools of ATP for phospholipid biosynthesis, and of potential modulation by Na+ ion, has important implications for understanding intracellular regulation of metabolism.
Effects of acetylcholine and norepinephrine on incorporation of [<sup>32</sup>P]orthophosphate into phospholipids of rabbit iridial processes and iris smooth muscle
1983, Experimental Eye ResearchWe have investigated: (a) phospholipid composition, inclusive of higher inositides, of rabbit iridial processes and iris smooth muscles; (b) 32Pi incorporation into their respective phospholipids, and (c) the effects of muscarinic cholinergic and adrenergic agonists and antagonists on 32P labelling of phospholipids of the iridial processes and iris smooth muscle. (1) Phosphatidylcholine, phosphatidylethanolamine, their respective plasmalogens and sphingomyelin, were found to be the major phospholipids in the iridial processes and iris smooth muscle. They constituted about 85% of the total phospholipids of these ocular tissues. (2) Both iridial processes and iris smooth muscles rapidly incorporated 32Pi and [1-14C]-arachidonic acid into their respective phospholipids, however this incorporation amounted to only 20% of that found for the whole iris-ciliary body. This could suggest a metabolic interrelationship between the iridial processes and the smooth muscle of the iris. (3) Addition of acetylcholine and norepinephrine to the iridial processes and iris smooth muscle increased 32P labelling of phosphatidic acid and phosphatidylinositol of the tissues. The increase in phospholipid labelling was higher in the iridial processes as compared to the iris smooth muscle. The effect of acetylcholine was blocked by atropine and that of norepinephrine was blocked by phentolamine and prazosin but not by yohimbine. This suggests that the observed effects of these neurotransmitters on phospholipid phosphorylation in the iridial processes and iris smooth muscle are mediated through muscarinic cholinergic and α1-adrenergic receptors, respectively. The data presented provide additional support for the concept that in the iris-ciliary body the neurotransmitter-induced 32P labelling of phosphoinositides is probably linked to the functional activities of this tissue.
Inositol phospholipids in stimulated smooth muscles
1982, Cell CalciumA variety of physiological stimulants, such as hormones and neurotransmitters, which exert physiological responses by increasing intracellular Ca2+ level, enhance phosphatidylinositol turnover in target organs. This reaction is characterized by an initial breakdown of phosphatidylinositol followed by a compensatory increase in its resynthesis. In many studies, experimental conditions adopted to demonstrate receptor-mediated increase of phosphatidylinositol turnover as the results of physiological stimuli, however, seem to be either unphysiological or inadequate. In some, only the secondary resynthesis of phosphatidylinositol was measured. In others, although the primary breakdown of phosphatidylinositol was measured, unphysiologically high concentration of stimulants or extended period of reaction was used. It is necessary to use physiological stimuli and methods to study physiological roles of “phosphatidylinositol response”. Therefore, in this review, discussions are focussed on relationships between physiological stimuli to smooth muscle tissues, alterations of phosphatidylinositol metabolism and their physiological roles.
Recent hypotheses regarding the phosphatidylinositol effect
1981, Life SciencesIn 1975, Michell first proposed that activation of phosphatidylinositol turnover provided a direct link between surface receptors and membrane Ca gates. Subsequently, a number of laboratories have begun to re-investigate this phenomenon first described by Hokin and Hokin some twenty years earlier. As would be expected, some new hypothesis have emerged, most being extensions or revisions of Michell's original concept.
Despite difficulties in obtaining direct proof, indirect evidence suggests that the plasma membrane is the primary locus of receptor-activated phosphatidylinositol turnover, at least for phosphatidylinositol breakdown and phosphatidic acid synthesis. The presence or absence of Na+ can markedly affect labelling of phosphatidylinositol by radioactive precursors but there is no compelling evidence that the initial events are mediated by Na+. Prostaglandins are apparently formed in some tissues on receptor activation, but in most instances the evidence suggests that these compounds are not obligatory intermediates in tissues that show the phosphatidylinositol effect. Several laboratories have obtained evidence that phosphatidic acid newly synthesized following phosphatidylinositol breakdown, may function as an endogenous Ca ionophore under neurohumoral control.
Sodium ion and the effect of acetylcholine on phospholipid and phosphoprotein phosphate turnover in the rabbit iris smooth muscle
1981, Biochemical Pharmacology