Mabel R. Hokin

The incorporation of 32P from triphosphate into polyphosphoinositides [γ-32P]adenosine and phosphatidic acid in erythrocyte membranes

Abstract 1. 1. Erythrocyte ghosts incubated with [γ- 32 P]ATP incorporate radioactivity into diphosphoinositide, triphosphoinositide, phosphatidic acid, and into at least one unidentified lipid. 2. 2. Over 90% of the radioactivity incorporated into the polyphosphoinositides is in the monoesterified phosphates. 3. 3. Exogenous phosphatidyl inositol increases the labeling in diphosphoinositide by about 30% and in the unidentified compound by about 200%. The labeling in triphosphoinositide is inhibited. 4. 4. Kinetic studies of the labeling of the polyphosphoinsitides fail to provide evidence that the exchange of phosphate in the monoesterified positions is an intermediate reaction in the magnesium-dependent, sodium and potassium-activated ATPase.

STUDIES ON A NA+ + K+-DEPENDENT, OUABAIN-SENSITIVE ADENOSINE TRIPHOSPHATASE IN THE AVIAN SALT GLAND.

Abstract 1. 1. A Na + + K + -dependent, ouabain-sensitive ATPase was found in homogenates of the avian salt gland. Some of its properties have been investigated. 2. 2. The levels of ouabain-sensitive and ouabain-insensitive ATPase activities were the same in homogenates of tissue from gulls maintained on fresh water and from gulls maintained on 1.5% salt water. 3. 3. The levels of ouabain-sensitive and ouabain-insensitive ATPase activities were the same in homogenates of slices which had been incubated in the absence of acetylcholine and slices which had been incubated in the presence of acetylcholine. (Acetylcholine stimulates NaCl secretion in this tissue.) 4. 4. The oxygen uptake in unstimulated salt-gland slices was reduced about 25% in the presence of 10 −4 M ouabain. The increase in oxygen uptake which occurs in response to acetylcholine did not occur if ouabain was also present. 5. 5. The level of 7-min acid-hydrolyzable phosphate esters in salt-gland slices fell in the presence of acetylcholine but this fall did not occur if ouabain was also present. 6. 6. These results, and the relationship between the rates of Na + transport and ATPase activity in the salt gland, are discussed.

The chromatographic separation of polyphosphoinositides and studies on their turnover in various tissues

Abstract 1. 1. 32 P-labeled spots obtained when total lipid extracts from tissue slices are chromatographed on silicic acid-impregnated paper with phenol-ammonia as the developing solvent been characterized as di- and triphosphoinositide. This has enabled us to develop a simple, rapid, quantitative method for assaying the radioactivity incorporated into di- and triphosphoinositide in large numbers of small samples of tissue. 2. 2. Slices of salts gland, brain cortex, kidney, liver, pancreas, and heart ventricle incorporated 32 P into di- and triphosphoinisitide when incubated in physiological saline containing [ 32 P]orthophosphate. With the exception of liver, the incorporation into triphosphoinositide was higher. The incorporation into diphosphoinositide generally paralled that into triphosphoinositide. 3. 3. Under conditions in which 32 P incorporation into phosphatidic acid and phosphatidyl inositol was stimulated by acitylcholine in salt-gland slices, there was an inhibition of incorporation into the polyphosphoinositides.

EFFECT OF NOREPINEPHRINE ON 32P INCORPORATION INTO INDIVIDUAL PHOSPHATIDES IN SLICES FROM DIFFERENT AREAS OF THE GUINEA PIG BRAIN1

Abstract— 1 Incorporation of 32P into phosphatidic acid was significantly increased in the presence of an appropriate concentration of norepinephrine, either 0.1 mm, or 1 mm, or both, in slices of guinea pig cerebellar cortex, olfactory bulbs, cerebral cortex, hypothalamus and thalamus; it was not significantly affected at either concentration of norepinephrine in slices of corpus striatum and the posterior and anterior colliculi. 2 Incorporation of 32P into phosphatidylinositol was significantly increased in the presence of norepinephrine in a concentration of either 0.1 mm, or 1 mm, or both, in three of the areas studied—the olfactory bulbs, cerebral cortex, and thalamus. 3 There was no significant effect of 0.1 mm‐norepinephrine on 32P incorporation into phosphatidylcholine or phosphatidylethanolamine plus phosphatidylserine in any of the areas studied. Norepinephrine (1 mm) significantly inhibited 32P incorporation into phosphatidylcholine in cerebral cortex, hypothalamus, corpus striatum and anterior colliculus; 32P incorporation into phosphatidylethanolamine plus phosphatidylserine was inhibited by 1 mm‐norepinephrine in cerebral cortex, hypothalamus, and posterior and anterior colliculi. 4 In slices of cerebellar cortex, 0.1 mm and 1 mm‐norpinephrine did not significantly affect either the specific activity or the level of nucleotide ∼ P in the tissue. 5 The physiological significance of changes in phosphatide metabolism in response to neurotransmitters in the central nervous system is discussed.

Diglyceride kinase and other pathways for phosphatidic acid synthesis in the erythrocyte membrane

Abstract Various pathways for the synthesis of phosphatidic acid in ghosts from human erythrocytes have been studied. The synthesis of phosphatidic acid by the diglyceride kinase reaction is 10–40 times more active than the synthesis of phosphatidic acid by phosphorylation of monoglyceride followed by acylation, and 2500 times more active than the synthesis by acylation of α-glycerophosphate. Diglyceride kinase activity is as great or greater than the Na + + K + -dependent, ouabain-inhibitable, ATPase in the erythrocyte membrane; this is compatible with its being a component of the ATPase. Various factors influencing diglyceride kinase activity have been studied, such as detergents, the state of dispersion of the diglyceride substrate, freezing of the ghosts, Na + and K + , and ouabain. The kinetic curve at 37° for phosphatidic acid synthesis from diglyceride shows an initial rapid component, followed after about 1 min by a slower component.

The Role of Phosphatidic Acid and Phospho-Inositide in Transmembrane Transport Elicited by Acetylcholine and Other Humoral Agents

Publisher Summary This chapter focuses on the group of phosphatides known as “diacyl glycerophosphatides.” The structural feature that all of these compounds share is that they are derivatives of α-glycerophosphoric acid, the two remaining free hydroxyl groups of glycerol being esterified with long-chain fatty acids predominantly of the C 16 and C 18 varieties. The degree of unsaturation of the fatty acids varies with the source of the lipid. Esterification of the phosphate of the phosphatidic acid with any one of a variety of substances, which contain an alcoholic group, gives rise to the other glycerophosphatides. An additional glycerophosphatide—called “diphosphoinositide” because it yields inositol metadiphosphate, glycerol, and fatty acid on acid hydrolysis—is isolated from the brain. Studies on the specific physiological functions of phosphatides in the brain and other organs are discussed in the chapter. Because the phosphatides are important constituents of the cytoplasmic membranes, intracellular organelles, and myelin sheaths, a structural role is assumed as one of their functions. The chapter describes a particular aspect of physiological function in which some phospholipids play an active role. Stimulation of the secretion of sodium chloride, catecholamines, polypeptides, and proteins in a variety of endocrine and exocrine glands is associated specifically with the stimulation of the turnover of phosphatidic acid and phosphoinositide. A similar effect occurs in cholinergic synaptic tissue in response to acetylcholine. The stimulation in synaptic tissue is believed to be associated with the pumping out of sodium that enters the post-synaptic cell body on depolarization by acetylcholine. The various studies suggest that phosphatidic acid functions as a sodium carrier in what has been termed the “phosphatidic acid cycle.”

Phosphatidic acid phosphatase in the erythrocyte membrane

A magnesium-dependent α-phosphatidic acid phosphatase is present in the erythrocyte membrane which is stimulated by sodium, and to a lesser extent by lithium, ammonium, potassium and rubidium. Cesium produces little or no stimulation. No magnesium-dependent, sodium-stimulated, activity was observed with β-phosphatidic acid or lysophosphatidic acid; but both of these substrates were hydrolyzed. α-Glycerophosphate was hydrolyzed very feebly. Beryllium inhibited the magnesium-dependent phosphatidic acid phosphatase 30% at 10−4 M and completely at 10−3 M. Cesium and fluoride inhibited the sodium stimulation of the magnesium-dependent phosphatidic acid phosphatase. The initial phosphatidic acid phosphatase activity was at least 41.6 mμmoles/mg dry weight of ghosts/h, which compares favorably with the activity of the Na+ + K+-dependent, ouabain-inhibitable ATPase in the erythrocyte membrane; this is compatible with the idea that phosphatidic acid phosphatase may function as part of the Na+ + K+-dependent ATPase.

INHIBITION BY γ-HEXACHLOROCYCLOHEXANE OF ACETYLCHOLINE-STIMULATED PHOSPHATIDYLINOSITOL SYNTHESIS IN CEREBRAL CORTEX SLICES AND OF PHOSPHATIDIC ACID-INOSITOL TRANSFERASE IN CEREBRAL CORTEX PARTICULATE FRACTIONS1

1 γ‐Hexachlorocyclohexane inhibits the ACh‐stimulated synthesis of phosphatidylinositol in guinea pig cerebral cortex slices, as measured either by the incorporation of [2‐3H]inositol or of 32P. Phosphatidylinositol synthesis in the control slices is not inhibited. 2 The synthesis of phosphatidylinositol from CDP‐diglyceride in cerebral cortex microsomal preparations is inhibited by γ‐hexachlorocyclohexane. The incorporation of [2‐3H]inositol into lipid in the absence of added cytidine nucleotide in these preparations is not inhibited. 3 δ‐Hexachlorocyclohexane profoundly inhibits phosphatide synthesis and phosphate metabolism in cerebral cortex slices both in the presence and absence of ACh. This isomer also inhibits the exchange reaction for the incorporation of [2‐3H]inositol into lipid in the microsomal preparations. 4 α‐, and β‐Hexachlorocyclohexanes do not inhibit either ACh‐stimulated or control synthesis of phosphatidylinositol in cerebral cortex slices; nor do they inhibit the exchange reaction for [2‐3H]inositol incorporation into lipid in the microsomal preparations. 5 The specific effects of γ‐hexachlorocyclohexane are taken as providing evidence that ACh‐stimulated phosphatidylinositol synthesis in cerebral cortex slices probably involves the CDP‐diglyceride pathway. The possibility is discussed that the primary action of ACh in this system is to cause an increased activity of diglyceride kinase to provide phosphatidic acid for this pathway.

Studies on chemical mechanisms of the action of neurotransmitters and hormones: II. Increased incorporation of 32P into phosphatides as a second, adaptive response to pancreozymin or acetylcholine in pigeon pancreas slices☆

Abstract The concentrations of pancreozymin or acetylcholine necessary to give hormone-responsive amylase extrusion, and those necessary to give hormone-responsive increases in 32 P incorporation into phosphatides in pigeon pancreas slices, have been investigated. Low concentrations of pancreozymin or acetylcholine stimulated amylase extrusion with little or no effect on 32 P incorporation into phosphatides. High concentrations of pancreozymin gave increased incorporation of 32 P into phosphatides without increasing amylase extrusion above that observed with lower concentrations of hormone. Amylase extrusion in response to 10 −4 or 10 −6 m acetylcholine (plus eserine) was less than in response to lower concentrations of acetylcholine, but 32 P incorporation into phosphatides was much higher with the higher concentrations of acetylcholine. These results provide further evidence that increased synthesis or turnover of phosphatidylinositol, phosphatidic acid, phosphatidylethanolamine, and phosphatidylcholine in response to hormone is not a secondary consequence of zymogen granule extrusion (reverse pinocytosis). In view of the requirement for higher concentrations of hormone than those necessary to elicit amylase extrusion, it is suggested that the increased incorporation of 32 P into phosphatides may represent a second, adaptive response to hormone—part of a gearing up of the cell to meet greater demands for the secretion of zymogens than it had previously been meeting.

Studies on chemical mechanisms of the action of neurotransmitters and hormones: I. Relationship between hormone-stimulated 32P incorporation into phosphatidic acid and into phosphatidylinositol in pigeon pancreas slices☆

Abstract In pigeon pancreas slices, pancreozymin evoked an increased incorporation of 32P into phosphatidylinositol, phosphatidic acid, phosphatidylethanolamine, and, to a lesser extent, phosphatidylcholine. The pattern of this response is the same as that observed previously in response to acetylcholine. Pancreozymin or acetylcholine did not significantly affect either the uptake of orthophosphate-32P or the specific activity and levels of nucleotide P in pigeon pancreas slices. Phosphatidic acid is the first phosphatide to show increased 32P incorporation in response to hormone. The kinetics of 32P uptake indicate that a fraction of phosphatidic acid is formed which undergoes continuous turnover. The kinetics of 32P incorporation into phosphatidylinositol are compatible with the interpretation that the fraction of phosphatidic acid which is formed is a pool of intermediate for the synthesis of phosphatidylinositol in response to hormone in the pancreas. The fraction of phosphatidic acid which is formed in response to hormone disappears on removal of the hormone and appears to be converted to phosphatidylinositol. A new fraction of phosphatidic acid is formed if the tissue is again exposed to hormone. The experimental data are interpreted as indicating that the point of action of pancreozymin and acetylcholine on this aspect of phosphatide metabolism in the pancreas may involve activation of diglyceride kinase.